CALCIUM RELEASE-ACTIVATED CALCIUM INFLUX IN CULTURED HUMAN MESANGIAL CELLS

Ca2+ influx is a major component of the response of cultured human mesangial cells (HMC) to vasoconstrictors. Activators of phospholipase C such as angiotensin II (Ang II) release Ca2+ from intracellular stores and enhance Ca2+ influx, which in turn is modulated by Na+/Ca2+ exchange. By microfluorometry we studied the mechanisms of Ca2+ entry in resting and stimulated fura-2-loaded monolayers or single HMC. Addition of 1 to 10 mM extracellular Ca2+ to cells equilibrated in Ca2+-free media resulted in a rapid, persistent elevation of free cytosolic Ca2+ ([Ca2+](i)), from 52 +/- 5 to 113 +/- 18 and 226 +/- 37 nM, respectively. Ca2+ influx was blocked by lanthanum or chelation with EGTA, while it was only partially inhibited by voltage-operated Ca2+ channel(VOC) blockers, such as nifedipine or verapamil. The rise of [Ca2+](i) at high external [Ca2+] was not due to a Ca2+-sensing mechanism with release of intracellularly stored Ca2+, since it was prolonged, and it was not seen in cells maintained in normal 1.25 mM [Ca2+] media. Moreover, it was not abolished by prior depletion of Ca2+ stores with 0.5 mu M thapsigargin or 5 mu M ionomycin in Ca2+-free media, which transiently increased [Ca2+](i) (to 281 +/- 39 and 380 +/- 51 nM, respectively). On the contrary, both agents markedly potentiated Ca2+ influx upon addition of 1 to 10 mM [Ca2+](c), (to a maximum of 686 +/- 111 and 633 +/- 150 nM, P < 0.05 vs. control). Prior stimulation of [Ca2+](i) with 1 mu M Ang II had similar effects, enhancing the subsequent Ca2+ influx to 241 +/- 42 (1 mM Ca2+) and 512 +/- 106 nM (10 mM Ca2+; P < 0.05). Enhancement of Ca2+ influx by thapsigargin, ionomycin and Ang II was confirmed by increased Mn2+ quenching of fura-2 fluorescence following addition of the agents in the absence of extracellular Ca2+. VOC activation by membrane depolarization was not responsible for such potentiation, since 50 mM KCl failed to modify Ca2+ influx. Na+/Ca2+ exchange was ruled out by persistence of influx after intracellular Na+ depletion. Thus, an initial elevation of [Ca2+](i) by vasoconstrictors, blockers of Ca2+ ATPase or Ca2+ ionophores enhances Ca2+ entry in HMC via a Ca2+ release-activated Ca2+ conductance, mostly independent of plasma membrane depolarization.