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 prolonged duration of the response. Phosphatidylcholine (PC) hydrolysis has been implicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefore, we have investigated the activation of PC-phospholipase D (PLD) and PC-phospholipase C (PLC) in intact rat mesenteric small arteries stimulated with 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 [3H] or [14C]myristate, was increased in a concentration- and time-dependent manner following ET-1 stimulation, as were [3H]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control levels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation reduced [3H]PA levels, but did not alter the time course of formation. In addition, [14C]choline release was increased, confirming PLD-mediated PC hydrolysis. In contrast, DAG levels, measured by [3H]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analysis of the subclasses of DAG demonstrated an increase in all types of DAG without any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D609, 2.5 microg/ml, completely abolished the ET-1-induced increase in [14C]DAG without affecting the increase in [14C]PA, PLD activity or the contractile response. In [14C]choline-labelled vessels, [14C]phosphocholine levels were increased by ET-1 with a similar time course to DAG production. Removal of extracellular Ca2+ and addition of 0.1 or 2 mM EGTA completely inhibited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 microM) abolished ET-1-induced [3H]PA, [3H]PEt and [14C]DAG increases, whilst the negative analogue A1 was without effect. These data suggest that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to PLD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to be 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.
Department of Medicine, Manchester Royal Infirmary, Manchester, UK.
G L
GL
Liu
L
L
Shaw
A M
AM
Heagerty
V
V
Ohanian
J
J
Ohanian
D000818:Animals; D002250:Carbon Radioisotopes; D004075:Diglycerides / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology*; D005260:Female; D020404:Glycerophospholipids / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008638:Mesenteric Arteries / Q000201:enzymology*; D009119:Muscle Contraction; D019814:Myristic Acid / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism*
D002250:Carbon Radioisotopes; D004075:Diglycerides; D019332:Endothelin-1; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; C051521:phosphatidylethanol; D019814:Myristic Acid; D014316:Tritium; D010738:Type C Phospholipases; D010739:Phospholipase D