Effect of different calcium modulators on motilin-induced contractions of the rabbit duodenum. Comparison with acetylcholine

Motilin and acetylcholine (ACh) have a direct contractile effect on rabbit small intestinal smooth muscle. To explore the role of calcium influx in these contractions, we studied the effect of extracellular calcium concentration and of calcium antagonists on the response of longitudinal muscle preparations from rabbit duodenum. Motilin- (10(-7) M) and ACh- (10(-4) M)-induced contractions were abolished in Ca2+-depleted medium. ACh (10(-4) M) or motilin (10(-8) and 10(-7) M) increased the contractile response to added Ca2+ to 130 +/- 6%, 129 +/- 10% and 145 +/- 5% of the maximal response to Ca2+ added alone (10 mM in a cumulative concentration response curve). The sensitivity to Ca2+ was greater in the presence of ACh and motilin (EC50 = 1.0 and 1.1 mM Ca2+) than in the absence of any agonist (1.7 mM). In cumulative concentration response (CCR) curves for motilin and ACh, pD2'-values were 7.0 and 6.6 for diltiazem, 8.4 and 7.8 for verapamil (two calcium entry blockers), 5.6 and 5.2 for TMB-8 (an inhibitor of intracellular calcium), 5.3 and 5.2 for TFP (a calmodulin-antagonist). All CCR-curves showed metactoid-like action of the antagonistic drugs. We conclude that ACh and motilin cause calcium to enter the smooth muscle cell. They are probably operating via separate channels, and use a mechanism which differs from K+-induced influx. Intracellular calcium stores appear to play a minor role in these contractions.     

Relationship between extra and intracellular sources of calcium and the contractile effect of thiopental in rat aorta

To evaluate the relationship between the vasocontractile effect of thiopental and the extra and intracellular sources of Ca2+, we analyzed both the contractile effect of the barbiturate on rat aortic rings and its ability to modify the intracellular calcium concentration in cultured rat aorta smooth muscle cells. Thiopental (10-310 microg/mL) contracted aortic rings only in the presence of extracellular Ca2+, and this effect was not blocked by verapamil or diltiazem. On the contrary, Ca2+ (0.1-3.1 mM) evoked contractions only when thiopental (100 microg/mL) was present. Although in calcium-free solution thiopental (100 microg/mL) did not contract aortic rings, it abolished the contractile effect of either phenylephrine (10(-6) M) or caffeine (10 mM). Finally, thiopental augmented the intracellular calcium concentration in cultured smooth muscle cells incubated either in the presence or absence of calcium. In conclusion, thiopental's vasocontractile effect depends on extracellular calcium influx, which is independent of L-calcium channels. The increase in intracellular Ca2+ concentration elicited by thiopental in Ca2+-free solution and its ability to block the effect of phenylephrine and caffeine suggest that this barbiturate can deplete intracellular pools of calcium. Therefore, the calcium entry pathway associated with the contractile effect of thiopental may correspond to the capacitative calcium entry model.     

Comparative effects of endothelin and phorbol 12-13 dibutyrate in rat aorta

The vasoconstrictive properties of endothelin (ET-1) and the protein kinase C activator, phorbol 12-13 dibutyrate (PDB) were comparatively investigated in isolated rat aorta. ET-1 (0.3-100 nM) and PDB (10 nM-3 microM) induced a slowly developing sustained contraction in endothelium denuded aorta. Maximal contractions induced by ET-1 and PDB were unaffected by diltiazem (10 microM). Substantial contraction to ET-1 (30 nM) and PDB (0.1 microM) remained in calcium-free medium. Contractions of ET-1 and PDB in calcium-free medium were unaffected by intracellular calcium depletion induced by phenylephrine. Following the response to ET-1 and PDB in a calcium-free medium, an additional sustained contraction was observed after calcium (2.5 mM) was added to the bath. The protein kinase C inhibitor, H7 (100 microM) was more potent in inhibiting contractions induced by phenylephrine and KCl than the ones elicited by ET-1 and PDB. The other protein kinase C inhibitors i.e. staurosporine (50 nM) and phloretin (100 microM) inhibited to a similar extent all the agonists tested. These results suggest that protein kinase C may play an important role in mediating the contraction to ET-1 in rat aorta.     

Ca2+ channel antagonist actions in bladder smooth muscle: comparative pharmacologic and [3H]nitrendipine binding studies

The actions of a series of 15 Ca2+ channel antagonists including D-600, nifedipine, and diltiazem were examined against K+ depolarization and muscarinic receptor induced responses in guinea pig bladder smooth muscle. Responses of bladder are very dependent upon extracellular Ca2+ and sensitive to the Ca2+ channel antagonists, the tonic component more than the phasic component of response. Regardless of stimulant, K+ or methylfurmethide (MF), or component of response, the same rank order of antagonist activities is expressed, suggestive of a single structure-activity relationship and the existence of a single category of binding site which may, however, exist in several affinity states. High affinity binding of [3H]nitrendipine (KD = 1.1 X 10(-10) M) occurs in bladder membranes, and similar high affinity binding was found in microsomal preparations from other smooth muscles including guinea pig and rat lung, rat vas deferens, uterus, and stomach. [3H]nitrendipine binding in the bladder was sensitive to displacement by other 1,4-dihydropyridines, paralleling their pharmacologic activities and showing excellent agreement with binding data previously obtained for guinea pig ileal smooth muscle. Comparison of pharmacologic data for inhibition of K+- and MF-induced responses by a common series of Ca2+ channel antagonists in bladder and ileum revealed excellent correlations. Neither pharmacologic nor binding studies suggest significant differences in Ca2+ channel antagonist properties in smooth muscle from bladder and intestine.     

Receptor-operated calcium-channels in isolated rabbit jejunum.

Calcium channels were studied in isolated spontaneously rhythmic rabbit jejunum using the muscarinic agonist carbachol as stimulant. Carbachol failed to produce the characteristic phasic and tonic components of smooth muscle contractions. A variety of chemically distinct calcium antagonists, viz. bepridil, diltiazem, isradipine (PN 200-110), nifedipine, and verapamil, non-competitively inhibited the contractions. Diltiazem was most potent (-logIC50 = 8.30) and bepridil least potent (-logIC50 = 6.19) in inhibiting the contractions. The findings conclude with the presence of pharmacologically distinct receptor-operated calcium-channels, besides the potential-dependent calcium-channels, in the rabbit jejunum.     

Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

Effect of lignocaine on intestinal smooth muscle of rat ileum

The effect of lignocaine on tone and contractility of intestinal smooth muscle, and on contractures produced by ACh or TEA, was studied in isolated ileum of the rat. Lignocaine (0.1-100 microM) produced concentration-dependent contractures in the rat ileum. In low concentrations, lignocaine increased the amplitude of spontaneous contractions and contractions produced by transmural stimulation. High concentrations of lignocaine abolished all contractile responses and produced a marked contracture in rat ileum. Lignocaine (10 microM) also reduced the contractures produced by ACh (0.01-10 microM). In contrast, the contractures produced by TEA (0.1-10 mM) were markedly increased by lignocaine. Furthermore, the contracture produced by lignocaine was reduced by lowering the external calcium from 2.5 mM to 1.5 mM. It was concluded that lignocaine in moderate and high concentrations produces a contracture in rat intestinal smooth muscle. Whereas lignocaine reduces the ACh-induced contracture, it increases that produced by TEA in the same preparation. The results further suggest that lignocaine modifies cholinergic responses and affects excitation-contraction coupling in rat intestinal smooth muscle.     

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta.

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.     

Sarmesin is a partial agonist of angiotensin-II receptors in rabbit, but not in rat, aortic rings

Sarmesin, [Sar1, Tyr(Me)4]angiotensinII], has been reported to be a competitive angiotensin II (AII) receptor antagonist in rat smooth muscle preparations (Scanlon et al., (1984), Life Science 34, 317-321). In the present study, sarmesin displaced AII from its binding sites in rat aortic smooth muscle cells and in a rabbit aorta membrane preparation (IC50 5 and 6 nM resp.; Ki 4.1 and 5.3 resp.) In rabbit aortic rings, sarmesin (0.003-3 microM) produced concentration-dependent contractions (ED50 89 nM) and this effect was inhibited by saralasin. No contraction was observed in the rat aorta up to 100 microM. In rabbit aortic rings, sarmesin, at the same concentrations that produced contraction, inhibited contractions induced by AII in a competitive manner (pA2 7, 26). These results indicate that, in rabbit aortic rings sarmesin is a partial agonist of AII receptors.     

Effect of nitrendipine on isolated uterus and other smooth muscles of the rat

Increasing concentrations of nitrendipine were found to inhibit various types of muscular activation (electrical stimulation, acetylcholine, oxytocin, potassium chloride), as well as the spontaneous rhythmic activity of the isolated rat uterus. The degree of the inhibitory effect of nitrendipine depends on the type of activation. Nitrendipine showed an exceptionally high efficacy in inhibiting contractions induced by electrical stimulation and of spontaneous rhythmic activity. For inhibition of these contractions even osmolar concentrations of nitrendipine were sufficient. The relaxant effect of nitrendipine depended on the concentration of extracellular calcium and the time of incubation of nitrendipine in the bathing medium. Nitrendipine showed high selectivity for the uterine smooth muscle because in a very high concentrations is exerted an insignificant relaxation of the other isolated smooth muscles (oesophagus, urinary bladder). Our experiments indicate that nitrendipine might have a role in therapy of premature delivery and abortion because of its great selectivity for the uterine smooth muscle. Addition of calcium into the medium restores completely all types of muscular activation after the inhibitory action of nitrendipine except its depressive action on the phasic component of oxytocin-induced contractions. These findings that individual types of activation, after inhibitory action of nitrendipine, are reestablished in various degrees by the addition of calcium into the medium, are also an additional confirmation about the existence of various types of calcium channels.     

Serotonin reuptake inhibitors fluoxetine and citalopram relax intestinal smooth muscle

Selective serotonin reuptake inhibitor antidepressants (SSRIs) exert depressant effects on cardiac myocytes and vascular smooth muscle cells by inhibiting Ca2+ channels. We hypothesized that the SSRIs fluoxetine and citalopram affect the contractile activity of intestinal smooth muscle by interfering with Ca2+ entry and (or) signaling pathways. The effects of fluoxetine and citalopram on contractions of guinea-pig ileum longitudinal muscle-myenteric plexus preparations (LMMP) were compared with the effects of the voltage-operated Ca2+ channel inhibitors nifedipine and diltiazem. In a concentration-dependent manner, nifedipine, diltiazem, fluoxetine, and citalopram elicited relaxation of LMMPs contracted by electrical field stimulation (EC50 values of 4 x 10(-7) M, 1.4 x 10(-6) M, 1.4 x 10(-5), and 6.8 x 10(-6) M, respectively). Nifedipine, diltiazem, fluoxetine, and citalopram also relaxed LMMPs contracted with a depolarizing concentration of KCl (48 mM; EC50 values of 1.8 x 10(-8) M, 1.4 x 10(-7) M, 3.7 x 10(-6) M, and 6.3 x 10(-6), respectively), a response that could be reversed by increasing the extracellular Ca2+ concentration (2.5-30 mM). These data suggest that fluoxetine and citalopram elicit relaxation of intestinal smooth muscle, likely by inhibiting Ca2+ channel(s). This effect may be of clinical importance.     

Mechanisms of the contractile effect of the hydroalcoholic extract of Cissampelos sympodialis Eichl. in the rat aorta.

In the present work the effect of the aqueous fraction of the ethanolic extract of the leaves (AFL) of Cissampelos sympodialis Eichl. was investigated in the rat aorta. In the presence of functional endothelium, AFL produced concentration-dependent contractions (EC50 value of 76.6 +/- 17.8 micrograms/ml). In the absence of functional endothelium, the concentration-response curves to AFL were significantly shifted to the left (EC50 values of 1.3 +/- 0.9 micrograms/ml) without modification of its maximal contractile effect. In the presence of L-NAME (300 microM) and of indomethacin (10 mM), the concentration-response curves produced by AFL were also shifted to the left (EC50 values of 21.8 +/- 6.2 and 24.3 +/- 13.2 micrograms/ml, respectively). The treatment of the aortas with L-NAME (300 microM) plus indomethacin (10 microM) produced a significant shift to the left of the concentration-dependent curves of AFL (EC50 value of 4.9 +/- 2.2 micrograms/ml), similar to that observed in the absence of the vascular endothelium. In addition, AFL-induced contraction was abolished in the presence of prazosin (1 microM), and significantly shifted to the right in the presence of yohimbine (EC50 value of 723.6 +/- 76.4 micrograms/ml). Thus, based on these results, it can be concluded that contractions induced by AFL in the rat aorta were due to activation of alpha-adrenoceptors. Furthermore, these results also showed that the AFL-induced contractions were modulated by the endothelium, via the release of NO and of a cyclooxygenase-derived relaxant product. Finally, it can be concluded that the contractile effects of AFL on vascular smooth muscle may play an important role in the hypertensive effects of this plant in vivo.     

Characterization of intestinal smooth muscle responses and binding sites for endothelin.

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k1 value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

DuP 532: a second generation of nonpeptide angiotensin II receptor antagonists

DuP 532 is a novel nonpeptide angiotensin II (AII) receptor antagonist under development for the treatment of hypertension. DuP 532 is a more potent antihypertensive agent in renal hypertensive rats (ED30 = 0.042 mg/kg, i.v.) and displays a similar or longer duration of action than the previously described AII antagonist, DuP 753. DuP 532, in contrast to DuP 753, is a noncompetitive antagonist of AII-induced contractions of rabbit aortic strips (KB = 1.1 x 10(-10) M). However, the inhibition of AII binding by DuP 532 in rat adrenal cortex does not correlate with either the aortic contractile response or with the hypotensive response. Assay conditions were evaluated and the presence or absence of BSA was shown to markedly affect the apparent binding affinity of DuP 532 and other 5-carboxylic acid derivatives. DuP 753 and other compounds were much less affected. The IC50 for DuP 532 was 4.7 x 10(-6) M with and 3 x 10(-9) M without BSA. The IC50s for DuP 753 were 1.7 x 10(-8) M with and 5 x -9 M without BSA. Both compounds with or without BSA did not completely inhibit AII binding which is characteristic of AT1 selectivity. BSA also reduced the effect of DuP 532 on the AII-induced contractions of rat main pulmonary artery preparations and the AII-induced Ca2+ mobilization in rat aortic smooth muscle cells. DuP 532 was very specific for AT1 receptors and did not interfere with receptors associated with neurotensin, prazosin, bradykinin, nitrendipine, or vasopressin. It is concluded that DuP 532 represents a new class of specific, but noncompetitive. AII receptor antagonists whose binding characteristics may provide new insight into AII receptor function.     

Contraction of cultured human uterine smooth muscle cells after stimulation with endothelin-1

To our knowledge, the problem of how to maintain isolated smooth cells in a "contractile" phenotypic state without deviation after subculturing has yet to be resolved. The present study characterized the in vitro contractile response of human uterine smooth muscle cell to endothelin-1, which induces contractions in isolated uterine strips. Contractile effects were qualitatively investigated using silicone rubber substrata. Endothelin-1 was able to distort and reduce the wrinkles in the silicone surface. Contractions were also quantified by measuring the resulting change in the collagen lattice area. Endothelin-1 significantly increased the contractile response in a dose-dependent manner by selectively activating endothelin A receptors. When myometrial cells were cultured within collagen lattices, a microfilament-disrupting agent, cytochalasin B, abolished contractions, and no change was observed in smooth muscle alpha-actin immunostaining. Taken together, these observations show that the uterine smooth muscle cells are contractile and respond appropriately to a potent uterotonic agent. Based on these findings, a cultured uterine smooth muscle cell model, which could be used to elucidate the mechanisms controlling uterine activity, is proposed.     

Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization

Cultured endothelial cells release a potent vasoconstrictor peptide, endothelin. Cumulative addition of synthetic endothelin to isolated rabbit aortic rings elicited a concentration-dependent increase in contractile tension which was endothelium-independent. In cultured rabbit vascular smooth muscle cells loaded with the fluorescent dye fura 2, endothelin induced a concentration-dependent increase in [Ca2+]i over the range of 0.01 to 100 nM. Moreover, in the absence of extracellular Ca2+, endothelin could still induce an increase in [Ca2+]i. In addition, endothelin stimulated 45Ca2+ efflux from preloaded vascular smooth muscle cells in the presence and absence of extracellular Ca2+, as well as stimulating 45Ca2+ influx in a concentration-dependent manner. Measurement of inositol phosphates in [3H]-myoinositol-labelled vascular vascular trisphosphate. Unlabelled endothelin inhibited (125I)-endothelin binding to cultured rabbit vascular smooth muscle cells in a concentration-dependent manner. Binding was not inhibited by other vasoactive hormones or calcium channel ligands, suggesting cell surface receptors specific for endothelin. We conclude that one of the initial membrane events in the action of endothelin is to induce phospholipase C-stimulated PIP2 hydrolysis and that this signalling mechanism is initiated by endothelin/receptor interaction at the plasma membrane.     

A comparative study of endothelin and sarafotoxin action in vascular and non-vascular smooth muscle

The effects of endothelin and sarafotoxin on smooth muscle tone have been examined in the rat aorta and anococcygeus muscle and their actions compared to those of norepinephrine. The contractions elicited by endothelin and sarafotoxin (10 nM), or norepinephrine (1 μM) were approximately equieffective in terms of tension development and correspond to EC₅₀ values and these concentrations were thus used throughout the study. In calcium-free Krebs the three agonists generated approximately similar levels of tone in the aorta and the anococcygeus corresponding to 20 and 5% of the maximum response, respectively. Nifedipine, 10 μM, significantly inhibited responses to endothelin and norepinephrine in the aorta but only norepinephrine in anococcygeus; the responses to sarafotoxin were however not significantly affected in either tissue. A combination of 10 μM ryanodine and nifedipine caused near complete inhibition of the response to endothelin in the aorta and also significantly reduced the response to both endothelin and norepinephrine in the anococcygeus. The lipoxgenase inhibitor, nordihydroguaiaretic acid, inhibited the response to endothelin in the aorta and endothelin and norepinephrine in the anococcygeus muscle. The cyclooxygenase inhibitor, indomethacin, however, had no effect on the responses to any of the three agonists in either the aorta or anococcygeus. At concentrations greater than 30 nM both endothelin and sarafotoxin induced myogenic activity in normally quiescent anococcygeus muscle. As determined by the loss of myogenic activity the tissues recovered more rapidly from sarafotoxin than endothelin with complete recovery apparent after 2.62 ± 0.85 and 5.22 ± 0.06 h respectively. Omitting Ca²⁺ from the Krebs solution reduced recovery times to 1.62 ± 0.2 and 2.4 ± 0.51 h respectively.

Overall the results suggest that endothelin and sarafotoxin activate different cell signaling systems to differing extents in rat aorta versus anococcygeus suggesting that the membrane receptors mediating the responses to endothelin and sarafotoxin are not necessarily identical.     

Dihydropyridine sensitive calcium channels in a smooth muscle cell line

The pharmacological properties of voltage sensitive calcium channels (VSCC) were examined in a rat aortic smooth muscle cell line (A10). The inorganic VSCC blockers Co2+ and Cd2+ blocked 45Ca2+ uptake into these cells in both 5 mM K+ and 50 mM K+ (depolarizing) conditions. The organic VSCC antagonists nitrendipine, nimodipine, D-600 and diltiazem also blocked 45Ca2+ uptake at low concentrations. The relative potencies of blockade were similar to those found in intact vascular smooth muscle. The VSCC "agonist" BAY K8644 enhanced 45Ca2+ uptake and this effect could be reversed by nitrendipine. These results indicate that A10 cells possess VSCC and that these VSCC behave similarly to those in authentic smooth muscle.     

Mechanisms of alpha-adrenoceptor mediated contractions of rat mesenteric artery

The nature of the calcium channels associated with the activation of alpha-adrenoceptors in vascular smooth muscle has been investigated. The inhibitory effects of nitrendipine, a calcium antagonist, were studied on the contractions elicited by alpha-adrenoceptor agonists in rat superior mesenteric artery. Responses to equieffective concentrations of phenylephrine (alpha 1-adrenoceptor agonist), clonidine and BHT-920, (alpha 2-adrenoceptor agonists), and noradrenaline (nonselective agonist) were inhibited differentially by the calcium antagonist, with the sensitivity order being as follows: BHT-920 = clonidine greater than phenylephrine greater than noradrenaline. When the contractions to two doses of noradrenaline were compared, the low dose response was more sensitive to nitrendipine inhibition than the high dose response. This differential inhibition was not seen for noradrenaline in the presence of verapamil or for phenylephrine in the presence of nitrendipine. The contractions of the vessel to the agonists in zero calcium conditions were not significantly different from each other. The sensitivity differences among the agonists to nitrendipine may arise from differences in the postreceptor mechanisms of activation. The differential sensitivity of noradrenaline responses suggests a greater heterogeneity of calcium channels than those available for the other agonists.     

Dissociation of the metabolic from the contractile response to muscarinic stimulation in the rabbit urinary bladder

The calcium dependence of contraction and NADH flurorescence was investigated in rabbit bladder stimulated with bethanechol or KCl. The absence of calcium in the bathing solution induced a rightward shift in the dose response to bethanechol for both contraction and NADH flurorescence. The contractile response was shifted to a greater degree than the fluorescence response and the maximal response to bethanechol was reduced by 80% for contraction but only 20% for NADH fluorescence. This rightward shift was also induced by the benzothiazepine calcium antagonist diltiazem (200 M) and again the contractile response was shifted significantly more than the fluorescence response. The combination of zero calcium and 200 M diltiazem virtually abolished contractions but only inhibited the NADH fluorescence by 65% at maximally effective bethanechol concentrations. Unlike the effect of diltiazem on the response to bethanechol, diltiazem (200 M) shifted both the contraction and fluorescence curves to the right equally in response to KCl stimulation. These results indicate that a metabolic response to muscarinic stimulation (decreased NADH) can occur in the absence of any observable contractile response. This metabolic response may be due to post receptor signal processing events. For KCl stimulation, the NADH response is probably secondary to and a result of the contractile response.Abbreviations ATP Adenosine Triphosphate - KCl Potassium Chloride - HPLC High Performance Liquid Chromatography - NADH reduced nicotinamide Adenine Dinucleotide - NAD Oxidized Nicotinamide Adenine Dinucleotide