INVOLVEMENT OF TYROSINE PHOSPHORYLATION IN ENDOTHELIN-1-INDUCED CALCIUM-SENSITIZATION IN RAT SMALL MESENTERIC-ARTERIES

1 We have studied the effect of endothelin-1 stimulation on protein ty rosine phosphorylation levels in intact small mesenteric arteries of t he rat and investigated the effects of tyrosine kinase inhibition on t he contractile response to this agonist. 2 Endothelin-1 stimulated a r apid (20 s), sustained (up to 20 min) and concentration-dependent (1-1 00 nM) increase in protein tyrosine phosphorylation levels which coinc ided temporally with the contractile response in intact and alpha-toxi n permeabilized small artery preparations. Tyrosine phosphorylation wa s increased in four main clusters of proteins of apparent molecular ma ss 28-33, 56-61, 75-85 and 105-115 kDa. Endothelin-1-induced protein t yrosine phosphorylation was independent of extracellular calcium, anta gonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin Al. 3 In intact small arteries tyrphostin A23 inh ibited the force developed to endothelin-1 at all concentrations studi ed; at higher concentrations (10 and 100 nM) the profile of contractio n was altered from a sustained to a transient response. Tyrphostin Al inhibited the contractile response to endothelin-1 at all concentratio ns except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endot helin-1 (100 nM) in the absence of extracellular calcium. 4 In rat alp ha-toxin permeabilized mesenteric arteries endothelin-1 caused a conce ntration-dependent increase in force in the presence of 10 mu M GTP an d low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin Al did not affect endotheli n-l-induced calcium sensitization of force. 5 We conclude that increas ed tyrosine phosphorylation is important in the contractile response i nduced by endothelin-1 in intact small mesenteric arteries. Furthermor e our data implicate activation of this signalling pathway in the toni c phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.