Mechanisms of serotonin-induced Ca2+ responses in mesangial cells

Abstract

Smooth muscle cell-like mesangial cells play an important role in the regulation of glomerular blood flow and are involved in renal inflammatory reactions, thereby interacting with circulating cells. The platelet products serotonin (5-HT) and ATP induce similar, e.g. mitogenic, effects in mesangial cells, but differentially activate and induce inflammation-related genes. To get an insight into intracellular signaling steps, a very early step in the signaling cascade, the biphasic Ca²⁺ signal elicited by 5-HT and ATP in rat mesangial cells was investigated. Both phases of the Ca²⁺ signal, release from internal stores as well as influx of extracellular Ca²⁺, were dependent on phospholipase C activation as shown by the specific inhibitor U73122 (complete inhibition at 10μM U73122). There was no evidence for voltage-gated L-type channels in these cells, suggesting that Ca²⁺ influx was mediated by Ca²⁺ release-activated channels. The L-type channel blocker verapamil, however, dose-dependently (0.1–10μM) and specifically inhibited 5-HT-elicited Ca²⁺ signals by interference with binding of 5-HT to 5-HT_2A receptors. 5-HT-mediated Ca²⁺ release was reduced by 80% when protein kinase C was activated by the phorbolester TPA (0.1μM). Interaction of 5-HT_2A receptors with phospholipase C was also inhibited by genistein (30% at 5μM; 100% at 50μM), an inhibitor of tyrosine kinases. Binding of 5-HT to its receptor reduced subsequent ATP-mediated Ca²⁺ signaling. The cross talk between the receptors was sensitive to genistein. ATP-mediated Ca²⁺ signaling was attributed to different types of P_2y receptors and/or multiple G-proteins coupled, because the signal was partially inhibited by pertussis toxin (50%). In accordance, modulation of the ATP-mediated signaling by phosphorylation was less tightly controlled than 5-HT-mediated Ca²⁺ release. These data indicate that although the Ca²⁺ responses elicited by the two stimuli are comparable, interactions between receptors, G-proteins and target enzymes are regulated differentially.