COUPLING BETWEEN [ARGININE(8)]-VASOPRESSIN-ACTIVATED INCREASES IN PROTEIN-TYROSINE PHOSPHORYLATION AND CELLULAR CALCIUM IN A7R5 AORTIC SMOOTH-MUSCLE CELLS

Effects of genistein, a tyrosine kinase inhibitor, on increases in [Ca 2+](i) and protein tyrosine phosphorylation induced by 20 nM [arginine s]vasopressin (AVP) were studied in A7r5 aortic smooth muscle cells, I n fura-2-loaded cells, AVP induced a rapid (0.5-2 min) transient incre ase in [Ca2+](i) that was followed by a smaller sustained increase in [Ca2+](i). In 66% of the cells, the transient response involved both i nflux of extracellular Ca2+ and release of intracellular Ca2+: influx accounted for 60% of the response, and release accounted for 40%. Howe ver, in 34% of the cells, the relative contribution of influx and rele ase during the transient could not be assessed. In all cells, the smal ler sustained response was entirely dependent on extracellular Ca2+. G enistein (148 mu M) always blocked the transient and sustained compone nts of the Ca2+ response showing that both influx and release were gen istein-sensitive. Isobestic fluorescence analysis, in medium containin g 0.5 mM Mn2+ in place of Ca2+, showed that the influx pathway was sel ective because it did not conduct Mn2+. It also confirmed that Ca2+ re lease was blocked by genistein, In contrast, 105 mu M lavendustin A, a different tyrosine kinase inhibitor, suppressed the transient by only 30%. Another inhibitor, tyrphostin 47 (80 mu M), did not alter the tr ansient or sustained components of the Ca2+ response, No AVP-induced i ncreases in tyrosine phosphorylation were detected unless special proc edures were used. When cells were preincubated in 10 mM vanadate, a ty rosine phosphatase inhibitor, AVP induced a transient increase in tyro sine phosphorylation (5-60 s). The time course for AVP-induced phospho rylation was similar to that for increases in [Ca2+](i). Vanadate alon e increased tyrosine phosphorylation and induced a slow small increase in [Ca2+](i) that was dependent on extracellular Ca2+. Genistein bloc ked tyrosine phosphorylation induced by AVP and vanadate, and it block ed the increase in [Ca2+](i) induced by vanadate alone. In contrast, l avendustin or tyrphostin unexpectedly enhanced tyrosine phosphorylatio n induced by vanadate alone and precluded assessment of AVP-induced ty rosine phosphorylation in the presence of vanadate. Levandustin produc ed time-dependent enhancement of vanadate-induced increase in [Ca2+](i ). These results underscored the need for measuring cellular changes i n protein tyrosine phosphorylation to assess potential functions of ty rosine kinase activity. Under conditions where changes in phosphorylat ion could be measured, the results suggested that AVP-activated increa ses in tyrosine phosphorylation may be coupled to AVP-activated mechan isms that regulate influx of extracellular Ca2+ and release of intrace llular Ca2+. (C) 1996 Academic Press, Inc.