Bradykinin B2 receptors and coupling mechanisms in the smooth muscle of the guinea-pig taenia caeci.

1. In the smooth muscle of the guinea-pig taenia caeci, bradykinin produces a relaxation followed by a contraction. In the presence of hexamethonium and guanethidine, both these phases of the response were insensitive to tetrodotoxin (100 nM), omega-conotoxin GVIA (100 nM) and ibuprofen (1 microM), suggesting that they are due to a direct action on the smooth muscle. 2. The B1 receptor-selective agonist, [des-Arg9]-BK (1-100 microM), was inactive in the taenia caeci, and the B1 receptor-selective antagonist, [Leu8,des-Arg9]-BK (1-10 microM), did not inhibit either phase of the bradykinin-induced response. The B2 receptor-selective antagonist, D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (Hoe 140) (30-300 nM), inhibited both the bradykinin-induced relaxation and contraction with a similar affinity (apparent pKB estimates of 8.5 +/- 0.1 and 8.4 +/- 0.1 respectively). 3. In a depolarizing high-K(+)-solution, bradykinin produced concentration-related contractions, though of diminished magnitude; but no relaxation was observed in such media. In Krebs solution, the Ca(2+)-activated K(+)-channel blocker, apamin (10 nM), abolished relaxant responses. These observations suggest that contraction results both from membrane potential-dependent, and membrane potential-independent, mechanisms; whereas relaxant responses result entirely from membrane potential-dependent mechanisms. Contractile responses obtained in the high K(+)-solution were inhibited by D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK with an apparent pKB value of 8.4 +/- 0.1. 4. In a Ca(2+)-free, EGTA-containing medium, relatively high concentrations of bradykinin (> 100 nM) produced transient contractions, suggesting that a component of the contractile response results from release of Ca2+ from an intracellular store. This intracellular Ca2+ store could be refilled in the presence of extracellular Ca2+. The B, receptor antagonist, [Leu8,des-Argj-BK (10 micro M), did not inhibit this bradykinin-induced contraction, whereas the B2 receptor antagonist, D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK(100 nM) markedly attenuated it (P<0.001; n = 6).5. Bradykinin (10 nM- 100 micro M) significantly elevated tissue levels of total [3H]-inositol phosphates in the presence of Li?, after incubation with myo-[3H]-inositol. The B, receptor-selective agonist, [des-Argl-BK(100IM) did not stimulate [3H]-inositol phosphate formation, and the B, receptor-selective antagonist,[Leu8,des-Argl-BK, did not inhibit the formation of [3H]-inositol phosphates in response to a submaximal concentration of bradykinin (1I0 1M; P> 0.05). Two B2 receptor antagonists, D-Arg-[Hyp3,DPhe7]-BK and D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK, inhibited bradykinin-induced accumulation of total[3H]-inositol phosphates with apparent pKB estimates of 5.4 +/0 0.3 and 8.4 +/- 0.1, respectively.6. These data suggest that in the guinea-pig taenia caeci, the five aspects of the action of bradykinin studied (the relaxant and the contractile elements of the biphasic mechanical response, the contractile response in a depolarizing high-K' solution medium and zero-Ca2+ media, and stimulation of phosphatidylinositol turnover), all result from activation of B2 receptors. A possible causal relationship is suggested between these B2 receptor-mediated membrane potential-dependent, and -independent events,and their roles in excitation contraction coupling.

Bradykinin receptors in the guinea-pig taenia caeci are similar to proposed BK3 receptors in the guinea-pig trachea, and are blocked by HOE 140.

1. Bradykinin (BK) receptors of the guinea-pig taenia caeci were compared with those of the guinea-pig trachea, a preparation proposed to possess novel BK3 receptors. 2. Bradykinin-evoked contractile responses were unaffected in both preparations by the selective BK1 receptor antagonist [des-Arg9,Leu8]-BK (1 microM-10 microM). The BK2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]-BK, both had low affinities (apparent pKB estimates less than 6) which did not differ significantly between the two preparations (P greater than 0.05). In contrast, the novel bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140) potently antagonized responses to bradykinin with relatively high affinity (apparent pKB = 8.42 +/- 0.15 and 8.94 +/- 0.16 in the taenia caeci, and trachea, respectively). 3. We conclude that the bradykinin receptors in the guinea-pig taenia caeci have similar recognition properties to those present in the guinea-pig trachea, and in this respect the taenia caeci represents a useful preparation for the further study of proposed novel BK3 receptors.

Putative novel bradykinin B3 receptors in the smooth muscle of the guinea-pig taenia caeci and trachea.

The bradykinin receptors mediating contraction in smooth muscle of the guinea-pig taenia caeci were compared with the proposed novel B3 receptors of the guinea-pig trachea. The activities of several antagonists in functional and binding studies were found to be very similar between these two guinea-pig preparations, but pKBs were markedly lower than in a number of typical B2 preparations from other species, suggesting that the characteristics of the proposed B3 receptor may be in part species-related.

On the histamine-induced depolarization of the isolated superior cervical ganglion of the rat.

1. Using a grease-gap technique, we studied the action of histamine on the d.c. potential recorded between the internal carotid nerve and the main body of the isolated superior cervical ganglion of the rat. 2. A small, slow depolarization was evoked by 10-300 microM histamine. This response was not reduced by lowering the calcium concentration in the superfusing medium (from 2.5 to 0.1 mM), or by superfusing tetrodotoxin, N-methylatropine, or propranolol (all at 1 microM). 3. Mepyramine (10 nM) antagonized this depolarization, but cimetidine (10 microM), metiamide (30 microM), burimamide (10 microM) and impromidine (1 microM) did not. Two other agonists also evoked a mepyramine-sensitive slow depolarization. The rank order of potencies was histamine greater than N alpha-methyl-histamine greater than 2-methyl-histamine. 4. At concentrations greater than 1 mM, histamine also evoked a larger, faster depolarization. This response was undiminished by reducing the calcium concentration of the medium to 0.1 mM or by adding 1 microM tetrodotoxin. The rank order of potency for the agonists was N alpha-methyl-histamine greater than histamine approximately 2-methyl-histamine. The histamine-induced fast response was not antagonized by any of the above-mentioned antagonists. It was slightly reduced by (+)-tubocurarine (100 microM) and N-methylbicuculline (100 microM) but such effects were not consistent with the blockade of nicotinic or GABAA receptor-mediated responses. 5. It was concluded that histamine depolarized the isolated superior cervical ganglion of the rat by activating H1 receptors. Relatively high concentrations of histamine also evoked a fast depolarization of this preparation, but this did not appear to be mediated by H1, H2 or H3 receptors.

Hexahydrodifenidol does not distinguish among M1 receptors in rat cerebral cortex, hippocampus and superior cervical ganglion.

We have determined the antagonist affinity of hexahydrodifenidol in a range of receptor assays in the rat:-radioreceptor binding and phosphatidyl-inositol turnover assays in cerebral cortex and hippocampus, and electrophysiological experiments on the superior cervical ganglion and hippocampus. We failed to detect any appreciable differences in the affinity of hexahydrodifenidol among any of these assays.

Methoctramine and hexahydrodifenidol antagonise two muscarinic responses on the rat superior cervical ganglion with opposite selectivity.

Two novel muscarinic antagonists, methoctramine and hexahydrodifenidol, have been assessed for their action against two muscarinic agonist-induced responses on the rat superior cervical ganglion in vitro. DC recordings were made between the desheathed ganglion and its internal carotid nerve using the grease-gap technique. Hexahydrodifenidol and methoctramine antagonised the muscarine-induced M1-mediated depolarisation of this preparation with estimated pA2 values of 7.5 and 6.5, respectively. In 0.3 microM pirenzepine and 0.1 mM CaCl2, 1 microM muscarine evoked a hyperpolarisation mediated by cardiac-like M2 receptors. Hexahydrodifenidol and methoctramine antagonised this response with pIC50 values (-log10IC50) of 5.7 and 7.4, respectively. The selectivity of methoctramine for cardiac-like M2 receptors over M1 receptors is therefore confirmed and extended to these two neuronal responses. The selectivity of hexahydrodifenidol was opposite to, and greater than, that seen with methoctramine.