Effects of endothelin on phospholipases and generation of second 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 A₂ (PLA₂), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol triphosphate (IP₃), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP₃ production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC₅₀ = 1.2 nM) than in activating PLC (EC₅₀ = 1.5 nM) or PLA₂ (EC₅₀ = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA₂ was stimulated first ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{s}$), followed by PLC ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 48}\text{s}$) and lastly PLD ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 106}\text{s}$). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ET_B receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ET_A receptor subtype to activate, via G proteins, phospholipases A₂, C and D in a temporal sequence.