Endothelin‐ and Sarafotoxin‐Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells

Abstract

The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphatase (IPs) and tracheal smooth muscle contraction. BQ-123, an ETA receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pKB values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca2+ and was blocked by treatment with EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca(2+)-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 microM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to downregulation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)