Angiotensin II and vasopressin modulate intracellular free magnesium in vascular smooth muscle cells through Na+-dependent protein kinase C pathways

Vasoactive peptides mobilize cytosolic free Mg2+ in vascular smooth muscle cells, It is unknown whether angiotensin II and arginine vasopressin, potent vaso constrictor agents, influence intracellular Mg2+, The effects of angiotensin II and vasopressin on intracellular free Mg2+ concentrations ([Mg2+](i)) were therefore investigated in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric arteries of Wistar Kyoto rats, and in an established cell line of rat thoracic aorta cells (A10 cells), Underlying mechanisms of agonist stimulated [Mg2+](i) changes were assessed in A10 cells by pharmacologically manipulating phospholipase C, protein kinase C, and the Na+/H+ exchanger. In addition, the dependence of [Mg2+](i) on intracellular Ca2+ was determined, [Mg2+](i) was measured in single cells by fluorescent digital imaging using mag-fura-2/AM. Basal [Mg2+](i) levels in Wistar Kyoto rat and A10 cells were 0.62 +/- 0.02 mmol/liter and 0.58 +/- 0.01 mmol/ liter, respectively, Angiotensin II and vasopressin induced a dose-dependent biphasic [Mg2+](i) response where [Mg2+](i) increased rapidly and transiently to a peak level and then declined to subbasal levels, which were sustained, Preexposure of cells to neomycin, a nonspecific phospholipase C inhibitor, U-73122, a selective phospholipase C inhibitor, calphostin C, a selective protein kinase C inhibitor, and 5-(N,N-hexamethylene)amiloride, a selective Na+/H+ exchange blocker, attenuated angiotensin II- and vasopressin-induced [Mg2+](i) responses in a concentration-dependent manner, Removal of extracellular Na+ completely inhibited agonist-elicited [Mg2+](i) transients. To determine whether intracellular free Ca2+ concentration ([Ca2+](i)) influences agonist-induced [Mg2+](i) changes, thapsigargin, a selective sarcoplasmic reticular Ca2+-ATPase inhibitor, was used to deplete intracellular Ca2+ stores, In thapsigargin-pretreated cells, angiotensin II-elicited [Ca2+](i) responses were significantly attenuated, whereas agonist-induced [Mg2+](i) responses were unchanged. These data demonstrate that in primary cultured VSMC and in an established VSMC line, angiotensin II and vasopressin modulate [Mg2+](i) through receptor-mediated pathways, which are [Ca2+](i)-independent but which involve phospholipase C, protein kinase C, and the Na+/H+ exchanger, These pathways are linked to a Na+-dependent Mg2+ transporter, which facilitates transmembrane Mg2+ transport.