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 ([Ca(2+)](i)) in rat afferent arterioles. [Ca(2+)](i) was measured in microdissected arterioles using ratiometric photometry of fura 2 fluorescence. The control [Ca(2+)](i) response to NE (1 microM) consisted of a rapid initial peak followed by a plateau phase sustained above baseline. Pretreatment with the tyrosine kinase inhibitor Tyr-23 (50 microM, 10 min) caused a slow 40% increase in baseline [Ca(2+)](i). Tyr-23 attenuated peak and plateau responses to NE, both by approximately 70%. In the absence of extracellular Ca(2+) (0 Ca), Tyr-23 reduced the immediate [Ca(2+)](i) response to NE by approximately 60%, indicative of mobilization of internal stores, and abolished the plateau phase. In other arterioles, the [Ca(2+)](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 Ca(2+) channel blocker nifedipine (1 microM) inhibited the NE response by approximately 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 Ca(2+) entry. Another tyrosine kinase inhibitor, Gen (5 and 50 microM), did not affect baseline [Ca(2+)](i). High-dose Gen inhibited the peak and plateau response to NE by 87 and 75%, respectively; low-dose Gen attenuated both responses by approximately 20%. In 0 Ca, Gen (50 microM) abolished the immediate [Ca(2+)](i) mobilization response. Combined nifedipine and Gen (50 microM) inhibited the rapid NE response by approximately 90% in the presence of extracellular Ca(2+). Gen (50 microM) also inhibited by 60% the [Ca(2+)](i) response to 50 mM KCl, indicating a direct interaction with voltage-sensitive, L-type Ca(2+) entry channels. These results indicate that tyrosine phosphorylation is an important link in the chain of events leading to alpha-adrenoceptor-induced Ca(2+) 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.
Salomonsson
Max
M
Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark.
Arendshorst
William J
WJ
D000818:Animals; D001160:Arterioles / Q000187:drug effects / Q000201:enzymology; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D019833:Genistein / Q000494:pharmacology; D007678:Kidney Glomerulus / Q000098:blood supply*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology; D009638:Norepinephrine / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D051381:Rats; D011921:Rats, Inbred WKY; D012079:Renal Circulation / Q000187:drug effects / Q000502:physiology; D020032:Tyrphostins / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D020032:Tyrphostins; D014662:Vasoconstrictor Agents; D019833:Genistein; D011505:Protein-Tyrosine Kinases; C060641:tyrphostin A23; D002118:Calcium; D016257:Fura-2; D009638:Norepinephrine