Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro.

The function of the bladder urothelium in modulating contractile responses of the underlying detrusor smooth muscle to muscarinic stimulation has been examined in the pig bladder. Saturation curves for [3H]-QNB binding demonstrated a greater muscarinic receptor density in the urothelium than in the detrusor smooth muscle. The presence of an intact urothelium on isolated bladder strips inhibited contractions induced by carbachol but not KCl. Contractions of a urothelium-denuded muscle strip were inhibited in the presence of a second bladder strip with an intact urothelium, but not if the second strip was denuded. The urothelium-induced inhibition of contractions was not prevented in the presence of L-NOARG, methylene blue, indomethacin, propranolol, suramin, TEA or apamin. The data suggest the presence of a diffusable, urothelium-derived inhibitory factor, which could not be identified but appears to be neither nitric oxide, a cyclo-oxygenase product, a catecholamine, adenosine, GABA nor an EDHF sensitive to apamin.

Chronic ethanol exposure potentiates muscarinic receptor and alpha 2-adrenoceptor-mediated inhibition of cAMP accumulation in PC 12 cells.

The effects of ethanol on receptor-mediated inhibition of cAMP production were investigated in PC 12 cells. The in vitro addition of ethanol enhanced N-ethylcarboxyadenosine (NECA)-stimulated cAMP production without altering the inhibitory action of carbachol or epinephrine. A 4 day exposure of PC 12 cells to ethanol decreased the stimulation of cAMP production by NECA, but increased the inhibition of NECA-stimulated cAMP production by carbachol and epinephrine.

Effect of an homologous series of aliphatic alcohols on neuronal and smooth muscle voltage-dependent Ca2+ channels.

The acute inhibitory actions of alcohol on K(+)-stimulated 45Ca2+ uptake into synaptosomes shows regional variation in sensitivity throughout the brain, suggesting the possibility of a selective action on a specific Ca2+ channel subtype. This was examined by comparing the effects of a homologous series of aliphatic alcohols on synaptosomal Ca2+ channels with their actions on K(+)-stimulated Ca2+ channels in guinea-pig intestinal longitudinal muscle, which have been demonstrated to be of the L-type. K(+)-stimulated contraction of and [3H]nitrendipine binding to smooth muscle were both inhibited by the alcohols at similar concentrations, with the potency increasing with chain length. In synaptosomes, however, K(+)-stimulated 45Ca2+ uptake was 5-30 times more sensitive to the inhibitory actions of alcohol than were [3H]nitrendipine and [125I]omega-conotoxin binding. These observations suggest that K(+)-stimulated 45Ca2+ uptake is mediated by a non-L non-N type channel which is more sensitive to the acute effects of alcohols. This is supported by the observation that K(+)-stimulated 45Ca2+ uptake which is insensitive to L- and N-channel antagonists was inhibited by funnel web spider venom.

Effect of thyroid status on beta-adrenoceptors and calcium channels in rat cardiac and vascular tissue.

To determine the influence of thyroid hormone on beta-adrenoceptors and Ca2+ channels, rats were treated with thyroxine (75 micrograms/100 g sc daily for 5 days) or propylthiouracil (0.05% in drinking water for 30 days). beta-Adrenoceptor density in ventricular tissue, measured by [125I]iodocyanopindolol binding, was significantly increased and decreased respectively, following thyroxine or propylthiouracil treatment to 124.7 +/- 7.11 fmol/mg protein and 71.98 +/- 5.37 fmol/mg protein from euthyroid (control) levels of 93.7 +/- 4.58 fmol/mg protein. Ca2+ channel density, measured by [3H]nitrendipine binding, was altered in the opposite direction; it was significantly decreased and increased to 324 +/- 24 fmol/mg protein and 691 +/- 31 fmol/mg protein from 562 +/- 35 fmol/mg protein after thyroxine or propylthiouracil treatment, respectively. No changes in affinity of either ligand were observed. Responses of isolated papillary muscles from propylthiouracil-treated animals accorded with changes seen in the binding studies. The geometric mean EC50 of isoproterenol increased from 9.5 x 10(-9) mol/l to 5.5 x 10(-8) mol/l, and the EC50 for calcium decreased from 3.16 x 10(-3) mol/l to 1.36 x 10(-3) mol/l; moreover, the responsiveness to the Ca2+ channel activator Bay K 8644 was increased. The corresponding responses in thyroxine-treated animals could not be examined because of prominent arrhythmic activity. As with papillary muscles the sensitivity of left atria to isoproterenol was decreased after treatment with propylthiouracil, with geometric mean EC50 values increasing from 3.21 x 10(-9) mol/l to 89.4 x 10(-9) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of adaptive supersensitivity in the left atrium of the guinea pig.

Chronic pretreatment of guinea pigs with reserpine (0.1 mg/kg/day for 7 days) induces inotropic supersensitivity in left atria. The sensitivity is increased to isoproterenol, norepinephrine, 5'-guanylylimidodiphosphate and forskolin, and the maximum response is increased to the partial agonist albuterol. These results, coupled with data in the literature, suggest that adaptive supersensitivity in the guinea pig heart is due to a change in one or more of the components of the adenylate cyclase system that is specifically coupled to beta adrenoceptors. The results indicate that the supersensitivity is demonstrable when the concentration-response curves for agonists are determined in isolated whole left atria but not when they are determined in strips cut from left atria. This explains a discrepancy in the literature. It is suggested that cellular changes, possibly in electrolyte distribution, resulting from the additional manipulation of cutting the atria into slices obscure the sensitivity difference between the control and experimental tissues.

The use of forskolin to investigate the site of cardiac beta-adrenoreceptor supersensitivity.

The positive inotropic responses of left atria and papillary muscles and the positive chronotropic responses of right atria of guinea-pigs to isoprenaline and forskolin were examined. An increase in sensitivity of the three preparations to isoprenaline was observed by lowering the bath temperature from 38 to 30 degrees C as demonstrated by a leftwards shift of the concentration-response curves. A similar degree of supersensitivity was observed for forskolin. Since forskolin is reputed to stimulate adenylate cyclase directly, whereas isoprenaline stimulates via the regulatory nucleotide Ns protein, this would suggest a common site for the supersensitivity at adenylate cyclase. However, the possibility that forskolin also stimulates via the Ns protein in producing cardiac stimulation and that this is the site of hypothermia-induced supersensitivity is discussed. Supersensitivity to isoprenaline was also observed in left atria and papillary muscles from guinea-pigs chronically pretreated with reserpine for 3 days (5 mg/kg at 72 h, 3 mg/kg at 48 and 24 h) or 7 days (0.1 mg/kg daily). In the same tissues, there was no change in the sensitivity to forskolin. The site of the supersensitivity can therefore be concluded to occur before the level of adenylate cyclase activation either directly or via the regulatory Ns protein; possibly at the beta-adrenoreceptor itself.

Examination of the bronchoconstrictor response of guinea-pig isolated lung to beta-phenylethylamine.

beta-Phenylethylamine (PEA) produced a contraction of the guinea-pig isolated lung parenchymal strip and bronchoconstriction of perfused lungs. Reserpine pretreatment had little effect on these responses indicating a substantial direct effect. Phentolamine (10(-6) and 10(-5) M) had minimal effect on PEA in the lung strip compared with that on noradrenaline, eliminating involvement of alpha-adrenoceptors. PEA was unaffected by atropine (10(-7)M) or a mepyramine (10(-7)M); metiamide (10(-4)M) mixture. The contraction was not therefore mediated via muscarinic or histaminergic receptors. 5-HT and dopamine receptors were also discounted. Possible stimulation of a phenylethylaminergic receptor and its relevance to bronchial asthma is discussed.

Reserpine-induced supersensitivity occurs for beta-adrenoceptor-mediated responses of heart and trachea but not of the uterus and lung.

This study was undertaken to determine whether reserpine-induced supersensitivity occurs in tissues containing beta1-adrenoceptors and in those with beta 2-adrenoceptors. Guinea-pigs and rats were pretreated with reserpine for either 3 days (5 mg kg-1 i.p. at 72 h, 3 mg kg-1 at 48 h and 3 mg kg-1 at 24 h before use) or 7 days (1 mg kg-1 daily). The sensitivities of left and right atria, papillary muscles, tracheal spirals, lung strips and uteri to isoprenaline were compared with those from untreated animals. The positive inotropic responses of left atria and papillary muscles and chronotropic responses of right atria from reserpine-pretreated animals were supersensitive to isoprenaline, the concentration-response curves being to the left. The relaxation response of the carbachol-contracted trachea also exhibited supersensitivity, but to a lesser extent. However, no supersensitivity occurred for the relaxation of carbachol-contracted lungs, K+-depolarized guinea-pig uteri or electrically stimulated rat uteri. As a pharmacological index of the presence of releasable noradrenergic stores, tyramine was added cumulatively to each tissue. Only cardiac and tracheal preparations yielded substantial responses, indicating the presence of sympathetic innervation. A relaxation of the rat uterus by tyramine was not attributable to releasable noradrenaline stores. The supersensitivity of the heart and trachea could therefore be associated with their sympathetic innervation and with the fact that their responses are mediated via beta 1-adrenoceptors; the trachea containing a small proportion of beta 1-adrenoceptors. The responses of the lung and uterus, however, are beta 2-adrenoceptor-mediated and failed to exhibit supersensitivity. Since the supersensitivity is a consequence of the neuronal depleting action of reserpine, these results are compatible with the concept that beta 1-adrenoceptors are associated with sympathetic innervation whereas beta 2-adrenoceptors are not.

Mechanisms of cardiac supersensitivity to sympathomimetic amines.

Supersensitivity of the beta-adrenoceptor-mediated positive inotropic and chronotropic responses of the heart to sympathomimetic amines is considered. The various types of supersensitivity of both pre- and post-synaptic origins are described and the possible mechanisms involved are discussed. Emphasis is given to the supersensitivity that arises from depletion of sympathetic catecholamine stores by reserpine and 6-hydroxydopamine. Results are presented which indicate that in both cases the supersensitivity develops slowly, is specific for the beta-adrenoceptor and is not associated with an increase in affinity of agonists for the receptor. Radioligand binding data is presented to show that there is no increase in the total number of beta-adrenoceptor binding sites in membrane fractions from ventricular homogenates of animals receiving reserpine. A review of the literature on binding studies indicates two opposing views with respect to the effects of reserpine on beta-adrenoceptor binding sites, one showing no change and the other showing an increase in total binding sites. Limited studies in the literature show that 6-hydroxydopamine may increase the number of sites. Binding data therefore remains inconclusive and must be interpreted with caution. The relationship between the development of depletion-induced supersensitivity and the innervation of the tissue and its receptor type is discussed.

beta-Adrenoceptor ligand binding and supersensitivity to isoprenaline of ventricular muscle after chronic reserpine pretreatment.

1. Isolated papillary muscles from guinea-pig hearts were paced at a constant frequency and isometric contractions reported. 2. Guinea-pigs were either untreated or pretreated with reserpine. Three pretreatment schedules were used; a) 0.5 mg kg-1 i.p. at 24 h, b) 5.0 mg kg-1 at 72 h and 3.0 mg kg-1 at 48 and 24 h, or c) 0.1 mg kg-1 daily for 7 days. 3. Cumulative concentration-response curves for the isoprenaline-induced increases in tension were obtained. The geometric mean EC50 values after the 3 and 7 day reserpine pretreatment schedules were significantly (P less than 0.05) less than for untreated guinea-pigs indicating a supersensitivity. 4. EC50 values for the positive inotropic responses to histamine and calcium in papillary muscles from reserpine-pretreated guinea-pigs did not differ significantly (P less than 0.05) from those from untreated animals. This suggests that the supersensitivity to isoprenaline is beta-adrenoceptor specific. 5. Membrane fractions were prepared from the ventricles of the untreated and reserpine-pretreated guinea-pigs from which papillary muscles had been removed. Binding of [3H]-dihydroalprenolol ([3H]-DHA) to beta-adrenoceptors of these membranes was determined. Equilibrium dissociation constants (KD) and total numbers of binding sites (Bmax) were determined by Scatchard analysis of the saturation curves for [3H]-DHA binding. 6. There was no increase in affinity (fall in KD value) or change in the total number of binding sites associated with reserpine-induced supersensitivity. The equilibrium inhibition constant (Ki) for the displacement of [3H]-DHA binding by isoprenaline was also identical in membranes from untreated and reserpine-pretreated animals. Thus reserpine-induced supersensitivity to isoprenaline does not appear to involve a change in affinity for the beta-adrenoceptor or in receptor numbers as determined by [3H]-DHA binding.