Activation of potentially mitogenic signalling pathways by endothelin-1 in rat small arteries

The vasoconstrictor peptide ET-1 is a potent mitogen in vascular smooth muscle cells. However, although the contractile response to this peptide has been studied, little is known of the signalling pathways activated by ET-1 in intact vascular preparations. We have determined the effects of ET-1 on protein tyrosine phosphorylation (Pty) and phospholipase D (PLD) activity in intact small mesenteric arteries from normotensive rats. Vessels were stimulated with ET-1, 1, 10 or 100nM for time periods up to 20 minutes. Total protein Pty levels were measured by Western blotting. The formation of [3H]-phosphatidylethanol (PET) or [3H]-phosphatidate (PA) in vessels labelled with [3H]-myristate and stimulated in the presence of 0.5% ethanol was used as a measure of PLD activity. ET-1 induced a dose related, rapid, within 1 minute, and sustained increase in [3H]-PET and [3H]-PA levels (5 fold and 3 fold increase respectively after 20 mins, ET-1 100nM N=3). ET-1 increased the Pty levels of two main clusters of proteins 65-85 and 115-125kDa. This effect was dose related, rapid (+64±18% 65-85kDa at 20sec) and sustained (+250±120% 65-85kDa 20 mins ET-1 100nM N=3). Removal of extracellular Ca2+ abolished the increase in [3H]-PET and [3H]-PA but did not affect the Pty levels of either the 65-85 or 115-125kDa proteins in response to ET-1 100nM. Tyrphostin A23, 100μM, a tyrosine kinase inhibitor blocked activation of PLD and Pty by ET-1 100nM at 10 mins. We conclude that ET-1 activates Ca2+-independent tyrosine kinases in intact small arteries. Activation of PLD occurs also leading to the accumulation of PA. ET-1 PLD activation is dependent on both Ca2+ and tyrosine kinases. The mitogenic potential of these effects suggests a role for ET-1 in vascular smooth muscle cell proliferation in intact tissue.