Pharmacological characterization of the phosphoinositide second messenger system in the rabbit kidney

MISSING IMAGE

Material Information

Title:
Pharmacological characterization of the phosphoinositide second messenger system in the rabbit kidney
Physical Description:
vii, 120 leaves : ill. ; 29 cm.
Language:
English
Creator:
McArdle, Shari, 1961-
Publication Date:

Subjects

Subjects / Keywords:
Phosphatidylinositols -- biosynthesis   ( mesh )
Kidney -- physiology   ( mesh )
Pharmacology and Therapeutics thesis Ph.D   ( mesh )
Dissertations, Academic -- Pharmacology and Therapeutics -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1988.
Bibliography:
Bibliography: leaves 112-119.
Statement of Responsibility:
by Shari McArdle.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001035364
oclc - 20606726
notis - AFB7727
System ID:
AA00011809:00001


This item is only available as the following downloads:


Full Text











PHARMACOLOGICAL CHARACTERIZATION OF THE PHOSPHOINOSITIDE SECOND MESSENGER
SYSTEM IN THE RABBIT KIDNEY












By

SHARI McARDLE






















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1988






















This dissertation is dedicated to my parents, John and Yuriko; my brothers, Takashi and

Jack; and my sister, Linda.
















ACKNOWLEDGEMENTS

I would like to thank Dr. Lal C. Garg, my advisor, for his support and attention

throughout my graduate training. I would also like to thank my dissertation committee:

Dr. Fulton Crews, Dr. Allen Neims, Dr. Christopher Wilcox, and Dr. Charles Allen. I

would like to acknowledge Dr. Noveen Das, Pauletta Sanders, Cindy Theiss, and Neil Ganz

for their technical assistance. I would like to thank Lynn, Sukanya, and Judy for their

help with the computer.

I would also like to thank other members of this laboratory: Neelam, Maciej,

Elzbieta, Dayna, Magda, Sherry, Mike, and Tony for making lab an enjoyable place to

work. Thanks are given to former fellow student and lunch escapee, Dr. Rita Bortell, for

tolerating my silliness and for being a special friend. Finally,l would like to thank the

students and staff for their support and friendship.
















TABLE OF CONTENTS

PAGE
ACKNOWLEDGEMENTS ...................................................................................................iii

A BS T R A C T ................................................................................. ......................................vi

CHAPTERS

1. INT RO D UC TIO N ............................................................... ........................... 1

The Phosphoinositide Second Messenger System........................................3
Diacylglycerol: A Second Messenger....................................... ..............6
Inositol Trisphosphate: A Second Messenger........................... ............. 6
The Kidney: Structure and Function...................................... ...............7
Control of Renal Function.....................................................................8

2. THE EFFECT OF VARIOUS HORMONES AND NEUROTRANSMITTERS
ON PHOSPHOINOSITIDE HYROLYSIS IN RABBIT KIDNEY...........................10

Introduction...................................................... .............................10
Materials and Methods........................................................................11
R esults..................................................................................................14
Discussion...........................................................................................16

3. CHOUNERGIC STIMULATION OF PHOSPHOINOSITIDE HYDROLYSIS
IN RABBIT KIDNEY..............................................................................25

Introduction...................................................................................25
Materials and Methods........................................................................25
R esults............................................................................ .....................35
Discussion...........................................................................................55

4. IMMUNOHISTOCHEMICAL LOCAUZATION OF PHOSPHATIDYUNOSITOL
4,5-BISPHOSPHATE IN RABBIT KIDNEY.................................................58

Introduction........................................................................................58
Materials and Methods................................. .......................................58
R esults..................................................................................................60
Discussion...........................................................................................60










5. CHOUNERGIC STIMULATION OF PHOSPHOINOSITIDE HYDROLYSIS IN
INNER MEDULLARY COLLECTING DUCT (IMCD) CELLS OF THE
RABBIT KIDNEY............................................................ ......................67

Introduction...................................................... ..................................67
Materials and Methods................................. ............... ........................68
Results.................... ............................................................ .............. 72
Discussion........................................................ 85

6. CHOUNERGIC RECEPTORS IN INNER MEDULLARY COLLECTING DUCT
CELLS OF THE RABBIT KIDNEY.......................................................................90

Introduction................................... .................... ... ..........................90
Materials and Methods................................. ............... ........................90
Results............... .................. ............. ................................................... 92
Discussion........................................................ ................................ 106

7. SUMMARY AND CONCLUSIONS.......................... .. ............ ............ 10

REFERENC ES.............................................................................. ................................ 12

BIOGRAPHICAL SKETCH.......................... ... ..................................................120















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

PHARMACOLOGICALCHARACTERIZATION OFTHE PHOSPHOINOSITIDE SECOND MESSENGER
SYSTEM IN THE RABBIT KIDNEY

By

Shari McArdle

August, 1988

Chairman: Lal C. Garg
Major Department: Pharmacology and Therapeutics

The cellular response to hormones and neurotransmitters is a result of receptor

activation of a second messenger system to initiate the intracellular cascade. In several

tissues, such as brain and liver, one of the second messenger systems involves the

hydrolysis of phosphoinositides (Pis) for the formation of inositol phosphates and

diacylglycerol as the intracellular messengers. In the present study, we examined the

effect of various agents on the hydrolysis of PIs in the rabbit kidney.

In the kidney, the effect of the various hormones and neurotransmitters was

region specific. Hydrolysis of PIs was stimulated in the inner medulla by [arg8]-

vasopressin, angiotensin II, and atriopeptin I, and in the outer medulla by histamine,

adenosine, and secretin. Only carbachol was able to stimulate the hydrolysis of Pis in

both the inner and outer medulla. None of the substances tested were able to stimulate

this response in the cortex. The following agents did not have an effect in any of the

three zones of the kidney: norepinephrine, dopamine, atriopeptins II, and Ill.

The effect of the cholinergic agonist, carbachol, on the kidney was further

examined. Cholinergic agents are known to cause renal vasodilation and diuresis. We

have shown that carbachol-stimulated hydrolysis of Pis in the inner medulla was










blocked by the muscarinic receptor antagonist, atropine. A similar effect was observed

with cholinergic-stimulation of hydrolysis of PIs in the inner medullary collecting duct

(IMCD) cells. This suggests that carbachol-stimulated PIs hydrolysis in the inner

medulla and IMCD cells was mediated by muscarinic receptors present in the rabbit

kidney.

Furthermore, we have directly demonstrated the presence of a high affinity

saturable binding site on IMCD cells with studies of binding characteristics of the

radiolabelled muscarinic antagonist, 1-quinuclidinyl (phenyl-4-3H) benzilate

([3H]QNB). The Kd of 0.27 nM and the Bmax of 27.5 fmol/mg protein were determined

from Scatchard analysis of the saturation data.

In summary, we have demonstrated that cholinergic muscarinic receptors are

present in the rabbit kidney, specifically in the IMCD cells. These receptors, which are

coupled to the hydrolysis of phosphoinositides, may be involved in the vasodilatory

and/or diuretic effects of cholinergic agents.
















CHAPTER 1
INTRODUCTION

The organization of multicellular organisms often requires individual cells to be

able to communicate with the other cells for coordination of different functions. In

higher organisms, intercellular communication is often the responsibility of the

nervous and endocrine systems which utilize neurotransmitters and hormones,

respectively, as primary messengers.

The specificity of the action on the target cell is dependent upon the presence of a

receptor for the hormone or neurotransmitter. In general, a receptor is a protein that

is located on the cell surface, but can also be found in the cytosol in some circumstances

(Ross and Oilman, 1985). The association or binding of a hormone or neurotransmitter

to its receptor is just the first step that culminates in the cellular response, such as

muscle contraction or fluid secretion. The interaction of the receptor with its ligand

activates transducer and other proteins within the cell membrane which results in the

formation and/or accumulation of an intracellular second messenger. This second

messenger, in turn, is the internal signal for the cellular response. In some cases, a

hormone or neurotransmitter is able to stimulate more than one response by binding to

Different, but specific receptor subtypes which may be coupled to different second

messenger systems (Table 1-1).

At present, studies indicate that there are at least two second messenger systems.

One involves receptor-activation of the enzyme, adenylate cyclase which results in the

formation of adenosine 3',5'-monophosphate (cyclic AMP) (Baxter and Funder, 1979).

The cyclic AMP is the second messenger in this system by activation of cyclic AMP-

dependent protein kinases. It is, however, noted that some receptors are coupled to the

inhibition of the enzyme, adenylate cyclase.











TABLE 1-1
SECOND MESSENGER SYSTEMS: THE RECEPTORS AND VARIOUS TARGET TISSUES.

Second
Messenger
System Stimulus Receptor Tissue

Stimulation of
cyclic AMP
formation NE beta skeletal muscle, fat
cells, heart, smooth
muscle

Vasopressin V2 kidney, liver

TSH thyroid

Serotonin salivary gland
(blowfly)

Prostaglandin 11 blood platelets

Inhibition of
cyclic AMP
formation NE alpha blood platelets

Adenosine fat cells

Pis response ACh muscarinic pancreas (1-cells),
smooth muscle, brain,
adrenal medulla

NE alphal liver, brain, aorta

Vasopressin V1 liver, smooth muscle,
sympathetic ganglion
Ang II liver, adrenal cortex

Histamine H1 smooth muscle, brain

Bradykinin Endothelial cells,
kidney
Abbreviations: NE, norepinephrine; TSH, thyroid stimulating hormone, ACh,
acetylcholine; Ang II, angiotensin II. From Berridge (1985).










Another system uses calcium as the second messenger. The interaction of calcium

with calmodulin results in modifications of activities of various proteins (Cheung,

1980). There is increasing evidence, however, that the hydrolysis of inositol-

containing phospholipids precedes the mobilization of calcium (Michell and Kirk,

1981).

The Phosohoinositide Second Messenger System

The possibility that the phosphoinositides play an important role in the effects of

a neurotransmitter, such as acetylcholine (ACh), was first described by Hokin and Hokin

(1955). They showed that ACh stimulated the metabolism of phosphoinositides in the

pancreas and suggested that phosphoinositides may play a role in cell function.

Increasing evidence suggested of a relationship between phosphoinositide metabolism and

calcium mobilization (Berridge, 1986; Michell, 1986a).

The phosphoinositides include, at least, three inositol-containing phospholipids:

phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and

phosphatidylinositol 4,5-bisphosphate (PIP2) (Figure 1-1). Of the total cell lipid, the

Pis are only a minor component, less than 5% in brain and iris muscle (Abdel-Latif,

1986).

The major contributor to cell phosphoinositides is PI which is synthesized by one

of the following pathways: 1) the reaction of inositol with CDP-diacylglycerol which is

Scatalyzed by the enzyme, PI synthetase or 2) the reaction of inositol with existing

phospholipid with a specific exchange protein (Holub, 1986). The

polyphosphoinositides and PI are interconverted by phosphomonoesterases and kinases.

Evidence suggests that it is PIP2 which plays the important role in receptor-

mediated events (Berridge, 1983). Binding of the external signal to its receptor

activates a phosphodiesterase, known as phospholipase C, which catalyzes the hydrolysis

of PIP2 and results in the formation of two second messengers, inositol trisphosphate
















80




o .0


RS?
.0 ci a---


C. Cl


_ c_.
C L



a.
E a
^."




o (L


CD
0 C









0
cm



cO
Q.C




















ow. 15
.E0 '


.0
0 L .




-r
0 a0


.0 0
6 CD



0 a
o:S







IL zs



























t 1




t I


I .) -
_^


auejqua|
i
I


lOSOl~A


I










(IP3) and diacylglycerol (DAG) (Figure 1-1) (Berridge, 1983). These two products

are discussed below.

The specificity of phospholipase C, however, is not absolute because PI and PIP

are also substrates (Majerus e al. 1986). The products of hydrolysis are DAG and

inositol monophosphate (IP) or inositol diphosphate (IP2). The importance of IP and

IP2 as second messengers has not been established.

Diacvlolvcerol: A Second Messenaer

Diacylglycerol acts as a second messenger by stimulation of protein

phosphorylation through protein kinase C (Berridge, 1984). Diacylglycerol or its

derivatives, phorbol esters, activates protein kinase C by increasing the affinity of the

enzyme for calcium (Kaibuchi al., 1981; Nishizuka, 1986).

Protein kinase C has a wide distribution in many tissues (Kuo eal., 1980). It

is known to phophorylate a variety of proteins, such as the alphal-adrenergic receptor

(Sibley etal., 1984) and IP3 phosphatase (Connolly and Majerus, 1986). The

phosphorylation of specific proteins by protein kinase C may play a role in cell function.

Diacylglycerol or its derivatives have been shown to alter different cell functions

(Abdel-Latif, 1986).

Under normal conditions, the increased levels of the second messenger is

transient. The actions of DAG are terminated by its rapid metabolism. Diacylglycerol is

S metabolized: 1) by DAG kinase to produce phosphatidic acid to be recycled into the

phospholipids, including PI or 2) by lipases to produce arachidonic acid which can

undergo further processing into such substances, as prostaglandins and leukotrienes

(Abdel-Latif, 1986).

Inositol Trisohosohate: A Second Messenger

Receptors that were previously thought to stimulate calcium fluxes may be

coupled to hydrolysis of phosphoinositides as the initial step leading to the cellular

response. Inositol trisphosphate is the other second messenger produced and is involved










in calcium mobilization (Berridge, 1986). Many studies have shown that IP3

stimulated the release of calcium in different cells, such as pancreatic cells (Streb et

al., 1985; Wolf e al., 1985) and hepatocytes (Burgess etlal., 1984; Joseph t al.,

1984). Inositol trisphosphate may even stimulate calcium mobilization via a receptor-

mediated process (Spat p al., 1986).

The IP3 that is formed from the hydrolysis of PIP2 is the inositol 1,4,5-

trisphosphate isomer (Berridge, 1983; Michell, 1986b). It has been discovered,

however, that there are several different isomers of the inositol phosphates that are

present in the cell (Michell, 1986b). The importance of each of the inositol phosphates

is not known.

The actions of IPs are terminated by sequential dephosphorylation to inositol by

phosphatases (Figure 1-1) (Abdel-Latif, 1986). The inositol, as with DAG, can be

reutilized for its incorporation into PI (Abdel-Latif, 1986; Holub, 1985). In this

sequence of events, lithium can inhibit the dephosphorylation of inositol 1-phosphate

(Hallcher and Sherman, 1980; Sherman eal., 1981). This discovery has been used as

a tool for the study of the phosphoinositide second messenger system. By inhibition of

the dephosphorylation of IP, one could prevent the recycling of IP into the

phosphoinositides. The IP formed from receptor activation of phospholipase C would

accumulate in the presence of lithium (Gonzales and Crews, 1984).

The Kidney: Structure and Function.

The kidneys are paired organs located in the posterior position of the abdomen

(Tisher and Madsen, 1986). The overall function of the kidneys is to maintain volume

and electrolyte concentrations of the extracellular fluid (Koushanpour, 1976). Through

a careful balance between conservation and elimination of water and electrolytes, the

kidneys are able to maintain homeostasis. This balance is achieved through control of

blood flow and reabsorption of electrolytes.










The kidney is a complex organ that can be separated into three zones: the cortex,

the outer medulla, and the inner medulla (Tisher and Madsen, 1986). This organ is

composed of many different cell types which make up the various structural units of the

kidney, such as the nephron, blood vessels, and interstitium. The functions of these

structural units are often interrelated.

The primary functional unit of the kidney is the nephron which is made up of the

renal corpuscle glomeruluss and Bowman's capsule), the proximal tubule, the thin

limbs, the distal tubule, and the connecting segment. The nephron is important in

filtering and processing of the plasma by mechanisms of reabsorption and secretion of

solutes and water along its length (Koushanpour, 1976). Each segment of the nephron is

composed of several cell types which account for different properties and functional

characteristics (Tisher and Madsen, 1986).

The kidney also contains blood vessels which originate from the renal artery

(Brenner etal., 1986). The circulation of the kidney is not homogeneous, but is

composed of several complex networks within the cortex and medulla. Through control

of blood flow in the kidney, the filtration and reabsorptive properties of the nephron can

also be controlled.

The interstitial cells of the kidney are also diverse in nature (Tisher and Madsen,

1986). The cortex contains two types of cells, one of which resembles fibroblasts. The

< medulla consists of three types which differ irt their structure. One of the interstitial

cells may be important in prostaglandin synthesis.

Control of Renal Function

The function of the kidney is controlled by several factors, such as hormones and

neurotransmitters which can alter blood flow or nephron reabsorption. Innervation of

the kidney has been examined in some detail (Gottschalk r al., 1985). Denervation or

nerve stimulation experiments demonstrated that renal innervation was important in

renal function. It is known that adrenergic neurons alter nephronal transport










properties, as well as renal blood flow. The importance of other neurotransmitters is

under investigation in several laboratories. It should be noted that cholinergic

innervation of the kidney has not been clearly demonstrated (Barajas and Wang, 1983;

Gattone et al., 1986).

Hormones, such as vasopressin and the adrenal steroids, are known to alter

various functions of the kidney (Kurokawa, 1985). These hormones can alter renal

blood flow and/or nephronal transport properties by acting on different cells.

The effect of hormones and neurotransmitters would depend upon the presence of

the receptor for the hormone on the target cell. In addition to this, the second messenger

system involved would be important. The effect of various hormones on the adenylate

cyclase system in the kidney has been examined in detail. Because of the lack of

information concerning the involvement of the phosphoinositide second messenger

system, we chose to examine the effect of a select group of hormones and

neurotransmitters on the hydrolysis of this lipid.















CHAPTER 2
THE EFFECT OF VARIOUS HORMONES AND NEUROTRANSMITTERS ON PHOSPHOINOSITIDE
HYDROLYSIS IN RABBIT KIDNEY

Introduction

The mammalian kidney not only excretes waste, but also maintains water and

electrolyte homeostasis. The functions of the kidney are regulated by a number of

different factors, such as hormones and neurotransmitters, which may alter renal

tubular reabsorption of electrolytes directly or through a change in renal blood flow.

Some of the hormones and neurotransmitters that are known to alter renal function are

acetylcholine (ACh) (Vander,1964; Hayslett gela., 1970; Lameire g ga., 1980;

Hartupee al., 1982), vasopressin (AVP) (Jard, 1983; Zimmerhackl, e al., 1985),

angiotensin II (Ang II) (Edwards, 1983; Harris and Navar, 1985), atriopeptins (Maack

eIal., 1985), histamine (Abboud-Dousa, 1983), adenosine (Osswald, 1987);

norepinephrine (Moss, 1982), secretin (Lamiere al., 1980), and dopamine

(Gottschalk ta g., 1985).

In most cases, neurotransmitters and hormones stimulate the cellular response

by activation of a second messenger system. One system involves receptor-coupled

adenylate cyclase which produces cyclic AMP. The regulation of this messenger system

in the kidney by AVP and other hormones has been studied extensively (Morel, 1981).

Another messenger system involves receptor-coupled phospholipase C which

preferentially hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two

second messengers: inositol trisphosphate (IP3) and diacylglycerol (Berridge, 1983).

Details of the mechanism of this biochemical pathway have been examined in greater

depth in such tissues such as brain and liver (Berridge, 1984), but not in the kidney.










In this study, we examined the effect of a variety of hormones and

neurotransmitters on phosphoinositide (Pis) hydrolysis in the three zones of the kidney:

the cortex (CTX), outer medulla (OM), and inner medulla (IM). Formation of inositol

phosphates (IPs) was measured as an indicator of the Pis hydrolysis by using a method

previously described for brain (Gonzales and Crews, 1984). The compounds that were

tested were chosen if 1) these were known to have an effect on kidney function and/or 2)

these were known to stimulate Pis hydrolysis in other tissues.
Materials and Methods

Animals Male New Zealand White rabbits (1-1.5 kg) were given Purina Rabbit

chow and water ad libitum.

Chemicals. Myo-2-[3H]inositol was obtained from Amersham Corp. (Arlington

Heights, IL). The following drugs were obtained from Sigma Chemical Co. (St. Louis,

MO): carbachol, [Arg8]- vasopressin (AVP), angiotensin II (human form, synthetic),

atriopeptins I,II and III, histamine, secretin, adenosine, norepinephrine, and dopamine.

All other chemical reagents were obtained from Fisher Scientific Co. (Orlando, FL).
Tissue preparation. The rabbits were killed by decapitation. The kidneys were

removed and the tissue separated into CTX, OM, and IM. The tissue was minced with a

Mcllwain tissue chopper to a thickness of 350 p.m in 2 directions.

The slices (approximately 0.5 g) were placed in a 50 ml polypropylene

centrifuge tube containing 30-40 ml of warm -(37 C) Krebs-Ringer bicarbonate

(KRB) buffer: NaCI, 118 mM; KCI, 4.7 mM; CaCI2, 0.75 mM; KH2PO4, 1.18 mM;

MgSO4, 1.18 mM; NaHC03, 24.8 mM and glucose, 10 mM. The tissue suspension was

bubbled continuously with 02:CO2 (95%:5%). The slices were washed four times with

KRB buffer by allowing the slices to settle and replacing the supernatant with 30-40 ml

of fresh KRB buffer to remove broken cells and intracellular debris.

Measurement of Pis hydrolysis. The method used for measuring Pis hydrolysis

in the rabbit kidney was developed earlier for studies with brain slices (Gonzales and










Crews, 1984). The assay procedure is based on the following two steps: 1) the

incorporation of [3H]inositol into Pis and 2) the stimulation and quantification of the

formation of total IPs (IP, IP2, IP3) (Figure 1-1) in the presence of different drugs.

Incorporation of [3H]inositol into Pis. This was achieved by incubating the

tissue slices with myo-2-[3H]inositol. One milliliter (0.5 g) of tissue slices was

mixed with 4 ml of warm KRB buffer. The [3H]inositol (specific activity of 12.3

Ci/mmol) was added to give a final concentration of 2.0 gCi/ml. The tubes containing

tissue were saturated with O2:CO2 (95%:5%). The tubes were placed at an angle in a

shaking water bath at 37 C for 60 minutes. After 60 minutes, the slices were washed

twice with warm, oxygenated KRB buffer to remove unincorporated [3H] inositol. The

final volume of the tissue suspension in each tube was three times the volume of the

packed slices.

Analysis of a few samples showed that total [3H]inositol incorporated into the

slices after 60 minutes of incubation was approximately 27,000 dpm/mg of protein in

the CTX, 11,000 dpm/mg of protein for the OM, and 17,000 dpm/mg in the IM. In these

samples the concentration of proteins was measured by the method of Lowry al.

(1951) by using bovine serum albumin as a standard and reading the absorbance at 750

nm in a Coleman spectrophotometer (model 620). The incorporation of [3H]inositol in

these samples was determined by measuring the radioactivity in the organic layer as

Described below for all other samples.

Release of [3HPs. While gently agitating to keep the [3H]inositol-loaded slices

in suspension, a 50 ll aliquot was added to a 12 x 75 mm polypropylene tube containing

190 i1 of KRB buffer with LiCI (final concentration, 8 mM). The reaction was started

by the addition of 10 pl of drug or buffer. LiCI was added to inhibit the

dephosphorylation of IP into inositol which would prevent the recycling of [3H]inositol

into Pis and result in the accumulation of IPs (Hallcher and Sherman, 1980; Sherman g











al., 1981). The tubes were incubated (at an angle) in a shaking water bath at 37 *C for

60 minutes.

The reaction was terminated by the addition of 1.0 ml of chloroform:methanol

(1:2 v/v) to the tubes. Also, 0.35 ml of water and 0.35 ml chloroform were added. The

tubes were capped and agitated for 10 minutes before centrifugation at low speed for 5

minutes to separate the two phases: organic (lower) phase and the aqueous (upper)

phase.

Quantitation of IPs. A 100 il aliquot of the lower organic phase that contained

unhydrolyzed 13H]PIs was placed into a borosilicate glass scintillation vial. The

chloroform was allowed to evaporate in a fume hood. Three milliliters of OCS

scintillation fluid (Amersham Corp., Arlington Heights, IL) were added as the

scintillant. The vials were vortexed and the radioactivity determined in a Beckman

LS7000 scintillation counter.

The aqueous phase was analyzed by anion exchange chromatography for IPs

(Berridge, 1983). A 0.750 ml aliquot was removed from the upper aqueous layer and

diluted to 3 ml with water. One milliliter of Dowex-1 (50% v/v; 100-200 mesh,

format form, Biorad Laboratories, Rockville Center, NY) was added. The slurry was

poured over polypropylene columns (Biorad Laboratories, Rockville Center, NY). After

the liquid was allowed to drain, the Dowex was washed with 10 ml of 5 mM cold inositol

to remove all free [3H]inositol. Total IPs (IP +IP2 + IP3; figure 1-1) were eluted into

scintillation vials with 5 ml of 0.1 M formic acid/1.0 M ammonium format. Ten

milliters of Liquiscint (National Diagnostics, Somerville, NJ) was added to the eluant.

The radioactivity of total IPs (IP + IP2 + IP3) was measured by a Beckman LS7000

scintillation counter. The recovery of [3H]IPs from the anion exchange under our

experimental conditions was 93%.

Calculation of IPs released. To eliminate the variation in pipetting and

incorporation of [3H]inositol into slices from experiment to experiment, the amount of










[3H]IPs (IP + IP2 + IP3) released in response to a drug or buffer was expressed as a

percentage of the total amount of [3H]inositol incorporated ([3HIIPs + [3H]PIs) into

the slices. The formula used was


[3H]IPs released (% total incorporated) =

dpm of total IPs in aqueous layer X 100

dpm in aqueous layer + dpm of total Pis in organic layer


The percentage of [3H]IPs released at time zero was subtracted from control and

experimental values. The zero time release is defined as the release of [3H]IPs in

samples that were removed after the incorporation of [3H]inositol into Pis, stored at

room temperature instead of incubating at 37 *C (for release of [3H]IPs) and analyzed

for radioactivity along with the other samples. These samples indicate [3H]IPs that may

have been released during the incorporation of [3H]inositol into Pis. The release of

[3H]IPs in zero time samples was usually low (2-5%). However, in some animals, the

release of [3H]IPs was as high as 10%.
Statistical analysis. Significance of experimental values was determined by

using the Student's t test. Differences were considered significant if P s 0.05.
Results

Table 2-1 shows the effect of drugs (that are known to affect renal function) on

SPis hydrolysis in three zones of the rabbit kidney. Except for atriopeptins II and III, all

of these drugs produced a significant increase in Pis hydrolysis in the IM, but not in the

CTX. Except carbachol, none of these drugs stimulated Pis hydrolysis in the OM. The

increase in Pis hydrolysis by AVP, Ang II, and atriopeptin I in the IM was approximately

100 to 300% over control values. On the other hand, the increase in Pis hydrolysis for

carbachol was greater than 600% in IM and greater than 200 % in the OM. The

response to carbachol and Ang II was dependent on the concentration of drug















IO
d







m a
I I
2 id


Io I

Z 6
z 1+.


2


0



o
o 0
















zI
z


0co -a
+1+ -H ++

c i c c m c o


in
oo
+-H +1


i c0 a 0)0
+1 +1 +1 +1

6- cj co c-l C


Co w


r" LO I,.

+1 +1 +1 +1

Ci CU C0 C
dc jl c'je-ji


0 +1 44 +1 -H
aE 't o q



1 5 cas co rco (D





E~ c


mm C

71 +1









ooa
+1 +1

(0(0









Co C-







00
99.,











o


a-
0 i -=
=<




CD

CL
Q)










(Figure 3-8, and Table 2-1, respectively). The response to AVP was the same with a

concentration of 10-7 M, as with 10-6 M, which suggested that maximal hydrolysis

was reached. Except for carbachol (Figure 3-8), a complete concentration curve was

not performed for any of the drugs.

Atriopeptin I (100uIM), but not atriopeptin II or atriopeptin III, slightly

stimulated Pis hydrolysis in slices from the IM (Table 2-1).

Table 2-2 shows the effect of drugs (that are known to affect blood flow in the

kidney and in several other organs) on Pis hydrolysis in the three zones of the rabbit

kidney. All of these drugs produced a significant stimulation of Pis hydrolysis in the OM,

but not in IM or CTX. Histamine and adenosine had the greatest effect at the

concentrations tested. Secretin had a relatively small effect on PIs hydrolysis in both

the IM and OM, but the difference compared to control was only significant in the OM.

Table 2-3 shows the effect of two well known neurotransmitters on PIs

hydrolysis in the three zones of the rabbit kidney. Neither norepinephrine nor

dopamine produced a significant stimulation of Pis hydrolysis in IM, OM, or CTX. The

lack of effect of these agents on Pis hydrolysis in renal tissues may be due to the

instability of these agents in itro or in vivo, lack of sensitivity of our method, or

mechanism of action through a second messenger system other than Pis messenger

system.

Discussion

PIs hydrolysis method. The method used for determination of drug-stimulated

release of IPs in the renal slices in the present study is essentially the same as reported

by Gonzales and Crews (1984) for cholinergic stimulation of PIs hydrolysis in brains

slices. The method is based on: 1) the incorporation of [3H]inositol into three

phosphoinositides (phosphatidylinositol, phosphatidylinositol 4-phosphate and PIP2) in

the absence of drugs and 2) the hydrolysis of PIP2 in the presence and absence of drugs













I

10





S I
i- Id


o .















+1


z im
0






0
4d

z


IN
0S in~


S.5"


o I I


qt

+1

IE

ic
I-
d 10
+1 I
" E

cI'

IZ

C, I
o U

r-
ri a
Im
4)
E

Q)


-H a
I>


.0_
on

O I
Ici








o


va
C IC



i v

+1 IT-










SIE


o in



lan-














C,
10


0

1 +1
io




CO

0 iC

Z <0,


0
I

























1 1+1
I
'5

o Io
0 I






o+


+ I









z I,
I


i
g0




I'


o IE


Iz




S1+
- I
I
I +1

NI
a I
lv,




0 I

- IC




Ic









1 7I
IC

SI.
- I

+1 I
- 0






0








"i u
'V










by (agonist-receptor stimulated) phospholipase C into diacylglycerol and IP3 (Figure

1-1). In our assay, we determined the hydrolysis of Pis by measuring the release of

total IPs. Although IP3 is an intracellular messenger that increases cytosolic Ca2+, it

is rapidly dephosphorylated to IP2 and IP (Berridge and Irvine, 1984). The presence of

LiCI in our assay prevents further degradation of [3H]IP to [3H]inositol that could be

recycled for synthesis of [3H]PIs (Figure 1-1). Therefore, our assay allowed us to
measure the accumulation of all three [3H]IPs (IP, IP2 andlP3) from radiolabeled Pis

in the absence of recycling [3H]inositol. The [3H]IPs (IP, IP2 and IP3) formed were

separated from unincorporated [3Hlinositol by anion exchange chromatography. By

using this method, we were able to determine the effects of several agents in the same

experiment.

The release of [3H]IPs from [3H]Pls will depend on the total incorporation of

[3H]inositol into [3H]PIs in the incubation medium. This may vary from animal to

animal. In addition some error can be introduced due to the presence of unequal amounts

of tissue slices in equal volumes (of radiolabeled tissue suspensions) taken in various

samples treated with different drugs for PIs hydrolysis. Therefore, we expressed the

release of [3H]IPs as a percentage of total [3H]inositol incorporated into PIs in each

sample. This was done: 1) by measuring the radioactivity in the organic layer (that

contained unhydrolyzed [3HIPI) and in the aqueous layer (that contained released

[3H]IPs) after incubating the radiolabeled renal slices with or without drugs and 2) by

dividing the radioactivity in the aqueous layer (representing total IPs) by the

radioactivity in the aqueous layer hydrolyzedd PIs) plus the organic layer

(unhydrolyzed PIs) in each sample.
Druas that stimulate PIs hydrolysis in the IM. Our results showed that

carbachol, AVP, Ang II, and atriopeptin I were able to stimulate Pis hydrolysis in the IM.

The IM is made up of many different cell types, such as vascular endothelial cells,
interstitial cells and collecting duct cells (Knepper etal., 1977). The collecting duct










cells are the major portion of the IM. The results of our study, however, do not

differentiate between these cell types.

Acetylcholine, a neurotransmitter, causes vasodilation and diuresis when infused

into the renal artery (Vander, 1964). The physiological significance of ACh in renal

function remains to be determined since parasympathetic innervation of the kidney has

not been demonstrated (Barajas and Wang, 1983). The cholinergic agonist, carbachol,

was able to stimulate Pis hydrolysis to the greatest extent in the IM and to a lesser

extent in the OM; no effect was observed in the CTX. These results indicate that

cholinergic receptors may be present in the IM and OM; however, further studies must

be performed to determine if cholinergic receptors are present (see Chapter 6). The

location of these receptors on vascular endothelial, tubular epithelial, or interstitial

cells is not known.

Vasopressin, a peptide, is also known as antidiuretic hormone, primarily because

of its effect on V2-activated adenylate cyclase in the kidney (Morel, 1981). Another

effect of AVP is vasoconstriction in the IM due to a direct effect on blood vessels through

V1 receptors (Zimmerhackl e al., 1985). In the liver, V1 receptors are coupled to Pis

hydrolysis (Creba eal., 1983). Our results indicate that V1 receptors may be found in

the kidney, in addition to V2 receptors. These V1 receptors may be localized on the blood

vessels which would be consistent with the studies on other tissues. However, only with

the use of specific Vi and V2 receptor antagonists to determine receptor specificity and

cell separation to determine localization can this hypothesis be proven.

Angiotensin II is a peptide synthesized in the kidney that is known to affect renal

vasculature and epithelium. The effects include: 1) increase in renal vascular

resistance in cortical microvessels (Edwards, 1983), 2) stimulation of gluconeogenesis

in the renal proximal tubule which may be due to Pis hydrolysis (Wirthensohn and

Guder, 1985), and 3) stimulation of prostaglandin formation in the IM which may also

be due to Pis hydrolysis (Benabe al., 1982). Angiotensin II stimulated Pis hydrolysis










in slices from the IM, but not the OM or CTX. Our studies are consistent with the studies

of Benabe gt a.(1982) which showed that Ang II stimulated the incorporation of [32p]

orthophosphate into Pis an indicator of increased hydrolysis, in the IM. Since no effect

of Ang II was observed in the CTX, the results presented here do not support the previous

study of Wirthensohn and Guder (1985). The inability to show Ang II stimulation of PIs

hydrolysis in the cortex proximall tubules are the major component ) may be due to the

method of measurement. Wirthensohn and Guder (1985) measured synthesis of Pis

(32P-incorporation into PIs) and not degradation, as was the case in this study. In some

cases, the increased synthesis of PIs does not correspond to the increased degradation of

Pis (Farese, 1983).

The atriopeptins are a group of peptides in the larger class of compounds called

the atrial natriuretic peptides that are involved in the regulation of blood pressure and

blood volume (Laragh, 1987). There are three atriopeptins that have natriuretic and

smooth muscle relaxant properties (Laragh, 1985). In general the peptides are

believed to act similarly. Like ACh, the natriuretic effects of these peptides are often

attributed to the vasodilatory effects (Maack ft al., 1985). These peptides, which affect

renal function, have binding sites present in the kidney, primarily in the outer cortex

and papilla (Maack e l., 1987). The receptors involved in vasodilation are coupled to

guanylate cyclase and the formation of cyclic guanosine monophosphate (Laragh, 1987).

S Our results showed that atriopeptin I (100 .M), not atriopeptin II or III, stimulated PIs

hydrolysis in the IM. Atriopeptin I, however, does not express the complete natriuretic

and vasodilatory effects observed with atriopeptin II (Laragh, 1987). The results

presented in this study suggest that there may be differences among the atriopeptins.

The importance of Pis hydrolysis in the natriurelic and vasodilatory effects of

atriopeptins remains to be determined.

Druas that stimulate Pis hydrolysis in the OM. The PIs response was stimulated

in the OM by histamine, adenosine, and secretin. The outer medulla is rich in blood










vessels and has, therefore, also been called as red medulla. In addition, it contains

epithelial cells ( thick ascending limb and collecting duct in the inner and outer zone,

proximal straight tubule only in the outer zone) and interstitial cells.

Histamine is a substance that is synthesized in the kidney, probably in the

glomeruli (Abboud and Dousa, 1983). The glomeruli have H2 receptors which are

coupled to cAMP formation (Torres g al., 1978). It also has effects on the blood vessels

where it can cause both vasodilation and vasoconstriction. These effects are mediated by

both H1 and H2 receptors. Histamine has been shown to stimulate PIs hydrolysis in

other tissues, such as brain and smooth muscle, through H1 receptors (Berridge,

1984). The results from this study indicates that H1 receptors are present in the OM of

the kidney. The location and role of these receptors in kidney function, however is not

completely known at the present time.

Adenosine is a metabolite of the renal tubular epithelium that is known to cause

vasoconstriction and inhibition of renin release (Osswald, 1986). We observed an

effect of adenosine in the OM which may be related to the effect of adenosine on the blood

vessels. Adenosine had no effect on PIs hydrolysis in the CTX. Renin is released from

the juxtaglomerular apparatus located in the cortex. This leads us to believe that

inhibition of renin release is not mediated by PIs hydrolysis. Stimulation of Pis

hydrolysis may be related to the vasoconstrictive effect of adenosine. The localization of

adenosine receptors and their functions remaina-to be determined.

Secretin is a vasodilator substance like ACh, but differs from ACh by its inability

to cause natriuresis (Lamiere e al., 1980). Secretin was able to stimulate Pis

hydrolysis in the OM, but did not have a significant effect in the IM. This second

messenger system may be involved in the vascular effects of secretin. The differences

between the pharmacological actions of secretin and ACh, may be due to the additional

action of ACh on renal tubules (see Chapter 5).










In this study, both vasoconstrictors (adenosine) and vasodilators secretinn)

were able to stimulate PIs hydrolysis in the OM. Differences in location of the vascular

receptor, such as on the endothelial or smooth muscle cells, could account for the

different responses to stimulation of PIs hydrolysis. It is also possible that adenosine

may have an additional action on renal tubules, such as the thick ascending limbs, that

form the bulk of OM and of CTX and are absent in the IM.

Druas that did not stimulate Pis hydrolysis in the rabbit kidney. Some

substances that were tested were not able to stimulate Pis hydrolysis in the IM, OM and

CTX. There are several possible explanations for the inability of a substance to

stimulate the hydrolysis of Pis as measured by this method. First, the substance may

not stimulate the Pis system in the kidney. The receptors may not be present or may not

be coupled to the Pis second messenger system. Second, the products of hydrolysis may

not have been detectable with this method. Receptors on a cell that comprise a small

population of all the cells in the slice could account for a small nondetectable increase in

Pis hydrolysis. Third, the substance is degraded enzymatically or nonenzymatically and

thus inactivated during our assay.

The substances that were not able to stimulate Pis hydrolysis in the three zones

of the kidney under our experimental conditions were norepinephrine, dopamine,

atriopeptin II and Ill. Norepinephrine is a neurotransmitter that is able to bind to four

receptor subtypes: alpha1, alpha2, betal, beta2. The kidney is innervated by adrenergic

neurons to renal vasculature, tubular epithelial cells, and juxtaglomerular granular

cells (Moss, 1982). The innervation is important in controlling renin release and

tubular transport systems. Norepinephrine was not able to stimulate Pis hydrolysis in

the kidney which could be a result of the reasons described above. Evidence from other

studies showed that increased synthesis of Pis (associated with increased Pis

hydrolysis) was observed with phenylephrine, an adrenergic agonist, in proximal

convoluted tubules, the major component of the kidney cortex (Wirthensohn and Guder,










1985). This discrepancy could be due to one of the four reasons for the inability of a

substance to stimulate Pis hydrolysis described above.

Dopamine is a neurotransmitter that is involved in renal function (Gottschalk et

al., 1985). In addition to circulating dopamine that arrives via the filtered load,

dopamine is synthesized within the kidney (Lee, 1982). It has a potent natriuretic

effect which is often ascribed to dopamine-induced vasodilation, but other mechanisms

have been proposed (Gottschalk t al., 1985). Dopamine may also have a direct effect on

proximal tubule fluid reabsorption (Bello-Reuss gIal., 1982). Our results indicate

that the effects of dopamine on renal vascular and epithelial function are not mediated by

Pis hydrolysis. It is, however, possible that an effect of dopamine on PIs hydrolysis was

not observed for the other reasons described above.

Atriopeptins II and III were not able to stimulate Pis hydrolysis in any of the

three zones of the kidney. Atriopeptin III is believed to be the active circulating

hormone (Schwartz e al., 1985). As discussed above, these peptides are known to cause

vasodilation and natriuresis (Laragh, 1987). Our results indicate that stimulation of

natriuresis and vasodilation by the atriopeptins does not involve the Pis second

messenger system and is probably mediated by cGMP (Laragh, 1987). Like

norepinephrine and dopamine, other reasons (described above) could account for the

inability of the atriopeptins to stimulate Pis hydrolysis.

Summary and Conclusions. We have shown that a variety of chemical agents

which include hormones and neurotransmitters were able to stimulate Pis hydrolysis in

the rabbit kidney. Stimulation of Pis hydrolysis showed regional specificity with

different drugs. This second messenger system may be involved in the physiological and

pharmacological actions of hormones and neurotransmitters. It should be pointed out

that stimulation of PIs hydrolysis is much greater than any other of the substances that

were tested. Therefore, in order to study the PIs second messenger system, we extended

our studies with carbachol.















CHAPTER 3
CHOUNERGIC STIMULATION OF PHOSPHOINOSITIDE HYDROLYSIS
IN RABBIT KIDNEY

Introduction

As described in Chapter 2, carbachol produced stimulation of phosphoinositide

(Pis) in renal slices to a much larger extent than any other hormone or

neurotransmitter. The effect was highest in the inner medulla (IM), less in the outer

medulla (OM), and nondetectable in the cortex (CTX).

The purpose of this study was: 1) to further characterize cholinergic stimulation

of hydrolysis of phosphoinositides in rabbit kidney and 2) to characterize the conditions

and types of receptors necessary for this response pharmacologically. We chose to use

carbachol in our studies because carbachol is an analog of ACh that is resistant to

hydrolysis by cholinesterases. Cholinesterases are present in the kidney, but their

importance in kidney function is not known (Marx and Carter, 1963; Barajas and Wang,

1983).

Materials and Methods

The methods of tissue preparation and PIs hydrolysis are essentially the same as

described in Chapter 2, except the time dependency of incorporation of [3H]inositol into

PIs and the elution of IPs which were performed with carbachol in the IM.

Chemicals. Myo-2-[3H]inositol was supplied by Amersham Corp. (Arlington

Heights, IL). Carbachol and other drugs were obtained from Sigma Chemical Co. (St.

Louis, MO), except pirenzipine, which was a gift from Dr. Lincoln Potter (Miami, FL).

All chemical reagents were obtained from Fisher Scientific Co. (Orlando, FL).

Tissue preparation. Male New Zealand white rabbits (body weight of 1-1.5 kg)

were killed by decapitation. The kidney tissue was prepared as described in Chapter 2.










The kidney was excised and separated into tissue from the CTX, OM, and IM. The tissue

was sliced to a thickness of 350 pm in 2 perpendicular directions with a Mcllwain

tissue chopper.

The slices (0.5 g) were placed in a tube containing 40 ml of Krebs-Ringer

bicarbonate (KRB) buffer (concentrations in millimolar): NaCI, 118; KCI, 4.7; CaC12,

0.75; KH2PO4, 1.18; MgSO4, 1.18; NaHCO3, 24.8; and glucose, 10, that was previously

warmed and bubbled with 02:CO2 (95%:5%). The tissue suspension was bubbled

continuously with the 02:CO2 mixture at 37 "C. In order to remove damaged cells and

subcellular debris, the slices were washed four times with KRB buffer by allowing the

slices to settle and then replacing the supernatant with fresh KRB buffer.

Measurement of Pis hydrolysis. We measured Pis hydrolysis in the rabbit

kidney by using the method that was described in chapter 2. The formation of IPs (IP +

IP2 + IP3; figure 1-1) in the presence or absence of cholinergic agents was the

indicator for Pis hydrolysis. The assay procedure involves two steps: 1) the

incorporation of [3H]inositol into Pis (IP + IP2 + IP3; figure 1-1) and 2) the

stimulation and quantification of the formation of IPs by receptor-activated

phospholipase C in the presence of cholinergic agents.

Incorporation of [3Hlinositol into Pis. This was achieved by incubating the

tissue slices with myo-2-[3H]inositol. One milliliter (0.5 g) of tissue slices was

S mixed with 4 ml of fresh KRB buffer. The [3H]itositol was then added to the incubation

mixture. Although the specific activity of [3H]inositol differed in two batches used

(12.3 and 16.3 Ci/mmol), the final concentration of [3H]inositol in the incubation

mixture was kept constant (2.0 pCi/ml). The tubes containing tissues were saturated
with 02:CO2 (95%:5%) and incubated for 60 minutes at 37 C. The tubes were placed at

an angle in a shaking water bath. After 60 minutes, the slices were washed twice with

warm, oxygenated KRB buffer to remove unincorporated [3H]inositol. The final volume

of the tissue suspension in each tube was three times that of the packed slices.










In a pilot experiment, we examined the effects of time on the incorporation of

[3H]inositol into IM slices (Figure 3-1). The IM slices were incubated with 2 i.Ci/ml

[3H]inositol at 37 OC for various times. We found that incorporation of [3H]inositol

increases with time, but the rate of incorporation decreases after 60 minutes. This

pattern is similar to that observed for brain slices (Gonzales and Crews, 1984). We,

therefore, chose 60 minutes for the incubation time for incorporation of [3H]inositol.

We also examined the effects of concentration of [3H]inositol on the incorporation

of [3H]inositol into IM slices (Figure 3-2). The incorporation of [3H]inositol began to

decline when the concentration of inositol was greater than 100 nM. In our

experiments, we chose concentrations of [3H]inositol between 100-200 nM.

In these samples, incorporation of [3H]inositol was determined by measuring the

radioactivity in the organic layer as described below. Protein was measured in similar

aliquots as determined by the method of Lowry gt al. (1951). The absorbance of the

samples at 750 nM was read on a Coleman spectrophotometer (model 620) along with

bovine albumin standards.

Release o [3H]LE. While gently agitating to keep the [3H]inositol-loaded slices

in suspension, a 50 ul aliquot was added to a 12 x 75 mm polypropylene tube containing

190 I1 of KRB buffer with UCI (final concentration, 8 mM). The reaction was started

by the addition of 10 Il of drug or buffer. Lithium chloride was added to inhibit the

S dephosphorylation of IP into inositol which results in the accumulation of IPs (Hallcher

and Sherman, 1980; Sherman geal., 1981). The tubes were saturated with 02:C02

(95%:5%) and capped. The tubes were incubated (at an angle) in a shaking water bath

at 37 C for 60 minutes. In a few samples, we determined the time dependency of the

release of [3H]IPs and incubated the samples from 5 to 120 minutes.

The reaction was terminated by the addition of 1.0 ml of chloroform:methanol

(1:2, v/v) to the tubes. Also, 0.35 ml of water and 0.35 ml of chloroform were added.














m
C ) ca

0






o


So (

E E

C) W

.0 w
E = .






|ii
^E

. "~
> 0








I. E



cM 0


- E
o 0

CE
-0


0.2Cu








o-








C),24
*IZ a 10
























































(uaelojd 6wu/uJdp)
paelJodJooul lOl!SOUI[H]














Sn


O .0
S Ea


S-S


S8
. EC 0





C.
co













o
0-0






-S-
-6 8m



o S?
C W -

0 .E E-








r r_


a)
"-




Ir10 5
LU|
coQ-








31














o
0
















0
.2
o



0


0
^ -


(u!elojd Bu6/wdp)
pelejodjooul IOI!SOUl[HSI










The tubes were capped and agitated for 10 minutes before centrifugation at low speed for

5 min to separate the two phases (organic phase and aqueous phase).

Quantitation of IPs. A 100 Il aliquot of the lower organic phase that contained

unhydrolyzed [3H]Pls was placed in a borsilicate glass scintillation vial. The

chloroform was allowed to evaporate in a fume hood. Three milliliters of OCS

scintillation fluid (Amersham Corp., Arlington Heights, IL) were added as the

scintillant. The vials were vortexed and the radioactivity determined in a Beckman

LS7000 scintillation counter.

The aqueous phase was analyzed by anion exchange chromatography for IPs

(Berridge, 1983). A 0.750 ml aliquot was removed from the upper aqueous layer and

diluted to 3 ml with water. One milliliter of Dowex-1 (50% v/v; 100-200 mesh,

format form, Biorad Laboratories, Rockville Center, NY) was added. The slurry was

poured over polypropylene columns (Biorad Laboratories, Rockville Center, NY). After

the liquid was allowed to drain, the Dowex was washed with 10 ml of 5 mM cold inositol

to remove all free [3H]inositol. Total IPs (IP + IP2 + IP3) were eluted into

scintillation vials with 5 ml of 0.1 M formic acid/1.0 M ammonium format. Ten

milliliters of Liquiscint (National Diagnostics, Somerville, NJ) were added to the eluant.

The radioactivity of total IPs (IP + IP2 + IPs) was measured by a Beckman LS7000

scintillation counter.

Berridge (1983) showed that IPs was the initial product of agonist-stimulated

Pis hydrolysis. We separated the IPs by elution with different buffers as described

previously (Figure 3-3) (Berridge, 1983; Gonzales and Crews, 1984). A 5 ml aliquot

of each of the buffers were added to the Dowex column in the order indicated: 5 mM

inositol (2 x); 5 mM disodium tetraborate/60 mM sodium format; 0.1 M formic

acid/0.2 M ammonium format; 0.1 M formic acid/0.4 M ammonium format; 0.1 M

formic acid/1.0 M ammonium format. These solutions will elute inositol,

glycerophosphoinositol, inositol 1-phosphate (IP), inositol 1,4-diphosphate, and














0


o-
0.









00
co





C
aE



O.
aS

w-5









C,




0
NE








0










co

0C
O.





















)U
O



is

















0 0
0 VC








0.










0.


: i
1^1
o2 -E B
= ^ E
Q_ t




















Edl




Zdl

E 2
o cc
dl







0 C0 0 0 0
n t Co CM -


(uo!loeJl/uJdp) Al!^!loeo!peIl


, _
(D
















E
z
o

LL







(11


I










inositol 1,4,5-triphosphate, respectively. Our results showed that the major product

of cholinergic stimulation of Pis hydrolysis was IP, not IP3. If IP3 was formed, it was

rapidly dephosphorylated to IP which accumulates in the presence of lithium. We,

therefore chose to measure total IPs, instead of separating the individual IPs after drug

treatment.

We also determined the completion of elution of inositol phosphates with 0.1 M

formic acid/1.0 M ammonium format (Figure 3-4). After 2 x 5 ml rinses with 5 mM

inositol, the eluants from 20 x 1 ml rinses with 0.1 M formic acid/1.0 M ammonium

format were collected. We demonstrated that after 4 ml of 0.1 M formic acid/1.0 M

ammonium format (fractions 7-22), no additional radioactivity was eluted from the

column (Figure 3-4). Results from fractions 8-22 are not shown. In our

experiments, inositol phosphates were eluted with 5 ml 0.1 M formic acid/1.0 M

ammonium format.

Calculation of IPs released. To eliminate the variation in pipetting and

incorporation of [3H]inositol into slices from experiment to experiment, the amount of

[3HilPs released in response to a drug or buffer was expressed as a percentage of the

total amount of [3H]inositol incorporated into the slices:

[3H]lPs released (%total incorporated) =

dpm in aqueous layer x 100

dpm in aqueous layer + dpm in organic layer

The percentage of [3H]IPs released at time zero was subtracted from control and

experimental values. The zero time release was defined in detail in chapter 2.

Statistical analysis. Significance of experimental values was determined using

the Student's t-test. Differences were considered significant if P < 0.05.

Results

Regional specificity of cholinergic stimulation of Pis hydrolysis. As described in

Chapter 2, when slices were taken from the three zones of the kidney: CTX, OM, and IM,


























- = VI
E-- -




a) CL0 C, C
E o -

o -o g -.




. : 0 0




C o,_-o --
E -
So .E



5 .P ,i 0









m (
So o a





C a N a







oB m-

- a>coS

3ct =
C 0 cu c
0:2 a!E L jz
iz -9 CLCO

















00 0 ca
E 0 0







0.0

z
t
0

Cu
U-


I -


(uoiloeji/wdp) Al!A!jlOo!peH)


0
CD
LO)


o 0 0 0
o 0 0
N -u


IONNE










the maximum effective concentration (1 mM) of carbachol stimulated the largest release

of IPs from the IM (from 3.93% release of IPs to 28.37%, an increase of 622% over

control levels) (Figure 3-5). The effect was less in the OM (from 1.77 to 8.63%, i.e.,

388% increase over control). No response was observed in slices from the cortex.

Effect of a-ketoalutarate. The renal CTX, OM, and IM utilized substrates

differently in the production of ATP (Klahr and Hammerman, 1985). In the rat, the CTX

utilizes a-ketoglutarate (a-kg) more readily than glucose as measured by oxygen

consumption (Klein lal., 1981). In order to determine if the lack of effect of

carbachol on stimulation of Pis hydrolysis in the renal CTX was due to a limitation of

glucose utilization, we measured the effect of carbachol in the presence of a-kg during

the incubations with [3H]inositol and with drug. First, we measured the incorporation

of [3H]inositol into cortical and inner medullary slices in KRB containing glucose or a-

kg (Table 3-1). No difference was observed between slices that has been incubated in

KRB with glucose or in KRB with a-kg. The differences of incorporation between CTX

and IM is due to differences in protein concentration per tube. When similar data were

normalized for protein, the difference was less than two-fold (Chapter 2). The

substitution of a-kg for glucose produced no change in the effect of carbachol on the

hydrolysis of Pis in the CTX (Figure 3-6). Therefore, the lack of effect of carbachol on

Pis metabolism in rabbit CTX was not a result of limitation of ATP supply due to

< substrate specificity. However, carbachol stirmulation of PIs hydrolysis in the IM was

attenuated with the substitution of a-kg for glucose (Figure 3-6). This effect supports

the finding that in rabbit, the CTX can utilize a variety of substrates, whereas the

medulla was dependent upon glucose (Lee t al., 1962).

Time dependency of release of IPs from the Pis. After incorporation of

[3H]inositol into the Pis for 60 minutes, we determined the release of IPs with 1 mM

carbachol for various time intervals in inner medullary tissue, which initial studies















E


co







E




c
0









03
ES a






E2
0





0(


sE






0
' i







0) 0
= i











0
-Cm
.0
*






















il
_if











































0 ?
(poeejodjooul i |O. %)
a3Sv313u
S3.LVHdSOHd IOIISONI [H,]



















TABLE 3-1
THE INCORPORATION OF [3H]INOSITOL INTO Pis
IN THE PRESENCE OF GLUCOSE OR a-KG.

[SH]lnositol Incorporated
(dpm/tube)

Cortex Inner Medulla

KRB with:

glucose, 10 mM 4733 + 1048 219 25

a-kg, 10 mM 3959+ 486 224 71

Cortical and inner medullary slices were incubated with 2 iCi/ml [3H]inositol at 37 "C
for 60 minutes. The slices were washed and treated as described in the methods for the
zero time samples. The total radioactivity in Pis (organic layer) and in IPs (aqueous
layer) represent the total [3H]inositol incorporated. Values are mean SEM from 9
samples from 3 animals.














-"



z
O
0.


o


08
E CZ
- SO





x a

8020




|gl
ca-




E c

C 0
S -= 0
c .2
- 0uw



E G"


U,


-5 E








cno



0 .




a. CO


^| ^
LL"S





















I-


n z
5 o












oz





(palejodjooul leoil %)
03SV313IU
S31VHdSOHd IOIlSONI [H1]










indicated was sensitive to carbachol. The data, shown in figure 3-7, indicate maximum

difference between control and carbachol stimulation at 60 minutes. The effect of

agonists on the formation of IPs from Pis is a matter of seconds (Berridge, 1983). The

maximum effect of carbachol was not observed until 60 minutes. In order to maximize

the response to carbachol, an incubation period of 60 minutes was used for drug

treatment.

Concentration-resoonse relationship. Stimulation of PIs hydrolysis in the inner

medullary slices by carbachol was dependent upon the concentration of carbachol

(Figure 3-8). The maximum response was observed with a carbachol concentration of 1

mM, with an apparent EC5o of approximately 10-5 M.

Receotor specificity. The effect of carbachol was blocked by 1 PM atropine, a

muscarinic antagonist, but not by 1 UM hexamethonium, a nicotinic antagonist (Figure

3-9). These results suggest that cholinergic stimulation of PIs turnover in the inner

medulla is mediated by muscarinic receptors. This statement is further supported by

the inability of the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide

(DMPP), to stimulate Pis turnover.

The muscarinic receptor class has been further separated into two subclasses,

M1 and M2, based on the selectivity of the M1 antagonist, pirenzipine. Pirenzipine

completely blocked carbachol-stimulated Pis hydrolysis (Figure 3-10). The EC5o

value for pirenzipine was approximately 4 UM and calculated Ki was 427 nM.

Calcium dependency. In order to determine if PI hydrolysis is dependent on Ca2+,

the effect of various concentrations of calcium on IP formation was measured in the

inner medulla (Figure 3-11). In the absence of calcium, the response to carbachol was

attenuated. The response was abolished completely by addition of 200 iM EGTA in

calcium-free buffer. Increasing the calcium concentration to 0.25 mM resulted in the

increased formation of IPs by carbachol.









































ca
a)





















C ,



















E
C)







































E
,-





CL











E



80
*0
5u
o































EO
E


c



.r




n.








-0
r,






Q)
Cru









Clu

0) C-












U- Cu
























































(paeDoodJooul lo 0l%)
]3SV3713 '7Od-1O0LISONI [HE

























Figure 3-8. Concentration response curve for the stimulation of Pls hydrolysis by
carbachol in the inner medulla of the rabbit kidney. Each point is the mean S.E.M. of 3
animals.


































C]
Q
UJ

a a

_1
a 20
Co"
a-5


II
,-J
d


-o

zo
I 10
I
x
Kl


LOG CARBACHOL CONCENTRATION [M]











'-(U



I I




O +
-CO
E



0.










C-"
fiE


.0





'-C











C c
*o






-S





.E






0






a
(0 <

















I lI


























0


0

(paleJOdJOU|l l|OJ. %)
a3SV313U
S31VHdSOHd 011OISONI [He]


















0
>-




CI,





5"0
..o



.~



C 8
5 P
i0
-0








E.0





-1







0.0





EC






7-0











3C
Li8
























'Ee
U U I~~---*i--
0 m







/ cc
| au
3 0
w






0
-- M----< .f

/
I
8






(paeJiodjooul lelo. %)
a3SV3'13
S31VHdSOHd 1011SONI [H1]













SC






.5
0o8W





o 0i
Cu


2o




.0












c a
CS
.O .0 (0








0E
7o E
X o
o)














0 00>
Esg












CDE '5 0
0 a0













LL .E 12 c
0 CTO 0,
) _

s C.












*1^?-

"^^O



















































0 0 0 0

(pa)BJodJooul le|O. %)
03SV313U
S31VHdSOHd 1011SONI [H,]


i
E

0r





E
O,
u


E
I





.2
Nu










Discussion

Regional soecificity. One of the goals of this study was to determine if cholinergic

agents (e.g., carbachol) were able to stimulate Pis hydrolysis in the rabbit kidney. We

have shown that carbachol stimulates Pis hydrolysis in kidney slices from the IM and to

a lesser extent in the OM.

The osmolality of the incubation media in the present study was 300 mOsm/kg of

H20. However, the IM is often exposed to an osmotic environment of more than 1000

mOsm/kg H20 (Schmidt-Nielsen e al., 1983). It should be pointed out that the effects

of osmolality on carbachol-stimulated Pis hydrolysis in the inner medullary slices of

the rabbit kidney have been determined. The increase in osmolality by NaCI and urea

(50:50 mixture) decreased the effect of carbachol on Pis hydrolysis in the IM (Garg e1

al., 1988). The effects of carbachol on stimulation of Pis hydrolysis was blocked

completely by increasing the osmolality of the media to 1200 mOsm/kg of H20. A

similar increase in osmolality by mannitol produced a smaller decrease in carbachol-

stimulated Pis hydrolysis than a mixture of NaCI and urea. Carbachol-stimulated Pis

hydrolysis was still significantly higher than the control even when the osmolality of

the incubation media was increased to 1200 mOsm/kg of H20 by mannitol. These results

suggest that the effect of carbachol on PIs hydrolysis depends on the osmolality as well as

the composition of the interstitial fluid of the inner medulla.

Concentration deoendencv of carbachol-action. Stimulation of Pis hydrolysis by

carbachol was dose-dependent. The maximum effect was observed with 1 mM carbachol.

This concentration was 1000 times greater than that which was commonly used to

stimulate diuresis when infused into the renal artery (Lameire e al., 1980). The

difference in concentrations required for the physiological response (diuresis) and PIs

hydrolysis is not uncommon (Abdel-Latif, 1986). The physiological response may

require the activation of a small fraction of receptors resulting in submaximal

formation of inositol phosphates. However, Pis hydrolysis is maximal when all










receptors are occupied; therefore, a higher concentration of carbachol is required. It

should be mentioned that the dose-response relationship for carbachol stimulation of Pis

hydrolysis in the kidney is similar to that for brain slices (Gonzales and Crew, 1984).

Receptor specificity. Cholinergic stimulation of Pis hydrolysis in the inner

medulla was blocked by atropine, but not by the nicotinic receptor antagonist,

hexamethonium. The nicotinic agonist, DMPP, did not stimulate Pis hydrolysis.

Therefore, stimulation of Pis hydrolysis by carbachol was mediated by the muscarinic

receptor.

Pirenzipine, a M1 receptor antagonist, was able to inhibit the response to

carbachol (Ki = 427 nM). The Ki value for pirenzipine is high which suggests that M2

receptors are involved in Pis hydrolysis since pirenzipine is believed to have a higher

affinity for MI receptors (Watson elal., 1984). This is consistent with recent reports

that M2 receptors are coupled to Pis hydrolysis in rabbit iris (Akhtar efal., 1987) and

chick heart cells (Brown rt al., 1985). However, this is not consistent with reports

that Pis hydrolysis is coupled to M1 receptors (Gonzales and Crews, 1984; Watson ft

al., 1984). Further studies are required to determine whether M1 or M2 receptors are

present in the kidney.

Physiological significance. Despite the inability to demonstrate cholinergic

innervation of the kidney, the physiological effects of ACh on renal function have been

.examined by several investigators (Vander, 1964; Hayslett al., 1970; Martinez-

Maldonado t al., 1972; Lameire p al., 1980; Fadem al., 1982; Hartupee ial.,

1982). Acetylcholine has been shown to stimulate diuresis and renal vasodilation as

well as increase interstitial pressure when infused into the renal artery (Vander,

1964; Lameire.tgal., 1980; Hartupee etal., 1982). Vasodilation and increased blood

flow were greater in the inner medulla (Lameire tal., 1980).










The effects of ACh in the kidney, such as vasodilation, diuresis and natriuresis,

were also blocked by atropine, a muscarinic receptor antagonist (Vander, 1964). This

suggests that these responses are mediated by the muscarinic receptor.

We have shown that carbachol, a cholinergic agonist, can stimulate Pis

hydrolysis in the kidney, specifically the inner and outer medulla. This is the first time

the biochemical effects of cholinergic agents on the kidney have been described.

However, a relationship between carbachol-stimulated Pis hydrolysis and diuretic and

vasodilatory actions of cholinergic agents remains to be elucidated.

Summary and conclusions. We have shown cholinergic stimulation of Pis

metabolism resulting in the formation of inositol phosphates is mediated by muscarinic

receptors in the kidney. Our results suggest that the PIs messenger system may be

involved in the pharmacological actions (diuresis) of ACh in the kidney; however, a

direct link between cholinergic-stimulated PIs messenger system and diuresis produced

by cholinergic agents remains to be established.















CHAPTER 4
IMMUNOHISTOCHEMICAL LOCALZATION OF
PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE IN RABBIT KIDNEY
Introduction

Inositol-containing phospholipids of cell membranes are believed to play an

important role in some receptor-mediated events (Berridge, 1984; Abdel-Latif,

1986). Phosphatidylinositol (PI) is the major contributor to this class of lipids, but

there are at least two minor components, phosphatidylinositol 4-phosphate (PIP) and

phosphatidylinositol 4,5-bisphosphate (PIP2) (Figure 1-1). The receptor is coupled

to the membrane associated enzyme, phospholipase C, which has been shown to
preferentially hydrolyze PIP2 to form inositol 1,4,5-trisphosphate (IP3) and

diacylglycerol (Berridge, 1983). These two compounds are the second messengers

which initiate the intracellular steps toward the cellular response (Berridge, 1984).

We have shown that various hormones and neurotransmitters were able to

stimulate the hydrolysis of phosphoinositides (PIs) in the rabbit kidney (Chapter 2)

which suggested that the Pis second messenger system is present in the kidney.

Stimulation of PIs hydrolysis by most of the drugs exhibited regional specificity in the

kidney (Tables 2-1 to 2-3) which may have been due to specificity in the distribution

of the PIP2. In addition to blood vessels and interstitium, each zone of the kidney

contains several types of epithelia. The purpose of this study was to determine the

distribution of PIP2 in three different zones of the rabbit kidney by

immunohistochemical localization of PIP2.

Materials and Methods

Chemicals. Rabbit immunoglobulin G, goat antirabbit immunoglobulin G, rabbit

peroxidase antiperoxidase and 3,3'- diaminobenzidine tetrahydrochloride were obtained










from Sigma Chemical Co. (St. Louis, MO). All other reagents were obtained from Fisher

Scientific Co. (Orlando, FL).
Tissue preparation. Male New Zealand white rabbits were killed by decapitation.

The kidneys were removed and cut into several cross sections of 0.5-1 mm thickness.

These sections were prepared as described earlier for immunohistochemical studies of

eye tissue (Das g lal., 1986). The kidney sections were immediately fixed in 4 %

paraformaldehyde in cacodylate buffer, pH 7.2 for 72 hours. The sections were washed

with 1.0 M sodium cacodylate overnight. The kidney sections were dehydrated with

increasing concentrations of ethanol before embedding in paraffin. Cross sections (4 umn

thickness) of the kidney were cut on a Reichert-Jung 2030 Biocut microtome (Reichert

Scientific Instruments, Buffalo, NY) and placed on clean microscope slides.
Preparation of PIPp antibody. The PIP2-specific antibody was obtained from Dr.

Tohru Yoshioka, Yokohama, Japan. The antibody was prepared as described earlier (Das

g al., 1986). New Zealand white rabbits were injected with PIP2-hapten (bovine

serum albumin) suspensions for 3 weeks. Antibodies to PIP2 were purified by affinity

absorption to Protein A. Immunoreactivity of the antibody to other lipids: PI, PIP,
phosphatidylcholine, phosphatidic acid, was less than 1% compared to PIP2 (100%).

Localization of PIP;. The method for localization was based on the peroxidase and

antiperoxidase method by Sternberger tal. (1971) and Das fal. (1986). Tissue

Sections were cleaned of paraffin and rehydrated before being placed in phosphate-

buffered saline for 10 minutes. In order to quench endogenous peroxidase activity, the

tissue sections were incubated with 3% hydrogen peroxide for 5 minutes. The sections

were rinsed in phosphate-buffered saline before incubation for 12 hours at 4 *C with

either rabbit immunoglobulin G (control) or rabbit anti-PIP2 antibody

(experimental). The washed sections were incubated at room temperature for 90

minutes with goat antirabbit immunoglobulin G, followed by an additional 90 minute

incubation with rabbit peroxidase-antiperoxidase. The sections were washed and










incubated with 3,3'-diaminobenzidine tetrahydrochloride as the chromagen. The stained

sections were examined and photographed with a UFX camera attached to a Labophot

microscope.

Results

Inner medulla. Figure 4-1 shows a cross section of the inner medulla of the

rabbit kidney that had been treated with PIP2-specific antibodies. Intense staining for

PIP2-specific antibody was observed primarily in the collecting duct cells of the inner

medulla. In addition, the papillary epithelium, which is an extension of the collecting

duct, was also stained. There was virtually no staining in sections that were treated with

rabbit immunoglobulin G for the control (not shown).

Because of the inability to duplicate the immunohistochemical staining in black

and white photographs, we have compared the intensity of PIP2 staining in the three

zones of the kidney, schematically (Figure 4-2). Figure 4-2A represents a portion of

the inner medulla shown in figure 4-1.

Outer medulla. Figures 4-2B shows a schematic drawing of the outer medulla

that had been treated with PIP2-specific antibody. Staining for PIP2-specific antibodies

was observed in the collecting duct cells.

Cotex. Figures 4-2C shows a schematic drawing of the cortex of the rabbit

kidney that had been treated with PIP2-specific antibody. None of the cells of the cortex

appeared to stain for PIP2.

Discussion

As stated in chapter 2, various hormones and neurotransmitters were able to

stimulate the hydrolysis of PIs in the rabbit kidney, specifically the medulla. In this

study, we have demonstrated that the distribution of PIP2 in the three zones of the

kidney is similar to the cholinergic stimulation of PIs hydrolysis in these zones.

Staining for PIP2 was greatest in the inner medulla, less in the outer medulla and

nondetectable in the cortex. These results indicate that PIP2 may be a substrate for


























7)
3.








E


.C

0
C









0.












E
C-



0.E
CO




C c


60







o 2
c &

so


E-a











E
0 w






62


































* ,_ 'p. Y *..i r

.Ao.

VI.























Figure 4-2. Schematic drawing of immunohistochemical staining for PIP2 -specific
antibodies in rabbit kidney. Shaded areas indicated cells that were stained for PIP2. A)
Inner medulla, B) Outer medulla, C) Cortex. C: collecting duct; PE: papillary
epithelium; 1: interstitial cells; V: blood vessel; T: thin limb of the loop of Henle; H:
thick ascending limb; P: proximal tubule; G: glomerulus; D: distal tubule.





64






A "







B v








C










stimulation of phospholipase C by cholinergic agents and other hormones in the rabbit

kidney. Berridge (1983) demonstrated that serotonin stimulated the hydrolysis of PIP2

and the formation of IP3 in insect salivary gland.

Some of the enzymes involved in Pis metabolism, including phospholipase C, have
been found in the kidney cortex and medulla (Speziale er al., 1982; Troyer i al., 1986;

Rogers and Hammerman, 1987). It should also be pointed out that compounds, such as

angiotensin II and phenylephrine, stimulate 32P incorporation into PIs (often considered

to be an indicator of Pis hydrolysis) in the proximal tubules of the kidney cortex
(Wirthensohn and Guder, 1985). In our studies, PIP2 levels were nondetectable in the

cortex. We, also, were not able to demonstrate agonist-stimulated hydrolysis of Pis in

the kidney cortex with any of the drugs tested (Chapter 2). There are two possible

reasons for the differences between our studies and those of others. First, there may be

low levels of PIP2 in the cortex which cannot be detected by our method. One problem

with the method used for immunohistochemical localization of PIP2, is that in order for

PIP2 to be detected, it must be accessible to the antibody. It is possible that PIP2 may

not be accessible to the antibody in the cortex. A second possibility is that even though

studies have shown that PIP2 is the preferential substrate for phospholipase C

(Berridge, 1983), it is not the only substrate. It appears that PI and PIP are also

substrates for phospholipase C leading to the formation of diacylglycerol and inositol

Sphosphates (Majerus lal., 1986). This suggests that the mechanism for hormone

stimulation of the hydrolysis of PIs in cortical elements is different from that in the

inner and outer medulla.

In any case we have shown by immunohistochemical techniques that both the
inner and outer medulla contain higher levels of PIP2 than the cortex. Staining of PIP2

was most prominent and distinct in the collecting duct cells of the inner medulla. This

suggests that the medulla and the collecting duct cells may be a site of action for

carbachol and various other agents that stimulate PIs hydrolysis.







66


Summary and conclusions. Immunohistochemical studies of PIP2 in the rabbit

kidney demonstrated differences in the regional distribution of this lipid in the cortex

and medulla. The lipid was not detected in the cortex. Higher levels of PIP2 were found

in the outer and inner medulla, specifically the collecting duct cells, suggesting that they

may be an site for receptor-stimulated PIs hydrolysis.















CHAPTER 5
CHOUNERGIC STIMULATION OF PHOSPHOINOSITIDE HYDROLYSIS IN
INNER MEDULLARY COLLECTING DUCT (IMCD) CELLS OF THE
RABBIT KIDNEY

Introduction

As stated in Chapter 1, acetylcholine (ACh) infusion into the renal artery

produces vasodilation of renal blood vessels and diuresis (Vander, 1964; Lameire etal.,

1980). Evidence from several studies suggests that diuresis is an indirect consequence

of the increase in blood flow, primarily in the papilla (Lameire lal., 1980). Some

investigators have questioned this hypothesis and proposed alternate explanations

(Earley and Friedler, 1965; Parmalee and Carter, 1968; Hartupee tal., 1982). No

one has, however, examined the biochemical actions of cholinergic agents on different

types of cells in the mammalian kidney.

As stated in Chapter 3, we have examined the effects of carbachol, a cholinergic

agonist, on phosphoinositide (PIs) hydrolysis in slices from the inner medulla (IM),

outer medulla (OM) and cortex (CTX) from rabbit kidney. Carbachol-stimulated PIs

hydrolysis was maximal in the IM, less in the OM and non-detectable in the renal CTX

(Table 2-1). Further characterization of cholinergic-stimulated PIs hydrolysis in IM

indicated that this effect of carbachol, like the vasodilation and diuresis, was mediated by

the muscarinic receptor (Figure 3-9). It is not known which cells of the IM and OM are

responsive to cholinergic agents to cause stimulation of PIs hydrolysis. The inner

medulla contains the following cells: cells of the Loop of Henle, collecting duct (IMCD)

cells, vascular endothelial cells, and interstitial cells (Knepper elal., 1977; Valtin,

1983). Binding of an agonist with the receptor results in activation of phospholipase C

which specifically hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two










second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which

initiates the cellular response (Berridge, 1983). Our immunohistochemical studies

showed a great quantity of the substrate, PIP2, in the IMCD cells (Chapter 4).

The purpose of this study was to determine if IMCD cells were responsible for the

effects of cholinergic agents in the IM. To do this, we measured the ability of carbachol

to stimulate PIs hydrolysis in IMCD cells isolated from the rabbit kidney.

Materials and Methods

Chemicals. All chemicals were obtained from Fisher Scientific Co., Orlando, FL,

unless otherwise specified.

Animals Male New Zealand white rabbits (body weight of 1- 1.5 kg) were fed

Purina Rabbit Chow and water ad libitum.

IMCD cell preparation. The method for isolation of IMCD cells was based on

methods described earlier (Grenier eal., 1981, Sato and Dunn, 1984).

Rabbits were decapitated. The kidneys from two rabbits were excised and placed

in cold 0.9% saline. The inner medulla including papilla (approximately 1 g tissue)

was separated and minced with a Mcllwain tissue chopper in two directions at a setting of

200 pm. The tissue was suspended in 20 ml of 0.1% collagenase (Type I, Sigma

Chemical Co., St. Louis, MO) in Krebs-Ringer bicarbonate (KRB) buffer: 118 mM NaCI,

4.7 mM KCI, 0.75 mM CaCI2, 1.18 mM KH2PO4, 1.18 mM MgSO4, 24.8 mM NaHCO3,

and 10 mM glucose. The suspension of IM slices was incubated in a 37 C water bath for

2 hours with continuous bubbling of 02: CO2 (95:5). Every 15-30 minutes, the slices

were mixed by taking the suspension up in a 15 ml syringe. After 2 hours, the

suspension was filtered through nylon filter with 70 p.m mesh opening (Fisher

Scientific Co., Orlando, FL). To lyse cells other than IMCD cells, 30 ml of distilled

water were added to the suspension. This mixture was immediately centrifugated in an










international clinical centrifuge (model CL, International Equipment Co.,Needham, MA)

at setting 5 for 5 minutes. The cells were resuspended in KRB buffer.

Cell purity and viability. We examined the purity of our IMCD cell preparation

by analysis of the staining for lipids with Sudan black. Lipid droplets, which would be

stained are characteristic of interstitial cells (Wuthrich tal., 1986). Over 96% of

the cells after hypotonic treatment remained unstained.

The viability of IMCD cells was determined by the method of trypan blue dye

exclusion. A 100 plI aliquot of the cell suspension was mixed with 5 PI of trypan blue

(0.6% wt/vol). The cells were examined on a hematocytometer with light microscopy.

Cell viability was the number of live intact cells (those that exclude the blue dye)

expressed as a percentage of total cells. Viability of IMCD cells in these experiments

varied from 70-95%.

Similar preparations of IMCD cells have been used by other investigators and did

not contain vascular endothelial cells or interstitial cells when examined with electron

microscopy (Wuthrich eal., 1986).

Determination of Na-K-ATPase activity in isolated IMCD cells. This enzyme

activity was determined by the method of Garg eal. (1981). In brief, the method was

based on the measurement of hydrolysis of ATP to ADP which was coupled to the oxidation

of NADH which was measured fluorometrically. The reaction mixture consisted of (in

* mM): NaCI, 100; NH4CI, 68.7; imidazole, 50.34 MgCI2, 3.7; EDTA, 0.33; Na2ATP, 1.1;

phosphoenolpyruvate, 0.6; NADH, 0.017; pyruvate kinase, 2.3 U/ml; and lactate

dehydrogenase 3.3 U/ml. Na-K-ATPase activity was the calculated difference between

total ATPase activity (in the absence of ouabain) and residual or Mg-ATPase activity (in

the presence of 1 mM ouabain) after incubation for 30 minutes. The results were

expressed as pmol of ADP formed per 1000 cells per minute or pmol of ADP formed per

ig protein per minute.










The total cell number was determined as described above by examination of the

cells on a hematocytometer with light microscopy. The amount of protein was

determined in similar aliquots by the method of Lowry i al. (1951). Bovine serum

albumin (Sigma Chemical Co., St. Louis, MO) was used as a standard. The absorbance at

750 nm was measured in a Coleman spectrophotometer (Model 620).

Measurement of Pis hydrolysis. Receptor-mediated Pis hydrolysis involves

activation of phospholipase C which preferentially hydrolyzes PIP2 into DAG and IP3

(Berridge, 1983). The hydrolysis of Pis was measured as described earlier for kidney

slices (Chapter 2) and neuronal cell cultures (Feldstein al., 1986). Stimulation of

Pis hydrolysis was determined by measuring the formation of IPs after incubating the

cells with [3H]inositol for its incorporation into the phosphoinositides.

Incorporation of 13Hinostol. The incubation of the cells with 1 .Ci/ml

[3H]inositol (19.0 Ci/mmol, Amersham Corp., Arlington Heights, IL) for its

incorporation into the membrane phospholipid was in two steps. Inner medullary slices

were initially incubated with [3H]inositol in addition to collagenase. After exposure to

the hypotonic medium and centrifugation, the cells were resuspended in KRB buffer

containing 1 .Ci/ml [3H]inositol and incubated for 60 minutes at 37 oC.

After this incubation the cells were washed twice by low speed centrifugation and

the pellet resuspended in KRB buffer (without [3H]inositol). The purpose of this step

< was to remove any extracellular [3H]inositol that was not incorporated into the

membrane lipids.

In some samples, the incorporation of [3Hjinositol into IMCD membranes was

measured by determining the radioactivity in the organic layer as described below.

Drug treatment. Cells were resuspended in KRB buffer (approximately 2 mg

protein/ml). Cells (50 il) were added to 12 x 75 mm polypropylene culture tubes that

contained up to 200 Il of KRB buffer with 10 mM LiCI substituted isotonically for NaCI.

LiCI inhibits the dephosphorylation of inositol monophosphate and thereby allowing the










accumulation of [3H]IPs and preventing the recycling of [3H]inositol (Hallcher and

Sherman, 1980; Sherman A al., 1981).

Buffer or drugs (10 ul volume) were added to the tubes to start the reaction.

The final volume of the assay mixture was 250 pl. The tubes were saturated with

02:C02 (95%:5%) and placed in a 37 *C shaking water bath for 60 minutes. The

reaction was stopped by the addition of 1 ml chloroform:methanol (1:2 v/v). An

additional 350 pil of chloroform and 350 0l of water were added. The tubes were agitated

for 10 minutes prior to low speed centrifugation to separate the two layers: aqueous

(upper) phase and organic (lower) phase. The aqueous layer contained [3H]IPs (IP,

IP2, IP3) and [3H]inositol. The organic layer contained the phospholipids that had

incorporated [3H]inositol.

At the start of the assay (time zero), 1 ml of chloroform: methanol mixture was

added to samples containing 200 Wl of KRB buffer with lithium and 50 AIl cells. These

zero time samples were processed for determination of radioactivity in the organic and

aqueous layers, like those samples that were incubated for 60 minutes with or without

drug. The IPs formed were measured as described below.

Measurement of inositol Dhosohates. The aqueous layer contains the [3H]IPs (IP,

IP2, IP3) and [3H]inositol. The inositol phosphates were separated from the inositol

with anion exchange chromatographic techniques (Berridge, 1983). A 1 ml aliquot of

Sthe upper aqueous phase was transferred to a -3 x 100 mm borosilicate glass test tube

and diluted to 3 ml with distilled water. One milliliter of Dowex-1 slurry (50% v/v in

water; 100-200 mesh, format form, Biorad Laboratories, Rockville Center, NY) was

added. This mixture was poured over polypropylene columns with fritted discs. The

Dowex in the column was rinsed with 10 ml of cold 5 mM inositol to remove any

[3H]inositol that may have been nonspecifically associated with the Dowex resin. Total

IPs were eluted with 5 ml 0.1 M formic acid/1.0 M ammonium format. The eluant was

collected in scintillation vials into which 10 ml Liquiscint scintillation fluid (National










Diagnostics, Somerville, NJ) were added. The radioactivity of these samples was

measured in a Beckman LS7000 scintillation counter.

A 525 0d aliquot of the lower organic phase was placed into a borosilicate glass

scintillation vial. The chloroform was allowed to evaporate in a fume hood before 3 ml of

OCS scintillation fluid (Amersham Corp., Arlington Heights, IL) were added. The

radioactivity was measured in a Beckman LS7000 scintillation counter.

Calculations. To eliminate variations of incorporation of [3H]inositol into IMCD

cells between experiments, the formation of [3H]IPs (dpm in aqueous layer) was

expressed as a percentage of the total [3H]inositol incorporated (dpm in organic layer

+dpm in aqueous layer).

The percent formation of [3HIlPs from zero time (non-incubated) samples were

subtracted from control and drug-treated (incubated) samples. The zero time values

varied from 5-10%.

Statistical Analysis. Significance of experimental values was determined using

the Student's t-test. Differences were significant if P < 0.05.

Results
Na-K-ATPase activity in IMCD cells. Table 5-1 shows that Na-K-ATPase

activity in the IMCD cells isolated from the rabbit was 49.0 pmol ADP formed per ig

protein per minute. The enzyme activity in rabbit IMCD cells was comparable to the

SNa-K-ATPase activity of 40.9 pmol ADP formed per gg protein per minute reported to

be present in IMCD cells isolated from the rat (Stokes p al., 1987). Results from the

same experiments above were also calculated with respect to the total cell number

instead of protein. The activity of Na-K- ATPase of IMCD cells was 18.5 pmol ADP

formed per 1000 cells per minute (or 0.02 pmol ADP formed per cell per minute)

which was similar to the value 0.03 pmol ADP formed per cell per minute reported for

cortical and outer medullary collecting duct of rabbit (Garg lal., 1981).























TABLE 5-1
ATPASE ACTIVITY OF IMCD CELLS ISOLATED FROM RABBIT KIDNEY

Enzyme Activity Activity
(pmol ADP formed per (pmol ADP formed per
gg protein per minute) 1000 cells per minute)

Total ATPase 181.0 + 46.2 83.5 33.3

Mg-ATPase 131.7 43.6 61.9 29.9

Na-K-ATPase 49.0 5.4 18.5 6.0

Values are the mean S.E.M. of 3 IMCD cell preparations. ATPase activity determined as
described in Materials and Methods.










[3H]lnositol incororation into IMCD cells. The incorporation of [3H]inositol

into isolated IMCD cells was examined in samples that were incubated with 1 gCi/ml for

various times after the initial incubation with collagenase. The radioactivity in the

organic layer was measured as described above. The incorporation of [3H]inositol was

erratic for the first 30 minutes probably due to incomplete mixing or slow diffusion of

[3H]inositol, but increased linearly with time up to 3 hours (Figure 5-1). We chose

60 minutes because the amount of radioactivity in the cells was sufficient and

reproducible. The incorporation of [3H]inositol increased linearly (r-0.97) with

increasing concentrations of [3H]inositol (Figure 5-2). We chose 1 pCi/ml (approx.

60 nM) because the amount of [3H]inositol incorporated was adequate and reproducible.

Time dependence of drug treatment. The effect of 1 mM carbachol on [3H]IPs

formation in IMCD cells was measured after treatment for various time intervals

(Figure 5-3). The maximum stimulation of [3H]lPs formation (carbachol-treated

minus control values) was observed after 60 minutes of incubation. Therefore, 60

minutes was chosen as the incubation time for drug treatment in all of our experiments.

Concentration dependency The formation of [3H]IPs was dependent upon the

concentration of carbachol (Figure 5-4). The maximum effective concentration was 1

mM and the EC50 was 4 gM.

Recentor specificity. Since carbachol is a nonspecific cholinergic agonist, it can

exert its effects through the muscarinic or nicotinic cholinergic receptor. The effects of

specific agonist or antagonists were examined (Figure 5-5). Atropine (1lM), a

muscarinic receptor antagonist, was able to completely block the effects of carbachol on
[3H]IPs formation in IMCD cells. The nicotinic receptor antagonist, hexamethonium (1

.M), did not alter cholinergic stimulation of this response. The nicotinic agonist, 1,1-

dimethyl-4-phenylpiperazinium iodide (DMPP; 1 mM) did not have an effect on

[3H]lPs formation in IMCD cells. These results support that cholinergic stimulation of














0



ou
=E









s0
I w







1a





O .D


o




Soo
cc a

o,
0-




Sc.


.0
SCO


088
C


"g







0
0E0





cw
* 0' (
X'
.-S Q

15 %

III
Sli






01 nLU
u- ic

































































Q Q
cn cli


(ue!lojd Bu6/udp)
plejodoo3ul Ol!SOUl[iH]


o i




o
E
a,
E
F




0















E
z 2
o U)
0


c E


8.











o



a-
(I














U;

D
0



E C
o a
ro








0 80
Cc-


cE
-u5
0 0







(-
CL
Ct







0


Co











L. 0 0
s-s











U O0








78














0
o















0 a



ID
CC
C





C,,


(u!aelod B6w/udp)
pelejodjooul |lO!SOUI[He]
















CL
0=






c



E
0












cE
a)















o
-e

i
E













a,


E
0



.2
CO
(I





0.
oP
ca)






80










o





o------------------- Hi- -^) "

I-.--
0
o 0





E




i i









ULO



(peleljodlooul lejol %)

paseelea saleqdsoqd |Ol!SOUl(HC]















CD








E
0





Z
U
0







a
t







i2








0




.0
A









I,
2U
0


a,
o
Xi
g














S.52
8































0.
.C
















o













0






-m
CI
M















0E
sUi
i .











U,
a,
r- W

o
Z-H
5
Eg





a Q

li
iT [i








82




























CD




o
I 0


-n
o
o o










\--



















(paleJodjooul IjIOI%)


peseelea seleqdso-d lOI!SOUI[HE]
















0




+E
o





am
- "















%E+I
E E







o0








i a ,
Es-




Sco
-1o



. .. 0
a










SE.
E 8
- +0,

















<0 E fe
*5.'2
c)^-0
c 2 ..
'0ff 0 i
a .Q
"CC~d



















0
(-






Ic +















Q





















Cl) 040




(paielodJooul leIlo%)


peseeaaI seleqdsoqtd IOl!SOUl[HE]










Pis hydrolysis in IMCD cells is mediated by the muscarinic receptor, as was the case

with IM slices.

Calcium dependency. It has been shown in brain (Gonzales and Crews, 1984) and

renal inner medullary tissues (Figure 3-11) that cholinergic stimulation of Pis

hydrolysis is calcium dependent. We, therefore, measured the response to carbachol in

KRB buffer to which calcium or ethylene glycol bis (B-aminoethyl ether)-N,N'-

tetraacetic acid (EGTA; 200 gM), a calcium chelator, had been added (Figure 5-6).

Carbachol (1 mM) had a small effect when no calcium had been added. When EGTA was

added, the response to carbachol decreased to a value not significantly different from

control. The cholinergic stimulation fof [3H]IPs formation by carbachol increases with

increasing Ca2+ concentration up to 1 mM. Increasing the calcium concentration to 2

mM resulted in a decrease in (3H]IPs formed by carbachol from the maximum at 1 mM

Ca2+ to that which was similar in the absence of Ca2+. The calcium concentration that

was used in our experiments of this study was 0.75 mM.

Discussion

Response in IM slices vs. IMCD cells. As stated in chapter 2, the effects of

carbachol in kidney slices showed the greatest stimulation of PIs hydrolysis in the inner

medulla (Table 2-1). The EC50 value for carbachol was 10 UM with a maximum

effective concentration of 1 mM.

The inner medulla consists of a number-.of cell types which include the vascular

endothelial cells, nephronal epithelial cells, and interstitial cells. Any of these

particular cells may be the site of action for cholinergic stimulation of Pis hydrolysis.

Immunohistochemical localization of PIP2-specific antibodies to the collecting duct cells

of the IM suggested that this may be an important target site for cholinergic agents and

possibly other hormones and neurotransmitters (Chapter 4).

In this study, we have shown that carbachol stimulates Pis hydrolysis in IMCD

cells in a concentration-dependent manner. The EC50 of 4 JLM and the maximum













< w






'm
LU



,o E
r en

aS






^|5
go




us (D
0 = a




0
o .2


c 8|
o



0- -
-a 0.0





0 D 0
E >

- sw>
(UI j-8





o "





t5 Ec






PP2







































E



c
0
E






o









LU
w
+
d


(paiejodJooul lelOl%)



pesealete seleqdsoid IO!SOUI[HCI










effective concentration of 1 mM are very similar to those for IM slices. The magnitude

of Pis hydrolysis expressed as a percentage of the total [3H]inositol incorporated is also

similar between IM slices and isolated IMCD cells (Table 2-1 vs. Figure 5-4).

The receptor specificity of the PIs hydrolysis in response to carbachol is

identical between IM slices and IMCD cells. The response in both tissues is blocked

completely by 1 UM atropine and not by 1 U.M hexamethonium, indicating the muscarinic

receptor is involved. Cholinergic stimulation in IM slices may be due totally or in part

to the effect on IMCD cells. The effects of cholinergic agents on PIs hydrolysis in the

other cells of the inner medulla remains to be determined.

The effect of calcium on this system, however, was slightly different between IM

slices and IMCD cells. The effects in IM slices and IMCD cells were both calcium

dependent. In IMCD cells, however, the response to carbachol decreased when the

calcium concentration was increased from 1 to 2 mM, whereas the response in IM slices

remained the same (Figure 5-6 vs Figure 3-11). The reason for this is unknown at

present. It is possible that the attenuation of carbachol-stimulated PIs hydrolysis seen

in IMCD cells may not have been detected in IM slices. Another possibility is that IMCD

cells may have become sensitive to the effects of calicum after treatment with

collagenase and hypotonicity.

Physiological significance. As noted earlier, ACh infusion into the renal artery

causes renal vasodilation as measured by an increase in blood flow and diuresis (Vander,

1964; Hayslett f al., 1970; Lameire p al., 1980; Fadem e al., 1982; Hartupee e al.,

1982). Both of these responses can be blocked by the muscarinic receptor antagonist,

atropine (Vander, 1964). Diuresis is most commonly believed to result indirectly from

changes that occur due to vasodilation and increased blood flow. The relationship between

vasodilation and diuresis is still questioned. Alternate explanations for the diuretic

effect of ACh include changes in interstitial pressure and in tissue hypertonicity which










could alter transport properties of the epithelial cells (Earley and Friedler, 1966;

Hartupee ital.,1982).

We have demonstrated that cholinergic agents act directly on IMCD cells to

stimulate PIs hydrolysis. Inner medullary collecting ducts cells have been shown to

absorb sodium (Diezi al., 1973; Stein ala., 1976; Higashihara l al., 1978;

Ullrich and Papavassilion, 1979; Rocha and Kudo, 1982). We have shown that IMCD

cells have Na-K-ATPase activity (Table 5-1) comparable to cortical and medullary

collecting duct cells. Furthermore, activation of protein kinase C has been shown to

inhibit sodium transport in other nephron segments of the rabbit kidney, such as the

cortical collecting duct (CCD) (Hays e al., 1987),as well as in cultured renal epithelial

cells, such as A6 cells (Yanase and Handler, 1986). One of the second messengers

formed upon stimulation of Pis hydrolysis is DAG which stimulates protein kinase C

(Abdel-Latif, 1986; Nishizuka, 1986). Thus, if cholinergic agents stimulate Pis

hydrolysis and the formation of DAG in IMCD cells, activation of protein kinase C could

result in inhibition of sodium transport in IMCD cells followed by diuresis. Therefore,

our results are consistent with the hypothesis that cholinergic agents not only increase

renal papillary blood flow but, may also affect electrolyte transport in IMCD (terminal

segment of the nephron).

Summary. We have shown that cholinergic stimulation of PIs hydrolysis in IMCD

cells is mediated by the muscarinic receptor, like that observed in IM slices. The IMCD

cells are a target site for cholinergic agents. The importance of this phenomenon as a

contributing factor to cholinergic-stimulated diuresis remains to be determined.















CHAPTER 6
CHOUNERGIC RECEPTORS IN INNER MEDULLARY COLLECTING DUCT CELLS
OF THE RABBIT KIDNEY

Introduction

The physiological effects of acetylcholine on vasodilation, diuresis, and other

aspects of renal function have been well documented (Vander, 1964; Hayslett rf aL.

1970; Martinez-Maldonado e al., 1972; Lameire ea al., 1980; Fadem 8 al., 1982;

Hartupee alg., 1982). Cholinergic stimulation of vasodilation and diuresis was blocked

by atropine, a muscarinic receptor antagonist (Vander, 1964).

The biochemical effects of cholinergic agents in the kidney were examined in

chapters 2, 3 and 5. Carbachol, a nonspecific cholinergic agonist, was able to stimulate

phosphoinositide (PIs) hydrolysis in the inner medulla (IM), specifically the inner

medullary collecting duct (IMCD) cells of the rabbit kidney. This biochemical process

like the vasodilatory and diuretic effects was blocked by atropine (IM slices: figure 3-9;

IMCD cells: figure 5-5), suggesting that the muscarinic receptors were present.

Muscarinic receptors, however, were not detectable in whole kidney membrane

preparations (Yamamura and Snyder, 1974b).

The purpose of this study was to determine if muscarinic receptors were present

in IMCD cells. We measured muscarinic receptors in IMCD cells with radioligand

binding technique using the muscarinic receptor antagonist, L-quinuclidinyl-[phenyl-

4-3H]-benzilate ([3H]QNB).

Materials and Methods

Chemicals. All chemical reagents were obtained from Fisher Scientific Co.,

Orlando, FL unless otherwise specified. The radiolabelled muscarinic receptor ligand,

[3H]QNB (44 Ci/mmol), was obtained from Amersham Corp., Arlington Heights, IL.










Animals. New Zealand white male rabbits were fed Purina rabbit chow and water

adlibitum.

Cell preparation. Inner medullary collecting duct cells were prepared as

described in Chapter 5.

Rabbits were killed by decapitation. The kidneys from two rabbits were removed

and placed in cold 0.9% saline. The IM (approximately 1 g tissue) was minced in two

directions with a Mcllwain tissue chopper at a setting of 200 pm. The tissue slices were

suspended in 20 ml 0.1% collagenase (Type I, Sigma Chemical Co., St Louis, MO) in

Krebs-Ringer bicarbonate (KRB) buffer (concentrations expressed in mM): NaCI, 118;

KCI, 47; CaCI2, 0.75; KH2PO4, 1.18; MgSO4, 1.18; NaHCO3, 24.8; and glucose, 10. The

suspension of IM slices was continuously bubbled with 02:CO2 (95%:5%) while in a 37

C water bath for 2 hours. Every 15-30 minutes the cells were resuspended. After 2

hours the suspension of cells was filtered over nylon gauze (70 pm opening; Fisher

Scientific Co., Orlando, FL). To lyse cells (vascular endothelial cells, interstitial cells,

other nephronal elements) other than IMCD cells, 30 ml of distilled water was added to

the filtrate. This mixture was immediately centrifugated at setting 5 for 5 min in a

clinical centrifuge (Model CL; International Equipment Co., Needham, MA). The cells

were resuspended in 2 ml KRB buffer. The cells were washed again by centrifugation

and resuspension in KRB buffer to the appropriate concentration of protein.

Protein assay. Prior to the binding assay, the protein concentration of the cell

suspension was measured based on the method of Lowry t al. (1951). Bovine serum

albumin (Sigma Chemical Co., St. Louis, MO) was used as the standard. The absorbance

was measured in a Coleman Jr. Spectrophotometer at a wavelength of 750 nm.

[3H]QNB binding assay. Muscarinic receptors of IMCD cells were measured by

radioligand binding techniques described earlier (Feldstein e al., 1986; Hynes t al.,

1986). Cell protein (0.1 mg) was incubated with [SH]QNB and KRB buffer in a final

volume of 2 ml in 13 x 100 mm borosilicate glass test tubes at 37 OC for 60 minutes.










Nonspecific binding was measured in similar tubes that contained 100 UM atropine. For

each cell preparation, the data were obtained from triplicate samples for each treatment.

The reaction was stopped by dilution of the assay mixture to approximately 8 ml with

cold KRB buffer. The mixture was immediately filtered through Whatman GF-B glass

fiber filter circles (Fisher Scientific Co., Orlando, FL) under vacuum in a Brandel M24

Cell Harvester (Brandel Biomedical Research and Development Laboratories,

Gaithersburg, MD) to remove unbound [3H]QNB. The filters were rinsed 3 times each

with 8 ml cold KRB buffer rapidly. The filters were placed in scintillation vials with

10 ml Liquiscint scintillation fluid (National Diagnostics, Somerville, NJ). The

[3H]QNB that remained on the filter was extracted by shaking the vials for 30 minutes.

Radioactivity of the samples was measured in a Beckman LS7000 scintillation counter.

Specific binding of [3H]QNB was calculated as the difference between total

binding in the absence of atropine and nonspecific binding. In pilot studies, we

determined that specific binding of [3H]QNB to IMCD cells was linear between 0.1 and

0.5 mg protein. Therefore, we chose to use 0.1 mg protein to increase the number of

assay tubes for each experiment.

Statistics. Significance of experimental values was determined using the

Student's t-test. Differences were significant if P < 0.05.

Results

Time dependency. The association (Figure 6-1) and the dissociation (Figure 6-

2) of the binding of [3H]QNB to IMCD cells was time dependent. Steady-state binding of

[3H]QNB was reached within 45 minutes at 37 C. Nonspecific binding was constant at

all time points (Figure 6-1A). Specific binding reached maximal levels at 45 minutes

(Figure 6-1B). After 60 minutes of incubation, addition of 200 UM atropine resulted

in a time dependent decrease in binding of [3H]QNB to IMCD cells (Figure 6-2). The

half-life of dissociation was 27 minutes.























Figure 6-1. Time course of [3H]QNB binding to IMCD cells isolated from rabbit kidney.
In A, IMCD cells (0.1 mg protein) were incubated with 1 nM [3H]QNB at 37 C in the
absence (total binding of [3H]QNB) or presence (nonspecific binding) of 100 PM
atropine for the times indicated. The suspensions were filtered and washed as described
in the Materials and Methods. Each point is the mean SEM of 3 cell preparations. In B,
the specific component of [3H]QNB binding to IMCD cells was calculated as described in
the Materials and Methods. Each point is the mean SEM of 3 cell preparations.




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EP79505UR_B2SW0V INGEST_TIME 2012-09-24T13:01:56Z PACKAGE AA00011809_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES