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Endothelin‐ and Sarafotoxin‐Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells
Author(s) -
Yang Chuen Mao,
Yo YingLing,
Ong Richard,
Hsieh JenTsung
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.62041440.x
Subject(s) - protein kinase c , pertussis toxin , staurosporine , medicine , endocrinology , egta , phorbol , endothelins , stimulation , contraction (grammar) , biology , inositol phosphate , muscle contraction , endothelin 1 , chemistry , inositol , receptor , biochemistry , calcium , signal transduction , g protein
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 phosphates (IPs) and tracheal smooth muscle contraction. BQ‐123, an ET A receptor antagonist, had a high affinity to block the ET‐1‐induced IP accumulation and tracheal smooth muscle contraction with pK B 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 Ca 2+ and was blocked by treatment with EGTA. The addition of Ca 2+ (3–620 n M ) 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 μ M ) 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 down regulation of PKC. Inactive phorbol ester, 4α‐phorbol 12, 13‐didecanoate at 1 μ M , did not inhibit this response. The site of this response was further investigated by examining the effect of PMA on AIF 4 − ‐induced IP accumulation in canine TSMCs. AIF 4 − ‐induced IP accumulation was inhibited by PMA treatment, suggesting that G protein(s) can be directly activated by AIF 4 − , which was uncoupled to phospholipase C by PMA treatment. These data conclude that ET‐stimulated PI hydrolysis and tracheal smooth muscle contraction are mediated by the activation of ET A receptors coupling to a G protein and dependent on the external Ca 2+ . The transduction mechanism of ETs is sensitive to feedback regulation by PKC.