Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology and Experimental Therapeutics

Abstract

Cerebral vasospasm, a grave sequel to subarachnoid hemorrhage (SAH), is characterized by prolonged severe constriction of arteries in the base of the brain, including the basilar artery. Spasmogens (serotonin, endothelin and vasopressin), elevated in response to SAH, induce persistent depolarization of the myocytes in the artery wall, leading to continuous influx of calcium (Ca2+) through voltage-sensitive Ca2+ channels (VSCC) resulting in hyperconstriction (spasm). The spasm of the arteries restricts blood flow to the brain, inducing ischemic neurological deficits and consequential high morbidity and mortality. The cellular pathogenesis of cerebral vasospasm is poorly understood and the therapeutic options are limited.

Kv7 (KCNQ) potassium channels are reported to be critical determinants of resting membrane voltage in several excitable cells including neurons and vascular myocytes. Recent findings from our laboratory suggested that arginine8-vasopressin exerts its vasoconstrictor effects on mesenteric arteries via suppression of Kv7 currents. Hence, we hypothesized that suppression of Kv7 currents by spasmogens is a central phenomenon in the pathogenesis of cerebral vasospasm and that Kv7 channel openers would be effective therapeutic agents to treat the vasospasm.

Expression of Kv7 channels evaluated by reverse transcriptase polymerase chain reaction revealed the presence of all five mammalian KCNQ channel subtypes (KCNQ1-5) in freshly isolated rat basilar artery myocytes. Kv currents recorded using the whole-

cell patch-clamp techniques were attributed to Kv7 channel activity based on their voltage dependence of activation, lack of inactivation, enhancement by flupirtine, retigabine (selective Kv7 channel activators), and inhibition by XE991 (a selective Kv7 channel blocker). XE991 depolarized the myocytes indicating that Kv7 channels are principal determinants of resting membrane voltage. Spasmogens suppressed Kv7 currents and depolarized the myocytes. The effects of spasmogens were significantly attenuated in the presence of retigabine. Celecoxib, a clinically used anti-inflammatory drug, not only enhanced Kv7 currents but also inhibited voltage-sensitive Ca2+ currents.

Functional responses to Kv7 channel modulators were studied in intact basilar artery segments using pressure myography. XE991 constricted basilar artery segments indicating that Kv7 channels are important determinants of contractile status. In the presence of XE991, spasmogens did not produce additive constrictor responses, suggesting that suppression of Kv7 currents is the predominant mechanism for spasmogen-induced membrane depolarization and constriction of basilar arteries. Kv7 channel activators dilated basilar artery segments pre-constricted with spasmogens, and were more effective than nimodipine, an L-type Ca2+ channel blocker used as standard care to relieve cerebral vasospasm in patients with SAH. Intraperitoneal administration of Kv7 channel openers, retigabine or celecoxib, significantly attenuated basilar artery spasm in rats with experimentally induced SAH. Both the Kv7 channel openers induced a transient drop in mean arterial pressure (MAP) generally lasting 30-60 min after administration, but mean 24h BP measured using radio-telemetry in normal rats was not altered.

In conclusion, Kv7 channels are expressed in basilar artery myocytes and function to maintain resting membrane voltage in basilar artery myocytes and hence oppose the constriction of the arteries. Suppression of Kv7 currents in myocytes is a mechanism by which spasmogens induce basilar artery constriction. Targeting vascular Kv7 channels using retigabine or celecoxib could provide a novel strategy to relieve basilar artery spasm without inducing hypotension.

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