Endothelin-1 stimulates hydrolysis of phosphatidylcholine by phospholipases C and D in intact rat mesenteric arteries

A characteristic of endothelin-1 (ET-1)-induced contraction is the prolonge d duration of the response. Phosphatidylcholine (PC) hydrolysis has been im plicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefor e, we have investigated the activation of PC-phospholipase D (PLD) and PC-p hospholipase C (PLC) in intact rat mesenteric small arteries stimulated wit h ET-1 and determined whether PC-derived DAG is necessary for ET-1-induced contraction. PLD activity, measured as phosphatidylethanol (PEt) production in vessels labelled with [H-3] Or [C-14]myristate, was increased in a conc entration- and time-dependent manner following ET-1 stimulation, as were [H -3]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control l evels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation r educed [H-3]PA levels, but did not alter the time course of formation. In a ddition, [C-14]choline release was increased, confirming PLD-mediated PC hy drolysis. In contrast, DAG levels, measured by [H-3]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analy sis of the subclasses of DAG demonstrated an increase in all types of DAG w ithout any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D 609, 2.5 mu g/ml, completely abolished the ET-1-induced increase in [C-14]D AG without affecting the increase in [C-14]PA, PLD activity or the contract ile response. In [C-14]choline-labelled vessels, [C-14]phosphocholine level s were increased by ET-1 with a similar time course to DAG production. Remo val of extracellular Ca2+ and addition of 0.1 or 2 mM EGTA completely inhib ited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 mu M) abolished ET-1-induced [H-3]PA, [H-3]PEt and [C-14]DAG incr eases, whilst the negative analogue Al was without effect. These data sugge st that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to P LD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to b e necessary for the contractile response, whereas sustained formation of PA , generated by PLD activity, implies that this lipid second messenger could be involved in the prolonged contractile response to this peptide.