Voltage-dependent K+ current in rat renal resistance arterioles

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Voltage-dependent K+ current in rat renal resistance arterioles
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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
    Abstract
        Page vi
        Page vii
    Chapter 1. Introduction
        Page 1
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    Chapter 2. Voltage-dependent K+ current in vascular smooth muscle cells from rat renal resistance arterioles: Evidence for heterotetramer formation
        Page 21
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    Chapter 3. Alterations in rat interlobar artery membrane potential and K+ channels in genetic and nongenetic hypertension
        Page 44
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    Chapter 4. Prevention of renovascular and cardiac pathophysiological changes in hypertension by AT1 receptor antisense gene therapy
        Page 66
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    Conclusions
        Page 89
    References
        Page 90
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    Biographical sketch
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Full Text










VOLTAGE-DEPENDENT K- CURRENT IN RAT RENAL RESISTANCE
ARTERIOLES: ALTERATIONS IN HYPERTENSION AND PREVENTION WITH
GENE THERAPY















By

JEFFREY R. MARTENS


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


1998






























This dissertation is dedicated to my wife and parents. To my parents, your love has
provided me with the confidence to pursue my goals. To my wife, Lori, your love and
support keeps me going. It is your presence in my life that gives me perspective on that
which is important during our time on this earth.













ACKNOWLEDGMENTS


I would like to express my sincere gratitude to Dr. Craig H. Gelband for his

support and guidance. He has been a friend and a teacher. His mentoring has established

the foundation for my scientific career. I would like to thank the members of my

committee, Drs. Luis Belardinelli, Jeffrey Harrison, Philip Posner, and Charles Wingo for

their encouragement, suggestions, and direction. Their contributions have lent valuable

perspectives to my growth as a scientist. I wish to thank Drs. Mohan Raizada and Colin

Sumners for their support and advice. In opening their laboratories to collaborative

projects, I have gained priceless knowledge and experience that will accompany me

throughout my research career. I would like to thank Dr. Sarah England for her work on

Figure 8 in Chapter 2 and Drs. Kathleen Berecek and Sanford Bishop for their work on

Figure 8 in Chapter 4. I would also like to thank Drs. Di Lu and Phyliss Reaves for their

work on the retroviral preparation and animal treatment in Chapter 4. Finally, I am

grateful for the support and patience of the members of Dr. Gelband's laboratory.

I would also like to acknowledge the financial support of Dr. Craig H. Gelband

and the American Heart Association, Florida Affiliate.













TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ................................................................................................. iii

A B S T R A C T ....................................................................................................................... vi

CHAPTERS

1. INTRODUCTION ......................................................................................................... 1

Voltage-Dependent Ca2-' Channels ................................................................................ 3
Smooth Muscle K" Channels.......................................................................................... 5
Smooth Muscle Cl Channels....................................................................................... 13
Ion Channel Alterations in Hypertension .................................................................... 15
Reversal of Ion Channel and Pathophysiological Alterations in Hypertension......... 17
R ation ale .... ................................................................................. ... 17

2. VOLTAGE-DEPENDENT K4 CURRENT IN VASCULAR SMOOTH
MUSCLE CELLS FROM RAT RENAL RESISTANCE ARTERIOLES:
EVIDENCE FOR HETEROTETRAMER FORMATION.......................................... 21

Introduction .................................................................................................................. 2 1
Materials and Methods................................................................................................. 22
R e su lts ..........................................................................................................................2 6
D iscu ssio n .................................................................................................................... 3 1

3. ALTERATIONS IN RAT INTERLOBAR ARTERY MEMBRANE
POTENTIAL AND K' CHANNELS IN GENETIC AND NONGENETIC
HYPERTENSION ....................................................................................................... 44

Introduction .................................................................................................................. 44
Materials and Methods................................................................................................. 46
R esu lts .......................................................................................................................... 4 9
D discussion .................................................................................................................... 52







4. PREVENTION OF RENOVASCULAR AND CARDIAC
PATHOPHYSIOLOGICAL CHANGES IN HYPERTENSION BY AT]
RECEPTOR ANTISENSE GENE THERAPY ........................................................... 66

In tro d u action .................................................................................................................. 6 6
M materials and M ethods................................................................................................. 67
R e su lts .......................................................................................................................... 7 1
D iscu ssio n .................................................................................................................... 74

CONCLUSIONS................................................................................................................ 89

REFERENCES .................................................................................................................. 90

BIOGRAPHICAL SKETCH ........................................................................................... 108













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

VOLTAGE-DEPENDENT K' CURRENT IN RAT RENAL RESISTANCE
ARTERIOLES: ALTERATIONS IN HYPERTENSION AND PREVENTION WITH
GENE THERAPY

By

Jeffrey R. Martens

May. 1998

Chairman: Craig H. Gelband, Ph.D.
Major Department: Pharmacology and Therapeutics

Essential hypertension is characterized by a near normal cardiac output but an

increase in total peripheral resistance. In turn, total peripheral resistance is controlled

directly by the diameter of the small arteries and arterioles like those in the kidney. The

dynamic regulation of renal vessel diameter is governed by the contractile state of the

vascular smooth muscle cells which line the artery walls. Voltage-dependent K' (Kv)

channels have been demonstrated to regulate smooth muscle membrane potential and

thereby control smooth muscle tone. However, little data are available on Kv channel

function in the renal vasculature and their potential role in essential hypertension. The

objectives of this study are (1) to biophysically. pharmacologically, and biochemically

identify and characterize Kv conductances in vascular smooth muscle cells from rat renal

resistance arterioles, (2) to detail potential changes in Kv current and membrane potential







in genetic and nongenetic models of hypertension, and (3) to determine the effects of

antihypertensive gene therapy on Kv current changes and renovascular alterations in

essential hypertension.

Electrophysiological experiments revealed the presence of two overlapping

components of Kv current. A transient component which activated at depolarized

potentials appeared superimposed on a non-inactivating delayed rectifier-like component.

The steady-state activation and inactivation, rate of recovery from inactivation, sensitivity

to 4-amionpyridine, and sensitivity to a-dendrotoxin and mast cell degranulating peptide

were similar for both components of Kv current. Western blot analysis demonstrated the

presence of Kvl.2 and Kvl.4 o-subunits in rat renal resistance vessels and co-

immunoprecipitation experiments confirmed the heteromultimeric association of these

proteins in vivo.

Comparison of K- currents and electrical properties between normotensive and

hypertensive rat models demonstrated that (1) smooth muscle cells from spontaneously

hypertensive rats (SHRs) and deoxycoritcosterone acetate (DOCA) treated rats were 20

mV more depolarized than control, and (2) the calcium activated K- current (Kca) was

increased and the Kv current was decreased in hypertensive models compared to

normotensive controls. The change in Kv current as well as the onset of other

renovascular and cardiac pathophysiological changes were prevented by treating pre-

hypertensive SHRs with angiotensin II type 1 receptor antisense.













CHAPTER 1
INTRODUCTION

The diagnostic measure of hypertension is an elevation in blood pressure which is

determined by cardiac output and total peripheral resistance (TPR). Essential

hypertension is characterized by a near normal cardiac output but an increase in total

peripheral resistance (Frohlich, 1973). TPR in turn is controlled directly by vessel

diameter particularly that of the small arteries and arterioles. The dynamic regulation of

vessel diameter is governed by the contractile state of the vascular smooth muscle cells

which line the vessel walls. Smooth muscle contraction, like that of striated muscle, is

controlled by increases in intracellular Ca2- concentration. Calcium binds to calmodulin

at four distinct "EF-hand" sites, inducing a conformational change in the binding protein

(Mani & Kay, 1996). The Ca2-calmodulin complex is then able to activate myosin light

chain kinase which transfers a phosphate from ATP to a serine at position 19 on the 20-

kD light chain of myosin (Somlyo & Somlyo, 1994). This allows the contractile

myofilaments actinn and myosin) to interact and the initiate crossbridge cycling. As long

as intracellular ATP is present and the intracellular free Ca2" concentration ([Ca2*],)

remains elevated over basal levels, contraction of the smooth muscle is maintained.

In vivo, vascular smooth muscle exists in a state of partial contraction. In this

state, termed myogenic tone, levels of [Ca2-], exceed that observed in the relaxed state

(Bolton, 1979: Marin. 1993). These levels of [Ca2>], are maintained by the balance







between calcium influx and extrusion (Khalil et al., 1989; Orallo, 1996; Karaki et a!.,

1997)). A stimulatory rise in [Ca2]i may lead to further contraction of the vascular

smooth muscle. This process is termed excitation-contraction coupling of which there are

two recognized mechanisms. The first, electromechanical coupling, operates through

changes in the vascular smooth muscle cell's membrane potential. The second,

pharmacomechanical coupling, operates through agonist stimulation of second messenger

signaling pathways to increase [Ca>], and does not necessarily involve prior membrane

depolarization. These mechanisms are not independent of each other (Somlyo & Somlyo,

1994); however, both require an increase in myoplasmic free Ca2'. The two pathways

leading to this increase in [Ca2"] are (1) release from intracellular stores, and (2) influx

from the extracellular space. The release of Ca2- from the sarcoplasmic reticulum is

associated with a transient contraction. Whereas, maintained contraction is dependent on

Ca2* influx across the sarcolemma. Although, it has been demonstrated that agonist

stimulated [Ca2>]- levels and contraction are altered in hypertension, it is the maintained

contraction that is important in the long term regulation of arterial pressure.

Two major routes exist for the stimulated entry of extracellular Ca2- in vascular

smooth muscle; voltage gated Ca2" channels and receptor operated channels (ROCs).

Other mechanisms of Ca2- entry have been identified (i.e. non-selective cation channels,

calcium release activated calcium channels, and stretch activated channels). However, the

nature of their role in the control of vascular tone is not clearly established and it has yet

to be determined if these mechanisms are truly separate and unique pathways of Ca2>

entry. For example, it has been proposed that Ca2> entry through ROCs may not be

distinct from that of voltage-dependent Ca2 channels or that the main function of ROC's







is to shift the membrane potential into the range where voltage-dependent Ca2 channels

have an increased conductance ( Bolton el al., 1988; Nelson et al., 1990b; Karaki et al.,

1997). Nevertheless, Ca>2 influx through voltage-dependent Ca2> channels has been

established as important in the control of vascular tone.

Since Ca2> channels in vascular smooth muscle are strongly voltage-dependent,

their function is closely linked to changes in membrane potential. Plasmalemma

membrane potential is determined by the permeability of several ions. While Na flux is

controlled mainly by pumps and exchangers (Na--Ca>2 exchanger or Na/K' ATPase), a

variety of ion channels permit the passage of Ca2'>. K- and CI across the membrane. This

review will focus on the nature of these channels in vascular smooth muscle and how

changes in their current may contribute to membrane potential alterations in hypertension.

Voltage-Dependent Ca2 Channels

To date, two subtypes of voltage-dependent Ca>2 channels have been identified in

vascular smooth muscle: L- and T-type (Orallo, 1996). L-type Ca>2 channels in vascular

smooth muscle are activated by relatively strong depolarizations, inactivate slowly and

incompletely, and are blocked by dihydropyridines. In contrast, T-type Ca>' channels are

activated at more negative potentials, exhibit rapid, voltage-dependent inactivation, and

are insensitive to dihydropyridines. L-type Ca2" channels appear to be the major Ca>2'

influx pathway in vascular smooth muscle. Entry through this channel is important in the

control of resting tone (Rubart et al., 1996). The influx through T-type Ca> channels is

small, transient, and likely to be inactivated at rest. Therefore, the remaining discussion

will focus on L-type Ca> channels.







In smooth muscle. L-type Ca2+ channels are composed of multiple subunits: a,,

ca/6, P (Hofmann & Klugbauer, 1996). The (x, subunit is the pore forming central

component, necessary for ion selectivity, voltage sensing, activation and inactivation

(Catterall, 1988). The x, also contains binding sites for the organic (dihydropyridines,

pheylalkylamines, and benzothiazepines) and inorganic (Cd2", Ni2) Ca2> channel blockers

(Bosse et al., 1992). The highly conserved cx/5 subunit is a dimer that is linked together

with a disulfide bond and may be involved in membrane anchoring. The P3 subunit

appears to be a modulatory subunit that shifts the voltage dependence of activation and

inactivation to more negative membrane potentials (Neely et al., 1993).

The dependence of the L-type Ca2- channels on membrane potential is important

in the control of vascular tone (Nelson et al., 1990b). The overlapping nature of the L-

type Ca2 channel activation and inactivation curves predicts a small sustained Ca2 influx

though this channel. This has been termed the Ca2- window current and its reported

values fall within the range of in vivo measured smooth muscle membrane potential.

Therefore, it may account for the steady-state levels of [Ca2], necessary for maintained

vascular tone ( Smimrnov & Aaronson, 1992b, Fleischmann et al., 1994). L-type Ca2-

channel open probability (P,) increases sharply with membrane depolarization, changing

approximately e-fold for 6-8 mV (Nelson et al., 1990b). This steep voltage dependence

allows the L-type Ca2" channels to respond to small graded changes in membrane

potential. Therefore, L-type Ca>2 channels provide a regulatory mechanism for the

interdependence of Ca2- influx, membrane potential, and vessel diameter (Khalil et al..

1987: Brayden & Wellman, 1989; Nelson et al., 1990b).







Smooth Muscle K4 Channels


While depolarization of vascular smooth muscle relies on the activation of Ca"

conductances, membrane repolarization is dependent on the opening ofplasmalemmal K'

channels. The opening of K- channels causes plasma membrane hyperpolarization and

inhibition of voltage-dependent Ca2 entry. Given the negative equilibrium potential for

K1, vascular smooth muscle K channels not only dampen membrane depolarization but

also play a prominent role in the maintenance of resting membrane potential and resulting

myogenic tone. Using the patch clamp technique, at least three types of calcium-

activated K' (Kca) channels, two types of voltage activated, ,calcium insensitive potassium

(K,) channels, and an ATP-dependent potassium (KAT-P) channel have been linked to

repolarization in vascular smooth muscle. In addition, an inwardly rectifying K' channel

(KIr) may play a role in regulation of membrane potential ( Tomita, 1988; Nelson &

Quayle, 1995; Brayden, 1996;).

Ca2-Activated K' Channels

Several types of Kc, channels that differ in unitary conductance (small,

intermediate, or large conductance) and pharmacology have been identified in various

non-vascular tissues. In vascular smooth muscle, the current most often described is a

large conductance (>200pS). Ca>2, time, and voltage-dependent K' current. It has been

identified in a number of vascular beds including: rabbit pulmonary artery (Okabe el al.,

1987), portal vein (Hume & Leblanc, 1989), ear artery (Benham & Bolton. 1986), canine

coronary (Wilde & Lee, 1989). renal artery (Gelband & Hume. 1992). rat renal resistance

vessels (Chapter 2, Figs. 2A.B,C), and cultured aortic cells (Toro & Stefani, 1987).







Membrane depolarization (- 2.7 fold increase per 12-14 mV depolarization) as well as

physiologically relevant increases in [Ca2"]i increase the Po of Kca channels (Nelson,

1997). Pharmacologically, external tetraethylammonium (TEA), charybdotoxin (ChTx),

iberiotoxin (IbTx). EGTA. and Ni2- inhibit channel activity. The molecular components

responsible for the large conductance Kca current have been described. The initial

cloning of this class of Ka channels was from the fruit fly Drosophila, and the gene has

been termed slowpoke (dslo) (Atkinson et al., 1991). Vogalis et al. (1996) recently

cloned and expressed cDNAs encoding the ax- and P-subunits of the dslo gene from

canine smooth muscle.

The physiological role of large conductance Kca channels in vascular smooth

muscle is well understood (Nelson & Quayle, 1995: Nelson, 1997). The membrane

depolarization and elevation of [Ca2']i associated with vasoconstriction will cause

activation of Kc, channels. This activation is under partial regulatory control by small,

concentrated bursts of Ca2- release (Ca2> sparks) from the sarcoplasmic reticulum

(Nelson et al., 1995). The resulting activation of these channels causes membrane

hyperpolarization and relaxation of arterial tone (Brayden & Nelson, 1992). Finally, Kca

channels are regulated by a number of endogenous vasoactive agents. Vasoconstrictors

such as angiotensin II, thromboxane A2, and muscarinic agonists have been linked to

inhibition of Kca channels (Toro et al., 1990; Kume & Kotlikoff, 1991; Scornik & Toro,

1992). Where as activators of protein kinase A (PKA) and G (PKG) appear to increase

channel activity (Brayden & Nelson, 1992). This further suggests a physiological role for

Kc, channels in the regulation of vascular tone.







Recently, a second type of Kca channel was found to coexist with the large

conductance Kca channel in vascular smooth muscle from rat renal arterioles

(Gebremedhin et al., 1996). This channel had a unitary conductance of 68 pS and was

selectively blocked by apamin (50 nmol/L). An apamin-sensitive K current has also

been identified in rabbit mesenteric arteries (Murphy & Brayden, 1995). However, the

role of this Kca channel needs to be elucidated.

Voltage-Dependent K4 Channels

Kv channels comprise the largest and most diverse class of ion channels. This

class can be subdivided into 3 major families based on their response to membrane

voltage, pharmacology, and molecular identity. These include (1) the delayed rectifier K

currents, (2) the A-type K currents, and (3) the inward rectifier K+ (Kir) currents, all of

which have been identified and characterized in vascular smooth muscle. As the name

implies, the Kir channels posses unique rectification properties which allow it to play a

separate function role in vascular smooth muscle. Therefore for the purpose of this

discussion, the Kir channels will be discussed separately.

Kv channels appear to be ubiquitously expressed. They have been identified and

the current characterized in a variety of vascular beds including; portal vein (Beech &

Bolton, 1989; Hume & Leblanc, 1989), coronary artery (Volk & Shibata. 1993). cerebral

artery (Bonnet et al., 1991), renal vasculature (Gelband & Hume, 1992; see Chapter 2),

pulmonary artery (Okabe et al., 1987), and the mesenteric vasculature (Smirnov &

Aaronson, 1992a). The outwardly rectifying current carried by delayed rectifier K+

channels turns on with a brief delay following membrane depolarization to potentials

positive to -40 mV. This current exhibits slow C-type inactivation which involves a







conformational change in the channel pore (Jan & Jan, 1992). In contrast, the A-type K*

current activates steeply with voltage between -90 and -60 mV and then decays

spontaneously and rapidly. Inactivation of the A-type, sometimes referred to as the

transient K-. current has been termed N-type because it involves a pore blocking particle

tethered to the N-terminus of the channel (Hoshi et al., 1990). Both currents are

insensitive to removal of extracellular Ca2' and are selectively inhibited by low

concentrations of 4-aminopyridine (4-AP).

The issue of multiple Kv channel subtypes within vascular smooth muscle is

complex and may not be easily classified into the above mentioned subfamilies.

Biophysical and pharmacological evidence suggests the existence of multiple components

of Kv current within a single tissue. Also the existence of different K- channel

distributions among various vascular beds or changes along an arterial tree have also been

shown (Albarwani et al., 1995; Evans et al., 1996). On a molecular level, K, channels are

traditionally represented in four gene families (Kv I -Kv4) based on homology to those

initially cloned from the Drosophila (Shaker, Shab. Shaw, and Shal genes). However,

new families of gene products are rapidly being identified. Within each family alternative

splicing and gene duplication give rise to many channel subtypes (Jan & Jan, 1992). In

addition, the possibility of heterotetramer formation in smooth muscle may also increase

diversity and further complicate the identification of such components (Russell et al.,

1994). To our knowledge only Kvl.2 (Hart et al., 1993), Kvl.5 (Overturf et al., 1994),

Kv2.2 and Kvl .1 (Horowitz. B., personal communication) have been cloned and

characterized in smooth muscle. However in vascular smooth muscle, the molecular







component for only Kvl.5 and Kv2.2 have been demonstrated using western blot

analysis.

Voltage-dependent K' channels have several physiological roles in vascular

smooth muscle. One important role is to dampen excitation by providing a mechanism of

membrane hyperpolarization. Nelson et al. (1 990b) calculated that due to the high input

resistance of vascular smooth muscle cells, only a small number of Kv channels need to

be open to limit membrane depolarization and facilitate vasorelaxation. A second

important role is the contribution of Kv channels to vascular smooth muscle resting

membrane potential. The steep relationship between steady state Po and membrane

voltage, as determined from the mean activation and inactivation curves, suggest the

presence of a significant sustainable Kv current at rest (Nelson & Quayle, 1995). In

addition, pharmacological blockade of Kv channels leads to membrane depolarization in

current clamped isolated vascular smooth muscle cells as well as constriction of intact

vessels (Yuan, 1995; Knot & Nelson, 1995; Gelband and Hume, 1995; Post et al., 1995;

Iyer et al., 1996). This provides additional evidence for the role of Kv channels in the

regulation of vascular smooth muscle membrane potential. Finally, it has been proposed

that Kv channels are regulated by a number of endogenous vasoactive agents.

Vasoconstrictors such as angiotensin II, histamine and hypoxia have been associated with

inhibition of Kv channels (Ishikawa et al., 1993; Gelband & Hume. 1995; Post et al.,

1995).

Inward Rectifier K' Channels

Kr current has been identified in several vascular beds including cerebral (Quayle

et al., 1993), coronary (Robertson et al., 1996), and mesenteric vasculature (Edwards &







Hirst, 1988a). Interestingly, these channels appear to be localized in small resistance size

vessels (i.e. < 200 lim). The channels display a time-independent rectification that is

opposite to the driving force established by the K- ion gradient. They open with steep

voltage dependence upon hyperpolarization, however pass a small sustained outward

current at potentials positive to the K" equilibrium potential (EK). The channel is also

very sensitive to changes in extracellular K' ([K']o) possessing the ability to shift its

voltage dependence of gating with changes in EK. Micromolar concentrations of

extracellular Ba2- cause a voltage dependent block of Kr. Inhibitors of other K' channels

(i.e. 4-AP. TEA, glibenclimide) appear to have no effect on Kr. However, millimollar

concentrations of Cs- also appear to block channel activity.

A Ki, channel is yet to be cloned from smooth muscle. However, it is expected to

share a similar structure to that of cloned Ki, channels from other tissues. K, is believed

to assemble as a tetramere with each subunit having two hydrophobic segments very

similar to the S5, S6 and H5 segments which comprise the pore region of the Shaker-like

K- channels.

Similar to the molecular identity, the physiological role of K,r in vascular smooth

muscle is not well defined. As mentioned above. K,, channels do conduct a small amount

of outward current at potentials positive to EK. Therefore, in the absence of depolarizing

factors they may contribute to the maintenance of resting membrane potential. Low

concentrations of Ba2. selective for K,, have been shown to depolarize vascular smooth

muscle from small cerebral and coronary arteries ( Hirst et al., 1986: Hirst & Edwards,

1989; Brayden, 1996). However, this is in the absence of transmural pressure which may







be significant (Brayden, 1990; McCarron & Halpern, 1990). The unique sensitivity of K,

to changes in extracellular K- may suggest another physiological role for this channel.

Several pathophysiological conditions such as hypoxia or ischemia result in the elevation

of extracellular KC ([K-].). Ki, is one mechanism of vasorelaxation by which vascular

smooth muscle cells may respond to this increase (Chen et al., 1972; Edwards et al.,

1988b; McCarron & Halpern, 1990). However, a second mechanism, activation of the

NaIK- pump, may underlie the vasodilatory response. Nevertheless, the physiological

role and molecular identity of KTr in vascular smooth muscle remains unclear.

ATP-Sensitive K' Channels

A potassium current that is voltage independent and inhibited by the noncovalent

binding of ATP has been identified in several vascular beds including the pulmonary

(Clapp & Gurney, 1992), coronary ( Xu & Lee, 1994; Dart & Standen. 1995), and

mesenteric vasculature ( Silberberg & van Breemen, 1992; Quayle et al., 1994). While

all of the currents from these tissues are blocked by glibenclimide, the nature of the single

channel conductance which underlies KATp is unclear. The literature describes two

populations of channels of varying unitary conductance (large; 130-260 pS : small; 10-30

pS) depending on the preparation, vascular bed, recording method, and Ca2- sensitivity

(Zhang & Bolton, 1996). KAT-p channels are composed of at least two subunits which are

believed to co-assemble as heterotetramers. Two subunits similar to the truncated

Shaker-like K, proteins combine with the sulfonylurea receptor to form functional KATrp

channels (Inagaki et al., 1995). These subunits have recently been cloned from smooth

muscle (Horowitz, B.; personal communication) and their heterologous expression will

certainly provide further insight into their unitary conductance and cellular regulation.







Although ATP inhibits channel activity, adenosine diphosphate (ADP) increases

KATP current, at least in the portal vein (Kajioka et al., 1991; Pfrunder et al., 1993;

Kamouchi & Kitamura, 1994). In addition adenosine has been shown to activate KAP and

cause vasodilation (Dart & Standen, 1995). Also, activation of KAT, via a cAMP/PKA

second messenger pathway contributes to the calcitonin gene-related (CGRP) mediated

relaxation of rabbit mesenteric arteries (Nelson et al., 1990a; Quayle et al., 1996).

Endothelial factors such as prostacyclin and nitric oxide (NO) also appear to activate KATP

channels (Murphy & Brayden, 1995). The NO dependent activation of KAp, like that of

CGRP appears to be transduced via a second messenger pathway, but in this case

involving cGMP (Miyoshi & Nakaya, 1994; Kubo et al., 1994). In contrast to the agents

that cause arterial relaxation, a number of endogenous vasoconstricting agents including

Ang II (Miyoshi & Nakaya, 1991), endothelin (Bymre & Large, 1987). and vasopressin

(Wakatsuki et al., 1992) have been shown to inhibit KATP. However, the mechanism of

this inhibition is yet to be elucidated.

The functional role of KATp in vascular smooth muscle is diverse. It has been

suggested that KAT-P plays an important role in reactive hyperemia, endotoxic shock, and

in the metabolic regulation of blood flow. Taking into account the large number of

endogenous vasoactive agents that target KATP. this channel must be important in the

regulation of vascular tone. However, this appears to be dependent on the vascular bed.

For example, glibenclimide constricts arteries from the coronary and mesenteric

vasculature but does not affect those from the renal, cerebral, or pulmonary vascular beds

(Nelson & Quayle, 1995). In addition, a number of K" channel opening drugs appear to







cause activation of KATP. These drugs including minoxidile, dioxide, pinacidil, and

cromakalim cause direct vasodilation and have been used as antihypertensive agents.

Smooth Muscle CI Channels

Two major types of chloride selective conductances have been identified in

vascular smooth muscle. One is a voltage-dependent chloride current that has been

reported in vascular smooth muscle cells (Soejima & Kokubun, 1988; Yamazaki et al.,

1998). The chloride conductance most often reported in vascular smooth muscle is that

of a Ca2' dependent Cl- (Ici(ca)) current with a small unitary conductance of- 2-3 pS

(Large & Wang, 1996). Patch clamp recordings first identified CI(ca) as one component

of current underlying the norepinephrine evoked depolarization of rat anaccoxycygeus

muscle cells (Bymrne & Large, 1987). Since then IcIca) has been identified in a number of

vascular tissues including; portal vein (Byrne & Large, 1988; Pacaud et al., 1989a;

Leblanc & Leung, 1995), ear artery (Amedee el al., 1990a), mesenteric artery (Klockner

& Isenberg, 1991). pulmonary artery (Leblanc & Leung, 1995), and rat renal resistance

arteries (Gordienko et al., 1994).

ICI(Ca) is strongly Ca2- dependent with this relationship varying between tissue and

species. One study in rat portal vein reports the half-maximal activation Of Il(ca) at 365

nmol/L (Pacaud et al., 1992). However, it appears that Iclca) is not as sensitive to [Ca2],

as Kca (Large & Wang, 1996). Also, it has been suggested that unlike Kca, activation of

ICi(Ca) may require both Ca2' and a second unknown substance. This claim is based on the

rapid rundown Of Icl(ca) in whole cell and excised patch recordings. This decrease in

channel activity was prevented using nystatin perforated patches (Amedee et al.. 1990b).

In addition Icl(ca) is weakly voltage dependent with an e-fold increase in Po for an 100







mV change in membrane potential (compare this to Ca" and Kv current) (Large & Wang,

1996).

In order to understand the functional role of Ic'(Ca) it is necessary to examine the

Cl- ion distribution in vascular smooth muscle cells. The presence of secondary active Cl

transport mechanisms (i.e. Cl/HCO3 exchanger and the Na'-K'-2Cl cotransporter)

within the plasmalemma of smooth muscle cells results in the accumulation of

intracellular chloride above that expected from passive distribution ( Casteels, 1971;

Jones, 1980; Koncz & Daugirdas, 1994). Therefore, the Cl equilibrium potential is -

20 mV. Since the resting membrane potential of vascular smooth muscle cells is more

negative than this (- -50 mV). activation of Cl channels will lead to a net Cl- efflux

resulting in membrane depolarization. Iclca) is activated in vascular smooth muscle by a

number of vasoactive agents including; norepinephrine (Byrne & Large, 1987; Byrne &

Large, 1988). ATP (Pacaud & Loirand, 1995; Wang et al.. 1997), endothelin (Klockner &

Isenberg, 1991; Gordienko et al., 1994), histamine (Wang & Large, 1993), and

vasopressin (Van Renterghem & Lazdunski, 1993). This activation appears to be

mediated by the stimulatory release of Ca2" from intracellular stores (Large & Wang,

1996). The resulting membrane depolarization may recruit more voltage dependent

calcium channels to open producing contraction. It has also been proposed that Ic(ca) may

be activated by Ca2' sparks from the SR. This current termed spontaneous transient

inward currents (STICS) has been recorded in several vascular tissues ( Wang et al..

1992; Hogg et al., 1993). Finally, Ic(c,) may play a role in the regulation of resting

membrane potential. It is hypothesized that membrane permeability to Cl- may explain

why the resting membrane potential of vascular smooth muscle is positive to EK.







Recently, Nelson et al. (1997) has suggested that a niflumic acid (niflumic acid is a

selective inhibitor of 'CI(Ca)) insensitive Cl current may contribute to myogenic tone in rat

cerebral arteries. However much work is needed to identify specific Cl channels

contributing to vascular tone. Nonetheless, the most probable role for Icl(ca,)is its

contribution to excitatory agonist induced vasoconstriction (Large & Wang, 1996; Yuan,

1997).

Ion Channel Alterations in Hypertension

As mentioned previously, essential hypertension involves a gradual and sustained

increase in total peripheral resistance. These changes in vessel diameter must involve

alterations in the contractile state of vascular smooth muscle. In both human and animal

models of essential hypertension it has been demonstrated that the resting vascular tone is

elevate and the contractile response to a number of physiological stimuli is enhance

compared to normotensive controls. Both of these changes will contribute to the

increased blood flow resistance. A number of cellular mechanisms in vascular smooth

muscle may contribute to these changes in hypertension (Fig. 1 -I1). The focus of this

discussion will be alterations in plasma membrane ion channels.

To date, the majority of fluorescence and electrophysiological studies of vascular

changes in essential hypertension have been performed on large conduit vessels.

Conflicting results regarding changes in contractility (Bohr, 1974), resting membrane

potential (Hermsmeyer. 1976; Steikel, 1989; Lamb & Webb, 1989; Rusch et al., 1992;

See Chapter 3), basal [Ca2,], (Sugiyama et al.. 1986; England et al., 1993; See Chapter 3),

and the onset of ion channel alterations (Ohya et al., 1996) in conduit vessels from

models of essential hypertension may be due to differences in the vascular bed studied.







Mulvaney et al. (1990) has suggested that the increased contractility observed in vascular

beds from hypertensive models must reside in the resistance vessels. Moreover, the large

conduit vessels play a less significant role in setting peripheral vascular resistance than do

smaller resistance vessels (Mulvany & Aalkjaer, 1990b). Studies of clinically relevant

resistance vessels (i.e. renal or cerebral vasculature) may provide further insight into the

role of ion channel alterations in the development and maintenance of essential

hypertension.

In the literature, a multitude of evidence suggests that altered ionic permeabilities

exist in vascular smooth muscle in hypertension (Jones, 1973; Frohlich, 1973). Using the

patch clamp technique and vascular smooth muscle cells dissociated from conduit arteries

it has been demonstrated that there is an increased L-type Ca2" current density in

vascular smooth muscle cells from the spontaneously hypertensive rat (SHR), a model of

human essential hypertension (Rusch & Hermsmeyer, 1988; Ohya et al.. 1993; Wilde et

al., 1994; Cox & Lozinskaya, 1995; Lozinskaya & Cox, 1997). In addition, it has also

been shown that the Kca current density in aorta from hypertensive rats is larger than

control (Rusch et al.. 1992). This alteration was observed in single-channel recordings of

the KYa current from excised aortic membrane patches in which a greater Ca2" sensitivity

of the SHR Kc was demonstrated (England et al., 1993). This increase in Ka may also be

due to an upregulation of channel expression resulting from a change in some post-

transcriptional event (Liu et al., 1997). Finally, Ohya et al. (1996) have reported an

impaired action of levocromakalim on KArp channels in smooth muscle cells from the

SHR. This data suggests that alterations in KAT function or density may exist in

hypertension.







Reversal of Ion Channel and Pathophysiological Alterations in Hypertension

Hypertension produces pathophysiological changes which are often responsible

for the mortality associated with the disease. However, it is unclear if normalizing blood

pressure with conventional therapy is effective in reversing the pathophysiological

damage. The duration and initiation of treatment, site of administration, and agent used

all appear to influence the reversal of the pathophysiological alterations associated with

hypertension. A major question regarding ion channel alterations in vascular smooth

muscle cells and the development of essential hypertension is one of cause vs. effect.

That is do the observed changes in ionic current underlie the development of

hypertension or do they simply follow changes in blood pressure. The relationship

between blood pressure changes and ion channel alterations has been addressed in a few

studies. Rusch and Runnells (1994) showed that normalization of high blood pressure in

SHR using ramipril, an angiotensin-converting enzyme inhibitor (ACE), reversed the

increase in Ka observed in the aorta. In addition, Ohya et al. (1996) reported that the

impaired action of levcromacalim on KATp channels in SHR was corrected with

pharmacological antihypertensive treatment including; hydralazine, Ca2- channel

blockers, and ACE inhibitors.

Rationale

Hypertension is a leading cause of death and disability among adults in the United

States. Despite years of intensive investigation, in most cases the etiology of

hypertension remains unknown. Essential hypertension is characterized by a near normal

cardiac output but an increase in total peripheral resistance (Frohlich, 1973). The

elevation in total peripheral resistance associated with hypertension may result from







alterations in vasomodulatory substances, passive or genetic modifications in blood

vessel structure, or intrinsic changes in the control of vascular tone (Khalil et al., 1989).

The dynamic regulation of vascular tone is governed by the contractile state of the

smooth muscle cells which line blood vessel walls. In turn, the excitation and contraction

of vascular smooth muscle depends on cellular membrane potential which is known to be

altered in the blood vessels of hypertensive animal models (Hermsmeyer, 1976). Kv

channels have been shown to regulate smooth muscle membrane potential and thereby

contribute to the control of vascular smooth muscle tone (Brayden et al., 1991; Nelson &

Quayle, 1995).

The results of earlier renal transplant studies in humans and animals suggest that

in a proportion of cases with essential hypertension there exists a defect in the structure

and/or function of the kidney (Dahl et al., 1972; Bianchi et al., 1979; Curtis et al., 1983).

The small arteries (< 200 ptm) of the kidney which provide the greatest resistance to

blood flow are an important vessel to study. Alterations in renal blood flow are known to

influence fluid volume regulation, secretion of a number of important neurohumoral

substances, and arteriolar resistance all of which may be involved in the maintenance of

hypertension. Given this, little data are available on Kv channel function in the renal

vasculature and its potential role in essential hypertension. This project has therefore

three specific aims.

Specific aim #1 In the first part of this project, the biophysical, pharmacological, and

biochemical characterization of Kv current in rat renal resistance arteries was carried out.







The results of this study titled "Voltage-dependent K+ current in rat renal resistance

arteries: Evidence for heteromultimeric formation" are presented in Chapter 2.

Specific Aim #2 In the second part of this project, we detailed changes in membrane

potential and K" current in vascular smooth muscle cells isolated from rat renal resistance

arteries of hypertensive animal models. Specifically, we studied Kv channel alterations in

genetic and nongenetic models of hypertension. The results of this study titled

"Alterations in rat renal resistance artery membrane potential and K+ channels in

genetic and nongenetic hypertension" are presented in Chapter 3.

Specific Aim #3 In the third part of this project, the effects of antihypertensive gene

therapy on renovascular alterations and Kv current changes in a genetic model of

essential hypertension are investigated. This study shows that interruption of the renin-

angiotensin system in pre-hypertensive animals attenuates the elevation in blood pressure

and prevents the development of Kv current alterations. The results of this study titled

"Prevention of renovascular and cardiac pathophysiological changes in hypertension by

angiotensin II type 1 receptor antisense gene therapy" are presented in Chapter 4.










2a (j,


2b


Figure 1-1. Cellular mechanisms in vascular smooth muscle cells which may
contribute to contractile alterations in essential hypertension: 1) increased
sensitivity to circulating hormones such as angiotensin II. 2) increased levels
of myoplasmic free Ca2+; this may result from 2a) increased Ca2 current,
2b) decreased K4 current, 2c) altered activity of pumps and exchangers in
the SL or SR. 3) increased Ca2+ loading of the SR, or 4) increased Ca2+
sensitivity of the contractile myofilaments.













CHAPTER 2
VOLTAGE-DEPENDENT K+ CURRENT IN VASCULAR SMOOTH MUSCLE
CELLS FROM RAT RENAL RESISTANCE ARTERIOLES: EVIDENCE FOR
HETEROTETRAMER FORMATION

Introduction

The presence of a voltage-dependent K- (Kv) current that is sensitive to 4-

aminopyridine (4-AP) appears to be ubiquitous in vascular smooth muscle cells.

However, the nature of this current has been shown to vary depending on the vascular bed

or segmental location along an arterial tree (Nelson & Quayle, 1995; Brayden, 1996;

Archer et al.. 1996; Patel et al., 1997). In addition, biophysical and pharmacological

evidence suggests the existence of multiple components of Kv current within single

smooth muscle cells (Carl. 1995). Both transient and delayed rectifier Kv currents have

been identified (Beech & Bolton, 1989; Lang, 1989; Clapp & Gurney, 1991; Gelband &

Hume, 1992; Smirnov & Aaronson, 1992a; Gordienko et al., 1994). While many studies

have focused on the macroscopic current properties, very little work has been aimed at

identifying Kv channel subtypes which may underlie these native currents.

Kv channels comprise the largest and most diverse class of ion channels. A large

number of Kv channel genes and their splice variants have been reported (Chandy &

Gutman, 1993; Roberds et al., 1993). This diversity complicates the correlation of Kv

channel cDNA's with endogenous currents within a vascular tissue. The process of

assigning molecular counterparts to Kv current components in vivo is further obscured by







the potential for heteromeric formation or modulation by accessory subunits. K* channel

stoichiometry was determined to be tetrameric with four a subunits combining to form a

functional channel (MacKinnon, 1991). Recent electrophysiological evidence shows that

both identical (homomeric) and non-identical (heteromeric) Kv ac subunits can co-

assemble ( Timpe et al., 1988; Christie et a!., 1990; Isacoff et al., 1990; Ruppersberg et

al., 1990). The biophysical and pharmacological properties of Kv heterotetramers are

often unique compared to homotetrameric channels (McCormack et al., 1990). In

addition, P3 subunits are now known to couple with Kv channels altering both membrane

expression and channel kinetic properties ( Rettig et al., 1994; England et al., 1995a;

England et al., 1995b; Majumder et al., 1995; McCormack et aL., 1995; Heinemann et al.,

1996). To date, direct in vivo evidence for heteromeric association between Kv at

subunits or with accessory P3 subunits has not been demonstrated in vascular smooth

muscle. In this study we have characterized the Kv current in rat renal resistance vessels

and provide the first in vivo evidence of heteromeric formation in vascular smooth

muscle.

Materials and Methods

Smooth Muscle Cell Isolation

Male rats were killed by decapitation and the right and left kidneys were removed

and placed in cold, oxygenated (95% 02-5% CO2) physiological saline solution (PSS).

Renal resistance vessels were dissected free from the kidney, cut into small pieces (- 4

mm), and incubated at room temperature in Ca2-free PSS for 30 minutes. The pieces

were subsequently resuspended in Ca2-free PSS digestion buffer for 20-30 minutes at







37C. The Ca2-free PSS digestion buffer contained (mg/15 ml) collagenase (151

units/mg Worthington Biochemical Corp., Freehold, NJ.) 4.5, bovine serum albumin 30,

trypsin inhibitor 30, ATP (sodium salt) 1.7, and protease (type XXIV, Sigma Chemical

Co., St. Louis, Mo.) 1.5. After the digestion, the pieces were gently triturated until a

large number of elongated smooth muscle cells were observed. The isolated cells were

collected and stored at 4C until used. Cells were used between 2 and 10 hours after

isolation.

Current Recording and Analysis

Single cells were voltage-clamped, and membrane currents were measured using

the whole-cell patch-clamp technique (Hamill el al., 1981). Voltage-clamp command

potentials were applied to the cells and membrane current recorded using an Axopatch-lD

patch-clamp amplifier (Axon Instruments, Burlingame, CA ). Membrane current was

digitized on-line (10.0 kHz) with in analog-to-digital interface (Labmaster TL-1 DMA

interface, Axon Instruments) and filtered at 2.0 kHz. Data analysis was performed with

pCLAMP 6.0 software (Axon Instruments). All experiments were performed at room

temperature. Gordienko et al. (1994) has demonstrated the presence of both a calcium

activated K' (Kc,) current and a Kv current in rat renal resistance arterioles. The Ki,,

current may be blocked using either tetraethylammonium (TEA) shown in Figures 2-1

and 2-2 or charybdotoxin (ChTx) as shown in Figure 2-3. For this study, isolation of the

Kv current was achieved using 10 mmol/L TEA in the bath solution and replacing Ca2

with equal concentrations of Mg>2 (see solutions).







Immunoprecipitation of Kvl1.2 Channels and Associated ca Subunits

Renal arterioles were hand homogenized on ice in homogenization buffer

containing (in mmol/L): 250 sucrose, 50 MOPS, 0.1 PMSF, 2 EDTA, and 2 EGTA, pH

7.4. To guard against proteolysis, additional protease inhibitors including leupeptin,

antipain, and aprotinin were added at a concentration of 5 Pg/ml. Cells were centrifuged

at 1000lxg to pellet cell debris and then at 14,000xg to pellet nuclear extract.

Supernatants from the 14,000xg spin were used for protein analysis quantitation using a

Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Membrane protein (500 kg) was detergent solubilized for 30 minutes with Triton

X-100 extraction buffer (1% w/v Triton X-100, 150 mmol/L NaCl. 50 mmol/L Tris (pH

7.5), 1 mmol/L ethylenediaminetetraacetic acid (EDTA). 0.2% bovine serum albumin

(BSA), 1 ug/ml leupeptin. 2 ug/ml aprotinin, 5 mmol/L phenylmethylsulfonyl fluoride

(PMSF), 2 mmol/L benzamidine, 5 mmol/L N-ethylmaleimide, and 1 mg/ml bacitracin)

and incubated on ice for 10 min. Insoluble cellular debris was removed by centrifugation

at 15,000xg for 15 min. Cellular extracts were kept at 4C throughout the solubilization

process. Eight ug of a monoclonal antibody against amino acids 428-499 of rat Kvl .2

(Upstate Biotechnology, Lake Placid, New York) was added to the solubilized extract and

incubated at room temperature for 2 hr on a rocking platform. Packed Protein A

Sepharose beads (3 pl). preblocked with 0.2% BSA. were added and the incubation

continued for an additional 2 hr at room temperature with rocking. Beads were then

sedimented and washed. The bound proteins were eluted from the beads by boiling for 2

min in SDS sample buffer.










In Vitro Translation of Kvl.2 and Kvl.4

Rat Kv 1.2 and Kv 1.4 were synthesized in an in vitro translation reaction using

previously described clones (Roberds & Tamkun. 1991) and the TUT-coupled

reticulocyte lysate system (Promega, Madison, WI) using the manufacturers protocol.

Western Analysis of Immunopurified Kvl.2 Channel Proteins

The purified protein and in vitro translated proteins were fractionated by SDS-

polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 3%

skim milk powder, 0.5% (w/v) Triton X-100. 0.1% (w/v) Tween 20 dissolved in 20 mM

Tris/HC1, pH 7.4, 150 mM NaCl (TBS) for 1 hr at 22C. Following block of the

membranes, blots were incubated with monoclonal antibodies generated against amino

acids 428-499 of Kv1.2 (1:10.000 dilution) and 13-37 of Kvl.4 (1:1.000 dilution). All

antisera was diluted in 3% skim milk powder, 0.5% Triton X-100, 0.1% Tween 20 in

TBS overnight at 4C. Blots were washed twice with H20 and incubated with

peroxidase-conjugated goat anti-mouse IgG at a 1:2000 dilution for 90 min at 22C.

Blots were then washed two times in H,0, once in TBS + 0.1% Tween, and five

additional times in HO20 and proteins identified using ECL detection methods (Amersham

Life Science Inc., Arlington Heights, IL).

Solutions

The PSS used during cell isolation procedures contained in mmol/L: NaCl 120.

KC1 4.2, KHPO4 1.2, MgCl 0.5, CaCI2 1.8, glucose 5.5, and HEPES 30 (pH 7.35 with

NaOH). For all whole-cell voltage-clamp experiments examining Kv currents, the bath

solution contained in mmol/L: NaCI 125, KCI 4.2. KH2PO4 1.2. MgCI2,2.3, D-glucose







5.5, TEA 10.0, and HEPES 30 (pH 7.4 with NaOH). The pipette solution for the whole-

cell experiments contained in mmol/L: KC1 140, MgC12 0.5, EGTA 1.0, ATP

(magnesium salt) 5.0, and HEPES 5.0 (pH 7.2 with KOH). Dendrotoxin and mast cell

degranulating peptide were obtained from Alomone Labs Inc. (Jerusalem, Israel). All

other chemicals were obtained from Sigma Chemical Co.

Statistics

Results are expressed as mean SEM. Statistical significance was evaluated

using repeated measures ANOVA and Student's T-test for unpaired data. Differences

were considered significant at P<0.05. Membrane currents were measured from the zero

current level and normalized to cell capacitance.

Results

Electrophysiological Characterization of Kv Current in Rat Renal Resistance
Arterioles

Whole cell patch clamp experiments on isolated renal arteriole vascular smooth

muscle cells revealed two components of Kv current. Kv current was measured in the

presence of TEA (10 mmol/L) and zero external calcium. Figure 2-4A shows a

representative example of current recordings following membrane depolarization from a

holding potential of-80 mV. Within the range of-40 to 0 mV the evoked response is that

of a slowly activating, non-inactivating, delayed rectifier-like K' current which we refer

to as the sustained component of Kv current. Further depolarization elicited a second

transient component of Kv current which activated and inactivated rapidly. This current

waveform was present in every cell examined (n=65). Similar currents were recorded

when ChTX (100 nmol/L) replaced the TEA or when it was applied in the presence of







TEA (data not shown). The mean current-voltage (I-V) relationship for both the transient

and sustained components of Kv current are plotted in Figure 2-4B (n=l 1). The

amplitude of the peak current component was measured immediately following the decay

of the capacitive transient whereas the amplitude of the sustained current component was

recorded at the end of the voltage pulse. Interestingly, compared to the sustained current

component, the amplitude of the transient component of Kv current increased much more

steeply with increasing depolarization. The nonlinearity of both I-V curves is indicative

of outward rectification. This was further supported by the smaller than expected ratio of

tail current amplitudes at -40 mV (observed ratio. 0.15; calculated ratio, 0.25). To verify

that K' was the major charge carrier of the outward current, external [K] was varied and

tail currents examined using a double-pulse protocol. The average tail current reversal

potential closely followed the predicted Ek values (data not shown).

The voltage dependence of activation and inactivation were measured and the

mean data is displayed in Figure 2-4C and D, respectively. Steady-state activation was

determined from the ratios of the tail currents recorded at -40 mV. The data were best fit

by a single Boltzmann function and the membrane potential at which one-half (V1/2)

activation occurs was calculated to be -26.5 1.8 mV with a slope factor (k) of 8.9 1.5

mV (n=9). The voltage dependence of steady state inactivation was determined using a

standard double pulse voltage clamp protocol. The conditioning pulse (potential reported

on the abscissa of Figure 2-4D) was followed by a 2 second test pulse to +80 mV to

assess open channel availability. The values for the transient and sustained current

components were measured at the start and end of the test pulse and both were fit with a







single Boltzmann function. Smimrnov and Aaronson (1992a) have suggested that the

transient and sustained components of the TEA-insensitive Kv current in human

messenteric arteries could be separated based on inactivation properties. However, in this

case there was no difference between the midpoints or slopes of the two inactivation

curves (transient component: V, = -38.8 + 0.96, k=-8.9 0.9 mV; sustained component:

V11/2 = -42.7 3.1, k= -7.8 2.7 mV, n=l 1).

Additional experiments were performed to separate the two components of Kv

current. The rate of recovery from inactivation was examined for both the transient and

sustained components of Kv current and the results are shown in Figure 2-5. A double

pulse protocol was used and a representative response is displayed in Figure 2-5A. A 10

sec depolarizing voltage step to + 80 mV was used to induce inactivation. This was

followed by a test pulse to +80 mV for 2 sec. The time course of recovery was assessed

by varying the interpulse interval and plotting this against the normalized current. The

results for the transient and sustained current components are shown in Figure 2-5B and

C, respectively. The mean values for both components were best fit using bi-exponential

functions each with a fast (milliseconds) and slow (several seconds) component of

recovery. This is consistent with reports of recovery from N-type (fast) and C-type (slow)

inactivation (Zagotta & Aldrich, 1990; Baukrowitz & Yellen, 1995). The time constants

for removal of inactivation determined from these fits were not significantly different

between the transient (T,= 19.0 7.9 msec, T2 = 2.47 1.4 sec. n=5) and sustained current

components (t,= 14.3 6.9 msec, T2 = 2.45 1.7 sec, n=5). This suggests that the two







components of Kv current could not be separated into two populations of channels based

on their inactivation properties.

Connor and Stevens (1971) first describe a transient K' current, termed the A-

current, which activated and inactivated in a voltage range that was negative to that of

delayed rectifier K- currents. This current has been described in vascular smooth muscle

and successfully isolated from delayed rectifier K' currents by altering the holding

potential (Beech & Bolton. 1989; Clapp & Gurney, 1991). We also tested the effect of

holding potential on Kv current in vascular smooth muscle cells isolated from rat renal

resistance vessels. Representative examples showing a family of currents elicited during

step depolarizations from a holding potential of-80, -60, and -40 mV are shown in Figure

2-5D, E, and F, respectively. Altering the holding potential did not provide separation of

the transient and sustained components of Kv current. Additional experiments employing

a number of prepulse voltage protocols designed to isolate the transient or sustained

current components also failed to separate the two current components (data not shown).

Pharmacological Characterization of Kv Current

4-AP is a well known inhibitor of voltage dependent K' channels. Differential

sensitivity to 4-AP has been used to identify dissimilar Kv current components (Rudy,

1988). In our experiments, the bath application of 4-AP blocked Kv current in a dose-

dependent manner. Figure 2-6 shows the dose response curve for the peak and sustained

components of Kv current. Data was collected before and during drug application using

successive 1 sec step depolarizations from a holding potential of-80 mV to + 80 mV with

a 20 sec interval between pulses. The inset shows a representative example of the current

recorded in the presence of 0, 1, and 30 mmol/L 4-AP. Data for both current components







were best fit with a single exponential function. The degree of inhibition at the maximal

drug concentration was greater for the transient component (69% of control) compared to

the sustained component (48% of control). However, the IC50 values for the two curves

generated from the mean data were not significantly different (transient, -300 ptmol/L,

sustained, -200 tmol/L, n=5). This suggests that the two components of Kv current

cannot be separated based on sensitivity to 4-AP.

We next examined the blocking effects of the K* channel toxins a-dendrotoxin

(aDTX) and mast cell degranulating peptide (MCDP). cxDTX, a peptide isolated from

green mamba snake venom, is known to block three Shaker Kv channels (Kvl. 1, Kvl .2,

Kvl.6, Kd< 20 nmol/L) (Harvey, 1997). Figure 2-7A shows a representative example of

the effects of 50 nmol/L caDTX on Kv current at a membrane potential of+80 mV. The

toxin inhibited the current without significantly changing the shape of the current trace.

This is reflected in Figure 2-7B which shows that caDTX (50 nmol/L) had similar effects

on the transient and sustained current components. The average degree of inhibition was

30.8 6.9% and 30.4 5.8%, respectively (n=4). Increasing the concentration of xDTX

to 100 nmol/L had no additional effect on the Kv current. MCDP inhibits delayed

rectifier type Kv channels (Kvl .1, Kvl .2, Kvl .6) (Grissmer et al., 1994). Figure 2-7C

shows an example of the inhibitory effects of 1 pmol/L MCDP on Kv current elicited

during a voltage step to +80 mV. The mean data is summarized in panel 2-7D. MCDP (1

p.mol/L) significantly reduced the Kv current for both the transient and sustained

components of Kv current but there was no significant difference in the percent inhibition

between the two (transient; 49.1 5.2%, sustained: 27.6 15.1%,n=4).







Characterization of Kv Channels in Rat Renal Resistance Vessels

Studies have demonstrated the presence of mRNA for multiple Kv channels in rat

vascular smooth muscle (Roberds & Tamkun, 1991). However, the presence of mRNA

provides information on the gene expression of these K' channel subunits and does not

confirm the presence of channel protein (Hales & Tyndale, 1994). Pharmacological

evidence showed that both the transient and sustained components of Kv current were

TEA-insensitive and were blocked, at least partially, by 4-AP, cDTX, and MCDP. The

inability to separate a distinct transient current component from that of the sustained

current suggested that heteromultimeric association may be occurring. This profile

limited the number Kv channel proteins which may underlie the current and provided the

framework for our initial protein screening strategy. Monoclonal Antibodies specific for

the Kvl.2 channel (anti-Kvl.2) were used to immunoprecipitate Kvl.2 and associated

proteins in rat renal arterioles. Anti-Kvl.2 is shown to be specific for Kvl.2 (Fig. 2-8 left

panel) since it immunoblots Kvl .2 in vitro translated core protein (-55 kDa) as well as

processed forms of Kvl .2 translated in the presence of canine microsomal membranes

(-80 kDa), but not a negative control or Kv1.4 in vitro translated protein.

Immunoprecipitation of Kvl.2 followed by immunoblotting with anti-Kvl.2 did

recognize a processed form of Kvl .2 in rat renal arterioles of-80 kDa. To determine

whether Kvl .2 heteromultimerizes with other shaker-type channels,

immunoprecipitations with Kvl .2 were then immunoblotted with antibodies specific for

Kvl .4 (anti-Kvl .4, right panel). Western blotting with a monoclonal antibody against

Kvl.4 recognized a Kvl.4 band of-96 kDa suggestive that Kvl.2 and Kvl.4







heteromultimerize in rat renal arterioles. Anti-Kvl .2 and anti-Kvl .4 were specific and

did not recognize a band in sham in vitro translated core proteins (control lanes).

Discussion

This study characterizes the biophysical and pharmacological properties of the Kv

current in vascular smooth muscle cells isolated from rat renal resistance arterioles. We

have identified two Kv channel proteins, Kvl .4 and Kvl .2, which may underlie the

observed Kv current and provide the first direct in vivo evidence for heteromeric

association of Kv channel cc-subunits in vascular smooth muscle.

First inspection of the K' current, elicited by step depolarizations, suggested the

presence of two overlapping voltage-dependent currents. A transient current component

which activated at depolarized potentials appeared superimposed on a non-inactivating

delayed rectifier-like current component (Fig. 2-4A). This was similar to that reported by

Smirnov and Aaronson (1992a) in human mesenteric arteries and by Gordienko et al.

(1994) in rat renal resistance arterioles. Both of these reports described a transient current

which may be classified as a "high-threshold subclass" of A-current (Rudy, 1988) and

which overlapped a slow activating, sustained component. However, unlike these reports

we were unable to separate the transient and sustained components based on kinetic or

pharmacological differences. Steady-state inactivation (Fig. 2-4D), rate of recovery from

inactivation (Fig. 2-5A-C), and sensitivity to 4-AP (Fig.2-6) were similar for both

components. In addition unlike other A-type currents described in smooth muscle, we

were unable to isolate the transient current by varying the holding potential (Beech &

Bolton, 1989; Clapp & Gurney, 1991).







When expressed in heterologous systems, Kvl .4 homomeric channels are known

to produce a TEA-insensitive transient current which recovers slowly from inactivation

and is sensitive to 4-AP ( Stuhmer et al., 1989; Ruppersberg et al., 1990). While this

current is similar to the transient component which we observed in native vascular

smooth muscle cells, differences in the voltage range of activation and recovery from

inactivation suggest that this channel alone may not account for the transient component

of Kv current. Furthermore, aDTX and MCDP which are toxins specific for the delayed

rectifier-like currents Kvl.1, Kvl .2, and Kvl .6 blocked both the transient and sustained

components of Kv current equally and incompletely. Of these three gene products, only

Kvl .2 encodes for a channel which produces a TEA-insensitive current suggesting that

this protein may also contribute to the subunit composition of Kv channel complexes in

rat renal arterioles. Western Blot analysis of immunopurified protein demonstrated the

presence Kvl .4 and Kvl .2 a-subunit immunoreactive-like protein in this tissue (Fig. 2-

8).

Our electrophysiological data suggested that Kvl .2 and Kvl .4 may associate in

rat renal resistance vessels to form a distinct current with properties that are different

from that displayed by either Kvl.2 or Kvl.4 homomers. The endogenous association of

these two channel proteins was confirmed by co-immunoprecipitation from rat renal

arteriole homogenate (Fig. 2-8). This biochemical strategy has been used in vivo to

demonstrate that Kv channels (including Kvl .2 and Kvl .4) assemble as heteromultimers

in rat brain (Sheng et al.. 1993; Wang et aL.. 1993: Shamotienko et al., 1997). In

addition, the heteromultimeric assembly ofKvl .4 and Kvl .2 has been study in vitro







using Xenopus oocytes (Po et al., 1993). Comparison of the properties between the

hybrid Kvl.4/Kvl.2 complex reported by Po et al. (1993) to those of the native Kv

current in rat renal resistance arteries reported herein reveals a strong similarity. Both

currents are TEA-insensitive, highly sensitive to 4-AP, exhibit fast inactivation, rapid

recovery from inactivation, and display the same relationship between steady-state

inactivation and membrane potential. This parallel provides further evidence that Kvl .4

and Kv1.2 act-subunits determine the subunit composition of at least one Kv channel

complex in rat renal arterioles. Recent evidence demonstrates that the presence of a

single inactivating Kvl.4 subunit confers N-type inactivation on heteromultimeric

channels (Lee et al., 1996). In addition, reports demonstrate that the biophysical

properties of heteromultimeric channels are quantitatively more similar to the most

abundant subunit (Hopkins et al.. 1994). However, it is important to note that the

difficulty in separating the transient and sustained components of Kv current does not

exclude the possibility of additional Kv channel complexes or the participation of kinetic

altering p3-subunits. Indeed, there is evidence for the presence of Kv channel p3-subunits

in vascular smooth muscle (mRNA, Morales et al., 1995; protein, S.K. England

unpublished observations in renal arterioles). The equal but incomplete blockade of both

the transient and sustained components by xDTX and MCDP may signal the presence of

additional components which we were unable to isolate with the methods presented here.

Alternatively, recent evidence suggests that high affinity binding of aDTX to Kv

channels requires that all four individual subunits of a heterotetramer posses an cXDTX

binding site (Tytgat et al., 1995). This would not be satisfied by a heterotetramer







containing Kvl.4 o-subunits. Ultimately, the contribution of Kv channel subunits and

multimeric complexes to native current requires specific approaches such as the use of

antisense strategies or gene knockout experiments.

The physiological role of a transient current in vascular smooth muscle is an

unanswered question and requires further study. The outward rectification and high

voltage threshold for activation suggests a role for the transient current in limiting

membrane depolarization. In small resistance arteries which respond to vasoconstricting

stimuli with graded depolarizations, the transient current would activate rapidly and carry

a large hyperpolarizing current. This may be important during times ofischemic insult or

in disease states such as hypertension which are characterized by increased

vasoconstriction. Recently, Roeper et al. (1997) have shown that phosphorylation of

Kvl.4 by Ca2/calmodulin dependent protein kinase leads to a slowing of inactivation

gating and accelerated recovery from N-type inactivation. In smooth muscle this would

result in an increased availability of hyperpolarizing current during times of elevated

intracellular Ca'- and may represent a modulatory mechanism for the transient current

during excitation-contraction coupling in health and disease states.

In summary, we have provided electrophysiological, pharmacological, and

biochemical evidence for the association ofKvl.2 and Kvl.4 a-subunits in rat renal

resistance arteries. This is the first direct in vivo evidence for the existence of

heteromultimeric Kv channels in vascular smooth muscle. This work suggests that

similar to neuronal and cardiac organ systems, vascular smooth muscle also use hetero-

oligomeric assembly to generate K- channel diversity.









A


500 VIMV




1.





-80 -60 -40


control

50 M

100 iM

500 'M
1 mM
400 pA
5mM


-20 0 20 40 60


Membrane Potential (mV)


1.0 -

0.9 -

0.8 -

0.7 -

0.6-

0.5 -
0.4 -


0.3-
0.2
0.1
0.0-- ---
-5 -4 [TEA] -3 -2
[TEA]


Figure 2-1. Effect of tetraethylammonium (TEA) on Kca current. (A) Representative
example of 4 sec. Ramp depolarization from a holding potential of -80 mv to +80 mV in
the presence of 4-AP and increasing concentrations of TEA. The inset shows a
representative example of step depolarizations from -80 mV to +40 mV in the presence
of 0, 500 pmol/L and 5 mmol/L TEA. (B) Dose response curve for TEA as measured
from step depolarizations to +40 mV (1C50 = 683.1 imol/L, n=4). Current is displayed
as a fraction of control (Imax).








control


10 mM TEA



00 ^hW4 00^^


'110"A pl,, !j LAW$
1, .W%0J0*


20 ms
710 pA


Figure 2-2. Effect of tetraethylammonium (TEA) on single channel Kca currents.
Shown are single channel recordings of Kca current at +40 mV in the absence
and presence of 10 mmol/L TEA.










control


100 nm ChTX



1000 pA


I 1 1 ..
30 -60 -40 -20 0 20 40 6'
Membrane Potential (mV)


Control


100 nM ChTX



A. - 1 i 1 1 IIT .


kAji~A~


22mSA
N 120 1 i


Figure 2-3. Effect of charybdotoxin (ChTX) on calcium activated K+ current. (A)
Representative example of a 4 sec. ramp depolarization from a holding potential
of -80 mV to +80 mV in the absence and presence of 100 nmol/L ChTX. (B)
Single channel recordings of Kca current at +40 mV using the cell attached patch
technique in the absence and presence of ChTX. The toxin inhibits most but not
all single channel activity.


















1.0
E
- 0.8

2 0.6

o 0.4

. 0.2-
E 0.0-
0
z


"" 250 pA
400 msec


* Transient
* Sustained


9-'. t


-0.2 -
-100-80-60-40-20 0 20 40 60 80
Conditioning Potential (mV)


1.0-

0.8-

0.6-


."-a
.8 -I



-80 -60 -40 -20 0 20 40 60 80
Membrane Potential (mV)


Transient
Sustained


* *U *-**.-
0 -m n -


0.2-

0.0- -. . .....
-120-100-80-60 -40 -20 0 20 40 60
Conditioning Potential (mV)


Figure 2-4. Electrophysiological characteristics of the Kv current in VSM cells
isolated from rat renal resistance arteries. (A) Representative recordings of current
elicited during voltage-step depolarizations in zero external Ca2+ and 5 mM TEA.
(B) Graph showing the mean current-voltage relationship for both the peak and
sustained component of Kv current (n=1 1). Current amplitude is normalized to each
cell's capacitance. (C) Voltage dependence of Kv current activation as determined
from the magnitude of the tail current (mean SE, n=9). (D) Voltage dependence
of Kv current inactivation determined using a double pulse protocol (mean SE,
n=11).


+80
80m +30-
-80 mV 1-- 1 L







A +80
-80 mV __ L



I I
MN'"ik


1.0-
0.8-
= 0.6-
0.4-
0.2


, ... J- 1200 pA
2 sec


1-20 60 mV[


-80 mV


T,=18.97 9 msec
S,=2.471.4 sec


2000 4000 6000
Time (msec)


8000


S-20 -40 mV


rff
T,=14.36.9 msec
T,=2.451.7 sec


0 2000 4000 6000 8000
Time (msec)


1 -20


J 100 pA
500 msec


Figure2-5. Time course of recovery from inactivation and the effect of holding potential on Kv current. (A)
Representative example showing recovery of current following inactivation. The experimental protocol is shown
above. (B,C) Graph showing the mean time course of recovery from inactivation for the transient (B) and sustained
(C) component. The data was best fit by a bi-exponential function. The two time constants for each fit are reported
on the graph (mean SE, n=5). (D,E,F) Within the same cell, representative examples of a family of currents evoked
from a holding potential of -80, -60, and -40 mV respectively. The voltage pulse protocol is shown above.


|! ....


I I





41



+80
-80 mYJlL- -80


Control

1 mM

30 mM


1.00-


0.75-



0.50-


0.25-


200 pA
500 msec


* Transient
* Sustained


U


Log M [4-AP]


Figure 2-6. Mean dose-response relations showing the effects of 4-
aminopyridine (4-AP) on Kv current. 1/Imax, percentage of current in the
presence of 4-AP as a fraction of its control value. Graph shows the effect
of 4-AP on both peak and sustained components of current (mean SE,
n=5). Currents were measured at +80 mV. Top shows a representative
recording of Kv current measured at +80 mV from a holding potential of -80
mV in the presence of 1 and 30 mmol/L 4-AP.


x
E


IC
,)

0













A B
100
Control
80-
0
DTX 60-
(50 nM) 0 E
.E40-
20
r r0
Transient Sustained

C 1s200pA D
1 sec 100-

Control 805
S60 *

MCDP 40-
(1 pM) 20-

0
Transient Sustained



Figure 2-7. Effect of xDTX and MCDP on Kv current. (A) Representative
recording of Kv current in response to a step depolarization from -80 mV to +80
mV in the absence and presence of aDTX (50 nmol/L). (B) Graph showing the %
inhibition by aDTX (50 nmol/L) of both the peak and sustained components of
Kv current (mean SE, n=4). (C) Representative recording of Kv current in
response to a step depolarization from -80 mV to +80 mV in the absence and
presence of MCDP (l1 mol/L). (D) Graph showing the % inhibition by MCDP
(1 .mol/L) of both the peak and sustained components of Kv current (mean SE,
n=4).








O

C% e4 N" O' (4 C1 (N
0 > > > > 05 > > >

0 > > 0 o > C"
kDa -
121
78 .

9 1 IgG


39.5


30.7

Western w/Kv1.2 Western w/Kvl .4

Figure 2-8. Co-immunoprecipitation of Kvl1.2 and Kvl1.4 from rat renal arterioles. Membranes isolated from rat renal
arterioles were immunoprecipitated with anti-Kvl.2 antibody and size fractionated by SDS-PAGE. Samples were
transferred to nitrocellulose and probed with anti-Kvl1.2 or anti-Kvl.4 antibodies and detected using ECL. A band
corresponding to a processed form of Kv1.2 of -80 kDa (left panel) was detected by the anti-Kv1.2 antibody (I.P. Kvl.2)
whereas a band of -96 kDa was recognized by the Kv1.4 antibody (I.P. Kvl1.2, right panel). Both antibodies were isoform
specific and only recognized their corresponding in vitro translated product on immunoblots (lanes marked i.v. Kvl1.2, i.v.
Kvl1.2 w/CMM, i.v. Kvl1.4). Neither antibody recognized sham in vitro translated proteins (control lanes). The position of
the IgG antibody from the immunoprecipitation as detected with goat anti-mouse secondary antibody is indicated by the
arrow.
















CHAPTER 3
ALTERATIONS IN RAT INTERLOBAR ARTERY MEMBRANE POTENTIAL
AND K1 CHANNELS IN GENETIC AND NONGENETIC HYPERTENSION

Introduction

The etiology of systemic hypertension involves a gradual and sustained increase

in total peripheral resistance (Bohr & Webb, 1988). A number of anatomical,

biochemical or biophysical mechanisms may underlie this phenomenon (Khalil et al.,

1989). Anatomically, rarefaction of the vascular beds and blood vessel hypertrophy

occurs in many hypertensive models. Biochemically, alterations in Na', K'-ATPase,

Ca2-ATPase and Na'-Ca2" exchange activity have been reported in blood vessels and

isolated cell preparations from various models of hypertension. Biophysical changes that

may occur in hypertension involve changes in ion channel function thereby causing

changes in vascular reactivity. Vascular smooth muscle contains at least two different

Ca2- channels, at least four different potassium channels, non-selective cation channels,

and Ca>-activated Cl channels ( Pacaud et al., 1989b: Nelson et al., 1990b; Lamb et al.,

1994; Nelson & Quayle, 1995). Therefore, alterations in ion channel function of any of

these channels could potentially change the contractile state of vascular smooth muscle

thereby altering blood pressure.

The use of fluorescent Ca2" indicators and electrophysiological techniques have

enabled studies of hypertension at the single cell level. However to date, the data from







various models have led to differing hypotheses regarding the roles of ion channels in the

regulation of intracellular free Ca2 concentration ([Ca2*]i) and in the etiology of

hypertension. For example, when SHR aortic smooth muscle cells were loaded with fura-

2, basal [Ca2]i was increased when compared to WKY cells (Sugiyama et al., 1986).

Furthermore, vasopressin (AVP) and angiotensin II (Ang II)-stimulated increases in

[Ca2]j were greater in SHR aortic cells when compared to WKY cells (Nabika et al.,

1985). Conversely, Storm et al. (1992) and England et al. (1993) show no change in

basal [Ca2,]i levels in hypertension. Using conventional microelectrodes, it was reported

that the ouabain-insensitive component of resting membrane potential is 10-15 mV more

depolarized in SHR blood vessels than WKY vessels (Hermsmeyer, 1976). However,

other laboratories report that no change in resting membrane potential exists in

hypertensive models (Lamb & Webb, 1989; Steikel, 1989; Rusch et al., 1992). Recently,

Rusch et al. (1992) and England et al. (1993) demonstrated that SHR aortic cells have

more Ca2-activated K" current (Kca) than WKY cells. However, no studies to date have

shown alterations in Kv in hypertensive models. To date, the fluorescence and

electrophysiological studies performed in various models of hypertension have been

obtained from larger conduit arteries which play a less significant role in setting

peripheral vascular resistance than blood vessels from the kidney or mesentery. Studies

on clinically relevant vessels from the renal vasculature should provide useful

information on the electrophysiological differences in hypertension at the single cell

level. Here we present data which for the first time demonstrates that alterations in Kv

channel activity, membrane potential, and [Ca2]i exist in small vessels of the genetic and

nongenetic hypertensive renal vasculature.







Methods

Animal Preparation

WKY, Sprague-Dawley (SD), and SHRs were bought from Charles River

Breeders (Boston, MA). Control SD rats were made hypertensive by implanting one 50

mm long silastic tube (Dow Coming), containing deoxycorticosterone acetate (DOCA,

100 mg/Kg), subcutaneously on the dorsal side of the rats. Rats were unilaterally

nephrectomized and given 0.15 mmol/L NaCI to drink as their sole drinking fluid.

Control blood pressures are taken two weeks before the above procedure and weekly

thereafter. Hypertension developed in 4-6 weeks after DOCA/salt/nephrectomy regime.

Adult male rats were used at the age of 15 weeks. The mean systolic blood pressure of

WKY rats wasl40 4 mm Hg (n=l 5) and was significantly greater in the SHRs, 195

11 mm Hg (n=15, p<0.01). Similar results were obtained for the SD and DOCA

hypertensive rats (SD: 115 7 mm Hg; DOCA: 165 10 mm Hg. (n=16, p<0.01). WKY

and SHR rats are used as the control and experimental model for genetic hypertension;

the SD and DOCA hypertensive rats are used as the control and experimental model for

nongenetic hypertension.

Electrophysiological Measurements

Single smooth muscle cells from WKY, SHR, SD, and DOCA hypertensive

interlobar artery were enzymatically dissociated using previously described methods.

Care was taken to minimize trauma to either arterial preparation. Cells from

normotensive and hypertensive animals were made on the same day using the same

procedure thereby ruling out nonspecific membrane effects that were due to the cell







isolation technique. All experiments were conducted blind thereby minimizing bias in

the results. However, there is a possibility that the connective tissue of the arterial wall is

altered in hypertension (Khalil et al., 1989) causing the effectiveness of the digestive

enzymes to be altered. Thus, smooth muscle cells isolated from the vessels of

hypertensive rats may have been in the presence of the enzymatic digestion cocktail for

longer periods of time than control, possibly altering sarcolemma proteins. Although

difficult to test, efforts were made to reduce the possible effects of variable digestion in

the tissues by examining reversibility of an angiotensin II (Ang II, 100 nmol/L) induced

contraction of isolated single smooth muscle cells. Only batches of cells that showed

contraction and relaxation to Ang II were used.

Single cells were voltage clamped and membrane currents were measured using

the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Patch

pipettes for whole-cell patch clamp recordings were made from borosilicate glass

capillaries and had resistances of 1-3 MO. Voltage clamp command potentials were

applied to the cells and membrane currents were recorded using an Axopatch-ID patch

clamp amplifier. Membrane current was monitored on a digital oscilloscope, digitized on-

line (0.5-2.0 kHz), and stored on a computer. Resting membrane potentials were

measured in the 1=0 position of the Axopatch 1-D. Input resistances were determined in

current clamp by applying a 1-5 pA hyperpolarizing current pulse and examining the

change in membrane potential. Any cell that did not have a seal resistance of greater than

5 GO was not used. Series resistance was in the range of 2-6 MQ and was compensated

for by approximately 80% using the Axopatch-ID. Junction potentials were zeroed at







the beginning of each experiment with Axopatch- 1 D. At the end of each experiment, no

change in the nulled junction potential occurred. Data analysis was performed with

pCLAMP 5.5.1 and 6.0 software (Axon Instruments). All experiments were performed at

room temperature and data illustrated in the figures were from different batches of cells

isolated from different animals.

Measurement of Intracellular Ca2"

The Ca2 indicator indo-1 I (pentapotassium salt, 100 pmol/L) was included in the

patch pipette solution and dialyzed into the cell as previously described (Gelband &

Hume, 1995). Background autofluorescence from the same cell was measured before

gaining access to the cell interior and subtracted from all fluorescence measurements.

Sufficient loading had taken place 5-10 minutes after access to the cell interior. A 10 Pm

diameter of the cell was irradiated with ultraviolet light at a wavelength of 340 nm with a

mercury lamp. The light emitted from the cell was collected using an epifluorescence

microscope (Nikon) and measured at 400 and 500 nm by means of a microfluorometer

and matched photomultiplier tubes. Changes in [Ca2*], were calibrated using the equation

of Grynkiewicz et al, 1985):

[Ca2"], =Kd[R-Rm ]J/R -R](Sf/Sb2)

where Kd is the dissociation constant of the Ca2"-Indo-1 complex; R is the F40oF5o

fluorescence ratio; Rn,, and Rk are the ratios measured by the addition of the Ca2-

ionophore ionomycin (10 pmol/L) to Ca2, free (10 mmol/L EGTA) solution and

Ca-containing solution respectively; and S%/Sb2 is the ratio of the 500 NM fluorescence

signal in Ca2' free and Ca2- containing solutions.







Solutions

The Krebs solution contained (in mmol/L): 120 NaCI, 25 NaHCO3, 4.8 KC1, 1.2

MgCl 2, 2.5 CaC12, 10 D-glucose. For all whole-cell voltage clamp experiments

examining outward currents, the bath solution contained (in mmol/L): 130 NaCl, 10

NaHCO3, 4.2 KC1, 1.2 KH2PO4, 0.5 MgCl2, 1.5 CaC12, 5.5 D-glucose, and 10 HEPES,

(pH 7.4 with NaOH). The pipette solution contained (in mmol/L): 140 KC1, 5 ATP

(potassium salt); 10 HEPES, (pH 7.2, with KOH). ChTX was obtained from Peninsula

Laboratories, Inc. and the stock was 0.1 M in 150 mmol/L NaCI. All other chemicals

were from Sigma Chemical Company.

Statistics

Results are expressed as mean S.E.M. Statistical significance was evaluated

using Student's t test for unpaired observations. Differences were considered significant

at p<0.05. n corresponds to the number of cells examined. Membrane currents were

measured from the zero current level and normalized to cell capacitance.

RESULTS

Electrical Properties of Single Rat Interlobar Artery Cells

Table 3-1 illustrates the electrical properties of isolated WKY, SD, SHR and

DOCA hypertensive interlobar artery cells. In 15-16 cells from 9 different preparations, a

significant difference was observed in the resting membrane potential of the cells. WKY,

SD, SHR and DOCA hypertensive cells had an average resting membrane potential of-52

+ 2.3, -50 3.8, -32 3.8* and -36 2.5 mV*. respectively (n=15 or 16, *p<0.01). All

cells had similar input resistances and membrane capacitances (Table 3-1). One







explanation of these results is that the altered contractile state of the hypertensive

interlobar artery may be due to alterations in K- permeability of the hypertensive single

smooth muscle cell.

Ionic Currents in WKY, SD, SHR and DOCA Hypertensive Cells

It has been reported that Kca was increased in hypertension, ( Rusch et al., 1992;

England et al., 1993) but to date no evidence for changes in Kv have been demonstrated

in hypertensive models. Figure 3-1 shows pharmacological isolation of Kv and Kca from

WKY and SHR cells. The use of pharmacological agents to isolate these two

components of K current was based on experimental results obtained from the canine

renal artery (Gelband & Hume, 1995). ChTX (100 nmol/L) and niflumic acid (100

mmol/L) were used to isolate Kv; 4-AP (10 mmol/L) and niflumic acid (100 mmol/L)

were used to isolate Kca; and 5 mmol/L ATP was present in the recording pipette to

inhibit KATr. Figure 3-1A (upper panel), shows representative ramp depolarizations from

a WKY and an SHR cell in the presence of 4-AP and niflumic acid. Under these

conditions Kv was significantly reduced, yet Kca was present. Figure 3-1 A (bottom panel)

shows the mean current-voltage relationship obtained for Kc, during voltage step

depolarizations (n=7). In 7 experiments, Kca in SHR cells was significantly increased

when compared to control (P<0.01, Table 2). Figure 3-1B illustrates the nature of Kv in

WKY and SHR cells in the presence of ChTX and niflumic acid. The upper panel shows

representative ramp depolarizations and under these conditions Kca was nearly abolished,

yet Kv was present. The lower panel shows the mean current-voltage relationship

obtained for Kv during voltage step depolarizations (n=7). In 7 cells tested, Kv was

decreased in SHR cells when compared to WKY cells during voltage step depolarizations







(P<0.01). Table 3-2 illustrates mean current density values for Kca and Kv in WKY and

SHR cells at membrane potentials of 0 and 40 mV.

Similar results were obtained when Kv and Kca were isolated in control and non-

genetic hypertensive cells. Figure 3-2 illustrates the characteristics of Kv in SD and

DOCA hypertensive cells. During voltage step depolarizations in the presence of

charybdotoxin and niflumic acid (Panel A), Kv in DOCA hypertensive cells is less than

control. During a voltage ramp depolarization (Fig. 3-2B), the DOCA hypertensive cells

possessed a decreased Kv when compared to control. In another set of experiments, the

nature of Kac, in SD and DOCA hypertensive cells was investigated (Fig. 3-3). Panel A

shows representative traces of step depolarizations from a SD and a DOCA hypertensive

cell in the presence of 4-AP and niflumic acid. Ka was significantly increased in DOCA

hypertensive cells when compared to control (n=7, P<0.01, Table 3-2). Panel B is a

representative trace of Kc, during voltage ramp depolarizations in both cell types. Again

it shows that the DOCA hypertensive Ka was greater when compared to control. Figure

3-4A and B illustrate mean current voltage relationships for Kv and Kc, during voltage

step depolarizations in SD and DOCA hypertensive cells (n=7, P<0.01). The DOCA

hypertensive mean Kv current-voltage relationship was right shifted and activated at

more positive potentials than control (Panel A). However, the DOCA hypertensive mean

Kc, current-voltage relationship was left shifted and activated at more negative potentials

than control (Panel B). Table 3-2 illustrates mean current density values at 0 and 40 mV

for Kca and Kv in SD and DOCA cells. These biophysical characteristics may be of some

importance since K- current has been suggested to play a role in regulating resting

membrane potential of vascular smooth muscle cells ( Fleischmann et al., 1993; Gelband







& Hume, 1995).

Regulation of Intracellular [Ca2] in WKY, SD, SHR and DOCA Hypertensive Cells

As illustrated in Table 3-1, the resting membrane potential of SHR and DOCA

hypertensive artery cells was more depolarized than control cells. Since [Ca2]j has been

suggested to inhibit K, channels and regulate membrane potential, (Gelband & Hume,

1995) a separate group of experiments were performed to investigate the regulation

[Ca2-], in single WKY, SD. SHR and DOCA hypertensive interlobar artery cells. Figure

3-5 shows that resting levels of [Ca2]i was significantly elevated in the hypertensive cells

when compared to control (WKY: 92 7 nmol/L; SHR: 125 9 nmol/L*; SD: 89 7

nmol/L; DOCA: 113 11 nmol/L*, n=6, p<0.05). Since circulating Ang II is a potent

renal vasoconstrictor and its circulating concentration is increased in various forms of

hypertension, (Khalil et al., 1989) we investigated the effects of Ang II on peak [Ca2]j.

Application of Ang II (100 nmol/L) significantly increased peak [Ca2]i over basal levels

in each cell type (n=6). This occurred within 5 sec of Ang II application. A significant

difference was also observed in the Ang II-stimulated increase in peak [Ca2]i when

hypertensive cells were compared to control cells (WKY: 450 25 nmol/L; SHR: 665

30 nmol/L*; SD: 369 31 nmol/L; DOCA: 595 20 nmol/L*, n=6, p<0.05). No

difference was observed in the time course of the Ang II effect in any cell type. When

combined with the results of Figures 3-1 thru 3-4, these data support the hypothesis that

[Ca21]i may modulate resting membrane potential by altering Kv (Gelband & Hume,

1995) and/or Kc, current (Brayden & Nelson, 1992).







DISCUSSION

Alterations in ion channel activity have been associated with an increased

vascular reactivity in hypertension (Khalil et al., 1989; Steikel, 1989). Specifically,

depolarization of the resting membrane potential, (Hermsmeyer, 1976; Tomobe et al.,

1991; Van-de etal., 1992) and increases in Ca2- current,( Rusch & Hermsmeyer, 1988;

Ohya et al., 1993; Wilde et al., 1994) Kca ( Rusch et al., 1992;England et al., 1993) and

[Ca2]i ( Losse et al., 1984; Sugiyama et al., 1986; Bukoski, 1990; Papageorgiou &

Morgan, 1991; Storm et al.. 1992) have been demonstrated in various forms of

hypertension. The present study describes the electrophysiological properties of a

physiologically relevant vascular preparation, the interlobar artery of the kidney, in

hypertension. Our results suggest that there are changes observed in both K' current and

Ca" homeostasis in a genetic and nongenetic model of hypertension. These effects may

contribute to the increase in vascular reactivity observed in hypertension.

Resting Membrane Potential and Hypertension

Normotensive vascular smooth muscle cells including those isolated from the

renal vasculature have resting membrane potentials between -50 and -60 mV ( Harder &

Hermsmeyer, 1983b; Gelband & Hume, 1992; Rusch et al., 1992; Fleischmann et al.,

1993; Gelband & Hume, 1995). In the present study, it was shown that SHR and DOCA

hypertensive interlobar artery cells have a resting membrane potential of approximately -

30 mV (Table 3-1). This was significantly depolarized when compared to the control cells

(approximately -50 mV). In a variety of conduit artery vascular preparations, significant

changes in passive membrane electrical properties have been reported in SHR cells when

compared to control (Hermsmeyer. 1976; Harder et al., 1983a; Harder & Hermsmeyer,








1983b; Lamb & Webb, 1989; Tomobe et al., 1991). However, there have been some

reports of an unaltered resting membrane potential in vascular smooth muscle cells from

the SHR and other models of hypertension ( Abel et al., 1981; Steikel, 1989; England et

al., 1993). One reason for the discrepancy in resting membrane potentials may be the site

and size of the vessel itself. There is ample evidence that larger capacitance vessels, like

the aorta and first branch coronary arteries, do not have similar excitation-contraction

coupling properties when compared to smaller resistance vessels (Van Breemen & Saida,

1989). This suggests that small arteries (<200 uim in diameter) from physiological

relevant vascular beds (e.g. renal, mesenteric and cerebral) are altered in hypertension

and play a significant role in the regulation of peripheral resistance.

K Channels and Hypertension

Membrane K permeability is the primary initiator of vascular smooth muscle

contractility (Nelson et al., 1990b; Nelson & Quayle, 1995). 45K+ effiux from conduit

artery hypertensive vascular smooth muscle was increased when compared to

normotensive vascular smooth muscle (Jones, 1973). To date there is no evidence that

alterations in voltage dependent K1 channels exist in any model of hypertension. Our

results are the first to show that in SHR and DOCA hypertensive interlobar artery cells,

Kv was significantly reduced when compared to control (Figs. 3-1 and 3-3). A

significant decrease of Kv current would cause membrane depolarization and an increase

in vascular smooth muscle tone. Indeed a significant depolarization of the resting

membrane potential of isolated cells from SHR and DOCA hypertensive animals was

observed when compared to control (Table 3-1). This would suggest that Kv channels







play a major role in the regulation of membrane potential and therefore tone in rat

interlobar arteries.

It was previously demonstrated there was an increased Kc, and open probability of

large conductance Kca channels in SHR cells isolated from the aorta ( Rusch et al., 1992;

England et al., 1993). It was suggested that this enhanced K' permeability may provide a

negative feedback mechanism by which an increased arterial contractility may be limited

in hypertension (Rusch et al.. 1992). The data described in this manuscript are consistent

with these conclusions. Basal [Ca2"]j is increased in the SHR and DOCA hypertensive

cells (Fig. 3-5), and this would lead to a greater Kc, current density in the SHR and

DOCA hypertensive cells (Figs. 3-1 and 3-2). However, based on the voltage-dependent

properties of this channel (i.e. extremely low open probability at more negative

membrane potentials), it would be difficult for this channel to be the sole channel protein

that causes the increased 45K efflux observed in SHR arterial smooth muscle.

[Ca2Ji and Hypertension

A number of studies have attempted to compare resting levels of [Ca2'] in SHR

and WKY artery preparations ( Losse et al.. 1984; Nabika et al., 1985; Sugiyama et al.,

1986; Erne & Hermsmeyer, 1989; Khalil et al., 1989; England et al., 1993; Rusch et al.,

1992Nelson & Quayle. 1995). These studies have shown differing results depending

mainly on the age of the animal used. In preparations of SHR arteries from young

animals (3 days to 5 weeks) which have not developed hypertension, resting [Ca2], levels

did not differ from the WKY (Khalil et al., 1989; Storm et al.. 1992; Nelson & Quayle.

1995). However, Ang II or vasopressin-stimulated changes in [Ca2], did show significant

differences in young SHR rats. suggesting a change in Ca2' homeostasis at an early age in







development before the onset of hypertension (Nabika et al., 1985). This is consistent

with our results (Figs. 3-3 to 3-5). In older rats (>8 weeks) an increase in resting levels

on [Ca2*] between control and hypertensive cells was observed ( Losse et a!., 1984;

Sugiyama et al.. 1986). However, there are studies stating that resting levels of [Ca2]

were not altered in adult SHR vascular smooth muscle cells from conduit arteries (Storm

et al., 1992; England et al., 1993). The present data using indo-1 fluorescence suggests

that [Ca21]i was significantly greater in SHR and DOCA hypertensive interlobar artery

cells than in control. This increase in resting [Ca2,]i levels can be directly correlated to

the changes observed in membrane potential and Kv current. Previous results in the

canine renal artery (Gelband & Hume, 1995)demonstrated the sensitivity of Kv channels

to increased [Ca2,]i which in turn caused membrane depolarization. These data are

consistent with our observations that cells from resistance blood vessels have different

excitation-contraction coupling pathways and Ca2' homeostatic mechanisms than cells

from conduit blood vessels.

Ion Channel Control of Vascular Smooth Muscle Tone in Hypertension

Recently, a number of K- channels have been suggested to regulate membrane

potential and tone of smooth muscle cells. It has been demonstrated that voltage

dependent delayed rectifier K- channels are important regulators of smooth muscle

resting membrane potential ( Fleischmann et al., 1993: Gelband & Hume, 1995). This is

consistent with our observations in the interlobar artery smooth muscle cells. Our results

in clinically relevant resistance vessels suggest that the inhibition of Kv and the

associated membrane depolarization would be of physiological importance to the

regulation of tone in normotensive and hypertensive states (Gelband & Hume. 1995).







However, other K- channels have also been suggested to play a role in the regulation of

smooth muscle tone. Large conductance Kc, channels may represent a negative feedback

mechanism for cerebral arteries when pressurized (Erne & Hermsmeyer, 1989). Kipn

(Clapp & Gumrney, 1992) and K,. channels (Quayle et al., 1993) also have been implicated

to play a role in regulating tone in the small vessels of cerebral, pulmonary and coronary

circulation. Therefore a combination of K' channels or variation in K' channel

distribution may explain why different K- channels are observed to regulate membrane

potential in various vascular beds.

Significance of the Kidney in Hypertension

Despite years of intensive investigation, the etiology of hypertension remains

unknown. Elevated blood pressure and total peripheral resistance associated with

hypertension may result from increases in neural, humoral, or vasoconstrictor substances,

passive or genetic structural alterations in blood vessels, or intrinsic changes in the

control of vascular tone. The interlobar artery of the kidney is a significant blood vessel

to study since alterations in renal blood flow are known to influence fluid volume

regulation, the secretion of a number of important neurohumoral substances, and

arteriolar resistance which may be involved in the etiology or maintenance of

hypertension. However, direct measurements of the major ionic conductances that may

underlie contraction of hypertensive rat interlobar arterial smooth muscle are lacking.

This is rather remarkable given the results of earlier renal transplant studies in humans

(Bianchi el al.. 1979; Curtis et a!.. 1983) and animals (Dahl et al.. 1972) which suggested

that the genetic defect in a proportion of the cases with essential hypertension is

expressed in the kidney. In these studies, kidney transplants from normotensive donors





58

into hypertensive recipients effectively reversed many of the abnormalities associated

with hypertension. In summary, the data demonstrate that the alterations in resting

membrane potential observed in the SHR and DOCA hypertensive interlobar artery could

be manifest through alterations in one or a combination of Kv channels or Kca channels.

These alterations in ionic conductances may play a role in the etiology and/or

maintenance of the hypertensive state.






Table 3-1. Electrical Properties of WKY, SD, SHR, and
DOCA Rat Interlobar Artery Cells


Vm(mV)


-52+2.3

-32+3.8

-50+3.6

-36+2.5


5.1+1.2

5.6+2.3

6.3+2.5

5.4+1.1


Values are mean SEM; n, number of cells
*P<0.01 when compared to control cells


Cell Type


WKY

SHR

SD

DOCA


Cm(pF)


25+2.6

26+1.8

292.1

31+2.3





60

Table 3-2. Mean Alterations in K Current at 0 and +40 mV


0 mV


2.5+0.03

0.1+0.01

2.9+0.09


0.2+0.03


40 mV


11.0+0.3

3.2+0.1

12.0+0.5

2.7+0.4
2.7+0.4


0 mV


1.9+0.1

2.8+0.2

2.3+0.1


3.7+0.4


Values are mean (pA/pF)+SEM; n, number of cells
*P<0.01 when compared to control cells


Cell Type


WKY

SHR

SD

DOCA


40 mV


14.2+1.5

35.0+1.8

12.6+0.8


42.0+1.0


















Ca2-activated K Current


K, Current


WKY u



J^ .^f^-SHR


SHR


500 pA 200 pA


-80 -40 0 40
Membrane Potential (mV)


100o

80

60

40I

20 -J
0


* WKY
* SHR


80 -80 -40 0 40
Membrane Potential (mV)


16
14
12
10 -
8
6
4
2
0 -


-80 -60 -40 -20 0 20 40 60 80
Membrane Potential (mV)


* WKY
* SHR


-80 -60 -40 -20 0 20 40 60 80
Membrane Potential (mV)


Figure 3-1. Isolation of Kv and Kca in WKY and SHR interlobar artery cells.
(A) In the presence of 4-AP (10 mmol/L) and niflumic acid (100 pmol/L), SHR Kca
was significantly greater than WKY. Similar results were obtained in seven cells.
Voltage ramp and step depolarizations are shown in the upper and lower panels,
respectively. (B) In the presence of charybdotoxin (100 nmol/L) and niflumic acid
(100 imol/L), WKY Kv was significantly greater than SHR Kv. Similar results
were obtained in seven cells. Voltage ramp and step depolarizations are shown
in the upper and lower panels, respectively. These two cells had similar
membrane capacitances (WKY, 26 pF and SHR, 27 pF).


I ...... I I ., I







K,, Current


Sprague-Dawley


| 300 pA
60 ms


SD
DOCA


I I I I I
-80 -40 0 40 80
Membrane Potential (mV)


0300 pA
300 pA


Figure 3-2. Isolation of Kv in SD and DOCA hypertensive interlobar artery cells.
In the presence of charybdotoxin (100 nmol/L) and niflumic acid (100 [.mol/L),
SD Kv was significantly greater than DOCA hypertensive Kv during (A) step
depolarizations (holding potential, -80 mV; test potential,-20 mV to 80 mV; 20
mV steps) and (B) ramp depolarizations (-80 to 80 mV, 4 seconds). The
membrane capacitances of the two cells were similar (SD, 30 pF; DOCA 29 pF).
Similar results were obtained in 7 cells.


DOCA


r







K(Ca) Current
Sprague-Dawley



W.'


DOCA


I 4uu p't
50 ms




DOCA


-80
-80


400 pA


I I I
-40 0 40
Membrane Potential (mV)


Figure 3-3. Isolation of Kca in SD and DOCA hypertensive interlobar artery cells.
In the presence of 4-AP (10 mmol/L) and nifiumic acid (100 [tmol/L), DOCA
hypertensive Kca was significantly greater than SD during (A) step (holding
potential, -80 mV; test potential, -20 to 70 mV; 10 mV steps) and (B) ramp
depolarizations (-80 to 80 mV, 4 seconds). These two cells had similar
membrane capacitances (SD: 29 pF; DOCA: 30 pF). Similar results were
obtained in seven cells.










K(Ca) Current


SD
DOCA


i I I 1 1
-80 -60 -40 -20 0


100

80-

60

40

20

0


I i I I
20 40 60 80


* SD
* DOCA


I I I 1
-80 -60 -40 -20


20 40 60 80
20 40 60 80


Membrane Potential (mV)


Membrane Potential (mV)


Figure 3-4. Mean current voltage relationships for Kv and Kca in SD and DOCA
hypertensive cells. DOCA hypertensive Kv (A) and Kca (B) are significantly
altered when compared to control (n=7). Error bars are the same size of the
symbol and therefore are not visible.


18-
16-
14-
12-
10-
8-
6-
4-


K(v) Current












P<0.05


P<0.05


800

700

600

500

400

300

200

100


SHR


S3
SD


iDOCA
DOCA


Figure 3-5. Basal and Ang 11-simulated [Ca2+],. Both basal and Ang 11-stimulated
[Ca21], are significantly different from control (n=7, *p<0.05).


7 ] Ibasal
- Ang II


WKY


P<0.05













CHAPTER 4
PREVENTION OF RENOVASCULAR AND CARDIAC
PATHOPHYSIOLOGICAL CHANGES IN HYPERTENSION BY AT,
RECEPTOR ANTISENSE GENE THERAPY

Introduction

The elevation of systemic blood pressure (BP) associated with hypertension is a

risk factor for cardiovascular disease and renal failure. Often it is the pathophysiological

alterations and impairments associated with hypertension that lessen life expectancy.

Pharmacological intervention has been relatively successful in normalizing the elevation

in BP. However, the assumption that reduction of BP will totally reverse hypertension-

induced pathophysiological changes remains unclear ( Ruilope et al., 1990; de Celentano

etal., 1993; Vogt et al., 1993: Ruilope et al.. 1994).

The duration of treatment, age at which the antihypertensive therapy is initiated,

site of administration, and specific agent used all appear to influence the reversal of the

pathophysiological alterations associated with the disease (Linz et al., 1989; Tschudi et

al., 1994; Schmieder et al., 1996). In some instances, reversal of pathophysiological

alterations may even be unfavorable such as when regression of left ventricular

hypertrophy (LVH) and peripheral resistance occur in a disproportionate manner (Simko,

1994). Similarly, other reports have indicated that traditional antihypertensive agents can

contribute to target organ injury by altering metabolic processes (i.e.

hypercholesterolemia, glucose intolerance, hyperkalemia).







A role for the renin-angiotensin system (RAS) in the development of hypertension

is well established in both humans and animal models such as the spontaneously

hypertensive rat (SHR). Interruption of the RAS pathway, either by preventing the

formation of Ang II (i.e. ACE inhibitor) or by blocking its actions at the level of the

peptide receptor (i.e. AT, receptor antagonists), has been shown to reduce BP and protect

against target-organ injury (MacGregor, 1992; van Zwieten, 1992; Kang et al., 1994;

Goldberg et al., 1995; Berecek & Zhang, 1995). However, the attenuation or delay of

non-hemodynamic pathophysiological impairments with these agents does not entirely

reduce the risk to hypertensive patients (Vogt et al., 1993; de Celentano et al., 1993). In

addition, chronic administration of traditional therapies is necessary for long term

antihypertensive benefits. Required daily dosing and undesirable side effects such as

sexual dysfunction, coughing, and lethargy diminish patient compliance.

We have previously established that retrovirally mediated delivery of AT,-

receptor antisense (ATR-AS) attenuates the development of high blood pressure on a

long term basis (lyer et al.. 1996; Lu el a!., 1997). A single injection of ATR-AS into

neonatal SHRs prevented the development of hypertension up to 90 days after injection.

Our objective in this study was to determine if this attenuation of high BP was associated

with the prevention of renal and cardiovascular pathophysiological changes induced by

the hypertensive state.

Materials and Methods

Preparation of Viral Particles Containing ATR-AS

A retroviral vector. LNSV. was used to deliver AT1BR-AS into the rats (Lu el a!.,

1995; Lu & Raizada. 1995). The ATIBR-AS was cloned in the LNSV vector. It was







transfected to packaging cell line PA3 17 (American Type Culture Collection). After

selection by G418, the medium containing viral particles that expressed ATIBR-AS

(LNSV-ATR-AS) was collected and used for all animal experiments. Viral particles that

did not contain ATIBR-AS (LNSV) were also prepared by the above protocol and used as

a control.

Animals and Experimental Protocols

Five-day-old Wistar Kyoto (WKY) and SHRs were used in this study (Harlan

Sprague Dawley). They were divided into three groups: vehicle (control), virus alone

(LNSV), or virus containing AT.R-AS (LNSV-ATR-AS). Treatments were injected via

the cardiac route under methoxyflurane (metofane, Pitmin-Moore, Mundelein, IL)

anesthesia. A bolus of 5 x 1010 plaque-forming units of viral particles in 10 il of

physiological saline was used per animal. There was an -95% survival rate 24-48 hr after

viral administration.

At the end of 120 days, rats from each group were used for determining the mean

BP through an indwelling catheter implanted in the carotid artery essentially as described

(Iyer et al., 1996). Rats were anesthetized with a mixture of ketamine (30 mg/kg),

xylazine (6 mg/kg), and acepromazine (1 mg/kg) administered intramuscularly (0.7

ml/kg). Direct blood pressure was recorded from the carotid artery catheter (PE-50) in

free-moving, nonrestrained animals with a pressure transducer coupled to a Digi-Med

blood pressure analyzer (Micro-Med. Louisville. KY).







Smooth Muscle Cell Isolation

Male rats were killed by decapitation and the right and left kidneys were removed

and placed in cold, oxygenated (95% 0,-5% CO,) physiological saline solution (PSS).

Renal resistance vessels were identified as the fourth to fifth branch distal to the renal

artery. These vessels were dissected free of fat and connective tissue under a dissecting

microscope. The vascular fragments were then cut into small pieces (- 4 mm) and

incubated at room temperature in Ca2-free PSS for 30 minutes. The pieces were

subsequently resuspended in Ca2-free PSS digestion buffer for 20-30 minutes at 37C.

The Ca>-free PSS digestion buffer contained (mg/15 ml) collagenase (151 units/mg

Worthington Biochemical Corp., Freehold, NJ.) 4.5, bovine serum albumin 30, trypsin

inhibitor 30. ATP (sodium salt) 1.7. and protease (type XXIV, Sigma Chemical Co., St.

Louis, Mo.) 1.5. After the digestion, the pieces were removed from the digestion buffer,

rinsed in Ca>-free PSS. and gently triturated until a large number of elongated smooth

muscle cells were observed. The isolated cells were collected and stored at 4C until

used. Cells were used between 2 and 10 hrs. after isolation.

Current Recording and Analysis

Single cells were voltage-clamped, and membrane currents were measured using

the whole-cell patch-clamp technique. Patch pipettes were made from borosilicate glass

capillaries, pulled on a vertical puller (PP-83. Narishige Scientific Instrument Laboratory,

Tokyo), and fire-polished with a microforge (model MF-83, Narishige); The pipettes had

resistances of 3-5 MQ when filled with the pipette solution.







Voltage-clamp command potentials were applied to the cells and membrane

current recorded using an Axopatch-ID patch-clamp amplifier (Axon Instruments,

Burlingame, CA ). Membrane current was digitized on-line (10.0 kHz) with in analog-to-

digital interface (Labmaster TL-1 DMA interface, Axon Instruments) and filtered at 2.0

kHz. Data analysis was performed with pCLAMP 6.0 software (Axon Instruments). All

experiments were performed at room temperature.

Tension Measurements

A segment of the rat renal artery was dissected free and cleaned of fat and

adventitia. Ring segments (3 mm long) were mounted onto two triangular tungsten wires

(35 gm in diameter) and hung vertically in an isolated organ chamber (10 ml). The

bottom triangle was mounted to a stable hook while the top triangle was attached to a

Gould strain gauge. The bath was maintained at 37C in PSS. A resting force of 1.5 g was

applied to the vessels. Vessel segments were equilibrated for 1 hr with three exposures to

phenylephrine (1 imol/L). KCL, Phe, and ACh dose response curves were performed in a

cumulative manner.

Assessment of Ventricular Hypertrophy and Cardiac and Perivascular Fibrosis

Animals were sacrificed, hearts excised, rinsed in 0.9% physiological saline, and

total heart and ventricular weights were recorded. These weights were normalized to

body weight (BW in g), and the ratios were compared among groups. In a separate series

of experiments, the heart, liver, kidney, and adrenal gland were removed during deep

anesthesia after arresting contraction by left ventricular injection of saturated KC1. These

tissues were fixed in 3% freshly prepared paraformaldehyde in 0.15 mol/L phosphate







buffer and stored at 4C for 24-72 hrs and then placed in phosphate buffer with 0.25

mol/L sucrose added (approximately 300 mOsM solution) at 4C. Slices of right and left

ventricles, liver, kidney and adrenal were dehydrated through a graded series of ethanol

and xylene, and embedded in paraffin. Sections were cut at 5 tm thickness, stained with

hemoatoxylin, eosin, and with aldehyde fuchsin Gomori trichrome and examined by light

microscopy. Heart tissue was qualitatively evaluated for type (perivascular, focal or

interstitial) and distribution (left or right ventricle, subendocardial, subepicardial) of

connective tissue.

Solutions

The PSS used during cell isolation procedures and contraction experiments

contained in mmol/L: NaCl 120, KC1 4.2, KHPO4 1.2, MgCI2 0.5, CaCI2 1.8, glucose

5.5, and HEPES 30 (pH 7.35 with NaOH). For all whole-cell voltage-clamp experiments

examining Kv currents, the bath solution contained in mmol/L: NaCl 125, KC1 4.2,

KH2PO4 1.2, MgCl22.3, D-glucose 5.5, TEA 5.0, and HEPES 30 (pH 7.4 with NaOH).

The pipette solution for the whole-cell experiments contained in mmol/L: KC1 140,

MgCL 0.5. EGTA 1.0. ATP (magnesium salt) 5.0, and HEPES 5.0 (pH 7.2 with KOH).

Statistics

Results are expressed as mean SEM. Statistical significance was evaluated

using repeated measures ANOVA and Student's T-test for unpaired data. Differences

were considered significant at P<0.5. Membrane currents were measured from the zero

current level and normalized to cell capacitance.







Results

Effect of ATR-AS Treatment on Blood Pressure

A single intracardiac injection of LNSV-ATR-AS to neonatal rats prevented the

increase in BP exclusively in SHRs at 120 days post-injection (fig. 4-1). The mean BP

was 28.9% lower in SHRs treated with LNSV-ATR-AS than in untreated SH rats (116.3

+ 16.6 vs. 163.7 + 6.0 mmHg; n=6, P< 0.05). In addition the mean BP measured in

LNSV-ATIR-AS treated SH rats was not significantly different from that of the

normotensive WKY control rats (116.3 16.6 vs. 115.6 8.3 mmHg; n=6, P>0.05). This

effect was specific for LNSV-ATR-AS treatment since treatment with LNSV alone had

no significant effect on mean BP compared with control SHRs (n=6). Antisense treatment

or vector alone did not affect the BP of WKY rats (n=6).

Effect of LNSV-ATR-AS Treatment on Renal Vascular Reactivity

Alterations in vascular contractile response are known to exist in the SHRs

(Triggle & Laher, 1985; Bohr & Webb, 1988). LNSV-ATR-AS treatment prevented the

alterations in renal artery reactivity measured in the SHRs. Enhanced contractile

responses to both potassium chloride (KC1. Fig. 4-2) and phenylephrine (Phe, Fig. 4-3)

were observed in the SHR model. A leftward shift in the KCI and Phe concentration-

response relationships were observed in SHRs (Figs. 4-5A and B) when compared to

WKY controls. The medium effective concentrations (EC50) for KC1 and Phe were 31.0%

and 46.8% lower (n=6) in the SHR (24 mmol/L. KC1 and 202 nmol/L, Phe) than in

WKY(35 mmol/L, KC1, and 380 nmol/L. Phe) rats, respectively. The shifts in the

concentration-response relationships for KC1 or Phe were not observed in LNSV-ATR-







AS treated SHRs (EC50: 34 mmol/L, KC1 and 470 nmol/L, Phe) and LNSV alone (EC50:

26 mmol/L, KC1 and 132 nmol/L, Phe) did not change the renal artery contractile

properties to KC1 or Phe. Finally, an impaired endothelial-dependent relaxation of pre-

contracted renal arteries was also observed in the SHR (Fig. 4-4). When the arteries were

pre-contracted with Phe (500 nmol/L), the ACh dose response curve was right shifted

and the maximal relaxation was decreased 36% (n=6) in the renal artery from the SHR

(Fig.4-5C). The EC50 value for ACh-induced relaxation in SHR (143 nmol/L) renal artery

was increased 4.7 fold compared to WKY (30 nmol/L). This shift was not seen in the

LNSV-ATR-AS treated animals (73 nmol/L). However it was still present in SHR

treated with LNSV alone (196 nmol/L).

Effect of LNSV-AT,R-AS on Kv Current Density

We have previously shown that Kv current density was decreased in vascular

smooth muscle cells from the interlobar arteries of the SHR when compared to WKY

control (see chapter 3). Figure 4-6 illustrates the characteristics of Kv current in single

cells isolated from renal resistance vessels of WKY and SHRs. During voltage step

depolarizations in the presence of 10 mmol/L TEA (to block the K,, ), Kv current

displayed two components. The first is a rapidly activating and inactivating component

which we termed the peak component of Kv current. The second is a slow inactivating

sustained component.

Under the above conditions, the Kv current was reduced in cells isolated from the

SHR. This decrease was not evident in SHRs treated with LNSV-ATR-AS but was

apparent in those treated with LNSV alone. Figure 4-7 shows the mean current voltage

relationship for both the peak (Fig. 4-7A) and sustained (Fig. 4-7B) component of Kv







current. In each experiment the Kv current, normalized to cell capacitance, was

significantly less in the cells from SHRs as compared to those obtained from WKY

controls. Again, this decrease was not observed in cells from LNSV-ATR-AS treated

SHRs. It should be noted that the cell capacitance was not significantly different between

all of the groups tested.

Effect of LNSV-ATR-AS on Heart Weights and Cardiac Morphology

Ventricular weights were recorded for all groups and normalized to body weight.

The ventricular weights of SHRs treated with LNSV alone were significantly greater than

the weights recorded for the normotensive WKY (Table 4-1). This increase was not seen

in SHRs treated with LNSV-AT]R-AS. There was no difference in the weights of the

LNSV-treated and LNSV-ATR-AS-treated WKY ventricles. Figure 4-8 shows the

effect of LNSV-ATR-AS on cardiac and perivascular fibrosis. Panel A shows

photomicrographs of representative sections taken from left ventricular

subendomyocardiurn from (top) WKY, SHR and LNSV-ATR-AS-treated SHRs,

respectively. Multiple focal areas of fibrosis were evident in hearts from SHRs (indicated

by arrows). This phenomenon was not seen in hearts from the WKY or LNSV-ATR-

AS-treated SHRs. Panel 4-8B depicts series of photomicrographs of sections from mid-

myocardium showing arterioles of less than 100 4m in diameter. Intramyocardial small

coronary arteries and arterioles from SHRs displayed dense perivascular fibrosis. This

was not seen in coronary vessels from WKY or LNSV-ATR-AS-treated SHRs. No

lesions were present in liver, kidney, or adrenal tissues from any animal.







Discussion

The results of this study demonstrate that delivery of ATR-AS prevents the

development ofhemodynamic and pathophysiological alterations associated with

hypertension in the SHR. Neonatal SHRs which were given a single intracardiac

injection of ATR-AS did not develop an elevation in mean BP, altered renal vascular

reactivity, decreased smooth muscle Kv channel current density, left ventricular

hypertrophy, or cardiac and perivascular fibrosis. These novel data suggest that

interruption of AT, receptor in the developing SHR, using a virally-mediated gene

delivery system, may be used to prevent hypertension and its associated renal and

cardiovascular risk factors.

The role of the kidney in the control of fluid volume and BP implies its

participation in the development of essential hypertension. The results of earlier renal

transplant studies in humans and animals suggest that in a proportion of hypertension

cases there exists structural or functional alterations in the kidney ( Dahl et al., 1972;

Kawabe et al., 1978; Curtis et al., 1983; Rettig et a!., 1990). In addition, evidence

indicates that regulation of renal blood flow is an important element of the hypertensive

process. Multiple studies have demonstrated that in hypertension there exists an

enhancement in renal vascular tone ( Guyton el a!., 1990; Ruilope et al., 1994). This

elevation in tone may lead to increased renal vascular resistance, a common observation

in hypertension, and an important factor in shifting pressure natriuresis and elevating BP.

Possible mechanisms responsible for the increase in renal vascular resistance include: (1)

an enhanced contractile sensitivity to vasoactive agonists; (2) an impaired endothelial

dependent relaxation; (3) an increased Ca'2 transport across vascular smooth muscle







membranes; (4) an altered ion channel activity in vascular smooth muscle: and (5)

smooth muscle hypertrophy or hyperplasia (Khalil et al., 1989). Our results show that

both KC1 and Phe produced an enhanced contractile response in renal arteries of SHRs.

However, this was not observed in SHRs treated with ATR-AS. The augmented

contractile response to vasoactive agents appears to depend on the vascular bed tested and

the type of vasoconstrictor agent (Berecek et al., 1980; Silva et al., 1996). However, this

does not diminish the significance of the result that alterations in both electro-mechanical

coupling, which is membrane potential-dependent (KC1), and pharmaco-mechanical

coupling, which is agonist-dependent (Phe). were prevented with AT)R-AS treatment.

A second mechanism which may contribute to increased renal vascular resistance

and/or decreased renal blood flow is endothelial dysfunction. It is well established that

endothelial-derived vasoactive agents regulate blood flow through vessels by controlling

the contractile state of vascular smooth muscle (VSM) cells (Vanhoutte et al., 1986).

One such substance, nitric oxide (NO), is released by the endothelium to cause relaxation

of the VSM cells (Furchgott & Zawadzki, 1980). It has recently been reported that ACh

induced relaxation of coronary arteries is mediated by endothelial-derived NO (Simko,

1994). Our results show that there exists an impaired endothelial dependent relaxation to

ACh in the renal artery of SHRs. This alteration is prevented in SH rats treated with

ATR-AS. Reports of altered endothelial function appear to depend on a number of

factors including vascular bed, model of hypertension, and constrictor agent used (

Luscher et al.. 1988: Li & Bukoski. 1993: Fuchs el al., 1996). Some reports suggest that

the altered endothelial relaxation in hypertension is not due to impaired NO release but

rather to changes in release of vasoconstricting prostaglandins ( Luscher et al., 1988; Li







& Bukoski, 1993). Regardless of the mechanism, our data demonstrate an impaired

endothelial relaxation in the SH rats which was prevented with ATR-AS treatment.

A third mechanism by which renal vascular resistance may be increased in

hypertension is altered ion channel function in VSM cells. It has been demonstrated that

Kv current modulates membrane potential (Fleischmann et al., 1993; Knot & Nelson,

1995). Tonic changes in membrane potential regulate Ca2 influx and hence contractile

tone in VSM cells. Blockade or reduction of this current may lead to increases in tone

and ultimately to an increase in vascular resistance. There is substantial evidence for

electrophysiological changes in hypertension (Hermsmeyer, 1976; Rusch & Hermsmeyer,

1988; Rusch et al.. 1992: England et al.. 1993: Ohya et al., 1993; Wilde et al., 1994).

We have previously demonstrated that the Kv current is decreased in rat renal resistance

vessels from both a genetic and nongenetic model of hypertension (see chapter 3). Herein

we show that this alteration can be prevented in the SHR by treatment with ATR-AS.

The parallel between the prevention of the Kv current alterations and normalization of

blood pressure suggests that, in hypertension, the alterations in VSM cell K' channel

function represents feedback mechanisms occurring secondary to the change in arterial

blood pressure (Rusch & Runnells. 1994) or distal to a RAS triggered event.

Cardiovascular ultrastructural changes are a major risk factor for morbidity and

mortality in hypertension. Ventricular hypertrophy is a compensatory response to the

increased pressure load attributable to the elevation in peripheral resistance (Vogt et al.,

1993). While traditional pharmacological therapies have been shown to be effective in

controlling and at times reversing these pathophysiological changes, there are often times

when this approach is not successful ( de Celentano et al., 1993; Vogt et al.. 1993). It is







important to note that regression of LVH has been reported in only 50% of patients

treated with traditional antihypertensive drugs and that it is difficult to determine which

patients will benefit from these therapies (de Celentano et al., 1993). Combination

therapies may improve benefits due to the additive BP lowering effects of some

antihypertensive agents. However, this may also increase the number and intensity of

undesirable side effects and hence further decrease patient compliance. We found an

increase in ventricular mass in SHRs treated with LNSV vector alone. However, this

ventricular hypertrophy was prevented with ATR-AS treatment. Other structural

alterations (i.e. cardiac and perivascular fibrosis) were also prevented with ATR-AS

treatment.

Finally, evidence suggests that simply normalizing the BP by pharmacological

means is not sufficient to completely regress pathophysiological changes associated with

the hypertension. ATR-AS gene therapy may be superior. It results in the prevention of

the increase in mean BP and the associated pathophysiological impairments in

hypertension. It may also offer an alternative to the compliance problem and

complications of vascular and target-organ injury. Finally, the ATR-AS therapy does

not produce a significant increase in plasma Ang II levels compared with the

antihypertensive effects of losartan, an AT, receptor antagonist (Lu & Raizada, 1995).

Therefore ATR-AS therapy may have prolonged antihypertensive effects without the

possible adverse side effects of elevated plasma Ang II.

A role for RAS in the development or maintenance of hypertension is well

established ( Berecek et al., 1987; Phillips. 1987; MacGregor. 1992; Wu & Berecek,

1993; Kang et al., 1994). Our results confirm a fundamental role for AT, receptors in







both the development of high BP and the production of pathological organ damage. The

interruption of the RAS pathway at an early age using AT|R-AS offers the potential to

prevent the development of hypertension and its associated pathophysiological alterations

with a single antisense treatment. Of course this approach depends on the identification

of genetic determinants of hypertension or upon the demonstration of reliable pre-

hypertensive risk factors. Studies are now underway to determine if this gene delivery

approach can be used to reverse the hemodynamic and pathophysiological alterations in

the adult hypertensive animal.









TABEL 4-1. Ventricular weights normalized to body
weights in WKY and SHR


Group n VW (g) VW/BW (mg/g)

WKY +ATR-AS 4 1.05 0.11 3.2 0.09


WKY + LNSV 3 1.07 0.20 3.0 0.32


SHR + ATR-AS 6 1.14 0.08 3.6 0.13


SHR+LNSV 3 1.54 0.15* 4.8 0.85*


Values are means SE, VW=ventricular weight,
BW= body weight, *P<0.05













180-



160-



140-


-5-
E
E
rE
(D

0)
0_
")
cCl
a)
V
0
0

a-
CD


100-


* *


Control
LNSV
LNSV+AT1R-AS
**P<0.01 vs WKY control
* P< 0.05 vs WKY control


I_


WKY


* *


SHR


Figure 4-1. Effect of LNSV-ATiR-AS treatment on mean blood pressure in WK
and SH rats. Five-day-old WKY and SH rats treated with physiological saline
(control), LNSV, LNSV+ATiR-AS were allowed to mature for 120 days. Mean
blood pressure was measured though catheters implanted in the carotid artery. A
single injection of LNSV+AT1R-AS into neonatal SH rats prevented the increase
in BP. There was not a significant effect of LNSV or LNSV+AT1R- AS on the
mean BP in the WKY. Data are expressed as mean SE (n=6).


120-1









KCI (mM)

90 120
60



WKY



i i i
SHR




SHR + AT,1R-AS




SHR + LNSV 4 grams

20 min


Figure 4-2. Prevention of the alteration in the KCL concentration-response
relationship of rat renal artery in hypertension with AT1R-AS. Shown are
representative examples of contractions in the rat renal artery from WKY, SHR,
SHR + AT1,R-AS, and SHR + LNSV. Arrows indicate time of intervention.







Phe (500 nM)


5000
10 50 100 500 10001
I I I -


WKY


1 I I


SHR


SHR + ATR-AS


I I I


SHR + LNSV


6 grams


15 min

Figure 4-3. Prevention of the alteration in the Phe concentration-response
relationship of rat renal artery in hypertension with AT1R-AS. Shown are
representative examples of contractions in the rat renal artery from WKY, SHR,
SHR + AT1iR-AS, and SHR + LNSV. Arrows indicate time of intervention.








Phe (500 nM)


10 nM 100 nM
I I


500 nM
I


1 PM
1 10PM


ATR-1 I

AT1R-AS ) I,


SHR + LNSV
1 gram
30 min

Figure 4-4. Prevention of the alteration in the acetylcholine concentration-
response relationship of rat renal artery in hypertension with ATjR-AS. Shown
are representative examples of contractions in the rat renal artery from WKY,
SHR, SHR + AT1,R-AS, and SHR + LNSV. Vessels were pre-contracted with
Phe. Arrows indicate time of Ach intervention


WKY



SHR


SHR +





85


A 120-
X
M 100-
80-
60- WKY
6 / SHR
40- SHR + AT1R-AS
20- SHR + LNSV
0- S_________
cr

Con 10 30 60 90120
[KCI, mM]

S 100-
x
M 80-
E
S 60- /WKY
o 40- / SHR
"t SHR + ATjR-AS
20- SHR + LNSV
O 0-
(.)
-9 -8 -7 -6 -5

log [Phe, M]

C 100-
80-
C
60-
X 1 WKY
40- SHR
X ^ 20-- XSHR + AT1R-AS
: SHR + LNSV
0-
-10 -9 -8 -7 -6 -5 -4
log [Ach, M]


Figure 4-5. Mean concentration-response relationship to KCI (A), Phe (B) and
ACh (C) in rat renal artery. The Phe and KCI dose response curve was shifted to
the left in SHR (0) and SHR + LNSV (*) when compared to WKY (*) and SHR +
AT1R-AS (A). The ACh dose response curve was shifted to the right and the
maximal relaxation was decreased in the SHR and SHR + LNSV when
compared to WKY and SHR + AT1R-AS. This shift was prevented in all
intervention with delivery of ATR-AS. Data are presented as mean SE (n=6).














80 mV
8i20 mV
-80 mV -----

WKY SHR








SHR + AT1R-AS SHR + LNSV









400pA
600ms



Figure 4-6. Characteristics of Kv current in single vascular smooth muscle cells
isolated from rat renal resistance arteries. The Kv current was reduced in cells
from SHR and SHR + LNSV-treated rats. Delivery of AT1R-AS prevented this
decrease. The voltage protocol is displayed at the top. The membrane
capacitances of all 4 cells were similar (WKY, 28 pF; SHR, 28 pF; SHR+AT1R-
AS, 29 pF; SHR+LNSV, 28 pF).




87


A
40
WKY
36 K SHR
32 A SHR + AT1 R-AS
28 V SHR + LNSV
28
:" 24 0
i 20 A
o 16
S 12 A*
8 A. -
4 lr
0
-80 -60 -40 -20 0 20 40 60 80
Membrane Potential (mV)


B
18 0 WKY
16 I SHR
U-_ 14 ASHR+AT1R-AS
vSHR +LNSVA
~12 A
- 10 A
Ao "















0
A *S *C

-80 -60 -40 -20 0 20 40 60 80

Membrane Potential (mV)











Figure 4-7. Mean current-voltage relationships for peak (A) and sustained (B)
B




















components of Ky current. There was no significant difference in current density
18 WKY
16 SHR









in the WKY (0, n7) and SHR+ATR-AS (A, n=6) treated rats. However, the KS









current density for both the peak and sustained components was significantly
lower in the SHR (, n=12) and SHR+LNSV treated (, n6) animals.
i 12
"E 10 A 0
8 _ .
"I 6 A i.
4 i O Rill
03 2 I i: !
o eef-ele

-80 -60 -40 -20 0 20 40 60 80
Membrane Potential (mV)


Figure 4-7. Mean current-voltage relationships for peak (A) and sustained (B)
components of Kv current. There was no significant difference in current density
in the WKY (*, n=7) and S.HR+AT1R-AS (,A, n=6) treated rats. However, the Kv
current density for both the peak and sustained components was significantly
lower in the SHR (*., n=12) and SHR+LNSV treated (V, n=6) animals.








SHR


WKY
PP701JgE-Now


SHR + AT1R-AS
I IM M u


Figure 4-8. Effect of AT1R-AS on cardiac and perivascular fibrosis. (A) Photomicrographs of
sections taken from left ventricular subendomyocardium from WKY, SHR, and SHR+ATR-AS,
respectively. Photographs were 50X magnification at 4X obj. Calibration bar 12 mm = 250 lm.
Arrows in SHR panel indicate multiple focal areas of fibrosis. This phenomenon was not seen in
hearts from WKY or SHR+AT1R-AS. (B) Sections from mid myocardium showing small arteries
and arterioles (<100 -im diameter) from WKY, SHR and SHR+AT1R-AS treated hearts.
Photographs were taken at X308 magnification at X40 objective. Calibration bar 15 mm = 50 Pm.
Vessels from SHR demonstrated dense perivascular fibrosis. This was not seen in vessels from
WKY or AT1,R-AS treated rats.


A













CONCLUSIONS

Results of this study characterize the Kv current in rat renal resistance arterioles

and provide the first in vivo evidence for heteromeric formation of K channels in

vascular smooth muscle. In addition, this study details changes in Kv current and

membrane potential in genetic and nongenetic models of hypertension. Finally, this work

demonstrates that the decrease in Kv current density as well as other renovascular and

cardiac pathophysiological alteration in hypertension, can be prevented using AT,

receptor antisense gene therapy.













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