The Pharmacology & Therapeutic Potential of Kv7 Channels in The

The Pharmacology & Therapeutic Potential of Kv7

Channels in The Pulmonary Circulation

A thesis submitted to the University of Manchester for the degree of

Doctor of Philosophy (PhD.) in the Faculty of Life Sciences

2013

BASMA GHAZI EID

List of Contents 2 Table of Contents 2

List of Figures 6

List of Tables 11

Abstract 12

Declaration 13

Acknowledgements 14

List of Abbreviations 15

Table of Contents Chapter 1: General Introduction

1.1 The pulmonary circulation ...... 21

1.2 Regulation of pulmonary artery tone ...... 22

1.3 The regulation of intracellular Ca2+ ...... 22

1.4 Hypoxic pulmonary vasoconstriction ...... 24

1.5 Membrane properties of PASMCs ...... 29

1.6 K+ channels in the pulmonary vasculature ...... 31

1.7 Kv7 potassium channels ...... 39

1.7.1 Structure of Kv7 channels ...... 40 1.7.2 Differential expression of KCNQ genes in smooth muscle organs ...... 40 1.7.3 Properties of Kv7 currents ...... 42 1.7.3.1 Homomultimers ...... 43 1.7.3.2 Heteromultimers & modulatory subunits ...... 44 1.7.4 Kv7 channel pharmacology ...... 45 1.7.4.1 Kv7 channel blockers ...... 45 1.7.4.2 Kv7 channel activators ...... 47 1.8. Evidence that Kv7 channels have a role in PASMCs ...... 51

1.9 Aims ...... 58

Chapter 2: Kv7 Channels in The Hypertensive Pulmonary Circulation

2.1 Animal models of pulmonary hypertension ...... 59

2.2 Mechanism of monocrotaline toxicity ...... 61

2.3 Alterations of endothelium & smooth muscle function ...... 63

2.4 Hypothesis and aims ...... 64

2.5 Methods...... 66

2.5.1 Contractile studies ...... 66 2.5.1.1 Tissue preparation ...... 66 2.5.1.2 Protocols used to study drug effects on PA tone ...... 67 2.5.1.3 Protocols used to study drug effects on pre-constricted PA ...... 70 2.5.1.4 Drugs ...... 70 2.5.1.5 Statistical analysis ...... 71 2.5.2 Molecular biology ...... 72 2.5.2.1 Polymerase chain reaction ...... 72 2.5.2.2 Tissue preparation ...... 72 2.5.2.3 Protocol for RNA isolation ...... 72 2.5.2.4 Protocol for first strand cDNA synthesis (reverse transcription) ...... 74 2.5.2.5 Primer design ...... 74 2.5.2.6 Gel electrophoresis ...... 75 2.5.2.7 Purification of PCR products ...... 77 2.5.2.8 Real time PCR ...... 78 2.5.2.9 Reference targets/housekeeping genes ...... 82 2.5.2.10 Analysis of KCNQ genes ...... 82 2.5.2.11 Factors influencing qPCR accuracy ...... 83 2.5.3 Structural characteristics of pulmonary arteries in MCT-induced PH .... 87 2.6 Results ...... 90

2.6.1 Effects of MCT on vessel responses to Kv7 channel blockers ...... 90 2.6.2 Effects of MCT on the responses of preconstricted vessels to Kv7 channel activators ...... 97 2.6.3 Intrinsic Tone ...... 103

2.6.4 Effects of membrane potential on vessel recovery from a contractile stimulus ...... 116 2.6.5 Effects of depolarization on contractile response to Kv7 modulators .... 117 2.6.6 KCNQ mRNA expression in monocrotaline and control pulmonary arteries ...... 124 2.6.7 Establishing house-keeping genes for real-time PCR ...... 124 2.6.8 KCNQ mRNA quantification by real-time PCR ...... 125 2.7 Discussion ...... 133

2.7.1 MCT enhances sensitivity to Kv7 channel blockers ...... 133 2.7.2 The effect of MCT on responses of preconstricted vessels to Kv7 channel activators ...... 134 2.7.3 The effect of vasodilators on intrinsic tone ...... 135 2.7.4 The effect of MCT on vessel recovery after a stimulus ...... 137 2.7.5 Membrane potential influences vessel responses to Kv7 modulators .... 139 2.7.6 MCT does not alter KCNQ mRNA expression in pulmonary and mesenteric arteries ...... 141 2.7.7 Conclusion ...... 142 Chapter 3: The Pharmacology of ZnPy in The Pulmonary Circulation

3.1. Introduction ...... 143

3.2 Identification of ZnPy as an opener of Kv7 channels ...... 143

3.3 The characterization of ZnPy modulation in expressed Kv7 channels ...... 145

3.4 Hypothesis and Aims ...... 146

3.5. Methods ...... 147

3.5.1 Patch clamp electrophysiology ...... 147 3.5.1.1 Tissue preparation ...... 147 3.5.1.2 PASMC isolation protocol for electrophysiology ...... 148 3.5.1.3 The patch clamp technique ...... 148 3.5.1.4 Preparation of pipettes and solutions ...... 150 3.5.1.5 Whole-cell recording ...... 154 3.5.1.6 Membrane potential recordings ...... 156 3.5.1.7 Current recordings ...... 157 3.5.1.8 Data analysis ...... 157

3.5.1.9 Limitations and problems in patch-clamp recordings ...... 158 3.5.2 Myography contractile studies ...... 159 3.5.2.1 Protocols used to study the effects of ZnPy ...... 159 3.5.2.2 Protocols used to study the effects of various blockers on the ZnPy induced relaxation ...... 160 3.5.2.3 Protocols used to study the mechanism of the ZnPy-induced relaxation ...... 160 3.5.2.4 Drugs ...... 160 3.6 Results ...... 162

3.6.1 Patch clamp electrophysiology ...... 162 3.6.1.1 Properties of rat PASMCs ...... 162 3.6.1.2 The effect of ZnPy on the membrane potential of PASMCs ...... 162 3.6.1.3 Effects of ZnPy on the voltage-activated K+ current in PASMCs ..... 171 3.6.1.4 Effects of ZnPy on the residual K+ current at 0 mV in PASMCs ...... 172 3.6.2 Myography contractile studies ...... 181 3.6.2.1 ZnPy-induced vasodilation ...... 181 3.6.2.2 Mechanism of ZnPy-induced relaxation ...... 181 3.6.2.3 The contribution of K+ channels to the ZnPy-induced dilation ...... 185 3.7 Discussion ...... 198

3.7.1 The effects of the Kv7 activator ZnPy on the pulmonary circulation ..... 198 3.7.2 Mechanism of ZnPy-induced vasodilation ...... 198 3.7.3 Mechanism of ZnPy-induced hyperpolarization ...... 200 3.7.4 Effects of ZnPy on K+ currents ...... 201 3.7.5 ZnPy and Kv7 channels ...... 203 3.7.6 Conclusion ...... 204 Chapter 4: Summary and Future Directions

4.1. Kv7 channels in the monocrotaline rat model of pulmonary hypertension . 205

4.2 The effects of the Kv7 activator ZnPy on the pulmonary circulation ...... 207

4.3 Concluding remarks ...... 209

Word Count: 57, 384

List of Figures:

Figure 1.1 The pulmonary circulation...... 23

Figure 1.2 Ca2+ mobilization in PASMCs...... 25

Figure 1.3 Ca2+ mobilization in HPV...... 28

Figure 1.4 Resting conductances in PASMCs...... 30

Figure 1.5 K+ channels in the pulmonary circulation...... 33

Figure 1.6 The non-inactivating current IKN...... 36

Figure 1.7 The structure of Kv7 channel subunits...... 41

Figure 1.8 Chemical structures of Kv7 channel modulators...... 53

Figure 1.9 Molecular determinants of ZnPy and retigabine sensitivity...... 54

Figure 1.10 Kv7 modulation in PASMCs...... 57

Figure 2.1 Monocrotaline toxicity...... 62

Figure 2.2 Changes in pulmonary artery behavior after MCT induced PAH... 65

Figure 2.3 Intrapulmonary artery preparation...... 68

Figure 2.4 Myography set-up...... 69

Figure 2.5 SYBR green detection of PCR amplification products...... 80

Figure 2.6 A typical amplification plot in a qPCR reaction...... 81

Figure 2.7 Bioanalysis of RNA integrity...... 85

Figure 2.8 Standard curve showing 100% PCR efficiency...... 86

Figure 2.9 Histological changes associated with MCT-induced PH...... 88

Figure 2.10 The elastic lamina in MCT-induced PH...... 89

Figure 2.11 The effect of linopirdine on pulmonary arteries from control and

MCT-treated animals...... 92

Figure 2.12 The effect of XE991 on pulmonary arteries from control and

MCT-treated animals...... 93

Figure 2.13 The effect of XE991 on mesenteric arteries from control and 94

MCT-treated animals......

Figure 2.14 The effect of chromanol 293B on pulmonary arteries from control

and MCT-treated animals...... 95

Figure 2.15 The effect of nifedipine on the XE991-induced constriction of

pulmonary artery...... 96

Figure 2.16 The effect of retigabine on pulmonary arteries from control and

MCT-treated animals...... 99

Figure 2.17 The effect of retigabine on mesenteric arteries from control and

MCT-treated animals...... 100

Figure 2.18 The effect of ZnPy on pulmonary arteries from control and MCT-

treated animals...... 101

Figure 2.19 The effect of BMS-204352 on pulmonary arteries from control

and MCT-treated animals...... 102

Figure 2.20 The effect of nifedipine on the basal tone of pulmonary arteries

from control and MCT-treated animals...... 106

Figure 2.21 The effect of retigabine on the basal tone of pulmonary arteries

from control and MCT-treated animals...... 107

Figure 2.22 Retigabine maximally abolished intrinsic tone...... 108

Figure 2.23 The effect of retigabine on the basal tone of mesenteric arteries

from control and MCT-treated animals...... 109

Figure 2.24 The effect of ZnPy on the basal tone of pulmonary arteries from

control and MCT-treated animals...... 110

Figure 2.25 The effect of levcromakalim on the basal tone of pulmonary

arteries from control and MCT-treated animals...... 111

Figure 2.26 The effect of sildenafil on the basal tone of pulmonary arteries

from control and MCT-treated animals...... 112

Figure 2.27 The effect of 2-ABP on the resting tone of pulmonary arteries

from MCT- treated animals...... 113

Figure 2.28 The effect of 2-ABP on preconstricted pulmonary arteries from

MCT-treated animals...... 114

Figure 2.29 The effect of vasodilators on the intrinsic tone of pulmonary

arteries from MCT-treated animals...... 115

Figure 2.30 Slowed recovery rates from 50mM KCl challenge in MCT

pulmonary arteries...... 118

Figure 2.31 Hyperpolarization enhances the rate of recovery of monocrotaline

pulmonary arteries...... 119

Figure 2.32 The effect of depolarization on the rate of recovery of control

pulmonary arteries...... 120

Figure 2.33 The effect of depolarization on Kv7 modulator activity...... 122

Figure 2.34 The effect of hyperpolarization on Kv7 modulator activity...... 123

Figure 2.35 KCNQ expression in pulmonary artery...... 126

Figure 2.36 Standard curves for HKGs...... 127

Figure 2.37 Dissociation curves for HKGs...... 128

Figure 2.38 GeNorm analysis of HKGs...... 129

Figure 2.39 Standard curves for KCNQ genes...... 130

Figure 2.40 Dissociation curves for KCNQ genes...... 131

Figure 2.41 Expression profile of KCNQ genes...... 132

Figure 2.42 Implications of depolarization on Kv7 and NCX function in

PASMCs...... 140

Figure 3.1 The concept of voltage-clamp...... 151

Figure 3.2 Patch-clamp configurations...... 152

Figure 3.3 An electronic model of the cell membrane...... 153

Figure 3.4 Equivalent circuit for the whole-cell patch clamp configuration.... 153

Figure 3.5 Capacitative and ionic currents in patch clamp...... 155

Figure 3.6 ZnPy-induced hyperpolarization of rat PASMCs...... 163

Figure 3.7 Effect of glibenclamide (10 µM) on The ZnPy-induced

hyperpolarization of rat PASMCs...... 164

Figure 3.8 Effect of TEA (10 mM) on The ZnPy-induced hyperpolarization of

rat PASMCs...... 167

Figure 3.9 The effect of paxilline (1 µM) on the ZnPy-induced

hyperpolarization of PASMCs...... 168

Figure 3.10 The effect of iberiotoxin (50 nM) on the ZnPy-induced

hyperpolarization of PASMCs...... 169

Figure 3.11 The effect of XE991 (10 µM) on the ZnPy-induced

hyperpolarization of PASMCs...... 170

Figure 3.12 The effects of ZnPy on the currents induced by a voltage step

from -80 to +40 mV...... 173

Figure 3.13 Time-course of the effect of ZnPy on the voltage-activated K+

current induced by a voltage step from -80 to +40 mV...... 174

Figure 3.14 The effects of paxilline (1 µM) on the ZnPy-induced currents...... 175

Figure 3.15 The effects of iberiotoxin (50 nM) on the ZnPy- induced currents. 176

Figure 3.16 The effects of XE991 (10 µM) on the ZnPy-induced currents...... 176

Figure 3.17 The voltage-dependence of ZnPy effects on the voltage activated

K+ current in PASMCs...... 177

Figure 3.18 Effect of ZnPy on the residual current recorded at 0 mV...... 179

Figure 3.19 Effect of ZnPy on the current recorded during voltage ramps 180

from 0 mV......

Figure 3.20 ZnPy provokes concentration-dependent relaxation of rat

pulmonary arteries...... 182

Figure 3.21 The effects of NaPy on rat intrapulmonary artery...... 183

Figure 3.22 The effect of ZnCl2 on rat intrapulmonary artery...... 184

Figure 3.23 The effects of ZnPy after preconstriction by different mechanisms 186

Figure 3.24 Effect of preconstricting agents on the response of PA to ZnPy..... 187

Figure 3.25 ZnPy provokes a concentration-dependent relaxation of

pulmonary arteries in the presence of TEA (10 mM)...... 191

Figure 3.26 ZnPy provokes a concentration-dependent relaxation of

pulmonary arteries in the presence of paxilline (1 µM)...... 192

Figure 3.27 ZnPy provokes a concentration-dependent relaxation of

pulmonary arteries in the presence of iberiotoxin (50 nM)...... 193

Figure 3.28 ZnPy provokes a concentration-dependent relaxation of

pulmonary arteries in the presence of 4-AP (1 mM)...... 194

Figure 3.29 ZnPy provokes a concentration-dependent relaxation of

pulmonary arteries in the presence of glybenclamide (10µM)...... 195

Figure 3.30 ZnPy provokes a concentration-dependent relaxation of

pulmonary arteries in the presence of XE991...... 196

Figure 3.31 Concentration-response curve for ZnPy in the presence of XE991 197

List of Tables:

Table 1.1 Pore-Forming K+ Channel α-subunits...... 32

Table 1.2 Modulatory K+ Channel β-subunits...... 31

Table 1.3 Kv7 channel blockers...... 46

Table 1.4 The selectivity and mechanism of action of Kv7 channel

activators...... 52

Table 2.1 Composition of PSS...... 68

Table 2.2 Drugs used to study vessel tone in the MCT model of PH...... 71

Table 2.3 KCNQ Primers...... 76

Table 2.4 House-keeping Gene Primers...... 76

Table 2.5 PCR reaction composition...... 77

Table 2.6 House-keeping genes and their roles in cells...... 83

Table 3.1 Composition of dissociation medium (DM) and pipette solution.... 147

Table 3.2 Drugs used to study the effects of ZnPy on the pulmonary

circulation...... 161

Abstract: The Pharmacology & Therapeutic Potential of Kv7 Channels in The Pulmonary Circulation. (The University of Manchester, Basma Ghazi Eid, Doctor of Philosophy (PhD.), 2013)

Pulmonary arterial tone is regulated in part by the membrane potential (Em) of pulmonary artery smooth muscle cells (PASMCs). The Kv7 family of K+ channels was recently implicated in regulating Em in rat PASMCs and expression of KCNQ1, KCNQ4 and KCNQ5 mRNA, which encode Kv7 channels, was reported. Kv7 activators were beneficial in two-independent mouse models of pulmonary hypertension (PH), which provides further evidence for their role in regulating pulmonary tone. The goals of this study were to: 1) Elucidate the role of Kv7 channels and Em in the hypertensive pulmonary circulation and 2) Study the effects and mechanism of action of a novel Kv7 modulator, zinc pyrithione (ZnPy) on the pulmonary circulation.

PH was induced in male Wistar rats by administering a single 60 µg/kg intraperitoneal injection of monocrotaline (MCT). The effects of Kv7 modulators on hypertensive and control pulmonary arteries (PA) were compared using small-vessel myography. The vasoconstrictor effect of the Kv7 blocker, XE991, was enhanced in MCT PA. The Kv7 activators retigabine and ZnPy showed enhanced efficacy in relaxing MCT PA and suppressed raised intrinsic tone identified in MCT PA relative to control PA. The effects of MCT in responses to Kv7 modulators were pulmonary specific as they were not seen in mesenteric arteries from the same animals. Real-time PCR studies revealed that PA from MCT and control rats showed a similar expression of KCNQ1, KCNQ4 and KCNQ5 mRNA transcripts. I propose that the enhanced effects of Kv7 modulators on PA from MCT rats were due to disease-induced depolarization of PASMCs, which raised intrinsic tone and increased Kv7 channel activation at rest. This is the first evidence that Kv7 channels are functional in this model of PH and may serve as potential drug targets.

The effects of ZnPy on PASMCs were studied by patch-clamp electrophysiology. ZnPy consistently hyperpolarized PASMCs and significantly increased the K+ current elicited by a voltage-step from -80 to +40 mV. ZnPy also increased the non-inactivating current recorded at 0 mV in some cells. The effects of ZnPy on Em and K+ currents were inhibited by 10 mM tetraethylammonium (TEA) and 1 µM paxilline but not by 50 nM iberiotoxin. XE991 (10µM) inhibited the ZnPy-induced hyperpolarization without altering its effects on K+ currents, suggesting that the current recorded was not responsible for its effect on Em. When tested on intact vessels, ZnPy consistently produced vasodilation. Its effects were unaffected by TEA, paxilline and iberiotoxin; however, XE991 (100 nM) had an inhibitory effect. The results suggest that ZnPy hyperpolarizes PASMCs by activating a TEA, paxilline and XE991 sensitive, but iberiotoxin insensitive channel, most likely a Kv7 channel. Its ability to dilate PA depended on pharmacologically distinct mechanisms, which are unlikely to involve Kv7 channels.

Declaration

I declare that no portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

Copyright Statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://www.campus.manchester.ac.uk/medialibrary/policies/intellectual- property.pdf), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on presentation of Theses.

Acknowledgements

I would like to sincerely thank my supervisor Prof. Alison Gurney for her tremendous help and support throughout the course of my studies. I am also thankful to my former advisor Dr. Paolo Tammaro for his feedback and support. I would like to thank all the present and past members of Prof. Gurney's lab, especially Sean Brennan, Katie Smith, Rebecca Brookfield and Roberta Oliviera for teaching me the technique of PCR and qPCR and for providing their positive input and moral support. I am grateful to Prof. Mark Boyett, Prof. George Hart, Ian Temple and Gillian Quigley for developing and validating the monocrotaline rat model which formed a substantial part of my project.

I would like to thank my parents, Ghazi Eid and Sawsan Khallaf, and my brother, Ahmed Eid, for their support and love throughout this journey. I am most grateful to my wonderful husband, Rasheed Jamjoom, who has been my main inspiration and strongest supporter throughout my studies. My beautiful son Yaser, who was born during the second year of my PhD. has been an incredible gift and a source of inspiration, joy and love. I would also like to thank the rest of my family members and friends for their support and love and especially my friend Douaa Sindi for cheering me up and helping me get through rough times.

I am thankful to all the members of the King Abdul-Aziz University Faculty of Pharmacy in Jeddah, Saudi Arabia. Finally, I would like to thank King Abdul-Aziz University and The Ministry of Higher Education of the Kingdom of Saudi Arabia for funding my research.

Abbreviations:

PH Pulmonary hypertension

PAH Pulmonary arterial hypertension

CTEPH Chronic thromboembolic pulmonary hypertension

ET-1 Endothelin-1

PDE Phosphodiesterase

Em Membrane potential

PASMC Pulmonary artery smooth muscle cell

PA Pulmonary artery

PAP Pulmonary arterial pressure

SMC Smooth muscle cell

SR Sarcoplasmic reticulum

VDCC Voltage-dependent calcium channels

ROC Receptor-operated cation channels

SOC Store-operated channels

PMCA Plasma membrane Ca2+-ATPase

NCX Sodium-calcium exchanger

PLC Phospholipase C

IP3 Inositol-3-phosphate

HPV Hypoxic pulmonary vasoconstriction

Kv Voltage-gated K+ channel

PIP2 Phosphatidylinositol 4,5-bisphosphate

GPCR G-protein coupled receptor

DAG Diacylglycerol

PKC Protein kinase C

ROS Reactive oxygen species

AMPK Adenosine monophosphate-activated kinase cADPR Cyclic ADP ribose

RyRs Ryanodine receptors

SERCA Sarcoplasmic reticulum Ca2+/Mg2+ ATPase

ABC ATP-binding cassette

SUR Sulfonylurea receptors

IKV Voltage-gated delayed rectifier K+ current

IKA A-like K+ current

IKN Non-inactivating K+ current

IKCa Large conductance Ca2+-activated K+ current

IKIR Inward rectifier K+ current

IKATP ATP-sensitive K+ current

TWIK Two-pore-domain weakly inwardly rectifying K+ channel

THIK Tandem pore domain halothane-inhibited K+ channel

TREK TWIK-related K+ channel

TASK TWIK-related, acid sensitive K+ channel

KIR Inward rectifier K+ channel

KATP ATP-sensitive K+ channels

TEA Tetraethylammonium ions

4-AP 4-aminopyridine siRNA Small-interfering RNA

IKM M-current

TMD Transmemrane domain

PKA Protein kinase A

MA Mesenteric artery

V0.5 Half-voltage of maximal activation

CHO Chinese hamster ovary

HEK Human embryonic kidney

FDA Food and drug administration

EMA European Medicines Agency

BKCa Big-conductance Ca2+-activated K+ channels

DIDS 4,4'-diidothiocyanateostilbene-2,2'-disulfonic acid

ZnPy Zinc pyrithione

MCT Monocrotaline

TRPC Canonical transient receptor potential cation channels qPCR Quantitative polymerase chain reaction

VEGF Vascular endothelial growth factor

IP Intra-peritoneal

CYP Cytochrome P

MCTP Monocrotaline pyrrole

RBCs Red blood cells

NO Nitric oxide

EDHF Endothelium-derived hyperpolarizing factor

PGF2α Prostaglandin F2 alpha

EGTA Ethylene glycol tetraacetic acid

PE Phenylephrine

PSS Physiological salt solution

PVAT Perivascular adipose tissue

HEPES NNN-[2-Hydroxyethyl]piperazine-N-[2-ethane-sulfonic acid]

2-APB 2-aminoethoxydipheyl borate

DMSO Dimethylsulfoxide

PCR Polymerase chain reaction

17 dNTPs Deoxynucleotide triphosphates

NCBI National Center for Biotechnology Information

β-actin Beta actin

Cyp Cyclophilin A

Hprt Hypoxanthine guanine phosphoribosyl transferase

Pgk1 Phosphoglycerate kinase 1

Ywhaz Tyrosine-3-monooxygenase/tryptophan 5 -monooxygenase

activation protein, zeta polypeptide

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

EDTA Ethylenediaminetetraacetic acid

CT Threshold cycle

ROX Passive reference

Rn Normalized reporter signal

Bn Normalized background signal

NTC No template controls

HKGs House-keeping genes

NF Normalization factor

RIN RNA integrity number

FU Fluorescence unit

Nt Nucleotides

DEL Double elastic lamina

PBS Phosphate buffered saline

H&E Hematoxylin and eosin

SEL Single elastic lamina

PDE5 Phosphodiesterase type 5 enzyme cGMP Cyclic guanosine monophosphate

T1/2 Time for half maximal recovery

PKG Protein kinase G

NaPy Sodium pyrithione

DEDTC Zinc diethyldithiocarbamate

DIQ 5,7-diiodo-8-hydroxyquinoline

Gmax Maximal conductance

Po Single-channel open probability

DM Dissociation medium

DTT Dithiothreitol

Cm Membrane capacitance

Rm Membrane resistance

Raccess Access resistance

Rseal Seal resistance

Rpipette Pipette resistance

Cpipette Pipette capacitance

FBA Feedback amplifier

Vp Voltage through pipette

Vc Command voltage

Rf Feedback resistor

Rs Series resistance

L-NAME NG-nitro-L-arginine methyl ester

Chapter 1: General Introduction

Pulmonary hypertension (PH) is a serious, progressive and potentially life-threatening condition. It is associated with vascular abnormalities that can cause various symptoms, such as shortness of breath, chest pain, dizziness, fainting, and peripheral oedema. PH was first described by Dr.Ernst von Romberg (a German physician) in 1891. The pathology of PH is multi-factorial and is usually a culprit of pre-existing disease states, genetic and/or exogenous factors. One of the hallmarks of this disease is vessel remodeling, leading to an increase in vessel resistance. The increased vascular resistance leads to the development of right ventricular dysfunction, heart failure and death.

PH may be classified by diagnostic category, functional class and/or pathologic findings (Badesch, 2011). The classification schemes have undergone tremendous changes over the years and according to the most recent Dana Point classification (2008), PH may be clinically divided into five categories, namely: pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease, pulmonary hypertension due to lung disease and/or hypoxia, chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary hypertension with unclear multi- factorial mechanisms (Simonneau et al., 2009). It is sometimes useful to classify PH at the cellular and molecular level, in which case it may be classified as either obliterative or secondary (Mandegar et al., 2002a). Obliterative PH occurs due to abnormalities at the level of the smooth muscle cells and endothelium (Rubin, 1997). This may be, for example, a mutation of a receptor protein of the smooth muscle cells of the pulmonary arteries (Yuan et al., 1998a). Secondary PH occurs due to an abnormality that is not triggered by any intrinsic pulmonary vascular disease (Mandegar et al., 2002a). This occurs for example in cystic fibrosis and after chronic exposure to hypoxia in high altitude (Mandegar et al., 2002a).

Over the past few years, there has been an increased understanding of the pathology of pulmonary hypertension, and as a consequence an increased interest in drug development. Although currently available treatments for this condition may reduce the symptoms, there remains no cure (Rhodes et al., 2009). The three main classes of drugs that are currently being used to treat PH include: prostanoids, endothelin-1 (ET-1) antagonists and phosphodiesterase (PDE) inhibitors (Rhodes et al., 2009). Many different therapeutic targets have been under investigation over the past few

20 years in the search for a cure. One of the proposed targets for the treatment of pulmonary arterial hypertension is potassium channels. Potassium channels appear to play a role in the pathophysiology of pulmonary hypertension and present an interesting area for research (Post et al., 1995; Yuan, 1995; Evans et al., 1996; Yuan et al., 1998a; Archer et al., 2000; Doi et al., 2000). In particular, the Kv7 family of potassium channels has been recently implicated in regulating the resting membrane potential (Em) of pulmonary artery smooth muscle cells (PASMCs) (Joshi et al., 2006; Joshi et al., 2009) and may therefore serve as a potential drug target for treating pulmonary hypertension.

1.1 The pulmonary circulation:

The pulmonary circulation is responsible for carrying de-oxygenated blood from the heart to the lungs, and returns oxygenated blood back to the heart. Deoxygenated blood leaves the heart via the pulmonary artery (PA), enters the lungs to become oxygenated, and returns to the heart via the pulmonary vein (Fig. 1.1A). In a healthy adult, the pulmonary circulation is a high-flow low-pressure system. The normal pulmonary arterial pressure (PAP) in a healthy adult has a mean value of 15 mmHg, with an upper limit of 20 mmHg (Yuan et al., 2001). Based on these values, pulmonary arterial hypertension is defined as an elevation of the mean pulmonary arterial pressure over 25 mmHg at rest or 30 mmHg during exercise (Rubin, 1997).

Like vessels in other parts of the body, pulmonary arteries are composed of three major layers: the tunica intima, tunica media and tunica adventitia (Fig. 1.1B). The tunica intima is the innermost layer and is composed of a single layer of endothelial cells. This layer is in direct contact with the lumen on one side and the elastic lamina and connective tissue (lamina propria) on the other side. The tunica media is the middle layer and forms a substantial component of the vessel. It is important in controlling vessel mechanics and is composed of smooth muscle cells (SMCs). Less well defined is the tunica adventitia, which forms the outer layer of the vessel. It is a layer composed mainly of connective tissue.

Normally, a regulated balance in cell proliferation and apoptosis maintains an optimal thickness of the pulmonary arterial wall (Mandegar et al., 2002a). If cell proliferation exceeds apoptosis, this balance is disturbed resulting in a thickened

21 vessel wall and a narrowed lumen which ultimately causes vessel obliteration (Mandegar et al., 2002a). In PAH, the proliferation and hypertrophy of PASMCs and endothelial cells leads to vessel remodeling that spans all three layers of the vessel (Stenmark et al., 1997). Some of the characteristic features include thickening of the medial and intimal layers as well as the presence of a double elastic lamina (Mandegar et al., 2002a). The complex molecular mechanisms involved in vascular remodeling seem to involve a disturbance in the intracellular Ca2+ levels, in addition to an altered expression and/or function of certain ion channels (Yuan et al., 1998b).

1.2 Regulation of pulmonary artery tone:

The pulmonary circulation is under the control of many different factors, which contribute to maintaining a low-pressure circuit. These factors may be broadly described as active and passive factors (Daly et al., 1966). Active factors cause relaxation or contraction of the vascular smooth muscle by changing the pulmonary vascular resistance and tone. Examples of such factors include humoral agents, autonomic nerves and respiratory gasses. Passive factors on the other hand, alter the pulmonary vascular resistance and blood flow independently of changes in the vascular tone. Examples of passive factors include: changes in cardiac output, gravitational force, airway and interstitial pressure, left atrial pressure and vascular obstruction or recruitment (Barnes et al., 1995). The pulmonary circulation is generally believed to be predominantly under active control (Barer, 1980).

1.3 The regulation of intracellular Ca2+:

Intracellular Ca2+ levels of PASMCs are absolutely critical for the regulation of vessel tone. PASMC contraction is dependent upon the interaction of the contractile proteins actin and myosin, which is triggered by a rise in intracellular Ca2+. Increased intracellular Ca2+ levels also regulate cell proliferation by increasing nuclear Ca2+ concentrations (Allbritton et al., 1994) and modulating various parts of the cell cycle and its signal transduction proteins (Means, 1994; Hardingham et al., 1997). Ca2+ levels in the sarcoplasmic reticulum (SR) were also found to be crucial for PASMC proliferation (Mogami et al., 1993; Short et al., 1993; Platoshyn et al., 2000; Golovina et al., 2001). Intracellular Ca2+ levels are intricately controlled by

Figure 1.1: The pulmonary circulation. a)Diagram showing the blood circulation between the heart and lungs adopted from http://www.usi.edu/science/biology/mkhopper/ap_labs/2402/Blood%20Vessels/Pa ges%20Linked%20to%20Objectives/Objective%206%20Pulmonary%20Circulation.ht m b)Diagram showing the major layers of pulmonary arteries and veins. (adapted from http://www.pearsonsuccessnet.com/snpapp/iText/products/0-13-115075- 8/text/chapter30/concept30.1.html).

23 several systems including: Ca2+ entry via voltage-dependant calcium channels (VDCC), receptor-operated cation channels (ROC), store-operated channels (SOC), Ca2+ release from and into the SR, and finally the plasma membrane Ca2+-ATPase (PMCA) and sodium-calcium exchanger (NCX) which are responsible for Ca2+ efflux (Fig. 1.2). The sarcoplasmic reticulum Ca2+/Mg2+ ATPase (SERCA) is responsible for the sequestration of Ca2+ into the SR and can be inhibited by various agents such as cyclopiazonic acid or thapsigargin. These systems are further controlled by membrane receptors and channels which all contribute to the regulation of intracellular calcium. For example, α1 adrenergic receptors present on pulmonary artery smooth muscle cells mediate vasoconstriction (Docherty et al., 1981) by activating G-protein which in turn activates phospholipase C (PLC) leading to an increase in inositol-3-phosphate (IP3) and intracellular calcium. Another example is the inhibition of potassium channels which indirectly increases intracellular calcium levels via depolarization and activation of VDCC.

1.4 Hypoxic pulmonary vasoconstriction:

Respiratory gases such as oxygen have a great influence on pulmonary vascular tone (Fishman, 1961). Since the function of the pulmonary circulation is to supply the body with oxygenated blood, the lung must be able to match its regional ventilation and perfusion. In other words, it is important that pulmonary arteries constrict in regions where oxygen levels are low so that blood is diverted to better ventilated areas of the lung. This phenomenon, which is unique to the pulmonary circulation, is known as hypoxic pulmonary vasoconstriction (HPV). It occurs mainly in the small intra-pulmonary arteries (100-300 µm in humans) (Yuan et al., 1990; Barnes et al., 1995; Weir et al., 2005) and ensures that gas exchange takes place in an efficient manner. However, if the response is global, it will ultimately lead to the development of pulmonary hypertension.

It is generally agreed that the PASMC is the primary locus of HPV, while the endothelium and other factors modulate the response (Holden et al., 1984; Ward et al., 2004). This is supported by the fact that this phenomenon can be observed in isolated pulmonary artery myocytes (Murray et al., 1990a; Murray et al., 1990b; Madden et al., 1992; Post et al., 1992; Archer et al., 1993). It had been proposed by

Figure 1.2: Ca2+ mobilization in PASMCs. Schematic diagram summarizing the main Ca2+ mobilization pathways in PASMCs. (Voltage dependent Ca2+ channels (VDCC), Receptor operated channels (ROC), phosphatidylinositol 4,5-bisphosphate (PIP2), phospholipase C (PLC), G-protein coupled receptor (GPCR), sarcoplasmic reticulum (SR), plasma membrane Ca2+-ATPase (PMCA), sodium-calcium exchanger (NCX), inositol-3-phosphate (IP3), diacylglycerol (DAG), protein kinase C (PKC), membrane potential (Em)).

25 some groups that K+ channels may contribute to HPV. Studies have shown that acute hypoxia can selectively inhibit voltage-gated K+ channels (Kv) while chronic hypoxia causes down-regulation of Kv channel expression (Smirnov et al., 1994; Wang et al., 1997; Osipenko et al., 1998; Sweeney et al., 2000; Platoshyn et al., 2001). It was initially proposed that this decreased activity of K+ channels leads to depolarization of PASMCs and the elevation of cytoplasmic Ca2+ concentrations, ultimately causing vasoconstriction (Osipenko et al., 1998; Mandegar et al., 2002b). However it is unlikely that inhibition of K+ channels is the principal mediator of HPV because it had been shown that this effect is not enough to elicit the depolarization required for constriction (Clapp et al., 1991b; Osipenko et al., 1997; Bakhramov et al., 1998; Cha et al., 2008). In addition, L-type Ca2+ channel blockers have been shown to have no inhibitory effect on HPV in various animal models (Robertson et al., 2000).

Although it is believed that the mitochondria of PASMCs act as oxygen sensors and are responsible for initiating HPV, the remaining signaling process is rather controversial (Ward et al., 2004; Waypa et al., 2008; Ward et al., 2009). The end result however, is as elevation of intracellular Ca2+ in PASMCs, which has been attributed to various calcium mobilization pathways, including voltage-dependent and voltage-independent Ca2+ entry, Ca2+ release from the ryanodine stores and store-operated Ca2+ entry (Ward et al., 2009) in addition to increased calcium sensitization by Rho-kinase (ROCK) (Aaronson et al., 2006). Currently, three main hypotheses are believed to underlie HPV, namely: 1) The redox hypothesis 2) The reactive oxygen species (ROS) hypothesis and 3) The energy state / adenosine monophosphate-activated kinase (AMPK) hypothesis. In the redox hypothesis a reduction of ROS is thought to initiate HPV by inhibiting Kv channels resulting in a depolarization that in turns activates voltage-gated Ca2+ influx and vasoconstriction (Weir et al., 1995; Michelakis et al., 2002; Moudgil et al., 2005). In contrast, the ROS hypothesis suggests that an increase in ROS shifts the cell to a more oxidized state ultimately leading to Ca2+ release from the intracellular stores, increased calcium sensitization as well as store operated calcium entry (Chandel et al., 2000; Waypa et al., 2005; Sylvester et al., 2012). Finally in the energy state/AMPK hypothesis the response is believed to be initiated mainly by a rise in AMP relative to ATP leading to the activation of AMPK. This in turn leads to the production of cyclic ADP ribose (cADPR) which activates ryanodine receptors (RyRs) in the sarcoplasmic reticulum

26 leading to Ca2+ release from the intracellular stores and store-operated Ca2+ entry (Evans, 2006). These three hypotheses are summarized in Fig. 1.3.

The crucial role of the ROCK-mediated Ca2+ sensitization in HPV is being increasingly recognized. It has been shown that inhibition of ROCK resulted in suppression of HPV in perfused lung preparations as well as in isolated vessels (Robertson et al., 2000). It is also believed that endothelial constricting factors increase Ca2+ sensitization via ROCK stimulation (Gaine et al., 1998). The activation of ROCK in HPV has been correlated with the Src-family kinases (Knock et al., 2008). It has been shown that the Src-family kinases are involved in ROCK activation by hypoxia as well as super-oxide (Knock et al., 2008), which is consistent with the ROS hypothesis.

Activation of ETA and 5-HT1B/1D by ET-1 and serotonin has been shown to activate ROCK in chronically hypoxic rats (Homma et al., 2007), which has also been linked with an increased production of ROS (Jernigan et al., 2008) . The role of ROCK in vascular remodeling is unclear, however it has been suggested that ROCK activates serotonin receptors (5-HT1B) which in turn stimulates PASMC proliferation. It has also been shown that resistance PA from idiopathic PAH lungs had increased ROCK expression (Laumanns et al., 2009).

Figure 1.3: Ca2+ mobilization in HPV. Schematic diagram showing Ca2+ mobilization in HPV and the proposed signaling pathways in PASMCs. Voltage-dependent Ca2+ channels (VDCC), receptor operated channels (ROC), store operated channels (SOC) and the Na+/Ca2+ exchanger (NCX), reactive oxygen species (ROS), Ryanodine receptor (RyR), sarcoplasmic reticulum (SR), adenosine monophosphate-activated kinase (AMPK). Adapted from (Ward et al., 2009)

1.5 Membrane properties of PASMCs:

The membrane of smooth muscle cells of pulmonary arteries can be described as quiescent in normal resting conditions (Casteels et al., 1977b; Casteels et al., 1977a). It can however respond to various pharmacological and electrical stimuli. Su et al. (1964) showed that noradrenaline can induce contraction in the smooth muscle of the rabbit main pulmonary artery without generating action potentials or causing depolarization. In a later study by Somlyo and Somlyo (1968), noradrenaline was found to depolarize and induce oscillations in the membrane potential. However, because there was only a limited correlation between the tension and depolarization and because contractions also occured in depolarized tissues, this type of excitation contraction coupling was termed pharmacomechanical coupling (Casteels et al., 1977b). Casteels et al. (1977b) elucidated the role of changes in membrane potential in the contractile response, by studying the effect of different concentrations of noradrenaline and external K+ on the tension and membrane potential in SMCs of the intact rabbit main pulmonary artery. They showed that increasing the external K+ depolarized the smooth muscle cells and increased Ca2+ influx. At low concentrations of noradrenaline there seemed to be an increase in Ca2+ influx, whereas higher noradrenaline concentrations caused a release of cellular Ca2+. All these findings support the idea that membrane potential is a factor in the regulation of pulmonary vascular tone, although it is only one of several mechanisms.

The concentration gradients of electrically charged ions (mainly K+, Na+ and Cl-) across the plasma membrane and their relative permeabilities are what largely determine Em of vascular smooth muscle cells (Fig. 1.4). The concentration of intracellular K+ ( 140 mM) is far greater than the extracellular concentration ( 5mM), and the permeability to K+ by far surpasses Na+ and Cl- permeabilities (Nelson et al., 1995; Yuan et al., 1999). This led to the conclusion that the K+ current generated by K+ efflux through K+ channels is a key determinant of resting Em (Yuan, 1995; Evans et al., 1996) in smooth muscle cells. However it is not the only determinant, because in that case the resting membrane potential would equal the equilibrium potential for K+. In contrast, the resting membrane potential of PASMCs is approximately -50 mV, whereas the equilibrium potential for K+ is about -80 mV (Casteels et al., 1977b). This difference can be attributed to Cl- and/or non-selective cation channels, which would oppose the effect of K+ efflux on Em.

Figure 1.4: Resting conductances in PASMCs. Em is the resting membrane potential. Ek, ENa, ECa, ECl are the reversal potentials for K+, Na+, Ca2+ and Cl- respectively. The red arrows represent the chemical gradients and the black arrows represent the electrical gradients for each ion. There is a strong chemical gradient favoring the movement of K+ out of cell at the resting membrane potential which is counteracted by the influx of Na+ and Ca2+ and possibly the efflux of Cl-.

1.6 K+ channels in the pulmonary vasculature:

K+ channels are membrane proteins that regulate the flow of K+ across the plasma membrane. K+ channels play critical roles in various physiological processes and have been recognized as potential drug targets for many years. To date, over 80 K+ channels and K+ channel related genes have been identified (Wickenden, 2002). They may be classified according to their structure, voltage-dependence, pharmacology and kinetics (Jan et al., 1997). Some K+ channels are activated by a change in membrane potential and are called voltage-gated K+ channels. Others are activated by an increase in intracellular Ca2+ concentration and are known as Ca2+-activated K+ channels. Another class is the inward rectifiers, which may be ATP-sensitive or G- protein regulated (Wickenden, 2002). Some K+ channels, such as TASK channels are voltage-independent and allow the efflux of K+ irrespective of the membrane potential. All K+ channels are composed of pore-forming α-subunits as well as modulatory cytoplasmic β-subunits. The α-subunits either contain six transmembrane segments and one pore-forming region (voltage-gated and Ca2+-activated channels), two transmembrane segments and one pore-forming region (inward rectifier channels) or four transmembrane segments and two pore-forming regions (two-pore-domain weakly inward rectifying K+ (TWIK)-related acid-sensitive K+channels (TASK)). The α- subunits are divided into subfamilies depending on their properties (ie. voltage-gated, inward rectifier, Ca2+-activated, etc.) as listed in table 1.1. Modulatory β-subunits are a diverse group of molecules (Table 1.2) and may be cytoplasmic proteins, single transmembrane spanning proteins or ATP-binding cassette (ABC) transport related proteins, such as sulfonylurea receptors (SUR) (Wickenden, 2002). They can confer characteristic features to the channel such as activation characteristics, drug sensitivity and can also regulate inactivation (Robbins, 2001). It is clear that many K+ channels are expressed in PA (Fig. 1.5), however the reason for such an expression profile is yet to be unveiled. Many of the channels expressed are not active at the physiological resting membrane potential. It is therefore essential that this expression profile is considered carefully to attribute the correct channel to its appropriate function.

K+ channels in smooth muscle have two important functions: 1) They are major determinants of the resting membrane potential. 2) They suppress membrane excitability.

Table 1.1: Pore-Forming K+ Channel α-subunits.

Table 1 (Continued) Subclass Channel Gene SIX TRANSMEMBRANE DOMAINS, ONE PORE Voltage-gated (shaker) Kv1.1 KCNA1 Subclass Channel Gene FOUR TRANSMEMBRANE DOMAINS, TWO PORES Kv1.2 KCNA2 Kv1.3 KCNA3 Two Pore TWIK-1 KCNK1 Kv1.4 KCNA4 TWIK-2 KCNK6 Kv1.5 KCNA5 KCNK7 KCNK7 Kv1.6 KCNA6 TASK KCNK3 Kv1.7 KCNA7 TASK-2 KCNK5 Voltage-gated (shab) Kv2.1 KCNB1 TASK-3 KCNK9

Kv2.2 KCNB2 TASK-5 KCNK15 Voltage-gated (shaw) Kv3.1 KCNC1 TRAAK KCNK4 Kv3.2 KCNC2 TREK-1 KCNK2 Kv3.3 KCNC3 TREK-2 KCNK10 Kv3.4 KCNC4 THIK-1 KCNK13 Voltage-gated (shal) Kv4.1 KCND1 THIK-2 KCNK12 Kv4.2 KCND2 TALK-1 KCNK16 Kv4.3 KCND3 TALK2 KCNK17 Voltage-gated (silent) Kv5.1 KCNF1 Kv6.1 KCNG1 Kv6.2 KCNG2 Table 1.2: Modulatory K+ Channel β- Kv6.3 KCNG3 subunits. Kv8.1 Kv9.1 KCNS1 Name Known Partners Gene Kv9.2 KCNS2 MinK KCNQ1, ERG1 KCNE1 Kv9.3 KCNS3 Mirp1 ERG1, KCNQ2, KCNE2 Voltage-gated (EAG) EAG KCNH1 KCNQ3, Kv4.2 EAG2 Mirp2 KCNQ1, KCNQ4, KCNE3 ERG1 KCNH2 Kv3.4 ERG2 Mirp3 KCNE4 ERG3 Bkβ1 Slo-1 KCNMB1 ELK1 KCNH4 Bkβ2 Slo-1 KCNMB2 ELK2 KCNH3 Bkβ3 Slo-1 KCNMB3 ELK3 Bkβ4 Slo-1 KCNMB4 Voltage-gated (KvLQT related) (Kv7) KCNQ1 KCNQ1 Kv β1 Kv1.x KCNQ2 KCNQ2 Kv β2 Kv1.x KCNAB1 KCNQ3 KCNQ3 Kv β3 Kv1.x KCNAB2 KCNQ4 KCNQ4 KCHIP1 Kv4.x KCNIP1 KCNQ5 KCNQ5 KCHIP2 Kv4.x KCNIP2 Ca2+ -activated (large conductance) Slo-1 KCNMA1 KCHIP3 Kv4.x Slo-2 KCNMA2 KCHAP Kv2.1, Kv4.3 Slo-3 KCNMA3 SUR1 Kir6.1, Kir6.2 ABCC8 Ca2+ -activated (intermediate conductance) IKCA KCNN4 SUR2 Kir6.1, Kir6.2 ABCC9 2+ Ca - activated (small conductance) SK1 KCNN1 SK2 KCNN2 SK3 KCNN3 TWO TRANSMEMBRANE DOMAINS, ONE PORE Inward rectifier (ROMK1) Kir1.1 KCNJ1 Kir2.1 KCNJ2 Kir2.2 KCNJ12 Kir2.3 KCNJ4 Kir2.4 KCNJ14 Inward rectifier (G-protein regulated) Kir3.1 KCNJ3 Kir3.2 KCNJ6 Kir3.3 KCNJ9 Kir3.4 KCNJ5 Inward rectifier Kir4.1 KCNJ10 Kir4.2 KCNJ15 Kir5.1 KCNJ16 Kir7.1 KCNJ13 Inward rectifier (ATP sensitive) Kir6.1 KCNJ8 Kir6.2 KCNJ11

Tables Adapted from (Wickenden, 2002).

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K+ channels contributing to the resting membrane potential must be active at voltages between -60 and -50 mV (Casteels et al., 1977b; Suzuki et al., 1982a). Depolarization above -35 mV activates both Ca2+ and K+ channels (Nelson et al., 1990). The activation of Ca2+ channels causes further depolarization and promotes Ca2+ entry and contraction. The activation of voltage-gated K+ channels on the other hand controls membrane excitability by limiting the initiation of an action potential by the much smaller Ca2+ channel currents (Gurney et al., 2010). This suggests that the presence of a large number of K+ channels in the pulmonary circulation may serve as a protective mechanism that prevents vasospasm and maintains a low intrinsic tone (Gurney et al., 2010).

Six main K+ currents have been reported in pulmonary arterial SMCs with varying contributions at different levels of the pulmonary tree (McCulloch et al., 1999):

1) Voltage-gated delayed rectifier (IKV) (Clapp et al., 1991a; Smirnov et al., 1994).

2) A-like (IKA) (Clapp et al., 1991a).

3) Non-inactivating (IKN) (Evans et al., 1996).

4) Large conductance Ca2+-activated (IKCa) (Clapp et al., 1991a; Clapp et al., 1996).

5) Inward rectifier (IKIR) (Clapp et al., 1991a).

6) ATP-sensitive (IKATP) (Clapp et al., 1992; Kitamura et al., 1993).

IKATP is a voltage-independent current that is activated in a time-independent manner (Clapp et al., 1991a; Clapp et al., 1992). Intracellular ATP concentrations and sulphonylurea compounds such as glibenclamide have been reported to inhibit this current, whereas the potassium channel opener levcromakalim was shown to activate it (Clapp et al., 1992). IKCa on the other hand is activated in both a voltage and time- dependent manner (Clapp et al., 1991b). Its activity increases when intracellular Ca2+ concentrations at the surface of the channel protein rise as well as at positive membrane potentials (Clapp et al., 1991a). Its inhibition by tetraethylammonium ions (TEA) (Clapp et al., 1991a; Robertson et al., 1992) and charybdotoxin (Yuan,

1995) enable it to be distinguished from other K+ currents in the PASMCs. IKA and IKV are both voltage-dependent currents that are insensitive to Ca2+ and to block by TEA and glibenclamide (Clapp et al., 1991a; Evans et al., 1994; Smirnov et al., 1994; Yuan, 1995). However, they are blocked by 4-aminopyridine (4-AP) (Clapp et al.,

1991a; Yuan, 1995). IKA and IKV have different rates of activation and inactivation, despite being pharmacologically similar (Evans et al., 1996). IKA activates in a rapid manner, peaks within 20 ms of a depolarizing voltage step and is a transient current that inactivates within 100 ms (Clapp et al., 1991a). Although IKA was found to be present in rabbit PASMCs (Clapp et al., 1991a), it is unclear whether this current is present in other species as well. IKV is slower in terms of activation and inactivation. It peaks within 100 ms (Evans et al., 1994; Smirnov et al., 1994; Yuan, 1995) of a depolarizing step and a minimum of 60 s is required for steady state inactivation (Evans et al., 1994). The voltage-activated current in rabbit PASMCs was shown to take many minutes (up to 45 min) to fully inactivate at 0 mV (Evans et al., 1996).

IKN is a voltage-gated current with a low threshold for activation (Evans et al., 1996), between -80 and -65 mV. It activates exponentially with a time constant of 1.6 s at - 60 mV and outward rectification (Fig. 1.6). Deactivation has a time constant of 107 ms at -60 mV. Similar to IKA and IKV, IKN showed sensitivity to 4-AP but with only

49% inhibition at 10 mM. However, IKN was distinguished from these currents as it was insensitive to 10 µM quinine, which inhibited both IKA and IKV by 51% and 47% respectively. IKN recorded from rabbit PASMCs can also be distinguished from IKATP and IKCa in that it lacks sensitivity to glibenclamide and TEA (Evans et al., 1996).

Although IKN, IKATP and IKv have been suggested to be involved in the control of Em, evidence suggests that the major contributor to the Em at rest is a voltage-gated K+ current (Yuan, 1995; Evans et al., 1996). There are differing opinions however, with regards to the nature of the voltage-gated channel(s) involved. There is strong evidence that the two pore domain channel, TASK-1, is also a major contributor to Em in rabbit and human PASMCs (Gurney et al., 2003; Olschewski et al., 2006).

Two voltage-gated channels that have been extensively studied as mediators of the resting membrane potential are Kv1.5 (Archer et al., 1998; Moudgil et al., 2006; Remillard et al., 2007) and Kv2.1/Kv9.3 (Patel et al., 1997). It has however been argued that Kv1.5 or Kv2.1 cannot be key determinants of IKN and the resting potential because of their high activation thresholds (Joshi et al., 2009). Kv1.5 channels activate above -50 mV and are blocked by concentrations of 4-AP much lower than the concentrations required to cause depolarization of PASMCs (Coetzee et al., 1999). Further evidence is seen in the comparison of wild type mice and mice

Figure 1.6: The non-inactivating current IKN. Current versus voltage relationship of IKN described by Evans et al. (1996). The trace shows the current recorded during a 1.2 s voltage ramp to -100 mV following a step to 60 mV. The holding potential of the cells was 0 mV and the experiments were carried out in the presence of extracellular TEA (10 mM) and glibenclamide (10 µM).

36 that lack the Kv1.5 subunit where despite a 30% reduction in delayed rectifier currents in the knockout animals, they had similar membrane potentials to the wild type (Archer et al., 2001). In addition, Kv1.5 knockout mice were not found to have any signs of pulmonary hypertension (Archer et al., 2001). Kv2.1 has an even more positive threshold for activation (Coetzee et al., 1999) and, similar to Kv1.5 a blockade of the channel by 4-AP was found to have minimal effect of membrane potential (Osipenko et al., 1998). Kv2.1 may be present as a complex with Kv9.3 which lowers the threshold for activation and decreases the sensitivity of the channels to 4-AP (Patel et al., 1997). Gurney et al. (2010) suggested that Kv1.5, Kv2.1 and the Kv2.1/Kv9.3 complex were poor candidates for mediating the resting membrane potential and regulating vascular tone because they are all closed at membrane potentials of -50 mV or lower. However loss of expression of these channels would increase the excitability of PASMCs enabling sustained depolarization and voltage- gated calcium entry (Gurney et al., 2010).

Reports have suggested that inward rectifier channels (KIR) may be present in rat PASMCs (Turner et al., 1996). In addition, cultured human PASMCs expressed

KIR2.1, KIR2.2 and KIR2.4 mRNA (Tennant et al., 2006). These were the only reports of inward rectifier channels in PA.

It is believed that IKCa is physiologically important because it has been widely identified (Evans et al., 1996), but it has been observed that IKCa blockers have no effect on the resting potential of PASMCs (Post et al., 1995; Yuan, 1995; Osipenko et al., 1997). They did however cause depolarization and contraction in pressurized systemic arteries, but this effect was negligible when the pressure was reduced to 15 mmHg (the mean PAP) (Brayden et al., 1992; Nelson et al., 1995). Evans et al. (1996) proposed that KCa channels activate as a result of stimulus-induced elevations of cytoplasmic [Ca2+] and cause repolarization, rather than controlling the resting membrane potential. KATP channels likely act as metabolic sensors or targets for endogenous vasodilators (Quayle et al., 1994), since glibenclamide was shown to have little effect on the resting tone and resting potential in pulmonary arteries in the presence of physiological levels of cytoplasmic ATP (Clapp et al., 1992; Yuan, 1995;

Evans et al., 1996). Evans et al. (1996) showed that the activation of IKN occurred at potentials close to the resting membrane potential of PASMCs and that its properties make it well suited to a role in the regulation of the resting membrane potential. Thus the resting membrane potential of PASMCs is probably determined largely by

IKN. However, the K+ channel or channels that mediate IKN are yet to be fully characterized.

TASK-1 channels are expressed in the PASMCs of many different species, namely rabbit, rat, mouse and human (Gurney et al., 2003; Gardener et al., 2004; Olschewski et al., 2006). Gurney et al. (2003), provided strong evidence that the two-pore domain K+ channels, TASK-1 channels, are major contributors to the resting membrane potential in rabbit PASMCs and may be one of the molecular correlates of IKN. An important role was subsequently demonstrated in human pulmonary artery (Olschewski et al., 2006), but TASK channels do not appear to be important in mouse (Gurney et al., 2009). The pharmacological profile of IKN closely resembles that of heterologously expressed TASK-1 channels, which are known to be highly sensitive to changes in extracellular pH (Lesage et al., 2000; Patel et al., 2001). Knockdown of TASK-1 mRNA in human pulmonary artery using small-interfering

RNA (siRNA), caused loss of IKN, depolarization and loss of responsiveness to hypoxia (Olschewski et al., 2006). TASK-2 is also expressed in rat pulmonary artery but thought to only have a minimal contribution as TASK-2 knockdown by siRNA caused only a slight depolarization (Gönczi et al., 2006).

IKN has voltage-dependent and independent components (Osipenko et al., 1997; Joshi et al., 2006). Although the voltage-independent component of IKN has been attributed to TASK channels (Gurney et al., 2003), the channels mediating the voltage- dependent component are yet to be unveiled. Evans et al. (1996) reported that the kinetic properties of IKN are strikingly similar to those of the neuronal M-current (IKM). Both currents have a threshold for activation in the region of -65 mV, activate slowly and are non-inactivating (Brown et al., 1980; Brown, 1988; Evans et al., 1996).

Similarities between IKN and IKM led to the speculation that the voltage-dependent component may be mediated by a voltage-sensitive potassium channel possibly linked to the M-current. The channels mediating the M-current are encoded by the genes of the Kv7 family of potassium channels (Wang et al., 1998; Robbins, 2001). This provided the basis for the hypothesis that the voltage-dependent component of IKN may be mediated by the Kv7 family of K+ channels.

1.7 Kv7 potassium channels:

Kv7 channels are merely a family of voltage-dependent K+ channels. Five genes KCNQ1-KCNQ5 encode the Kv7 channels α-subunits (Wang et al., 1996; Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998; Yang et al., 1998; Kubisch et al., 1999; Lerche et al., 2000a; Schroeder et al., 2000a). As all Kv channels they consist of four α-subunits that surround a K+ selective pore (MacKinnon, 1991). These subunits may all be identical (homomultimers), or may be a combination of two or more different Kv7 α-subunits (heteromultimers). Each α-subunit contains six transmembrane α-helical segments (S1-S6), and a membrane entering P-loop which contributes to the channel pore (Fig. 1.5). The diversity of this group is further increased, as α-subunit complexes can also be modified by association with intracellular β-subunits (encoded by KCNE genes) (Wang et al., 1996) and by various chemical interactions such as phosphorylation and dephosphorylation.

Kv7 channels have gained tremendous interest over the past few years, due to the presence of a direct link between mutation and disease (Jentsch, 2000; Rogawski, 2000). Mutations in KCNQ genes have been linked to various diseases, such as cardiac arrythmia (Wang et al., 1996), epilepsy (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998), deafness (Neyroud et al., 1997; Kubisch et al., 1999) and most recently autism (Gilling et al., 2013). Most of the expressed channel genes have clear physiological correlates and the resulting channels show sensitivity to certain pharmacological compounds, which may serve as clinically useful drugs (Wang et al., 1996; Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998; Yang et al., 1998; Kubisch et al., 1999; Lerche et al., 2000a; Schroeder et al., 2000a).

Kv7 channels were recently suggested to be involved in regulating the tone of pulmonary artery smooth muscle cells in rodents (Joshi et al., 2009). More recently, there has been evidence for their involvement in the myogenic control of rat cerebral arterial diameter (Zhong et al., 2010; Mani et al., 2013). In addition, they may help to regulate contractility of the rat bladder (Rode et al., 2010) & myometrium (McCallum et al., 2009; McCallum et al., 2011).

1.7.1 Structure of Kv7 channels:

Kv7 channel proteins share 30-65% amino acid identity, and this homology is particularly noticeable in the transmembrane regions (Lerche et al., 2000a; Schroeder et al., 2000a). The fourth transmemrane domain (TMD) is thought to be the voltage sensor domain and has a regular distribution of six positively charged amino acids, with the exception of Kv7.1, which has four (Fig.1.7). The P-loop hosts the K+ pore signature, which has a TxxTxGYG sequence and determines K+ selectivity. The length of the C-terminus is different between subtypes and hosts the "A-domain", which is a highly homologous region in all five subtypes (Schroeder et al., 2000a; Schroeder et al., 2000b; Schwake et al., 2000). All five subtypes show a similar length of their N-termini, which is in of the order of 100 amino acids (Robbins, 2001). The N-termini of Kv7.1 and Kv7.2 have been identified as having putative protein kinase A (PKA) phosphorylation sites. It has been suggested that Kv7.1 contains a glycosylation site on the extracellular loop between TMDs 5 and 6 (Barhanin et al., 1996; Wang et al., 1996). These sites are likely to be important for the regulation of Kv7 channel selectivity.

1.7.2 Differential expression of KCNQ genes in smooth muscle organs:

KCNQ gene expression in vascular smooth muscle represents a new area of research. There are very few cases where the expression of functional Kv7 channels has been confirmed. Ohya et al. (2003) were first to identify KCNQ expression at the mRNA level in mouse portal vein smooth muscle, with a greater expression of KCNQ1 over other subtypes in this tissue. It was later shown by Yeung et al. (2008) that mRNA for KCNQ4 and KCNQ5 was also present. They also studied KCNQ mRNA in mouse thoracic aorta, carotid artery and femoral artery (Yeung et al., 2007) and reported a predominant expression of KCNQ1 and KCNQ4 mRNA, with KCNQ5 transcripts also detected. The adult rat aorta and mesenteric artery smooth muscle cells also express KCNQ4 and KCNQ5 transcripts (Brueggemann et al., 2007). KCNQ5 expressed in vascular smooth muscle was a shorter spliced variant, also found in certain visceral tissues (Yeung et al., 2008). This alternative splicing is also seen in a truncated form of KCNQ1 (termed KCNQ1b) which is found in blood vessels (Ohya et al., 2003). Joshi et al. (2009) showed that KCNQ1, KCNQ4 and KCNQ5 transcripts were also expressed in PASMCs obtained from rats, with KCNQ4 being the most abundant. Interestingly,

Figure 1.7: The structure of Kv7 channel subunits. The six transmembrane dgomains (S1– S6), the P-loop (P), glycosylation ( ), protein kinase A (PKA) phosphorylation ( ), and A-domains ( ) locations are shown. (Adapted from (Robbins, 2001))

41 the level of expression was significantly higher in pulmonary arteries than in mesenteric arteries (MA). Less is known of the expression of the KCNE β subunits, however, the expression of KCNE genes seems to be vessel-specific (Ohya et al., 2002a; Ohya et al., 2002b; Yeung et al., 2007).

Functional Kv7 channels have also been found in non-vascular smooth muscle. KCNQ4 and KCNQ5 were the main transcripts present in the smooth muscle of the murine gastrointestinal tract (Jepps et al., 2013), where they are thought to decrease activity, especially in the colon. KCNQ expression has also been demonstrated in the smooth muscle of the rat stomach and the modulation of Kv7 channels affected muscle tone in the stomach (Ohya et al., 2002a; Ipavec et al., 2011). In addition to effects on the gastrointestinal tract, Kv7 activators were found to cause relaxation of rat bladder, suggesting a role for Kv7 channels in the regulation of the excitability of SMCs in the bladder (Streng et al., 2004; Rode et al., 2010). A Kv7 activator was also found to augment Kv7 currents in bladder interstitial cells of the Cajal (Anderson et al., 2009). A more recent study by the same group confirmed the expression of KCNQ transcripts in guinea pig bladder and provided evidence for a role of Kv7 currents in setting the resting membrane potential of detrusor smooth muscle cell in the bladder (Anderson et al., 2013). KCNQ expression has also been established in the uterus of mice and humans, where KCNQ1 was the main transcript throughout the oestrus cycle (McCallum et al., 2009). The expression of KCNQ genes was found to vary during pregnancy, with a decreased expression noted in early pregnancy (McCallum et al., 2011). Kv7 activators were found to relax the human and mouse uterus and were more effective during late pregnancy. This suggests that Kv7 channels may potentially be used to treat preterm labour. Expression of Kv7 channels has also been established in the airways where KCNQ1, KCNQ4 and KCNQ5 were the main transcripts seen in humans (Brueggemann et al., 2012). Guinea pig airways show a different expression profile, where KCNQ2 was the main transcript present and KCNQ1 was not detected (Brueggemann et al., 2012). Kv7 modulation in both species was found, however to have an effect on airway diameter, suggesting the potential use Kv7 activators to treat airway diseases (Brueggemann et al., 2012; Jepps et al., 2013).

1.7.3 Properties of Kv7 currents:

Kv7 currents are outwardly rectifying voltage-dependent K+ currents, which activate above -60 mV and show minimal or no inactivation (Robbins, 2001). Whether

42 expressed as homomultimers or heteromultimers is what ultimately dictates the current characteristics.

1.7.3.1 Homomultimers:

Kv7.1 homomeric channels show sigmoidal activation kinetics and take 100-200 msec to reach 90% activation. The current has a half activation voltage (V0.5) of approximately -10 to -20 mV (Robbins, 2001). In Xenopus oocytes, the current showed slow activation and does not reach the maximum in 2-3s (Barhanin et al., 1996; Tristani-Firouzi et al., 1998; Abitbol et al., 1999; Schroeder et al., 2000b), whereas in mammalian cell lines the current displayed a rapid activation and peaked within 1 second (Sanguinetti et al., 1996; Yang et al., 1997; Wang et al., 1998).

The properties of the current resulting from homomeric Kv7.2 channel expression appears to differ based on the expression system and/or the voltage protocol used (Robbins, 2001). Kv7.2 has been expressed in chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, oocytes and COS cells where it has corresponding V0.5 of -14,-21,-37, and -38 mV respectively (Wang et al., 1998; Shapiro et al., 2000; Tinel et al., 2000; Selyanko et al., 2001).

Kv7.3 homomeric expression has been disputed. Some studies show very low expression (Schroeder et al., 1998; Wang et al., 1998; Main et al., 2000; Wickenden et al., 2000). In other studies, when expressed successfully, Kv7.3 currents showed a sigmoidal activation that began positive to -60 mV (V0.5 = -37 mV) (Yang et al., 1998; Selyanko et al., 2000). Kv7.3 currents exhibited a "crossover" phenomenon at membrane potentials >0 mV, due to a characteristic inward rectification at these potentials. This means that the current decreased with incremental voltage steps above 0 mV despite an increasing driving force for K+ ions (Robbins, 2001).

When expressed in CHO cells, homomeric Kv7.4 channels had an exponential activation (with an exception at very positive voltages) with V0.5 = -19 mV and they displayed little or no inactivation (Selyanko et al., 2000). In Xenopus oocytes, however, the activation was slow ( = 600 msec at +40 mV, V0.5 = -10 mV) (Kubisch et al., 1999; Schroeder et al., 2000a).

Kv7.5 homomultimeric channels activate bi-exponentially in a very slow manner (0.1 and 1.0 sec) (Schroeder et al., 2000a). They are similar to Kv7.3 in that they show an inward rectification at very positive potentials and different from Kv7.1 in that they deactivate in an exponential manner, showing M-current-like behavior (Robbins, 2001).

1.7.3.2 Heteromultimers & modulatory subunits:

Although Kv7.1 does not co-assemble with any other members of the Kv7 family (Schroeder et al., 1998; Kubisch et al., 1999; Lerche et al., 2000a; Schroeder et al., 2000a; Schroeder et al., 2000b), it can co-assemble with members of the KCNE family of β-subunits. This was especially noted for KCNE1 (Barhanin et al., 1996; Sanguinetti et al., 1996; Tristani-Firouzi et al., 1998; Wang et al., 1998) and KCNE3 (Schroeder et al., 2000b). The co-assembly of Kv7.1 with KCNE1 produced a current that has a greater amplitude, slower activation, no inactivation and a more positive activation range than Kv7.1 alone. Its co-assembly with KCNE3, however, gave a current with an immediate activation and a linear current-voltage relationship (Schroeder et al., 2000b; Robbins, 2001).

Kv7.2 and Kv7.3 can co-assemble to form a heteromultimer. Their combination resulted in a current with ten times the amplitude of the summation of the individual currents (Wang et al., 1998; Yang et al., 1998; Main et al., 2000). This increase in current was attributed to an increase in channel expression (Schwake et al., 2000), which required an intact carboxyl terminus in the channel subunits. The "A domain" was also reported to be important for this interaction (Lerche et al., 2000a; Schwake et al., 2000). Kv7.2 was also found to co-assemble with KCNE1 (Robbins, 2001).

Kv7.3 is of particular interest. This is because out of all the Kv7 members it is able to form the most interactions with the other subtypes, and the least able to form homomultimers (Robbins, 2001). Kv7.3 can functionally interact with all Kv7 subunits (except Kv7.1) (Schroeder et al., 1998) as well as with KCNE1. KCNE2, which is linked with hERG in the heart (Abbott et al., 1999) interacts functionally with both Kv7.2 and Kv7.2+Kv7.3 heteromultimers (Tinel et al., 2000). Kv7.4 seems to co-assemble with Kv7.3, giving a current with greater amplitude than the sum of the individual homomeric currents (Kubisch et al., 1999). It is also able to interact

44 with KCNE β-subunits in Xenopus oocytes (Strutz-Seebohm et al., 2006). It is apparent that Kv7.5 also functionally interacts with Kv7.3, but not with Kv7.1, Kv7.2 or any of the KCNE members (Lerche et al., 2000a; Schroeder et al., 2000a).

1.7.4 Kv7 channel pharmacology:

1.7.4.1 Kv7 channel blockers:

Various known K+ channel blocking agents have been examined on the Kv7 family (Table 1.3). 4-AP, charybdotoxin and 1-[2-(6-methyl-2-pyrydinil)ethyl]-4(4 methylsuphonylaminobenzoyl) piperidine (E-4031: hERG blocker) did not block most Kv7 currents, while Ba2+ ions produced 50% inhibition at 1 mM (Robbins, 2001). TEA sensitivity appears to be dependent on the Kv7 subtype involved with the order of sensitivity of homomeric channels being as follows: Kv7.2>Kv7.4=Kv7.1>Kv7.5>Kv7.3 (Hadley et al., 2000; Schroeder et al., 2000a; Shapiro et al., 2000). This differential sensitivity depends on the nature of the amino acids in the P-loop of the channel. Kv7.1 has a valine (V) residue at position 284, while Kv7.3-Kv7.5 have threonine (T). The superior sensitivity of Kv7.2 has been suggested to be attributed to the presence of a tyrosine (Y) residue at the corresponding position, which renders shaker channels highly sensitive to TEA (Heginbotham et al., 1992). This proposition was tested in CHO cells (Hadley et al., 2000) and Xenopus oocytes (Schroeder et al., 2000a) expressing Kv7.3 (T323Y) mutants. However, in both studies, they were unsuccessful in producing sufficient currents to test the blocker.

Heteromeric channels displayed varying sensitivities to TEA. Kv7.3+Kv7.5 heteromultimers had a weak sensitivity to TEA (Schroeder et al., 2000a) and there is conflicting data with regards to Kv7.2+Kv7.3 heteromultimeric sensitivity. Some studies indicate an IC50  3-4 mM (Wang et al., 1998; Hadley et al., 2000), whereas another study reports it with a ten-fold difference giving IC5030 mM (Shapiro et al., 2000). It remains a mystery whether this discrepancy is attributable to differential expression levels of the two subtypes or to the nature of the cell expressing the channel (Robbins, 2001).

Table 1.3: Kv7 channel blockers (adapted from (Robbins, 2001))

Compound Kv7.1 Kv7.2 Kv7.3 Kv7.4 Kv7.5 Kv7.2+7.3 Kv7.3+7.4 Kv7.3+7.5 Ba2+ IC50 = 1mM 50% 60% 67% 46mM 5mM 80% 10mM 70% TEA 5.0 0.3 >30 3 71 3.8 ND 200 (IC50mM) Linopirdine 8.9 4.8 4.8 >200 16 4 <200 15 (IC50 µM) 51 XE991 0.8 0.7 <50 ND 65 0.6 ND ND (IC50 µM) Clofilium ND ND ND 10 µM 70% NE 30% 10% 30µM 40% Chromanol ND ND ND ND ND ND 293B 80% 45% 100 µM NE= No Effect; ND = Not Determined; % refers to % inhibition; IC50 is the 50% inhibitory concentration. (Wang et al., 1998; Hadley et al., 2000; Lerche et al., 2000b; Schroeder et al., 2000b; Tinel et al., 2000; Wang et al., 2000; Wickenden et al., 2001; Heitzmann et al., 2004)

Linopirdine has been used as a marker for Kv7 currents (Wang et al., 1998; Selyanko et al., 1999). It was shown to facilitate the release of acetylcholine, among other neurotrasmitters, from the rat brain (Nickolson et al., 1990) and ganglia (Kristufek et al., 1999) by inhibiting the M-current (Costa et al., 1997; Lamas et al., 1997; Noda et al., 1998). It also blocks nicotinic and GABAA receptors (Lamas et al., 1997; Schnee et al., 1998). It has a 10-20 fold lower selectivity for other voltage- and Ca2+- gated K+ currents (Robbins, 2001). Linopirdine was found to inhibit recombinant Kv7 channels, with an order of potency of Kv7.2=Kv7.3>Kv7.1>Kv7.5>Kv7.4 in homomeric channels, and Kv7.2+Kv7.3>Kv7.3+Kv7.5>>Kv7.3+Kv7.4 in heteromultimeric channels (Robbins, 2001). Two different IC50 values for linopirdine on Kv7.5 channels expressed in Xenopus oocytes have been reported. Lerche et al. (2000a) report

IC50=16 µM, while Schroeder et al. (2000a) report it to be three-fold greater (IC50= 51 µM). Whether this discrepancy was due to differences in the size of the expressed protein in the two studies, requires further investigation. Linopirdine has also been shown to inhibit Kv7 currents in rat stomach smooth muscle (Ohya et al., 2002a) and murine portal vein myocytes (Yeung et al., 2005).

The compound 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE991) is a more potent (10 fold) analogue of linopirdine (Zaczek et al., 1998). It has a different potency profile than linopirdine, in that the potency order for homomultimers is Kv7.1=Kv7.2>Kv7.5=Kv7.3 (Robbins, 2001). XE991 has the same sensitivity for the Kv7.2+Kv7.3 heteromultimer as Kv7.2, but a lower sensitivity for the Kv7.1+KCNE1 heteromultimer compared with Kv7.1 (IC50=11 µM vs. 0.8 µM) (Wang et al., 2000; Robbins, 2001). When XE991 was studied on Kv7.1 channels expressed in Xenopus oocytes, it was found to block Kv7 currents in a voltage- and time-independent manner. Both linopirdine and XE991 have been reported to block Kv7 channels by directly interacting with the channel protein rather than via a second messenger pathway (Costa et al., 1997; Lamas et al., 1997; Wang et al., 2000).

Chromanol 293B (293B, trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy- 2,2-dimethyl-chroman) is a Kv7.1 selective blocker (Bleich et al., 1997). It was originally developed as a lead compound of potential class III antiarrythmic drugs that act by inhibiting the cardiac IKs channel, which is formed by a co-assembly of Kv7.1 and KCNE1 (Lerche et al., 2000b; Seebohm et al., 2001; Lerche et al., 2007). It has been suggested that chromanol 293B inhibits Kv7 channels by blocking the channel pore in a voltage-independent manner and without altering channel kinetics (Yang et al., 2000; Seebohm et al., 2001). The residues T312S, I337V, and F340Y were found to be important for chromanol 293B sensitivity (Lerche et al., 2007).

1.7.4.2 Kv7 channel activators:

The effects of a number of Kv7 activators are listed in table 1.4. The anti-convulsant retigabine (N-[2-amino-4-(4-fluorobenzylamino)-phenyl] carbamic acid ethyl ester) (ezogabine in the US) activates Kv7.2, Kv7.3, Kv7.2+Kv7.3, Kv7.4 and Kv7.5 at concentrations of 1 to 10 µM (Main et al., 2000; Schrøder et al., 2001; Tatulian et al., 2001; Passmore et al., 2003). It was found to have no effect on Kv7.1 (Tatulian et al., 2001). It has been reported to shift the activation of Kv7.2/Kv7.3 currents to more negative potentials (at 10 µM), ultimately leading to an increase in the rate of activation and a decrease in the rate of deactivation (Main et al., 2000; Rundfeldt et al., 2000; Wickenden et al., 2000). It has also been shown that retigabine increases the single channel open probability (Tatulian et al., 2003). Retigabine is also known to potentiate other channels, such as the GABAA receptor channels (van Rijn et al., 2003). Retigabine (Trobalt®) has been approved by the American food and drug

47 administration (FDA) and the European Medicines Agency (EMA) as adjunct therapy for the treatment of refractory focal seizures (Porter et al., 2007; Brodie et al., 2010). Its less potent analogue, flupirtine, has been used as a centrally acting non-opoid analgesic and showed muscle relaxing properties, as well as anticonvulsant activity (Miceli et al., 2008).

BMS-204352 (5-Chloro-2-methoxyphenyl)-3-fluro-6-(trifluoromethyl)-2,3-dihydro-1H- indol-2-one )(Maxipost®) was found to activate all neuronal Kv7 channels expressed in HEK cells apart from Kv7.1 (Schrøder et al., 2003; Korsgaard et al., 2005). The order of sensitivity to BMS-204352 to the different Kv7 subtypes was Kv7.5 = Kv7.3/Kv7.5 > Kv7.4 = Kv7.3/Kv7.4 > Kv7.2/Kv7.3. BMS-204352 was found to activate voltage-dependent and voltage-independent currents (Korsgaard et al., 2005). When tested on Kv7 channels expressed in HEK293 cells it caused a hyperpolarizing shift in the voltage of activation and increased the activation rate while decreasing deactivation (Korsgaard et al., 2005). It was reported to have an EC50 of 2.4 µM on Kv7.4 and Kv7.5 (Schrøder et al., 2001; Dupuis et al., 2002). Its R-enantiomer however was found to have opposite effects. Both BMS-204352 and its R- enantiomer were found to activate big-conductance Ca2+-activated K+ channels (BKCa) channels. They were also shown to have opposite effects on GABAA receptors (Korsgaard et al., 2005).

The effects of the compound (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenylacrylamide (S-1) on Kv7.2 channels were initially investigated by Wu et al. (2003) in a rat model of migraine. S-1 was found to activate Kv7.2 channels expressed in Xenopus laevis oocytes and HEK cells. It caused a hyperpolarizing shift in the threshold of current activation, decreasing it from -70 mV to -85 mV. It also significantly increased the outward K+ current at voltages between -70 and +10 mV in this expression system. At membrane potentials greater than 10 mV the current amplitude decreased in the presence of S-1. When the compound was tested on HEK cells it produced a hyperpolarizing shift in the threshold of current activation and increased the single channel open probability. The effects of this compound on other Kv7 subtypes were more extensively studied a few years later by Bentzen et al. (2006).

In their study Bentzen et al. showed that S-1 potentiated Kv7.2, Kv7.4 (EC50 = 10.4 µM) and Kv7.5 in addition to Kv7.2/Kv7.3 expressed in Xenopus laevis oocytes. They reported however that at voltages greater than -20 mV, S-1 had an inhibitory effect

48 on Kv7.2 and Kv7.2/Kv7.3. This effect was found to be previously reported for retigabine (Tatulian et al., 2001) and may be due to the compound interacting with a different site on the channel (Schenzer et al., 2005). S-1 was found to inhibit homomeric Kv7.1 channels and Kv7.1 channels co-expressed with KCNE1 without shifting the voltage-dependence of activation (Bentzen et al., 2006). Another acrylamide, (S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)- ethyl]-3-(2-fluoro-phenyl)-acrylamide (S-2) was investigated on Kv7.1-5 channels expressed in Xenopus laevis oocytes by Blom et al. (2009). S-2 was reported to weakly inhibit Kv7.1, while activating Kv7.2-Kv7.5. It caused a hyperpolarizing shift in the activation voltage and increased current amplitude (Blom et al., 2009). It was also found to decrease inactivation while speeding up the rate of activation and slowing deactivation (Blom et al., 2009).

Mutagenesis studies revealed that a tryptophan residue in the fifth transmembrane domain is critical for the activity of retigabine on Kv7 channels (Schenzer et al., 2005; Wuttke et al., 2005) as well as the activity of S-1 (Bentzen et al., 2006) and S-2 (Blom et al., 2009) on Kv7.4 channels expressed in Xenopus laevis oocytes. It was proposed by Bentzen et al. (2006) that the aromatic tryptophan side chain may be an important binding site for Kv7.4 activators. This binding site was also found to be critical for the activity of BMS-204352 which is also capable of activating Kv7.4 channels (Bentzen et al., 2006). The same study also showed that retigabine and S-1 or BMS-204352 had no additive effects suggesting the presence of a common binding site. Interestingly, it was found that this residue was highly conserved from Kv7.2- Kv7.5 and there was a corresponding leucine in Kv7.1. This correlates with the sensitivity profile of retigabine, S-1, and BMS-204352 on Kv7 channels. In addition, a glycine residue in the sixth transmembrane domain was found to affect the efficacy of retigabine.

The compounds 4,4'-diidothiocyanateostilbene-2,2'-disulfonic acid (DIDS) and mefenamic acid are both Cl- channel blockers, but they were also reported to activate Kv7.1 channels (Busch et al., 1994; Busch et al., 1997). The effect of Cl- channel blockers was even greater on Kv7.1+KCNE1 heteromultimers, producing an instantaneous current with exponential activation (Robbins, 2001). Another fenamate, niflumic acid (0.5 mM), was reported to activate Kv7.5 (Schroeder et al., 2000a). Both meclofenamic acid and diclofenac are relatively potent activators of

Kv7.2 (EC50 = 25 µM) and Kv7.3 (EC50 = 2.6 µM) (Miceli et al., 2008). Unfortunately fenamates have many different pharmacological effects, so lack selectivity.

A more recently discovered Kv7 activator is bis(1-hydroxy-2(1H)-pyridineselonato- O,S)zinc, more commonly known as zinc pyrithione (ZnPy). This compound, which has been widely used for psoriasis (Crutchfield et al., 1997) and dandruff (Marks et al., 1985), was recently shown by Xiong et al. (2007) to be a strong activator of all Kv7 channels, apart from Kv7.3. When studied on neuronal Kv7 channels, ZnPy caused a hyperpolarizing shift in the voltage dependence of activation and an increase in current amplitude (Xiong et al., 2007). This compound is structurally different from retigabine and flupirtine (Fig. 1.8). Studies by Xiong et al. (2007) on Kv7.2 channels, revealed that Leu249, Leu275 and Arg306 are critical residues for ZnPy sensitivity (Figure 1.9A-C). It was assumed that Leu249 and Leu275 are on two separate α- helices but facing the same side. Arg306 is found between Gly301, known as the gating hinge, and the conserved putative Pro-Ala-Gly bend of the sixth transmembrane domain. These two components are important for voltage-gating in Kv7 channels (Xiong et al. 2007). Another study by Gao et al. (2008) on Kv7.1 channels, suggested that Ser338 and Leu342 are important interaction sites for ZnPy activity (Fig. 1.9D). These residues are also located in the sixth transmembrane domain, suggesting that this domain clearly played a critical role in the modulation of these channels by ZnPy. These regions were also found to be important for the binding of KCNE1 and KCNE3 subunits. The association of KCNE1 or KCNE3 subunits with Kv7.1 channels resulted in a loss of the sensitivity of these channels to ZnPy (Gao et al., 2010).

N-(6-Choloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243) was identified as a selective Kv7.2+Kv7.3 activator by Wickenden et al. (2008). This compound was found to cause a hyperpolarizing shift in the voltage-dependent activation of the Kv7.2+Kv7.3 channel (Wickenden et al., 2008). The selectivity of ICA-27243 for the Kv7.2+Kv7.3 and its ability to discriminate among Kv7 subtypes sparked interest in investigating its binding site. It was later discovered by Padilla et al. (2009) that ICA- 27243 indeed acted on a novel voltage-sensor domain binding site. This compound was found to have anticonvulsant activity in certain models of epilepsy (Wickenden et al., 2008) and may therefore be used as a template for future drug development.

Drug screens by Gao et al. (2010) have led to the discovery of more modulators, such as ztz240 (N-(6-chloro-pyridin-3-yl)-4-fluorobenzamide). This compound was found to be different from ZnPy and retigabine in that it prolonged the deactivation rate of expressed Kv7.2 channels at a far more significant level. It also had a different selectivity profile than ZnPy and retigabine in that it potentiated Kv7.4 and Kv7.5 more than Kv7.2 while having no effects on Kv7.1 and Kv7.3. It was proposed by Gao et al. (2010) that ztz240 acted on a different channel binding site than ZnPy and retigabine.

A very recently discovered activator of Kv7 channels is the retigabine analogue NS15370 (2-(3,5-difluorophenyl)-N-[6-[(4-fluorophenyl)methylamino]-2-morpholino-3- pyridyl]acetamide)hydrochloride) (Dalby-Brown et al., 2013). This compound was similar to retigabine in that it activated Kv7.2-Kv7.5 channels. However unlike retigabine, it was found to have no effect on GABAA receptor channels (Dalby-Brown et al., 2013). NS15370 was found to be 10-100 times more potent than retigabine when tested on HEK293 cells expressing Kv7 channels (Dalby-Brown et al., 2013). NS15370 caused a hyperpolarizing shift of activation at much lower concentrations than retigabine, BMS-204352, S-1 and ICA-27243 (Dalby-Brown et al., 2013).

1.8. Evidence that Kv7 channels have a role in PASMCs:

Joshi et al. (2006) used the selective Kv7 blockers, linopirdine and XE991, as markers for Kv7 channels to investigate their potential role in the regulation of pulmonary vascular tone in rats and mice. Both drugs were found to act as potent pulmonary vasoconstrictors. Linopiridine had an IC50  1 µM, while XE991 had a 10- fold lower IC50 (0.6-0.8 µM) for vasoconstriction. These effects were mediated by a direct action on the smooth muscle as an intact endothelium was not a prerequisite. The vasoconstriction was dependent on Ca2+ influx from the extracellular space into the smooth muscle, because it was abolished in a Ca2+-free medium and in the presence of the calcium antagonist, nifedipine. Levcromakalim, a KATP channel opener that causes hyperpolarization of PASMCs (Clapp et al., 1992; Clapp et al., 1993), abolished the responses as well, further supporting the mechanism underlying the vasoconstriction caused by linopirdine and XE991 (Fig. 1.10A). It was concluded

Table 1.4: The selectivity and mechanism of action of Kv7 channel activators (Adapted from (Xiong et al., 2007))

Compound Target Channels Effects Acrylamides Acrylamide (S)-1 Kv7.2,Kv7.3,Kv7.4, Hyperpolarzing shift of V1/2 Kv7.5 and Kv7.2+Kv7.3 Acrylamide (S)-2 Kv7.2 Hyperpolarzing shift of V1/2 DIDS Kv7.1+KCNE1 Decrease IKS deactivation rate. Increase the time-dependent outward current. Fenamates Diclofenac Kv7.2 and Kv7.3 Hyperpolarizing shift of V1/2 Kv7.2+Kv7.3 Slow deactivation rate

Flufenamic acid Kv7.1+KCNE1 Decrease IKS deactivation rate. Decrease the time-dependent Meclofenamic acid Kv7.2 and Kv7.3 outward current. Kv7.2+Kv7.3 Hyperpolarizing shift of V1/2 Slow deactivation rate Mefenamic acid Kv7.1+KCNE1 Decrease IKS deactivation rate. Niflumic acid Kv7.1+KCNE1 Increase the time-dependent outward current. Hyperpolarizing shift of V1/2 Decrease IKS deactivation rate. Decrease the time-dependent outward current. Retigabine Kv7.2,Kv7.3,Kv7.4, Hyperpolarizing shift of V1/2. Kv7.5 and Kv7.2+Kv7.3 Accelerate activation rate and slow deactivation rate. Flupirtine Kv7.2 Hyperpolarizing shift of V1/2. Accelerate activation rate and slow deactivation rate. Zinc Kv7.1,Kv7.2,Kv7.4, Hyperpolarizing shift of V1/2. Slow Pyrithione Kv7.5 and Kv7.2+Kv7.3 deactivation and activation rate, increase single channel open probability Po. ICA-27243 Kv7.2+Kv7.3 Hyperpolarzing shift of V1/2 Ztz-240 Kv7.4,Kv7.5>Kv7.2 Hyperpolarizing shift of V1/2. Slow deactivation rate. BMS- Kv7.5 = Kv7.3+ Kv7.5 > Hyperpolarizing shift of V1/2. 204352 Kv7.4 = Kv7.3+Kv7.4 > Accelerate activation rate and slow Kv7.2+Kv7.3 deactivation rate. NS15370 Kv7.2,Kv7.3,Kv7.4 and Hyperpolarizing shift of V1/2. Kv7.5 Accelerate activation rate and slow deactivation rate.

Figure 1.8: Chemical structures of Kv7 channel modulators. A) The structures of the Kv7 channel activators retigabine, flupritine, BMS-204352 and ZnPy. B) The chemical structures of the Kv7 channel blockers linopirdine and XE991 and the Kv7.1 selective blocker chromanol 293B.

B) C)

Figure 1.9: Molecular determinants of ZnPy and retigabine sensitivity. A) Kv7.2 channel showing the proposed interaction sites of retigabine (red) and ZnPy (blue) in recombinant Kv7.2 channels. B) Model showing the S1-S6 segments in a Kv7.2 channel with essential residues for channel modulation by retigabine (red) and ZnPy (blue). C) Enlarged S5-S6 segments of Kv7.2 from different views showing the essential residues for channel modulation by retigabine (red) and ZnPy (blue). (adapted from (Gao et al., 2008)). D) Model structure for the S5-S6 domain of Kv7.1. The left panel shows the residues essential for ZnPy modulation. The right panel shows the enlarged S6 domain. (Adopted from (Xiong et al., 2007)).

54 therefore that the responses of these two drugs were dependent upon the membrane potential, K+ flux and voltage-gated Ca2+-influx. A much smaller response was obtained in mesenteric arteries and this suggested that this response may be selective for the pulmonary circulation (Joshi et al., 2009).

In a later study, the activity of the Kv7 activators, retigabine and flupirtine, was studied on rat pulmonary artery (Joshi et al., 2009). These agents produced an endothelium-independent vasodilation, which was decreased by reducing the K+ gradient across the cell membrane. This is consistent with the hypothesis that their action involved the activation of smooth muscle K+ channels (Fig. 1.10B). Since their action was not prevented by glibenclamide, KATP channels do not play a role in the mediation of the response. The study also looked at the electrophysiological effects of the Kv7 blockers and activators. XE991 and linopirdine reduced IKN and caused membrane depolarization, whereas retigabine and flupirtine had the opposite effects. The actions of these modulators were consistent with modulation of a low threshold

K+ channel. However, even at maximal concentrations of the blockers, IKN was only reduced by 40% (at 0 mV), presumably because additional channels, including TASK-

1, were involved in mediating IKN.

Because of the relatively higher expression of KCNQ4 in rat PASMCs (Joshi et al., 2009), the study concluded that Kv7 channels and especially Kv7.4, were involved in controlling the resting membrane potential of PASMCs. A contribution of Kv7.1 may be ruled out, because it is insensitive to retigabine (Tatulian et al., 2001). Homomeric

Kv7.4 channels are less sensitive to linopirdine and XE991 than IKN (Robbins, 2001), so if they are involved, they may not function as homomers. Interactions with other subunits may be the answer, but whether other α-subunits and/or KCNE β-subunits are involved is yet to be determined. Kv7.4 can co-express with Kv7.5 as a heteromeric channel (Xu et al., 2007). It is present in the auditory pathway as variety of splice variants (Beisel et al., 2005), but nothing is known of splice variants in the pulmonary artery.

Although the study by Joshi et al. (2009) found predominant expression of KCNQ4 in the pulmonary circulation, other studies have demonstrated systemic expression (Yeung et al., 2007; Mackie et al., 2008). They also found that Kv7 blockers have effects on systemic vessels, although the responses reported were small. One study reported that intravenous linopiridine raised the mean arterial pressure (Mackie et

55 al., 2008), while other reports found that linopirdine had no effect on systemic blood pressure (Saletu et al., 1989; Pieniaszek et al., 1995; Joshi et al., 2009). Although there is a discrepency in these reports, it has been suggested that Kv7 modulators could affect Kv7 currents in the baroreceptors of the aortic arch which could have an effect on blood pressure (Wladyka et al., 2008).

The ability of Kv7 activators to dilate pulmonary arteries suggests that they could possibly be developed as drug candidates for treating pulmonary hypertension. The first direct evidence was shown in a recent study by Morecroft et al. (2009), where the effects of flupirtine on PAH in two independent mouse models was studied. In one model, PAH was induced in mice by chronic exposure to hypoxia. In the other model, spontaneous PAH developed in mice that over expressed the 5-HT transporter (SERT+ mice). The study showed that flupirtine reduced the development of chronic hypoxia-induced PAH and reversed PAH in SERT+ mice, significantly. It still has to be confirmed, however, that the effect was due to the activation of Kv7 channels.

Figure 1.10: Kv7 modulation in PASMCs. Schematic diagram showing the mechanism by which Kv7 blockers (A) and activators (B) are proposed to induce PASMCs contraction and relaxation respectively. Voltage-dependent Ca2+ channels (VDCC).

1.9 Aims:

There is a substantial amount of evidence linking the background K+ conductance, the regulation of the resting membrane potential and vascular tone in PASMCs. The background K+ conductance has been resolved into voltage-dependent and voltage- independent components, the latter being attributed to mediation by TASK channels (Gurney et al., 2003; Olschewski et al., 2006). The voltage-dependent component remains to be fully elucidated, but its similarity to the neuronal M-current led to investigations implicating Kv7 channels.

My first goal was to establish the functional role and therapeutic potential of Kv7 channels in the hypertensive pulmonary circulation, using the monocrotaline (MCT) rat model of pulmonary hypertension. The aim was to determine whether Kv7 modulators retained their ability to affect PA tone, and whether depolarization of PASMCs, which is known to occur in PH (Suzuki et al., 1982b; Yuan et al., 1998a; Ito et al., 2000), had any effect on their activity. I hypothesized that Kv7 channel activators would cause vasodilation in preconstricted PA vessels and would suppress the raised intrinsic tone which has been reported in MCT PA (Ito et al., 2000). In addition, we predicted that depolarization of PASMCs would enhance the activity of Kv7 modulators as the activity of these agents is known to be voltage-dependent. As K+ channel expression is known to be affected in PH, the expression of KCNQ genes in untreated and diseased PA was compared using quantitative polymerase chain reaction (qPCR) to establish whether or not their expression had been altered.

My second goal was to identify the effects and mechanism of action of the newly discovered Kv7 activator, ZnPy on rat PA. The effects of ZnPy on rat PASMCs and intact vessels were investigated using patch-clamp electrophysiology and small-vessel myography respectively. I predicted that ZnPy would produce hyperpolarization of PASMCs and therefore vasodilation of PA via Kv7 channel activation.

Chapter 2: Kv7 Channels in The Hypertensive Pulmonary Circulation

2.1 Animal models of pulmonary hypertension:

Pulmonary hypertension is a condition that is associated with a variety of abnormalities and histological findings. Various ion channels have been shown to be affected in PH. Idiopathic pulmonary hypertension has been associated with enhanced store-operated calcium entry and an increased expression of the canonical transient receptor potential cation (TRPC) channels TRPC3/C6 (Golovina et al., 2001). Enhanced store-operated calcium entry and TRPC channel expression has also been shown in MCT-treated rats (Liu et al., 2012). In addition, down-regulation of Kv channels has been shown to affect Ca2+ signaling in PAH. Changes in the expression of Kv1.5, Kv1.5/Kv1.2, Kv7, Kv2.1 and Kv3.1b channels have been implicated in PH (Kuhr et al., 2012).

Due to the complexity of this disease, it is important that we have valid animal models that help explain the pathophysiology of the disease as well as develop new drug treatments. Newer models are constantly being developed as our knowledge of PH expands. The chronic hypoxia exposure model, the sugen model, genetic models of PH and the monocrotaline injury model are a few examples of commonly used animal models of PH. It is important to bear in mind that although these models can be very useful, each of these models has certain limitations and none of these models exactly reproduce the human disease.

As the name suggests, in the hypoxia model, rodents are exposed to hypoxia for a few weeks to induce PAH. Vasoconstriction is initially observed, followed by progressive structural changes to the pulmonary vessels (Rabinovitch et al., 1979). The walls of the arterioles of the lungs and the alveoli undergo structural changes such that there is muscularization just three days after exposure to hypoxia (Rabinovitch et al., 2007). This muscularization is followed by hypertrophy without neointimal proliferation. The hypoxia model offers several advantages such as reproducibility and predictability, however there are species as well as age differences in the responses to chronic hypoxia (Stenmark et al., 2009). In addition, the plexiform lesions which occur in human PAH do not appear in this model of PH (Stenmark et

59 al., 2009). Thus this model may be used to investigate PH linked to hypoxia as is seen in COPD and at high altitudes (Stenmark et al., 2009).

Although the chronic hypoxia model is generally considered a model for less severe forms of PH, it is possible to induce a more severe form of PH in hypoxic rats by using additional triggers. Taraseviciene-Stewart et al. (2001) showed that the vascular endothelial growth factor (VEGF) receptor inhibitor Sugen 5416 (SU-5416) caused a severe and irreversible form of PH in chronically hypoxic rats. In this model there was marked vascular remodeling that was characterized by increased proliferation of smooth muscle cells and endothelial cells. This model offers the unprecedented advantage of the appearance of intimal lesions similar to those seen in the human disease. In addition, it has been reported that SU-5416 caused selective toxicity to the lungs without affecting other organs and without perivascular infiltration of monocytes and macrophages (Stenmark et al., 2009). However studies comparing gene expression in the lungs of this model to human PAH lungs found very little correlation, with only four genes in common (Moreno-Vinasco et al., 2008).

Genetic models are useful for studying cases where there is a greater genetic predisposition for developing PH. An example is seen in transgenic mice overexpressing the serotonin transporter (SERT+) who spontaneously develop PH (MacLean et al., 2004) . Another example is seen in fawn-hooded rats who are genetically predisposed to have a low uptake of serotonin into their platelets. This leads them to develop PH when they are just 4 weeks old (Kentera et al., 1988; Sato et al., 1992). Genetic models permit the use of very specific stimuli to trigger the development of PH and provide a means of studying specific pathways implicated with the disease.

A study by Morecroft et al. (2009), provided the first direct evidence of the involvement of Kv7 channels in PH. The study looked at the effects of the Kv7 activator flupirtine, in two different animal models of pulmonary hypertension: 1) a mouse hypoxia model of pulmonary hypertension and 2) mice over-expressing the serotonin transporter (SERT+ mice). It was found that flupirtine was able to prevent the development of PH in mice exposed to hypoxia. In addition, flupirtine was able to reverse the condition in SERT+ mice, where the disease was pre-existing. Whether this effect was due to an inhibition of vasoconstriction via channel activation, or due to the prevention of Kv7 channel down-regulation remains unclear. Although the

60 study looked at the role of Kv7 channels using two valid models of PH, it is important to study the role of Kv7 channels in a different model of pulmonary hypertension which may be more pertinent to the human disease.

The monocrotaline model of pulmonary hypertension is a relatively simple, cost- effective model. It has been widely used for over 40 years to study the pathogenesis of pulmonary hypertension and to develop new therapies. In this model, a single intra-peritoneal (IP) injection of monocrotaline in rats leads to the progressive development of PH with vascular lesions similar to those seen in the human disease (Kay et al., 1967; Mattocks, 1986; Cheeke, 1988; Wilson et al., 1992). Unlike the hypoxia model, it is considered a model of severe pulmonary hypertension. There is however a large variation seen in the response of various species to MCT (Wilson et al., 1992). In addition, this model has been criticized for having an acute nature which is very different from the human disease. It also lacks the plexiform lesions seen in severe forms of human PAH (Stenmark et al., 2009).

2.2 Mechanism of monocrotaline toxicity:

Monocrotaline is an 11-membered macrocyclic pyrrolizidine alkaloid found in the seeds of the plant Crotalaria spectabilis (Smith et al., 1981; Mattocks, 1986) (Fig. 2.1). Acute toxicity with this compound is known to induce the development of periacinar hepatic necrosis in both humans and animals (McLean, 1970; Huxtable et al., 1977; Wilson et al., 1992). It is of particular interest in rats, because although it causes hepatotoxicity in high doses, a single low dose consistently leads to the development of PH and cor pulmonale with no hepatic side effects (TURNER et al., 1965; Mattocks, 1986; Cheeke, 1988; Wilson et al., 1992).

The exact mechanism by which monocrotaline induces PH is unclear. It has been hypothesized however, that it causes pneumotoxiciy by conferring irreversible, non- cytotoxic endothelial injury that ultimately leads to the development of pulmonary hypertension (Wilson et al., 1992). Monocrotaline in itself is an inactive compound that is taken up by the liver where it is converted by dehydrogenation to its reactive metabolite monocrotaline pyrrole (MCTP) (also known as dehydromonocrotaline) (Mattocks et al., 1971; Mattocks, 1986). This product is taken up by red blood cells (RBCs) and transported to the lung, where it causes endothelial injury by a

Figure 2.1: Monocrotaline toxicity. Diagram showing the structure of MCT (extracted from the plant Crotalaria spectabilis (inset)) and its conversion to the toxic metabolite monocrotaline pyrrole by Cytochrome P (CYP) dehydrogenation in the liver.

62 mechanism that remains elusive, but is likely an inflammatory process involving platelet activation (Lalich et al., 1961; Wilson et al., 1989; Wilson et al., 1992). MCT is structurally different from other toxic plant alkaloids and this is believed to play a role in the compound's unique toxicity profile (Estep et al., 1990). Since the pulmonary circulation is the first arterial bed to be exposed to the metabolite when it is given systemically (Lafranconi et al., 1984; Yan et al., 1994), this leads to a preferential injury of the pulmonary endothelium. MCT causes inhibition of the proliferation of endothelial cells within hours of administration (Jago, 1969; Reindel et al., 1991; Lappin et al., 1998). This is accompanied by vascular leak, pulmonary edema and inflammation of the adventitia (Meyrick et al., 1980; Reindel et al., 1990). Smooth muscles cells proliferate into the non-muscular arterioles within one week of administration. Hypertrophy of arteries occurs next with loss of pulmonary perfusion within a couple of weeks (Meyrick et al., 1980; Ghodsi et al., 1981; Hilliker et al., 1982; Roth et al., 1991; Lappin et al., 1997; Lappin et al., 1998).

2.3 Alterations of endothelium & smooth muscle function:

The resting membrane potential of PASMCs is a key determinant of vascular tone. The endothelium contributes to the resting membrane potential of PASMCs by releasing substances such as nitric oxide (NO) (Tare et al., 1990), prostacyclin (Murphy et al., 1995) or endothelium-derived hyperpolarizing factor (EDHF) (Nagao et al., 1991) that give rise to a hyperpolarization. In addition, coupling between smooth muscle cells and the endothelium also influences pulmonary vascular tone (Yamamoto et al., 1999).

A study by Ito et al. (2000), examined the alterations in the function of the endothelium and smooth muscle in pulmonary arteries obtained from rats treated with monocrotaline. They found that there was development of basal tone in arteries at 3 weeks after being subjected to a single subcutaneous injection of monocrotaline in comparison to control vessels (Fig. 2.2A). Pulmonary arteries normally show no basal tone in the absence of constricting agents. They also found that the resting membrane potential of PASMCs was depolarized in vessels after 2-3 weeks of treatment with monocrotaline and noted spontaneous spike activity that increased progressively between 2 and 3 weeks of treatment. In the first three weeks after MCT was administered, the removal of the endothelium was found to cause further

63 depolarization of the resting membrane potential. After three weeks, the removal of the endothelium had no effect on the resting membrane potential. They concluded that due to the endothelial injury, the endothelium exerted less of a hyperpolarizing influence on the smooth muscle cells and therefore led to more depolarized membrane potentials. Since the resting membrane potential of PASMCs is important in regulating vascular tone, it may be possible to reverse or inhibit this deploarization by Kv7 channel activation.

In their study, Ito et al. (2000) also noted that PA from MCT-treated animals exhibited slower washout after being challenged with a contractile stimulus using high KCl or prostaglandin F2 alpha (PGF2α), in comparison to PA from animals that had not been treated with MCT (Fig. 2.2B). They speculated that depolarization of PASMCs in MCT-treated vessels may have contributed to the slowed recovery times by reducing the activity of the NCX.

2.4 Hypothesis and aims:

The aim of this part of my project was to determine whether the effects of Kv7 modulators on PA were altered in PAH. The effects of Kv7 modulators on PA were also compared with mesenteric arteries, which are known to be unaffected by the disease, (Wilson et al., 1992) in order to determine tissue specificity. I also aimed to determine if the depolarized condition of the PASMCs had any influence on the response to Kv7 modulators. I predicted that Kv7 modulators would have enhanced sensitivity in MCT vessels due to disease-induced depolarization. Therefore hyperpolarization of MCT vessels would render then less sensitive to Kv7 modulators by counter-acting the disease-induced depolarization. I established the role of membrane potential on the sensitivity of pulmonary arteries to Kv7 modulators using a 15 mM K+ physiological salt solution (PSS) to depolarize control PA and the KATP channel opener, levcromakalim, to hyperpolarize MCT PA. I was particularly interested in determining if Kv7 activators could reduce intrinsic tone in the absence of a stimulus and hypothesized that Kv7 channel activators would suppress intrinsic tone by causing vasodilation via hyperpolarization and closure of voltage-gated Ca2+ channels. To determine the role of KCNQ expression in PAH, I used qPCR studies to compare the expression of KCNQ genes in pulmonary and mesenteric arteries from MCT and control animals in order to detect any changes that may have occurred as a

64 result of the development of PH. This will help us to understand whether Kv7 modulation is altered in this model of PH and will shed some light on the utility of Kv7 openers as potential drug treatments.

Figure 2.2: Changes in pulmonary artery behavior after MCT induced PAH. A) Basal tone developed as shown by vessels treated with Ca2+-free ethylene glycol tetraacetic acid (EGTA) at 3 weeks after treatment with either saline (control) or monocrotaline (MCT). B) Decline in tension after washout of KCl from MCT and control arteries using Ca2+ free EGTA. (adapted from Ito et al. (2000)).

2.5 Methods:

2.5.1 Contractile studies:

2.5.1.1 Tissue preparation:

Male Wistar rats (250-300 g) were divided into control and monocrotaline groups. The rats in the MCT group were given a single MCT injection intraperitoneally (60 µg/kg) and generally developed PAH within 3-4 weeks. The rats in the control group were given saline in lieu of the MCT. The animals were weighed daily and observed for any clinical symptoms of pulmonary hypertension. They were subjected to various cardiovascular measurements (echocardiography) to confirm that they had indeed developed pulmonary hypertension. They were subsequently killed by cervical dislocation in accordance with the UK Scientific Procedures (Animals) Act 1986. The animals in the control group were also sacrificed at the same time in order to serve as controls. The procedures until this point were performed by Ian Temple and Gillian Quigley under the supervision of Prof. George Hart and Prof. Mark Boyett.

The heart and lungs from both groups of animals were removed as one unit and placed into PSS (Table 2.1). The pulmonary artery running along the length of a lobe was dissected from the lungs and cleaned of connective tissue and blood (Fig. 2.3). The vessel was then cut into rings ( 5 mm in length) and slices were mounted on the pins of a myograph chamber (Danish Myo Technology Multi-myograph 610M and 620M) for isometric tension studies (Fig. 2.4). The vessels were bathed in PSS and aerated at 37ºC. A basal tension of 5 mN was applied, as this tension was found to produce a maximal response to agonists and was found to be equivalent to the pulmonary arterial pressure (15 mmHg) for the size of the vessels used. The vessels were left to equilibrate for 45 min. The vessels were washed with fresh PSS every 15 min during equilibration. The response to various drugs was measured as a change in tension recorded by the myograph. Intracept-chart software V.4.9.0 (developed by Dr. John Dempster (University of Strathclyde)) was used with a National Instruments analogue to digital interface (model AT-M10-16L-9) to record the tension. In some experiments the intestine was removed with its blood supply intact and the mesenteric arteries were obtained by careful removal of the perivascular adipose tissue (PVAT). The mesenteric artery was cut into rings ( 5mm in length). Tungsten wires (2.2 cm in length, 40 µm in diameter) were used to mount the vessel onto the jaws of a wire-myograph. While the average diameter of rat pulmonary arteries is  2

66 mm (Hislop et al., 1978), mesenteric arteries are much smaller with an average diameter of 240 µm (Zhang et al., 2007a). In these vessels a basal tension of 5 mN was applied and the tension was recorded as for the pulmonary artery.

Once the vessels were equilibrated they were challenged with 50 mM KCl. This was done in order to sensitize the tissue and was repeated three times to test the reproducibility of the response. The vessels were exposed to 50 mM KCl for 5 min and this generally produced a steady contractile response. The vessels were then washed with PSS until the tension returned to base-line.

2.5.1.2 Protocols used to study drug effects on PA tone:

The effects of various blockers on the tone of pulmonary and mesenteric arteries were investigated. The Kv7 blockers linopirdine (1 nM-100 µM), XE991 (0.1 nM-10 µM), and chromanol 293B (10 nM-100 µM) were applied in a cumulative manner with at least 10-15 min between applications. The effects of XE991 on the vessels were also tested in the presence of the Ca2+ channel blocker nifedipine in order to determine whether or not the observed effects were dependant on voltage-gated Ca2+ entry.

To assess the presence of a raised intrinsic tone, the vessels were exposed to several vasodilators. Nifedipine (1 µM) and increasing concentrations of retigabine (10n M- 100 µM), ZnPy (100 nM-100 µM), sildenafil (0.1 nM-10 µM), levcromakalim (1 nM-100 µM) or 2-aminoethoxydipheyl borate (2-APB) (10 µM, 50 µM and 100 µM) were used. The effects of the Kv7 activator retigabine on basal tone were further investigated in the presence of the Ca2+ channel blocker nifedipine and in a Ca2+-free solution.

The role of depolarization on contractile responses to the Kv7 blocker XE991 (0.1 nM- 10 µM) and the Kv7 activator retigabine (10 nM-100 µM) was tested by exposing control pulmonary arteries to an osmotically balanced depolarizing solution of 15 mM K+ PSS. After being exposed to this solution for 20-30 min, the vessels exhibited a small but sustained constriction. They were then treated with the blocker XE991 or pre-constricted with PE in order to test the effects of retigabine.

Table 2.1: Composition of PSS: This table shows the composition of the PSS used in my experiments.

Salt Concentration (mM) NaCl 122 KCl 5 NNN-[2-Hydroxyethyl]piperazine-N-[2-ethane-sulfonic 10 acid] (HEPES)

KH2PO4 0.5

NaH2PO4 0.5

MgCl2 1 Glucose 11

CaCl2 1.8 *pH adjusted to 7.3 with 1M NaOH

A) B)

Figure 2.3: Intrapulmonary artery preparation. Figure shows the dissection of the main intrapulmonary artery (A) and a ring of artery mounted on the pins of a myoagraph chamber (B). Average diameter of PA is  2 mm (Hislop et al., 1978).

Figure 2.4: Myography Setup. Schematic diagram showing a myograph chamber with either wires or pins. The micropositioner is used to apply tension. The fixed jaw is attached to the computer through an analogue to digital interface and detects changes in tension which are displayed as a recording on the computer screen. The myograph had multiple chambers and drugs were added directly to the bath and washed by replacement of the bathing solution.

In another set of experiments control pulmonary arteries were challenged three times with 50 mM KCl and washed, followed by two challenges with PE (1µM), with a wash between each to establish controls. The vessels were then exposed to 15 mM K+ PSS and challenged once with 50 mM KCl and once with 1µM PE. Recovery times in 15 mM K+ PSS were compared to those in normal PSS to determine how depolarization affected the washout kinetics.

The KATP opener levcromakalim (1 µM) was used to test the effect of hyperpolarization on vessel recovery rates after stimulation with 50 mM KCl, as well as the contractile responses to the Kv7 blocker XE991 (0.1 nM-10 µM) and the Kv7 activator retigabine (10 nM-100 µM). After being exposed to levcromakalim (1 µM), MCT pulmonary arteries were challenged with KCl and the recovery times were studied. Subsequently the vessels were either pre-constricted with PE to study the effects of retigabine or exposed directly to XE991.

2.5.1.3 Protocols used to study drug effects on pre-constricted PA:

The α-agonist, PE (1 µM in pulmonary arteries and 10 µM in mesenteric arteries) was used to preconstrict the vessels. After a 20-45 min period the response to PE was stable and the vessels were subsequently exposed to increasing concentrations of the Kv7 openers retigabine (10 nM-100 µM), ZnPy (100 nM-100 µM) or BMS-204352 (10 nM-100 µM) applied cumulatively with at least a 15-20 min interval between applications. 2-APB was also tested at 10 µM, 50 µM and 100 µM concentrations.

2.5.1.4 Drugs:

The drugs used in this study are summarized in Table 2.2 below. Dimethylsulfoxide (DMSO) was used to dissolve drugs with low water solubility. This may have an effect on the response of the vessels especially at higher concentrations. However, DMSO controls were carried out and it was shown that the effects of DMSO were negligible.

Table 2.2: Drugs used to study vessel tone in the MCT model of PH:

Drug Name Source Action Stock Solution Retigabine Toronto Research Kv7 activator 100 mM in H2O Chemicals Inc. ZnPy Sigma-Aldrich Kv7 activator 100 mM in DMSO (UK) BMS-204352 Axon Med Chem Kv7 and BKCa 100 mM in DMSO (Netherlands) activator XE991 Tocris (UK) Kv7 blocker 10 mM in H2O Linopiridine Tocris Kv7 blocker 100 mM in H2O Chromanol 293B Tocris Kv7.1 blocker 100 mM in DMSO Sildenafil Pfizer PDE5 inhibitor 1 mM in H2O Levcromakalim Tocris KATP activator 10 mM in DMSO Nifedipine Sigma-Aldrich L-type Ca2+ channel 1 mM in DMSO blocker Hydralazine Sigma-Aldrich Blocker of Ca2+ 100 mM in H2O release from intracellular stores 2-APB Calbiochem (UK) Blocker of Ca2+ 10 mM in DMSO release from intracellular stores and IP3 receptors

2.5.1.5 Statistical analysis:

The responses to vasodilators applied to PE-constricted vessels were measured as the change in tension expressed relative to the response produced by PE. Responses to agents applied at resting tone were measured as the change in tension expressed relative to the maximal constriction induced by 50 mM KCl. Chart software (V 4.9.0, The University of Strathclyde) was used to record and measure the tension developed in the vessels. OriginPro software (version 8.1, OriginLab Corporation), Excel (2007, Microsoft), Powerpoint (2007, Microsoft) and Graphpad Prism 5 (Graphpad) were used for statistical analysis and figure preparation. The data are expressed as means ± S.E.M of n animals and were compared using Student's t-test, where p<0.05 was considered to be statistically significant. Two-Way ANOVA followed by Bonferroni post test was used to determine statistical significance where appropriate. However, caution must be taken in the interpretation of the results of experiments with low n numbers as the power calculation of the statistical test has not been carried out. This implies that in certain cases the n numbers were too low to detect statistical significance.

2.5.2 Molecular biology:

2.5.2.1 Polymerase chain reaction

The principle of a polymerase chain reaction (PCR) is to increase a specific target DNA from a very small amount of starting material (Mullis et al., 1986). Any PCR reaction requires the presence of target DNA, primer pairs, a heat-resistant polymerase enzyme, deoxynucleotide triphosphates (dNTPs), a cofactor (MgCl2) and a reaction buffer (Saiki et al., 1988). The primers are complimentary to the target DNA sequence and are extended by the action of a thermostable Taq DNA polymerase enzyme (Saiki et al., 1988). Changes in temperature during a PCR reaction control the activity of the enzyme and the binding of the primers to the target DNA. At the onset of the reaction, the temperature is raised to 95ºC. At this temperature the DNA is "melted" into single strands, a step known as denaturation. The temperature is then lowered to 50-65ºC to allow the primers to attach to the gene of interest, a step known as annealing. Finally the temperature is raised to 72-78ºC, which is the optimal temperature for the DNA polymerase to work. The polymerase begins its action on the annealed primer:DNA complex, a step known as extension or elongation. At the end of this step the amount of starting material has doubled. The cycle is repeated 25-40 times, resulting in the effective amplification of starting material.

2.5.2.2 Tissue preparation:

Pulmonary artery and/or mesenteric artery were cut into slices ( 5 mm in length) and placed in an eppendorf containing RNA later (Ambion, UK), which preserves RNA integrity. The tubes used were all sterile and RNAase/DNAase free. The tissue was left in RNA later overnight in the fridge (-4ºC). The following day, either RNA isolation was performed or the eppendorf containing the tissue was transferred for storage at - 80ºC for use at a later date.

2.5.2.3 Protocol for RNA isolation:

RNA was isolated following the manufacturer's fibrous tissue protocol with an RNA Mini Kit (Qiagen, UK). Each sample was transferred from RNA later to a 2 ml tube

72 containing 150 µl RLT buffer (supplied with the kit and containing 1% β- mercaptoethanol) and 295 µl DNA-RNase free water (Qiagen). The tissue was homogenized in the RLT buffer solution using a tissue homogenizer, while gently scraping the tissue against the inside walls of the tube, using 50-80% speed. Care was taken to avoid the production of too much froth and to prevent the tube from warming up. At this stage the homogenization was only partial. The tube was centrifuged briefly to separate the homogenate. Next, 5 µl of proteinase K (Qiagen) was added to the tube, which was then incubated for 10 min at 55ºC using a heating block or a water bath. Meanwhile the homogenizer was washed using fresh DNA- RNAse free water. The homogenization of the sample was then repeated and the homogenate transferred to a labelled QIA-shredder column and centrifuged at full speed for 3 minutes. The supernatant was collected carefully and transferred into a new 1.5 ml tube. Subsequently, 225 µl of 96% ethanol was added to the sample and mixed by pipetting. The mixture was then transferred into a labelled RNeasy column placed in a 2 ml collection tube (both supplied with the kit) and centrifuged for 15 s at 10,000xg.

Buffer RW1 (350 μl) was added to the RNeasy spin column and centrifuged for 15 s at 10,000xg in order to wash the spin column membrane. Next a DNA digestion step was performed to remove genomic DNA. DNase I stock solution (10 μl) (Qiagen) was added to 70 μl Buffer RD and mixed by gently flicking the tube or by pipetting. The tube was then centrifuged briefly and the mix added directly to the RNeasy spin column membrane, which was placed on the bench top at room temperature for 15 min. Buffer RW1 (350 μl) was then added to the RNeasy spin column and the tube was centrifuged for 15 s at 10,000xg. Next 500 μl of Buffer RPE (with ethanol added) was added to the RNeasy spin column, followed by centrifugation for 15 s at 10,000xg. Buffer RPE (500 μl) was added once more to the RNeasy spin column and centrifuged for 2 min at 10,000xg to wash the spin column membrane. The RNeasy spin column was placed in a new 2 ml collection tube and centrifuged at full speed for 1 min to ensure that no ethanol was carried over. The RNeasy spin column was placed in a new 1.5 ml collection tube and 40 μl RNase-free water was added directly to the spin column membrane. The tube was then centrifuged for 1 min at 10,000xg to elute the RNA. The concentration of RNA was subsequently checked using a Nanodrop spectrophotometer (ND-1000). Absorbance was measured at 260 nm and

280 nm and the ratio A260/A280 was used to assess sample purity (1.9-2.1 was

73 considered as pure). RNA integrity was also checked using a bioanalyzer machine (Agilent 2100, The Faculty of Life Sciences Genomic Technologies Core Facility).

2.5.2.4 Protocol for first strand cDNA synthesis (reverse transcription):

The experiments were designed such that for pulmonary arteries 600 ng of RNA went into the reaction mix and for mesenteric arteries 150 ng was used. A lower amount was used for the mesenteric arteries because the RNA extraction yields were lower than for pulmonary arteries. For each sample, two tubes were prepared such that each contained the sample RNA, 0.5 µl dNTP mix (Qiagen), 1 µl random hexamers (Applied Biosystems-Roche, UK) and RNAase free water to make up a total volume of 13 µl. The tubes were vortexed and centrifuged at 10,000xg for 15 sec then transferred into a thermal cycler (Techne TC-512), where they were heated to 65ºC for 5 minutes. The samples were then left on ice for at least one minute. A 'buffer mix' (6 µl) composed of 4 µl 5x first strand buffer (Invitrogen, UK), 1 µl Dithiothreitol (DTT) (Invitrogen) and 1 µl RNAse out (Invitrogen) was added to each sample. The two tubes prepared for each sample were now labelled as the RT(+) and RT(-) sample. The RT(-) sample served as a negative control and lacked the reverse transcriptase enzyme necessary for the synthesis of cDNA. 1 µl of reverse transcriptase SSIII (Invitrogen) was added to the RT(+) samples, and RNAse free water was added to the RT(-) samples. The samples were mixed by low speed vortex or pipetting, then centrifuged briefly. Samples (20 µl) were then loaded onto the heating plate of the thermal cycler. The program was as follows: 65ºC, 5 min; 25ºC; 5min and 50ºC, 1 hour. When the run was complete the samples were labelled and stored at -20ºC.

2.5.2.5 Primer design:

The careful design of primers is one of the most important steps in any PCR reaction. and numerous factors must be taken fully into account. These include: specificity, annealing temperature, primer length, GC content and possibility of the formation of primer dimers. The KCNQ1 and KCNQ4 primers used in this study were taken from Joshi et al. (2009), which investigated the expression of Kv7 channels in the pulmonary arteries of sprague dawley rats. The KCNQ5 primer was designed using the National Center for Biotechnology Information (NCBI)-Primer Blast online tool. The primers selected were all intron spanning to prevent the amplification of genomic DNA. The sequences for the housekeeping genes (beta actin (β-actin), cyclophilin A

(also known as peptidyl prolyl isomerase A) (Cyp), hypoxanthine guanine phosphoribosyl transferase (Hprt), phosphoglycerate kinase 1 (Pgk1) and tyrosine-3- monooxygenase/tryptophan 5 -monooxygenase activation protein, zeta polypeptide) (Ywhaz)) were taken from a study by Langnaese et al. (2008), which investigated the selection of reference genes for quantitative real-time PCR studies on rat cardiac tissue. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a sixth housekeeping gene and its sequence was taken from Joshi et. al (2009). The sequences (Tables 2.3 and 2.4) were checked for specificity using the NCBI-Primer Blast online tool. Primers were ordered from Integrated DNA Technologies (USA) and prepared as 100 µM stock solutions in nuclease free water.

In the PCR protocol used, 1 µl cDNA was included in a 50 µl reaction mixture as defined in table 2.5. The cycling protocol programmed in a thermal cycler (Techne TC- 512) was optimized to the following conditions: 95ºC for 15 min in order to activate the HotStarTaq DNA Polymerase (Qiagen), followed by 35 cycles at 95ºC for 1 min (denaturation), 53ºC for 30s (annealing), and 70ºC for 1 min (extension). At the end of the protocol the samples were heated for a further 10 min at 70ºC to allow for the complete extension of the product. After amplification, the samples were stored overnight at 2-8ºC or at -20ºC for future use.

2.5.2.6 Gel electrophoresis:

PCR reaction products are commonly analyzed by gel electrophoresis, a technique that enables the separation of proteins and nucleic acids based on their molecular weight and charge. The molecules are separated by applying an electrical field across a gel (most commonly agarose) in the presence of a buffer solution, the negatively charged DNA moves towards the anode. The smaller molecules move faster through pores in the gel so that the distance moved depends upon the molecular weight of the DNA species (Sambrook et al., 1989). The DNA molecules are subsequently stained using ethidium bromide, which is a fluorescent dye that intercalates between the base pairs of the DNA molecule (Sharp et al., 1973). Images of the gel are produced by irradiation with UV light. The ethidium bromide-DNA complex emits at 590 nm when excited at 302 nm (Sambrook et al., 1989).

Table 2.3: KCNQ primers. The table below gives details of the KCNQ primers used in the study. F: denotes the forward primer and R: denotes the reverse primer. References give the accession numbers for each gene and the position of the gene and amplicon lengths are also given.

Gene Sequence Reference Position Amplicon (cDNA) length (bp) KCNQ1 F: GGCTCTGGGTTTGCACTG NM_0320773 1042-1059 106 R: CATAGCACCTCCATGCAGTC 1128-1147 KCNQ4 F: CCCCGCTGCTCTACTGAG XM_233477 1753-1770 86 R: ATGACATCATCCACCGTGAG 1819-1838 KCNQ5 F: TACCGGAGGGTGCAGAACTA NM_001134643.2 495-514 146 (qPCR) R: GCCAACTTTGTGTGCTCAGG 621-640 KCNQ5 F: CGAGACAACGACAGATGACC XM_237012 1842-1861 77 (RT-PCR) R: TGGATTCAATGGATTGTACCTG 1997-2018

Table 2.4: House-keeping gene primers. The table below gives details of the HKG primers used to in the study. F: denotes the forward primer and R: denotes the reverse primer. References give the accession numbers for each gene and the position of the gene and amplicon lengths are also given.

Gene Sequence Reference Position Amplicon (cDNA) length (bp) GAPDH F: CAACTCCCTCAAGATTGTCAGCAA NM_017008 493–516 118 R: GGCATGGACTGTGGTCATGA 591-610 Β-Actin F: AAGTCCCTCACCCTCCCAAAAG V01217 3474–3495 97 R: AAGCAATGCTGTCACCTTCCC 3550-3570 CycA F: TATCTGCACTGCCAAGACTGAGTG M19533 381–404 126 R: CTTCTTGCTGGTCTTGCCATTCC 485-507 Hprt F: CTCATGGACTGATTATGGACAGGAC NM_012583 179–203 123 R: GCAGGTCAGCAAAGAACTTATAGCC 277-301 Ywhaz F: GATGAAGCCATTGCTGAACTTG NM_013011 955–976 117 R: GTCTCCTTGGGTATCCGATGTC 1050-1071 Pgk1 F: ATGCAAAGACTGGCCAAGCTAC NM_053291 969–990 104 R: AGCCACAGCCTCAGCATATTTC 1051-1072

Table 2.5: PCR reaction composition The table below shows the components of a 50 µl PCR reaction.

Component Volume per reaction Final concentration 10xPCR Buffer 5 µl 1x dNTP mix 1 µl 200 µM of each dNTP Forward primer 1.5 µl 300 nM Reverse primer 1.5 µl 300 nM HotStarTaq DNA polymerase 0.25 µl 2.5 units per reaction Nuclease-free water 39.75 µl - Template cDNA 1 µl 30 ng/µl

My experiments used a 1.5% agarose gel in TAE buffer, containing 45 mM Tris- acetate (pH 8.0) and 1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich). Ethidium bromide (Promega, UK, 0.5mg/ml) was added to the gel and the mixture was poured into an electrophoresis plate. A comb was positioned approximately 1 mm above the plate in order to form wells while the gel sets. After the gel had completely set, TAE buffer was poured over it and the comb was carefully removed. Aliquots of the PCR product (15 µl) were mixed with 3 µl of loading dye (New England Biolabs, UK), which gave a color to the samples, making it easier for them to be added to the wells and ensuring that they spread evenly. A 50 bp molecular weight ladder was also used (New England Biolabs) to allow for determination of product size. After loading the wells with the appropriate samples, the electrophoresis plate was covered with a lid attached to a power supply and 100 mV was applied for 75 min. When the electrical field was applied, bubbles appeared in the gel and the DNA molecules started to move towards the anode. When electrophoresis was complete, the gel was removed and viewed by applying UV light, using a transilluminator (Uvitec) and photographed using a camera.

2.5.2.7 Purification of PCR products:

PCR products were purified using a PCR and gel ISOLATE kit (Bioline, UK). Binding buffer A (500 µl) was added to the PCR product sample. The solution was then transferred to spin column A (provided in the kit) and mixed by pipetting. The spin columns were centrifuged at 10,000xg for 2 min. The collection tube was discarded

77 and spin column A was transferred to a 1.5 ml elution tube also provided in the kit. Elution buffer (15 µl) was added directly to the spin column membrane and left to stand at room temperature for 1 min. The column was then centrifuged at 6,000xg for 1 min to elute the product. The solution produced represented the purified PCR product and was ready to use in other applications.

2.5.2.8 Real time PCR:

In the PCR experiments described above, mRNA expression was measured at the endpoint of the PCR reaction and was not quantitative. Real-time PCR permits the simultaneous amplification and detection of the product in a single tube throughout the PCR reaction and allows the mRNA to be quantified. The amount of DNA in each PCR cycle doubles, so that after n cycles, there will be 2n times as much DNA present. The reaction is initially exponential, but eventually reaches a plateau phase, when one of the reagents in the reaction becomes limiting. In real time PCR fluorescent technologies are used to detect the presence of amplicons. At the end of each PCR cycle, the amount present is proportional to the amplitude of the fluorescence signal detected (Wittwer et al., 1997). Some of the commonly used detection methods include SYBR Green, high resolution melting dyes, TaqMan probes, locked nucleic acid probes and molecular beacon probes. In this study SYBR Green was used as a fluorescent dye for detection of the target sample. It is an intercalating dye that binds to double-stranded DNA and emits fluorescence at 520 nm when excited at 497 nm. The fluorescence emitted is detected by the thermocycler PCR machine and is directly related to the amount of target in the sample. As the reaction progresses more target DNA is produced, with more SYBR Green binding and increasing fluorescence (Fig. 2.5).

SYBR green is known to bind to any double-stranded DNA. Since different DNA products dissociate at different characteristic melting temperatures, a melt curve is run at the end of the PCR reaction to assess the purity of the products. A single peak on the melt curve indicated a single product.

In a qPCR assay, the results are usually displayed in the form of an amplification plot, also known as a growth curve, where the increase in fluorescence (Rn) is plotted versus the cycle number (Fig.2.6). The baseline is the average background noise level and is determined from the early cycles, where the increase in fluorescence is too small to be detected. The threshold level of fluorescence is where the signal begins

78 to increase above background. The threshold cycle (CT value) is the cycle at which the curve crosses the threshold. The CT value is inversely proportional to the amount of target sample. In other words, samples with a low CT value have more expressed target than samples with a higher CT value, in which more cycles are required to detect the target. Many thermocyclers, including the one used in my experiments, use a passive reference known as ROX, which is a fluorescent dye included in the mastermix but does not interact with DNA. SYBR Green fluorescence was normalized to the ROX signal and is known as the normalized reporter signal (Rn). When there is variation in background noise between PCR reactions, comparisons can be made using the difference in signal obtained by subtracting the normalized background signal (Bn) from the normalized reporter signal (Rn = Rn - Bn).

SYBR Green master mix (Applied Biosystems, UK) containing ROX was used in the study. The PCR reaction consisted of 2xSYBR Green master mix (10 µl), 300 nM primer mix, cDNA (1 µl) and nuclease free water (3.8 µl). This gave a total reaction volume of 20 µl. The mixture was pipetted into the wells of a 96-well plate (Primer Design, UK), with triplicates for each sample. No template controls (NTC) were included, in which water was added in place of cDNA. Negative controls, lacking reverse transcriptase in the cDNA preparation step, were also used in order to check for genomic DNA contamination. The plate was sealed using an adherent transparent sheet (Applied Biosystems) and centrifuged briefly (2-4 min) at 22ºC and 1,000xg. The qPCR was then performed in accordance with the manufacturer's instructions for the Applied Biosystems 7500 system. The cycling protocol consisted of holding at 50ºC for 2 min, 95ºC for 10 min, followed by 40 cycles at 95ºC for 15 s and 60ºC for 1 min. An additional dissociation step was added at the end of the protocol, which permitted melt curve analysis to confirm that a single product was produced.

stranded stranded DNA are - of the PCR reaction

At At the start

ceeds ceeds and more copies of double

amplification products

free free in solution. As the reaction pro

SYBR Green is generated, SYBR green intercalates between the two strands of DNA giving fluorescence, which can be sample. the in ofDNA amount the detect to used Figure 2.5: SYBR green detection of PCR

Figure 2.6: A typical amplification plot in a qPCR reaction. This figure shows a plot of the normalized reporter signal (Rn) versus the cycle number. The threshold cycle CT is the cycle number at which the Rn exceeds the threshold. The difference in Rn and the baseline signal (Bn) can be used to calculate the variation in background noise between PCR reactions.

2.5.2.9 Reference targets/housekeeping genes:

Quantitative real-time PCR is a valuable method for evaluating changes in mRNA expression that occur in different disease states, but it is important to measure expression relative to genes that are stably expressed in each tissue. These genes are known as house-keeping genes (HKGs) and serve different functions (Table 2.6). For many years it was assumed that HKGs were stably expressed in different tissues and in different disease conditions. This however is certainly not the case as several studies have shown that the expression levels of HKGs can vary under certain disease states and/or treatments in the same samples (Schmittgen et al., 2000; Bonefeld et al., 2008). Several criteria must be fulfilled when selecting a HKG for normalization in qPCR studies (Bonefeld et al., 2008). It should have stable expression in the samples to be studied. It should have a similar expression level to the target gene. Its amplification should be RNA-specific and genomic DNA should be removed by DNAse digestion.

The selection of appropriate HGKs has become increasingly easier with the development of software that simplifies the process. The geNorm method, which was put forward by Vandesompele et al. (2002), relies on a pair-wise comparison of HKGs and gives a list of the most stable HKGs, also giving a normalization factor (NF) that is used to normalize the data. Pfaffl et al. (2004) developed another program called Bestkeeper. This also relies on pair-wise comparison and can be used to analyze HKGs and target genes. Normfinder is another tool introduced by Andersen et al. (2004), which selects the most stable HKG and also suggests the best combination of two HKGs to use. In this study, the software Biogazelle qbasePLUS (Biogazelle), which relies on the geNorm method, was used to select the most stable HKGs.

2.5.2.10 Analysis of KCNQ genes:

The 7500 system software (Applied Biosystems) was used in conjunction with Excel (Microsoft 2007) and Biogazelle qbasePlus for data analysis. Relative quantification was used in which the change in expression of a gene was measured relative to another reference sample. A comparative CT method was used in which the CT values for the target gene are compared with the CT values for the reference gene.

Table 2.6: House-keeping genes and their roles in cells. The table below lists the six house-keeping genes used in this study and describes their roles in cells. (adapted from Langnaese et al. (2008)).

Abbreviation Name Role GAPDH glyceraldehyde-3-phosphate Glycolytic enzyme dehydrogenase β-actin Β-actin Cytoskeletal structural protein CycA (Ppia) cyclophilin A Catalyzes the cis-trans (peptidyl prolyl isomerase A) isomerization of proline imidic peptide bonds in oligopeptides, accelerating folding Hprt hypoxanthine guanine Purine synthesis in salvage phosphoribosyl transferase pathway Ywhaz tyrosine 3- Signal transduction by binding to monooxygenase/tryptophan 5 - phosphorylated serine residues monooxygenase activation on a variety of signalling protein, zeta polypeptide molecules Pgk1 phosphoglycerate kinase 1 Glycolytic enzyme

Biogazelle qbasePlus was also used to validate the HKGs used in the study using the geNorm method.

In order to validate the HKGs, the software calculates the ratio of expression of a particular gene and each of the other genes. The software then determines the pair- wise variation by giving the internal control gene stability value M, which is defined as the standard deviation of the logarithmically transformed expression ratios (Vandesompele et al., 2002). The lower the M value the more stable the gene. The software then gives a normalization factor based on the geometric mean of expression of the most stable house-keeping genes (Vandesompele et al., 2002). At least three stably expressed HKGs must be used to calculate the normalization factor. The software calculates the optimum number of HKGs to use by determining whether the addition of more HKGs is going to have a significant contribution to the newly calculated normalization factor (Vandesompele et al., 2002).

2.5.2.11 Factors influencing qPCR accuracy:

The quality of the RNA can affect qPCR results. Poor quality RNA samples may be samples co-extracted with proteins and RNAses, degraded RNA templates, genomic DNA or chemicals carried over from the RNA isolation. All these may lead to adverse effects on the reaction, including inhibition of PCR, further RNA degradation and

83 contamination. There are different methods for assessing RNA integrity, but my study employed a Bioanalyzer machine, which utilizes capillary electrophoresis and fluorescence on a small amount of RNA (25 ng). Distinct peaks of fluorescence in a 2:1 ratio for 28S and 18S rRNA indicate a sample of high integrity. RNA integrity is often expressed in the form of an RNA integrity number (RIN), varying from 1 to 10 with 1 being the most degraded and 10 indicating a sample with intact RNA. The samples used in this study had RIN values between 8.3 and 9.9 (Fig. 2.7), indicating that the samples had a high RNA integrity. In addition to having good quality RNA, an adequate amount of sample must be included in the qPCR reaction. It is important to select a sample mass that will amplify within 34 cycles of the reaction.

CT values between 34-40 are considered to have poor precision. PCR efficiency is another important factor influencing qPCR. It is defined as the rate at which the PCR product is generated and is commonly expressed as a percentage. In a 100% efficient PCR, the PCR product doubles with every cycle (Fig. 2.8). The efficiency (E) of a particular assay can be calculated using the slope of the PCR standard curve using the following equation:

E = (10-1/slope - 1) X 100 (2.1)

A slope of -3.32 indicates 100% efficiency. Slopes that are more negative indicate a lower efficiency. It is impossible for any given PCR reaction to be more than 100% efficient and calculated efficiencies of more than 100% signify poor sample quality or more likely pipetting errors. In general practice, primer efficiencies of 90-110% are considered acceptable.

Figure 2.7: Bioanalysis of RNA integrity. Electropherogram showing the 18S and 28S ribosomal RNAs for RNA isolated from pulmonary arteries (A) and mesenteric arteries (B). RNA with good integrity is represented by two clear peaks. [FU] = fluorescence units, [nt] = nucleotides.

Figure 2.8: Standard curve showing 100% PCR efficiency. The figure shows a plot of a standard curve obtained by plotting the CT values against log[concentration] of cDNA serial dilutions. R2 is the coefficient of determination of the linear regression and is an indicator of linearity. The equation for the linear regression is given in the figure.

2.5.3 Structural characteristics of pulmonary arteries in MCT-induced PH:

PA remodeling with increased medial thickness is a key finding in PH. It has been reported that MCT increased the media to lumen ratio in pulmonary arteries with an external diameter of the order of 30-200 µm (van Suylen et al., 1998). In addition there was a greater percentage of vessels with a double elastic lamina (DEL) in the chronic hypoxia and MCT models of PH (Sheedy et al., 1998; van Suylen et al., 1998). These variables were examined in lung sections from control and MCT-treated rats to confirm the morphological changes that were expected to occur due to the development of PH.

The cranial lobe of lungs from control and MCT-treated rats was fixed by infusing the airway with a 4% paraformaldehyde solution. The lobe was incubated in this solution over-night and then washed several times with phosphate buffered saline (PBS). The lung lobes were then embedded in paraffin and sections were cut and stained using either hematoxylin and eosin (H&E) or aldehyde fuchsin. The medial wall thickness of arteries (30-100 µm) in the H&E stained sections was measured by subtracting the length of the outer diameter and the inner lumen and dividing the result by two. The media to vessel diameter ratio was calculated by dividing the medial wall thickness by the length of the vessel. The percentage of DEL was calculated blindly from the elastin stained sections by counting the number of vessels with a DEL versus a single elastic lamina. The variables from control lung sections were then compared with MCT lung sections using unpaired Student's t-test.

The media to diameter ratio of pulmonary arteries (30-100 µm) in control animals was found to be 0.16 ± 0.01 (n=8). There was a significant increase in the media to lumen ratio in MCT PA, where it was 0.27 ± 0.02 (n=8, p<0.001)(Fig. 2.9). In addition, the percentage of DEL measured in proportion to the total number of PA vessels and was found to be 6 ± 1 % in control animals and 19 ± 2 % in MCT animals. The proportion of DEL was significantly higher in MCT animals (n=8, p<0.001) (Fig. 2.10). This is a similar profile to the study by Van Suylen et al. (1998) and Sheedy et al. (1998) and the findings are consistent with PH.

Figure 2.9: Histological changes associated with MCT-induced PH. H&E stained sections of PA from control (left column) and MCT-treated (right column) rats. Arteries are circled in red. The black horizontal bar represents 100 µm.

Figure 2.10: The elastic lamina in MCT-induced PH. Aldehyde fuchsin elastin stained sections of PA showing examples of the presence of single (SEL) and double (DEL) elastic laminae which are stained in black.

2.6 Results:

2.6.1 Effects of MCT on vessel responses to Kv7 channel blockers:

The Kv7 channel blockers linopirdine and XE991 were found to produce a concentration-dependent constriction of rat intra-pulmonary arteries obtained from both monocrotaline-treated and control rats (Fig. 2.11A) . 50 mM KCl was initially applied to both vessels for 5 min and produced a marked constriction. Subsequent washout of the KCl returned the tone back to baseline, after-which linopirdine was applied cumulatively, starting at a concentration of 1 nM and going up to 100 µM. Increasing drug concentrations were applied as soon as the constriction reached a steady level and the tension was measured prior to the addition of the higher concentration. In control vessels, constriction was first noted at 1 µM concentrations and increased thereafter with amplitudes comparable to the constriction caused by 50 mM KCl. In the example shown, 10 µM linopirdine gave a constriction that was greater than that induced by 50 mM KCl suggesting that the KCl response had not reached its maximum during its brief application. Concentrations of 100 µM caused partial inhibition of the constriction. In MCT vessels, constriction was first noted at 100 nM concentrations of linopirdine and the addition of 100 µM concentrations caused a reversal of the constriction similar to control vessels. In both animal groups the maximal constriction was seen at 10 µM linopirdine. Figure 2.11B shows the concentration-response curves for the linopirdine-induced constriction. The concentration-response curve was however shifted to the left in the case of MCT PA.

The pEC50 was determined as the logarithm of the concentration giving 50% constriction and was found to be -6 ± 0.3 (n=4) in control vessels and -5.7 ± 0.2 (n=4) in MCT vessels.

Figure 2.12A shows the effects of the Kv7 channel blocker XE991 on monocrotaline and control pulmonary arteries. Similar to the linopirdine experiments, the vessels were first challenged with 50 mM KCl for 5 min leading to an increase in the tension recorded and recovery on washout with PSS. Subsequently XE991 was applied cumulatively at concentrations between 0.1 nM and 10 µM. In control vessels constriction was first noted at 1 µM concentrations of XE991 and a maximal constriction, comparable to that induced by 50 mM KCl, was seen at 10 µM. In MCT vessels constriction first appeared at 100 nM concentrations of XE991 and increased progressively with maximal constriction also seen at 10 µM. The concentration-

90 response curves for XE991 are given in figure 2.12B. The figure shows a leftward- shift of the concentration-response curve for MCT PA. The pEC50 for XE991 was found to be significantly lower in MCT PA, being -5.7 ± 0.2 (n=4) for control PA and - 7 ± 0.2 for MCT PA (p<0.01, n=4). To determine whether this vasoconstrictor effect was pulmonary specific the effects of XE991 were tested on mesenteric arteries (Fig. 2.13A). It was found that XE991 had no vasoconstrictor effects on mesenteric arteries obtained from either control or MCT rats over 0.1 nM-10 µM concentrations. The concentration-response curves for XE991 tested on mesenteric arteries are shown in figure 2.13B.

The effects of chromanol 293B (as a racemate), which is a Kv7.1 specific blocker, were also tested on the intra-pulmonary arteries from control and MCT animals. After sensitizing the tissue using 50 mM KCl, chromanol 293B was applied cumulatively to the arteries at concentrations between 10 nM and 100 µM (Fig. 2.14A). Chromanol 293B had no vasoconstrictor effect on the vessels from control animals. It also did not have any vasoconstrictor effect on the vessels from MCT rats, and although it appeared to cause a very small dilation when applied at 10-100 µM concentrations, this was not statistically significant. The concentration-response curves for chromanol 293B are given in figure 2.14B.

XE991 was chosen in order to explore the mechanism of the constriction induced in pulmonary artery vessels. The effects of XE991 on the intrapulmonary artery were tested in the presence of the Ca2+ channel blocker nifedipine, to assess the importance of voltage-gated Ca2+ influx in inducing the observed constriction. In these experiments nifedipine (1 µM) was applied to the vessels for 15-20 min. XE991 was subsequently applied cumulatively at concentrations between 0.1 nM and 10 µM (Fig. 2.15A). It was found that in the presence of nifedipine, XE991 failed to constrict pulmonary arteries obtained from either control or MCT rats. The concentration response curves for XE991 tested on PA in the presence of nifedipine are shown in figure 2.15B.

Figure 2.11: The effect of linopirdine on pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by linopirdine (1 nM - 100 µM) in control and MCT rat pulmonary arteries. B) Concentration-response curve for linopridine plotted as the % constriction measured relative to 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments for each animal group.*p<0.05, **p<0.01, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

Figure 2.12: The effect of XE991 on pulmonary arteries from control and MCT- treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by XE991 (0.1 nM - 10 µM) in control and MCT rat pulmonary arteries. B) Concentration-response curves for XE991 plotted as the % constriction measured relative to 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments for each animal group. **p<0.01, ***p<0.001, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

Figure 2.13: The effect of XE991 on mesenteric arteries from control and MCT- treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by XE991 (0.1 nM - 10 µM) in control and MCT rat mesenteric arteries. B) Concentration-response curves for XE991 plotted as the % constriction measured relative to 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments for each animal group (error bars are within symbols for some points).

Figure 2.14: The effect of chromanol 293B on pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by chromanol 293B (10 nM - 100 µM) in control and MCT rat pulmonary arteries. B) Concentration-response curve for chromanol 293B plotted as the % constriction measured relative to 50 mM KCl. Results are shown as mean ± S.E.M of 3-4 experiments for each animal group (error bars are within symbols for some points).

Figure 2.15: The effect of nifedipine on the XE991-induced constriction of pulmonary artery. A) Raw trace showing the change in tension caused by the addition of XE991 (0.1 nM - 10 µM) in the presence of the Ca2+ channel blocker nifedipine (1 µM) in pulmonary arteries from MCT rats. B) Concentration-response curve for XE991 in the presence of nifedipine (1 µM) plotted as the % constriction measured relative to 50 mM KCl. Results are shown as mean ± S.E.M of 3-4 experiments for each animal group. (error bars are within symbols for some points).

2.6.2 Effects of MCT on the responses of preconstricted vessels to Kv7 channel activators:

The Kv7 activators retigabine, ZnPy and BMS-204352 were tested on intrapulmonary arteries from control and MCT rats. The vessels were pre-constricted with 1 µM phenylephrine to allow us to measure the dilation caused by the activators. The addition of phenylephrine produced a constriction that was maintained for 20-30 min (Fig. 2.16A). Retigabine (10 nM-100 µM) was subsequently added and it produced a concentration-dependent relaxation of intrapulmonary arteries from both control and MCT rats. In control vessels, relaxation was first noted at 1 µM and a maximal concentration of 100 µM caused only a partial relaxation of the vessel (43 ± 13%). In MCT vessels relaxation was also first noted above 1 µM. Unlike control vessels, the application of 100 µM to MCT vessels produced a full relaxation. The concentration- response curves for retigabine in intra-pulmonary arteries are shown in figure 2.16B.

The pEC50 value in this case was defined as the logarithm of the retigabine concentration giving a 50% relaxation relative to PE. The pEC50 values were -3.6 ± 0.5 for control PA (n=6) and -4.6 ± 0.3 for MCT PA (n=6). Retigabine was also tested on the mesenteric arteries of control and MCT treated rats. It was found that retigabine produced a concentration-dependant relaxation similar to that seen in the pulmonary artery. Unlike the pulmonary arteries however, relaxation was first noted at 10 nM. In addition, the efficacy of retigabine was similar in mesenteric arteries from control and MCT animals. The pEC50 values were -5 ± 0.3 (n=4) and -5.9 (n=4) µM for control and MCT mesenteric arteries respectively. The effect of retigabine on the mesenteric arteries is shown in figure 2.17.

ZnPy is a Kv7 activator that is known to act at a different site on the Kv7 channel from retigabine. When tested on preconstricted intrapulmonary arteries from control and MCT rats, ZnPy (100 nM - 100 µM) produced a concentration-dependent relaxation similar to retigabine (Fig. 2.18A). In control vessels relaxation was first noted at 5 µM concentrations and the pEC50 value for ZnPy was -4.6 ± 0.2. In MCT

PA, relaxation was first noted at 1 µM concentrations and the pEC50 value was -4.8 ± 0.6. ZnPy produced a 74 ± 5% relaxation of control vessels at 100 µM, while fully relaxing MCT vessels at the same concentration. Apart from these differences, the effects of ZnPy on the vessels were similar in control and MCT rats. The concentration-response curves for ZnPy are shown in figure 2.18B.

BMS-204352 is another activator of Kv7 channels. When tested on preconstricted intrapulmonary arteries from control and MCT rats it had no vasodilatory effect on the vessels at concentrations lower than 100 µM (Fig. 2.19A). The addition of 100 µM produced a relaxation of 61 ± 9% in control vessels and 75 ± 11% in MCT vessels. The vasorelaxant effects of BMS-204352 were similar in both control and MCT pulmonary arteries. The pEC50 for BMS-204352 was -4.1 ± 0.1 for control animals and -4.3 ± 0.1 for MCT animals. The concentration-response curves for BMS-204352 are shown in figure 2.19B.

Figure 2.16: The effect of retigabine on pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the addition of PE (1 µM) followed by retigabine (10 nM - 100 µM) in control and MCT rat pulmonary arteries. B) Concentration-response curve for retigabine plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 6 experiments for each animal group. **p<0.01, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

Figure 2.17: The effect of retigabine on mesenteric arteries from control and MCT- treated animals. A) Raw traces showing the change in tension caused by the addition of PE (10 µM) followed by retigabine (10 nM - 100 µM) in control and MCT rat mesenteric arteries. B) Concentration-response curve for retigabine plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 4 experiments for each animal group. **p<0.01, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

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Figure 2.18: The effect of ZnPy on pulmonary arteries from control and MCT- treated animals. A) Raw traces showing the change in tension caused by the addition of PE (1 µM) followed by ZnPy (100 nM - 100 µM) in control and MCT rat pulmonary arteries. B) Concentration-response curve for ZnPy plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 5 experiments for each animal group.

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Figure 2.19: The effect of BMS-204352 on pulmonary arteries from control and MCT-treated animals. Raw traces showing the change in tension caused by the addition of PE (1 µM) followed by BMS-204352 (10 nM - 100 µM) in control and MCT rat pulmonary arteries. B) Concentration-response curve for BMS-204352 plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 4 experiments for each animal group.

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2.6.3 Intrinsic Tone:

It has been shown that the pulmonary arteries from the pulmonary hypertensive MCT-treated rats had a raised intrinsic tone relative to pulmonary arteries from control rats (Ito et al., 2000). This raised intrinsic tone was shown to be dependent on voltage-gated calcium influx (Ito et al., 2000). The effects of nifedipine on pulmonary arteries from control and MCT-treated animals were investigated to test for the presence of a raised intrinsic tone in these vessels and to assess the involvement of voltage-gated calcium channels. When nifedipine (1 µM) was applied to control pulmonary arteries it produced no change in the resting tone (Fig. 2.20A). However when applied to MCT vessels, it produced a relaxation that first appeared within 1-3 min and reached a maximum after 20-30 min. Figure 2.20B is a histogram outlining the effects of nifedipine on control and MCT PA. The response was measured as a percentage of the constriction caused by 50 mM KCl.

Having established dependence of intrinsic tone on voltage-gated Ca2+ influx, it was important to determine whether drugs that acted via hyperpolarization, such as K+ channel activators, were able to abolish it. The Kv7 channel activator retigabine was first tested on intrapulmonary arteries obtained from both MCT and control rats. It was found that in control PA, retigabine had no effect on the basal tone (Fig. 2.21A). On the other hand, when retigabine was applied to pulmonary arteries from MCT rats it produced a concentration-dependent relaxation of the vessels. The relaxation first appeared when 1 µM concentrations of retigabine were applied and 65 µM gave 50% relaxation. The concentration-response curve for retigabine on the intrinsic tone of pulmonary arteries is shown in Fig. 2.21B.

To determine whether the effects of retigabine on the intrinsic tone of MCT pulmonary arteries were maximal, it was applied at a maximal concentration of 100 µM, followed by 1 µM nifedipine and the removal of external calcium. The removal of external calcium was expected to produce a maximal dilation of the vessels. The addition of 100 µM retigabine caused a relaxation of PA vessels from MCT-treated animals that was not enhanced after the addition of 1 µM nifedipine nor the removal of external calcium (Fig. 2.22A). Figure 2.22B is a histogram showing the relaxation caused by retigabine under control conditions, in the presence of nifedpine and in the presence of nifedipine and 0 Ca2+. The relaxation caused by retigabine was similar under these three conditions.

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In order to determine whether the raised intrinsic tone was pulmonary specific, the effects of retigabine were tested on mesenteric arteries obtained from both control and MCT-treated rats. If the mesenteric vessels from MCT animals had a raised intrinsic tone, we would expect retigabine to have a vasorelaxant effect on these vessels. Figure 2.23 shows retigabine had no effect on the basal tone of the mesenteric arteries obtained from control and MCT rats.

The ability of another Kv7 channel activator, ZnPy, to abolish the intrinsic tone of MCT pulmonary arteries was also tested. It was found that ZnPy had no effect on the tone of pulmonary arteries from control animals, except at 100 µM where it caused a constriction of the vessels (Fig. 2.24A). On the other hand, when tested on MCT pulmonary arteries it produced a relaxation when used at concentrations of 10 µM- 100 µM. The effects of ZnPy on the intrinsic tone of pulmonary arteries from control and MCT animals are summarized in Fig.2.24B. Similar to retigabine these results suggest that there is a raised intrinsic tone in MCT pulmonary arteries that is sensitive to Kv7 modulation.

Levcromakalim is another agent that causes hyperpolarization by opening KATP channels. It was tested in a similar manner to retigabine and ZnPy and was found to have no effect on the tone of pulmonary arteries from control animals (Fig. 2.25A). It did however produce a concentration-dependent relaxation when applied to MCT pulmonary arteries at concentrations of 100 nM or higher. The concentration- response curve for levcromakalim on the intrinsic tone of pulmonary arteries is shown in Fig. 2.25B. The dilation caused by 10 µM levcromakalim was similar to the relaxation caused by 100 µM ZnPy but lower than that caused by 100 µM retigabine.

It was important to determine whether the effects on intrinsic tone were specifically inhibited by hyperpolarization or would be inhibited by any vasodilator regardless of its mechanism of action. Sildenafil, is an agent that causes relaxation by inhibiting phosphodiesterase type 5 enzyme (PDE5). This enzyme is responsible for the degradation of cyclic guanosine monophosphate (cGMP). In PASMCs, nitric oxide stimulates guanylate cyclase leading to the production of cGMP which is known to produce vasodilation by altering calcium signalling (Schoeffter et al., 1987; Komas et al., 1991; Stoclet et al., 1995; Polson et al., 1996; Wagner et al., 1997; Carvajal et al.,

2000). Sildenafil was reported to produce its vasodilation primarily by inhibiting IP3 calcium release from the intracellular Ca2+ stores (Pauvert et al., 2003), and there is mixed evidence for the involvement of potassium channels and hyperpolarization

104 caused by cGMP (Kannan et al., 1995; Trongvanichnam et al., 1996; Yamakage et al., 1996; Zhou et al., 1996). When tested on control pulmonary arteries sildenafil had no effect on the resting tone (Fig. 2.26A). Sildenafil was able to inhibit the intrinsic tone in MCT pulmonary arteries when applied at 10 µM concentrations. The concentration-response curves for sildenafil are shown in Fig. 2.26B.

2-APB is a useful probe for the investigation of IP3 calcium signalling. It has been reported that 2-APB blocks store-operated calcium entry and inhibits IP3 dependent calcium release (Bootman et al., 2000). It was also found to modulate members of the transient receptor potential (TRP) channels. 2-APB is reported to activate TRPV1, TRPV2, TRPV3, TRPA1, TRPM6 and inhibit TRPC4, TRPC5, TRPC6, TRPM2 and TRPM6 (Clapham, 2007). In addition, it has been reported that 2-APB weakly inhibits voltage gated K+ channels in vascular smooth muscle cells (Ma et al., 2011). When tested on MCT pulmonary arteries, 2-APB produced a concentration-dependant relaxation that was detected at 10 µM and was maximal at 100 µM (Fig. 2.27A). A histogram showing the effect of 2-APB on the resting tone of MCT pulmonary arteries is given in Fig. 2.27B. When tested on vessels preconstricted with phenylephrine (1µM), it also produced a concentration-dependant relaxation (Fig.2.28A). The relaxation was first detected at 10 µM and a full relaxation was seen at 100 µM. The results are given as a histogram in Fig. 2.28B. The effects of 2-APB were not investigated on control PA.

Although all of the drugs tested were able to relax unstimulated pulmonary arteries from MCT-treated animals, the effectiveness of the drugs were compared in order to determine the importance of membrane potential and hyperpolarization for the observed effects. Figure 2.29 shows a histogram comparing the effects of the various vasodilators on the intrinsic tone of MCT-treated pulmonary arteries. The Kv7 channel modulator retigabine was as effective as nifedipine in inhibiting the resting tone. The ability of ZnPy and levcromakalim to cause relaxation was similar. The effect of sildenafil on resting tone was found to be significantly lower than nifedipine. Finally, 2-APB was found to be as effective as the K+ channel activators and nifedipine in inhibiting the resting tone.

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Figure 2.20: The effect of nifedipine on the basal tone of pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by nifedipine (1 µM) in control and MCT pulmonary arteries. B) Histogram showing the nifedipine relaxation measured as the % relaxation relative to the constriction induced by 50 mM KCl. Results are shown as mean ± S.E.M of 3-4 (C,M) experiments for each animal group. *p<0.05, unpaired comparison.

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Figure 2.21: The effect of retigabine on the basal tone of pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by retigabine (10 nM-100 µM) in control and MCT pulmonary arteries. B) Concentration-response curves for retigabine plotted as the % relaxation measured relative to the constriction induced by 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments for each animal group. **p<0.01, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

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Figure 2.22: Retigabine maximally abolished intrinsic tone. A) Raw trace showing the change in tension induced by the addition of retigabine (100 µM), followed by nifedipine (1 µM) and the removal of external Ca2+ in MCT rat intrapulmonary artery. B) Histogram showing the retigabine relaxation under control conditions and in the presence of nifedipine before and after the removal of external calcium. The retigabine relaxation was measured as the % of the constriction induced by 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments.

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Figure 2.23: The effect of retigabine on the basal tone of mesenteric arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by retigabine (10 nM-100 µM) in control and MCT mesenteric arteries. B) Concentration-response curves for retigabine plotted as the % relaxation measured relative to 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments for each animal group (error bars are within symbols for some points).

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Figure 2.24: The effect of ZnPy on the basal tone of pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by ZnPy (100 nM-100 µM) in control and MCT pulmonary arteries. B) Concentration-response curves for ZnPy plotted as the % relaxation measured relative to 50 mM KCl. Results are shown as mean ± S.E.M of 4,5 (C,M) experiments for each animal group. ***p<0.001, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

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Figure 2.25: The effect of levcromakalim on the basal tone of pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by levcromakalim (1 nM - 10 µM) in control and MCT pulmonary arteries. B) Concentration-response curves for levcromakalim plotted as the % relaxation measured relative to the constriction induced by 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments for each animal group (error bars are within symbols for some points).

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Figure 2.26: The effect of sildenafil on the basal tone of pulmonary arteries from control and MCT-treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by sildenafil (0.1 nM -10 µM) in control and MCT pulmonary arteries. B) Concentration-response curves for sildenafil plotted as the % relaxation measured relative to 50 mM KCl constriction. Results are shown as mean ± S.E.M of 4 experiments for each animal group. **p<0.01, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

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Figure 2.27: The effect of 2-APB on the resting tone of pulmonary arteries from MCT- treated animals. A) Raw traces showing the change in tension caused by the brief addition of 50 mM KCl followed by 2-APB (10-100 µM) in control and MCT pulmonary arteries. B) Histogram showing % relaxation caused by 2-APB relative to the constriction induced by 50 mM KCl. Results are shown as mean ± S.E.M of 4 experiments

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Figure 2.28: The effect of 2-APB on preconstricted pulmonary arteries from MCT treated animals. A) Raw traces showing the change in tension caused by the addition of PE (1 µM) followed by 2-APB (10-100 µM) in control and MCT rat pulmonary arteries. B) Histogram showing the % relaxation caused by 2-APB measured relative to the constriction induced by 1 µM PE. Results are shown as mean ± S.E.M of 4 experiments.

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Figure 2.29: The effect of vasodilators on the intrinsic tone of pulmonary arteries from MCT-treated animals. Histogram showing the maximal inhibition of intrinsic tone by a range of vasodilators: nifedipine (1 µM), retigabine (100 µM), ZnPy (100 µM), levcromakalim (100 µM), sildenafil (10 µM) and 2-APB (100 µM). Results are shown as mean ± S.E.M of 4-5 experiments for each animal group and for each drug compared with nifedipine. *p<0.05, two-way ANOVA followed by Bonferroni's post hoc test.

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2.6.4 Effects of membrane potential on vessel recovery from a contractile stimulus:

PASMCs from pulmonary hypertensive animals, including MCT-treated animals, are more depolarized than PASMCs from healthy animals (Ito et al., 2000). This depolarization can affect the activity of voltage-gated ion channels as well as certain electrogenic membrane transporters. The sodium calcium exchanger is an electrogenic membrane transporter that is important for returning intracellular Ca2+ levels to normal following a contractile stimulus. It introduces 3 Na+ ions into the cytoplasm for each Ca2+ ion expelled from the cell . It has been suggested that the slowed recovery of MCT PA from a contractile stimulus was due to the inhibition of the NCX by depolarization (Ito et al., 2000).

The addition of 50 mM KCl to pulmonary arteries from control and MCT-treated animals produced a rapid constriction that was reversible on washout (Fig. 2.30A). It was noted that the washout was significantly slower in MCT-treated vessels than in control vessels. The time for half maximal recovery (T1/2) was increased 13 fold in MCT pulmonary arteries, being 38 ± 3 s (n=10) in control vessels and 498 ± 23 s (n=13) in MCT vessels (p<0.001). Similarly, mesenteric arteries from control and MCT-treated rats produced a rapid constriction when treated with 50 mM KCl, that was reversible on washout (Fig 2.30B). However, the recovery was similar in mesenteric vessels from both animal groups, with T1/2 being 18 ± 3 s for control vessels and 19 ± 4 s for MCT vessels (n=4).

In order to test whether depolarization of PASMCs in MCT PA may have contributed to their slower recovery, levcromakalim (1 µM) was used to hyperpolarize PASMCs in

MCT vessels and T1/2 was measured under these conditions. The addition of levcromakalim caused a relaxation of the basal tone, suggesting effective hyperpolarization (Fig. 2.31). Once the response had stabilized, the vessels were challenged twice with 50 mM KCl for 5 min. The addition of 50 mM KCl in the presence of levcromakalim produced a rapid constriction. It was found that the recovery of the vessels was significantly quicker in the presence of levcromakalim, with T1/2 being 546 ± 64 s under control conditions and 65 ± 3 s in the presence of levcromakalim (n=3, p<0.05).

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To further elucidate the role of depolarization, control PA were exposed to a depolarizing solution of 15 mM K+ PSS. The T1/2 for the recovery of the vessels from KCl and PE challenges, was measured under these conditions and compared with the

T1/2 under physiological conditions to determine whether depolarization would slow the recovery of the vessels from these stimuli. In this experiment, the vessels were first challenged with 50 mM KCl for 10 minutes. This produced a constriction that recovered completely on washout (Fig. 2.32). This step was subsequently repeated and the average T1/2 was calculated to be 40 ± 5 s (n=6). Phenylephrine (1 µM) was then added for 10 min and it also produced a constriction with complete recovery on washout. This was repeated and the average T1/2 was calculated as 61 ± 10 s (n=6). The vessels were then exposed to a 15 mM K+ PSS solution, which gave a constriction that stabilized within 20-30 min. The vessels were re-challenged with 50 mM KCl and recovered completely on washout with a T1/2 of 58 ± 22 s (n=6). Next, PE (1 µM) was applied and produced a constriction that showed only partial (60%) recovery on washout. The T1/2 for recovery from PE was calculated as 84 ± 22 s (n=6).

2.6.5 Effects of depolarization on contractile response to Kv7 modulators:

As Kv7 channels are voltage-dependent, and the ability of Kv7 modulators to influence Kv7 channel activity may be voltage-dependent, then depolarization of SMC in MCT pulmonary arteries may have influenced the ability of Kv7 modulators to affect tone. This was investigated by exposing PA vessels from control animals to a 15 mM K+ PSS depolarizing solution. Exposure to this solution produced a small constriction that was steady after 20-30 min (Fig. 2.33A). Once the constriction reached a plateau the vessels were exposed to increasing concentrations of XE991 or preconstricted with PE (1 µM) to test the effects of retigabine. The concentration- response curves for XE991 and retigabine under these conditions are shown in Fig. 2.33B,C. The curve for XE991 was shifted to the left indicating that it was effective at lower concentrations. The dotted line in Fig. 2.33B represents the response of MCT PA to increasing concentrations of XE991 (refer to Fig. 2.12B). The concentration- response curve for constriction of control PA exposed to 15 mM K+ PSS was shifted towards the response seen in MCT vessels. After depolarization the pEC50 values for XE991 and retigabine were -7 ± 0.2 (n=4) and -4.6 (n=4) respectively.

In separate experiments, MCT vessels were exposed to levcromakalim (1 µM) in order to induce hyperpolarization. Levcromakalim, decreased basal tone, providing

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Figure 2.30: Slowed recovery rates from 50 mM KCl challenge in MCT pulmonary arteries. A) Raw traces of tension recorded from pulmonary arteries from control and monocrotaline animals in response to 50 mM KCl applied to the recording chamber. B) Raw traces of tension recorded from mesenteric arteries from control and monocrotaline animals in response to 50 mM KCl applied to the recording chamber.

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Figure 2.31: Hyperpolarization enhances the rate of recovery of monocrotaline pulmonary arteries. Raw trace of tension recorded in response to the addition of 50 mM KCl to MCT PA under control conditions and in the presence of levcromakalim (1 µM).

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Figure 2.32: The effect of depolarization on the rate of recovery of control pulmonary arteries. Raw trace of tension recorded in response to brief applications of 50 mM KCl followed by phenylephrine (1 µM), under control conditions and in the presence of 15 mM K+ PSS.

120 assurance that hyperpolarization had occurred. After the response to levcromakalim had stabilized, the vessels were exposed to increasing concentrations of XE991 or preconstricted with PE (1 µM) to test the effects of retigabine. It was found that in the presence of levcromakalim, the concentration-response curve for XE991 was shifted to the right (Fig. 2.34A). The dotted line in Fig. 2.34A represents the response of control PA to increasing concentrations of XE991 in the absence of levcromakalim (refer to Fig. 2.12B). In the presence of levcromakalim the concentration-response curve for XE991 in MCT vessels was shifted towards the responses seen in control vessels. The pEC50 for XE991 in the presence of levcromakalim was -5.4 ± 0.1 (n=4). It was difficult to preconstrict vessels using PE in the presence of levcromakalim in order to study the effects of retigabine. This was because levcromakalim causes hyperpolarization which in turn opposes the effect of PE. The effects of retigabine were tested on 2 vessels where preconstriction with PE in the presence of levcromakalim was successful. Figure 2.34B shows the concentration-response curves for retigabine under these conditions. There appears to be no change in the sensitivity of MCT vessels to retigabine in the presence and absence of levcromakalim.

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Figure 2.33: The effect of depolarization on Kv7 modulator activity. A) Raw trace showing the tension recorded in response to the brief addition of 50 mM KCl followed by 15 mM K+ PSS. B) Concentration-response curves for XE991 in 15 mM K+ PSS plotted as the % constriction measured relative to 50 mM KCl. The dotted line represents the response of MCT PA to XE991 under control conditions (refer to Fig. 2.12B) C) Concentration-response curves for retigabine in 15 mM K+ PSS plotted as the % residual constriction measured relative to PE (1 µM). The dotted line represents the response of MCT PA to retigabine under control conditions (refer to Fig. 2.16B) Results are shown as mean ± S.E.M of 4 experiments for each drug. **p<0.01, ***p<0.001, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

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Figure 2.34: The effect of hyperpolarization on Kv7 modulator activity. A) Concentration-response curves for XE991 in the presence of levcromakalim (1 µM) plotted as the % constriction measured relative to 50 mM KCl. The dotted line represents the response of control PA to XE991 in the absence of levcromakalim (refer to Fig. 2.12B) B) Concentration-response curves for retigabine in the presence of levcromakalim (1 µM) plotted as the % residual constriction measured relative to PE (1 µM) (n=2). The dotted line represents the response of control PA to retigabine in the absence of levcromakalim (refer to Fig. 2.16B). Results are shown as mean ± S.E.M of 2-4 experiments **p<0.01, ***p<0.001, two-way ANOVA followed by Bonferroni's post hoc test (error bars are within symbols for some points).

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2.6.6 KCNQ mRNA expression in monocrotaline and control pulmonary arteries:

Endpoint RT-PCR was performed on pulmonary arteries from control and monocrotaline animals using the KCNQ primers listed in table 2.3 and GAPDH as the reference gene (table 2.4). Fig. 2.35 shows the expression of mRNA for KCNQ1, KCNQ4 and KCNQ5 from both control and monocrotaline rat pulmonary arteries when resolved on an agarose gel using ethidium bromide as a stain. All three transcripts were detected in the vessels from both groups of animals. The generated products had sizes in accordance with the expected sizes for the genes, as listed in tables 2.3 and 2.4. The products in this particular study were not verified by sequencing, but similar bands for the same experiment on rat PA were previously sequenced and matched with their respective primers.

2.6.7 Establishing house-keeping genes for real-time PCR:

The efficiencies of the house-keeping genes HPRT, Pgk1, YWHAZ, β-actin, CycA and GAPDH were first investigated in order to validate their use for real-time PCR. 10 times serial dilutions of the purified PCR products were used starting from a 1:105 dilution to a 1:1010 dilution. Figure 2.36 shows the standard curves for each house- keeping gene obtained by plotting of the CT values from a real-time PCR reaction using these dilutions versus log[concentration]. In each case, the result was a straight line whose slope was used to calculate the efficiency of the primer. For HPRT, PGK1, YWHAZ, β-actin, CYCA and GAPDH the efficiencies were found to be 94%, 93%, 94%, 95%, 98% and 95% respectively. A dissociation step was added at the end of the real-time PCR protocol that generated a melt-curve that was used to confirm the specificity of the primers. As shown in Figure 2.37, all the HKG genes had a single peak in the melt-curve confirming that the primers were specific and that a single product was formed.

The HKGs were then tested in a real-time PCR reaction using the control and monocrotaline PA and MA cDNA as the template. The CT values were then input into Biogazelle qbasePLUS software that generated a plot of the average stability value M for the HKGs (Fig. 2.38A). The lower this value the more stable the genes. Pgk1 had the lowest geNorm M value, followed by GAPDH, β-actin, YWHAZ and HPRT. CycA had

124 the highest M value and was therefore the least stable. In order to determine the optimal number of HKGs to use, the software also generated a plot of the geNorm V value for each HKG (Fig. 2.38B). This measure is known as the pairwise variation and a value of 0.15 or less is considered optimum. In this study the optimum number of house-keeping genes for normalization was 3. Therefore Pgk1 ,GAPDH and β-actin were used as they were the three most stably expressed HKGs.

2.6.8 KCNQ mRNA quantification by real-time PCR:

In order to quantify KCNQ mRNA expression it is necessary to perform the PCR in real-time. Before carrying out the qPCR experiments, it was necessary to confirm that the KCNQ gene primers used in the study had the appropriate efficiency. They were tested is a similar manner to the HKGs and standard curves were constructed by plotting the CT values versus log[concentration] (Fig. 2.39). The efficiencies for the KCNQ1, KCNQ4 and KCNQ5 primers were 99%, 95% and 95% respectively. The primers generated a single product as confirmed by melt curve analysis (Fig. 2.40). Subsequently, the expression of KCNQ1, KCNQ4, KCNQ5 mRNA was studied in pulmonary and mesenteric arteries from control and MCT animals. The normalized relative expression of KCNQ1, KCNQ4 and KCNQ5 in pulmonary and mesenteric arteries from MCT and control animals is shown in figure 2.41. Statistical analysis revealed that there was no significant difference in the expression of KCNQ1, KCNQ4 and KCNQ5 mRNA in pulmonary and mesenteric arteries from MCT and control animals.

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Figure 2.35: KCNQ mRNA expression in pulmonary artery. RT-PCR detection of KCNQ1, KCNQ4 and KCNQ5 mRNA transcripts in pulmonary arteries from control and MCT rats. GAPDH was used as a reference gene. There were two reactions for each gene, the (+RT) indicates the presence of reverse transcriptase and the (-RT) is the negative control lacking reverse transcriptase. M is the marker.

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Figure 2.36: Standard curves for housekeeping genes. The figure shows a plot of the standard curves for the HKGs used in the study, obtained by plotting the CT values against log[concentration] of rat pulmonary artery PCR products diluted 1:105, 1:106,1: 107, 1:108, 1:109 and 1:1010. R2 is the coefficient of determination of the linear regression and is an indicator of linearity. The equations for the linear regression of each curve are given in the figure.

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Figure 2.37: Dissociation curves for HKGs. The figure shows the dissociation curves for the HKGs used in the study. The presence of a single peak confirms the presence of a single product and the absence of primer-dimers.

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Figure 2.38: GeNorm analysis of HKGs. A) A plot of the internal control gene- stability value M for the HKGs used in the study. B) A plot of the pair-wise variation V and the number of house HKGs used for normalization.

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Figure 2.39: Standard curves for KCNQ genes. The figure shows a plot of the standard curves for KCNQ1, KCNQ4 and KCNQ5 obtained by plotting the CT values against log[concentration] of rat pulmonary artery PCR products diluted 1:105, 1:106,1: 107, 1:108, 1:109 and 1:1010. R2 is the coefficient of determination of the linear regression and is an indicator of linearity. The equations for the linear regression of each curve are given in the figure.

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Figure 2.40: Dissociation curves for KCNQ genes. The figure shows the dissociation curves for the KCNQ genes used in the study. The presence of a single peak confirms the presence of a single product and the absence of primer-dimers.

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Figure 2.41: Expression profile of KCNQ genes. Histogram showing the relative expression of KCNQ1, KCNQ4 and KCNQ5 mRNA in pulmonary and mesenteric arteries from control and MCT-treated animals. Data are expressed as mean ± S.E.M (n=4 for PA and n=3 for MA). Expression is normalized to the HKGs β-actin, GAPDH and Pgk1.

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2.7 Discussion:

This findings of my study provide the first evidence that Kv7channels are functional in the monocrotaline rat model of pulmonary hypertension, suggesting that they may be targeted to treat the disease. The expression of KCNQ genes was not altered in this model of PH, therefore any changes in the responses of PA vessels to Kv7 modulators was not due to a change in channel expression. The sensitivity of PA vessels to the Kv7 blocker XE991 was greatly enhanced in this model. In addition, Kv7 channel activators were effective in abolishing the raised intrinsic tone seen in MCT-PA. These findings are different from other studies where loss of function has been reported for some K+ channels in PAH (Yuan et al., 1998a; Morecroft et al., 2009) and in systemic hypertension where Kv7 channel expression was down-regulated (Jepps et al., 2011).

2.7.1 MCT enhances sensitivity to Kv7 channel blockers:

The Kv7 channel blockers XE991 and linopirdine were found to produce concentration-dependent constriction of pulmonary arteries from both control and MCT animals. This is consistent with previous studies were the blockers were found to cause profound constriction of pulmonary arteries from mouse and rat (Joshi et al., 2006). The pEC50 values for XE991 and linopridine were slightly higher than those reported by Joshi et al. (2006). In particular XE991 was about 10-fold more effective in the previous study. These discrepancies may be due to differences in the species used. In this study Wistar rats were used, whereas the studies by Joshi et al. (2006, 2009) used Sprague Dawley rats. On the other hand the studies in chapter 3 of this thesis were on Sprague-Dawley rats, which showed similar XE991 sensitivity to the Wistar rats. It was found that the pulmonary arteries from MCT animals were more sensitive to XE991 than pulmonary arteries from control animals.

Chromanol 293B was tested as a selective blocker of Kv7.1 channels (Jentsch, 2000; Yang et al., 2000; Robbins, 2001), and was found to have no constrictor effect on the pulmonary arteries from either control or MCT animals. This finding is consistent with reports by Yeung et al. (2007), where chromanol 293B had no effect on mouse aorta. It is also consistent with previous reports where chromanol 293B had no effects on murine gastrointestinal smooth muscle (Greenwood et al., 2009) and no effects on the tone of visceral adipose arteries or mesenteric conduit arteries in

133 humans (Ng et al., 2011). These results suggest that Kv7.1 channels are not likely to be functionally involved in regulating the tone of pulmonary or other arteries, at least in rat.

The blocker XE991 had no effect on mesenteric arteries from either control or MCT rats. This finding is consistent with the findings of Joshi et al. (2006), where XE991 and linopirdine were found to have pulmonary specific effects. It differs from reports of a vasoconstrictor effect of XE991 on mesenteric arteries in the presence of pretone (Yeung et al., 2007). Nevertheless, the constrictions reported by that group were only a small percent of the response to 50 mM KCl. So there does appear to be a large difference in the sensitivities of pulmonary and mesenteric arteries to Kv7 blockers, at least where the vessels are at resting tone in the absence of any other vasoconstriction. The pulmonary vasoconstrictor activity of XE991 was abolished in the presence of the calcium channel blocker nifedipine. This implies that the constriction induced by XE991 was dependent upon voltage-gated calcium influx as previously found by Joshi et al. (2006).

2.7.2 The effect of MCT on responses of preconstricted vessels to Kv7 channel activators:

The vasodilator effects of the Kv7 activators retigabine, ZnPy and BMS-2042352 were also compared in control and MCT pulmonary arteries. It was confirmed that retigabine (an activator of all Kv7 channels apart from Kv7.1), caused relaxation of pulmonary arteries preconstricted with PE with similar potency to that previously reported by Joshi et al. (2009). It was found that ZnPy, which activates all Kv7 channels except Kv7.3, caused concentration-dependent relaxation of pulmonary arteries, as described in detail in chapter 3. Since retigabine and ZnPy have a different spectrum of selectivity for Kv7 channel subtypes, the results suggest that Kv7.1 and Kv7.3 were not likely involved in mediating vasodilation in these vessels.

BMS-204352 is known to activate Kv7 and BKCa channels. The effects of BMS- 204352 have not been previously investigated on pulmonary arteries and it was found that BMS-204352 caused dilation of pulmonary arteries at concentrations above 100 µM.

In MCT pulmonary arteries there was a significant increase in the maximal relaxation caused by retigabine, suggesting that MCT had increased the drug efficacy. The

134 effects of ZnPy appeared similar in control and MCT pulmonary arteries, but like retigabine there was a significant increase in the efficacy of the drug in MCT vessels. In contrast, the effects of BMS-204352, were unaltered by MCT. This may be because BMS-204352 caused vasodilation through activation of BKCa rather than Kv7 channels.

The vasodilator effect of retigabine was also tested in mesenteric arteries from both control and MCT animals. Retigabine was found to produce concentration-dependent dilation of mesenteric arteries from both control and MCT animals. Retigabine has been reported to dilate human and rat mesenteric arteries at concentrations between 1 µM and 10 µM (Jepps et al., 2011; Ng et al., 2011). It has been proposed that this dilation is due to activation of Kv7 channels (Jepps et al., 2011; Ng et al., 2011). In the present study retigabine produced dilation of mesenteric arteries at concentrations of 100 nM or above. It was also found to fully relax these vessels, whereas the study by Jepps et al. (2011) showed a partial relaxation. These discrepancies may be due to differences in experimental protocols. In the study by

Jepps et al. (2011), the α1-adrenoceptor agonist methoxamine was used to preconstrict the vessels, whereas in my experiments I used phenylephrine.

2.7.3 The effect of vasodilators on intrinsic tone:

It was reported by Ito et al. (2000) that there was raised intrinsic tone in pulmonary arteries from MCT-treated animals in comparison to control animals. In this study it was confirmed that PA from MCT animals indeed exhibited raised intrinsic tone. This was shown by the ability of various vasodilators to dilate pulmonary arteries without the need for any preconstriction. This was not the case with pulmonary arteries from control animals, where dilation was only observed if the vessels were first stimulated to constrict with PE. Mesenteric arteries from control and MCT animals were also tested and neither showed intrinsic tone. This was confirmed by the inability of retigabine to relax these vessels when they were not pre-constricted. These findings suggest that the raised intrinsic tone in PAH is specific to pulmonary arteries.

The calcium channel blocker nifedipine, relaxed unstimulated pulmonary arteries from MCT rats, while having virtually no effect on control arteries. Retigabine also relaxed intrinsic tone in MCT PA. Moreover, nifedipine and removal of extracellular Ca2+ had no further effect. This suggested that hyperpolarization of PASMCs in MCT

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PA is enough to maximally dilate the vessels and that the raised intrinsic tone was dependent on voltage-gated calcium influx. It also supports a previous study proposing that the raised intrinsic tone resulted from depolarization (Ito et al., 2000). We predicted that other drugs that acted by hyperpolarization would have similar effects. This was indeed the case with the Kv7 channel activator ZnPy and the KATP channel activator levcromakalim which relaxed MCT PA to a similar level as retigabine and nifedipine. Therefore K+ channel activators effectively abolished intrinsic tone.

It was important to determine whether hyperpolarization, in particular, was effective at reversing the raised intrinsic tone, or whether any vasodilator, irrespective of its ability to cause hyperpolarization could produce a similar effect. Sildenafil is a PDE5 inhibitor that causes vasodilation through cGMP-dependent pathways. Several mechanisms have been proposed to underlie the cGMP-induced vasodilation, although it is acknowledged that it is due to a reduction in the intracellular Ca2+ concentration and sensitivity (Carvajal et al., 2000). Some groups propose that the activation of protein kinase G (PKG) by cGMP activates BKCa channels giving rise to hyperpolarization and vasodilation (Kannan et al., 1995; White et al., 1995; Peng et al., 1996; Trongvanichnam et al., 1996; Yamakage et al., 1996; Zhou et al., 1996; Mikawa et al., 1998; Tanaka et al., 1998; Zhou et al., 1998). Other groups suggest that there is a direct inhibition of Ca2+ channels by PKG (Clapp et al., 1991a; Quignard et al., 1997; Tewari et al., 1997). These mechanisms predict that cGMP would be effective at suppressing intrinsic tone. It has also been proposed that PKG activation of the Ca2+/ATPase pumps in the plasma membrane and sarcoplasmic reticulum are responsible for the reduction of intracellular Ca2+ caused by cGMP (Rashatwar et al., 1987; Furukawa et al., 1988; Raeymaekers et al., 1988; Vrolix et al., 1988; Clapp et al., 1991a; Cornwell et al., 1991; Yoshida et al., 1991; Horowitz et al., 1996; Karczewski et al., 1998). Another way that cGMP reduces intracellular Ca2+ is by inhibiting phospholipase C (PLC), (Rapoport, 1986; Hirata et al., 1990; Lincoln et al., 1993; Pfeifer et al., 1995; Baines et al., 1996) which is essential for the formation of IP3 and therefore release of Ca2+ from the intracellular stores. PKG activation by cGMP may also directly inhibit IP3 receptors by phosphorylation to reduce the intracellular Ca2+ concentration and cause smooth muscle relaxation (Komalavilas et al., 1994; Komalavilas et al., 1996). These pathways are all independent of voltage-gated Ca2+ influx suggesting that they would not block

136 intrinsic tone. The mechanism of action of sildenafil likely involves several of these pathways, however a study of its effects on vascular tone and Ca2+ signaling in rat pulmonary arteries suggested that it acted predominantly by altering the IP3 calcium signaling pathway (Pauvert et al., 2003). In the present study, sildenafil was found to relax unstimulated pulmonary arteries from MCT-treated rats. However, the relaxation was significantly smaller than that caused by nifedipine and retigabine. This finding supports the hypothesis that intrinsic tone largely reflects depolarization of the smooth muscle cells and voltage-gated calcium influx.

2-APB is a drug that is known to inhibit IP3 receptors and store operated Ca2+ channels (Bootman et al., 2000). It therefore causes relaxation of SMCs by a mechanism that is independent of voltage-operated Ca2+ channels and K+ channels. When tested on unstimulated MCT pulmonary arteries, 2-APB produced concentration-dependent relaxation of the vessels. Moreover, 2-APB was as effective as nifedipine and retigabine in abolishing the intrinsic tone. This suggests that perhaps enhanced store-operated Ca2+ entry may have contributed to the raised intrinsic tone. It does not rule out the original hypothesis that depolarization was responsible. Enhanced store-operated Ca2+ entry and TRPC1 and TRPC4 channel expression has been reported in pulmonary arteries of MCT-treated rats (Liu et al., 2012). 2-APB has been reported to block TRPC4 channels and therefore can inhibit their depolarizing effects on the membrane. It is possible that TRPC channels contribute to the depolarization in MCT PA, and hence 2-APB may have been as effective as nifedipine and retigabine by inhibiting the channels that caused depolarization. Therefore the hypothesis that raised intrinsic tone may be due to depolarization remains a valid one. Although the results are not shown, the effects of hydralazine, which acts by inhibiting Ca2+ release from the SR (Gurney et al., 1995), were investigated. These experiments were disregarded though, because of the inability of the drug to relax vessels pre-constricted with PE.

2.7.4 The effect of MCT on vessel recovery after a stimulus:

Ito et al. (2000) reported a slower relaxation of MCT versus pulmonary arteries after a contractile stimulus with high KCl or PGF2α was removed. The same study also reported that the intracellular Ca2+ concentration in PASMCs from MCT animals was significantly higher than in controls consistent with the raised intrinsic tone and sustained Ca2+ influx at rest. I also found that after stimulating pulmonary arteries

137 with KCl, the washout was significantly slower in MCT pulmonary arteries than in control arteries. This effect was specific for pulmonary arteries as similarly treated mesenteric arteries did not show a change in the recovery rate after being stimulated with 50 mM KCl. The elevated Ca2+ and slow recovery was proposed to be due to inhibition of NCX exchanger, caused by the depolarized Em of PASMCs (Ito et al., 2000). Consistent with this, the rate of recovery from a KCl stimulus in MCT PA returned towards the rate seen in control PA when the membrane potential was hyperpolarized by levcromakalim. It is also possible that altered activity of SERCA may have contributed to the slowed recovery rates after washout, as this pump is responsible for sequestering intracellular Ca2+ into the SR and plays a role in the regulation of intracellular Ca2+ concentrations.

The NCX, in conjunction with the PMCA, is responsible for maintaining low intracellular Ca2+ concentrations. In addition to removing Ca2+ from the cell (forward mode), the NCX can mediate the entry of Ca2+ (reverse mode) in parallel with some ion channels, depending on the Na+, Ca2+ and K+ gradients across the plasma membrane (Yuan et al., 1999). When the intracellular Ca2+ rises, for example due to closure of Kv7 channels and opening of voltage-gated calcium channels, the Na+/Ca2+ exchanger extrudes one Ca2+ ion in exchange for the entry of 3 Na+ ions. This exchanger is therefore electrogenic in nature meaning that its activity is dependent on the resting membrane potential of the smooth muscle cell in which it is present (Aaronson et al., 1989; McCarron et al., 1994). It has been reported that an upregulation of NCX1 contributes to the enhanced Ca2+ entry seen in PASMCs from patients with idiopathic pulmonary hypertension mainly via the reverse mode of the NCX (Zhang et al., 2007b). This may contribute to the characteristic depolarized membrane potentials seen in pulmonary hypertension.

Depolarization of PASMCs has various implications (Fig. 2.42). If membrane potentials are more depolarized, we would expect an inhibition of the NCX making it less effective in removing intracellular Ca2+. This could account for the significantly slower recovery rates from 50 mM KCl seen in MCT pulmonary arteries in comparison to control pulmonary arteries. The effect of depolarization on the slower recovery of PA vessels from 50 mM KCl is also supported by the finding that hyperpolarization using levcromakalim significantly speeded up their recovery rate. The effect of depolarization on the recovery rate of the vessels must be accepted with caution because the exposure of control vessels to a depolarization solution of 15 mM K+ PSS

138 did not slow down their recovery rates. It would be useful to repeat this experiment in the absence of the endothelium because the endothelium is known to be severely impaired in the MCT-treated vessels and its removal may have an effect on these results.

2.7.5 Membrane potential influences vessel responses to Kv7 modulators:

Depolarization is characteristic of pulmonary hypertension. It has been shown that PASMCs are depolarized in vessels from MCT-treated rats (Ito et al., 2000), as well as in hypoxia induced PH (Smirnov et al., 1994; Osipenko et al., 1998) and in patients with PH (Yuan et al., 1998a). This depolarization may be responsible for the differences in responses to Kv7 modulators seen in MCT and control animals. This was shown by the effect of depolarizing PASMCs in control vessels using a 15 mM K+ PSS solution, which rendered them more sensitive to the Kv7 blocker XE991.

Moreover, hyperpolarizing PASMCs of MCT vessels using the KATP opener levcromakalim rendered them less sensitive to XE991. The results obtained in this experiment were similar to findings by Joshi (2007), where the contractile effect of XE991 was blocked by levcromakalim. These results support the hypothesis that depolarization of PASMCs in MCT pulmonary arteries plays a pivotal role in their response to Kv7 modulators.

The greater activation of Kv7 channels at depolarized Em could account for the enhanced response of MCT PA to the Kv7 blocker XE991, as there would be more channels open and amenable to the drug. It is less clear how a depolarized membrane would affect the response to retigabine as it is active over a narrow range of membrane potentials (Joshi et al., 2009), but the increased Kv7 channel activity might promote its effects. The lack of effect of MCT on the sensitivity to retigabine, was however reproduced when control vessels were exposed to a 15 mM K+ PSS solution, implying that the sensitivity is unaffected by Em. Further support for this comes from the lack of effect of levcromakalim on the sensitivity of MCT vessels to retigabine.

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Figure 2.42: Implications of depolarization on Kv7 and NCX function in PASMCs. Schematic diagram showing the effects of depolarization on Kv7 channels, voltage-gated Ca2+ channels and the sodium calcium exchanger (NCX). Depolarization leads to the activation of more Kv7 channels which in turn are more accessible to Kv7 modulators. Depolarization also activates voltage-gated Ca2+ channels leading to increased Ca2+ influx. The removal of intracellular Ca2+ is slower at depolarized membrane potentials due to inhibition of the NCX.

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The efficacy of the Kv7 openers retigabine and ZnPy, was increased in MCT PA in comparison to control PA. A possible explanation for this is the presence of the raised intrinsic tone, which means that these agents not only removed the tone developed due to the addition of PE but also the initial raised basal tone. Thus the apparent percentage inhibition of PE constriction was increased

2.7.6 MCT does not alter KCNQ mRNA expression in pulmonary and mesenteric arteries:

Responses to Kv7 activators were found to be suppressed in mouse models of PAH (Morecroft et al., 2009). Rats exposed to hypoxia for several days were found to express a lower level of Kv7.4 in pulmonary arteries than matched controls (Sedivy et al, 2013). It was also reported that the expression of Kv7 channels in systemic arteries was down-regulated in systemic hypertension (Jepps et al., 2011). Other K+ channels are also down regulated in models of PAH. For example, Kv1.5 and Kv2.1 were down regulated in SERT+ mice (Guignabert et al., 2006). This study is the first to look at the expression of KCNQ mRNA in the MCT model of PH. It was found that KCNQ1, KCNQ4 and KCNQ5 mRNA are all expressed in pulmonary and mesenteric arteries of MCT and control rats. The expression profile was similar to that reported by Joshi et al. (2009) for pulmonary arteries and by Mackie et al. (2008) for mesenteric arteries. It was found that there was a similar expression of KCNQ1, KCNQ4 and KCNQ5 mRNA in PA. The expression of KCNQ mRNA was similar in both MCT and control groups of animals for both types of vessels. These findings were different from reports by Joshi et al.( 2009), where expression of KCNQ4 mRNA was higher than KCNQ1 and KCNQ5 in PA and where KCNQ4 mRNA expression was higher in PA in comparison to MA. The study by Joshi et al. (2009) used a single house-keeping gene to normalize expression, whereas in the present study the expression was normalized to three HKGs using the geNorm method. This could account for the differences in the expression profile. The results of this study suggest that the enhanced Kv7 modulation seen in MCT rats was not due to altered expression of Kv7 channels. Unlike other K+ channels which were found to be down- regulated in the PA of MCT rats, Kv7 mRNA expression is maintained and therefore these channels may serve as potential drug targets to treat pulmonary hypertension.

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2.7.7 Conclusion:

The findings of this study suggest that Kv7 channels remain functional in the MCT model of PH and therefore may serve as future therapeutic targets to treat the condition. Previous reports that KATP channel activators JTV-506 (Tsutsumi et al., 2004), pinacidil (Wanstall et al., 1994) and nicorandil (Sahara et al., 2012) attenuated the development of PH in the MCT rat model support idea that K+ channels may be effective drug targets in treating PH.

Pulmonary arteries from MCT-treated rats exhibited raised intrinsic tone in comparison to control PA. This tone was maximally inhibited by Kv7 activators and other agents that cause inhibition of voltage-gated Ca2+ influx either directly or indirectly. The expression of KCNQ mRNA, which encode Kv7 channels, was not altered in this model suggesting that the enhanced sensitivity of the vessels to Kv7 modulators was largely dependent on SMC membrane depolarization which occurs during the development of PAH. Depolarization was found to have a major effect on the sensitivity of the vessels to the Kv7 blocker XE991 and accounted for the slower recovery rates of MCT PA after stimulation with 50 mM KCl.

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Chapter 3: The Pharmacology of Zinc Pyrithione in The Pulmonary Circulation

3.1. Introduction:

Pulmonary arterial tone is regulated in part by the resting membrane potential of PASMCs. The resting membrane potential is regulated by a K+ conductance, such that, an inhibition of the K+ conductance would give rise to a depolarization. This depolarization in turn activates voltage dependent Ca2+ channels giving rise to vasoconstriction (Post et al., 1992; Nelson et al., 1995; Yuan et al., 1998b). On the other hand, the opening of K+ channels would give rise to a hyperpolarization and inhibition of voltage dependent Ca2+ entry. This inhibition ultimately leads to vasorelaxation (Clapp et al., 1993).

Different classes of K+ channels were found to be expressed in PASMCs. These include: the large-conductance calcium and voltage-activated potassium channels

(BKCa), ATP sensitive potassium channels (KATP) channels, A-like potassium channels

(KA), two-pore domain channels (TASK), and more recently Kv7 potassium channels (Clapp et al., 1991a; Clapp et al., 1992; Evans et al., 1996; Gardener et al., 2004;

Joshi et al., 2009). Em is determined by a non-inactivating current, which is partially mediated by TASK channels and also depends on a voltage-gated potassium channel.

The exact voltage-gated K+ channel(s) that regulate IKN and Em remain debated. However, the properties of these channels closely resemble the neuronal M-current, which is known to be mediated by channels encoded by the KCNQ genes. The Kv7 family of potassium channels was recently implicated in the regulation of Em in rat PASMCs, and KCNQ1, KCNQ4 and KCNQ5 were found to be the main mRNA transcripts present. It has been proposed that Kv7.4 makes the greatest contribution to the resting membrane potential (Joshi et al., 2009).

3.2 Identification of ZnPy as an opener of Kv7 channels:

Zinc pyrithione (ZnPy), was recently identified as an activator of Kv7 channels and was shown by Xiong et al. (2007) to be a strong activator of all Kv7 channels apart from Kv7.3. In their study, Xiong et al. (2007) screened more than 20,000 molecules at a concentration of 10 µM and identified ZnPy as an unusually potent synthetic

143 activator of Kv7 channels. ZnPy was able to act on native M channels as well as heterologous Kv7 channels. Further tests in their study also revealed that ZnPy had no effects on hERG, Kv2.1, Kv4.2 and N-type calcium channels.

To ascertain that the effects caused by ZnPy were due to an opening of K+ channels, Xiong et al. (2007) tested the effects of ZnPy on CHO cells expressing Kv7.1-Kv7.5 using the whole-cell voltage clamp technique. They studied the currents in the presence of TEA, which is a commonly used inhibitor of K+ channels. They found that in the presence of 20 mM TEA the currents induced by ZnPy were completely abolished, and the subsequent removal of TEA allowed for complete recovery. They also tested the effects of ZnPy in the presence of the Kv7 blocker linopirdine and found that 30 µM linopirdine similarly blocked the effects of ZnPy with complete recovery after removal of the blocker. They thus concluded that ZnPy was acting on a current that was mediated by Kv7 channels.

The study by Xiong et al. (2007) also revealed that both Zn2+ and pyrithione in a preferential stoichiometry were required for the observed potentiation. They studied the potentiation produced in an aqueous solution in the presence of different ratios of sodium pyrithione (NaPy) and ZnSO4. They concluded that in a 2:1 ratio of NaPy:

ZnSO4 the observed potentiation was maximal. They also found that this potentiation was comparable to that caused by 10 µM preformed ZnPy. It was also determined that 20 µM NaPy did not cause a change in the amplitude of the currents in CHO cells expressing Kv7.2. The subsequent application of ZnSO4 resulted in a potentiation of the current, which was not seen when CuSO4 and CdCl2 were added.

ZnPy is an ionophore (Kimura et al., 2004), therefore it was important to determine whether the potentiation caused by this drug was largely due to a chemical interaction with the channel or due to an ionophore effect. In their study, Xiong et al. (2007) ruled out that ZnPy was mainly producing the potentiation via an ionophore effect. This was determined after three different zinc ionophores, namely zinc diethyldithiocarbamate (DEDTC), 5,7-diiodo-8-hydroxyquinoline (DIQ) and (±) α- tocopherol (Schroeder et al., 1998; Jentsch, 2000; Hille, 2001) had no effect on the current amplitude in CHO cells expressing Kv7.2 (Xiong et al., 2007).

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3.3 The characterization of ZnPy modulation in expressed Kv7 channels:

In order to characterize the observed effects of ZnPy, Xiong et al. (2007) compared voltage-dependant activation in heterologously expressed Kv7.2 and Kv7.3 channels.

They found that the voltage required for half-maximal activation (V0.5) of Kv7.2 was shifted from -18.9 ± 0.6 mV to -44.4 ± 0.9 mV, whereas Kv7.3 did not show any current potentiation or change in V0.5. ZnPy did however, cause a consistent hyperpolarizing shift in the V0.5 of Kv7.2/Kv7.3 channels, which can therefore be mainly attributed to Kv7.2. ZnPy was also found to reduce the activation and deactivation rates of the Kv7.2 and Kv7.2/Kv7.3 channel. Not only was ZnPy found to have an effect on V0.5 but it was also found to augment the maximal conductance

(Gmax) of the channels. Both these factors contributed to an overall increase in the current amplitude. This study by Xiong et al.(2007) also showed that ZnPy increased the single-channel open probability (Po) for Kv7.2 and Kv7.4. It is believed that the increase in maximal conductance was largely due to this increase in Po.

ZnPy is a particularly interesting compound because it has a different spectrum of activity from retigabine, which is considered a more classical Kv7 opener. Both chemicals are Kv7 channel activators, however ZnPy opens all Kv7 channels apart from Kv7.3. On the other hand, retigabine opens all Kv7 channels apart from Kv7.1. ZnPy and retigabine are also structurally different. Crystallographic studies of ZnPy revealed that it is a complex of one zinc atom chelated by two pyrithione units via a sulphur and oxygen atom (Barnett et al. (1977), whereas retigabine is an N-(2-amino- 4-(4-fluorobenzylamino) phenyl carbamic acid ethyl ester (Fig. 1.8 - Chapter 1).

When an activator binds to a channel, it triggers a series of conformational changes essential for channel opening. Mutagenesis and recombinant channels provide a means of identifying sites of action of various chemical compounds. A study by Xiong et al. (2008) found that ZnPy and retigabine had distinct interaction sites and therefore acted non-competitively on different sites of Kv7.2 channels expressed in CHO cells. It was reported that although both compounds caused a hyperpolarizing shift of V1/2, ZnPy but not retigabine, caused a five-fold increase in maximal conductance. In the same study, the co-application of these openers was also found to restore a Kv7.2 disease mutant channel (I238A). This mutant channel is not activated by membrane depolarization. In the presence of ZnPy, but not retigabine, I238A responded to depolarization and showed similar properties to wild type Kv7.2

145 channels. The now functional ZnPy bound I238A channel regained sensitivity to retigabine. This suggested that it was possible to alter membrane excitability using small molecules such as ZnPy.

3.4 Hypothesis and Aims:

The effects of ZnPy on PASMCs and pulmonary artery vessels have not been previously investigated. As ZnPy was shown to activate recombinant Kv7 channels, we hypothesized that ZnPy would activate K+ channels in PASMCs resulting in hyperpolarization of the resting membrane potential and closure of L-type Ca2+ channels giving rise to a vasodilation. Since various K+ channels are indeed expressed in PASMCs, it was important to determine whether or not ZnPy was acting on Kv7 channels in particular or whether other channels were involved.

The main aim of this part of my project was to determine if ZnPy affected freshly isolated PASMCs and intact pulmonary arteries. The ability of ZnPy to hyperpolarize PASMCs was investigated using the whole-cell patch clamp technique. The roles of different K+ channels expressed in PASMCs in the ZnPy-induced hyperpolarization were assessed, including : Kv7, KATP and BKCa channels. Glibenclamide was used as a blocker of KATP channels, TEA as a blocker of multiple K+ channels, paxilline and iberiotoxin as BKCa blockers and XE991 as a Kv7 selective blocker. The aim was to identify the nature of key ion channels modulating the effects of ZnPy on Em.

Parallel experiments on intact vessels were designed to address the physiological role of Kv7 channels in the intact artery. I investigated the mechanism of the ZnPy- induced dilation in vessels preconstricted with drugs that cause constriction via different mechanisms. These included Bay K-8644 as Ca2+ channel opener, PE as an α-adrenoceptor agonist and ionomycin which is a Ca2+ ionophore. Similar to the electrophysiology experiments, the effects of ZnPy were also tested in the presence of various K+ channel blockers to determine the pharmacological profile of this drug. This was important not only to determine the selectivity of the drug but also to determine whether the effects seen in the intact artery may be attributed to the effects on the membrane potential recorded from PASMCs.

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3.5. Methods:

3.5.1 Patch clamp electrophysiology

3.5.1.1 Tissue preparation:

Male Sprague-Dawley rats (250-300 g) were killed by cervical dislocation in accordance with the UK Scientific Procedures (Animals) Act 1986. The heart and lungs were removed and placed into PSS (See Table 3.1). The pulmonary artery running along the length of a lobe was dissected from the lungs and cleaned of connective tissue and blood. Slices were then used for myography or cut into 3-4 mm pieces and used for PASMC isolation.

Table 3.1: Composition of PSS, dissociation medium (DM) and pipette solution. The table below shows the constituents of DM used in cell isolation and PSS and pipette solution used in patch clamp experiments.

Salt Concentration (mM) PSS DM Pipette Solution NaCl 122 110 - KCl 5 5 130 NNN-[2-Hydroxyethyl]piperazine-N- 10 10 20 [2-ethane-sulfonic acid] (HEPES)

KH2PO4 0.5 0.5 -

NaH2PO4 0.5 0.5 -

NaHCO3 - 10 - Taurine - 10 - EDTA - 0.5 - EGTA - - 1

MgCl2 1 2 1 Glucose 11 10 -

CaCl2 1.8 160 µM - Phenol Red - 0.03 -

Na2GTP - - 0.5 *pH adjusted to 7.3 *pH adjusted to 7.3 *pH adjusted to with 1M NaOH with 1M NaOH. 7.3 with 1M KOH

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3.5.1.2 PASMC isolation protocol for electrophysiology:

Pieces of isolated artery were placed in dissociation medium (Table 3.1) and the vessel was cut open longitudinally then sliced into 3-4 mm length pieces. Papain (1.5 mg, 9.94U/mg, Sigma-Aldrich) was dissolved in 1 ml DM to produce a 1.5 mg/ml papain solution.

Slices of PA were then transferred into the papain solution and incubated for 1 hour at 4ºC. Dithiothreitol (5 µl, DTT, Sigma-Aldrich) was then added to the papain solution in order to activate it (Lewis et al. 1976) and the tissue was incubated at 37ºC for 6 min while being shaken at 60 rpm. The tissue was then transferred quickly into 1 ml of DM containing 20 mg/ml collagenase IA (Sigma-Aldrich) and incubated again at 37ºC for a further 4-5 min, while being shaken at 60 rpm. Individual cells were obtained by triturating the tissue, using a wide bore (3 mm) glass Pasteur pipette in 0.5 ml fresh DM. The cells obtained were verified to have the characteristic spindle-shape of SMCs under the microscope (Fig. 3.2). They were stored at 4ºC and were used over the next 4-6 hours.

3.5.1.3 The patch clamp technique:

The notion that electrical phenomena existed in the human body has been recognized ever since the nineteenth century, where muscle movements in frogs were evoked by applying electrical stimuli. The concept of voltage clamp was developed by Cole in 1949 and forms the basis of the patch-clamp technique which was developed by Neher and Sakmann in 1976. When an ion channel is open, the ions move across this ion channel like an electric current. This current will in turn result in a change in the resting membrane potential of the cell which ultimately alters the flow of ions across the membrane. This is because the driving force for the flow of the ions across the membrane is different at different membrane potentials and also because there are voltage activated ion channels present in the membrane which may also be affected. The change in flow of ions again results in a change in the resting membrane potential and the cycle continues making it impossible to measure the current which changes constantly due to this feed-forward cycle. Therefore to be able to measure the activity of an ion channel, it is important to clamp the voltage at a constant level. Figure 3.1 illustrates how voltage clamp is applied to study the

148 currents in a particular cell. The cell is impaled with two microelectrodes connected to an electric circuit. An amplifier is connected to the electrode and measures the membrane potential relative to ground. The voltage controller applies the desired voltage and this voltage is measured by a second amplifier. This amplifier compares the applied voltage with the membrane potential and injects a current that compensates for any differences in the measurements. This current is equal and opposite to the current that flows through the ion channels which is needed to keep the resting membrane potential constant. In current clamp the current is set at a zero and this permits recording the resting membrane potential which is defined as the potential at which there is no net flow of ions across the membrane.

The patch-clamp technique is an extension of voltage-clamp which involves forming a very tight seal with the cell under study using a glass pipette. The patch clamp technique has undergone many developments since its advent and represents one of the most powerful tools to study the activity of ion channels. There are several configurations which may be used in the patch clamp technique, including: cell- attached, whole-cell, inside-out, outside-out and perforated patch (Fig. 3.2).

The cell-attached mode is when the micropipette forms a very tight seal with the cell membrane without actually penetrating into the cell. This means that the small patch of membrane that is surrounded by the pipette can now be studied. This configuration permits the study of single-channels and is therefore also known as the single-channel configuration. In whole-cell mode, the pipette physically ruptures the patch of membrane and makes direct contact with the cytoplasm. This permits the study of currents across the whole membrane. In the case of perforated patch, contact with the cytoplasm is achieved by adding agents (usually nystatin or amphoteracin B) to the pipette solution that perforate the membrane and allow ions to pass through without altering the composition of the cytoplasm. The outside-out and inside-out configurations are two single-channel configurations where the patch of membrane is excised and studied. To achieve an outside-out configuration, the pipette is simply pulled away from a whole-cell configuration. The intracellular side of the membrane makes contact with the inside of the pipette and the extracellular side is in contact with the bath. The inside-out configuration can be achieved by pulling the pipette away from a cell-attached configuration. This configuration allows the study the effects of cytoplasmic factors on ion channels.

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From an electronics point of view, the cell membrane, together with the intracellular and extracellular media, form a capacitor which has the ability to store charge. The physical dimensions of the cell membrane are one of the key factors in determining the capacitance of the membrane (Cm), which can be measured as an indicator of the size of the cell membrane. The ion channels act as resistors (Rm), and the more channels that are open, the lower the resistance. These features can be represented by a simple circuit diagram (Fig. 3.3). In all my experiments, the whole-cell patch- clamp configuration was used. This means that the pipette solution and electrode made direct electrical contact with the cytoplasm. In this configuration the patch resistance becomes very low and is referred to as the access resistance (Raccess) (Fig . 3.4)

The transmembrane currents are carried from the cells via the pipette solution, through a Ag/AgCl electrode to a headstage (Axopatch 201A, Axon instruments, USA). A feedback amplifier (FBA) which is found in the headstage, serves to amplify the received current as well as to clamp the membrane at a specific voltage. The FBA maintains the voltage through the pipette (Vp) equal to a command voltage (Vc), imposed by the experimenter, by constantly passing current through a feedback resistor (Rf) in order to compensate for any current that would change the membrane potential. The voltage drop across Rf is directly proportional to and provides a measure of ionic current across the membrane. After amplification of the current, it passes through a low pass Bessel filter (4.0 kHz) and is then digitized and displayed as a record on a computer screen.

3.5.1.4 Preparation of pipettes and solutions:

The pipette electrodes used for patch-clamping were made from borosilicate glass capillaries (Harvard Apparatus Ltd, external diameter 1.5 mm, internal diameter 1.17 mm), using a vertical, multi-stage pipette puller (Narishige, Japan, Model PB-7) that pulled the capillaries in two steps. The pipettes had blunt tips and were fire-polished using a V-shaped platinum-iridium filament coated with glass, mounted on a microforge (MF-380, Narishige, Japan). The shank was then coated with beeswax close to the tip. This causes thickening of the shank, and prevents the bath solution from creeping up the sides of the pipette, therefore minimizing the pipette capacitance.

150

clamp voltage how illustrating diagram Schematic

clamp. voltage of concept The 3.1: Figure achieved. is

151

Figure 3.2: Patch-clamp configuration: A) Figure illustrating the different patch- clamp configurations and how they are achieved. B) Image of isolated rat PASMC used in my experiments, after the cell was patched-clamped effectively. The average length of PASMCs is 50-80 µm.

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Figure 3.3: An electronic model of the cell membrane. Rm = membrane resistance, Cm = membrane capacitance and Em = membrane potential.

Figure 3.4: Equivalent circuit for the whole-cell patch clamp configuration. Rm = membrane resistance, Raccess = access resistance, Rseal = seal resistance, Rpipette = pipette resistance, Cm = membrane capacitance, Cpipette = pipette capacitance. Adapted from Molleman (2009).

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The pipettes were filled with pipette solution whose ionic composition was close to that of the normal intracellular solution (Table 3.1). The pipettes were filled using a microsyringe with a fine-gauge needle, and air bubbles were removed by flicking the shank of the pipette. The patch electrode was mounted on the headstage, and the circuit was completed by a Ag/AgCl2 pellet electrode that connected the bath solution to the ground.

The Ag/AgCl2 wire used with the pipette was regularly dipped into bleach to maintain the chloride coat and the reversibility of the reaction at the metal-liquid junction. As Ag+ ions leaking from the electrodes may affect the cells, this was prevented by positioning the wire in the pipette well away from the cells and keeping the reference electrode in a side chamber.

3.5.1.5 Whole-cell recording:

After carrying out the cell isolation, a drop of the solution containing isolated PASMCs was placed on the glass bottom of the recording chamber, mounted on an inverted microscope as a part of the patch-clamp set up. The cells were left to settle for about 10-15 min after which they were super-perfused with PSS (composition in table 3.1) at a rate of about 0.5 ml/min. A pipette was slowly lowered into the chamber towards a cell using a micromanipulator. When the pipette made contact with the bathing solution a junction potential was recorded and was effectively cancelled using the pipette offset control. A 2 ms long hyperpolarizing step from -80 to -90 mV, was applied and repeated every 10 ms, leading to the appearance of a rectangular waveform in the current trace. The pipette was slowly moved closer to the cell and as the pipette made contact with the cell, there was a reduction in the current amplitude. In order to increase the resistance of the seal, suction was applied to the inside of the pipette via a 2 ml syringe connected to the pipette holder. When successful, this caused abolition of the current response to the voltage step, leaving small current transients at the start and end of the voltage step, and giving rise to a 'Giga seal'. The application of more suction finally ruptured the membrane just underneath the pipette tip, while minimally affecting the seal and the cell. Large current transients now appeared at the start and end of the voltage step reflecting the increased membrane capacitance as the current crossing the entire cell membrane was recorded (Fig. 3.5).

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Figure 3.5: Capacitative and ionic currents in patch clamp: Current trace showing the capacitative and ionic currents.

155

The area under this current was used to calculate the cell capacitance, which is given by:

C = Q/V (3.1) where Q is the charge (area under the current versus time curve) and V is the change in the voltage (10 mV).

The current induced by the voltage step was due to the flow of ions through ion channels and the seal between the pipette and cell. Provided the seal had a large resistance, then the current was a direct reflection of the ions flowing across the membrane.

3.5.1.6 Membrane potential recordings:

The membrane potential was recorded in current-clamp mode. This means that the current injected into the cell was kept constant at zero (I=0), while the resting membrane potential was recorded. No correction was made for junction potentials, which were no more than 3.1 ± 0.5 mV (n=4) (Joshi 2007). The membrane potential was continuously monitored before and after drug application. In the initial experiments testing the effect of ZnPy on Em, the drug was applied using a slow- perfusion system that replaced the PSS with drug-solution. In the remaining experiments, the drugs were applied onto the cells by a multi-barrel pipette which was positioned >100 µm from the cells, using a manually controlled, gravity flow channel system. The rate of flow was approximately 2 ml/min. To ensure that the effects observed were entirely due to the drug and not induced from the flow, a control solution was applied before switching to the drug and control Em recorded. Next the cell was exposed to 10 µM ZnPy and the resulting membrane potential was recorded. ZnPy was then washed using PSS and the membrane potential was recorded. After the resting membrane potential recovered, the cell was exposed to a blocker and the membrane potential noted. The cell was then exposed to ZnPy in the presence of the blocker and the effect recorded. Next ZnPy was washed off using a solution of the blocker. Finally the blocker was washed off using PSS. The blockers used in these experiments were: 10 µM glibenclamide (to block KATP channels), 10 mM TEA (to block BKCa as well as KV channels including some Kv7 channels), 50 nM iberiotoxin and 1 µM paxilline (both as blockers of BKCa channels) and 10 µM XE991

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(as a blocker of Kv7 channels). WinWCP software (University of Strathclyde) was used with a BNC-2090 digitizer (National instruments USA) to record membrane potential.

3.5.1.7 Current recordings:

K+ currents were studied in voltage-clamp mode. The voltage-activated K+ current was studied by clamping the cells at -70 mV and then stepping to increasingly depolarized potentials up to +50 mV, in 10 mV increments. Each step was 300 ms in duration and there was a 5 s interval between sweeps. The average current was measured between 250 to 265 ms, where it had reached steady-state level. The effect of ZnPy (10 µM) on the current caused by a single voltage step from -80 to +40 mV was also recorded. The drug was then washed off and the current was measured. This was also repeated in the presence of TEA (10 mM), iberiotoxin (50 nM), paxilline (1 µM) and XE991 (10 µM), where after washing off the ZnPy with PSS, the cells were exposed to the blocker and the effect of the blocker on its own were measured. Next the cell was exposed to 10 µM ZnPy in the presence of the blocker. After recording the current, ZnPy was washed off using a solution of the blocker and the current recorded. Finally the blocker was washed off using PSS and the current recorded.

In another protocol, the cell was clamped at 0 mV for ≥ 5 min and the residual current at 0 mV studied. Clamping the cells at 0 mV for over 5 min inactivated the majority of Kv channels (Evans et al., 1996). The current at 0 mV was continuously recorded in order to study the drug effects and their time-course. ZnPy (10 µM) was tested on its own as well as in the presence of TEA, paxilline and XE991 applied in an order similar to the membrane potential and voltage-activated K+ current recordings. To investigate the voltage-dependence of the residual current, the membrane potential was stepped to +60 mV, then ramped to -100 mV over a period of 1.5 s.

3.5.1.8 Data analysis:

The current and membrane potential recordings were analyzed by WinWCP software (Version 4.1.3, University of Strathclyde). Data are expressed as mean ± s.e.m of n cells and were compared using paired Student's 't' test when from individual cells, or unpaired t-test for populations of cells. In myography experiments n was the number of animals. Two-way ANOVA followed by Bonferroni post test was performed when

157 there was more than one variable in the data. Data were subsequently analyzed and plotted using Microsoft Excel, Origin 8.1 software (OriginLab Corporation) and Graphpad Prism 5 (Graphpad). Concentration-response curves were fitted using a

Boltzmann equation and pEC50 values were calculated using the Hill-equation.

3.5.1.9 Limitations and problems in patch-clamp recordings:

There are various factors that can influence the accuracy of patch-clamp recordings. These include poor seals, clamping speed, junction potentials and series resistance. It is important that these factors are taken into account in order to reliably perform the experiments and best interpret the results.

The actual current that crosses the cell membrane may not be exactly the same as that injected to control voltage. This is due to the fact that the injected current has to flow through a resistance that is in series with the membrane (Rs). Remnants of the cell membrane that may be present at the pipette tip, as well as the size of the pipette, lead to a voltage drop across Rs that is not recorded. This error (V) is calculated using Ohm's law as:

V = I Rs (3.2)

When Rs is large, the error is large. Since Rs affects the time constant () of the capacitative transient, it can be calculated by dividing the time constant by the capacitance, as follows:

Rs = /C (3.3)

In reference to Fig. 3.4, we can see that Raccess and Rpipette are in series and that Raccess is in parallel with Rm. The input resistance in the circuit may be calculated as the sum of all the resistances in the circuit.

Rin = Rseries + Rparallel (3.4)

1/ Rparallel = 1/Rm + 1/Rseal (3.5)

By substitution, Rin = Rseries + 1/(1/Rm +1/Rseal) (3.6)

This input resistance was calculated from the change in ionic current caused by a 10 mV hyperpolarization from -80 mV and was found to be 2 ± 0.1 GΩ (n=116).

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A junction potential is formed when two different conductors come into contact. Two types of junction potentials are encountered in the whole-cell configuration. The first is a liquid-metal junction potential, which is formed when the Ag/AgCl electrodes come in contact with the solution. These are formed both at the pipette electrode as well as at the reference electrode. During experiments, these potentials remained constant and were compensated for prior to recording. The second junction potential is a liquid-liquid junction potential, which forms at the tip of the pipette when the pipette solution comes in contact with the bathing solution, with a different ionic composition. This junction potential disappeared when a seal was formed between the pipette and the cell leading to an error in the measurement of membrane potential, which was not corrected in my experiments.

Although ideally the voltage applied would be reached immediately as the current is injected into the cell, this practically is not the case. The injected current has to first charge the membrane and there is a slight delay while the membrane voltage reaches the applied voltage exponentially with a time constant () as described before. The recording of fast currents is affected by this delay. The currents recorded in this study were relatively slow to activate, so it is likely that the membrane was effectively clamped before there was significant current activated.

3.5.2 Myography contractile studies:

Rings of PA ( 5 mm in length) were mounted on the pins of a myograph chamber (Danish Myo Technology, Model 610M, 620M) for isometric tension studies. The vessels were bathed in PSS and aerated at 37ºC. A basal tension of 5 mN was applied and the vessels were left to equilibrate for 45 minutes. The vessels were washed with fresh PSS every 15 minutes during equilibration. The response to various drugs was measured as a change in tension recorded by the myograph. Intracept-chart software V.4.9.0 (developed by Dr. John Dempster (University of Strathclyde)) was used with a National Instruments analogue to digital interface (model AT-M10-16L-9) to record the tension (for tissue preparation please refer to chapter 2).

3.5.2.1 Protocols used to study the effects of ZnPy:

Once the vessels were equilibrated they were challenged with 50 mM KCl solution. This was done in order to sensitize the tissue and was repeated three times in order

159 to test the reproducibility of the response in these vessels. The vessels were then washed with PSS until the tension returned to base-line. The α-adrenoceptor agonist PE (1 µM) was used to preconstrict the vessels and produced a steady response after 20-45 min. The vessels were subsequently exposed to increasing concentrations of ZnPy (100 nM - 100 µM) applied cumulatively with a 15-20 min interval between applications. NaPy and ZnCl2 were investigated in the same manner to determine their ability to cause relaxation. This will highlight the importance of the Zn2+ and/or pyrithione moieties for the ZnPy-induced relaxation.

3.5.2.2 Protocols used to study the effects of various blockers on the ZnPy induced relaxation:

TEA (100 mM), paxilline (1 µM), iberiotoxin (50 nM), 4-AP(1 mM), glibenclamide (10 µM) and XE991 (1 nM,5 nM,10 nM and 100 nM) were each tested for their ability to inhibit the responses to ZnPy. In one approach the blockers were applied before preconstricting with PE to see if they had any effect on the resting tone. In the second approach they were applied after preconstriction with PE but before ZnPy was applied. The responses to ZnPy (100 nM -100 µM) were measured as above.

3.5.2.3 Protocols used to study the mechanism of the ZnPy-induced relaxation:

To determine if the effects caused by ZnPy were mediated by voltage-dependent or voltage-independent Ca2+ influx, its ability to dilate PA constricted using PE (1 µM), BayK-8644 (1 µM) (as an agonist of Ca2+ channels) and the calcium ionophore ionomycin (3 µM) was compared. In these experiments the vessels were challenged with 50 mM KCl three times, then preconstricted and exposed to 5 µM and 10 µM concentrations of ZnPy and the change in tension was measured. In the ionomycin experiments, the endothelium was removed by rubbing the inside of the vessel with a nail in a circular motion. The vessels were also treated with NG-nitro-L-arginine methyl ester (L-NAME) (300 µM) to block the effects of endothelial factors. The removal of the endothelium is important because ionomycin also acts as an endothelium-dependant vasodilator.

3.5.2.4 Drugs:

The drugs used in this study are summarized in Table 3.2 below.

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Table 3.2: Drugs used to study the effects of ZnPy on the pulmonary circulation:

Drug Name Source Form Stock Solution

ZnPy Sigma-Aldrich Powder 100 mM in DMSO

TEA Sigma-Aldrich Powder Added directly to PSS

Paxilline Tocris Powder 10 mM in H2O

Iberiotoxin Tocris Powder 100 µM in H2O

Glibenclamide Sigma-Aldrich Powder 10 mM in DMSO

Phenylephrine Sigma-Aldrich Powder 10 mM in H2O

XE991 Tocris Powder 10 mM Stock in H2O

4-AP Sigma-Aldrich Powder 100 mM in HEPES (10 mM)

(pH 7.3)

NaPy Sigma-Aldrich Powder 100 mM in DMSO

ZnCl2 Fluka, UK Powder 100 mM in H2O

BayK-8644 Tocris Powder 100 mM in DMSO

Ionomycin Sigma-Aldrich Powder 10 mM in DMSO

DMSO was purchased from Sigma-Aldrich

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3.6 Results:

3.6.1 Patch clamp electrophysiology:

3.6.1.1 Properties of rat PASMCs:

Isolated rat PASMCs were found to have a resting membrane potential of -44 ± 3 mV (n=14). The mean membrane capacitance was 18.1 ± 0.8 pF (n=116) and the mean input resistance was 2 ± 0.1 GΩ (n=116).

3.6.1.2 The effect of ZnPy on the membrane potential of PASMCs:

A 10 µM concentration of ZnPy was found to produce a vasodilatory response of 74% (see myography experiments) and therefore this concentration was used in patch clamp experiments. The effects of ZnPy on the resting membrane potential were recorded in current clamp mode with current set at zero. After obtaining a stable recording for 1-2 min, ZnPy (10 µM) resulted in a significant hyperpolarization of 11 ± 1 mV (n=12, p<0.001) in a matter of 1-2 min (using the slow perfusion system to apply the drug) (Fig. 3.6A). The results are shown as a histogram in Fig. 3.6B. In two of the cells tested the drug failed to show any hyperpolarization.

Since the pipette solution lacked ATP, it is possible that the depletion of the cellular

ATP led to the activation of KATP channels in the cells. KATP channels can contribute to Em of PASMCs (Clapp et al., 1992) and can be activated by K+ channel opener drugs

(Clapp et al., 1993). Therefore, it was necessary to determine whether KATP channels would contribute to the observed hyperpolarization. This was carried out using glibenclamide (10 µM) as a KATP blocker. In the presence of glibenclamide, the resting membrane potential was found to be -30 ± 1.6 mV (n=47). Therefore at the concentration used, glibenclamide caused a significant depolarization of PASMCs (p<0.001). The addition of 10 µM ZnPy produced hyperpolarization as before (Fig. 3.7A). The mean hyperpolarization was 12 ± 1.5 mV (n=47, p<0.001) (Fig. 3.7B). This was not different from the response measured in the absence of glibenclamide. In both the absence and presence of glibenclamide, the effects of ZnPy were observed within a few seconds and the membrane potential recovered completely after washing within 1-2 min (using a multi-barrel pipette to apply the drug). In four cells out of the 47 tested in the presence of glibenclamide, ZnPy had no effect on the membrane potential and in one cell it produced a small depolarizing effect of 3.5 mV. Having

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Figure 3.6: ZnPy-induced hyperpolarization of rat PASMCs. A) Typical trace of membrane potential recorded in current-clamp mode. After obtaining a stable recording for 1 min, ZnPy (10 µM) was applied and remained in the recording chamber until the end of the experiment. B) Histogram illustrating the mean membrane potential of PASMCs in the absence (control) and presence of 10 µM ZnPy (n=12, *** p<0.001, paired comparison)

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Figure 3.7: Effect of glibenclamide (10 µM) on The ZnPy-induced hyperpolarization of rat PASMCs. A) Typical trace of membrane potential recorded in current-clamp mode in the presence of glibenclamide (10 µM). After obtaining a stable recording, ZnPy (10 µM) was applied and produced a steady response with complete recovery on washout. B) Histogram illustrating the mean membrane potential of PASMCs in the absence (control) and presence of 10 µM ZnPy recorded with glibenclamide (10 µM) in the bathing solution (n=21, *** p<0.001, paired comparison).

164 established that glibenclamide did not affect the response to ZnPy, it was included in the PSS for the remaining experiments.

At a 10 mM concentration, TEA is a blocker of BKCa channels as well as some Kv channels, including TEA-sensitive Kv7 channels (Hadley et al., 2000). It was therefore used to investigate the role of BKCa channels and Kv7 channels in the ZnPy- induced hyperpolarization on PASMCs. The effects of TEA (10 mM) were first investigated by applying it to cells in the absence of ZnPy. When the membrane potential recorded just before the application of TEA (-25 ± 5 mV, n=10) was compared with that in the presence of TEA (21 ± 6 mV), the difference was not significant implying that TEA had no effect. When applied in the presence of 10 mM TEA, ZnPy (10 µM) did not cause any statistically significant hyperpolarization (n= 10) (Fig. 3.8). Therefore TEA inhibited the ZnPy-induced hyperpolarization. In two out of the ten cells studied, a very slight depolarizing effect of ZnPy was observed in the presence of TEA.

It was important to determine whether the inhibition of the ZnPy-induced hyperpolarization by TEA was due to a block of BKCa or Kv channels. Since TEA is known to block both of these channels, the BKCa selective blocker paxilline (1 µM) was used to assess the involvement of BKCa channels. The effects of paxilline were first investigated by applying it to cells in the absence of ZnPy. When the membrane potential recorded just before the application of paxilline (-34 ± 3.6 mV, n=11) was compared with that in the presence of paxilline (-34 ± 3.8 mV), there was no significant difference. When applied in the presence of paxilline, ZnPy (10 μM) did not cause any statistically significant hyperpolarization (n= 11) (Fig. 3.9).

Iberiotoxin, which a highly selective blocker of BKCa channels, was also used to establish the role of these channels in the ZnPy-induced hyperpolarization. The effects of iberiotoxin (50 nM) were first investigated by applying it to cells in the absence of ZnPy. When the membrane potential recorded just before the application of iberiotoxin was compared with that in the presence of iberiotoxin, the difference was not significant (n=5) (Fig. 3.10). When applied in the presence of iberiotoxin, ZnPy (10 μM) caused a significant hyperpolarization of 14.1 ± 3.3 mV (n= 5, p=0.01). Therefore iberiotoxin did not inhibit the ZnPy-induced hyperpolarization.

The Kv7 blocker XE991 (10 µM) was used to assess the role of Kv7 channels on the ZnPy-induced hyperpolarization. Its effects were first investigated by applying it to

165 cells in the absence of ZnPy. XE991 caused a significant depolarization of 6 ± 1.5 mV (n=10, p<0.01) from -33 ± 3.4 mV to -27 ± 3.6 mV. When applied in the presence of XE991, ZnPy (10 μM) did not cause any statistically significant hyperpolarization (n= 10) (Fig. 3.11).

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Figure 3.8 Effect of TEA (10 mM) on The ZnPy-induced hyperpolarization of rat PASMCs. Typical trace of membrane potential recorded in current-clamp mode. After obtaining a stable recording, ZnPy (10 µM) was applied and produced a steady response with complete recovery on washout. TEA (10 mM) was then applied and the effects of ZnPy were tested in its presence. B) Histogram illustrating the mean membrane potential of PASMCs in the absence (control) and presence of ZnPy (10 µM) before and after the application of TEA (10 mM) (n=10, ** p<0.01, paired comparison).

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Figure 3.9: The effect of paxilline (1 µM) on the ZnPy-induced hyperpolarization of PASMCs. A)Typical trace of membrane potential recorded in current-clamp mode. After obtaining a stable recording, ZnPy (10 µM) was applied and produced a steady response with complete recovery on washout. Paxilline (1 µM) was then applied and the effects of ZnPy were tested in its presence. B) Histogram illustrating the mean membrane potential of PASMCs in the absence (control) and presence of ZnPy (10 µM) before and after the application of paxilline (1 µM) (n=11, ***p<0.001, paired comparison).

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Figure 3.10: The effect of iberiotoxin (50 nM) on the ZnPy-induced hyperpolarization of PASMCs. A)Typical trace of membrane potential recorded in current-clamp mode. After obtaining a stable recording, ZnPy (10 µM) was applied and produced a steady response with complete recovery on washout. Iberiotoxin (50 nM) was then applied and the effects of ZnPy were tested in its presence. B) Histogram illustrating the mean membrane potential of PASMCs in the absence (control) and presence of ZnPy (10 µM) before and after the application of iberiotoxin (50 nM). (n=11, *p<0.05, **p<0.01, paired comparison)

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Figure 3.11: The effect of XE991 (10 µM) on the ZnPy-induced hyperpolarization of PASMCs. A)Typical trace of membrane potential recorded in current-clamp mode. After obtaining a stable recording, ZnPy (10 µM) was applied and produced a steady response with complete recovery on washout. XE991 (10 µM) was then applied and the effects of ZnPy were tested in its presence. B) Histogram illustrating the mean membrane potential of PASMCs in the absence (control) and presence of ZnPy (10 µM) before and after the application of XE991 (10 µM). (n=10, **p<0.01, paired comparison)

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3.6.1.3 Effects of ZnPy on the voltage-activated K+ current in PASMCs:

In order to investigate the effects of ZnPy on the voltage activated K+ current, a standard voltage protocol was used. This protocol consisted of a 300 ms voltage step from -80 to +40 mV, which was applied to the cell at 5 s intervals. The current reached maximum in <50 ms of the 300 ms sweep (Fig. 3.12A). The control current amplitude and the effects of ZnPy (10 µM) were measured both in the absence and in the presence of TEA (10 mM). It was found that in the absence of TEA, ZnPy caused an average increase in the current of 344%, the current amplitude increasing significantly from 532 ± 84 pA to 2000 ± 331 pA (n= 33, p <0.001). Figure 3.12B shows the ZnPy-induced current obtained by digitally subtracting the current recorded under control conditions from the current in the presence of ZnPy. The maximum effect was reached within 35-45 s of applying ZnPy. The time constant for activation of the ZnPy-induced currents at +40 mV was 39 ± 0.5 ms (n=8). The effect was also reproducible and the reapplication of ZnPy 4 min after recovery from the first application, produced similar results. The effect was also readily reversible and the drug effect washed off in a matter of 20-25 s. The time course for the ZnPy- induced current is shown in figure 3.13.

TEA was found to decrease the voltage activated K+ current by an average of 104 ± 34 pA, from 651 ± 196 pA to 547 ± 183 pA (n=6). This difference was statistically significant (p<0.05), although it is not apparent in the trace in figure 3.12A due to the size of the current scale. It was found that in the presence of TEA, ZnPy caused an average increase in the current of only 1%, from 547 ± 183 pA to 617 ± 171 pA (n=6). The effect of ZnPy in the presence of TEA was not significant.

Paxilline (1 µM) had no significant effect on the voltage activated K+ current (n=10) (Fig. 3.14A). In the presence of paxilline, it was found that ZnPy caused an average decrease in the current of only 1%, from 479 ± 136 pA to 393 ± 96 pA (n=10). The effect of ZnPy in the presence of paxilline was not significant. The time-course is shown in figure 3.14B.

Iberiotoxin was found to decrease the voltage activated K+ current by an average of 57 ± 15pA, from 668 ± 164 pA to 611 ± 160 pA (n=6) (Fig. 3.15). The effect of iberiotoxin on the delayed rectifier current was statistically significant (n=6, p<0.05). In the

171 presence of iberiotoxin, it was found that ZnPy caused a significant increase in the current by 241%, from 611 ± 160 pA to 1470 ± 254 pA (n=6, p<0.05).

XE991 was found to decrease the voltage activated K+ current by an average of 127 ± 48 pA, from 707 ± 116 pA to 587 ± 87 pA (n=11) (Fig.3.16). This difference was statistically significant (p<0.05). In the presence of XE991, it was found that ZnPy caused a significant increase in the current by 345%, from 587 ± 87 pA to 2028 ± 343 pA (n=11, p<0.01).

In order to examine the voltage-dependence of this effect of ZnPy, a series of voltage steps were applied using the following protocol: 12 voltage steps were applied in 10 mV increments, from -70 mV to +50 mV, at 5 s intervals (Fig.3.17A). A family of currents recorded using this protocol, in control conditions and in the presence of ZnPy, are shown in figure 3.17A. The effects of ZnPy on the current density, measured in pA/pF of the cell membrane at each voltage were calculated. This normalization was done in order to account for the differences in the sizes of the cells and to minimize the variability in the current amplitude. It was found that ZnPy caused an increase in the current density at voltages above -20 mV and this effect became more profound as more positive potentials were reached (Fig. 3.17B).

3.6.1.4 Effects of ZnPy on the residual K+ current at 0 mV in PASMCs:

Since Em has been proposed to be mediated by a K+ current that fails to inactivate at

0 mV (IKN), the effects of ZnPy were tested against the residual current after clamping the cell at 0 mV for ≥5 min. ZnPy (10 µM) was tested both on its own and in the presence of drugs that block various ion channels. Clamping the cells at 0 mV for ≥ 5 min inhibited 76 ± 2 % (n=22) of the initial outward current. This was due to the inactivation of voltage-gated channels. The average residual current in the presence of 10 µM glibenclamide was found to be 41 ± 6 pA (n=31) at 0 mV.

ZnPy (10 µM) sometimes increased the current recorded at 0 mV in 1-2 min and the current recovered completely and immediately after washing (Fig. 3.18A). ZnPy produced an average 6-fold increase in the current from 41 ± 6 pA to 261 ± 79 pA (n=31, p<0.001). This effect was however, variable with many cells showing no effect at 0 mV. The effects of TEA on the ZnPy-induced currents were investigated to assess the potential roles of BKCa and Kv7 channels. TEA (10 mM) did not produce any significant change in the current recorded at 0 mV, which was 85 ± 25 pA (n=5) both

172

Figure 3.12: The effects of ZnPy on the currents induced by a voltage step from -80 to +40 mV. A) Typical trace showing the voltage-activated K+ current before and during the application of ZnPy (control) and in the absence and presence of TEA (10 mM). B) Trace showing the net increase in current caused by ZnPy (10 µM). This was obtained by digitally subtracting the ZnPy current and the control current. The baseline is shown as a dotted line.

173

Figure 3.13: Time-course of the ZnPy effect on the voltage activated K+ current induced by a voltage step from -80 to +40 mV. Amplitude of currents recorded at 5 s intervals before and during the application of ZnPy (control) and in the absence and presence of TEA (10 mM). The voltage protocol was as in Fig.3.12. Each point represents the maximum amplitude of the current recorded at +40 mV.

174

Figure 3.14: The effects of paxilline (1 µM) on the ZnPy-induced currents. A) Typical trace showing the voltage-activated K+ current before and during the application of ZnPy (control) and in the absence and presence of paxilline (1 µM). The baseline is shown as a dotted line. B) Amplitude of currents recorded at 5 s intervals before and during the application of ZnPy (control) and in the absence and presence of paxilline (1 µM). Each point represents the maximum amplitude of the current recorded at +40 mV.

175

Figure 3.15: The effects of iberiotoxin (50 nM) on the ZnPy- induced currents A) Typical trace showing the voltage-activated K+ current before and during the application of ZnPy (control) and in the absence and presence of iberiotoxin (50 nM). The baseline is shown as a dotted line.

Figure 3.16: The effects of XE991 (10 µM) on the ZnPy-induced currents A) Typical trace showing the voltage-activated K+ current before and during the application of ZnPy (control) and in the absence and presence of XE991 (10 µM). The baseline is shown as a dotted line.

176

Figure 3.17: The voltage-dependence of ZnPy effects on the voltage activated K+ current in PASMCs. A) The inset shows the voltage protocol from -70 to +50 mV. The figure shows traces of the delayed rectifier K+ currents before (left panel) and after (right panel) the application of 10 µM ZnPy. B) Current densities as a function of voltage before and after the application of ZnPy (10 µM). Data are expressed as mean ± s.e.m of current density (n=3)(*p<0.05,**p<0.01). Two-way ANOVA followed by Bonferonni post test.

177 in its absence and its presence. In the presence of TEA however, ZnPy failed to produce any significant increase in the current recorded at 0 mV, which remained at 83 ± 24 pA in the presence of ZnPy (n=5).

Paxilline (1 μM) was used as a more selective blocker of BKCa than TEA. It also did not produce any significant change in the current recorded at 0 mV, with the current being 20 ± 6 pA in its absence and 18 ± 6 pA in its presence (n=7). In the presence of paxilline, ZnPy failed to produce any significant increase in the current recorded at 0 mV, which remained at 17 ± 7 pA after the application of ZnPy (n=7).

XE991 was chosen as a blocker of Kv7 channels to determine their role if any on the ZnPy induced current. XE991 (10 μM) did not produce any significant change in the current recorded at 0 mV, with the current being 29.5 ± 6 pA in its absence and 22.5 ± 7 pA in its presence (n=4). In the presence of XE991, the currents were 107 ± 45 pA after applying ZnPy (n=4). The effects of ZnPy on the residual current at 0 mV in the presence of TEA, paxilline and XE991 are shown in Fig. 3.18B. There appears to be no significant difference in the effects of ZnPy under control conditions and in the presence of the blockers. It is possible however, that the low n numbers and the variability in the responses of the cells to ZnPy under these conditions may have had an effect on the results of the statistical analysis.

The voltage dependence of the residual current at 0 mV was investigated by stepping the membrane potential to from 0 mV to +60 mV, followed immediately by a ramp to - 100 mV. Each sweep lasted for 1.5 s and the time between each sweep was 1 minute. This protocol has been previously used to investigate IKN (Evans et al., 1996). ZnPy (10 µM) was applied to cells both in the absence and presence of TEA (10 mM) and the effect on the current generated were studied. It was found that the current amplitude at positive potentials increased with the application of ZnPy, but in the particular cell shown in Fig. 3.19, the effect was only apparent at positive potentials. This is an example of a cell in which ZnPy had no effect on the current at 0 mV, however it did cause an increase in current at more positive potentials. In the presence of TEA the current was not increased by ZnPy. The effect of ZnPy was readily reversible and the cell recovered completely after washing the drug. It was not possible to calculate statistical significance, as this experiment was only performed on a single PASMC.

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Figure 3.18: Effect of ZnPy on the residual current recorded at 0 mV. A) Typical trace showing the current remaining after clamping the cell at 0 mV for 5 min, followed by the application of ZnPy (10 µM) on its own and in the presence of TEA (10 mM). B) Histogram showing the change in current caused by ZnPy (10 µM) in the absence and presence of TEA, paxilline and XE991 (n=4-7).

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Figure 3.19: Effect of ZnPy on the current recorded during voltage ramps from 0 mV. Trace showing the currents recorded when the cell was stepped to +60 mV, followed by a ramp to -100 mV under control conditions and in the presence of ZnPy, TEA and ZnPy+TEA.

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3.6.2 Myography contractile studies:

3.6.2.1 ZnPy-induced vasodilation:

The ability of ZnPy to relax pulmonary arteries was assessed by small-vessel myography. In these experiments, the vessels were preconstricted using PE (1 µM) which produced a steady contraction within 20-40 min. ZnPy was then applied in increasing concentrations with dilation first detected at 1 µM concentrations. ZnPy immediately relaxed the pulmonary artery in a concentration-dependant manner (Fig. 3.20A). 10 µM concentrations of ZnPy produced a 74% relaxation relative to PE. The addition of 100 µM ZnPy produced a further relaxation that was always followed by an irreversible constriction. Figure 3.20B shows the concentration-response curve obtained from the mean data from 8 PA vessels. The pEC50 value for ZnPy was -5.5 ± 0.1 (n=8).

In order to determine whether ZnPy as a complex was responsible for producing the observed dilation, the ability of NaPy and ZnCl2 to dilate preconstricted pulmonary arteries was investigated. These compounds were chosen because they contain Zn2+ and pyrithione moeities separately and would address the question whether both moeities are needed in concert to produce the observed dilation. It has been suggested in the literature that both moieties should be present in order to cause potentiation of Kv7 channels (Xiong et al., 2007). In these experiments, vessels were preconstricted with PE (1 µM) and NaPy was applied cumulatively at increasing concentrations. NaPy had inconsistent effects on the vessels as reflected in Fig.3.21A. NaPy in some instances produced a dilation followed by a constriction, in some cases a full dilation or no dilation at all. The concentration-response curve for

NaPy is shown in Fig.3.21B and the pEC50 value for NaPy was- 5.7 ± 0.4 (n=7). ZnCl2 was tested in a similar manner, where it was applied at increasing concentrations after preconstricting with PE (Fig. 3.22A). ZnCl2 had no relaxant effect on pulmonary arteries. The concentration-response curve for ZnCl2 is shown in Fig. 3.22B.

3.6.2.2 Mechanism of ZnPy-induced relaxation:

If the dilation caused by ZnPy was due to activation of Kv7 channels, this would in turn give rise to an inhibition of voltage-gated Ca2+ influx. The mechanism of ZnPy

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Figure 3.20: ZnPy provokes concentration-dependent relaxation of rat pulmonary arteries. A) Raw trace showing the change in tension caused by the addition of PE (1 µM) followed by ZnPy (100 nM - 100 µM) in pulmonary arteries. B) Concentration-response curve for ZnPy plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 8 experiments.

182

Figure 3.21: The effects of NaPy on rat intrapulmonary artery. A) Raw trace showing the change in tension caused by the addition of PE (1 µM) followed by NaPy (100 nM - 100 µM) in pulmonary arteries. B) Concentration-response curve for NaPy plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 7 experiments.

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Figure 3.22: The effect of ZnCl2 on rat intrapulmonary artery. A) Raw trace showing the change in tension caused by the addition of PE (1 µM) followed by ZnCl2 (100 nM - 100 µM) in pulmonary arteries. B) Concentration-response curve for ZnCl2 plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 4 experiments.

184 relaxation was tested by investigating its ability to dilate vessels constricted using agents that constrict PA by different mechanisms. This will help to determine whether ZnPy is having a direct effect on Ca2+ influx or is acting indirectly via hyperpolarization.

PE is an α-adrenoceptor agonist that gives rise to constriction by IP3 calcium release from the intracellular Ca2+ stores in addition to increasing Ca2+ influx. After preconstricting the vessel with 1 µM PE, ZnPy was applied at 5 µM and 10 µM concentrations and was found to cause an immediate concentration-dependent relaxation (Fig. 3.23A) with almost a full relaxation observed at 10 µM.

BayK-8644 is a calcium channel agonist that directly increases intracellular Ca2+ concentrations giving rise to vasoconstriction. The addition of 5 µM and 10 µM concentrations of ZnPy to vessels contracted with BayK-8644 resulted an immediate concentration-dependent relaxation of PA (Fig. 3.23B) with almost a full relaxation observed at 10 µM.

Ionomycin is a Ca2+ ionophore that increases Ca2+ concentrations by a voltage independent mechanism. It also acts as an endothelium-dependant vasodilator and therefore the endothelium was removed and vessels were treated with L-NAME in order to block any endothelial effects. The vessels were then preconstricted using ionomycin and the subsequent application of 5 µM ZnPy produced no relaxation, but rather it further increased tension (Fig. 3.23C). When 10 µM ZnPy was applied it eventually caused a dilation of the PA, however the onset was delayed with dilation first noted 20-30 min after applying the drug. These vessels also responded much more slowly to ZnPy as is apparent in the time scale in Fig. 3.23C. The effects of ZnPy on vessels treated with PE, BayK-8644 and ionomycin are shown in Fig. 3.24.

3.6.2.3 The contribution of K+ channels to the ZnPy-induced dilation:

The experiments investigating the mechanism of the ZnPy-dilation support the hypothesis that ZnPy is at least partially producing its dilatory response by inhibiting voltage-gated calcium influx. This merits the investigation of the contribution of different K+ channels to the ZnPy-induced dilation. Therefore, the ability of ZnPy to cause dilation in the presence of various K+ channel blockers was investigated. Two approaches were taken, in the first the blocker was applied before preconstricting the vessel with PE, in order to determine the effect of the blockers on the resting tone of

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Figure 3.23: The effects of ZnPy after preconstriction by different mechanisms. Raw traces showing the change in tension caused by the addition of PE (1 µM) (A), Bay-K 8644 (1 µM) (B) or ionomycin (3 µM)(C) followed by ZnPy (5 µM and 10 µM) in pulmonary arteries.

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Figure 3.24: Effect of preconstricting agents on the response of PA to ZnPy. The bars show the ZnPy-induced relaxation of rat intrapulmonary arteries relative to PE (1 µM), Bay-K8644 (1 µM), and ionomycin (3 µM). The results are shown as mean ± S.E.M of 6 experiments for each agent.

187 the vessels. Alternatively, the blocker was applied after the vessel had been preconstricted with PE, but before ZnPy application. This was undertaken to assess the ability of the blockers to influence the tone of preconstricted vessels as well as the response to ZnPy. TEA was used to assess the involvement of BKCa channels and Kv7 channels in the ZnPy induced dilation. 10 mM TEA was applied to the vessels prior to preconstriction with PE and was found to have no effect on the basal tone (Fig. 3.25A). Its ability to affect the response of the vessels to ZnPy was also tested by using the approaches described above. After applying TEA for 10-15 min then preconstricting with PE, ZnPy was added in increasing concentrations. It was found that ZnPy produced a concentration-dependent relaxation of the vessels with relaxation first noted at 1 µM concentrations (Fig. 3.25A). When TEA was applied after preconstricting the vessels with PE, it sometimes produced an increase in tension (Fig. 3.25B) and when this occurred the resulting tension was considered the baseline for measuring ZnPy effects. When TEA was applied after preconstriction with PE, ZnPy retained its ability to produce a concentration-dependent relaxation of the vessels. The concentration-response curves are shown in Fig. 3.25C. The pEC50 for ZnPy in the presence of TEA applied prior to preconstriction was -5.4 ± 0.2 (n=7).

When TEA was applied after preconstricting the vessel, the pEC50 for ZnPy was -5.4 ± 0.1 (n=5).

The involvement of BKCa channels in the ZnPy-mediated dilation was assessed using the two BKCa channel blockers paxilline and iberiotoxin. These blockers were tested in a similar manner to TEA. Paxilline (1 µM) was applied to the vessel prior to preconstriction and was found to cause no change in the resting tone. The vessel was then preconstricted using PE and increasing concentrations of ZnPy were applied. ZnPy produced a concentration-dependent relaxation of the vessel with relaxation first detected at 100 nM ZnPy. 100 µM ZnPy produced a dilation that was followed immediately by a constriction. Unlike TEA, when paxilline was applied after preconstriction it did not produce any change in the tone of the vessel (Fig 3.26B) and the vessel was readily relaxed with increasing concentrations of ZnPy. The concentration-response curve for ZnPy in the presence of paxilline is shown in Figure

3.26C. The pEC50 for ZnPy in the presence of paxilline applied prior to preconstriction was -5.4 ± 0.2 (n=6). When the blocker was applied after preconstricting the vessel, the pEC50 for ZnPy was -5.2 ± 0.2 µM (n=5). Iberiotoxin being the more selective blocker of BKCa channels allowed the verification of the

188 involvement of BKCa channels to a higher degree of confidence. Iberiotoxin applied before preconstriction had no effect on resting tone (Fig.3.27A). The subsequent preconstriction with PE and the application of increasing concentrations of ZnPy led to the full relaxation of the vessel in a concentration-dependent manner. Similar to TEA however, when iberiotoxin was applied after preconstriction with PE a slight increase in tension was seen in some vessels (Fig. 3.27B) and this resulting tension was considered baseline for the ZnPy-induced dilation. The subsequent application of ZnPy gave rise to a concentration-dependent relaxation with relaxation first detected at 1 µM. The concentration-response curve for ZnPy in the presence of iberiotoxin is shown in Fig. 3.27C. ZnPy had a pEC50 of -5.4 ± 0.1 (n=5) in the presence of iberiotoxin applied before PE, and a pEC50 of -5.3 ± 0.1 (n=4) when iberiotoxin was applied after preconstriction.

4-Aminopyridine is a blocker of voltage-gated potassium channels with a different selectivity profile than TEA. This drug was used in conjunction with ZnPy to assess the involvement of Kv channels in mediating the ZnPy-induced dilation. 4-AP was applied to the vessel prior to preconstriction with PE and had no effect on the resting tone (Fig. 3.28A). Subsequently the vessel was preconstricted with PE and the effects of ZnPy were tested. ZnPy was found to produce a concentration-dependant relaxation of the vessels similar to above. When 4-AP was applied to preconstricted vessels it always produced an immediate dip in the tension which recovered quickly within 2-5 min. When the tension recovered ZnPy was applied in increasing concentrations and was found to produce a concentration-dependent relaxation with relaxation first noted at 1 µM concentrations. The concentration-response curves for

ZnPy in the presence of 4-AP are shown in Fig. 3.28B. The pEC50 for ZnPy was -5.7 ± 0.1 (n=5) when 4-AP was applied before PE, and -5.4 ± 0.2 (n=5) when 4-AP was applied after preconstriction.

Glibenclamide was used to test the involvement of KATP channels in the vasodilatory effect caused by ZnPy. 10 µM glibenclamide was applied to the vessel and had no effect on the resting tone (Fig. 3.29A). Subsequently the vessel was preconstricted with PE and increasing concentrations of ZnPy were applied cumulatively. ZnPy produced a concentration-dependent relaxation of the artery with relaxation first noted at 1 µM concentrations and a pEC50 value of -5.2 ± 0.1 (n=6). The concentration-response curve is shown in figure 3.29B.

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It was demonstrated by Joshi et al. (2006) that the Kv7 blocker XE991 caused a concentration-dependent constriction of PA vessels with constriction readily detected at 10 nM concentrations. In order to investigate the involvement of Kv7 channels in the ZnPy-induced dilation, it was important to choose appropriate concentrations of XE991. In other words, the concentrations used should be high enough to block some Kv7 channels but low enough so as not to completely inhibit them. This ensured that some channels would remain active and therefore amenable to ZnPy allowing us to measure the response. The effects of ZnPy were tested in the presence of XE991 at 1 nM, 5 nM, 10 nM and 100 nM so as to cover a broad spectrum of concentrations that enabled the detection of any shift in the activity of ZnPy. 1 nM, 5 nM, 10 nM and 100 nM concentrations of XE991 were applied to different pulmonary artery vessels prior to preconstriction with PE and had no effect on the resting tone of the vessels. After being exposed to XE991 the vessels were preconstricted using PE and ZnPy was applied in increasing concentrations. In the presence of 1 nM XE991, ZnPy was found to produce a concentration-dependant relaxation first detected at 1 µM concentrations (Fig. 3.30A). In the presence of 5 nM and 10 nM concentrations of XE991, ZnPy produced a concentration-dependent relaxation that was detected at slightly higher concentrations of ZnPy than in the presence of 1nM XE991 (Fig. 3.30B,C). In the presence of 100 nM XE991, ZnPy only produced a dilation at concentrations of 10 µM or higher (Fig. 3.30D). In the example shown in Fig. 3.30D ZnPy was only effective at dilating the vessels at 100 µM. The concentration-response curves for ZnPy under control conditions and in the presence of 100 nM XE991 are shown in figure 3.31. In the presence of 100 nM XE991, the concentration-response curve for ZnPy was shifted to the right. The pEC50 values for ZnPy in the presence of 1 nM, 5 nM and 10 nM were -5.3 ± 0.4 (n=5), -4.9 ± 0.3 (n=4) and -5 ± 0.3 (n=5). In the presence of 100 nM XE991, the pEC50 for ZnPy was -4.8 ± 0.2 (n=6), which was significantly higher than the pEC50 for ZnPy in the absence of the drug.

190

Figure 3.25: ZnPy provokes a concentration-dependent relaxation of pulmonary arteries in the presence of TEA (10 mM). A) Raw trace showing the change in tension caused by the addition of TEA (10 mM) followed by PE (1 µM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. B) Raw trace showing the change in tension caused by the addition of PE (1 µM) followed by TEA (10 mM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. C) Concentration-response curve for the ZnPy-induced relaxation under control conditions and in the presence of TEA applied either before or after preconstriction with PE; plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 5-8 experiments.

191

Figure 3.26: ZnPy provokes a concentration-dependent relaxation of pulmonary arteries in the presence of paxilline (1 µM). A) Raw trace showing the change in tension caused by the addition of paxilline (1 µM) followed by PE (1 µM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. B) Raw trace showing the change in tension caused by the addition of PE (1 µM) followed by paxilline (1 µM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. C) Concentration-response curve for the ZnPy-induced relaxation under control conditions and in the presence of paxilline applied either before or after preconstriction with PE; plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 5-8 experiments.

192

Figure 3.27: ZnPy provokes a concentration-dependent relaxation of pulmonary arteries in the presence of iberiotoxin (50 nM). A) Raw trace showing the change in tension caused by the addition of iberiotoxin (50 nM) followed by PE (1 µM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. B) Raw trace showing the change in tension caused by the addition of PE (1 µM) followed by iberiotoxin (50 nM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. C) Concentration-response curve for the ZnPy-induced relaxation under control conditions and in the presence of iberiotoxin applied either before or after preconstriction with PE; plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 4-8 experiments.

193

Figure 3.28: ZnPy provokes a concentration-dependent relaxation of pulmonary arteries in the presence of 4-AP (1 mM). A) Raw trace showing the change in tension caused by the addition of 4-AP (1 mM) followed by PE (1 µM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. B) Raw trace showing the change in tension caused by the addition of PE (1 µM) followed by 4-AP (1 mM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. C) Concentration-response curve for the ZnPy-induced relaxation under control conditions and in the presence of 4-AP applied either before or after preconstriction with PE; plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 5-8 experiments.

194

Figure 3.29: ZnPy provokes a concentration-dependent relaxation of pulmonary arteries in the presence of glibenclamide (10 µM). A) Raw trace showing the change in tension caused by the addition of glibenclamide (10 µM) followed by PE (1 µM) and ZnPy (100 nM - 100 µM) in pulmonary arteries. B) Concentration-response curve for the ZnPy-induced relaxation under control conditions and in the presence of glibenclamide applied before preconstriction with PE; plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 6-8 experiments.

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Figure 3.30: ZnPy provokes a concentration-dependent relaxation of pulmonary arteries in the presence of XE991. Raw traces showing the change in tension caused by the addition of XE991 (1 nM (A), 5 nM (B), 10 nM (C) or100 nM (D)) followed by PE (1 µM) and ZnPy (100 nM - 100 µM) in pulmonary arteries.

196

Figure 3.31: Concentration-response curve for the ZnPy-induced relaxation in the presence of XE991. Concentration-response curve for the ZnPy-induced relaxation under control conditions and in the presence of XE991 (100 nM) plotted as the % residual constriction measured relative to PE. Results are shown as mean ± S.E.M of 6-8 experiments.

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3.7 Discussion:

3.7.1 The effects of the Kv7 activator ZnPy on the pulmonary circulation:

This part of the study investigated the effects of the Kv7 activator ZnPy on PASMCs using the whole-cell patch clamp technique and its effects on intact pulmonary arteries using small-vessel myography. ZnPy was found to cause hyperpolarization of PASMCs and dilation of pulmonary arteries preconstricted with phenylephrine. The mechanisms by which ZnPy caused hyperpolarization of PASMCs and vasodilation of PA were investigated and found to differ. Although ZnPy consistently hyperpolarized PASMCs and caused vasodilation of pulmonary arteries, the responses to ZnPy in the presence of various blockers were different when studied at the level of isolated cells or at the level of the intact pulmonary artery. The inhibition caused by TEA was clear when PASMCs were studied using the patch clamp technique, however, in contractile studies ZnPy was able to maximally dilate pulmonary artery vessels in the presence of TEA. This suggests that ZnPy may have multiple actions in producing its vasodilatory response and the hyperpolarization may be a small contributor. ZnPy was able to relax vessels preconstricted with the calcium agonist BayK-8644, which may have been brought about by the activation of K+ channels and the inhibition of voltage-gated Ca2+ influx.

3.7.2 Mechanism of ZnPy-induced vasodilation:

The vasodilation caused by ZnPy may not be entirely due to the presence of ZnPy as a complex. This is because NaPy sometimes produced full relaxation of the vessels. There is however a possibility that Zn2+ contamination in the bath may have led to the formation of ZnPy, which in turn caused the dilation. We can however conclude that the effects were not mediated by Zn2+ itself, because ZnCl2 failed to dilate preconstricted vessels. These results suggest that both the Zn2+ and pyrithione moieties are required to effectively produce vasodilatation. It has been shown that both moieties are required for potentiation of Kv7.2 currents in CHO cells expressing Kv7.2 channels (Xiong et al. 2007).

The mechanism by which ZnPy dilated PA was investigated on arteries preconstricted by different stimuli. When tested on endothelium denuded vessels preconstricted with the calcium ionophore ionomycin, ZnPy produced a further constriction at 5 µM.

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The constriction may have been due to additional Ca2+ influx. If ZnPy acted by opening K+ channels then the efflux of K+ would have caused hyperpolarization. This in turn would increase the driving force for the positively charged Ca2+ ions to enter the cell and could have therefore caused further constriction. At higher concentrations, ZnPy caused a dilation of vessels preconstricted with ionomycin, albeit with slow onset, suggesting that perhaps ZnPy acted by a different mechanism at these concentrations. The finding that ZnPy relaxed vessels preconstricted with Bay-K8644 suggests that the vasodilatory effect of ZnPy, is at least in part due to closure of voltage-gated Ca2+ channels. This could be due to a direct inhibition of Ca2+ channels or an indirect effect via activation of K+ channels and hyperpolarization.

Since electrophysiology experiments showed that ZnPy could activate K+ channels and hyperpolarize PASMCs, the ability of ZnPy to dilate PA in the presence of various K+ channel blockers was investigated. This allowed us to determine the contribution of different K+ channels to the observed dilation. The application of TEA, paxilline, iberiotoxin, 4-AP and glibenclamide prior to preconstriction with PE had no effect on the resting tone of the vessels. XE991 had no effect on resting tone when used at concentrations up to 100 nM. This differs from reports by Joshi et al. (2006) where 100 nM XE991 was found to cause constriction of nearly 40% of the response to 50 mM KCl. The reason for this discrepancy is unknown, but means that in the current study 100 nM was a threshold concentration for constriction. When applied after preconstriction, TEA and iberiotoxin sometimes caused a further increase in the constriction elicited by PE. This may be attributed to the blockade of BKCa channels which normally respond to the rise in intracellular [Ca2+] in PASMCs during contraction.

Regardless of the order in which the blockers were applied, they had the same effect on the response to ZnPy. ZnPy retained its ability to dilate PA preconstricted with PE in the presence of TEA, paxilline and iberiotoxin. These findings suggest that BKCa channels are not likely to be mediators of the dilation caused by ZnPy. In addition the involvement of KATP channels may be ruled out because the KATP blocker glibenclamide had no effect on the ZnPy-induced dilation. 4-AP is a K+ channel blocker that has no inhibitory effect on Kv7 channels but blocks several other voltage-gated K+ channels (Kv1.1, Kv1.5, Kv2.1, Kv3.1, Kv1.2, Kv1.3, Kv1.4 and Kv1.6) (Albrecht et al., 1993; Smirnov et al., 1994; Bouchard et al., 1995; Post et al.,

199

1995; Yuan, 1995; Archer et al., 1998). ZnPy retained its ability to dilate PA vessels in the presence of 4-AP supporting a possible role for Kv7 channels in mediating the dilation. XE991 was used to more specifically study the role of Kv7 channels. There was a gradual rightward shift in the concentration response curve for ZnPy in the presence of increasing concentrations of XE991 suggesting that blocking Kv7 channels decreased the ability of ZnPy to dilate the PA vessels. This was confirmed by a significant increase in the pEC50 value of ZnPy in the presence of 100 nM XE991. Higher concentrations of XE991 could not be tested, due to its profound constrictor effect, which precluded the use of PE as the pre-constricting agent. Thus Kv7 channels could be contributing to the dilation caused by ZnPy. ZnPy is however clearly having additional effects and this is supported by its ability to relax vessels treated with 90 mM K+ PSS (Sean Brennan - oral communication). At this concentration of K+ we would not expect to see any dilation if ZnPy was indeed acting solely on K+ channels. The ability of ZnPy to dilate PA at such a high concentration of K+ suggests that the ZnPy-induced dilation was not due to activation of K+ channels. We cannot however rule out a contribution of Kv7 channels because 100 nM XE991 partially inhibited the ZnPy effect.

3.7.3 Mechanism of ZnPy-induced hyperpolarization:

As ZnPy was found to cause dilation of PA vessels, it was important to investigate its effects on freshly isolated PASMCs. In this study, Em of PASMCs was found to be -44

± 3 mV. This is consistent with several previous studies where Em of rat PASMCs was found to be between -45 to -65 mV in similar conditions (Clapp et al., 1992; Smirnov et al., 1994; Osipenko et al., 1997; Patel et al., 1997; Joshi et al., 2009). ZnPy was found to cause a consistent hyperpolarization of PASMCs by 11 ± 1 mV (n=12, p<0.001) at 10 µM concentrations. This hyperpolarization was blocked by TEA, paxilline and XE991, but not by glibenclamide or iberiotoxin.

Glibenclamide caused depolarization of PASMCs, suggesting that KATP channels were open under resting conditions and that they contributed to Em. This is consistent with what has been previously reported with regards to the contribution of KATP channels to the resting membrane potential when the intracellular solution contained no ATP, as in my experiments (Clapp et al., 1992). More importantly, the fact that glibenclamide had no effect on the ZnPy-induced hyperpolarization, implies that KATP channels, although present, were not involved in mediating the actions of ZnPy. The

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BKCa blockers TEA, iberiotoxin and paxilline had no effect on the resting membrane potential where [Ca2+] were low. This correlates with the relatively high threshold for activation of these channels under resting conditions which means they would be closed at potentials more negative than -40 mV (Coetzee et al., 1999). It is also consistent with previous findings in rabbit and rat PASMCs, where the resting membrane potential was found to be TEA insensitive (Archer et al., 1996; Evans et al., 1996).

At a 10 mM concentration, TEA is known to block BKCa channels and some Kv channels including TEA-sensitive Kv7 channels. Among these channels, Kv2.1,

Kv1.2, BKCa, Kv7.1, Kv7.4 and Kv7.5 are all known to be expressed in PASMCs (Sweeney et al., 2000; Joshi et al., 2009). ZnPy failed to produce a hyperpolarization in the presence of TEA, consistent with it acting on at least one of these K+ channels. This is similar to the findings in recombinant Kv7.2 channels where ZnPy effects were also blocked by TEA (Xiong et al., 2007). In contrast, ZnPy retained its ability to hyperpolarize PASMCs in the presence of the BKCa blocker, iberiotoxin. This suggests that BKCa channels were not involved in mediating the ZnPy-induced hyperpolarization. This also implies that the TEA effects on the ZnPy-induced hyperpolarization were not mediated by BKCa channels. Although the hyperpolarization was blocked by paxilline, iberiotoxin is considered the gold- standard blocker of BKCa. As the effects of paxilline on Kv7 channels have not been studied, we cannot rule out that it blocked these channels in PASMCs. The ZnPy- induced hyperpolarization was also abolished in the presence of the Kv7 blocker XE991. This finding provides strong evidence that ZnPy was acting via activation of these channels. Studies of paxilline effects on Kv7 channels would be helpful in clarifying the channels involved.

3.7.4 Effects of ZnPy on K+ currents:

Having established that ZnPy hyperpolarizes PASMCs, its effects on K+ currents that may underlie this effect were investigated. ZnPy was found to enhance the voltage- activated K+ current. In recombinant channels, the time for maximum activation of the ZnPy-induced current in Kv7.1, Kv7.4 and Kv7.5 channels upon depolarization to +50 mV was 300 ms, 675 ms and 375 ms respectively (Xiong et al., 2007). In this study the time for maximal activation of the ZnPy-induced current using a similar protocol was found to most closely resemble the activation of Kv7.1 channels. ZnPy

201 increased the amplitude of the maximum current at voltages of -20 mV or above. This suggests that ZnPy may have acted on channels that are closed at the resting membrane potential. This effect was different from that observed with the Kv7 openers flupirtine and retigabine, neither of which had any effect on the current recorded at 0 mV or above (Joshi et al. 2009). It is known that retigabine does not affect recombinant Kv7 currents at positive potentials (Tatulian et al., 2001; Joshi et al., 2009). This is because Kv7 channels are maximally active between -10 and -20 mV, as reported by several studies (Barhanin et al., 1996; Kubisch et al., 1999; Selyanko et al., 2001). Retigabine and flupirtine shift the voltage dependence of activation to more negative potentials, without a change in maximum conductance (Xiong et al., 2007). Thus the effects of Kv7 openers on PASMCs fits with their differential effects on recombinant channels and the native M-current (Xiong et al., 2007). Unlike retigabine, ZnPy produces its effects predominantly via an increase in the maximum conductance of the channels, as well as a shift of the voltage sensitivity (Xiong et al., 2007). It causes a marked reduction of the deactivation rate and increases open channel probability (Xiong et al., 2007).

In an attempt to dissect the channels mediating the effects of ZnPy on K+ currents, it was re-tested in the presence of different K+ channel blockers. The ZnPy-induced potentiation of the voltage-activated K+ current at +40 mV was inhibited by TEA and paxilline, but not by XE991 or iberiotoxin. This pharmacology is different from that seen when the effects of ZnPy were studied on the resting membrane potential, suggesting that these currents were not responsible for the ZnPy-induced hyperpolarization. When the blockers were first applied on their own, TEA, XE991 and iberiotoxin were found to decrease the voltage-activated K+ current showing that they were all active. TEA and XE991 had no effect on the residual current at 0mV. Paxilline was found to have no effect on the voltage-activated K+ current on its own, which conflicts with the effect of iberiotoxin. It is not clear why that should be but suggests that paxilline did not block BKCa channels in these cells. ZnPy also increased the residual current at 0 mV, although this effect was variable, being seen in only a few cells. Since the threshold for the ZnPy enhancement of Kv was above - 20 mV, it may be that this variable effect reflects variability between cells in the threshold voltage for activation. This current displayed a similar pharmacology to the voltage-activated K+ current in that it was sensitive to TEA and paxilline, but not XE991.

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Since iberiotoxin did not block the ZnPy-induced current, it is unlikely to be mediated by BKCa channels. It is also unlikely to be mediated by a Kv7 channel because XE991 had no inhibitory effect. The current is a TEA and paxilline sensitive current.

3.7.5 ZnPy and Kv7 channels:

The sensitivity of the ZnPy effects to TEA, is consistent with the hypothesis that it activated Kv7 channels in PASMCs, as TEA sensitivity is a characteristic of most channels encoded by KCNQ genes. The KCNQ expression profile in PASMCs showed that only KCNQ1, KCNQ4 and KCNQ5 were expressed, with KCNQ4 being the most predominant (Joshi et al., 2009). Kv7.4 is more sensitive to TEA than Kv7.1 (IC50 (mM) values of 0.3 and 5 respectively) (Hadley et al., 2000). On the other hand, Kv7.5 channels require very high concentrations of TEA to be blocked (IC50 (mM) = 71) (Hadley et al., 2000; Robbins, 2001). At the concentration of TEA used in my experiments (10 mM), we would expect Kv7.1 and Kv7.4 to be completely blocked, while Kv7.5 would remain active. Since the activity of ZnPy was completely abolished in the presence of TEA, it is unlikely that it was acting on homomeric Kv7.5 channels in PASMCs. ZnPy therefore could be producing its effect by acting on Kv7.4 or Kv7.1 channels.

Although ZnPy does activate Kv7.1, this is only the case when it is not in a complex with the β-subunit KCNE1. KCNE expression is highly vessel-specific (Greenwood et al., 2009), and it is unknown whether KCNE1 is expressed in PASMCs. KCNE1 transcripts were found in rat gastric antral smooth muscle layers, but have not been reported in blood vessels (Ohya et al., 2002a). Retigabine also activates IKN and hyperpolarizes Em of PASMCs (Joshi et al., 2009). It does not open Kv7.1 channels (Tatulian et al., 2001) suggesting that Kv7.4 channels are the most likely to contribute to Em. Joshi et al. (2009), provided strong evidence that Kv7.4 is predominately responsible for setting the resting membrane potential in PASMCs and mediates the effect of Kv7 modulators. The effects of TEA on the actions of ZnPy are consistent with TEA inhibiting the effects of other Kv7 openers, such as retigabine, in neuronal tissue (Rundfeldt, 1999), but retigabine did cause a small increase in IKN in PASMCs in the presence of 10 mM TEA (Joshi et al., 2009) suggesting that other Kv7 channels may also contribute.

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In a study by Xiong et al. (2007), ZnPy was found to activate all of the Kv7 channels, apart from Kv7.3. This activation was readily reversible. Additional studies by the same group showed that ZnPy has no effect on the activity of Kv2.1 and actually appeared to decrease the activity of hERG and Kv4.2 channels (Xiong et al. 2007). Although not much is known about the actions of ZnPy on other ion channels, the finding that ZnPy had no effect on Kv2.1 channels is an important one, because

Kv2.1 has been proposed to be a major contributor to the voltage-activated K+ current and resting membrane potential in PASMCs (Patel et al., 1997; Archer et al., 1998). Another channel proposed to be an important candidate in the mediation of the resting membrane potential is Kv1.5 (Archer et al., 1998; Moudgil et al., 2006; Remillard et al., 2007), but it is not blocked by TEA (Coetzee et al., 1999) so is unlikely to mediate the TEA-sensitive effect in this study. These data all support the idea that Kv7 channels are at least partially responsible for mediating the effects of ZnPy in the pulmonary circulation.

3.7.6 Conclusion:

ZnPy served as a tool to help us expand our knowledge of Kv7 channels in the pulmonary circulation. Although ZnPy produced its effects on the pulmonary circulation mainly through mechanisms that do not appear to depend on Kv7 channels, it is nevertheless another drug that is known to open Kv7 channels and hyperpolarizes PASMCs. Other Kv7 openers, including flupirtine, retigabine, meclofenamic acid, mefanamic acid and diclofenac are all Kv7 openers that hyperpolarize PASMCs and dilate pulmonary arteries (Dong et al., 2009; Joshi et al., 2009; Brookefield, 2010). These observations, combined with the depolarization and constriction caused by the Kv7 blockers, linopirdine and XE991, all support the hypothesis that Kv7 channels can modulate the resting membrane potential of PASMCs.

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Chapter 4: Summary and Future Directions

The main aims of this study were divided into two major parts:

1) To determine the role and expression of Kv7 channels in the MCT rat model of PH.

2) To identify the effects and mechanism of action of the newly identified Kv7 activator, ZnPy, on PASMCs and intact PA.

4.1. Kv7 channels in the monocrotaline rat model of pulmonary hypertension:

The role of Kv7 channels in PH has not been previously studied using the MCT rat model of PH. Since PH is a complex condition that is not represented in a single animal model, the MCT model provided a new way to explore the function and therapeutic potential of Kv7 channels in PH.

The main findings of this part of the study were:

1) MCT enhanced the sensitivity of PA to the Kv7 blocker, XE991, and enhanced the efficacy of the Kv7 activators, retigabine and ZnPy.

2) PA vessels from MCT-treated rats exhibited raised intrinsic tone that was maximally abolished by retigabine and nifedipine, indicating an important role for voltage-operated calcium channels.

3) The effect of retigabine on intrinsic tone was mimicked by other drugs that act via hyperpolarization of PASMCs.

4) PA vessels from MCT-treated rats exhibited slower recovery times after being challenged with 50 mM KCl than vessels from control rats, and this was reversed by hyperpolarizing PASMCs using levcromakalim.

5) Depolarization of PASMCs with 15 mM K+ PSS was able to mimic the raised intrinsic tone and enhanced sensitivity of the vessels to XE991.

6) The expression of KCNQ1, KCNQ4 and KCNQ5 was unaltered by MCT.

The enhanced sensitivity to Kv7 blockers and efficacy of the Kv7 activators may be due to depolarization of PASMCs, which occurs during the development of PAH (Suzuki et al., 1982b; Yuan et al., 1998a; Ito et al., 2000). Depolarization could be

205 brought about by decreased K+ conductance or increased Na+ and Cl- conductances. It was initially suggested that depolarization of PASMCs in MCT-induced PH was due to increased Cl- conductance (Suzuki et al., 1982b). This was supported by a recent study which found the activity of the calcium-activated chloride channel TMEM-16A to be increased in the MCT model of PH (Forrest et al., 2012). It has also been suggested that depolarization may be due to reduced Na+/K+ ATPase activity (Suzuki et al., 1982b; Shubat et al., 1990). This electrogenic pump removes three Na+ ions from the cell while transporting 2 K+ ions into the cell. A study of the Na+/K+ ATPase activity in MCT-PA reported that this pump is an early target in MCT-intoxication (Shubat et al., 1990).

Depolarization may have several adverse effects. For example, inhibition of the NCX, which suppressed Ca2+ removal from the cell and may be responsible for the slowed recovery of tension following a stimulus (Ito et al., 2000). Depolarization would affect any electrogenic transport process, for example the Na+/K+ ATPase which would be inhibited (Nakao et al., 1986). The activity of voltage-gated ion channels, such as Kv1.5 and Kv1.2 is enhanced by depolarization (Coetzee et al., 1999), as is that of voltage operated calcium channels (Clapp et al., 1991b), which appear to be responsible for intrinsic tone. These factors together must be considered when studying the effect of membrane potential on vessel tone.

The hypothesis that the different responsiveness of MCT and control PA to Kv7 modulation were due to depolarization needs further study. In order to further elucidate the role of membrane potential it is important to study the effects of Kv7 modulators at the level of the smooth muscle cells using patch-clamp electrophysiology. This technique enables the experimenter to record Em of control and MCT using current clamp or to directly control the membrane potential using voltage-clamp mode. A primary step would be to confirm that membrane potentials were depolarized in MCT PASMCs in comparison to control PASMCs. Next, testing the effects of XE991 on membrane potential and determining if the effects were enhanced in MCT PASMCs as they were in the intact vessels. Current could be injected to vary the membrane potential in control PASMCs and test how MCT affects membrane potential responses to Kv7 modulators.

MCT-induced PAH is thought to begin with damage to the endothelium. Although depolarization with 15 mM K+ PSS mimicked some of the effects of MCT on responses

206 of vessels to Kv7 modulators, it did not mimic the slowed recovery from a contractile stimulus. A possible explanation is that control vessels contained an intact endothelium which could have counteracted the K+ induced depolarization. A recent study showed that when PA were cultured, the PASMCs became depolarized, but that can be prevented by removing the endothelium before culture, suggesting that a damaged endothelium itself may be responsible for membrane depolarization (Manoury et al., 2009). It would be useful to investigate the role of the endothelium by removing it and seeing if that would mimic the effect of MCT.

4.2 The effects of the Kv7 activator ZnPy on the pulmonary circulation:

The effects of ZnPy, identified as an opener of Kv7 channels by Xiong et al. (2007), on the pulmonary circulation were studied because of its unique mode of activation, binding site and selectivity profile. The main findings were:

1) ZnPy (10µM) consistently hyperpolarized PASMCs.

2) The ZnPy-induced hyperpolarization was blocked by TEA, paxilline and XE991 but unaffected by iberiotoxin and glibenclamide.

3) ZnPy (10µM) significantly increased the K+ currents activated at potentials above 0 mV and occasionally increased the non-inactivating current at 0 mV.

4) The effects of ZnPy on the voltage-activated K+ current were inhibited by TEA and paxilline, but unaffected by XE991 and iberiotoxin.

5) ZnPy produced concentration-dependent relaxation of PA vessels pre-constricted using PE or Bay-K8644.

6) The ZnPy-induced relaxation was unaffected by TEA, paxilline, iberiotoxin, glibenclamide and 4-AP, but partially inhibited by low concentrations of XE991.

There is a clear distinction between the pharmacological properties of the currents activated by ZnPy and the ZnPy-induced hyperpolarization, implying that the hyperpolarization was mediated by a current that has not yet been identified electrophysiologically. For the K+ current mediated by ZnPy to cause hyperpolarization, it needs to be activated at voltages near the resting membrane potential. It is important to study the effects of ZnPy at the appropriate potentials,

207 but at the resting membrane potential K+ currents are very small and difficult to resolve. This could be done using high K+ solution to amplify the current at negative potentials. It is important to determine whether or not a change in current could be detected at these voltages, which would ultimately alter the resting membrane potential. Retigabine was found to act over a narrow range of potentials and induced very small currents, which could only be detected between -60 and -30 mV when K+ was raised (Joshi et al., 2009). Although ZnPy activated recombinant channels at negative voltages, we do not know the identity of the Kv7 channels present in PASMCs. Indeed it has yet to be proven that Kv7 channels are present in PASMCs. We need to also consider its potential effects on non K+ channels, although the effects of the blockers suggest that it is acting on a K+ conductance.

Although paxilline and iberiotoxin are both BKCa channel blockers, they had different effects on the actions of ZnPy. The effects of paxilline on Kv7 channels have not been reported and my data would be easier to interpret if it were known whether it potentially blocks Kv7 channels. Thus experiments on recombinant Kv7 channels aimed at establishing whether or not they can be blocked by paxilline would help in the interpretation of my results.

The voltage sensitivity of ZnPy to TEA and XE991, is consistent with the hypothesis that it was activating Kv7 channels in PASMCs. It is safe to rule out the involvement of BKCa channels, because iberiotoxin did not block any of the effects of ZnPy. Despite identifying this possible action of ZnPy on PASMC Kv7 channels, it did not appear to be the mechanism by which ZnPy caused vasodilation. This is because the ZnPy-induced dilation displayed a pharmacologically distinct profile in the intact vessels. It is possible that ZnPy was inhibiting Ca2+ channels and this is consistent with the findings that it fully dilated vessels in the presence of 90 mM K+ PSS and reversed the Bay-K8644-induced constriction. One way to test this hypothesis was to test the ability of ZnPy to inhibit agonist induced constriction in Ca2+ free medium. Separate studies from our lab found ZnPy to have little effect in these conditions implying it inhibited influx rather than Ca2+ release (Sean Brennan - oral communication). Microelectrode studies would help explain whether the effects seen in the intact vessel were associated with hyperpolarization.

The ability of Kv7 modulators to affect other targets such as non-selective cation channels or gap junctions has not been previously investigated. It is possible to test

208 the selectivity of these compounds using molecular biology approaches by using siRNA to knock out the KCNQ genes and subsequently testing the drugs. This will require culturing the cells. It also not known whether endothelial cells express Kv7 channels. However the ability of most Kv7 modulators to influence tone were independent of the endothelium, suggesting that even if Kv7 channels were expressed in endothelial cells they had little or no contribution.

4.3 Concluding remarks:

My results provide new information on the role of Kv7 channels in the pulmonary circulation. I have shown that Kv7 channels remain functional in MCT-induced pulmonary hypertension. Indeed modulators of these channels have an enhanced action, possibly due to depolarized PASMCs. Although Kv7 activators were also able to dilate systemic arteries, some selectivity of action over the pulmonary circulation may be afforded by this enhanced activity in PH. On the other hand, as Kv7 channel activity appears to be reduced in other models of PAH, how well these models reproduce the human condition in this respect will need to be tested.

Kv7 activators are currently being developed and many structural approaches have been taken. ZnPy, provides a template for Kv7 activator development. My studies suggest, however, that its actions on PA were largely independent of Kv7 channel activation. Therefore, it does not appear to be a good starting point for drugs used to treat PAH.

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