Bradykinin‐stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells

Stimulation of bradykinin (BK) receptors coupled to phosphoinositide (PI) hydrolysis was investigated in canine cultured tracheal smooth muscle cells (TSMCs). BK, kallidin, and des‐Arg⁹‐BK, stimulated [³H]‐inositol phosphates (IPs) accumulation in a dose‐dependent manner with half‐maximal responses (EC₅₀) at 20 ± 5, 13 ± 4, and 2.3 ± 0.7 nM, (n = 5), respectively. d‐Arg[Hyp³, d‐Phe⁷]‐BK and d‐Arg[Hyp³, Thi^5,8, d‐Phe⁷]‐BK, BK B₂ receptor antagonists, were equipotent in blocking the BK‐induced IPs accumulation with pK_B = 7.1 and 7.3, respectively. Short‐term exposure of TSMCs to phorbol 12‐myristate 13‐acetate (PMA, 1 μm), attenuated BK‐stimulated IPs accumulation. The concentrations of PMA that gave half‐maximal and maximal inhibition of BK‐induced IPs accumulation were 15 ± 4 nM and 1 μm, n = 3, respectively. The inhibitory effect of PMA on BK‐induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA for 24 h, resulted in a recovery of receptor responsiveness which may be due to down‐regulation of PKC. The inactive phorbol ester, 4α‐phorbol 12, 13‐didecanoate at 1 μm, did not inhibit this response. The site of this inhibition was further investigated by examining the effect of PMA on AIF₄⁻‐induced IPs accumulation in canine TSMCs. AIF₄⁻‐stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the G protein(s) can be directly activated by AIF₄⁻, which is uncoupled from phospholipase C by PMA treatment. Incubation of TSMCs in the absence of external Ca²⁺ or upon removal of Ca²⁺ by addition of EGTA, caused a decrease in IPs accumulation without changing the basal levels. Addition of Ca²⁺ (3–620 nM) to digitonin‐permeabilized TSMCs stimulated IPs accumulation was obtained by inclusion of either guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) or BK. The combination of GTPγS and BK caused an additive effect on IPs accumulation. Pretreatment of TSMCs with cholera toxin enhanced BK‐stimulated IPs accumulation, whereas there was no effect with pertussis toxin. These data suggest that BK‐stimulated PI metabolism is mediated by the activation of BK B₂ receptors coupling to a G protein which is not blocked by cholera toxin or pertussis toxin treatment and dependent on external Ca²⁺. The transduction mechanism of BK coupled to PI hydrolysis is sensitive to feedback regulation by PKC. 1994 British Pharmacological Society