PubMed1996
Effects of genistein, a tyrosine kinase inhibitor, on increases in [Ca2+]i and protein tyrosine phosphorylation induced by 20 nM [arginine 8]vasopressin (AVP) were studied in A7r5 aortic smooth muscle cells. In fura-2-loaded cells, AVP induced a rapid (0.5-2 min) transient increase in [Ca2+]i that was followed by a smaller sustained increase in [Ca2+]i. In 66% of the cells, the transient response involved both influx of extracellular Ca2+ and release of intracellular Ca2+: influx accounted for 6% of the response, and release accounted for 40%. However, in 34% of the cells, the relative contribution of influx and release during the transient could not be assessed. In all cells, the smaller sustained response was entirely dependent on extracellular Ca2+. Genistein (148 microM) always blocked the transient and sustained components of the Ca2+ response showing that both influx and release were genistein-sensitive. Isobestic fluorescence analysis, in medium containing 0.5 mM Mn2+ in place of Ca2+, showed that the influx pathway was selective because it did not conduct Mn2+. It also confirmed that Ca2+ release was blocked by genistein. In contrast, 105 microM lavendustin A, a different tyrosine kinase inhibitor, suppressed the transient by only 30%. Another inhibitor, tyrphostin 47 (80 microM), did not alter the transient or sustained components of the Ca2+ response. No AVP-induced increases in tyrosine phosphorylation were detected unless special procedures were used. When cells were preincubated in 10 mM vanadate, a tyrosine phosphatase inhibitor, AVP induced a transient increase in tyrosine phosphorylation (5-60 s). The time course for AVP-induced phosphorylation was similar to that for increase in [Ca2+]i. Vanadate alone increased tyrosine phosphorylation and induced a slow small increase in [Ca2+]i that was dependent on extracellular Ca2+. Genistein blocked tyrosine phosphorylation induced by AVP and vanadate, and it blocked the increase in [Ca2+]i induced by vanadate alone. In contrast, lavendustin or tyrphostin unexpectedly enhanced tyrosine phosphorylation induced by vanadate alone and precluded assessment of AVP-induced tyrosine phosphorylation in the presence of vanadate. Lavendustin produced time-dependent enhancement of vanadate-induced increase in [Ca2+]i. These results underscored the need for measuring cellular changes in protein tyrosine phosphorylation to assess potential functions of tyrosine kinase activity. Under conditions where changes in phosphorylation could be measured, the results suggested that AVP-activated increases in tyrosine phosphorylation may be coupled to AVP-activated mechanisms that regulate influx of extracellular Ca2+ and release of intracellular Ca2+.