Effect of tyrosine kinase blockade on norepinephrine-induced cytosolic calcium response in rat afferent arterioles

We used genistein (Gen) and tyrphostin 23 (Tyr-23) to evaluate the importance of tyrosine phosphorylation in norepinephrine (NE)-induced changes in intracellular free calcium concentration ([Ca2+]i) in rat afferent arterioles. [Ca2+]i was measured in microdissected arterioles using ratiometric photometry of fura 2 fluorescence. The control [Ca2+]i response to NE (1 μM) consisted of a rapid initial peak followed by a plateau phase sustained above baseline. Pretreatment with the tyrosine kinase inhibitor Tyr-23 (50 μM, 10 min) caused a slow 40% increase in baseline [Ca2+]i. Tyr-23 attenuated peak and plateau responses to NE, both by ∼70%. In the absence of extracellular Ca2+ (0 Ca), Tyr-23 reduced the immediate [Ca2+]i response to NE by ∼60%, indicative of mobilization of internal stores, and abolished the plateau phase. In other arterioles, the [Ca2+]i response to depolarization induced by KCl (50 mM) was not attenuated by Tyr-23, indicating no direct effect on L-type Ca+ channels activated by depolarization. The Ca2+ channel blocker nifedipine (1 μM) inhibited the NE response by ∼50%; the effects of nifedipine and Tyr-23 were not additive. Nifedipine had no inhibitory effect after Tyr-23 pretreatment, indicating Tyr-23 inhibition of Ca2+ entry. Another tyrosine kinase inhibitor, Gen (5 and 50 μM), did not affect baseline [Ca2+]i. High-dose Gen inhibited the peak and plateau response to NE by 87 and 75%, respectively; low-dose Gen attenuated both responses by ∼20%. In 0 Ca, Gen (50 μM) abolished the immediate [Ca2+]i mobilization response. Combined nifedipine and Gen (50 μM) inhibited the rapid NE response by ∼90% in the presence of extracellular Ca2+. Gen (50 μM) also inhibited by 60% the [Ca2+]i response to 50 mM KCl, indicating a direct interaction with voltage-sensitive, L-type Ca2+ entry channels. These results indicate that tyrosine phosphorylation is an important link in the chain of events leading to α-adrenoceptor-induced Ca2+ recruitment (both entry and release) in afferent arteriolar smooth muscle cells. Furthermore, different blockers of tyrosine kinase appear to have different modes of action in renal microvessels.