<%BANNER%>

Targeting the Nitric Oxide Signal Transduction Pathway as a Potential Ocular Hypotensive

Permanent Link: http://ufdc.ufl.edu/UFE0041178/00001

Material Information

Title: Targeting the Nitric Oxide Signal Transduction Pathway as a Potential Ocular Hypotensive
Physical Description: 1 online resource (96 p.)
Language: english
Creator: Dismuke, William
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Pharmacodynamics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nitric oxide donors decrease intraocular pressure by increasing aqueous outflow facility in the trabecular meshwork and/or Schlemm s canal however the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study we investigated the effects of nitric oxide donors on outflow facility. Additionally, we examined the nitric oxide -induced effects on trabecular meshwork cell volume, the signal transduction pathway(s) and ion channel involved. A novel protocol was used to measure cell volume in individual primary human and porcine trabecular meshwork cells. Cell volume was measured using Calcein AM fluorescent dye, detected by confocal microscopy and quantified using NIH ImageJ software. Inhibitors and activators were used to characterize the involvement of nitric oxide, soluble guanylate cyclase, cyclic GMP, protein kinase G and BKCa channel. An anterior segment organ perfusion system measured outflow facility. Exposure of trabecular meshwork cells to nitric oxide resulted in decreased cell volume and these decreases were abolished by ODQ and the BKCa channel inhibitor, IBTX, suggesting the involvement of soluble guanylate cyclase and K+ eflux respectively. The nitric oxide -induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the protein kinase G inhibitor, (RP)-8-Br-PET-cGMP-S suggesting the involvement cGMP and protein kinase G pathway. Additionally, the time course for the nitric oxide -induced decreases in trabecular meshwork cell volume correlated with nitric oxide -induced increases in outflow facility suggesting that the nitric oxide -induced alterations in cell volume may influence outflow facility.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by William Dismuke.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Ellis, Dorette.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041178:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041178/00001

Material Information

Title: Targeting the Nitric Oxide Signal Transduction Pathway as a Potential Ocular Hypotensive
Physical Description: 1 online resource (96 p.)
Language: english
Creator: Dismuke, William
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Pharmacodynamics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nitric oxide donors decrease intraocular pressure by increasing aqueous outflow facility in the trabecular meshwork and/or Schlemm s canal however the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study we investigated the effects of nitric oxide donors on outflow facility. Additionally, we examined the nitric oxide -induced effects on trabecular meshwork cell volume, the signal transduction pathway(s) and ion channel involved. A novel protocol was used to measure cell volume in individual primary human and porcine trabecular meshwork cells. Cell volume was measured using Calcein AM fluorescent dye, detected by confocal microscopy and quantified using NIH ImageJ software. Inhibitors and activators were used to characterize the involvement of nitric oxide, soluble guanylate cyclase, cyclic GMP, protein kinase G and BKCa channel. An anterior segment organ perfusion system measured outflow facility. Exposure of trabecular meshwork cells to nitric oxide resulted in decreased cell volume and these decreases were abolished by ODQ and the BKCa channel inhibitor, IBTX, suggesting the involvement of soluble guanylate cyclase and K+ eflux respectively. The nitric oxide -induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the protein kinase G inhibitor, (RP)-8-Br-PET-cGMP-S suggesting the involvement cGMP and protein kinase G pathway. Additionally, the time course for the nitric oxide -induced decreases in trabecular meshwork cell volume correlated with nitric oxide -induced increases in outflow facility suggesting that the nitric oxide -induced alterations in cell volume may influence outflow facility.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by William Dismuke.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Ellis, Dorette.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041178:00001


This item has the following downloads:


Full Text

PAGE 1

1 TARGETING THE NITRIC OXIDE SIGNAL TRANSDUCTION PATHWAY AS A POTENTIAL OCULAR HYPOTENSIVE By WILLIAM MICHAEL DISMUKE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF TH E REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

PAGE 2

2 2009 William Michael Dismuke

PAGE 3

3 To my parents Bill and Nancy

PAGE 4

4 ACKNOWLEDGMENTS I thank my parents

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF FIGURES .........................................................................................................................8 ABSTRACT .....................................................................................................................................9 CHAPTER 1 INTRODUCTION ..................................................................................................................11 Intraocular Pressure and Glaucoma ........................................................................................11 Cellular Mechanisms that Regulate Outflow Facility ............................................................12 Nitric Oxide Regulation of Intraocular Pressure ....................................................................13 Other Activators of Soluble Guanylate Cyclase .....................................................................15 Direct Activation of the Large conductance, Calcium activated Potassium Channel ...........15 2 MATERIALS AND METHODS ...........................................................................................17 Cell Culture .............................................................................................................................17 Outflow Facility Measurements .............................................................................................18 Cell Volume Measurements ...................................................................................................19 Drug Preparation .....................................................................................................................22 3 NOINDUCED REGULATION OF HUMAN TRABECULAR MESHWORK CELL VOLUME AND AQUEOUS HUMOR OUTFLOW FAC ILITY INVOLVE THE BKCa ION CHANNEL .....................................................................................................................24 Introduction .............................................................................................................................24 Materials and Methods ...........................................................................................................26 Tissue ...............................................................................................................................26 Cell Culture .....................................................................................................................26 Measurement of Cell Volume .........................................................................................27 Outflow Facility Measuremen ts ......................................................................................28 Materials and Reagents ....................................................................................................29 Statistics ...........................................................................................................................30 Results .....................................................................................................................................30 NO Donors Increase Outflow Facility .............................................................................30 NO Decreases TM Cell Volume ......................................................................................31 Changes in Cell Volume in Response to Changes in Osmolarity ...................................32 The NOinduced Decrease in Cell Volume Involves Activation of sGC and cGMP .....33 Involvement of Protein Kinase G in the NO Induced Decreases in TM Cell Volume ...34 BKCa Channels are Involved in the NO Induced Decreases in TM Cell Volume ...........34

PAGE 6

6 Discussion ...............................................................................................................................35 Outflow Facility ...............................................................................................................35 Cell Volume Studies ........................................................................................................37 Acknowledgements .................................................................................................................41 Grant .......................................................................................................................................41 4 HUMAN TRABECULAR MESHWORK CELL VOLUME DECREASE BY NOINDEPENDENT SOLUBLE GUANYLATE CYCLASE ACTIVATOR YC 1 AND BAY582667 INVOLVE THE BKCa ION CHANNEL ........................................................49 Introduction .............................................................................................................................49 Materials and Methods ...........................................................................................................50 Cell Culture .....................................................................................................................50 Measurement of Cell Volume .........................................................................................51 cGMP Assay ....................................................................................................................52 Materials and Reagents ....................................................................................................52 Statistics ...........................................................................................................................52 Results .....................................................................................................................................52 YC1 and BAY582667 Induced Regulation of TM cell Volume are Biphasic ...........52 The YC1induced Decrease in Cell Volume Involves Activation of Soluble Guanylate Cyclase and cGMP .....................................................................................53 The YC1 and BAY 582667induced Increases in Cell Volume Do Not Involve cGMP ...........................................................................................................................54 PKG is Involved in the YC 1 and BAY 582667 induced Decreases in TM Cell Volume .........................................................................................................................54 BKCa Channels are Involved in the YC 1 and BAY 582667 induced Decreases in TM Cell Volume ..........................................................................................................55 Discussion ...............................................................................................................................55 Funding ...................................................................................................................................59 5 ACTIVATION OF THE BKCa CHANNEL INCREASES OUTFLOW FACILITY AND DECREASES TRABECULAR MESHWORK CELL VOLUME ........................................66 Introduction .............................................................................................................................66 Materials and Methods ...........................................................................................................67 Cell Culture .....................................................................................................................67 Measurement of Cell Volume .........................................................................................68 Outflow Facility Measurements ......................................................................................68 Materials and Reagents ....................................................................................................68 Statistics ...........................................................................................................................69 Results .....................................................................................................................................69 NS1619 Increases Outflow Facility .................................................................................69 NS1619induced Decrease in TM Cell Volume is Concentration Dependent ................70 The BKCa Channel is Involved in TM Cell Regulatory Volume Decrease .....................70 Effects of NS1619 and DETA NO on Cell Volume Are Not Additive ..........................71 Discussion ...............................................................................................................................71

PAGE 7

7 6 CONCLUSION .......................................................................................................................79 Activation of Soluble Guanylate Cyclase and IOP .................................................................79 YC1 and BAY 582667 .........................................................................................................82 YC1 ................................................................................................................................82 BAY582667 ..................................................................................................................83 Direct Activation of the Large Conductance, Calcium Activated Potassium Channel .........84 Physiological Significance ......................................................................................................85 Future Directions ....................................................................................................................85 LIST OF REFERENCES ...............................................................................................................87 BIOGRAPHICAL SKETCH .........................................................................................................96

PAGE 8

8 LIST OF FIGURES Figure page 31 NO donors increase outflow facility in porcine anter ior organ culture perfusion. ..........42 32 NO decreases TM cell volume. ........................................................................................43 33 Changes in osmolarity effect changes in TM cell volume. ................................................44 34 sGC mediates the NO induced decreases in TM cell v olum e ...........................................45 35 Effects of PKG inhibitor (RP)8Br PETcGMP S (PKGi) on 8 Br cGMP and DETA NO induced decreases in TM cell volume. .........................................................46 36 BKCa channels mediate the NO induced decreases in TM cell volume. ...........................47 37 Summary diagram of the pathway of NO regulation of TM cell volume. .........................48 41 YC1 and BAY 582667induced regulation of TM cell volume is biphasic. ..................60 42 Involvement of sGC/cGMP in the YC 1 and BAY 582667ind uced decrease in TM cell volume. ......................................................................................................................61 43 Low concentration YC 1 and BAY58 2667 induced increases in cell volume are not associated with significant increases in cGMP. ...........................................................62 44 PKG is involved in the YC 1 and BAY 582667induced decreases in TM cell volume. ...............................................................................................................................63 45 The YC1 and BAY 582667 induced dec reases in TM cell volume involve the BKCa channel. .....................................................................................................................64 46 Summary diagram of the pathway of NO dependent and NO independent regulation of TM cell volume. ............................................................................................................65 51 NS1619 increases outflow facility. ....................................................................................75 52 NS1619induced decreases in TM cell volume are concentration dependent. ..................76 53 NS1619 abolishes the hypotonic induced increases in TM cell volume. ..........................77 54 Actions of NS1619 and DETA NO on TM cell volume are not additive and are abolished by IBTX. ............................................................................................................78

PAGE 9

9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TARGE TING THE NITRIC OXIDE SIGNAL TRANSDUCTION PATHWAY AS A POTENTIAL OCULAR HYPOTENSIVE By William Michael Dismuke December 2009 Chair: Dorette Z Ellis Major: Pharmaceutical Sciences Pharmacodynamics Nitric oxide donors decrease intraocular pressure by i ncreasing aqueous outflow facility in the trabecular meshwork and/or Schlemms canal however the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility In this study we investigated the effects of nitric oxide donors on outflow facility. Additionally, we examined the nitric oxide induced effects on trabecular meshwork cell volume, the signal transduction pathway(s) and ion channel involved. A novel protocol was used to measure cell volume in individual primary human and porcine trabecular meshwork cells. Cell volume was measured using Calcein AM fluorescent dye, detected by confocal microscopy and quantified using NIH ImageJ software. Inhibitors and activators were used to characterize the involvement of nitric oxide soluble guanylate cyclase, cyclic GMP, protein kinase G and BKCa channel. An anterior segment organ perfusion system measured outflow facility. Exposure of trabecular meshwork cells to nitric oxi de resulted in decreased cell volume and these decreases wer e abolished by ODQ and the BKCa channel inhibitor, IBTX, suggesting the involvement of soluble guanylate cyclase and K+ eflux respectively. The n itric oxide induced decreases in cell volume were mimicked by 8 Br cGMP and abolished by the protein kinase G inhibitor, (RP) 8Br PETcGMP S suggesting the involvement cGMP and

PAGE 10

10 protein kinase G pathway. Additionally, the time course for the nitric oxide induced decreases in trabecular meshwork cell vol ume correlated with nitric oxide induced increases in outflow facility suggesting that the n itric oxide induced alterations in cell volume may influence outflow facility.

PAGE 11

11 CHAPTER 1 INTRODUCTION Intraocular Pressure and Glaucoma Elevated intraocular pressure (IOP) put s a patient at a n increased risk for de veloping visual field loss, in the progressive blinding disease glaucoma. IOP results from the balance between aqueous humor secr etion by the ciliary processes and outflow through the trabecular mesh work and Schlemms canal The major route for the outf low of aqueous humor is the trabecular meshwork 1 2 com prising of uveal and corneoscleral trabecular meshwork 1 and the juxtacanalicular cells (JCT trabecular meshwork ) 3 in conjunction with the Schlemms canal. An improper balance of aqueous humor secretion and outflow will yield high pressures within the eye. Through an unknown mechanism, this elevated IOP increases the likely hood of retinal ganglion cell death. As retinal ganglion ce lls are lost, the patients vision is progressively lost as well, both of which are irreversible. Since we are currently unable to repair the damage from the loss of the retinal ganglion cells, the only viable therapy for patients with ocular hypertension i s management of IOP. Currently IOP can be managed by two major methods, ocular hypotensive drugs or surgery. The surgical methods to reduce IOP are numerous and well outside the scope of these studies. In general they are intended to increase the drainage of aqueous humor from the eye, but from here on will not be mentioned. Our focus will be on pharmacological methods to reduce IOP. To lower IOP, ocular hypotensive drugs generally work by reducing the production of aqueous humor or increasing the drainage of aqueous humor. Inhibitors of carbonic anhydrase, an enzyme involved in aqueous humor secretion, lower IOP by reducing aqueous humor production. Conversely, prost a glandin analogs lower IOP by increasing the drainage of aqueous humor from the eye. While both of these IOP lowering strategies are effective, these studies will

PAGE 12

12 focus on the outflow of aqueous humor, as dysfunctions in aqueous humor outflow are thought to underlie the IOP increases in the most common form of glaucoma, primary open angle Cellu lar Mechanisms that Regulate Outflow Facility Once thought to be a passive process, the outflow of aqueous humor is now known to be actively controlled by the cells in the aqueous humor outflow pathway. Several mechanisms have been characterized by which c hanges in aqueous humor outflow can occur. These include trabecular meshwork contractility and cell volume changes, ciliary muscle contractility, alterations of the ECM in the trabecular meshwork and pore formation and vacuoles in the inner wall of the Sch lemm s canal4 10 This research focus es on trabecular meshwork cell volume changes and the role it plays in regulating outflow facility. Our goal is to identify a signal transduction pathway in trabecular meshw ork cells by which nitric oxide and NO independent sGC activators mediate increases in aqueous humor outflow. Cell volume changes in the aqueous humor outflow pathway are of interests to study as they have been correlated with changes in outflow facility. Previous studies demonstrate that a hypotonic or hypertonic challenge to a perfused eye anterior segment will decrease or increase outflow facility respectively7 To begin to hypothesize a mechanism by which changes in trabecular meshwork cel l volume would affect aqueous humor drainage we must understand the architecture of the aqueous humor outflow pathway. The trabecular meshwork is composed of interwoven collagen covered elastin beams. Trabecular endothelial cells (trabecular meshwork cel ls) line the outflow pathway and have been suggested to perform a number of functions including phagocytosis11 and extracellular matrix turnover12 As aqueous humor flows deeper into the trabecular meshwork, toward the S chlemms canal, the open spaces available for fluid to flow through decrease. As the space for fluid flow decreases, the volume of the trabecular meshwork cells lining these openings would begin to exert more influence over outflow facility.

PAGE 13

13 Increasing or decreasi ng the volume of these cells would therefore decrease or increase the rate by which fluid flows exits the eye, respectively. To understand the components involved in maintaining and/or returning to resting cell volume and basal outflow facility investigat ors have utilized perfused eye anterior segments. Using this technique, s everal ion channels have been implicated in mediating the recovery to baseline outflow facility following a hypotonic or hypertonic challenge. R ecovery from a hypotonic challenge was slowed and the magnitude of the decrease in outflow facility was greater in the presence of the BKca channel inhibitor iberiotoxin. The opposite effect was seen when the BKca channel activator NS1619 was included with the hypotonic challenge. Recovery to baseline outflow quickened and the magnitude of the outflow facility reduction was decreased. This study also implicated the chloride swell channel in mediating a recovery for hypotonic challenge as the channel blocker tamoxifen produced effects similar to iberiotoxin5 Additionally, the Na, K, 2Cl cotransporter blocker bumetanide, which has been shown to decrease TM cells volume6 i ncreased outflow facility in an eye anterior segment perfused with isotonic medium7 While these mechanisms have been experimentally shown to contribute to the regulation of aqueous humor outflow, the possibility exist that other unknown mecha nisms also contribute. Nitric Oxide Regulation of Intraocular Pressure The gaseous signaling molecule nitric oxide (NO) has been shown to regulate both the production and outflow of aqueous humor. NO mediates many of its effects through soluble guanylate cyclase (sGC) and increases in cyclic GMP (cGMP). Because the binding of NO to sGC results in decreased IOP, the study of the activation sGC in the tissues of the aqueous humor outflow pathway, becomes critically important since it may provide new targets for the development of ocular hypotensive drugs.

PAGE 14

14 NO is a diatomic gas with a very short half life of just a few seconds 13 It is produced by a family of enzymes, the nitric oxide syntheses (NOS), through the conversion of L Arginine to NO and L citrulline. The active enzyme is composed of a hemebound homodimer assembled from heme deficient monomers. There are 3 major types of NOS in mammalian tissue; endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). eNOS and nNOS are expressed constitutively, and are capable of producing low levels of NO, <1uM 14 NO production it thought to have two distinct roles in the body; production of an autocrine/paracrine signaling molecule and production of a toxic free radical gas. With all three isoforms producing the same molecule, the pathophysiological implications of any inappropriate NO production must be kept under consideration. NO generated by nNOS and eNOS has been shown to activate cyclic nucleotide gated channels, protein kinases, and phosphodiesterases 15 These p hysiological processes may be considered low concentration effects, requiring less than 1 micromolar of NO 14 High concentrations of NO produced by iNOS can result in auto oxidation and the production of dinitrogen trioxide 16 which is the primary mechanism of nitrosylation in which nitrosothiols of cysteines are formed. In addition, strong oxi dants such as superoxide ( a reactive oxygen species) can act with NO to form peroxynitrite 17 which reacts with the phenol moiety of tyrosine resulting in nitration of tyrosine residues in proteins. Both nitrosylation and nitration of tyrosine residues have been show n to affect protein function. The implications of these findings on the effect of high concentrations of NO to increase aqueous humor outflow and NOs effect on retinal ganglion cells are unknown. This is outside the scope of our study, but must a lways remain under consideration. Here we will focus on the effects of low, physiologically relevant concentrations on NO such as produced by nNOS or eNOS

PAGE 15

15 NO mediates its physiological effects by binding to sGC. Once bound to sGC, it causes the conversion of GTP to 3 5 cyclic guanosine monophosphate (cGMP). cGMP will then go on to interact with various cyclic nucleotide gated channels, protein kinases and protein phosphodiesterases to produce physiological effects. Other Activators of Soluble Guanylate Cyclase In addition to the endogenous sGC activator NO, compounds such as YC 1 have been shown to activate sGC independent of NO18 and decrease IOP19 Though the mechanism by which YC1 activates sGC is not fully understood, it thought to affect the positioning of the heme in the enzyme20 or enhance the activation of sGC by NO21 With evidence of a decreased ability to produce NO in the trabecular meshwork of glaucomatous eyes22 it is also of interest to note the reported synergistic effect of YC 1 on the NO induced stimulation of sGC23 This suggests the possibility that drugs like YC 1 may be used, not only to activate sGC, but also to amplify the diminished, endogenous NO production capacity suggested by the above mentioned study. In addition to YC 1like, heme dependent sGC activators, several hemeindependent sGC activators have been characterized. These compounds, like BAY 582667, are reported to activate sGC even though the heme may be absent or oxidized24 a state in which NO cannot activate sGC. With the complexities of NOs participation in the redox state of a cell, as well as the ability of YC 1 to act synergistically with NO and BAY58 2667 to activate sGC independent of heme it is of significant clinical interest to investigate the ability of NO independent sGC activators to affect outflow facility and volume changes in trabecular meshwork cells. Direct Activation of the Large conductance, Calcium activated Potas sium Channel With the above mentioned studies on the influence of the BKca channel on recovery of basal outflow facility following a hypo or hypertonic challenge, studies demonstrating the

PAGE 16

16 cGMP induced activation of this channel in bovine trabecular meshw ork cells 25 and studies demonstrating the channels involvement in cGMP induced relaxation of isolated bovine trabecular meshwo rk strips26 we are interested in investigating the role BKca channel activation plays in r egulating trabecular meshwork cell volume and outflow facility. The BKca channel is a potassium channel, with its activity regulated endogenously by membrane potential, intracellular calcium concentration27 and cGMP dependent protein phosphorylation events28, 29 By allowing a large efflux of potassium, when opened, the channel is capable of hyperpolarizing the cell. That and the ability of cGMP dependent pro tein phosphorylation to modulate channel activity led investigators to test and demonstrate the involvement of the BKca channel in the NO induced relaxation of smooth muscles. With this in mind, we examine the role the BKca channel plays in the NO \ cGMP induced increases in outflow and decreases in trabecular meshwork cell volume, as well as the role the channel plays in restoring resting cell volume following a hypoor hypertonic challenge. To do this we utilize a well characterized BKca channel blocker i beriotoxin (IBTX)30 In addition to the endogenous BKca activity modulators mentioned above, several compounds have been synthesized that are reported to directly activate the channel. For our studies we will use the benzimidazolone derivative, NS1619 as our BKca channel activator31

PAGE 17

17 CHAPTER 2 MATERIALS AND METHODS Cell Culture For our in vitro studies we obtained h uman donor eyes with no history of ocular disease or surgery from Lions Eye Institute (Tampa, Fl) within 24 30 hours postmortem. Primary human TM cell lines were developed and named according to the age of the donor (ie HTM80). Prior to dissection, eyes were stored in a moist en vironment at 4C. Porcine eyes were procured from the local abattoir within 1 hour postmortem, stored in PBS and kept on ice. To kill any containments on the exterior of the eye, the eye is submerged in a 1:1 solution of phosphate buffered saline solution (PBS) and Betadine (providone iodine, 7.5%) for 3 minutes, then rinsed three times in PBS. Following that, s tandard ophthalmic microsurgery instruments were used to hemi sect the eye and remove the lens, iris and ciliary body. The remaining anterior portion of the eye is bisected and the TM was removed from both halves using forcepts The TM stripes were them placed in a filtered solution containing 5mL PBS, 5mg collagenase type IV and 75uL human albumin. This solution containing TM explants was vigorously shaken and placed in a 37C water bath for 30 minutes, with additional vigorous shaking at 10 minute intervals. This process is intended to breakdown the type 4 collagen, an ECM component upon which trabecular meshwork cells are thought to directly attach32 thus increasing t he likelihood trabecular meshwork cells will migrate off the dissected TM strips and onto culture dishes. However, long term exposure to the collagenase solution may have negative effects on the proliferation of the cells in the culture dish and needs to be removed. To remove the collagenase, the tube containing TM explants and collagenase solution was centrifuged at 800 x g for 8 minutes. The resulting supernatant was aspirated and the pellet resuspended in 5mL of low glucose (1 g/L) DMEM (Mediatech, Herdo n, VA) containing 10% (v/v) FBS, 100 U/mL penicillin and 100 ug/mL

PAGE 18

18 streptomycin and transferred to one well of a gelatin coated 6 well plate. Cells were grown in a tissue culture incubator at 37C in 5% CO2. Confluent cells were trypsinized and passaged. To ensure the human cells we have cultured are indeed from the trabecular meshwork they were validated by their morphology and the presence of dexamethasone induced myocilin expression. Specifically trabecular meshwork cells in vivo produce a mutant form o f the protein myocilin when exposed to synthetic glucocort i coids such as dexamethasone. This is the etiology underlying the steroidinduced form of glaucoma. To use the trabecular meshwork cells sensitivity to dexamethasone to validate their origin, we use immunocytochemistry and a polyclonal myocilin antibody33 We grew the suspected TM cells on chambered slides to 80% confluency. Dexamethasone (100nM) in DMEM with 10% fetal bovine serum was added to the cells. The cells were exposed to dexamethasone for 7 days washed in PBS, fixed in ice cold methanol for 7 minutes, washed in PBS again and stored at 20C. The sli des were thawed on the benchtop, washed with TBST and blocked in 5% goat serum for 1 hour. The polyclonal myocilin antibody in TBST (1: 1000) was added to the slide and incubated at 4C in a moist environment overnight. The slides were washed three times in TBST and a Cy3 conjugated goat anti mouse secondary antibody in TBS T (1: 1000) was incubated for two hours at room temperature. Porcine TM cells were validated by their ability to take up acetylated LDL labeled with BODIPY FL and secrete tissue plasminogen activator. Human and porcine TM cells were serum starved for 48 hours prior to being used in experimental protocols. For our experiments, cells from passage 35 were used Outflow Facility Measurements Porcine eyes were obtained from a local abattoir within 1 hours of the animals death and kept in DPBS on ice prior to dissection. Standard ophthalmic microsurgery instruments were used to hemi sect the eye and remove the lens, iris and ciliary body. The dissected anterior

PAGE 19

19 portion of the eye was mounted in an anterior segment organ culture perfusion chamber. The anterior segment is then placed in a cell culture incubator kept at 37C in 100% humidity with a 5% CO2 atmosphere and perfused at a constant 14mmHg. The perfusate for these studies was l ow glucose (1g/L) DMEM with 100 U/ml penicillin and 100 g/ml streptomycin O utflow facility is calculated as the loss of perfusion media from the supply bottle, determined by weight, over time divided by the constant pressure of 14mmHg. The anterior segments were allowed to perfuse for 12 to 48 hours prior to drug treatments to al low for the establishment of a stable basal outflow facility. To administer experimental treatments to the anterior segment, a constant pressure fluid exchange system was used. This system allows for the complete exchange of the media contained in the arti ficial anterior chamber (~600 L) without a change in pressure, giving us the ability to apply a bolus of the experimental treatment. Outflow facility measurements are recorded by a computer every minute for the entire duration of the anterior segments perf usion. For the analysis, each perfusion experiment is normalized to its mean baseline outflow facility, giving us a ratio of the pre and post treatment outflow facilities (C0/CD) facility 7, 34, 35 The data from several perfusion experiments are pooled and an ANOVA and Holm Sidak post hoc tests are performed to determine any statistical ly significant effect of the experimental treatment on outflow facility. Cell Volume Measurements Previous studies examining changes in trabecular meshwork cell volume could not measure the volume of a single trabecular meshwork cell over time that was fi rmly attached to its substrate6, 36 the normal state for a trabecular meshwork cell in vitro To achieve this, we utilized cultured human and porcine trabecular meshwork cells and a laser scanning confocal microscope. Low passage human and porcine TM cells (passage 2 5) in either collagen coated

PAGE 20

20 Lab Tek II chambered coverglass (Nalge Nunc) or collagen coated 35mm FluoroDishes (WPI Inc) were grown to confluency. These cells were then serum st arved for 48 hours prior to the start of the cell volume measurements. The TM cells were incubated with the fluorescent dye Calcein AM (Molecular Probes) at a concentration of 1 M for 1 hour in a tissue culture incubator at 37C in 5% CO2. The original, membrane permeable, nonfluorescent acetylmethyl ester (AM) form of the dye diffuses into the cells where it undergoes acetylmethyl ester hydrolysis, becoming membrane impermeable and fluorescent. We use this intracellular fluorescence as a marker for intracellular volume. Low passage TM cells incubated with calcein were removed from the tissue culture incubator and placed on the microscope stage. Cells were visualized with a 20X objective and a region of cells was selected for the capturing of confocal image stacks. Individual confocal images were captured in an 8 bit, 1024x1024 or 512x512 pixel format. Spacing between images in the image stack was set at 1 m. To ensure that both the upper and lower portions of the cells being imaged were included in the image stack, we were mindful to set the upper and lower bounds of the area to be scanned, at least 1 m above and below where the cells upper and lower porti on appeared to be. To capture images of the same cells over time, we set the microscope to capture image stacks at 5 minute intervals for 20 minutes. The first image stack is captured and upon completion, our experimental compound is carefully pipetted int o the dish containing our cells. The microscope will capture four additional image stacks 5, 10, 15 and 20 minutes following the addition of our experimental compound. For studies involving enzyme or ion channel inhibitors, the inhibitor was added 5 minute s prior to capturing the initial image stack. However, for experiments involving a hypoor hypertonic challenge a modification of the above protocol was needed as five minute intervals for volume measurements were too long to capture

PAGE 21

21 the rapid volume cha nges induced by tonicity changes. The need to quickly capture image stacks for each time point required we reduced our resolution to 512x512 and increased the scan speed from 400hz to 700hz. This increase in scan necessitates a 1.7 ratio digital zoom, reducing the number of cells per treatment we could image. By altering our protocol this way, we can capture a complete zstack with a 1 m z spacing in less than 1 minute allowing us to take an initial, resting cell volume measurement, add our hypoor hypert onic solution then scan the cells at a 1 minute interval for 10 minutes. To quantify cell volume measurements from the confocal image stacks we utilize NIH ImageJ software and a custom voxel counting macro. First we must determine a fluorescence intensity threshold value to allow us to mark the boundary between intracellular and extracellular space. To do this we image fluorescent microspheres of known diameter and volume that have similar fluorescence intensity as our calcein incubated cells. Our confocal images were captured with an intensity resolution of 8bits or 256 (ie 2^8) shades from light to dark. In defining a threshold value to use in our cell volume analysis, we selected a brightness between 0 and 255, which will thereafter define the intracellu lar (bright from calcein fluorescence) and extracellular (dark) space. To select this threshold value, we analyze the volume of a number of the fluorescent microspheres using all 256 possible threshold values. The threshold value yielding the most accurate microsphere volume (known from the manufacture) will then be used as the threshold value for further cell volume analysis. Image stacks from the experiment are then imported into ImageJ and the outer, lateral boundary of each cell in the first image stack (the initial or 0 time point, without drug) is defined by hand (known as a region of interest or ROI) and saved. The threshold value determined above is then applied to the image stack, yielding a black and white or binary image and all thresholded pi xels (black) within each ROI

PAGE 22

22 are counted for each image in the image stack. The resulting number is the voxel count, a number representing the volume of each cell in the image stack. This number is used as the initial, resting cell volume, prior to drug treatment. Similarly, we imported the image stack from the next time point and ran the voxel counting macro again, using the saved RIOs from the initial (0 time point) image stack. By ensuring that the microscope stage did not shift during our experiment the RIOs defined in the 0 time point image stack should also accurately define the outer boundary of each cell in the subsequent image stacks. The voxel counts for each cell, at each time point was then imported to a spreadsheet. To represent this data as a percentage change in cell volume, we divide the voxel count for each cell at each time point by the voxel count of that cell at the 0 time point. Statistical comparisons were performed by ANOVA followed by the Holm Sidak or Fisher least significant differe nce method for the comparison among different means. Drug Preparation DETA NO (1 [N (2 Aminoethyl) N (2 ammonioethyl)amino]diazen1ium 1,2diolate) o Solid dissolved in deionized water, 296mM stock ODQ ( 1H [1,2,4]oxadiazolo[4,3a]quinoxalin1one) o Solid dissolved in DMSO, 27mM stock Rp 8Br PETcGMPS o Solid dissolved in deionized water, 4.36mM stock IBTX (Iberiotoxin) o Solid dissolved in deionized water, 10 M stock Hypertonic solution o D mannitol dissolved in DMEM, 300mM stock Hypotonic solution o DMEM:deionized water (2:3, v:v) YC1( 3(5' hydroxymethyl 2' furyl) 1benzylindazole) o Solid dissolved in DMSO:Ethanol (1:1), 30mM stock

PAGE 23

23 o Working solution, 30mM stock:DMEM:DMSO:Ethanol (2:2:1:1) o Working solution kept at 37C until use o Use immediately BAY582667 o Solid dissolved in DMSO:Ethanol (1:1), 30mM stock o Working solution, 30mM stock:DMEM:DMSO:Ethanol (2:2:1:1) o Working solution kept at 37C until use o Use immedi ately NS1619 ( 1,3Dihydro1 [2 hydroxy5(trifluoromethyl)phenyl] 5(trifluoromethyl)2H benzimidazol 2one) o Solid dissolved in Ethanol 13.8mM stock

PAGE 24

24 CHAPTER 3 NOINDUCED REGULATION OF HUMAN TRABECULAR MESHWORK CELL VOLUME AND AQUEOUS HUMOR OUTFLOW FAC ILITY INVOLVE THE BKCA ION CHANNEL Introduction Maintenance of correct intraocular pressure (IOP) is a requirement for good vision. Two major factors contribute to IOP, production of aqueous humor by the ciliary processes and the outflow of aqueous humor through the trabecular meshwork (TM) and Schlemms canal; thus increases in production and/or decreased in outflow facility of aqueous humor could result in high IOP. In fact, i ncreased resistance to aqueous humor outflow through the juxtacanalicular regio n of the TM has been implicated in primary open angle glaucoma 37 a blinding disease that affects millions of people worldwide. Decreasing IOP is a v iable strategy for preventing blindness caused by glaucoma and slowing its progression. The TM is comprised of three anatomical r egions, the uveal, corneoscleral and the juxtacanalicular regions 3, 38 ; with two distinct cell p opulations 39 The cellular mechanisms underlying changes in a queous humor outflow througth the TM are not well understood: however, s everal cellular mechanisms have been proposed 40 The TM is thought to be a smooth musclelike tissue with contractile properties 41, 42 Contraction and relaxation of the cells are thought to regulate aqueous humor outf low 4346 Similarly, increases or decreases in the volume of TM cells could influence outflow. 5 7, 4749 Trabecular meshwork cell volume is influenced by the activities of the Na K 2Cl exchanger 4, 6, 7 the Na+/H+ transporter 36 and the K+ and Cl channels 5, 36 Further, it is possible that both the cellular contractile mechanisms and the cell volume regulatory mechanisms are functionally linked 50 52 as the large conductance calcium activated potassium channel (BKCa), have been shown to regulate TM cell volume and contractility 5, 45 and outflow facility 5

PAGE 25

25 The BKCa channels are activated by increased calcium 53, 54 changes in cell membrane voltage 55 or neurotransmitters including NO 28, 56 In cerebral artery smooth muscle cells the phosphorylation of the subunit of the BKCa channel by PKG has been shown to mediate the NOinduced activation the BKCa channel 28 The human TM is enriched with the NO producing enzym e, nitric oxide synthase (NOS) particularly endothelial NOS (NOS III) 57 and nitrinergic nerve terminals 58 NO donors reduced IOP in both normal 59 and glaucomatous rabbit eyes 60 without systemic effects 59 Additionally, intracameral injection of NO donors decreased IOP by increasing outflow of aqueous humor 61 Similarly i ntravitreal and intracameral injections of NO donors in rabbits caused drastic decrease s in IOP, concurrent with nitrite production the measurable result of NO production 62 Other studies have shown that NO donors reduce IOP in monkeys through an action on outflow resistance 35 While NO donors effectively reduce IOP, the signaling cascade mediating the cellular response in this tissue is unknown. The present study was designed to elucidate the mechanism of response of the TM to NO We tested the hypothesis that NO donors regulat e TM cell function by decreasing TM cell volume. We tested the involvement of a regulatory pathway by which NO acting via activation of sGC, cGMP, and PKG decrease TM cell volume. We also examined the role of BKCa channel in the NO induced decreases in c ell volume and the NO induced increases in outflow facility and determined if the time course for the NO induced decreases in TM cell volume correlated with the NOinduced increases in outflow facility.

PAGE 26

26 Materials and Methods Tissue Because morphologica l and biochemical studies suggested that the porcine anterior chamber perfusion model can be correlated with the human perfusion system 63 perfusion studies were performed using porcine eyes. Cellular studies w ere performed in human primary TM cells and parallel studies were performed in primary porcine TM cells. Cell Cultur e Eyes from human donors with no history of ocular disease or surgery were obtained from Lions Eye Institute ( Tampa, FL) within 2430 hour s postmortem. Primary human TM cell lines (numbers representing ages of the donors) (HTM44; generous gift of Dr. D. Stamer, HTM26, HTM71, HTM36, HTM80 and HTM86) were developed. For our experimental protocols cells from early passages (3 5) were used. H uman TM explants were obtained either from whole eyes that were stored in a moist environment at 4oC or from corneal scleral rims stored in O ptisol (Dexol; Chiron Ophthalmics, Irvine, CA) at 4oC. Porcine eyes were obtained from the local abattoir within 1 hour postmortem and maintained on ice. We used standard ophthalmic microsurgery instruments to bisect the eyes and remove the cornea, iris, lens and ciliary body. TM cells were isolated after collagenase digestion of TM explants 64 Collagenasetreated cells were grown in low glucose (1g/L) DMEM (Mediatech, Herdon VA.) in the presence of 10% f etal bovine serum (Mediatech, Herdon VA.), 100 U/ml penicillin and 100 g/ml streptomycin (Mediatech, Herdon VA.). Cells were grown in 6well culture dishes (Nalge Nunc International, Rochester, NY) in a tissue culture incubator @ 37oC in 5% CO2. Confluent cells were trypsinized and passaged. We validated human TM cells by their morphology and the presence of dexamethasone induced myocillin expression 33 To identify porcine TM cells we used the ability of TM cells to take up acetylated low density lipoprotein and secrete tissue plasminogen

PAGE 27

27 activator. For experimental protocols, TM cells were grown on Lab Tek ll chambered cover glass (Nalge Nunc International, Rochester, NY) in low glucose DMEM as described above to 100% confluency, after which the y were exposed to serum free media for 2 days prior to performing the experiments. Measurement of Cell Volume Cell volume measurements were performed using the modified protocols of Mitchell et al and Bush et al 36, 65, 66 P rior to any drug treatments, the cells were loaded with the fluorescent dye Calcein AM in DMEM at 37C, in 5% CO2 incubator for 60 minutes to ensure a stable baseline. The cove rslips containing the cells were subjected to confocal microscopy using a Leica confocal microscope. For some experiments a Leica confocal microscope with a platform containing a 37C, 5% CO2 incubator was used. We developed a technique of drug delivery t o the TM cells on the cover slips to ensure that the slides did not shift during imaging and that images would be taken of the same cells. Specifically, several ports were drilled in the covers of the glass chambers. Tubes attached to syringes were insert ed into each port allowing for the exchange of media and drugs. Images were taken without drug treatment, (0) time point, this served as the experimental control. Drugs were then added to the cells through the ports without shifting the cover slip. Image s were taken of the same cell (s) at varying time periods following application of the drugs. Additionally, images were taken of cells that were not exposed to drugs at the time periods indicated above to serve as controls for evaluating the stability of the dye. In some experiments media containing drugs were carefully removed from the cover slip and fresh media was added. Images were taken of the same cells and changes in cell volume were quantified. To assess the functioning of the volume regulatory mechanisms in TM cells the osmolarity of the media was changed. Hypertonic medium was made by addition of 150 mM mannitol to DMEM (~469 mOsm/kg) and

PAGE 28

28 hypotonic medium was made by addition of deionized water to DMEM for a final concentration of 30% water and 70% DMEM (~208 mOsm/kg). For our experiments, the microscope captured either a 1024 x 1024 or a 512 x 512 pixel image with 8 bits of resolution (256 colors). The confocal microscope captures images in three dimentions, allowing t he NIH ImageJ software t o identify the top and bottom edges of the cell. I mages were converted from 8 bit to binary values using a threshold that was determined by analysis of fluorescent Fluoresbrite latex beads (Polyscience Inc., Warrington) of known diameter and volume that we re imaged under conditions identical to those used for TM cells. A region of interest was then selected around each cell and t he ImageJ software was used to calculate the number of voxels in the region of interest in the image stack Changes in cell volume were determined by dividing the voxel count with drug treatment by the voxel count without drug treatment. Outflow Facility Measurements Anterior segment perfusion organ culture was used to measure outflow as described by Johnson et al 67, 68 Porcine eyes were obtained from the local abattoir and maintained on ice following enucleation and bisected within 2 hours postmortem. Eyes were bisected at the equator and the iris, le ns and ciliary processes were removed. The anterior segments were cultured at 37C in 100% humidity a t 5% CO2 atmosphere and perfused at constant pressure of 14 mmHg 69, 70 The outflow rates were determined gravimetrically as the changes in weight of the medium as the eyes were perfused over time The data were captured at one minute intervals by WinWedge software attached to the balance and recorded in an Excel spread shee t; outflow facility was expressed as l/min/mmHg perfusion pressure. Organ perf usions were performed with isotonic DMEM (~309 mOsm/kg) to establish baseline facility followed by perfusion with

PAGE 29

29 several experimental conditions: hypotonic, and hypertonic media (to establish that the volume regulatory mechanisms are functional) and the N O donor, diethylenetriamine nitric oxide (DETA NO) (100 M). Hypotonic and hypertonic media were made as described above. After a stable baseline was established, the drugs were delivered via a drug exchange system which supplied a bolus of drug. Specifically, a 10 ml syringe, held at a height above the mounted anterior segment and containing DMEM + drugs was attached to one of the perfusion chambers ports. Another 20 ml waste syringe, held below the mounted anterior segment, was attached to a chamber port and this line was clamped. By slightly opening the clamp to the waste syringe, the anterior chamber medium was slowly exchanged with the medium in the drug supply syringe in a manner that prevented any changes in the perfusion pressure. After medium containing drugs was in the chamber, entry to this port was clamped and flow of medium without drugs was restarted through a third port. The viability of the tissue was evaluated using live dead stain (Molecular Probes, Carlsbad, CA), which stained for the total number of nuclei in the TM, following perfusion. Specifically, flatmount intact TM tissue was treated with live dead stain and visualized using confocal microscopy. Live cells were stained with the green fluorescence and dead cells were stained with red fluorescence. Cellular viability was determin ed to be good and data usable when 8590% of the total cells stained with green fluorescence 9 Materials and Reagents Routine reagents and iberiotoxin (IBTX) were purchased from Sigma (St. Louis, MO). Others were obtained as follows: 8 bromo cGMP sodium salt, 1 H [1,2,4]oxadiazolo[4,3a]quinoxalin 1one (ODQ), and diethylenetriamine NO (DETA NO) from Sigma RBI (Natick, MA ); (RP)8Br PETcGMP S from Calbiochem (La Jolla, CA)

PAGE 30

30 Statistics Statistical anylasis was performed using ANOVA, followed by Holm Sidak method for comparison of significant difference among different means. Results NO Donors Increase Out flow Facility Outflow facility was measured in porcine anterior eye segments as described. Freshly dissected eye anterior segments were allowed to adapt to their new environment during which time outflow facility increased for the first 3 8 hours a phenomenon referred to as washout 70, 71 after which outflow facility remained stable 63 Basal outflow facility (pre drug treatment) was 0.23730.5220 l/min/mmHg among experiments and was stable for several hours prior to drug treatment and remained stable post drug effect. Because of the stability of the outflow facility baseline after the initial wash out period, it was not necessary to correct for nondrug related changes in outflow facility in our experimental protocol. After a stable baseline was established DETA NO (100 M) was added to the perfusate and resulted in a significant increase in outflow facility Figure 3 1A. Outflow facility was increased at 10 minutes and reached its maximal level at 20 minutes following application of the DETA NO. The maximal effect of the drug was sustained for 1.5 hours a fter which outflow facility returned to values similar to baseline outflow facility between 5 6 hours post drug application. We observed significant increase in outflow facility (0.4884 1.3956 l/min/mmHg; mean 0.8635 l/min/mmHg + SEM 0.1029) over baseline values in response to DETA NO in 8 separate experiments. We tested the ability of the outflow pathway to respond to changes in osmolarity preceding NO treatment. This allowed us to determine that the perfused eye segments were healthy and

PAGE 31

31 responded with expected changes in outflow facility when challenged with changes in osmolarity. Figure 31B demonstrates that exposure of porcine eye anterior segments to perfusion with hypertonic media resulted in a significant increase in outflow facility (40% above baseline) after which outflow facility returned to baseline. The eyes were perfused for 19 hour s with DMEM only and the baseline values decreased slightly but remained constant for several hours Subsequently a bolus of DETA NO was added to the porcine anterior segment and allowed to perfuse Figure 31B demonstrates that as with figure 31A, DETANO increased outflow facility after the eye had previously responded to hyperosmotic challenge. NO donors are known to activate the BKCa channels resulting in the efflux of K+ from the cell. We wished to determine the involvement of BKCa channels in the NO induced regulation of outflow facility. After baseline outflow facility was determined, anterio r segments were perfused with DETA NO as described. IBTX (50 nM) 5, 25 was added to the perfusate after the NOinduced incr ease in outflow facility was observed. Addition of IBTX resulted in a significant decrease in the NO induced response in outflow facility within 10 minutes after the IBTX was added (Figure 31C). NO Decreases TM Cell Volume Agents known to decrease TM cel l volume also increase outflow facility. Others have demonstrated that NO decreases ventricular cell volume 72 Therefore, we wanted to de termine if changes in cell volume are cellular mechanisms by which NO modulated TM cell function. To quantitatively measure changes in cell volume, HTM cells (HTM26, HTM44 and HTM86) were incubated in Calcein AM dye and subsequently exposed to the NO donor DETA NO. Z stack images demonstrate that DETA NO (100 M) decreases TM cell volume (Figure 3 2A) To determine the concentration needed to decrease cell volume, early passage human TM cells were

PAGE 32

32 exposed to varying concentrations of DETA NO (1 300 M). Images were taken without drug treatment (0) time poi nt which served as a control for the treatment groups. Drugs were then added to the cells and images were taken of the same cells at 20 minutes. Figure 32B demonstrates that DETA NO elicited a dosedependent decrease in human TM cell volume with maximal effects procduced by 100 and 300 M. We also tested the reversibility of the DETA NO induced decreases in TM cell volume. In our hands Calcein AM is stable for up to 1 hour post Calcein AM incorporation into cells and exposure to laser treatments. Therefore all experimental treatments needed to be done within 1 hour time period. Because in our preliminary studies we had observed significant decreases in TM cell volume at 10, 15 and 20 minutes of exposure to DETA NO, TM cell were for 10 minutes. TM cells were incubated with Calcein AM as previously described and images were captured without drugs. Subsequently, the cells were treated with DETA NO (100 M), and images were captured at 10 minutes post drug treatment. The medium containing drug was removed and replaced with fresh medium and the cells were then incubated for 30 minutes in DMEM only at 37C in 5% CO2 and images were captured. Figure 32C de monstrates that cells exposed to DETA NO resulted in significant decreases in cell volume which were reversed following removal of the drug. Changes in Cell Volume in Response to Changes in Osmolarity To assess the function of volume regulatory mechanisms in TM cells we exposed both early passage human (HTM80, 86) and porcine TM cells to hypoand hypertonic DMEM which would be expected to cause swelling and shrinkage of the cells respectively. TM cells were incubated with Calcein AM as described and stab le baselines were established. Images were captured at 0 time point in isotonic medium (control) after which the osmolarity of the medium

PAGE 33

33 was altered. Porcine TM cell volume was altered in response to hypotonic as well as hypertonic medium (Figure 33A). There was a 12.4 % increase in TM cell volume after the medium was changed from isotonic to hypotonic. Thereafter, TM cell volume gradually decreased without changes in osmolarity of the medium. Exposure of TM cells to hypertonic medium resulted in 16.2 % decrease in cell volume post isotonic changes and cell volume increased after hypertonic medium was exchanged with isotonic medium at 37oC in 5% CO2 (Figure 33A). Figure 33B demonstrates that exposure of human TM cells to hypotonic medium resulted in 11 % increase in cell volume. Images were captured of cells treated with isotonic medium, after which cell were exposed to hypotonic medium and images were captured within 1 minute of exposure of cells to anisosmotic medium. There was an increase in TM cell volume which peaked within 3 minute after the medium was changed from isotonic to hypotonic and returned to baseline. We then tested the involvement of the BKCa channel in the TM volume regulatory mechanism. Calcein AM loaded cells were exposed to hy potonic medium in the presence or absence of IBTX (50 nM). Figure 33B demonstrates that maximum cell volume increase was observed in the presence of IBTX. Additionally, IBTX inhibited the regulatory volume decrease and allowed for a sustained increase in cell volume in response to hypotonicity when compared to cells that were not treated with IBTX. Exposure of TM cells to hypertonic medium resulted in a 31 % decrease in cell volume. Hypertonic medium was then exchanged with isotonic medium at 37oC in 5% CO2 after which cell volume increased (Figure 33B). The NO induced Decrease in Cell Volume Involves Activation of sGC and cGMP To test the involvement of sGC in the NO induced decreases in cell volume, primary human TM cells (HTM44 and HTM86) were inc ubated with Calcein AM as described above. Images were taken at 0 time point, without drug, then DETA NO (100 M) was added to the

PAGE 34

34 cells in the presence or absence of ODQ (1 M) 73 the specific sGC inhibitor, and images were taken at 5, 10, 15 and 20 min time points. Figures 34A and B demonstrate that ODQ abolished the NOinduced decr eases in cell volume in both human and porcine TM cells. As with primary human TM cells (Figure 34A), decreases in cell volume in response to DETA NO in porcine TM cells are also timedependent (Figure 34B). ODQ (1 M) abolished this time dependent decrease in TM cell volume suggesting that the NO induced decreases are mediated by sGC and cGMP. Involvement of Protein Kinase G in the NOInduced Decreases in TM Cell Volume The pathway downstream of sGC was tested us ing the cGMP analog, 8Br cGMP. Primary human TM cells (HTM86, HTM44) were incubated with 8 Br cGMP (2 mM) 74 and cell volume was determined. Figure 15 demonstrates that as with DETA NO, 8bromo cGMP significantly reduced TM cell volume suggesting the involvement of cGMP and possible involvement of protein kinase G in regulating TM cell volume. To further determine if protein kinase G is involved in the NO induced decreases in TM cell vo lume, human TM cells (HTM26, HTM86) were incubated with DETA NO (100 M) or 8 Br cGMP (2 mM) 75 with or without the specific inhibitor of protein kinase G, (RP) 8Br PETcGMP S (50 M ) 76 Figure 5 shows that (RP)8Br PETcGMP S partially inhibited the DETA NOand 8Br cGMP induced decreases in TM cell volume suggesting a role for protein kinase G in regulating cell vo lume. BKCa Channels are Involved in the NO Induced Decreases in TM Cell Volume To test whether or not activation of the BKCa channel was involved in the NO induced decreases in cell volume, HTM cells (26 and 80) were preincubated with IBTX (100 nM), and i mages were captured at 0 time point. DETA NO (100 M) was then added to the cells and images were captured at 5, 10, 15, 20 minutes time period post DETA NO exposure. Figure 36

PAGE 35

35 (open circles) demonstrates that NO was unable to cause decreases in TM cell volume in cells that were pretreated with IBTX. We next wanted to determine the effects of IBTX on cells that had experienced decreased cell volume in response to NO treatment. Images were captured without drugs at 0 time point. Cells were then treated with DETA NO (100 M) and images were captured at 5, 10 and 15 minutes. IBTX (100 nM) was then added at 15 minutes post DETA NO treatment and images were captured at 20 minute time point. Figure 36 demonstrates that decreases in cell volume were timedependent, with significant decreases observed at 10 and 15 minutes post drug incubation. Addition of IBTX 15 minutes post incubation with DETA NO, reduced the NO induced decreases in cell volume when compared with only DETA NO treated cells. Additionally, IBTX alone had no significant effect on HTM cell volu me (Figure 3 6). Discussion In this study we provide evidence that NO decreases TM cell volume by activation of the sGC/cGMP/PKG pathway in a manner dependent on BKCa channels (Figure 37). We also show that the time course for increased outflow facility in response to NO correlates with changes in cell volume in response to NO. Outflow Facility In our experimental protocol an acute application of DETA NO increased outflow facility in porcine eye anterior segments. Other studies in monkeys 77 and rabbits 61 demonstrate the involvement of the NO/sGC and cGMP pathway in increasing outflow facility. The NO effect was immediate, transient and the degree of increases in outflow facility varied among experiments. We do not know the reason for this variability in response to NO. The possibility exists that lower flow rates allow for increased time for medium to be he ld in the anterior segment of the eye, thus bathing the tissue with the drug for a longer period of time, hence

PAGE 36

36 producing a more sustained drug effect. The ability of NO to increase outflow facility is corroborated by reports from Kotikoski et al. demonst rating the ability of NO donors to increase outflow facility in rabbit eyes 61 Additionally, we demonstrated the ability of IBTX to inhibit the NO induced increases in outflow facility. The rapid reversal of the NOinduced increases in outflow facility by IBTX would suggest that inhibition of the BKCa channel would possibly stop cell shrinkage and allow for a rapid regulatory volume increase. Other studies have demonstrated that the BKCa channels are downstream effectors of NO and are involved in mediating the NO/cGMP induced smooth muscle relaxation 78 In TM, the BKCa channel is expressed and is involved in the NO induced relaxation of precontracted bovine TM muscle strips 25 These data suggest that in addition to i t s ability to regulate the NO induced decreases in cell volume, the BKCa channel may be involved in regulating the contractile states of cells in the outflow pathway. In physiological states, NO could cause previously contracted cells to relax thus increasing outflow facility. Blocking the BKCa channel would potentially cause the cells to contract, thus causing outflow facility to return to base line values. Taken together these data suggest the possible involvement of both NOinduced decreases in cell volume and/or changes in the cells contractile states as cellular functions by which TM cells are altered. The proposed role of NO in regulating IOP is not without controvers y, however. One report by Krupin et al demonstrated an increase in IOP in rabbits in response to the NO donor sodium nitroprusside 79 While we do not understand the reason for this discrepancy is is possibly explained by dose dependent effects of NO on IOP. Higher doses of NO donors result in increases in IOP, while lower doses resu lt in decreases in IOP 59, 80 Additionally, repeated

PAGE 37

37 use of the organic nitrat e, nitroglycerin, resulted in tolerance, where as chronic usage of the nucleophile, hydralazine, did not result in tolerance 59 Increases in the osmolarity of the perfusi on medium resulted in increases in outflow facility which mimicked the effects of DETA NO. Consistent with the literature, anterior eye segments perfused with hypertonic medium 7, 48 resulted in increases in outflow facility while hypotonic medium 5, 7, 48 resulted in decreases in outflow facility. Cell Volume Studies Our protocol a llowed us to quantify changes in cell volume in hundreds of intact, adherent cells in their native states and each cell was able to serve as its own control. While we allowed for Calcein AM to achieve a stable baseline after which cell volume was measured in response to drug treatment in isotonic medium, we also imaged cells that were not treated with drugs to assess any changes in fluorescence in response to laser exposure. Additionally, because during the experimental protocol these cells were in their native state and were not harvested, we did not experience the movement of cells from the region of study or observe the rapid contraction and relaxation phenomenon as previously described 4, 81 It has been observed that TM cell cultu re contains two distinct cell populations 39, 82 which is consistent with the identified regions of the TM, the cribr iform or juxtacanalicular region, the uveal and the corneoscleral regions 3, 38 In our hands we were able to visually identify the different cell types, however, as with reports by Mitchell et al 36 all cells that were analyzed did not experience decreases in cell volume in response to drug treatment. The juxtacanalicular regio n, and hence the juxtacanalicular cells are regions of high resistance to aqueous humor outflow and may constitute the area where changes in cell volume may affect outflow resistance. Together, these data would suggest that modulation of the volume of TM cells may modify outflow resistance of aqueous humor, and may alter IOP.

PAGE 38

38 We tested the validity of our protocol with agents and conditions known to alter the osmolarity of the medium and subsequently alter TM cell volume. Both swelling and shrinkage of TM cell after exposure to hypotonic or hypertonic media respectively demonstrated that TM cells have the ability to respond to osmotic changes in their extracellular environment. Our initial studies were performed in porcine cells where we observed that i ncreases in cell volume were detectable at 1 minutes post osmotic changes and cell volume decreased within 3 minutes post hypotonic exposure. Porcine cell volume in response to hypertonic changes decreased gradually over a 7.5 minute period and remained c onstant for the duration of the experiment. Because of these observations we performed similar experiments in human TM cell. Cell volume changes in response to hypotonic treatment mimicked the hypotonic effects observed in porcine TM cell line. Exposur e of TM cells to hypotonic medium resulted in 11 % increase in cell volume. These results mimic similarly treated TM cells 36, 49 To date the correlation between amount of changes in cell volume in response to changes in osmolarity or drugs and the physiological relevance of these changes to cell function has not been ascertained. As with these studies, other studies by Mitchell et al, demonstrated that human TM cells exposed to hypotonic solution experienced regulatory volume decrease within 4 minutes of treatment with hypotonic medium 4 In our experimental protocols neither porcine nor human cells treated with mannitol or NaCl experienced regulatory volume increase in the presence of hypertonic medium. Immediate regulatory volume increase was observed when hypertonic medium was exchanged with isotonic medium at 37 C 6 with cell volume restored to baseline values within 6 minutes of medium exch ange.

PAGE 39

39 In our hands, TM cells exposed to hypotonic medium experienced a peak cell volume increase when treated with IBTX. This suggests that in physiological states the BKCa channel may be involved in the regulatory volume decrease mechanism in the cell i nhibition of which may potentiate cell swelling. IBTX abolished the regulatory volume decrease that occurred spontaneously in hypotonic treated cells suggesting a role for the BKCa channel in the cells regulatory volume decrease mechanism. NO donors can ac tivate sGC in a number of tissues, presumably through release of NO. The ability of the specific sGC inhibitor ODQ to antagonize the actions of DETA NO on TM cell volume would suggest that a direct consequence of NO stimulation is the activation of sGC. T echnical constraints did not allow us to correlate changes in endogenous cGMP levels with changes in TM cell volume. However, 8 Br cGMP mimicked the actions of DETA NO suggesting that cGMP is involved in TM cell volume regulation. In fact our studies involving 8Br cGMP corroborated previous results published by ODonnell et al. In these studies exposure of bovine TM cells to 8 Br cGMP (50 M) resulted in an 8% decrease in TM cell volume 6 These studies were performed using electronic cell sizing of suspended TM cells. Similar changes in cell volume were observed in human TM cells in our experiments using fluorescent probes validating our protocol. In other studies, exposure of TM cells to 8 Br cGMP (50 M) resulted in inhibition of the bumetanide sensitive K+ influx demonstrating the involvement of cGMP in the Na K 2Cl co transport regulation 6 In our hands, the decrease s in TM cell volume in response to DETA NO were similar to decreases in cell volume in response to 8 Br cGMP suggesting that cGMP maybe the second messenger mediating the effects of NO on cell volume. Our studies demonstrated that IBTX inhibited the NO in duced decreases in TM cell volume suggesting the involvement of

PAGE 40

40 the BKCa channel and K+ efflux in regulating the NO induced decreases in TM cell volume. We also demonstrated that BKCa channel is necessary for the NO induced response in TM cells. Other st udies demonstrated that cGMP generated by activation of the atrial natriuretic peptide receptor, and by the NO donor, sodium nitroprusside, decreased cardiac cell volume by inhibiting ion uptake by the Na K 2Cl co transporter 83 These observations suggest that NO regulation of K+ transport and cell volume are bidirectional, facilitating both K+ efflux via BKCa channel and K+ influx via bumetanide se nsitive K+ cotransporter. Protein kinase G inhibitors were able to inhibit the NO and 8Br cGMP induced changes, demonstrating the role of protein kinase G and protein phosphorylation events in regulating TM cell volume. Our studies, however, do not prec lude the involvement of other second messengers, including cAMP 49, 84 or protein kina se C 85 or the involvement of other ion transporters and co transporters in modulating cell volume 4 We were not able to demonstrate that the NO induced increases in outflow facility occurred as a result of changes in TM cell volume. However, we have demo nstrated that the time course for the DETA NO induced increases in outflow facility correlate with the time course for the DETA NO induced decreases in cell volume. Changes in TM cell volume induced by changes in tonicity correlate with tonicity induced c hanges in outflow facility. Studies have demonstrated that drugs known to cause cell swelling reduce outflow facility while drugs know to shrink cells increase outflow facility in human and bovine eyes 5, 7 While the results of a study by Gabelt et al., demonstrated that bumetanide, an inhibitor of the Na K 2Cl co transporter had no effect on outflow facility in living primate eyes (suggesting that alterations in cell volume had no effect on outflow facility) 86 the preponderance of electrophysiological, biochemical and pharmacological studies demonstrate a correlation between cell volume changes

PAGE 41

41 and outflow facility. Together these studies suggest that there might be multiple transporters involved in cell volume regulation and subsequent regulation of outflow facility. We conclude that NO decreases TM cell volume, that cellular response to NO is mediated by sGC, cGMP, PKG and BKCa channels, and that changes in cellular volume are correlated with changes in outflow facility. We are mindful that there might not be a direct cause and effect relationship between TM cell size and outflow facility because we have not accounted for possible involvement of TM contractile mechanisms 5052 Acknowledgements We thank Drs. T. Acott and D. Stamer for teaching us the anterior segment organ perfusion protocol and TM cell culture techniques, Mr. Douglas Smith for technical assistance with the Confocal Microscope and Drs. Charles Wood and Elaine Summer for helpful discussion of the manuscript. Grant This work was supported by a grant from the American Health Assista nce Foundation, National Glaucoma Research.

PAGE 42

42 Figure 31. NO donors increase outflow facility in porcine anterior organ culture perfusion. A) A stable baseline was achieved after which the anterior chamber perfusate was rep laced with an acute treatment of DETA NO (100 M) dissolved in DMEM. The drug entry port was clamped, and perfusion continued with DMEM alone. Data shown is representative of 8 experiments. *Significantly different from baseline values at 30 and 50 minutes, P<0.05; ANOVA and the Holm Sidak method. B) A stable baseline was achieved after which the isotonic medium was exchanged with hypertonic DMEM. Following the effects of the hypertonic DMEM, a stable baseline was reestablished and DETA NO (100 M) was then added to the perfusate. Data shown is repre sentative of 3 experiments. C. A stable baseline was established and anterior eye segment was perfused with DETA NO (100 M). Subsequently, IBTX (50 nM) was added to the perfusate. Data shown is representative of 3 experiments. *Significantly different f rom baseline at 50 and 100 minutes and # significantly different from DETA NO treated samples P<0.05; ANOVA and the Holm Sidak method.

PAGE 43

43 Figure 32. NO decreases TM cell volume. A) Thresholded zstack images of a TM cell. At 0 minutes (without drug), the thresholded voxels are qualitatively and quantitatively greater than at 20 minutes post DETA NO (100 M) treatment. Voxel count for cell at 0 minute, 27650 and at 20 minutes, 23342. Scale bar = 20 m. B) NOinduced decreases in cell volume are concentration dependent. Confocal images of the same cells were acquired with a 20x objective lens at 1 m z step intervals to a depth of 15 m. Human TM cells were exposed to varying concentrations of DETA NO (1 300 M). Images were captured at 0 and 20 minute time points. Data shown for 1, 10, 30, 50, 100 and 300 M DETANO represent the mean + SEM for 23, 31, 19, 22, 44 and 23 cells respectively and are expressed as % of initial volume at 0 time point without drugs. Average voxel count is 25,449 + 4398 (*Significantly different from control P<0.05; # significantly different from 30 and 50 M DETA NO, P<0.05; ANOVA and the Holm Sidak method). 2C). Decreases in human TM cell volume are reversible.. Data are expressed as % of initial volume at 0 time point mean SEM; n=23 cells. Voxel count for 0 time point is 11606 + 1155 (*Significantly different from contr ol (0 time point) P<0.001, # significantly different from DETA NO treated cells P<0.05).

PAGE 44

44 Figure 33. Changes in osmolarity effect changes in TM cell volume. A) In porcine TM cells hypotonic DMEM increased cell volume (mean SEM; n=55 cells) and hyperto nic DMEM decreased cell volume (mean SEM; n= 47 cells). Data are expressed as % volume at 0 time point. Voxel count at 0 time point for hypotonic treatment 2385.1+ 290 (*significantly different from 0 time point, P<0.05 and # significantly different fr om 1,2,3 and 4 min P<0.05). Voxel count for hypertonic treatment 2827.5 + 274 (*significantly different from 0 time point P<0.05;). B) In human TM cells hypotonic DMEM increased cell volume (mean SEM; n=19 cells) while hypertonic DMEM decreased cell vol ume (mean SEM; n=17 cells). Hypotonic + IBTX allowed for a sustained increase in cell volume (mean + SEM; n=66 cells). There were no changes in cell volume in cells incubated in isotonic medium (mean + SEM; n=39 cells). Data are expressed as % volume at 0 time point. Voxel count at 0 time point for hypotonic, hypertonic, isotonic and hypotonic + IBTX treatments are 3273 + 533; 7921 + 1230; 4392 + 278; and 5408 + 286 respectively (* Significantly different from control (0 time point) and # significantl y different from anisosmotic treated cells, P<0.05; ANOVA and the Holm Sidak method.

PAGE 45

45 Figure 34. sGC mediates the NO induced decreases in TM cell volume. A). Data are expressed as % of initial volume at 0 time point and for: DETA NO treated group represents the mean SE M; n=28 cells and for DETA NO + ODQtreated group represents the mean SE M; n=33 cells. Voxel count for 0 time point: DETA NO, 16855 + 3185; DETA NO + ODQ, 11923 + 908 (* Significantly different from 0 time point P<0.001). B) A s with human TM cells, porcine TM cells were exposed to DETA NO and ODQ as described. Data are expressed as % of initial volume at 0 time point and for DETA NOtreated group represents the mean SE M; n=111 cells and for DETA NO + ODQtreated group repr esents the mean SEM; n=11 cells. Voxel count for 0 time point: DETA NO, 2994 + 171; DETA NO + ODQ, 2288 + 81 (* Significantly different from 0 time point P<0.001).

PAGE 46

46 Figure 35. E ffects of PKG inhibitor (RP) 8Br PETcGMP S (PKGi) on 8Br cGMP and DETA NO induced decreases in TM cell volume. C ells were incubated with 8Br cGMP (2 mM) or DETA NO (100 M) in the presence or absence of (RP) 8Br PET cGMP S (50 M). Images were taken and cell volume measured. Data are expressed as % of initial volum e at 0 time point and represents the mean + SEM for: 8 Br cGMP, n=100 cells; 8 Br cGMP+PKGi, n=86 cells; DETA NO mean SEM, n=89 cells; DETA NO + PKGi mean SE M, n= 97 cells. Voxel count for 0 time point: 8Br cGMP, 2771+ 102; 8 Br cGMP + PKGi, is 4742 155; DETA NO, 4125 + 123; DETA NO + PKGi, 3422 + 131. Significantly different from 0 time point and # significantly different from 8 Br cGMP group P<0.001 and ## significantly different from DETA NO treated cells P<0.001.

PAGE 47

47 Figu re 36. BKCa channels mediate the NO induced decreases in TM cell volume. IBTX only: data are expressed as % of initial volume at 0 time point and represents the mean SEM; n= 58 cells. DETA NO + IBTX: images were captured at 0 time point without drugs, then DETA NO (100 M) was added and images were captured at 5, 10 and 15 minutes. IBTX (100 nM) was added at 15 minutes and images were taken at 20 minutes. DETA NO: cells were incubated with DETA NO only and data at 20 minute time point was compared with data obtained at 20 minute time point for the DETA NO + IBTX treated cells. Data are expressed as % of initial volume at 0 time point and represents the mean SE M; n= 46 cells. DETA NO + IBTX was significantly different from DETA NO treated cells P<0.05. IBTX + DETA NO: TM cells were preincubated with IBTX then images were captured. The cells were then exposed to DETA NO (100 M) and images were captured at 5, 10, 15 and 20 minutes. Data are expressed as % of initial volume at 0 time point and represents the mean SE M; n= 51 cells. Voxel count for 0 time point: IBTX, 13147 865; DETA NO + IBTX, 9202 625; IBTX + DETANO, 1 9202 925.

PAGE 48

48 Figure 37. Summary diagram of the pathway of NO regulation of TM cell volume. NO donors cause the formation of NO, which then binds to and activates soluble guanylate cyclase (sGC), the synthetic enzyme of cGMP. cGMP and it s analogue 8Br cGMP activate protein kinase G (PKG) which may, directly or indirectly phosphorylate BKCa channels, with subsequent K+ efflux and decreases in cell volume.

PAGE 49

49 CHAPTER 4 HUMAN TRABECULAR MESHWORK CELL VOLUME DECREASE BY NOINDEPENDENT SOLUBLE GUANYLATE CYCLASE ACTIVATOR YC1 AND BAY582667 INVOLVE THE BKCA ION C HANNEL Introduction Aqueous humor exits the eye through the TM and Schlemms canal. Activation of sGC by NO dependent donors increase the rate at which aqueous humor flow s through the TM and Schlemms canal. These changes in outflow facility occur concomitant with sGC induced decreases in TM cell volume. Soluble guanylate cyclase comprises an subunit and a smaller heme containing subunit 87, 88 both of which constitute the active enzyme. H eterodimers are activated by NO binding to the heme moiety, whereas homodimers exhibit little or no synthetic activity, even in the presence of the ligand. Binding of NO to sGC, results in the format ion of 3,5 cyclic guanosine monophosphate (cGMP) from guanosine 5 triphosphate (GTP). Increased cGMP activates protein kinase G (PKG) 89 with subsequent phosphorylation of target proteins. NO acting through the sGC, cGMP and PKG pathway decreased TM cell volume in a time course that correlated with the NO induced increases in outflow f acility in perfused eye anterior segments 90 Although NO is a potent regulator of IOP, chronic administration of NO donors to eyes result in lack of responsiveness and the develo pment of tolerance 59 Therefore, the need to identify other activators of sGC that regulate TM cell function is of vital interest. YC1 [3 (5 hydroxymethyl 2furyl) 1be nzyl indazole) 18 a benzyl indazole derivative and BAY 582667 91 are NO independent activators of sGC. As with NO activation of sGC, YC 1 and BAY582667 activation of sGC also results in increases in cGMP and PKG phosp horylation events. Alterations of the contractile states and volume of the TM cells would regulate aqueous humor outflow 5 7, 4349 Changes in ce ll volume are influenced by the activities of the Na K 2Cl

PAGE 50

50 co transporter 6, 7, 36 the Na+/H+ transporter 36 the K+ and Cl channels 5, 36 and the large conductance calci um activated potassium channel (BKCa) 90 Further, it is possible that both the cellular contractile mechanisms and the cell volume regulatory mechanisms are functionally linked 50 52 ; as the BKCa channels have been shown to regulate TM cell volume and contractility 5, 45 and outflow facility 5 In these studies we will test the hypothesis that YC 1 and BAY582667 regulate TM cell function. Specifically, we will test the ability of YC 1 and BA Y 582667 to regulate TM cell volume, and test the involvement of sGC, cGMP, PKG and BKCa channel in the YC1 and BAY582667 induced response. Materials and Methods Cell Culture Eyes from human donors with no history of ocular disease or surgery were obtained from Lions Eye Institute ( Tampa, FL) within 2430 hours postmortem. Primary human TM cell lines (numbers representing ages of the donors) (HTM26, HTM71, HTM36, HTM80 and HTM86) were developed. For our experimental protocols cells from early passag es (3 5) were used. Human TM explants were obtained either from whole eyes that were stored in a moist environment at 4oC or from corneal scleral rims stored in O ptisol (Dexol; Chiron Ophthalmics, Irvine, CA) at 4oC. TM cells were isolated after collagena se digestion of TM explants 64 Collagenase treated cells were grown in low glucose (1g/L) DMEM (Mediatech, Herdon VA.) in the presence of 10% fetal bovine serum (Mediatech, Herdon VA.), 100 U/ml penicillin and 100 g/ml streptomycin (Mediatech, Herdon VA.) and then passaged into 6 well culture dishes (Nalge Nunc International, Rochester, NY) in a tissue culture incubator @ 37oC in 5% CO2. We validated human TM cells by their morphology and the presence of dexametha sone induced myocillin expression 33 For experimental protocols, TM cells were grown on Lab Tek ll chambered cover glass (Nalge Nunc International, Rochester, NY) in low glucose DMEM as

PAGE 51

51 described above to 100% confluency, after which they were exposed to serum free media for 2 days prior to performing the experiments. Measurement of Cell Volume Cell volume measurements were performed as previously described 90 P rior to any drug treatments, the cells were loaded with the fluorescent dye Calcein AM (2 M) in DMEM at 37C, in 5% CO2 incubator for 60 minutes to ensure a stable baseline. The coverslips containing the cells were subjected to confocal microscopy using a Leica confocal microscope, thermostated at 37oC and images of the same cells were acquired with a 20x objective lens at 1 m z step intervals to a depth of 15 m. Because during the experimental protocol these cells were in their native state and were not harvested, we did not experience the movement of cells from the region of study or observe the rapid contraction and relaxation phenomenon as previously described 36, 81 .The confocal microscope calculated the number of voxels and cell volume was quantified using NIH ImageJ software. T he NIH ImageJ software was used to identify the top and bottom edges of the cell. I mages were converted from 8 bit to binary v alues using a threshold that was determined by analysis of fluorescent Fluoresbrite latex beads (Polyscience Inc., Warrington) of known diameter and volume that were imaged under conditions identical to those used for TM cells. A region of interest was th en selected around each cell and t he ImageJ software was used to calculate the number of voxels in the region of interest in the image stack Changes in cell volume were determined by dividing the voxel count with drug treatment by the voxel count without drug treatment. Unless otherwise stated in the text, for studies involving drug treatments, images were taken without drug treatment (0) time point which served as controls for the treatment groups. Because our preliminary data demonstrated that the maxim um decrease in TM cell volume in response to drug treatment was achieved at 20 minutes, images were taken of the

PAGE 52

52 same cells at 20 minutes post drug treatment. For all experiments, YC 1 (10 mM) and BAY 582667 (10 mM) were solublized in a DMSO ethanol mixture for a final concentration of 0.1%. cGMP Assay For cGMP measurements, cells were grown in 12 well culture dishes (Nalge Nunc International, Rochester, NY) in a tissue culture incubator @ 37oC in 5% CO2 as described above. Two days prior to experiments, the cells were exposed to serum free media. Cyclic GMP was assay ed by an Enzyme Imm uno Assay ( EIA ) (Amersham Biosciences, Piscataway, NJ) according to the manufactures protocol. Materials and Reagents Routine reagents, YC 1 [3 (5 hydroxymethyl 2furyl) 1benzyl indazole) and iberiotoxin (IBTX) were purchased from Sigma (St. Louis, MO). Others were obtained as follows: 8 bromocGMP sodium salt, 1 H [1,2,4]oxadiazolo[4,3a]quinoxalin1one (ODQ), from Sigma RBI (Natick, MA ); (RP)8 Br PETcGMP S from Calbioc hem (La Jolla, CA) Bay 582667 was obtained from Alcon Research, Ltd.,(Fort Worth, TX). Statistics Statistical comparisons were performed by ANOVA, followed by Holm Sidak method or Fisher LSD method for comparison of significant difference among differe nt means. Results YC1 and B AY582667 Induced Regulation of TM cell Volume are Biphasic To quantitatively measure changes in cell volume, HTM36 and HTM80 cells were exposed to varying concentrations of YC 1 (10 nM 200 M) 92 Figure 41A demonstrates that the action of YC 1 is biphasic in HTM cells; 1 M significantly increased TM cell volume while higher concentrations (50 200 M) decreased TM cell volume. We also observed that there were no changes in cell volume over time in cells incubated in Calcein AM only (Figure 41A).

PAGE 53

53 Similarly, varying concentrations of BAY 582667 (10 nM 100 M) were added to TM cells and images were captured with or without drugs. Figure 41B demonstrates that as with figure 41A, the action of BAY 582667 on TM cell volume is biphasic; 100 nM caused increases in TM cell volume, while higher concentrations resulted in decreases in TM cell volume. The YC1induced Decrease in Cell Volume Involves Activation of Soluble Guanylate Cyclase and cGMP To test the involvement of sGC in the YC 1induced decreases in cell volume, primary human HTM36 and HTM80 cells were incubated with YC 1 (150 M) in the presence or absence of ODQ (500 nM 5 M) 73 the specific sGC inhibitor, and images were taken at 20 min. ODQ at 500 nM and 1 M had no effect on the YC 1induced decreases in TM cell volume while 5 M significantly attenuated the YC 1 effect (Figure 4 2A). Because activation of sGC results in increased cGMP levels, we exami ned the ability of exogenously applied cGMP to decrease TM cell volume. Figure 4 2A demonstrates that the nonhydrolyzable analog of cGMP, 8bromocGMP, mimics the action of YC 1 in decreasing TM cell volume. To further determine if sGC is involved in the YC 1induced decreases in HTM cell volume, t he ability of YC1 (50 150 M) to increase cGMP l evels was tested There was a concentration dependent increase in cGMP levels in cells treated with varying concentrations of YC1 that was saturating at 100 M (Figure 42B). Additionally, ODQ (5 M) abolished the ability of YC 1 (150 M) to increase cGMP levels (Figure 42B). Unlike YC1 however, ODQ (5 and 10 M) potentiated the BAY 582667 induced decreases in TM cell volume (Figure 42C). While we were unable to pharmacologically determine sGC involvement, we tested downstream ef fects. Cyclic GMP levels were measured in high concentration exposed BAY 582667 samples. Figure 42D demonstrates that BAY 58-

PAGE 54

54 2667 (10 and 100 M) increased cGMP levels. Similarly, 8 Br cGMP mimicked the actions of BAY582667 in decreasing TM cell volume (Figure 42C). The YC1 and BAY 582667induced Increases in Cell Volume Do Not Involve cGMP To determine if sGC is involved in the low concentration YC 1 and BAY 582667 induced increases in cell volume, HTM36 and HTM80 cells were incubated with Calcei n AM then exposed to YC 1 (10 nM 25 M) and BAY58 2667 (100 nM) and cGMP levels were then measured. Addition of 10 nM 25 M YC1 to HTM cells did not result in a statistically significant increase in cGMP level s when compared to control samples (Figure 43A). There were no alterations in cGMP levels in TM cells incubated with YC 1 in the presence of ODQ (5 M) (Figure 43A). Although it appears as if there is a trend for decreases in cGMP levels when ODQ (5 M) was added in the presence of YC 1(1 M), when all concentrations of YC 1 were included in the data analysis of the cGMP assay, the decrease was not statistically significant. As with low concentrations of YC 1, BAY 582667 (100 nM) did not result in a statistically significant increase in cGMP levels (Figure 4 3B). PKG is Involved i n the YC 1 and BAY582667 in d u c ed Decreases in TM Cell Volume The pathway downstream of sGC was tested by exposure of HTM cells to varying concentrations of the PKG inhibitor, (RP) 8Br PET cGMP S (25 100 M). HTM cells were incubated with YC 1 (150 M) in the presence of (RP) 8Br PET cGMP S (25 100 M) and imaged. Addition of (RP) 8Br PET cGMP S (50 and 100 M) resulted in the attenuation of the YC1 induced decreases in TM cell volume (Figure 44A). TM cells were also exposed to BAY582667 (10 M) in the presence of varying PKG inhibitor concentrations (25 100 M) that resulted in concentration dependent attenuation of the BAY 582667 effect (Figure 44B).

PAGE 55

55 This provides evidence for the involvement of protein phosphorylation in mediating the YC1 and BAY58 2667 induced decreases in TM cell volume. BKCa Channels are Involved in the YC1 and BAY582667 induced Decreases in TM Cell Volume To test if activation of the BKCa channel is obligatory for the YC1 and BAY582667 induced decreases in TM cell volume, HTM cells were pre incubated with IBTX (100 nM) 5, 25 and im ages were captured at 0 time point. YC 1 (150 M) and BAY582667 (10 M) were then added to the cells and images were captured at 20 minutes post drug exposure. Figure 45 demonstrates that IBTX attenuated both the YC 1 and BAY 582667 induced decreases in TM cell volume. Additionally, IBTX alone ha d no significant effect on HTM cell volume (Figure 4 5). Discussion In this study we provide evidence that YC 1 and BAY 582667, nitric oxide independent sGC activators decrease human TM cell volume through the involvement of the BKCa channel. Specifically YC 1 at concentrations of 50 200 M and BAY 582667 at concentrations of 10100 M decreased TM cell volume and these decreases are mediated by the sGC/cGMP/PKG pathway in a manner dependent on the BKCa channel (Figure 46). The actions of YC 1, however, are biphasic, with 1 M causing i ncreases in TM cell volume, while higher YC 1 concentrations elicit a cell volume reduction. As with YC 1, BAY582667, at higher concentrations (10 M 100 M) significantly decreased TM cell volume while exposure to BAY582667 (100 nM) resulted in a significant increase in cell volume. The data observed at the concentrations used are consistent with the know potency of YC 1 and BAY 582667; as BAY582667 has previously been shown to have higher potency than YC 1 in activating sGC 93 The biphasic effects of YC 1 and BAY582667 could be explained by the possible existence of

PAGE 56

56 two binding sites for these compounds on sGC that may involve heme dependent and independent moieties of the sGC 91 Cell volume was measured in adherent cells in their native states and each cell was able to serve as its own control. After Calcein AM dye achieved a stable baseline, cell volume was measured in response to drug treatment in isotonic medi a. Additionally, cells that were not treated with drugs were also imaged to assess any changes in fluorescence in response to laser exposure. It has been observed that TM cell cultures contain two distinct cell populations 82 which is consistent with the identified reg ions of the TM, the cribriform or juxtacanalicular region and the uveal \ corneoscleral region 3, 38 The juxtacanalicular region, and hence the juxtacanalicular cells are regions of high resistance to aqueous humor outflow. While the cells in the juxtacanalicular tissue contribute very little to total tissue volume, the changes in cell volume in this area may have a large contribution to outflow resistance. While we were able to visually identify the two cell populations, we were unable to determine if the two cell populations respond to low or high YC 1 or BAY 582667 concentrations similarly. The a bility of the specific sGC inhibitor ODQ, to antagonize the actions of YC1 on TM cell volume would suggest that a direct consequence of YC 1 stimulation is the activation of sGC. We measured alterations in cGMP levels in response to varying concentratio ns of YC 1 and demonstrated a concentration dependent increase in cGMP levels. Higher concentrations of YC1 caused significant increases in cGMP levels that correlated with decreases in TM cell volume; however, increased cell volume in response to 1 M YC1 is cGMP independent. The physiological and pharmacological significance of this observation is unclear at present. ODQ abolished the YC 1 induced increases in cGMP, but had no effect on basal cGMP levels suggesting that ODQ acts by inhibiting the inter action of YC 1 with sGC. Further evidence for

PAGE 57

57 the involvement of cGMP in the YC 1induced response was demonstrated by the ability of 8Br cGMP to mimic the actions of YC 1 in decreasing TM cell volume. In our hands, the decreases in TM cell volume in response to YC 1 were similar to decreases in cell volume in response to 8Br cGMP suggesting that cGMP maybe the second messenger mediating the effects of YC 1 on cell volume. Unlike ODQs attenuation of the YC 1induced decreases in TM cell volume, ODQ potentiated the BAY 582667 effects. While the precise mechanisms are unclear, experimental evidence suggests that removal of the heme prosthetic group or oxidation to its ferric form by ODQ causes conformational changes in sGC such that it no longer responds to NO or YC1 but does respond to BAY 582667 94 While we did not demonstrate sGC involvement in the BAY 582667induced decre ases in TM cell volume, other studies have demonstrated that BAY 582667 binds to and activates sGC with subsequent increases in cGMP levels 93 Similarly, we demonstrated that higher concentrations of BAY 582667 caused increases in cGMP levels that correlated with decreases in TM cell volume. Additionally, the PKG inhibitor was able to inhibit both the YC 1and BAY 582667induced cell volume changes, further demonstrating the role of PKG and protein phosphorylation events in regulating TM cell volume. Our studies, however, do not preclude the involvement of other second messengers, including cAMP 49, 84 and protein kinase C 85 IBTX inhibited the YC 1 and BAY 582667induced decreases in TM cell volume suggesting the involvement of the BKCa channel and also the role of K+ efflux in regulating the YC1 and BAY 582667induced decreases in TM cell volume. Similar observations were made in studies involving TM cells treated with NO in the presence of IBTX; preincubation of TM cells with IBTX abolished the NO induced decreases in TM cell volume, suggesting that the

PAGE 58

58 BKCa channel is obligatory for the sGC/cGMP induced decreases in TM cell volume 90 While we do not know the mechanism(s) by which the sGC/cGMP/PKG system regulates the BKCa channel, other studies demonstrate that PKG phosphorylation of the subunit of the BKCa channel results in its activation 28, 29 Thus, the possibility exists that YC1 or BAY582667 activation of PKG in TM cells could result in phosphorylation of BKCa channels and subsequent decreases in TM cell volume. While this study suggests the involvement of the BKCa channel and K+ efflux in the BAY 582667 and YC 1induced decreases in TM cell volume, other studies have demonstrat ed that cell volume decrease is accompanied by both K+ and C lefflux 95 induced by activation of K+ and C lchannels and/or K+ and C lsymport. This suggests that K+ efflux may initiate a parallel C lefflux in TM c ells. In other studies, exposure of TM cells to 8Br cGMP (50 M) resulted in inhibition of the bumetanide sensitive K+ influx demonstrating the involvement of cGMP in Na K 2Cl co transport regulation 6 This suggests that the Na K 2Cl co transporter may be regulated by YC 1 and BAY 582667. However, its ability to decrease TM cell volume is dependent on low bicarbonate levels 6, 36 or blockade of the Na/H exchanger 36 experimental conditions that were not manipulated in these studies. Furthermore, increased cGMP levels resulting from both sGC and membrane guanylate cyclase a ctivation r esulted in decreased cardiac cell volume through inhibition of K+ influx via the bumetanide sensitive K+ cotransporter 83 These observations sugge st that NO dependent and NO independent regulation of K+ transport and cell volume are bidirectional; facilitating K+ efflux via BKCa channel and inhibiting K+ influx by the Na K 2Cl co transporter. Additionally, the NO dependent sGC/cGMP system plays an important role in regulating aqueous humor dynamics by regulating; aqueous humor production in the ciliary processes 96, 97

PAGE 59

59 and aqueous humor outflow via the TM/Schlemms canal 90 with subsequent decreases in IOP 19, 59 Therefore, these data would suggest that modulation of t he volume of TM cells by YC 1 and BAY582667 via the sGC/cGMP/PKG system may modify aqueous humor outflow resistance and thus may alter IOP. Funding Supported in part by a grant from the American Health Assistance Foundation, National Glaucoma Research, and Alcon Research Laboratories, Ltd.

PAGE 60

60 Figure 41. YC1 and BAY 582667induced regulation of TM cell volum e is biphasic. A) YC1induced changes in cell volume are concentrationdependent. Human TM cells were exposed to varying concentrations of YC 1 (10 nM 200 M). Images were captured at 0 and 20 minute time points. Data shown represent the mean + SEM for; control n=52 cells, 10 nM n=22 cells, 100 nM n=35 cells, 1 M n=58 cells, 25 M n=47 cells, 50 M n=65 cells, 75 M n=11 cells, 100 M n=40 cells, 150 M n= 51 cells and 200 M n=49 cells. Data are expressed as % of initial volume at 0 time point without drugs. #Significantly different from control at P<0.05; ANOVA and the Holm Sidak method. *Significantly different from 1 M YC 1 at P<0.05; ANOVA and the Ho lm Sidak. B) Cells were exposed to varying concentrations of BAY582667 (10 nM 100 M) and images were captured at 0 and 20 minutes. Data are expressed as % of initial volume at 0 time point without drugs and represent the mean + SEM for; control n=22 c ells, 10 nM n=26 cells, 100 nM n=17 cells ,1 M n=17 cells, 10 M n=13 cells and 100 M n=17 cells. #Significantly different from control at P<0.05; ANOVA and the Holm Sidak method. *Significantly different from 100 nM BAY 582667 at P<0.05; ANOVA and the Holm Sidak.

PAGE 61

61 Figure 42. Involvement of sGC/cGMP in the YC 1 and BAY 582667induced decrease in TM cell volume. A) ODQ inhibition of the YC 1induced decrease in TM cell volume is concentration dependent. Data are expressed as % of initial volume at 0 time point and for: YC 1 (150 M) treated group represents the mean SEM, n=24 cells; 8 Br cGMP (2 mM) treated group represents the mean SEM, n= 71 cells; YC 1 + ODQ (500 nM) treated group represents the mean SE M, n=35 cells; YC 1 + ODQ (1 M) treated group represents the mean S E M, n=31 cells; YC 1 + ODQ (5 M) treated group represents the mean SEM, n=32 cells. *Significantly different from control at P<0.05; ANOVA and the Holm Sidak method. #Significantly different from YC 1 (150 M) treated group at P<0.05; ANOVA and the Hol m Sidak method. B) YC 1 induced decrease in cell volume is associated with increases in cGMP. Levels of cGMP in TM cells after incubation with YC1 ( 50 150 M) and YC 1 (150 M) plus ODQ ( 5 M) Results measured are expressed as mean + SEM f mo l of cGMP d one in triplicate *Significantly different from control at P<0.05; ANOVA and the Holm Sidak method. C) ODQ potentiates the BAY 582667induced decreases in TM cell volume. Data are expressed as % of initial volume at 0 time point and for: BAY 582667 (10 M) treated group represents the mean SEM, n=38 cells; 8 Br cGMP (2 mM) treated group represents the mean SEM n= 30 cells; BAY 582667 + ODQ (5 M) treated group represents the mean SE M, n= 48 cells and BAY 582667 + ODQ (10 M) treated group repre sents the mean SEM, n= 44 cells. *Significantly different from control at P<0.05; ANOVA and the Holm Sidak method. #Significantly different from BAY 58 2667 (10 M) treated group at P<0.05; ANOVA and the Holm Sidak method. D) BAY 582667induced decrease in cell volume is associated with increases in cGMP. Levels of cGMP in TM cells after incubation with BAY582667 ( 1100 M). Results measured are expressed as mean + SEM f mo l of cGMP done in quadruplicate

PAGE 62

62 Figure 43. Low concentration YC 1 and BAY58 2667 induced increases in cell volume are not associated with significant increases in cGMP. A) Levels of cGMP in TM cells after incubation with YC1 ( 10 nM 25 M) and YC 1 (1 M) plus ODQ ( 5 M) Results measured are expressed as mean + SEM f mo l of cGMP done in triplicate B) Levels of cGMP in TM cells after incubation with BAY582667 (100 nM). Results measured are expressed as mean + SEM f mo l of cGMP done in quadruplicate

PAGE 63

63 Figure 44. PKG is involved in the YC 1 and BAY 582667induced decreases in TM cell volume. A) Cells were incubated with YC1 (150 M) in the presence or absence of varying concentrations of (RP) 8Br PETcGMP S a PKG i nhibitor (PKGi) ( 25 1 00 M). Images were taken, and the cell volume was measured. Data are expressed as % of the initial volume at the 0min time point and are means SEM; n = 22 cells for the YC1 treated group, n=21 cells for the YC1 + PKGi (25 M) treated group, n =14 cells for the YC1 + PKGi (50 M) treated group and n= 14 cells for the YC1 + PKGi (100 M) treated group. *Significantly different from Control p<0.05 by ANOVA and the Holm Sidak method; #significantly different from the 150 M YC1 treated group ( P < 0.05) by ANOVA and the Holm Sidak method. B) Cells were incubated with BAY582667 (10 M) in the presence or absence of varying concentrations of PKGi ( 25 100 M). Images were taken, and the cell volume was measured. Data are expressed as % of the initial volume at the 0 min time point and are the means SEM; n = 37 cells for the BAY582667 treated group, n =31 cells for the BAY582667 + PKGi (25 M) treated group, n =84 cells for the BAY582667 + PKGi (50 M) treated group, and n= 25 cells for the BAY582667 + PKGi (100 M) treated group. *Significantly different from Control p<0.05; #significantly different from the 10 M BAY582667 treated group ( P < 0.05) by ANOVA and the Holm Sidak method.

PAGE 64

64 Figure 45. The YC 1 and BAY 582667 induced decreases in TM cell volume involve the BKCa channel. TM cells were incubated with YC 1 (150 M) and BAY58 2667 (10 M) in the presence or absence of IBTX (100 nM). Data are expressed as % of initial volume at 0 time point and represents the mean SEM; n = 27 cells for the control group, n = 23 cells for the YC1 treated group, n= 37 cells for the BAY 582667 treated group, n = 19 cells for the IBTX treate d group, n = 41 cells for the IBTX+YC1 treated group and n=69 cells for the IBTX+BAY 582667treated group. *Significantly different from the Control group and #significantly different from the YC1 or BAY 582667treated group ( P < 0.05) ANOVA and the Fi sher LSD method.

PAGE 65

65 Figure 46. Summary diagram of the pathway of NO dependent and NOindependent regulation of TM cell volume. Increases in [Ca2+]i result in activation of nitric oxide synthase (NOS), and the subsequent formation of NO, which then binds to and activates soluble guanylate cyclase (sGC). YC 1 and BAY 582667, NOindependent activator s of sGC also bind to sGC and cause increases in cGMP cGMP then activates PKG which may, directly or indirectly through other proteins phosphorylat e the BKCa channels, with subsequent K+ efflux and decreases in cell volume.

PAGE 66

66 CHAPTER 5 ACTIVATION OF THE BKCA CHANNEL INCREASES OUTFLOW FACILITY AND DECREASES TRABECULAR MESHWORK CELL VOLUME Introduction Aqueous humor is secreted by the ciliary processes and exits the eye via the conventional pathway, consisting of the trabecular meshwork (TM) and Schlemms canal. Increased resistance to outflow of aqueous humor in the TM and/or the Schlemms canal results in increased intraocular pressure (IOP); a major risk factor for the development of openangle glaucoma 37 Viable treatment known to reduce the development and progression of blindness caused by glaucoma is decreasing IOP. The mechanism (s) by which TM cells influence aqueous humor outflow is still not completely understood. I t is postulated that TM cells may influence the tissue permeability, as agents that cause changes in cell volume 5, 7 contractile states 44, 98 and cell shape 52 also modulate outflow facility. The large conductance calcium activated potassium channel (BKCa) consists of a tetramer of pore forming subunits associated with the regulatory subunits in a 1:1 stoichiometry that when open allows for potassium efflux from the cell. The B Kca channel has been shown to be activated by changes in membrane potential and intracellular calcium concentration 27 cGMP dependent protein phosp horylation events 28, 29 and by activators, including the benzimidazolone derivative, NS1619 31 Conversely, the channel is inhibited by charybdotoxin 99 and iberiotoxin (IBTX) 30 The BKCa channel has been shown to be involved in TM cell volume regulation and contractility. Specifically, it mediates the recovery of a hypotonic induced decrease in outflow facility 5 suggesting its role in the TM cells regulatory volume decrease. The BKca channel also regulate s the NO/cGMP induced decreases in TM cell volume and increases in aqueous humor

PAGE 67

67 outflow facility 90 Additionally, cGMP increases the charybdotoxinsensitive outward currents in bovine TM cells 45 suggesting the involvement of the BKca channel in the cGMP mediated relaxation of isolated bovine TM strips 26 These events may not be mutually exclusive as both the cellular contractile mechanisms and the cell volume regulatory mechanisms may be functionally linked 50 52 In these studies we determined if direct activation of BKCa channel increases outflow facility. Additionally, we also tested the hypothesis that activation of the BKCa channels decrease TM cell volume in a time course that corresponds with the time course fo r activated BKCa channel induced increase in outflow facility. Materials and Methods Cell Culture Eyes from human donors with no history of ocular disease or surgery were obtained from Lions Eye Institute (Tampa, FL) within 24 30 hours postmortem. Prima ry human TM cell lines (numbers representing ages of the donors) (HTM26, HTM71, HTM36, HTM80 and HTM86) were developed as previously described 90 For our experimental protocols c ells from early passages (3 5) were used. TM cells were isolated after collagenase digestion of TM explants 64 Collagenase treated cells were grown in low glucose (1g/L) DMEM (Mediatech, Herdon VA.) in the presence of 10% fetal bovine serum (Mediatech, Herdon VA.), 100 U/ml penicillin and 100 g/ml streptomycin (Mediatech, Herdon VA.). Cells we re grown in 6well culture dishes (Nalge Nunc International, Rochester, NY) in a tissue culture incubator @ 37oC in 5% CO2. Confluent cells were trypsinized and passaged. Prior to drug treatment, cells were transferred to a FluoroDish (World Precision I nstruments, Inc) in low glucose DMEM as described above after which they were exposed to serum free media for 2 days prior to performing the experiments.

PAGE 68

68 Measurement of Cell Volume Cell volume measurements were performed as previously described 90 Prior to any drug treatments, the cells were loaded with the fluorescent dye Calcein AM in DMEM (~309 mOsm/kg) at 37C, in 5% CO2 incubator for 60 minutes to ensure a stable baseline. For changes in osmolarity, hypotonic media was made by addition of 30% deionized water to DMEM (~208 mOsm/kg). The coverslips containing the cells were subjected to confocal microscopy using a Leica confocal microscope. Changes in cell volume were determ ined by dividing the voxel count with drug treatment by the voxel count without drug treatment. Outflow Facility Measurements Outflow facility was measured using the anterior segment perfusion organ culture 67, 68 Porcine eyes were obtained from the local abattoir within 1 hour postmortem and maintained on ice. Because morphological and biochemical studies suggested that the porcine anterior chamber perfusion model can be corre lated with the human perfusion system 63 perfusion studies were performed using porcine eyes. Eyes were bisected at the equator and the iris, lens and ciliary processes were removed. The anterior segments were cultured at 37C in 100% humidity at 5% CO2 atmosphere and perfused at constant pressure of 14 mmHg 69, 70 The outflow rates were determined gravimetrically as the changes in weight of the media as the eyes were perfused over time and outflow f acility was expressed as l/min/mmHg perfusion pressure. Organ perfusions were performed with isotonic DMEM (~309 mOsm/kg) to establish baseline facility followed by perfusion with the drugs. Materials and Reagents Routine reagents, iberiotoxin (IBTX), and 1,3Dihydro1[2 hydroxy5(trifluoromethyl)phenyl] 5(trifluoromethyl)2H benzimidazol 2one (NS1619) were purchased

PAGE 69

69 from Sigma (St. Louis, MO). Diethylenetriamine NO (DETA NO) from Sigma RBI (Natick, MA). Statistics Statistical analysis was performed using ANOVA, followed by Holm Sidak and Fisher LSD method for comparison of significant difference among different means. Results NS1619 Increases Outflow Facility Outflow facility was measured in porcine anterior eye segments as described. Prior to drug treatment, eye anterior segments adapted to their new environment during which time outflow facility increased for several hours, a phenomenon referred to as wash out 70, 71 after which outflow facility rema ined stable 63 Basal outflow facility was 0.38150.4745 l/min/mmHg among experiments and was stable for several hours prior to drug treatment and remained stable post drug effect. Because of the stability of the outflow facility baseline after the initial wash out period, it was not necessary to correct for nondrug related changes in outflow facility in our experimental protocol. Previous studies have demonstrated that NS1619 dose dependently activated the outward current in aortic smooth muscle cells (3 30 M) 31 relaxes precontracted rat basilar artery rings (EC50=12.45+2.0 M) 100 and partially attenuated a hypotonic induced decrease in outflow facility in perfused bovine anterior segments (30 M) 5 To test the effect of NS1619 on outflow facility, NS1619 (30 M) was added to the perfusate. This resulted in a significant increase in outflow facility Figure 51. Outflow facility was increased immediately following drug treatment and reached statistical significance after 40 minutes Outflow facility reached it s maximal level 0.7816+0.0528 l/min/mmHg at 80 minutes foll owing application of the

PAGE 70

70 NS1619 and t he maximal effect of the drug was sustained for 3 hours after which outflow facility returned to values similar to baseline outflow facility. V alues are representative of three separate experiments. NS1619induced Decrease in TM Cell Volume is Concentration Dependent Quantitative measurements of changes in cells volume in response to NS1619 were made in low passage human TM cells. Cells were exposed to varyin g concentrations of the drug (300 nM 30 M). Images were taken without drug treatment (0) time point, drugs were then added to the cells and images were taken of the same cells at 20 min as described. Using this protocol, each cell serves as its own control. Cells treated with vehicle only are also imaged at 20 min to ensure that the effects observed are as a result of changes in cell volume in response to drug treatment and not because of photo bleaching or the vehicle. Figure 52 demonstrates that there is a concentration dependent decrease in TM cell volume in response to NS1619. While 300 nM NS1619 caused only a 3% decrease in cell volume, this decrease was significant when compared with the same cells before drug treatment. Addition of 3 M and 30 M NS1619 also resulted in significant decreases in cell volume, when compared with the same cells before drug treatment (7 and 10% changes respectively). The BKCa Channel is Involved in TM Cell Regulatory Volume Decrease To test that the effects of the BKCa channel on TM cell volume are not artifactual, cells were exposed to hypotonic media in the presence or absence of NS1619. Hypotonic media was made by addition of deionized water to DMEM for a final concentration of 30% water and 70% DMEM (~20 8 mOsm/kg). Images were captured of cells in isotonic media, after which the media were exchanged with hypotonic media and images were captured of the same cells at 1, 2, 3, 4 and 5 min. Figure 5 3 demonstrates that exposure of cells to hypotonic media res ulted in a

PAGE 71

71 7% increase in cell volume within 1 minute following application of hypotonic media after which cell volume returned to baseline without removal of the media. Cells exposed to NS1619 in the presence of hypotonic media resulted in a slight 1% inc rease in cell volume within 1 minute of changes in media conditions. One minute after the peak increase in cell volume, the cells experienced a significant decrease in cell volume after which cell volume returned to baseline conditions. Effects of NS1619 and DETANO on Cell Volume Are Not Additive We had previously demonstrated that the BKCa channel mediates the NO induced increases in outflow facility and the NO induced decreases in TM cell volume. Therefore in these studies we wanted to determine if the effects of NO on cell volume were additive in the presence of NS1619. TM cells were incubated with NS1619 only, with DETA NO only, or with NS1619 in the presence of DETA NO. Images were captured of the cells before drug treatment, after which the drugs were added and images captured at 20 minutes. There was a significant decrease in cell volume in response to both NS1619 and DETA NO treatment. When both NS1619 and DETA NO were added, cell volume decreases were equivalent to cell volume changes with either N S1619 or DETA NO alone (Figure 5 4). To determine that the effects of NS1619 are occurring through activation of the BKca channel, TM cells were incubated with NS1619 in the presence of IBTX 30 The NS1619 induced decrease in TM cell volume was abolished by preincubation with IBTX (Figure 54). Discussion To elucidate the role BKca channel activity plays in outflow facility and trabecular meshwork cell function, we utilized a direct activator of the BKca channel, NS1619. In these studies we demonstrate the ability of NS1619 to increase outflow facility in a time course that correlates with the NS1619 induced decreases in TM cell volume. Additionally, the decreases in

PAGE 72

72 TM cell volume in response to NS1619 were equivalent to the decreases in cell volume observed in response to DETA NO only, but in combinati on, NS1619 and DETA NO produced no additive cell volume decrease. Previous studies demonstrated the specificity of NS1619 to the BKca channel; exposure of bovine aortic smooth muscle cells 31 and rat cerebral arterial smooth muscle cells 100 to NS1619 resulted in increased open probability and outward currents that were blocked by t he potassium channel blockers tetraethylammonium and charybdotoxin 31 or IBTX 100 suggesting the outward current and subsequent hyperpolarization of these cells are due to BKca channel activation. Additionally, NS1619 dose dependently relaxed human coronary arterioles which was blocked by IBTX 101 Furthermore, NS1619 selectively activates BKca channels and has been shown to have no effect on calcium, sodium, ATP sensitive potassium or voltage gated potassium channels 31 ; 27 The selectivity of NS1619 for activation of the BKca channel allowed us to determine the role BKca channel activity plays in determining outflow facility. The ability of NS1619 to increase outflow facility in porcine anterior segment provides new insight into the role of the BKCa channel in aqueous humor outflow. The NS1619induced increases in outflow facility w ere immediate and transient and were sustained for 3 hours follow ing bolus application of the drug after which outflow facility returned to baseline. These experiments are the first, to our knowledge, to demonstrate that BKCa channel opening regulate s aqueous humor outflow. Other studies demonstrated that DETA NO incre ased aqueous humor outflow facility, and this increase was abolished by the BKCa channel inhibitor, IBTX 90 suggesting endogenous physiological regulation of the BKCa channel by the NO system.

PAGE 73

73 To better understand the cellular mechanism by which BKca channel activity affects outflow facility, we examined the ability of NS1619 to cause changes in TM cell volume, a mechanism by which TM cells are thought to influence outflow 7 ;47 ;48 ;6 ;5 ;49 Exposure of TM cells to NS1619 in isotonic media dose dependently decreased cell volume in a time course that correlates with the NS1619 induced increases in outflow facility. While we did not determine the mechanism u nderlying the NS1619induced cell volume decrease, electrophysiological studies in bovine aortic smooth muscle cells have demonstrated a NS1619induced, dose dependent leftward shift in the currentvoltage relationship with the drug suggesting increased potassium efflux with increasing drug concentration 31 To further demonstrate the ability o f the BKca channel to affect changes in TM cell volume, we included NS1619 in a hypotonic challenge and examined the drugs effect on regulatory volume decrease. NS1619 reduced the hypotonic induced increase in TM cell volume. Previously, we demonstrated t hat inhibition of the BKca channel with IBTX potentiated the hypotonic induced increases in cell volume and delayed the recovery to resting cell volume 90 Similar results were de monstrated in another study; NS1619 when included in a hypotonic challenge to a perfused bovine eye reduced the decrease in outflow facility and quickened recovery to baseline. Conversely, IBTX potentiated the hypotonic induced decreases in outflow facilit y and delayed recovery to baseline 5 Taken together, these data provide further evidence that BKca channel activity modulates TM cell volume and this modulation of TM cell volume may affect outflow facility. Incubation of TM cells with DETA NO only, NS1619 only or DETA NO in the presence of NS1619 resulted in decreased cell volume of similar magnitude suggesting that DETA NO and NS1619 are not additive in decreasing cell volume. Released NO from DETA NO is thought

PAGE 74

74 to activate the BKCa channel via protein phosphorylation through protein kinase G 28, 29 This PKGdependent phosphorylation of the BKca channel increased the outward currents in rabbit arterial smooth muscle cells by increasing the voltage dependent open probability of the channel 28 A similar mechanism is suggested to underlie the actions of NS1619 on BKca channel activity 31, 100 DETA NO and NS1619 individually caused similar decreases in TM cell volume, but together showed no additive effect on cell volume, suggesting that activating the BKca channel with either DETA NO or NS1619, presumably by altering the open probability of the channel, is sufficient to elicit a reduction in cell volume. Additionally, previous studies showed that IBTX attenu ated the NO induced decreases in TM cell volume, suggesting that the BKca channel is involved in the cell volume decrease 90 Similar results were demonstrated in these studies, a s IBTX abolished the NS1619induced decrease in TM cell volume. This provides another line of evidence suggesting the NS1619induced reductions in TM cell volume are due to BKca channel activity. The se reductions in TM cell volume with NS1619 in our experi ments may be due to potassium efflux, as studies in the TM have demonstrated that inhibition of the BKca channel with IBTX is associated with inhibition of potassium conductance 102 Potassium efflux through the BKca channel with possible parallel chloride efflux would tend to drive water out of the cytosol thus decreasing cell volume 95 We are mindful that there may not be a direct linkage between NS1619induced cha nges in TM cell volume and outflow facility as in these studies we have not accounted for the possible involvement of the BKca channel in TM contractile mechanisms 45, 52

PAGE 75

75 Figure 51. NS1619 increase s outflow facility. A stable baseline was achieved after which the anterior chamber perfusate was replaced with an acute treatment of NS1619 ( 30 M) dissolved in ethanol (0.1 %, final concentration). Data shown is mean + SEM for 3 experiments. *Significantly different from baseline values, P<0.05; ANOVA and the Fisher LSD method.

PAGE 76

76 Figure 52. NS1619induced decreases in TM cell volum e are concentration dependent. Data are expressed as % of initial volume at 0 time point and for NS1619: 300 nM, mean + SEM, n= 42 cells; 3 mean + SEM, n= 28 cells; and 30 M, mean + SEM, n= 34 cells *Significantly different from control at P<0.05; ANOVA and the Holm Sidak method.

PAGE 77

77 Figure 53. NS1619 abolishes the hypotonic induced increases in TM cell volume. H ypotonic DMEM incr eased cell volume (mean SEM; n =36 cells) Cells were exposed to hypotonic medium in the presence of NS1619 (30 M) and images were captured (mean SEM; n =35 cells) Data are expressed as % volume at 0 time point (isotonic medium) *S ignificantly differ ent from 0 time point, P<0.05 and # significantly different from hypotonic treatment at 1 and 2 minutes, P<0.05 by ANOVA and the Holm Sidak method.

PAGE 78

78 Figure 54. Actions of NS1619 and DETA NO on TM cell volume are not additive and are abolished by IBTX. Cells were incubated with NS1619 (30 M) alone, with DETA NO (100 M) alone, with NS1619 (30 M) and DETA NO (100 M) or with NS1619 (30 M) and IBTX (100 nM). Images were taken, and the cell volume was measured. Data are expressed as % of the ini tial volume at the 0 min time point and are means SEM; n= 24 cells for the NS1619 treated group, n=28 cells for the DETA NO treated group, n=13 cells for the NS1619 + DETA NO treated group, n=17 cells for the NS1619 + IBTX treated group. *Significantly different from vehicle p<0.05; by ANOVA and the Holm Sidak method. #Significantly different from NS1619 p<0.05; by ANOVA and the Holm Sidak method.

PAGE 79

79 CHAPTER 6 CONCLUSION Activation of Soluble Guanylate Cyclase and IOP In this study we demonstrated; 1. The signal transduction pathway mediating the NO induced increases in outflow facility and decreases in TM cell volume (figure 6 1) 2. The ability of the NO independent sGC activators YC 1 and BAY 582667 to cause decreases in TM cell volume and increases in cG MP, involving activation sGC, PKG and the BKca channel. 3. Direct activation of the BKca channel mimics NO in increasing outflow facility and decreasing TM cell volume. These provide the mechanism by which soluble gu anylate cyclase activators regulate IOP in humans, rabbit and monkey 35, 59, 61, 62, 103111 Studies have demonstrated that topical application of the NO donors to rabbit eyes 59 reduced IOP by increasing the rate of AH outflow through the TM 61 Studies have also shown that intravitrea l and intracameral injections of NO donors in rabbits caused a drastic decrease in IOP, which was correlated with nitrite production indicating that NO was released 62 Additionally it has been shown that NO donors reduce IOP in monkeys through an action on outflow resistance 35 While these actions on IOP are attributable to NO activating sGC, i t is important to note that activators of both membrane guanylate cyclase112, 112 and sG C have the ab ility to lower IOP. H owever, due to the pharmacokinetics of the peptide activators of membrane guanylate cyclase, they must be intravitreally applied to have an effect. This makes the IOP lowering effects mediated through sGC noteworthy, because many sGC a ctivators can be topically applied to the eye to achieve an effect. Although the IOP lowering ability of NO donors are known, little was known about the specific signal transduction pathway and cellular mechanism mediating the NO induced increase

PAGE 80

80 in outf low facility. Using perfused eye anterior segments, we found that the NO donor DETA NO increased outflow facility within twenty minutes of the drugs application. Based on the literature, this rapid time course for outflow facility changes eliminated two ce llular mechanisms thought to contribute to outflow regulation, alteration of the TM cytoskeleton and changes in the extracellular matrix of the TM. Both of these cellular mechanisms have been shown to affect outflow too slowly to account for our findings w ith DETA NO. The other identified cellular mechanisms by which TM cells affect outflow facility, changes in cell volume and tissue contractility, do fit with our rapid changes in outflow facility with NO. We decided to examine the effect of DETA NO on TM c ell volume in vitro using low passage TM cells and a confocal microscope. We measured the volume of TM cells over twenty minutes in five minute intervals and found DETA NO dose dependently lowered TM cell volume. To further ensure that the magnitude of ce ll volume change we were seeing with NO was appropriate to elicit a change in outflow facility, we treated perfused anterior segments with a hypertonic medium and measured the affect on outflow. Similarly, we treated our cultured cell with the same medium and measured cell volume. The results were consistent with our DETA NO studies, reduced TM cell volume correlates with increases in outflow facility. Using pharmalogical tools, we examine d the signal transduction pathway mediating these cell volume decreases. By inhibiting sGC, protein kinase G or the BKca channel we block ed the ability of DETA NO to cause TM cell volume decreases. Conversely, the cGMP analog 8 Br cGMP mimicked the effects of DETA NO on TM cell volume. With this knowledge in hand, we used the anterior segment perfusion system to demonstrate the requirement for BKca channel activity in the NO induced increases in outflow facility.

PAGE 81

81 With all of the evidence that NO acting on the TM lowers resistance to fluid flow, thus lowering IOP it was su rprising that sGC in the TM had not been very well characterized. Without knowing the sGC isoforms and the relative abundance of sGC in the TM we have no ground to look at potential alterations in the enzyme that may have implications in the pathophysiology of open angle glaucoma. Prior to our studies of NO independent sGC activators, we began to characterize sGC in cultured TM cells. In the low passage cell lines we tested we found consistent expression of the 1 and 1 sGC isoforms in an equivalent ratio. The ratio of to subunit expression is important as the functional enzyme consist of an heterodimer113 In these cells, DETA NO trea tment resulted in significant increases in cGMP. Interestingly, we could not detect a significant DETA NO induced increase in cGMP in transformed human TM cells and demonstrated the these cells do not express the and sGC subunits in equal abundance114 Still, further characterization of sGC in both the physiological and pathophysiological state is nee ded. It has been suggested that a subpopulation of patients with systemic hypertension have compromised NO sGC signaling due to oxidation of or decreased abundance of the heme moiety in sGC, a state rendering the enzyme incapable of being activated by NO115 Logically, a similar situation regarding the state of the sGC heme in the cells of the aqueous humor outflow pathway is possible, but yet unproven. Additionally, there are reports of reduced NOS abundance in the outflow pathway of patients with glaucoma22 In either of the cases, the ability to activate sGC in the TM would be diminished. A patient with a reduced ability to activate sGC in TM cells may experience reduced aqueous humor outflow and increase d IOP. Logically, adding e xogenous NO to the anterior chamber may overcome the lack of endogenous NO production or activate the remaining,

PAGE 82

82 undamaged sGCs, but may also have unintended, detrimental consequences detailed in chapter 1. Additionally, tolerance to the NO donor may deve lop, eliminating its ocular hypotensive properties59 Therefore we examine d the effects of NO independent activators of sGC. YC1 and BAY 582667 YC1 In our studies we de monstrate the ability of YC 1, the first reported NO independent activator of sGC18 to increase cGMP concentration and decrease TM cell volume in a manner dependent on activation of sGC, PKG and the BKca channel. Our data and data from the literature on YC1 suggest YC 1 like compounds may offer interesting properties as ocular hypotensive drugs. For example, YC 1 alone c an stimulate sGC activity but, NO and YC1 stimulate sGC activity synergistically Similar to NO, YC 1 cannot activate a heme independent or oxidizedheme sGC. Based on the similarities between YC 1 and NO in activating sGC work with YC1 has led to a better understanding of how NO activates sGC116 While we are interested in examining sGC activation in TM cells, o ur study of YC 1 on the cells of the outflow pathway is not without precedent. Topically applied YC 1 has been shown to reduce IOP in normotensive rabbits19 Since the discovery of YC 1s potential to activate sGC, a number of other compounds have been synthesized which show sGC stimulating properties. These compounds fall into two groups, heme dependent and heme independent sGC activators. As stated above, YC 1 is a heme dependent sGC activator and cannot activate sGC if the he me moiety is oxidized or absent. In our study of NO independent sGC activators in addition to YC 1, we selected a hemeindependent sGC activator BAY 582667 and examined its effects on TM cell volume.

PAGE 83

83 BAY582667 In our studies we demonstrate the ability of BAY582667 to increase cGMP concentration, and dose dependently reduce TM cell volume through activation of PKG and the BKca channel. Unlike YC 1, cells treated with ODQ experienced a potentiation of the BAY 582667 induced cell volume decrease. BAY582667 was first described in 200293 In this study, the investigators demonstrate the sGC activating properties of BAY 582667 alone. T his sGC activation was addititive with BAY 582667 and a NO donor. Inte restingly, when the sGC inhibitor ODQ was added with the BAY 582667 there was a potentiation of the BAY 582667 induced stimulation of sGC activity as opposed to the sGC inhibition seen with NO or YC1 in the presence of ODQ Additionally, BAY 582667 could stimulate heme free sGC activity unlike NO or YC 1. These heme independent sGC activating properties made BAY 582667 an interesting compound to include in our study. The results we obtained with both YC 1 and BAY 582667 fit well with studies on these compounds outside the eye. Both dose dependently reduced TM cell volume, although BAY 582667 appeared to be more effacious and potent. Both increase cGMP levels in cultured TM cells. They both appeared to activate the same signal transduction pathway as inhibitors of PKG and the BKca channel attenuated their effects on TM cell volume. In line with the literature, we could inhibit the YC 1 induced cell vo lume decrease with ODQ, while ODQ potentiated the cell volume reduction seen with BAY 582667 alone. Based on our findings and those in the literature, we can conclude that these NO independent sGC activating compounds may have ocular hypotensive prope rties of clinical significance. The interesting implication is that heme dependent sGC activators like YC 1 may benefit a patient with reduced endogenous NO production, but functional sGC. In this patient, the synergy of a YC 1like compound with the reduc ed NO production may restore sGC activity

PAGE 84

84 to normal levels. This has an advantage over simply adding exogenous NO to the eye, as there is a much lower potential for deleterious sGC independent NO effects. The possibility also exist that a n ocular hyper tens ive patient may have normal NOS activity in the outflow pathway, but a compromised sGC system, possibly due to oxidation of the heme moiety. In this patient, adding excess NO would have very little effect on increasing cGMP and decreasing IOP. However, the use of a NO independent, heme independent sGC activator like BAY 582667 could restore normal sGC cGMP activity and lower IOP. While YC 1 has been shown to lower IOP in normotensive rabbits, to date no one has demonstrated the ocular hypotensive effects o f BAY 582667. More importantly, neither compound has been shown to lower IOP in an ocular hypertensive patient or animal model, a critical step in gaining traction to become a clinically beneficial ocular hypotensive treatment. Direct Activation of the L arge Conductance, Calcium Activated Potassium Channel We demonstrated that in a perfused eye anterior segment that the well characterized, direct BKca channel activator NS1619 alone was capable of significantly increasing outflow facility. Similar to DETA NO, NS1619 reduced TM cell volume in a dose dependent manner. We also found that in combination, NS1619 and DETA NO gave no additive reduction in TM cell volume when compared to either alone. In chapter 3, we found that applying the BKca channel blocker I BTX following a NOinduced increase in outflow facility could immediately return outflow facility to baseline values, indicating sustained BKca channel activation is required for the NO induced increase. Our results indicate activation of the BKca channel in TM cells is all that is required to initiate a reduction in cell volume or increase in outflow facility. This raises t wo interesting questions; do direct activators of the BKca channel have clinically significant ocular hypotensive properties and what r ole, if any, does the BKca channel have in the pathophysiology of open angle

PAGE 85

85 glaucoma? While our findings indicate BKca channel activation increases outflow facility, our data does not account for actions on BKca channel stimulation in other tissue of the eye, which may interfere with our goal of lowering intraocular pressure. These concerns could be addressed with topically applied NS1619 in an animal model. We could then begin to understand the net effect of BKca channel stimulation in the eye. Secondly, it would be of interest to examine the functioning of the BKca channel in TM cells from a glaucomatous patient. The possibility exists that in some forms of ocular hypertension the BKca channel may be in reduced abundance or not responsive to the physiological means of activation. Physiological Significance The mechanism by which aqueous humor drainage from the eye and ultimately IOP is controlled is, to date, not fully understood. We have begun to dissect the cellular mechanisms mediating pharmacological changes in outflow facility, but much work must still be done. Our work in the TM has added to the understanding of how one endogenous modulator of outflow facility, NO, affects the TM cells. By identifying the signal transduction pathway mediating the NOinduced increases in outflow and decreases in TM cell volume, we were able to identify and characterize the role sGC and the BKca channel play in affecting outflow facility. Our work indicates that these two pharmacological targets h old promise for the development of novel ocular hypotensive strategies. Future Directions The studies presented here have only addressed the trabecular meshwork, one of the components associated with aqueous humor drainage. We have now begun to look at the effect of NO on the S chlemms canal. The relationship between the S chlemms canal the TM and how they in combination control aqueous humor drainage is not well understood. The data obtained regarding NO and the cells of the S chlemms canal may help unravel the contribution ea ch

PAGE 86

86 provides in resistance to aqueous drainage as well as how they work in combination. Additionally, our data are from in vitro studies and perfused porcine anterior eye segments. The next step towards determining the ocular hypotensive potential for compounds like BAY 582667 and NS1619 would involve topical application to the eye in a normotensive and ocular hypertensive animal model while monitoring IOP and outflow.

PAGE 87

87 LIST OF REFERENCES 1. Lee WR, Grierson P, McMenamin PG. The Morp hological Response of the Primate Outflow System. In: LutjenDrecoll E (ed), Basic Aspects of Glaucoma Research Stuttgart: F.K. Schattauer; 1982:123 139. 2. Kaufman PL. Aqueous humor outflow. Curr Top Eye Res. 1984;4:97138. 3. Krupin T, Civan MM. The physiological basis of aqueous humor formation. In: Ritch R, Shields MB (eds), The Glaucomas St. Louis: Mosby; 1995:251280. 4. Mitchell CH, Fleischhauer JC, Stamer WD, Peterson Yantorno K, Civan MM. Human trabecular meshwork cell volume regulation. Am J Phy siol Cell Physiol 2002;283:C315C326. 5. Soto D, Comes N, Ferrer E, et al. Modulation of aqueous humor outflow by ionic mechanisms involved in trabecular m eshwork cell volume regulation. Invest Ophthalmol Vis Sci 2004;45:36503661. 6. O'Donnell ME, Brandt JD, Curry FR. Na K Cl cotransport regulates intracellular volume and monolayer permeabilit y of trabecular meshwork cells. Am J Physiol. 1995;268:C1067C1074. 7. Al Aswad LA, Gong H, Lee D, et al. Effects of NaK 2Cl cotransport regulators on outflow faci lity in calf and human eyes in vitro. Invest Ophthalmol Vis Sci 1999;40:16951701. 8. Stumpff F, Wiederholt M. Regulation of trabecular meshwork contractility. Ophthalmologica. 2000;214:3353. 9. Bradley JM, Vranka J, Colvis CM, et al. Effect of matrix metalloproteinases activity on outflow in perfused human organ culture. Invest Ophthalmol Vis Sci 1998;39:26492658. 10. Grierson I, Johnson NF. The post mortem vacuoles of Schlemm's canal. Albrecht Von Graefes Arch Klin Exp Ophthalmol 1981;215:249264. 11. Polansky JR, Wood IS, Maglio MT, Alvarado JA. Trabecular meshwork cell culture in glaucoma research: evaluation of biological activity and structural properties of human trabecular cells in vitro. Ophthalmology 1984;91:580595. 12. Alexander JP, Samples JR, Van Buskirk EM, Acott TS. Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork. Invest Ophthalmol Vis Sci 1991;32:172180. 13. Thomas DD, Liu X, Kantrow SP, Lancaster JR, Jr. The biological lifetime of nitric oxide: impl ications for the perivascular dynamics of NO and O2. Proc Natl Acad Sci U S A 2001;98:355360.

PAGE 88

88 14. Murad F. Nitric oxide signaling: would you believe that a simple free radical could be a second messenger, autacoid, paracrine substance, neurotransmitter, and hormone? Recent Prog Horm Res. 1998;53:4359. 15. Lucas KA, Pitari GM, Kazerounian S, et al. Guanylyl cyclases and signaling by cyclic GMP Pharmacol Rev 2000;52:375 414. 16. Ford PC, Wink DA, Stanbury DM. Autoxidation kinetic s of aqueous nitric oxide FEBS Lett 1993;326:13. 17. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol. 1996;271:C1424C1437. 18. Ko FN, Wu CC, Kuo SC, Lee FY, Teng CM. YC 1, a novel activator of platelet guanyl ate cyclase. Blood 1994;84:42264233. 19. Kotikoski H, Alajuuma P, Moilanen E, et al. Comparison of nitric oxide donors in lowering intraocular pressure in rabbits: role of cyclic GMP J Ocul Pharmacol Ther 2002;18:1123. 20. Martin E, Lee YC, Murad F. Y C 1 activation of human soluble guanylyl cyclase has both heme dependent and heme independent components. Proc Natl Acad Sci U S A 2001;98:1293812942. 21. Mulsch A, Bauersachs J, Schafer A, Stasch JP, Kast R, Busse R. Effect of YC 1, an NOindependent, s uperoxide sensitive stimulator of soluble guanylyl cyclase, on smooth muscle responsiveness to nitrovasodilators. Br J Pharmacol. 1997;120:681689. 22. Nathanson JA, McKee M. Alterations of ocular nitric oxide synthase in human glaucoma. Invest Ophthalmol Vis Sci 1995;36:17741784. 23. Lamothe M, Chang FJ, Balashova N, Shirokov R, Beuve A. Functional characterization of nitric oxide and YC 1 activation of soluble guanylyl cyclase: structural implication for the YC 1 binding site? Biochemistry. 2004;43:30393048. 24. Stasch JP, Alonso Alija C, Apeler H, et al. Pharmacological actions of a novel NO independent guanylyl cyclase stimulator, BAY 418543: in vitro studies Br J Pharmacol 2002;135:333343. 25. Stumpff F, Strauss O, Boxberger M, Wiederholt M. Char acterization of maxi K channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate. Invest Ophthalmol Vis Sci 1997;38:18831892. 26. Wiederholt M, Sturm A, Lepple Wienhues A. Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide. Invest Ophthalmol Vis Sci 1994;35:25152520.

PAGE 89

89 27. Ghatta S, Nimmagadda D, Xu X, O'Rourke ST. Large conductance, calcium activated potassium channels: structural and functional implications. Pharmacol Ther 2006;110:103116. 28. Robertson BE, Schubert R, Hescheler J, Nelson MT. cGMP dependent protein kinase activates Ca activated K channels in cerebral artery smooth muscle cells. Am J Physiol. 1993;265:C299C303. 29. Alioua A, Tanaka Y, Wallner M, et al. The large conductance, voltage dependent, and calcium sensitive K+ channel, Hslo, is a target of cGMP dependent protein kinase phosphorylation in vivo. J Biol Chem 1998;273:3295032956. 30. Galvez A, GimenezGallego G, Reuben JP, et al. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem 1990;265:1108311090. 31. Olesen SP, Munch E, Moldt P, Drejer J. Selective activation of Ca(2+) dependent K+ channels by novel benzimidazolone. Eur J Pharmacol 1994;251:5359. 32. Zhou L, Zhang SR, Yue BY. Adhesion of human trabecular meshwork cells to extracellular matrix proteins. Roles and distribution of integrin receptors. Invest Ophthalmol Vis Sci 1996;37:104113. 33. Stamer WD, Roberts BC, Howell DN, Epstein DL. Isolation, culture, and characterization of endothelial cells from Schlemm's canal. Invest Ophthalmol Vis Sci 1998;39:18041812. 34. Kaufman PL, BARANY EH. Cytochalasin B reversibly increases outflow facility in the eye of the cynomolgus monkey. Invest Ophthalmol Vis Sci 1977;16:4753. 35. Schuman JS, Erickson K, Nathanson JA. Nitrovasodilator effects on intraocular pressure and outflow facility in monkeys. Exp Eye Res 1994;58:99105. 36. Mit chell CH, Fleischhauer JC, Stamer WD, Peterson Yantorno K, Civan MM. Human trabecular meshwork cell volume regulation. Am J Physiol. 2002;283:C315C326. 37. Lutjen Drecoll E. Importance of trabecular meshwork changes in the pathogenesis of primary openangle glaucoma. J Glaucoma 2000;9:417418. 38. Fuchshofer R, Welge Lussen U, LutjenDrecoll E, Birke M. Biochemical and morphological analysis of basement membrane component expression in corneoscleral and cribriform human trabecular meshwork cells. Invest O phthalmol Vis Sci 2006;47:794 801. 39. Coroneo MT, Korbmacher C, Flugel C, Stiemer B, LutjenDrecoll E, Wiederholt M. Electrical and morphological evidence for heterogeneous populations of cultured bovine trabecular meshwork cells. Exp Eye Res 1991;52:375388.

PAGE 90

90 40. Fleischhauer JC, Mitchell CH, Stamer WD, Karl MO, Peterson Yantorno K, Civan MM. Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport. J Membr Biol. 2003;193:121136. 41. B arany EH. The mode of act ion of pilocarpine on outflow resistance in the eye of a primate (Cercopithecus ethiops). Invest Ophthalmol 1962;1:712727. 42. Lepple Wienhues A, Stahl F, Wiederholt M. Differential smooth muscle like contractile properties of trabecular meshwork and cil iary muscle. Exp Eye Res 1991;53:3338. 43. Wiederholt M. Direct involvement of trabecular meshwork in the regulation of aqueous humor outflow. Curr Opin Ophthalmol 1998;9:4649. 44. Llobet A, Gual A, Pales J, Barraquer R, Tobias E, Nicolas JM. Bradykini n decreases outflow facility in perfused anterior segments and induces shape changes in passa ged BTM cells in vitro Invest Ophthalmol Vis Sci 1999;40:113125. 45. Wiederholt M, Thieme H, Stumpff F. The regulation of trabecular meshwork and ciliary muscle contractility. Prog Retin Eye Res. 2000;19:271 295. 46. Rao PV, Deng PF, Kumar J, Epstein DL. Modulation of aqueous humor outflow facility by the Rho kinase specific inhibitor Y 27632. Invest Ophthalmol Vis Sci 2001;42:10291037. 47. Freddo TF, Patterson MM, Scott DR, Epstein DL. Influence of mercurial sulfhydryl agents on aqueous outflo w pathways in enucleated eyes. Invest Ophthalmol Vis Sci 1984;25:278 285. 48. Gual A, Llobet A, Gilabert R, et al. Effects of time of storage, albumin, and osmolality cha nges on outflow facility (C) of bovine anterior segment in vitro. Invest Ophthalmol Vis Sci 1997;38:21652171. 49. Srinivas SP, Maertens C, Goon LH, et al. Cell volume response to hyposmotic shock and elevated cAMP in bo vine trabecular meshwork cells. Exp Eye Res. 2004;78:1526. 50. Ziyadeh FN, Mills JW, Kleinzeller A. Hypotonicity and cell volume regulation in shark rectal gland: role of organic osmolytes and F actin. Am J Physiol. 1992;262:F468F479. 51. Henson JH, Roesener CD, Gaetano CJ, et al. Confocal microscopic observation of cytoskeletal reorganizations in cultured shark rectal gland cells following treatment with hypotonic shock and high external K+. J Exp Zool 1997;279:415424. 52. Tian B, Geiger B, Epstein DL, Kaufman PL. Cytoskeletal involveme nt in the regulation of aqueous humor outflow. Invest Ophthalmol Vis Sci 2000;41:619623.

PAGE 91

91 53. Benham CD, Bolton TB, Lang RJ, Takewaki T. Calcium activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea pig mesenteric artery. J Physiol 1986;371:4567. 54. Nelson MT, Cheng H, Rubart M, et al. Relaxation of arterial smooth muscle by calcium sparks. Science 1995;270:633637. 55. Singer JJ, Walsh JV, Jr. Characterization of calcium activated potassium channels in single smooth m uscle cells using the patch clamp technique. Pflugers Arch 1987;408:98 111. 56. Sausbier M, Schubert R, Voigt V, et al. Mechanisms of NO/cGMP dependent vasorelaxation. Circ Res 2000;87:825830. 57. Nathanson JA, McKee M. Identification of an extensive sy stem of nitric oxide producing cells in the ciliary muscle and outflow pathway of the human eye. Invest Ophthalmol Vis Sci 1995;36:17651773. 58. Selbach JM, Gottanka J, Wittmann M, Lutjen Drecoll E. Efferent and afferent innervation of primate trabecular meshwork and scleral spur. Invest Ophthalmol Vis Sci 2000;41:21842191. 59. Nathanson JA. Nitrovasodilators as a new class of ocular hypotensive agents. J Pharmacol Exp Ther 1992;260:956 965. 60. Giuffrida S, Bucolo C, Drago F. Topical application of a nitric oxide synthase inhibitor reduces intraocular pressure in rabbits with experimental glaucoma. J Ocular Pharmacol Ther 2003;19:527534. 61. Kotikoski H, Vapaatalo H, Oksala O. Nitric oxide and cyclic GMP enhance aqueous humor outflow facility in rabbits. Curr Eye Res. 2003;26:119123. 62. Behar Cohen FF, Goureau O, d'Hermies F, Courtois Y. Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye. Invest Ophthalmol Vis Sci 1996;37:17111715. 63. Bachmann B, Birke M, Kook D, Eichhorn M, Lutjen Drecoll E. Ultrastructural and biochemical evaluation of the porcine anterior chamber perfusion model. Invest Ophthalmol Vis Sci 2006;47:20112020. 64. Stamer WD, Seftor RE, Williams SK, Samaha HA, Snyder RW. Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion. Curr Eye Res. 1995;14:611 617. 65. Bush PG, Hall AC. The volume and morphology of chondrocytes within nondegenerate and degenerate human articular cartilage. Osteoarthritis Cartilage 2003;11:242 251.

PAGE 92

92 66. Bush PG, Hodkinson PD, Hamilton GL, Hall AC. Viability and volume of in situ bovine articular chondrocytes changes following a single impact and effects of medium osmolarity. Osteoarthritis Cartilage 2005;13: 54 65. 67. Johnson DH, Tschumper RC. Human trabecular meshwork organ culture. A new method. Invest Ophthalmol Vis Sci 1987;28:945 953. 68. Johnson DH, Tschumper RC. The effect of organ culture on human trabecular meshwork Exp Eye Res 1989;49:113127. 69. EricksonLamy K, Rohen JW, Grant WM. Outflow facility studies in the perfused bovine aqueous outflow pathways Curr Eye Res. 1988;7:799 807. 70. Overby D, Gong H, Qiu G, Freddo TF, Johnson M. The mechanism of increasing outflow facility during washout in the bovine eye. Invest Ophthalmol Vis Sci 2002;43:34553464. 71. EricksonLamy K, Schroeder AM, Bassett Chu S, Epstein DL. Absence of time dependent facility increase ("washout") in the perfused enucleated human eye Invest Ophthalmol Vis Sci 1990;31:23842388. 72. Clemo HF, Feher JJ, Baumgarten CM. Modulation of rabbit ventricular cell volume and Na+/K+/2Cl cotransport by cGMP and atrial natriuretic factor. J Gen Physiol 1992;100:89 114. 73. Garthwaite J, Southam E, Boulton CL, Nielsen EB, Schmidt K, Mayer B. Potent and selective inhibition of nitric oxide sensitive guanylyl cyclase by 1H [1,2,4]oxadiazolo[4,3a]quinoxalin1 one. Molec Pharmacol 1995;48:184 188. 74. Ellis DZ, Nathanson JA, Sweadner KJ. Carbachol inhibits Na+K+ATPase activity in chor oid plexus via stimulation of the NO/cGMP pathway. Am J Physiol. 2000;279:C1685C1693. 75. Scavone C, Scanlon C, McKee M, Nathanson JA. Atrial natriuretic peptide modulates sodium and potassium activated adenosine triphosphatase through a mechanism involvi ng cyclic GMP and cyclic GMP dependent protein kinase. J Pharmacol Exp Ther 1995;272:10361043. 76. Butt E, Pohler D, Genieser HG, Huggins JP, Bucher B. Inhibition of cyclic GMP dependent protein kinase mediated effects by (Rp)8bromoPETcyclic GMPS. Br J Pharmacol 1995;116:31103116. 77. Kee C, Kaufman PL, Gabelt BT. Effect of 8Br cGMP on aqueous humor dynamics in monkeys. Invest Ophthalmol Vis Sci 1994;35:27692773. 78. Tanaka Y, Koike K, Toro L. MaxiK channel roles in blood vessel relaxations induc ed by endothelium derived relaxing factors and their molecular mechanisms. J Smooth Muscle Res. 2004;40:125 153.

PAGE 93

93 79. Krupin T, Weiss A, Becker B, Holmberg N, Fritz C. Increased intraocular pressure following topical azide or nitroprusside Invest Ophthalmol Vis Sci 1977;16:10021007. 80. Feelisch M. The use of nitric oxide donors in pharmacological studies. Naunyn Schmiedebergs Arch Pharmacol 1998;358:113122. 81. Comes N, Abad E, Morales M, Borras T, Gual A, Gasull X. Identification and functional charac terization of ClC 2 chloride channels in trabecular meshwork cells. Exp Eye Res 2006;83:877889. 82. Acott TS, Kingsley PD, Samples JR, Van Buskirk EM. Human trabecular meshwork organ culture: morphology and glycosaminoglycan synthesis. Invest Ophthalmol Vis Sci 1988;29:90100. 83. Clemo HF, Baumgarten CM, Ellenbogen KA, Stambler BS. Atrial natriuretic peptide and cardiac electrophysiology: autonomic and direct effects. J Cardiovasc Electrophysiol. 1996;7:149162. 84. Putney LK, Brandt JD, O'Donnell ME. N a K Cl cotransport in normal and glaucomatous human trabecular meshwork cells. Invest Ophthalmol Vis Sci 1999;40:425 434. 85. Larsen AK, Jensen BS, Hoffmann EK. Activation of protein kinase C during cell volume regulation in Ehrlich mouse ascites tumor ce lls. Biochim Biophys Acta. 1994;1222:477482. 86. Gabelt BT, Wiederholt M, Clark AF, Kaufman PL. Anterior segment physiology after bumetanide inhibition of Na K Cl cotransport. Invest Ophthalmol Vis Sci 1997;38:17001707. 87. Garbers DL. Purification of s oluble guanylate cyclase from rat lung. J Biol Chem 1979;254:240243. 88. Wedel B, Harteneck C, Foerster J, Friebe A, Schultz G, Koesling D. Functional domai ns of soluble guanylyl cyclase J Biol Chem 1995;270:2487124875. 89. Arnold WP, Mittal CK, Katsu ki S, Murad F. Nitric oxide activates guanylate cyclase and increases guanosine 3':5' cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci U S A 1977;74:32033207. 90. Dismuke WM, Mbadugha CC, Ellis DZ. NO induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel. Am J Physiol Cell Physiol. 2008;294:C1378C1386. 91. Schmidt PM, Schramm M, Schroder H, Wunder F, Stasch JP. Identification of residues crucially involved in the binding of the heme moiety of soluble guanylate cyclase. J Biol Chem 2004;279:30253032.

PAGE 94

94 92. Wu CC, Ko FN, Kuo SC, Lee FY, Teng CM. YC 1 inhibited human platelet aggregation through NO independent activation of soluble guanylate cyclase Br J Phar macol 1995;116:19731978. 93. Stasch JP, Schmidt P, onsoAlija C, et al. NO and haem independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle. Br J Pharmacol. 2002;136:773783. 94. Schmidt P, Schramm M, Schroder H, Stasch JP. Mechanisms of nitric oxide independent activation of soluble guanylyl cyclase. Eur J Pharmacol 2003;468:167174. 95. Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S. Receptor mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 2001;532:316. 96. Ellis DZ, Nathanson JA, Rabe J, Sweadner KJ. Carbachol and nitric oxide inhibition of Na,K ATPase activity in bovine ciliary processes. Invest Ophthalmol Vis Sci 2001;42:26252631. 97. Shahidullah M, Delamere NA. NO donors inhibit Na,K ATPase activity by a protein kinase G dependent mechanism in the nonpigmented ciliary epithelium of the porcine eye. Br J Pharmacol. 2006;148:871880. 98. Krauss AH, Wiederh olt M, Sturm A, Woodward DF. Prostaglandin effects on the contractility of bovine trabecular meshwork and ciliary muscle. Exp Eye Res. 1997;64:447 453. 99. Miller C, Moczydlowski E, Latorre R, Phillips M. Charybdotoxin, a protein inhibitor of single Ca2+ a ctivated K+ channels from mammalian skeletal muscle. Nature 1985;313:316318. 100. Holland M, Langton PD, Standen NB, Boyle JP. Effects of the BKCa channel activator, NS1619, on rat cerebral artery smooth muscle. Br J Pharmacol 1996;117:119129. 101. Fen g J, Liu Y, Clements RT, et al. Calcium activated potassium channels contribute to human coronary microvascular dysfunction after cardioplegic arrest. Circulation. 2008;118:S46S51. 102. Gasull X, Ferrer E, Llobet A, et al. Cell membrane stretch modulates the highconductance Ca2+ activated K+ channel in bovine trabecular meshwork cells. Invest Ophthalmol Vis Sci 2003;44:706714. 103. Millar JC, Shahidullah M, Wilson WS. Atriopeptin lowers aqueous humor formation and intraocular pressure and elevates ciliary cyclic GMP but lacks uveal vascular effe cts in the bovine perfused eye J Ocul Pharmacol Ther 1997;13:111.

PAGE 95

95 104. Mittag TW, Tormay A, Ortega M, Severin C. Atrial natriuretic peptide (ANP), guanylate cyclase, and intraocul ar pressure in the rabbit eye Curr Eye Res. 1987;6:1189 1196. 105. Nathanson JA. Direct application of a guanylate cyclase activat or lowers intraocular pressure. Eur J Pharmacol 1988;147:155156. 106. Diestelhorst M, Krieglstein GK. [The intraocular pressure lowering ef fect of human a trial peptide] Fortschr Ophthalmol 1989;86:89 91. 107. Fernandez Durango R, Moya FJ, Ripodas A, de Juan JA, Fernandez Cruz A, Bernal R. Type B and type C natriuretic peptide receptors modulate intraocu lar pressure in the rabbit eye Eur J Pharmacol 1999;364:107113. 108. Goldmann DB, Waubke N. [A pilot study on the effect of atrial natriuretic peptide on intraocular pre ssure in the human] Fortschr Ophthalmol 1989;86:494496. 109. Korenfeld MS, Becker B. Atrial natriuretic peptides. Effects on intraocul ar pre ssure, cGMP, and aqueous flow Invest Ophthalmol Vis Sci 1989;30:23852392. 110. Wolfensberger TJ, Singer DR, Freegard T, Markandu ND, Buckley MG, MacGregor GA. Evidence for a new role of natriuretic peptides: c ontrol of intraocular pressure Br J O phthalmol 1994;78:446448. 111. Yang L, Guan J, Gao S. [An experimental study on effect of atrial natriuretic peptide on intraocular pressure of white rabbits] Zhonghua Yan Ke Za Zhi 1997;33:149151. 112. Mittag TW, Tormay A, Ortega M, Severin C. Atrial natriuretic peptide (ANP), guanylate cyclase, and intraocul ar pressure in the rabbit eye Curr Eye Res. 1987;6:1189 1196. 113. Foerster J, Harteneck C, Malkewitz J, Schultz G, Koesling D. A functional hemebinding site of soluble guanylyl cyclase requires intact N termini o f alpha 1 and beta 1 subunits Eur J Biochem 1996;240:380386. 114. Ellis DZ, Dismuke WM, Chokshi BM. Characterization of Soluble Guanylate Cyclase in NOInduced Increases in Aqueous Humor Outflow Facility and in the Trabecular Meshwork Invest Ophthalmol Vis Sci 2008. 115. Stasch JP, Schmidt PM, Nedvetsky PI, et al. Targeting the heme oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels. J Clin Invest 2006;116:25522561. 116. Andersson RM, Aizman O, A peria A, Brismar H. Modulation of Na+,K+ ATPase act ivity is of importance for RVD Acta Physiol Scand. 2004;180:329334.

PAGE 96

96 BIOGRAPHICAL SKETCH William Michael Dismuke was born in Jacksonville, FL in 1980 to Bill and Nancy Dismuke. He managed to graduate Mandarin High School in 1999 and surprisingly was accepted to attend the University of Florida in the fall of 1999. He graduated in the fall of 2003 with a BS in Botany.