The effect of ablation and acute inhibition of plasma membrane calcium ATPase 4 (PMCA4) with a novel inhibitor on isolated mouse mesenteric resistance arterial contractility

ABSTRACT OF THESIS submitted by Sophronia Amabel Lewis for the degree of Doctor of Philosophy (PhD), entitled ? The effect of ablation and acute inhibition of plasma membrane calcium ATPase 4 (PMCA4) with a novel inhibitor on isolated mouse mesenteric resistance arterial contractility? December 2012Plasma membrane calcium ATPase 4 (PMCA4) is a calcium extrusion pump which may also modulate Ca2+-triggered signal transduction pathways. Previous studies postulate that PMCA4 modulates signalling via an interaction with neuronal nitric oxide synthase (nNOS) in localised plasmalemmal microdomains. The effect of PMCA4 on vascular contractility is unclear. This project has utilised PMCA4 ablated mice (PMCA4 KO (-/-)) and a novel specific PMCA4 inhibitor (termed AP2) to study the role of PMCA4 in mouse resistance artery contractility.Immunohistochemistry, Western blotting and polymerase chain reaction (PCR) confirmed the absence of PMCA4 in the brain, vasculature and ear snips obtained from PMCA4 KO (-/-) mice whereas it was present in those from wild type (WT (+/+)) mice. Pressure myography was employed to assesss contractile function of isolated, pressurised (to 60 mmHg) mesenteric resistance arteries from 3 months old male PMCA4 KO (-/-) and WT (+/+) mice, in response to high K+ physiological salt solution (KPSS) (40mM & 100mM) and noradrenaline (NA) (Log[NA] -9.0 to -5.0M). Passive lumen diameter and left and right wall thicknesses of arteries from PMAC4 KO (-/-) and WT (+/+) mice were taken at transmural pressures of 5-140 mmHg. Effects of acute PMCA4 inhibition with AP2 (10�M and 1�M), nitric oxide synthase (NOS) inhibition with LNNA (100�M) and specific nNOS inhibition with Vinyl-L-Nio (10�M) were also investigated. Effects of PMCA4 ablation and AP2 (10�M) on global intracellular Ca2+ changes ([Ca2+]i) in pressurised mesenteric arteries were assessed after loading arteries with the Ca2+-sensitive indicator indo-1. PMCA4 ablation had no effect on the magnitude of arterial constrictions or on the changes of [Ca2+]i in response to KPSS (40mM & 100mM) or to noradrenaline. The passive intra-lumen diameter, wall thickness, wall to lumen diameter and cross sectional area of mesenteric arteries across the intravascular pressure range studied were also not modulated by PMCA4 ablation. A leftwards shift in the stress to strain relationship and significant increase in beta elastic modulus (? ) were revealed in arteries from PMCA4 KO (-/-) mice compared to those from WT (+/+) mice, suggesting that PMCA4 ablation reduces mesenteric arterial distensibility. Acute PMCA4 inhibition with AP2, significantly reduced arterial constrictions and the increase in [Ca2+]i in response to noradrenaline in arteries from WT (+/+) mice, but had no effect on arterial constrictions elicited by arteries from PMCA4 KO (-/-) mice. Inhibitory effects of AP2 were not present in arteries after NOS inhibition by LNNA and also after nNOS inhibition with Vinly-L-Nio. Hence, PMCA4 inhibition with AP2 reduces vascular constriction by a nNOS-dependent mechanism.In conclusion, the main findings of the study were that ablation and acute inhibition of PMCA4 with AP2 have different effects on mouse mesenteric resistance arterial contractility. This study provides more insight into PMCA4 as a significant modulator of signalling within the vasculature via effects on nNOS.