Genistein sensitivity of calcium transport pathways in serotonin-activated vascular smooth muscle cells.

Recent studies showed that serotonin-activated increases in intracellular Ca2+ in vascular smooth muscle cells are associated with enhanced protein tyrosine phosphorylation. These responses were blocked by inhibition of tyrosine kinase activity with genistein, suggesting that the increases in Ca2+ and tyrosine phosphorylation are functionally coupled. Therefore, we sought to characterize genistein-sensitive Ca2+ transport pathways in rat aortic A10 cells loaded with fura-2. In the presence of extracellular Ca2+, serotonin evoked a transient increase in [Ca2+]i that was followed by a smaller sustained increase. The transient was inhibited 25-40% by L-type Ca2+ channel antagonists and inhibited 90-95% by genistein. The sustained response was unaffected by L-channel antagonists and only slightly inhibited by genistein. In the absence of extracellular Ca2+, the transient was reduced by 50%, while the sustained component was virtually abolished. These results suggest that influx and release pathways are major contributors to the transient component, whereas the lower sustained component is largely limited to influx pathways. The influx pathway during the transient probably involves an L-type Ca2+ channel that is regulated by tyrosine kinase activity. The pathways that participate in the sustained response are different because they are insensitive to l-channel antagonists and only slightly inhibited by genistein. The transient evoked in Ca2+-free media was blocked by genistein, inhibited by caffeine, and prevented by thapsigargin. Ionomycin-induced release of Ca2+ was unaffected by genistein, reduced by caffeine, and essentially eliminated by thapsigargin. Therefore, thapsigargin-mediated suppression of serotonin-activated release probably reflects depletion of Ca2+ from the sarcoplasmic reticulum, whereas genistein-mediated suppression probably reflects inhibition of tyrosine kinase linked release. Caffeine-mediated suppression appears to involve both partial depletion of Ca2+ and interference with release. Each A10 cell expressed at least two different ryanodine receptors and two different receptors for inositol 1,4,5-trisphosphate.