GENISTEIN SENSITIVITY OF CALCIUM-TRANSPORT PATHWAYS IN SEROTONIN-ACTIVATED VASCULAR SMOOTH-MUSCLE CELLS

Recent studies showed that serotonin-activated increases in intracellu lar Ca2+ in vascular smooth muscle cells are associated with enhanced protein tyrosine phosphorylation. These responses were blocked by inhi bition of tyrosine kinase activity with genistein, suggesting that the increases in Ca2+ and tyrosine phosphorylation are functionally coupl ed. Therefore, we sought to characterize genistein-sensitive Ca2+ tran sport pathways in rat aortic A10 cells loaded with fura-2. In the pres ence of extracellular Ca2+, serotonin evoked a transient increase in [ Ca2+](i) that was followed by a smaller sustained increase. The transi ent was inhibited 25-40% by L-type Ca2+ channel antagonists and inhibi ted 90-95% by genistein. The sustained response was unaffected by L-ch annel antagonists and only slightly inhibited by genistein. In the abs ence of extracellular Ca2+, the transient was reduced by 50%, while th e sustained component was virtually abolished. These results suggest t hat influx and release pathways are major contributors to the transien t component, whereas the lower sustained component is largely limited to influx pathways. The influx pathway during the transient probably i nvolves an L-type Ca2+ channel that is regulated by tyrosine kinase ac tivity. The pathways that participate in the sustained response are di fferent because they are insensitive to L-channel antagonists and only slightly inhibited by genistein. The transient evoked in Ca2+-free me dia was blocked by genistein, inhibited by caffeine, and prevented by thapsigargin. Ionomycin-induced release of Ca2+ was unaffected by geni stein, reduced by caffeine, and essentially eliminated by thapsigargin . Therefore, thapsigargin-mediated suppression of serotonin-activated release probably reflects depletion of Ca2+ from the sarcoplasmic reti culum, whereas genistein-mediated suppression probably reflects inhibi tion of tyrosine kinase linked release. Caffeine-mediated suppression appears to involve both partial depletion of Ca2+ and interference wit h release. Each A10 cell expressed at least two different ryanodine re ceptors and two different receptors for inositol 1,4,5-trisphosphate. (C) 1997 Academic Press.