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Title: Novel Ca2+ signalling pathways in vascular smooth muscle and endothelial cells
Author: Lim, Chloe Siew Suan
ISNI:       0000 0004 5361 8269
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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Novel Ca2+ signalling pathways in both endothelial cells and smooth muscle cells of rat small resistance arteries were investigated using a combination of confocal imaging, isometric tension recordings, and electrophysiology to study freshly isolated arteries and cells. We first examined the hypothesis that hyperpolarization could alter endothelial cell Ca2+ events. Hyperpolarization evoked by direct opening of KATP channels in the smooth muscle with levcromakalim triggered an increase in the frequency of Ca2+ events in the endothelium of rat cremaster arterioles. These Ca2+ events were discrete in nature, requiring subcellular regions of interest to reliably identify them. Opening of KATP channels indirectly through β-adrenoceptor stimulation with isoprenaline, caused a similar increase in the frequency of endothelial cell Ca2+ events in rat mesenteric third order arteries. These events also had a similar, focal profile. Pharmacological investigation suggested that the response to isoprenaline was receptor-mediated, and dependent on Ca2+ influx and opening of KATP channels. The presence of β-adrenoceptors on endothelial cells was confirmed using fluorescently-tagged β-adrenoceptor ligands, which showed punctate labelling in smooth muscle and endothelial cells of rat mesenteric arteries. Freshly isolated endothelial cells also showed Ca2+ increases to isoprenaline, although this was not consistently observed. Following on from the observed endothelial cell Ca2+ response to hyperpolarization, we tested the hypothesized involvement of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels using the channel inhibitor, ZD7288. Pre-treatment with ZD7288 (1 μM) reduced both the endothelial cell Ca2+ response to isoprenaline (in mesenteric arteries) and levcromakalim (in cremaster arterioles). HCN channel subtypes were identified in cremaster arterioles through immunolabelling. We also observed an interesting effect of higher concentrations of ZD7288 to potentially inhibit K+ channels, including endothelial cell KCa channels, since hyperpolarization to isoprenaline, levcromakalim or acetylcholine (ACh) was reduced by 10 μM ZD7288, and relaxation to ACh was partially inhibited. ACh-mediated relaxation was also partially inhibited by the clinically used HCN channel blocker, ivabradine (0.3-30 μM). Finally, we identified an interaction of the Ca2+-releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) with BKCa channels in the smooth muscle. NAADP-mobilised Ca2+ has been reported to interact with ryanodine receptors hence we hypothesized an interaction with BKCa channels via Ca2+ sparks. We found that NAADP-AM relaxed and hyperpolarized rat mesenteric arteries, which was blocked by iberiotoxin (BKCa channel inhibitor) and high extracellular [K+] (45 mM). Furthermore, NAADP increased paxilline-sensitive K+ currents and the frequency and amplitude of spontaneous transient outward currents (STOCs) in freshly isolated vascular smooth muscle cells patched in the whole-cell configuration, further supporting an action at BKCa channels. All together these data identify novel Ca2+ signalling pathways in resistance arteries that are both activated by and promote hyperpolarization, which is a key determinant of vascular tone.
Supervisor: Dora, Kim Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Medical Sciences ; Pharmacology ; nicotinic acid adenine dinucleotide phosphate ; hyperpolarization ; endothelium ; smooth muscle ; calcium