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 (ECm) at 20 ± 5, 13 ± 4, and 2.3 ± 0.7 nM, (n = 5), respectively. 3 Short-term exposure of TSMCs to phorbol 12-myristate 13-acetate (PMA, 1 1M), attenuated BKstimulated IPs accumulation. The concentrations of PMA that gave half-maximal and maximal inhibition of BK-induced IPs accumulation were 15 ± 4 nM and 1 fLM, 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, 4oc-phorbol 12, 13-didecanoate at 1 tlM, 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. A1F4 -stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the G protein(s) can be directly activated by A1F4-, 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) (GTPyS) or BK. The combination of GTPyS 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.

1 The relationship between muscarinic receptor-mediated phosphatidylinositol 4, breakdown and the increase of intracellular Ca2+ ([Ca2+])i has been examined in canine cultured tracheal smooth muscle cells (TSMCs

The interaction of the myelin basic protein (MBP) and the major endogenous ganglioside GM1 in myelin of the central nervous system has been investigated using both 500-MHz 1H and 67.89 MHz 13C NMR. Titration of MBP by GM1 resulted in 13C NMR signal shifts for the I1e and His residues of MBP at a GM1/MBP mole ratio of one or less. The carbohydrate head group of GM1 was also found to be perturbed. 1H NMR results obtained in a similar manner demonstrated the perturbation of His and Phe residues. At a GM1/MBP mole ratio of 0.5, small perturbation of Trp #116 was observed, and at mole ratios of two and beyond significant involvement of Phe residues and methylated Arg #107 was found. Met #167 was more perturbed than Met #20; hence, more extensive interaction of the lipid is occurring with the C-terminus of the protein than with the N-terminus. No resonances from GM1 bound to MBP at mole ratios of up to one appeared in the spectra. However, as the GM1/MBP mole ratio was increased to eight or greater a major conformational change of MBP was detected. An upfield shift of the GM1 midchain methylene resonance was observed for the GM1/MBP complex. This observation provides strong evidence that the state of GM1 interacting with MBP is different from that of GM1 micelles. The number of saturable GM1 binding sites on MBP is estimated to be four. The data also favor a rapid exchange between bound GM1 and GM1 micelles. Interaction of MBP with the oligosaccharide derived from GM1 was found to be weaker than with GM1. Based on our data, a model for the interaction can be proposed: the first GM1 molecule is bound to the protein molecule through its head group and hydrocarbon chains, followed by the formation of a GM1/MBP complex with a concomitant conformational change of MBP as more GM1 is added.

1 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time-and dose-dependent with a half-maximal response (EC") and a maximal response at 0.38 ± 0.05 and 10 gM, respectively. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 ± 0.4 nM and 1 tM, n = 3, respectively. The protein kinase C (PKC) activator, 4x-phorbol 12,13-didecanoate, at 1 pLM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6 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 effect of PMA is distal to the 5-HT receptor. 7 Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine. 8 These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin-and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HTstimulated contraction of tracheal smooth muscle.

mobilization produced by CTX, forskolin and dibutyryl cyclic AMP involves a cyclic AMP-dependent mechanism which is induced by a sustained increase in the level of intracellular cyclic AMP. CTX and forskolin may promote an increase of the synthesis of BK receptors, and thereby enhance BK-induced responses.