The role of cyclic AMP in the positive inotropic effect mediated by β1- and β2-adrenoceptors in isolated human right atrium

Summary The time course of the effects of isoprenaline (3 × 10^–7 mol/l) on contractile force and on the cyclic AMP level was studied in the electrically driven isolated muscle strip of the human right atrium. Isoprenaline produced a rise in cyclic AMP content (maximum increase after 60 s) preceding the increase in contractile force. The effects of isoprenaline on contractile force and on the intracellular level of cyclic AMP were enhanced in the presence of the phosphodiesterase inhibitor papaverine (10^–5 mol/l). On the other hand, the -adrenoceptor antagonist propranolol (10^–7 mol/l) suppressed isoprenaline-induced cyclic AMP increases, but reduced the increase in force of contraction by only 35%. In addition, both the ₁-selective antagonist bisoprolol (3 × 10^–9 × 10^–8 mol/l) and the ₂-selective antagonist ICI 118,551 (3 × 10^–9 × 10^–8 mol/l) inhibited the isoprenaline-induced cyclic AMP increase concentration-dependently; ICI 118,551 produced more pronounced inhibition than bisoprolol. It is concluded that cyclic AMP is involved in the positive inotropic action of isoprenaline evoked by ₁- and ₂-adrenoceptor stimulation in isolated human right atrium; however, an additional cyclic AMP independent mechanism cannot be ruled out. Send offprint requests to O.-E. Brodde at the above address     

Responses to catecholamines of the rat isolated uterus throughout the natural oestrous cycle

Summary The effects of noradrenaline (NA), adrenaline (Adr) and isoprenaline (Iso) on rat isolated uterus were studied, throughout the natural oestrous cycle. The -inhibitory effects of the catecholamines were measured as a percentage inhibition of a standard acetylcholine (ACh)-induced contraction. Iso produced approximately 80% maximum inhibition of the standard ACh-contraction in all 4 stages of the oestrous cycle. Adr and NA produced 80% maximum inhibition in oestrus only, and 50–60% maximum inhibition in proestrus, metoestrus and dioestrus. The differences in the degree of inhibition produced by the catecholamines were overcome (i.e. the maximum inhibition produced by Adr and NA was increased to become equal to that produced by Iso) when experiments were repeated in the presence of the uptake inhibitors desmethylimipramine (DMI) and normetanephrine (NMN), but not in the presence of an -adrenoceptor antagonist (azapetine 10^–7 M). Variations in the uptake of³H-Iso and³H-NA were observed in the different stages of the oestrous cycle. Small -adrenoceptor mediated motor responses to NA and Adr were observed only in the presence of a -antagonist (propranolol 10^–5 M), in uteri from rats in oestrus, metoestrus and dioestrus, but not proestrus. It is concluded that in the rat isolated uterus, -inhibitory receptors predominate throughout the natural oestrous cycle, although the existence of -excitatory receptors has been shown in 3 of the 4 stages.     

Smooth muscle of rabbit aorta contains α1- but not α2-Adrenoceptors

Summary We have investigated the residual contractile response to noradrenaline remaining after phenoxybenzamine (3×10^–7 mol/l) in rabbit aorta, since it has been reported that phenoxybenzamine at low doses completely and irreversibly blocks ₁- but not ₂-adrenoceptors. The contraction elicited by noradrenaline slowly recovered with time after it had been almost abolished by phenoxybenzamine. This residual response was blocked by the ₁-selective antagonist prazosin (3×10^–8 mol/l) but not by the ₂-selective antagonist rauwolscine (3×10^–7 mol/l). The results confirm that the smooth muscle of rabbit aorta contains ₁- not ₂-adrenoceptors.     

Binding of 3H-clonidine to an α-adrenoceptor in membranes of guinea-pig ileum

Summary The binding of ³H-clonidine to membrane particles from guinea-pig ileum was investigated. The specific binding, i.e. the binding that could be inhibited by high concentrations of unlabeled clonidine or noradrenaline, was of high affinity, K _D3 nM. The number of sites was approximately 25 fmol/mg protein. Rate constants of association and dissociation were 5.3×10⁷ M^–1 min^–1 and 0.18 min^–1, respectively. Affinites of various drugs to the binding site were determined by measuring their effect on the binding of ³H-clonidine. The affinity of adrenergic agonists decreased in the order clonidine = tramazoline > (–)-erythro--methylnoradrenaline > (–)-noradrenaline (–)-phenylephrine. (–)-Noradrenaline had about 20 times more affinity than the (+)-isomer. The affinity of -adrenoceptor antagonists decreased in the order phentolamine > rauwolscine = yohimbine > WB 4101 > pseudoyohimbine > prazosin = corynanthine. Yohimbine and rauwolscine had about 100 times more affinity than their stereoisomer corynanthine. Serotonin 10 M and metiamide 10 M did not affect the binding, and propranolol inhibited it only at high concentrations. — The results indicate that ³H-clonidine labels an ₂-adrenoceptor in guinea-pig ileum. The orders of affinity of -adrenoceptor agonists and antagonists agree well with their orders of potency in functional tests, namely as modulators of cholinergic transmission in the guinea-pig ileum and as modulators of noradrenaline release in the rabbit pulmonary artery. An -adrenoceptor should be classified as ₂ when the affinities of clonidine, tramazoline and -methylnoradrenaline greatly exceed the affinity of phenylephrine, and when the affinities of rauwolscine and yohimbine exceed those of prazosin and corynanthine.     

Studies on alpha adrenoceptors in guinea-pig peripheral lung strips

Summary Contractile responses of guinea-pig peripheral lung strips to noradrenaline were determined in the presence of propranolol (2.5 × 10^–6 mol/l). All strips (n = 44) contracted to noradrenaline and a geometric mean EC₅₀ of 1.4 × 10^–6 mol/l (1.1 × 10^–6 mol/l, 1.8 x 10^–6 mol/l 95% confidence limits) was obtained. Contractions were antagonised by phentolamine (5 × 10^–7–10^–5 mol/l) and by prazosin (10–10^–7 mol/l). Dose-ratios (DR) were calculated and log (DR-1) was plotted against log concentration of antagonist to yield slopes (± SE) of 0.84 ± 0.14 and 0.73 ± 0.22 respectively which were not significantly different from unity. A pA₂ value (± SE) of 6.7 ± 0.2 was obtained for phentolamine and 7.5 ± 0.1 for prazosin. Yohimbine (10^–7–10^–5 mol/l) did not significantly affect the maximal tension generated or the EC₅₀ values for noradrenaline. These results suggest that ₁ adrenoceptors are mediating the contractile responses to noradrenaline. In the presence of cocaine (10^–5 mol/l, n=18), normetanephrine (2 × 10^–5 mol/l, n = 15), hydrocortisone (2.5 × 10^–5 mol/l, n = 15) and normetanephrine (2 × 10^–5-5 mol/l) plus cocaine (10^–6 5 mol/l, n=15) pA₂ values for phentolamine of 6.9, 6.7, 6.6, and 6.3 respectively were obtained. Since these pA₂ values are not significantly different from 6.7, it is unlikely that this original pA₂ value, which is lower than values obtained with phentolamine at -adrenoceptors in other tissues, may be explained by neuronal or extraneuronal uptake of noradrenaline. A possible explanation may be that more than one population of -adrenoceptors contribute to changes in tension in peripheral lung strips. Send offprint requests to J. P. Seale at the above address     

The β-adrenergic receptor-adenyl-cyclase system of rat reticulocytes

Summary Non-nucleated red blood cells from rats contain adenyl cyclase, the activity of which is predominantly localized in the reticulocytes. Basal enzyme activities in membrane preparations from reticulocyte-rich blood (pretreatment of rats with acetyl-phenylhydrazide: about 60% reticulocytes) are about 5 times higher than in preparations from reticulocyte-poor blood (untreated animals: 2–3% reticulocytes). The enzyme activities are stimulated 10-fold by sodium fluoride (10^–2 M) and 6 to 8-fold by isoprenaline (10^–4 M).Adenyl cyclase activities in membrane preparations from reticulocyte-rich and reticulocyte-poor blood can be ascribed to identical enzymes since identical apparent K _m (ATP; 3×10^–4 M), K _a (isoprenaline; 3×10^–6 M) and K _i (propranolol vs. isoprenaline; 3×10^–7 M) values were obtained in both preparations.Besides NaF, only phenylethanolamine derivatives with -adrenergic receptor stimulant properties were effective as stimulators of adenyl cyclase activity. The affinities (apparent K _a values) of the investigated compounds decreased in the order isoprenaline—hexoprenaline—fenoterol—salbutamol—adrenaline—terbutaline—noradrenaline—phenylephrine. For maximal intrinsic activity, the catechol structure was essential; the relative intrinsic activities of resorcinol derivatives did not exceed 0.6.The isoprenaline-stimulated adenyl cyclase activities in erythrocyte membrane preparations were competitively inhibited by -adrenergic blocking drugs, the affinities (apparent K _i values) decreasing in the order prindolol—penbutolol—propranolol—practolol. The dextrorotatory enantiomers of penbutolol and propranolol were 1/100 to 1/200 as active as the resp. levorotatory enantiomers.From experiments with -adrenergic agonists (e.g. phenylephrine) and antagonists (e.g. phentolamine), it is concluded that -adrenergic receptors do not interfere with the -adrenergically-mediated cAMP formation in these particular membranes.A variety of hormones and drugs known to stimulate adenyl cyclase activities in various tissues, e.g. ACTH, glucagon, STH, erythropoietin, prostaglandin E ₁ etc. did not affect adenyl cyclase activity in reticulocyte-rich erythrocyte membrane preparations.In contrast to adenyl cyclase activity, phosphodiesterase activities in erythrocyte membrane and cytoplasmic fractions were only twice as high in reticulocyterich as in reticulocyte-poor preparations.From the experiments described, it is obvious that the adenyl cyclase of the rat reticulocyte is subject to monovalent-hormonal, i.e. -sympathomimetic stimulation. Moreover, the premature red blood cell provides a useful model for quantitative studies of the interaction of drugs with the -adrenergic receptor.This study was supported by the Deutsche ForschungsgemeinschaftPreliminary accounts were presented at meetings of the Deutsche Pharmakologische Gesellschaft (Gauger et al., 1972; Gauger and Palm, 1973; Quiring et al., 1974a).     

Minimal α1- and α2-adrenoceptor-mediated coronary vasoconstriction in the anaesthetized swine

Summary -Adrenoceptor-mediated coronary vasoconstriction contributes to the initiation and aggravation of experimental and clinical myocardial ischaemia. However, the extent of ₁- and ₂-adrenoceptor-mediated constriction has not been characterized in the porcine coronary circulation despite the frequent use of this experimental model.Fifteen swine were anaesthetized with either -chloralose, enflurane or isoflurane to determine the amount of -adrenoceptor-mediated coronary constriction elicited by either the selective ₁-adrenoceptor agonist methoxamine or the selective ₂-adrenoceptor agonist azepexole. The left anterior descending coronary artery was cannulated and perfused by an external pump delivering constant blood flow from the carotid artery. Following bilateral cervical vagotomy and ß-adrenoceptor blockade with propranolol (2 mg kg^–1), graded dosages of either one of the -adrenoceptor agonists (9–45 g kg^–1 min^–1) were infused into the coronary perfusion line while coronary arterial pressure (CAP) was measured through a distal side arm of the cannula to detect changes in coronary vascular resistance. Infusion of the -adrenoceptor agonists was terminated when systemic arterial pressure increased. Sonomicrometers were used to measure anterior left ventricular wall thickening for the assessment of regional contractile function. During methoxamine infusion, no increase in vascular resistance was observed during -chloralose, enflurane or isoflurane anaesthesia, whereas the infusion of azepexole increased CAP from 103 ± 31 mmHg to 120 ± 35 mmHg (-chloralose), from 101 ± 16 mmHg to 122 ± 11 mmHg (enflurane) and from 84 ± 20 mmHg to 94 ± 19 mmHg (isoflurane), respectively. In four additional swine anaesthetized with enflurane, the intracoronary infusion of the full catecholamine agonist noradrenaline in the presence of propranolol (6 mg kg^–1) increased CAP from 98 ± 10 to 105 ± 10 mmHg prior to an increase in regional left ventricular function or systemic arterial pressure.These results indicate that there are no ₁- and relatively little ₂-adrenoceptor-mediated coronary constrictive effects in swine. Furthermore, neither -adrenoceptor agonist produced any detectable change in regional myocardial contractile function, regardless of the anaesthesia used.Supported by the German Research Foundation (He 1320/3-2). Dr. Guth is the recipient of a scholarship from the Alexander von Humboldt-Foundation. Send offprint requests to G. Heusch at the above address     

β-Adrenoceptor studies

Summary Propranolol, pindolol, practolol and metoprolol were investigated for -adrenoceptor blocking activities in the guinea-pig right atrium and tracheal strip preparation, both in the absence and presence of 2.8% bovine serum albumin. Similarly, the influence of 2.8% albumin on the antagonism of ouabain-induced arrhythmias in the guinea-pig right atrium was studied. Local anesthetic activities were measured on the isolated, partially desheathed, frog sciatic nerve.Incubation with albumin decreased the in vitro atrial -adrenoceptor blocking potency of propranolol against (-)-isoprenaline-induced positive chronotropism by a factor of 12 whereas the activity on tracheal receptors was 4.5 times lower. The activities of pindolol, practolol and metoprolol both on atrial and tracheal -receptors were not significantly influenced by the presence of albumin. The results demonstrate the important role of albumin binding in causing differences in the in vivo and in vitro cardiac -adrenoceptor blocking potency ratios of propranolol compared to the other -receptor antagonists studied.The large difference between -adrenoceptor blocking and minimal antiarrhythmic concentrations, the approximately 1000 times lower antiarrhythmic activity of practolol compared to propranolol and the very significant correlation between the antiarrhythmic and local anesthetic activities demonstrate that the antagonism of ouabain-induced arrhythmias in the isolated right atrium of the guinea pig is solely due to the membrane-stabilizing properties of the -receptor antagonists. The moderate decrease of the antiarrhythmic potency of propranolol by 2.8% albumin was concluded to have no relevance for the in vivo situation.Binding to 2.8% albumin was investigated using the ultracentrifugation technique. For propranolol a linear relationship was found between log concentration and percent binding which amounted to 88.2% at 10^–6 M. In contrast, protein binding of pindolol, practolol and metoprolol was low and concentration independent.     

Effects of compound 48/80 on mast cells,histamine and cyclic AMP in isolated superior cervical ganglia

Summary Superior cervical ganglia of the rat contain mast cells which are sensitive to degranulation by compound 48/80. The granulation process is shown to the independent of the ATP content of the ganglion. Compound 48/80 released histamine into the incubation medium, thereby decreasing the histamine content of the ganglia. Moreover, the release of ³H-noradrenaline was accelerated by the compound. Histamine and adrenaline induced a rapid accumulation of cyclic AMP in the ganglia. This effect of the amines was specifically blocked by diphenhydramine or propranolol with an ID₅₀ of 1.5×10^–9 M and 2.2×10^–7 M, respectively.In contrast to other findings with isolated mast cell preparations, compound 48/80 induced a rapid and marked accumulation of cyclic AMP in intact ganglia and an enhanced release of cyclic AMP into the incubation fluid. Diphenhydramine prevented the accumulation in the tissue but only partly inhibited the enhanced appearance of cyclic AMP in the medium. The accumulation of the cyclic nucleotide in the tissue was partly blocked by propranolol, suggesting an additional action of compound 48/80 on cyclic AMP through catecholamines.The cyclic nucleotide phosphodiesterase activity in homogenates of superior cervical ganglia was completely inhibited by compound 48/80 at 7 g/ml when low cyclic AMP concentrations were used.In addition to cyclic AMP release, rapid and marked efflux of ATP into the medium was observed during incubations with compound 48/80. The lactate dehydrogenase activity in the incubation medium was significantly enhanced with incubation periods of 40 to 60 min indicating rather slowly occurring toxic damage to cell membranes by compound 48/80.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 70).     

α2-Adrenoceptor mediated inhibition of forskolin-stimulated cyclic AMP accumulation in isolated porcine palmar lateral veins

The aim of this study was to use a ³H-adenine pre-labelling technique to characterise the effect of ₂-adrenoceptor activation on forskolin-stimulated cyclic AMP accumulation in the isolated porcine palmar lateral vein. Forskolin (10^–7–10^–4 M) stimulated ³H-cyclic AMP accumulation in the isolated porcine palmar lateral vein in a biphasic and concentration-dependent manner. In the absence of the cyclic AMP-selective phosphodiesterase inhibitor rolipram, forskolin stimulated ³H-cyclic AMP accumulation approximately 7–8 fold. The response reached a peak after 5 min. In the presence of rolipram (10^–5 M), basal ³H-cyclic AMP levels were approximately 70% higher than in its absence (basal: 1823 ± 57 dpm; rolipram: 3088 ± 229, n \2 = 3) and forskolin (3 × 10^–5 M) stimulated ³H-cyclic AMP accumulation approximately 8 fold. The latter response reached a plateau 10 min after the addition of forskolin. In all subsequent studies, the tissues were incubated with forskolin (3 × 10^–5 M) for 5 min in the absence of rolipram. Noradrenaline (NA; 10^–9–10^–4 M) and UK14304 (10^–9–10^–4 M) inhibited forskolin-stimulated ³H-cyclic AMP accumulation in a concentration-dependent manner with mean pIC₅₀ values of 7.61 ± 0.37 (n \s = 4) and 7.76 ± 0.23 (n \s = 5), respectively. With either NA or UK14304, the maximal inhibition of the forskolin response obtained was approximately 75%. Neither NA (10^–4 M) nor UK14304 (10^–4 M) altered basal ³H-cyclic AMP levels. Phenylephrine (10^–4 M) had no effect on basal ³H-cyclic AMP levels and produced a 25.4 ± 7.1% inhibition of the forskolin-stimulated response, an effect that was reversed by 10^–6 M rauwolscine. Rauwolscine (10^–9–10^–6 M) produced a concentration-dependent reversal of the inhibitory effect of UK14304 10^–6 M on forskolin-stimulated ³H-cyclic AMP accumulation with a mean pK _i of 8.35 ± 0.39 (n = 3), but had no effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. Similarly, prazosin (3 × 10^–8–3 × 10^–5 M) or imiloxan (3 × 10^–8––3 × 10^–5 M) produced a concentration-dependent reversal of the UK14304 (10^–7 M)-induced inhibition of forskolin-stimulated ³H-cyclic AMP accumulation, with mean pK _i values of 6.32 ± 0.22 (n = 4) and 6.01 ± 0.30 (n = 3), respectively; neither drug had any effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. This suggests that the receptor is of the _2A-adrenoceptor subtype. It can be seen from these studies that it is possible to measure changes in cyclic AMP accumulation in porcine vascular smooth muscle using a pre-labelling technique, and it has been possible to demonstrate the presence of functional ₂-adrenoceptors, stimulation of which results in inhibition of forskolin-stimulated cyclic AMP formation.     

Effects of dopamine on whole kidney function and proximal transtubular volume fluxes in the rat

Summary The renal effects of dopamine were studied using clearance and micropuncture techniques in rats. Intravenous infusion of dopamine (4.7 · 10^–6 mol · kg^–1 · h^–1) increased glomerular filtration rate and renal blood flow. Renal blood flow was measured by an electromagnetic flowmeter. The increase in filtered fluid and sodium was nearly completely matched by increased tubular reabsorption. Thus, only a small rise in urine flow and in urinary sodium excretion was observed.The micropuncture experiments using the split oil droplet method of Gertz demonstrated a stimulation of the transepithelial fluid transfer after injection of dopamine (10^–4M) into the proximal tubular lumen. This effect was abolished by simultaneous injection of propranolol (10^–3 M) which, by its own, did not affect transtubular volume fluxes. It is concluded that dopamine, by stimulation of -adrenoceptors, may increase reabsorptive capacity of the proximal tubular epithelium independent of changes in renal hemodynamics.Supported by Deutsche Forschungsgemeinschaft     

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

Summary To determine the influence of thyroid hormone on -adrenoceptors and Ca²⁺ channels, rats were treated with thyroxine (75 g/100 g sc daily for 5 days) or propylthiouracil (0.05% in drinking water for 30 days). -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 × 10^–9 mol/1 to 5.5 × 10^–8 mol/l, and the EC₅₀ for calcium decreasedfrom 3.16 × 10^–3 mol/1to 1.36 × 10^–3 mol/1; moreover, the responsiveness to the Ca ²⁺ 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 × 10^–9 mol/1 to 89.4 × 10^–9 mol/l. This was however, associated with a decrease in the sensitivity to calcium with geometric mean EC₅₀ values increasing from 2.43 × 10^–3 mol/1 to 5.33 × 10^–3 mol/1 and a reduction in the maximal response to Bay K 8644. Treatment with thyroxine had no effect on tissue sensitivity. The responses of isolated tail artery to phenylephrine, calcium and Bay K 8644 were not significantly different in propylthiouracil- or thyroxine-treated animals. Send offprint requests to D. J. Triggle at the above address     

β-Adrenergic receptors in guinea-pig myocardial tissue

Summary Stereospecific binding sites for (–) [³H]-alprenolol, a -adrenergic antagonist, have been identified in guinea-pig myocardial broken cell preparations. The concentration of the sites was 0.3 pmoles per mg of protein and the dissociation constant (at 37°C) 10^–8 M. A close correlation between the ability of various -adrenergic antagonists to compete with tracer alprenolol binding and to block the response of isoprenaline-stimulated myocardial adenylate cyclase has been found. Low affinity sites for the labelled -adrenergic antagonist in contrast to stereospecific sites are heat stable and do not discriminate between the (–) and the (+) forms of the -adrenergic antagonists. Adenylate cyclase in guineapig myocardial tissue is poorly stimulated by isoprenaline or 5-guanylylimidodiphosphate. This is attributed to a high basal activity which could be lowered by a preincubation at 37°C.     

Calcium channels at the adrenergic neuroeffector junction in the rabbit ear artery

Summary Neurotransmitter release is dependent on influx of Ca²⁺ through voltage-operated calcium channels (VOCCs). These channels may be divided into L, N, T and P subtypes. To investigate the subtypes of VOCC involved in transmitter release from adrenergic nerves in the isolated rabbit ear artery, the effects of some subtype selective VOCC antagonists were examined on contractile responses induced by electrical field stimulation (EFS), and exposure to an isosmolar (low Na⁺, normal Cl^– content) or a hyperosmolar (normal Na^]+, high Cl^– or 60 mM K+ solution). Tetrodotoxin (TTX) and the L channel blocker nimodipine were present in the latter experiments to inhibit sodium-dependent action potential discharge and the direct contractile effect of K⁺ depolarization on the smooth muscle cells. Prazosin abolished the contractile effect of EFS, indicating that the response was elicited by activation of adrenergic nerves. The EFS-induced contractions were concentration-dependently inhibited by the N channel blocker -conotoxin (PIC₅₀ = 9.0) and the proposed L channel blocker T-cadinol (pIC₅₀ = 4.5), while nimodipine and the T channel blocker tetramethrin had no effect. The isosmolar and hyperosmolar K⁺ solutions induced a prazosin-sensitive contraction, amounting to 46% and 10% of the response to 10^–5 M noradrenaline (NA), respectively. -Conotoxin inhibited the contractile response to the hyperosmolar K⁺ solution, but not that to the isosmolar K⁺ solution. T-cadinol preferentially inhibited the response to the hyperosmolar K⁺ solution. Tetramethrin had no effect on contractions induced by either type of K⁺ solution. The contractile response to exogenous NA was unaffected by -conotoxin and tetramethrin, whereas the response was partially inhibited by both nimodipine and T-cadinol. These results suggest that NA release from adrenergic nerves in the rabbit ear artery, depend on Ca²⁺ influx through VOCCs of the N type, whereas L and T channels seem to be of minor importance. Calcium influx into the nerve terminals via a tentative Na⁺/Ca²⁺ exchange mechanism may explain the failure of -conotoxin to inhibit the adrenergic response to the isosmolar K⁺ solution.Correspondence to P. Zygmunt at the above address     

Characterization of the antagonism with metoprolol,ICI 147,798, pindolol,mepindolol and bopindolol on the responses of the rat left atria to isoprenaline

Summary The aim of the study was to determine whether the antagonism with pindolol, mepindolol and bopindolol at the ₁-adrenoceptor of the rat left atria, a tissue with plenty of spare ₁-adrenoceptors for isopren aline maximum responses, was readily reversible or not. The effects of these drugs were compared to those of metoprolol, a readily reversible, and of ICI 147,798, an irreversible -adrenoceptor antagonist. Metoprolol at 10^–7 and 10^–6 M, ICI 147,798, pindolol, bopindolol (all at 10^–8 and 10^–7 M) and mepindolol at 10^–9 and 10^–8M inhibited the cardiac stimulation responses to a small extent, which is indicative of membrane stabilizing activity, and also caused surmountable antagonism of isoprenaline responses. The inhibitory effects on the isoprenaline responses of metoprolol and pindolol were readily reversible, that of mepindolol was slowly reversible and those of ICI 147,798 and bopindolol were not reversed in 3 h. The inhibitory effects on isoprenaline responses of metoprolol at 10^–6 M, pindolol and bopindolol at 10^–7 M and mepindolol at 10^–8 M were at equilibrium, which is indicative of reversible, whereas the inhibitory effects of ICI 147,798 were increased, which is indicative of irreversible antagonism, when the -blocker treatment time was increased from 1 to 2 h. We conclude that the antagonism with pindolol at the ₁-adrenocep-tors of the rat left atria is readily reversible, that of mepindolol is slowly reversible and that of bopindolol is very slowly reversible.Correspondence to S. A. Doggrell at the above address     

Celiprolol exerts microvascular dilatation by activation of β2-adrenoceptors

Summary In order to clarify the question whether the ₁-selective adrenoceptor antagonist celiprolol possesses vasodilating properties, isolated vascular networks were perfused with increasing concentrations of celiprolol (in a cumulative manner) ranging from 10^–8 to 10^–4 mol/l. The study was carried out using the isolated mesenteric vascular bed of the guinea pig mesenterium coli. Vascular diameters of four different vascular regions [vessels classified as G1 (585 ± 30 m), G2 (403 ± 25 m), G3 (282 ± 27 m) and G4 (197 ± 13 m)] were assessed by means of microscopic videoangiometry. Perfusion with celiprolol resulted in concentration dependent vasodilation which was more pronounced in G3 and G4 vessels. In addition, cumulative concentration-response curves were determined from responses obtained in the presence of 10^–8, 10^–7, 10^–6 and 10^–4 mol/l ICI 118,551 (a highly selective adrenoceptor antagonist). In the presence of ICI 118,551 at concentrations 10^–6 mol/l, no celiprolol response could be observed. Lower concentrations of ICI 118,551 shifted the celiprolol concentration-response curve to the right in a concentration-dependent manner. Therefore, it is concluded (a) that celiprolol has a vasodilating effect, (b) that this vasodilation is produced by stimulation of ₂-adrenoceptors and (c) that the vasodilating effect is more pronounced in smaller than in larger vessels (G3, G4 vs G1, G2). Send offprint requests to S. Dhein at the above address     

Interaction of enantiomers of hydroxy tolazoline with adrenoceptors

Summary Adrenoceptor-mediated effects of the enantiomers of hydroxytolazoline and tolazoline (i. e., desoxy derivative) have been investigated in vitro. The enantiomers and tolazoline were partial agonists of postjunctional ₁-adrenoceptors in rat aorta. The rank order of potencies of the compounds in this system was as follows: tolazoline > R(–)-hydroxytolazoline > S(+)-hydroxytolazoline. The efficacy of R(–)-hydroxytolazoline was higher than that of tolazoline, though its affinity for the receptor was less. The K _B values for prazosin against these agonists were nearly equal, which indicated that these imidazolines activate the same type of receptor in rat aorta. The S(+)-isomer, however, produced both a prazosin sensitive and resistant component of the response. The interactions of the derivatives with presynaptic ₂-adrenoceptors were studied in field-stimulated myenteric plexus-longitudinal muscle of guinea-pig ileum. These substances were blockers at presynaptic ₂-adrenoceptors. Based on K _B values, the order of affinity in this system was as follows: tolazoline > S(+)isomer R(–)-isomer. -Adrenoceptor mediated activity was quantitated in guinea-pig and rat atria. R(–)-hydroxytolazoline lacked chronotropic effects either in guinea pig or rat atria. At 3 × 10^–4 M the isomer did not antagonize the effect of isoproterenol in the atria. On the other hand, S(+)-hydroxytolazoline produced a variable chronotropic effect in guinea-pig atria, but failled to show any significant activity in rat atria. Thus, the -adrenoceptor mediated action appears to be insignificant. Steric aspects of -adrenoceptor mediated events are discussed.This investigation was, in part, supported by a grant from the United States Public Service, National Institutes of Health, GM 29358 Send offprint requests to P. N. Patil at the above address     

Preponderance of β- over α-adrenoceptors in mediating the positive inotropic effect of phenylephrine in the ferret ventricular myocardium

Summary [³H]prazosin bound to the membrane fraction derived from the ferret ventricular muscle with high affinity in a saturable manner (K _d = 0.25 nmol/l and B _max = 27 fmol/mg protein in the right ventricle). [³H]CGP-12177, a -adrenoceptor ligand, bound to the membrane fraction with a _{K d} value of 0.29 nmol/l and a B _max of 42 fmol/mg protein. In the isolated ferret papillary muscle driven at 1 Hz at 37°C, phenylephrine elicited a concentration-dependent positive intropic effect. The maximal effect of phenylephrine was comparable to that of isoprenaline. Prazosin (0.3 ol/l) shifted the concentration-response curve for phenylephrine slightly but significantly to the right, the maximal response being unaffected. In contrast, bupranolol (0.3 gmol/l) shifted the curve for phenylephrine markedly downwards: the maximal response was depressed significantly to 40% and the curve became less steep. In the presence of prazosin and bupranolol the curve was shifted to the right, being essentially parallel to the control curve. These results indicate that in the ferret ventricular myocardium both - and -adrenoceptors mediate the positive inotropic effect of phenylephrine. The extent of contribution of the two classes of adrenoceptor is quite different from that in other mammalian species. In the ferret heart, -adrenoceptors predominate over -adrenoceptors in mediating the positive inotropic effect of phenylephrine, although the number of -adrenoceptors is not especially high when compared with other species. Send offprint requests to M. Endoh at the above address     

Cardiac Ca current does not run down and is very sensitive to isoprenaline in the nystatin-method of whole cell recording

Summary The conventional l type Ca²⁺ channel current (I_Ca.L) was measured in single atrial and ventricular myocytes, with a new whole-cell recording technique using an ionophore, nystatin. The membrane of a cell-attached patch was gradually permeabilized by nystatin (100–200 gg/ml), added to the pipette solution, within 2–5 min after formation of a G-seal. The electrical activity of the cells was measured through the pipette. I_Ca.L, measured with the nystatin-whole cell recording technique, did not exhibit the so-called run-down phenomenon for up to 90 min. The response of I_Ca.L to isoprenaline was also well preserved during the measurement. The half maximal concentration for the isoprenaline-induced increase of I_Ca.L was 8.2 × 10^–9 M, which is a much smaller value than that reported previously. Thus, the nystatin-whole cell clamp recording is a useful technique to measure membrane currents of cardiac myocytes with preserving the physiological intracellular milieu. Send offprint requests to Y. Kurachi at the above address     

Pharmacological characterization of the postjunctional alpha-adrenoceptors of the rat isolated seminal vesicle

Summary In an attempt to define the pharmacological characteristics of the postjunctional -adrenoceptors of the rat seminal vesicle, responses to certain phenylethanolamine and imidazoline agonists were investigated, in vitro, under experimental conditions outlined by Furchgott (1972), using ₁-selective, non-selective and ₂-selective adrenoceptor antagonists. Adrenaline (ADR), noradrenaline (NA) and phenylephrine (PE) produced concentration-dependent contractions. In many experiments the concentration-response (C-R) curves had a distinct shoulder at the level of 60–80% of the maximum response (E_max), a situation reminiscent of the rat anococcygeus muscle and the rat basilar artery. The relative potencies of ADR:NA:PE, derived from their EC₅₀ values, were 4.07:1:0.26. In contrast clonidine, oxymetazoline and naphazoline failed to contract the tissue even in concentrations up to 1 × 10^–3 M. In fact the imidazoline derivatives prevented responses to the phenylethanolamines. The antagonist action of clonidine, against phenylephrine, was studied in detail.Prazosin, phentolamine, yohimbine, corynanthine and clonidine all caused a rightward displacement of the C-R curves for NA without depressing E_max. The Arunlakshana and Schild plots of the data were linear and had slopes not significantly different from unity. The pA₂ estimates obtained were 9.17 (9.13–9.21) for prazosin, 8.58 (8.07–9.09) for phentolamine, 6.70 (6.44–6.98) for yohimbine and 7.05 (6.81–7.30) for corynanthine. Clonidine had a pA₂ value of 6.60 (6.55–6.67) against phenylephrine.On the basis of results obtained with antagonists, the postjunctional -adrenoceptors of the rat seminal vesicle could be firmly placed in the gross category of ₁. The concept of heterogeneity of ₁-adrenoceptors is discussed in the light of the profiles of the phenylethanolamine and imidazoline agonists as well as the antagonist potencies of prazosin and yohimbine. Send offprint requests to S. I. Sharif at the above address