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

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Abstract
1 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. 2 d-Arg[Hyp3, d-Phe7]-BK and d-Arg[Hyp3, Thi5,8, d-Phe7]-BK, BK B2 receptor antagonists, were equipotent in blocking the BK-induced IPs accumulation with pKB = 7.1 and 7.3, respectively. 3 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. 4 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. 5 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. 6 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. 7 Pretreatment of TSMCs with cholera toxin enhanced BK-stimulated IPs accumulation, whereas there was no effect with pertussis toxin. 8 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.