ALTERATION OF INTRACELLULAR CALCIUM RELEASE MEDIATED BY CD38 UNDER CHRONIC HYPOXIA IN PULMONARY ARTERIAL SMOOTH MUSCLE CELL
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Prolonged exposure to hypoxia due to high altitude or pulmonary diseases like chronic obstructive pulmonary disease causes chronic hypoxia-induced pulmonary hypertension (CHPH), which results in right heart hypertrophy, worsens the prognosis of the underlying pulmonary diseases, and even death. Chronic hypoxia (CH) impacts various targets in the pulmonary vasculature such as pulmonary artery (PA), leading to complex physiologic responses during the development of CHPH. It is well established that CH alters calcium (Ca2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs) due to the enhancement of extracellular Ca2+ influx as the result of changes in the expression and activities of various membrane channels, transporters, and exchangers. However, the evidence for alterations of intracellular Ca2+ release in PASMCs caused by CH is scanty to date. CD38 is a multifunctional enzyme that synthesizes the endogenous Ca2+ mobilizing messengers cyclic adenosine diphosphate-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), which are potent regulators of Ca2+ release via ryanodine receptor (RyR)-gated sarcoplasmic reticulum and NAADP-sensitive endolysosomal Ca2+ stores, respectively. CD38 is thought to play important roles in [Ca2+]i regulation via Ca2+ release, contributing to diverse physiologic responses in many different cell types. However, its functions and regulatory mechanisms in PASMCs are still unclear. In particular, there is no systematic study on the mechanism of agonist-induced activation of CD38 in PASMCs. Furthermore, the effect of CH on CD38-dependent Ca2+ release in PASMCs has not been examined. The objective of this thesis research is to examine systematically the mechanism of agonist-induced CD38 activation and the CH-induced alteration of CD38 expression and activity in PASMCs, as to provide novel insight into the contribution of CD38 in the development of CHPH. The first part of this thesis research determined the expression of CD38, and its roles in angiotension II (Ang II)-induced vasoconstriction in PAs, and Ang II-induced Ca2+ release (AICR) in PASMCs. Examination of the expression profile of CD38 in various rat arteries indicates a relatively high level of CD38 expression in PA smooth muscle and PASMCs. Application of Ang II to PASMCs elicited Ca2+ response composed of both extracellular Ca2+ influx and intracellular Ca2+ release AICR activated in the absence of extracellular Ca2+ was significantly reduced by pharmacological or by siRNA inhibition of CD38, implying that CD38 mediates AICR in PASMCs. AICR was suppressed by the cADPR antagonist cADPR 8-Br-cADPR or the inhibition of the RyR-gated Ca2+ released with ryanodine. It was also suppressed by the NAADP-antagonist Ned-19 or the disruption of endolysosomal Ca2+ stores by the vacuolar H+-ATPase inhibitor bafilomycin A1. Suppression of AICR by the inhibition of cADPR- and NAADP-dependent pathways was non-additive, indicating inter-dependence between RyR- and NAADP-gated Ca2+ release in PASMCs. Furthermore, AICR was inhibited by the protein kinase C (PKC) inhibitor staurosporine, the non-specific NADPH oxidase (NOX) inhibitor apocynin and DPI, the NOX2 specific inhibitor gp91ds-tat, and the reactive oxygen (ROS) species scavenger TEMPOL. These results provide direct evidence that Ang II activates CD38-dependent Ca2+ release via the PKC-NOX2-ROS pathway in PASMCs. The second part of the research characterized the CH-induced alterations in the expression and functions of CD38 in PASMCs. The expression of CD38 protein and mRNA were both significantly upregulated in the PA smooth muscle of CH rats. The upregulation of CD38 in PA of CH rats was time-dependent, observed after 3–7 days of hypoxic exposure and declining after 3 weeks of CH. NADase activity of CD38 in PA smooth muscle was significantly increased, whereas the activity in whole lung was decreased after 1-week hypoxia, suggesting specific CD38 upregulation in hypoxic PA. AICR was significantly increased in PASMCs of CH rat. The CH-induced enhancement in AICR was completely vanished by the inhibition of CD38, indicating that CH triggers augmentation of CD38 activity in PASMCs. The CH-induced upregulation of CD38 expression and activity were due to the direct effect of hypoxia on PASMCs. In vitro exposure of PASMCs from normoxic rats to hypoxia caused significant increase in CD38 expression and enhancement in AICR, which was abolished by the inhibition of CD38 activity. Moreover, the CH-induced upregulation of CD38 expression in PASMCs was inhibited by calcineurin/NFAT inhibitors, indicating that CH induces CD38 upregulation in PASMC mediated by the calcineurin/NFAT-pathway. Furthermore, the NAADP-dependent Ca2+ channel TPC1 and TPC2 was increased in CH rat PA. Hence, these results suggest that CH increases the expression and activity of CD38 and the CD38-dependent Ca2+ release in PASMCs. Collectively, the results indicate the CH-induced enhancement of CD38 expression and activity in PASMCs may contribute to the alteration of Ca2+ homeostasis in PASMCs. It also implies that CD38 could be a possible therapeutic target for the treatment of this dreadful disease.