2015welismphd.pdf (3.31 MB)
Regulation of L-Type Ca2+ Channels by Nitric Oxide Signalling in Guinea Pig Ventricular Myocytes
thesisposted on 2015-10-01, 15:01 authored by Sardar Mohammed Weli
Nitric oxide (NO) is constitutively generated by cardiac myocytes and has important roles in cardiac function, including modifying L-type Ca2+ currents (ICa,L). The precise nature of this modification remains elusive with NO reported to increase, reduce or have biphasic effects on ICa,L. Here I explored the effects of NO signalling on ICa,L in both active period and resting period myocytes recorded from guinea pig ventricular myocytes using the perforated whole-cell switched voltage-clamp technique to maintain intracellular signalling pathways. Both cGMP-dependent and S-nitrosylation pathways were investigated. Isoprenaline (100 nM) significantly increased peak ICa,L by about two fold. Subsequent application of NO, using the NO donor SNAP, significantly decreased this enhanced ICa,L but had little effect on basal ICa,L. In contrast to these results obtained from active period myocytes, NO did not inhibit isoprenaline enhanced ICa,L in resting period myocytes. In active period myocytes NO inhibition of isoprenaline enhanced ICa,L was maintained in the presence of ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one), a soluble guanylyl cyclase (sGC) inhibitor. Direct activation of sGC or peripheral guanylyl cyclase independently of NO by BAY 60-2770 or ANP respectively, however, gave results similar to those observed with NO; although in some cells, BAY 60-2770 did not reduce isoprenaline enhanced ICa,L. Thus direct activation of sGC mimics the effect of NO, yet inhibiting sGC did not abolish NO reduction of isoprenaline enhanced ICa,L. These results suggest that NO modulates ICa,L through more than one mechanism. To investigate the S-nitrosylation pathway, denitrosylation was inhibited using N6022, a blocker of S-nitrosoglutathione reductase, an enzyme involved in denitrosylation. This treatment, either completely abolished or slowed significantly the rate of development of isoprenaline enhancement of ICa,L in cells exposed previously to NO. In conclusion, NO inhibition of isoprenaline enhanced ICa,L involves at least two signalling pathways; a cGMP-dependent and the S-nitrosylation pathway in active period myocytes.
Supervisor(s)Davies, Noel; Mitcheson, John
Date of award2015-08-21
Author affiliationDepartment of Cell Physiology and Pharmacology
Awarding institutionUniversity of Leicester