Won Joon Kim

Increase of [Ca2+]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and Rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.

The Action of Diazepam in the Isolated Rat Detrusor Muscle

Diazepam is one of the benzodiazepines, a group of drugs that depresses the central nervous system. It also inhibits the contractility of smooth muscles in the periphery, but the mechanism of this inhibitory action has not been clarified. Our study was undertaken to investigate the effect of diazepam on the contractility of the detrusor muscle. Detrusor muscle strips isolated from rat urinary bladder were examined by isometric myography. Diazepam, as well as baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, reduced the electric field stimulation-induced contractions; delta-aminovaleric acid, a GABAB receptor antagonist, completely antagonized the inhibitory effect of baclofen, but not the inhibitory action of diazepam. Diazepam reduced the basal tone of detrusor muscle concentration dependently, and this inhibitory action was not affected by tetrodotoxin. Diazepam suppressed the contractile responses to bethanechol, adenosine triphosphate and potassium chloride. Diazepam diminished the calcium-induced recovery of tension in calcium-free PSS. A23187, a calcium ionophore, partially recovered the basal tone which had been reduced by diazepam in normal physiologic salt solution (PSS). The loss of tension in calcium free PSS containing diazepam could not be recovered by addition of A23187. On the other hand, the loss of tension in calcium-free PSS containing 3,4,5-trimethoxybenzoic acid 8(diethylamino)octyl ester (TMB-8), an inhibitor of intracellular calcium release, was considerably recovered by addition of A23187. Based on these results, it is suggested that diazepam inhibits the contractility of detrusor muscle acting directly on the smooth muscle cell, which is unrelated to the activation of GABA receptors. Its inhibitory action appears to be mediated through interference with the influx of extracellular calcium.

THE EFFECTS OF DIAZEPAM ON THE CARBACHOL INDUCED CONTRACTION OF THE ISOLATED RAT ILEUM

To investigate the effect of diazepam on the contractility of the intestinal smooth muscle, longitudinal muscle strip isolated from rat ileum was prepared for myography in isolated organ bath. 1) Basal tone of ileal muscle was reduced by diazepam concentration-dependently. 2) Higher concentrations(30 and 100 microM) of diazepam inhibited(p-induced tension recovery in calcium-free solution was inhibited in the presence of diazepam concentration-dependently. These results suggest diazepam reduces the contractility of the longitudinal muscle isolated from rat ileum via interference with influx of calcium into the muscle cells.