EFFECTS OF ENDOTHELIN ON PHOSPHOLIPASES AND GENERATION OF 2ND MESSENGERS IN CAT IRIS SPHINCTER AND SV-CISM-2 CELLS

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A(2) (PLA(2)), as measured by the rel ease of arachidonic acid (AA), phospholipase C (PLC), as measured by t he production of inositol trisphosphate (IP3), and phospholipase D (PL D), as measured by the formation of phosphatidylethanol (PEt). In SV-C ISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showe d that the peptide is more potent in activating PLD (EC(50) = 1.2 nM) than in activating PLC (EC(50)= 1.5 nM) or PLA(2) (EC(50) = 1.7 nM). T he time course studies revealed that ET-1 activated the phospholipases in 3. temporal sequence in which PLA(2) was stimulated first (t(1/2) = 12 s), followed by PLC (t(1/2) = 48 s) and lastly PLD (t(1/2) = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sara fotoxin-c, an ET(B) receptor agonist, had no effect on the phospholipa ses, and indomethacin, a cyclooxygenase inhibitor, had no effect on th e stimulatory effect of ET-1 on the phospholipases. These results sugg est that in this smooth muscle cell line, ET-1 interacts with the ET(A ) receptor subtype to activate, via G proteins, phospholipases A(2), C and D in a temporal sequence.