Vascular smooth muscle : phenotypic diversity and calcium signaling

(eng) Vascular smooth muscle cells (VSMCs) exhibit different physiological properties when they are localized in the wall of large conductance artery like the aorta, or small resistance arteries as resistance mesenteric artery (RMA). Cytosolic Ca2+ increase is crucial for controlling vascular tone. Contractile tension of arteries is mainly regulated by agonist acting on G protein-coupled receptors. In addition, in small RMA, cytosolic Ca2+ increase triggers a myogenic contraction in response to increased intravascular pressure. Cytosolic Ca2+ increase occurs through voltage-operated Ca2+ channels (VOCC) or receptor- or store-operated Ca2+ channels. Mechanisms of cytosolic Ca2+ increase are still not completely elucidated. It has been previously shown that Rho kinase (ROCK) contributes to non-voltage-dependent and non-capacitative Ca2+ entry in rat aorta, and that the Ca2+-dependent kinase of myosin light chain (MLCK) is involved in Ca2+ channels regulation in HEK293 cells. Furthermore, in pathological conditions such as hypertension or atherosclerosis, VSMCs change from a contractile to a non-contractile synthetic proliferative phenotype that can be mimicked by culturing VSMCs. Ca2+ signaling pathways in cultured VSMCs differ from the situation in whole artery. The aim of the present work was to investigate and compare the role of ROCK and MLCK in cytosolic Ca2+ increase regulatory mechanisms between rat large and small arteries, and between whole rat aorta and cultured rat aortic smooth muscle cells. In addition, particular attention was paid to the role of ROCK in Ca2+ signaling and myogenic contraction in response to increased intravascular pressure in RMA. We showed that in fura-2 loaded small RMA, ROCK is not involved in the regulation of cytosolic Ca2+ increase in response to noradrenaline. Conversely, the myogenic response and Ca2+ entry following pressure elevation in pressurized-RMA is dependent on ROCK activity. We also demonstrated that MLCK participates in the VOCC-mediated Ca2+ entry in response to noradrenaline in RMA. In aorta, we further characterized the Ca2+ entry induced by noradrenaline and concluded that actin cytoskeleton is not involved in the ROCK-dependent Ca2+ entry. However, myosin cytoskeleton is a potential candidate for Ca2+ influx regulation as the inhibition of MLCK by ML-7 depresses the VOCC-insensitive Ca2+ entry. MLCK could be therefore responsible for Ca2+ channels trafficking to the plasma membrane in arteries. Additionally, although cultured cells are usually used to investigate cellular functions, we highlighted that in cultured VSMCs, neither ROCK nor MLCK contribute to the vasopressin-induced Ca2+ signaling pathways. The lack of sensitivity to ROCK and MLCK inhibition could result from lower MLCK expression in cultured VSMCs. Eventually, we observed that MLCK regulates differentiation markers as well as Ca2+ channels mRNA expression in the rat aortic cell line A7r5, where MLCK could be involved in the regulation of transcription. Together, our results stress important differences in Ca2+ signaling between conductance and resistance arteries and between whole artery and cultured VSMCs.