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.

Differential effects of chronic ethanol exposure on ATP- and bradykinin-induced increases in intracellular calcium levels in PC-12 cells.

The present study investigated the regulation of intracellular calcium levels ([Ca2+]i) by ethanol, ATP, and bradykinin (BK) in PC-12 cells. Acute addition of 150 mM ethanol increased [Ca2+]i but did not alter ATP- and BK-induced increases in [Ca2+]i. After a 4-day exposure to 150 mM ethanol, the maximal response to ATP was decreased 39.7 +/- 8.12% (p < 0.01), whereas that to BK was increased 43.8 +/- 6.81% (p < 0.01). There was no change in the EC50 values for either ATP or BK after chronic ethanol exposure. Addition of excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to remove extracellular free calcium prevented the ATP-induced increase in [Ca2+]i. In contrast, BK produced an increase in [Ca2+]i in the presence of excess EGTA, suggesting that BK releases calcium from internal stores. Consistent with this suggestion, chronic ethanol exposure enhanced BK-induced phosphoinositide hydrolysis. ATP, however, did not increase phosphoinositide hydrolysis. Pretreatment with 10 microM nifedipine, which blocked depolarization-evoked increases in [Ca2+]i, reduced ATP- and BK-induced increases in [Ca2+]i but did not alter the response to chronic ethanol exposure. Although acute addition of ethanol reduced KCl-stimulated increases in [Ca2+]i and 45Ca2+ uptake, chronic ethanol exposure did not alter the depolarization-induced increase in [Ca2+]i or 45Ca2+ uptake. The present study demonstrates that chronic ethanol exposure inhibits calcium influx through voltage-independent cationic channels associated with purinergic receptors and enhances BK-stimulated phosphoinositide hydrolysis, with a subsequent release of calcium from internal stores.

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.

Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4-dihydropyridine receptors.

The kinetic and equilibrium binding properties of the 1,4-dihydropyridine activator [3H](-)-S-Bay K 8644 and the antagonist [3H](+)-PN 200-110 were determined in rat heart membrane particulate preparations at temperatures between 4 and 37 degrees C. The binding of [3H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with KD = 3.57 nM and Bmax = 330 fmol/mg.protein at 25 degrees C. The association and dissociation rate constants were 3.4 x 10(7) min-1 M-1 and 0.095 min-1 respectively at 25 degrees C and decreased slightly at lower temperatures. In contrast, [3H](+)-PN 200-110 bound to high (KD(H) = 0.032 nM, Bmax(H) = 316 fmol/mg.protein) and low affinity sites (KD(L) = 27.6 nM and Bmax(L) = 6432 fmol/mg.protein) at 25 degrees C in rat heart preparation. A similar two-site binding of [3H](+)-PN 200-110 was found in rat brain preparation, but only a single binding site was detected in rat skeletal muscle. Binding of [3H](+)-PN 200-110 to the high and low affinity sites in cardiac membranes was sensitive and insensitive respectively to temperature. Association and dissociation rates of [3H](+)-PN 200-110 at the high affinity binding sites were best fitted as mono-exponential functions. Association and dissociation rates of [3H](+)-PN 200-110 were 3.94 x 10(8) min-1 M-1 and 7.86 x 10(-3) min-1 at 25 degrees C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4 degrees C. Thermodynamic analysis of equilibrium binding showed that the binding of activator was enthalpy driven, whereas the binding of antagonist to the high affinity site was both entropy- and enthalpy-driven and to the low affinity site was totally entropy-driven.

Iminodipropionitrile-induced dyskinesia in mice: striatal calcium channel changes and sensitivity to calcium channel antagonists.

Administration of 3,3'-iminodipropionitrile (IDPN) (1 g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN-induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200-110 binding sites (Bmax) in whole brains of IDPN-treated mice. Similarly, the K(+)-depolarization-dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 +/- 7 fmol/mg to 237 +/- 31 fmol/mg in control and treated groups, respectively) of PN 200-110 binding to the striatum without change of KD value (38 +/- 4.7 pM versus 33 +/- 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 +/- 10.1 pM to 45 +/- 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 +/- 51 fmol/mg versus 485 +/- 41 fmol/mg) of PN 200-110 binding to the cortex. IDPN did not alter omega-conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment as inhibited by L-type calcium channel antagonists and this may be associated with the observed increase in striatal L-type calcium channels.

Pharmacologic and radioligand binding analysis of the actions of 1,4-dihydropyridine activators related to Bay K 8644 in smooth muscle, cardiac muscle and neuronal preparations.

The structure-activity relationships of a series of 1,4-dihydropyridine Ca2+ channel activators, including Bay K 8644, have been determined by pharmacologic and radioligand binding techniques. Pharmacologic techniques included tension responses and the measurement of pA2 values for nifedipine antagonism of Bay K 8644 responses in guinea pig ileal, rat femoral and rat atrial and papillary muscle preparations. Radioligand binding experiments employed competition against [3H]nitrendipine binding in ileal smooth muscle and rat ventricular membranes and rat brain synaptosomal preparations. The series of compounds was employed as the racemates. Binding affinities were not significantly different between smooth muscle, cardiac muscle and brain preparations and the same rank order of pharmacologic activities is observed in smooth and cardiac muscle, where the effects of the 4-phenyl substituents, o greater than or equal to m greater than p, parallel those observed for 1,4-dihydropyridine antagonists. In the ileal and femoral artery smooth muscle preparations a 1:1 correlation is observed between pharmacologic and radioligand binding affinities. However, in the cardiac muscle preparations, left atrium and papillary muscle, there is an approximately 10-fold difference between the binding affinities and the lower pharmacologic affinities. A similar difference between smooth and cardiac muscle is observed with the pA2 values of 6.97 and 7.06 in atrial and papillary muscle respectively, which are significantly lower than the values of 8.54 and 8.72 measured in ileal and femoral artery respectively. The structure-activity expressions measured for this small series of 1,4-dihydropyridine activators parallel those observed in the larger series of 1,4-dihydropyridine antagonists. This is consistent with proposals that activators and antagonists interact at common binding sites that are components of a voltage-dependent Ca2+ channel.

Kainic acid lesions decrease striatal dopamine receptors and 1,4-dihydropyridine sites.

The effects of intrastriatal injection of kainic acid (2 microliters, 1 mg/ml) in the rat were determined. Four weeks after the lesioning, striatal dopamine receptors and 1,4-dihydropyridine sites were measured by radioligand binding with [3H]spiperone and [3H]nimodipine, respectively. Dopamine receptor and 1,4-dihydropyridine binding densities were decreased by 58% and 43% respectively, with no change in binding affinity for either ligand. 1,4-Dihydropyridine-sensitive Ca2+ channels may be located primarily on postsynaptic elements.

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)

The actions of peppermint oil and menthol on calcium channel dependent processes in intestinal, neuronal and cardiac preparations.

The activities of menthol and peppermint oil were determined in guinea-pig ileal smooth muscle, in rat and guinea-pig atrial and papillary muscle, in rat brain synaptosomes and in chick retinal neurones by pharmacological 45Ca2+ uptake and radioligand binding assays. Menthol is a major constituent of peppermint oil and is approximately twice as potent as peppermint oil as an inhibitor of K+ depolarization-induced and electrically stimulated responses in ileum and electrically stimulated atrial and papillary muscles. IC50 values in the ileal preparation ranged from 7.7 to 28.1 micrograms ml-1 and in the cardiac preparations from 10.1 to 68.5 micrograms ml-1. Similar potencies were demonstrated against K+ depolarization-induced 45Ca2+ uptake in synaptosomes and against K+ depolarization and Bay K 8644-induced uptake in chick retinal neurons. IC50 values for menthol inhibition of K+ and Bay K 8644 responses in the retinal neurons were 1.1 x 10(-4) M (17.2 micrograms ml-1) and 1.75 x 10(-4) M (26.6 micrograms ml-1), respectively, and for peppermint oil were 20.3 and 41.7 micrograms ml-1 respectively. Both menthol and peppermint oil inhibited specific [3H]nitrendipine and [3H]PN 200-110 binding to smooth and cardiac muscle and neuronal preparations with potencies comparable to, but slightly lower than, those measured in the pharmacological and 45Ca2+ uptake experiments. Binding of menthol and peppermint oil, studied at 78 micrograms ml-1, was competitive against [3H]nitrendipine in both smooth muscle and synaptosome preparations. The data indicate that both menthol and peppermint oil exert Ca2+ channel blocking properties which may underlie their use in irritable bowel syndrome. Ca2+ channel antagonism may not be the only pharmacological effect of menthol and peppermint oil contributing to intestinal smooth muscle relaxation.

Regulation by chronic drug administration of neuronal and cardiac calcium channel, beta-adrenoceptor and muscarinic receptor levels.

Chronic administration of atropine (40-100 mg/kg, 23 days) produced a 29-33% increase in muscarinic receptors, measured by [3H]quinuclidinyl benzilate binding, in rat brain. Diisopropyl phosphorofluoridate (0.9 mg/kg, 14 days) produced a 35% decrease in muscarinic receptors. Propranolol administration (800 micrograms/kg/hr, 10 days) increased beta-adrenoceptors, measured by [3H]dihydroalprenolol binding, by 69 and 50% in brain and heart respectively. Isoproterenol administration (800 micrograms/kg/hr, 10 days) produced a 50% reduction in cardiac beta-adrenoceptors but did not alter brain receptors. These drug treatments were without effect on binding of the Ca2+ channel ligands, [3H]nimodipine and [3H]nitrendipine, to brain or heart respectively. However, chronic administration of nifedipine for 20 days (36 and 360 micrograms/kg/hr) did produce down-regulation of both cardiac and neuronal Ca2+ channels and a similar down-regulation of beta-adrenoceptors. Co-regulation of Ca2+ channels and neurotransmitter receptors may occur but may not be an automatic consequence of either receptor or channel regulation.

Potential-dependent interactions of nitrendipine and related 1,4-dihydropyridines in functional smooth muscle preparations.

Interaction of nitrendipine and other Ca2+ channel antagonists including nifedipine, diltiazem, and D600 with intestinal smooth muscle was shown to depend on membrane potential. In the absence of extracellular Ca2+, guinea pig ileal longitudinal muscle does not contract and can be incubated at various K+ concentrations in the presence or absence of antagonist. Preincubation with elevated K+ prior to admission of Ca2+ and challenge with K+ to a total of 100 mM increased the activity of the antagonist. The IC50 for nitrendipine incubated in the presence of 5 mM K+ was 5.36 X 10(-9) M and in the presence of 40 mM K+ was 0.53 X 10(-9) M. Other 1,4-dihydropyridines showed similar potency shifts.

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.