Erythromycin and motilin stimulate sphincter of Oddi motility and inhibit trans-sphincteric flow in the Australian possum

Summary The actions of erythromycin lactobionate and porcine motilin on trans-sphincteric flow and simultaneous sphincter of Oddi motility were studied in 15 anaesthetized Australian Brush-tailed possums (Trichosurus vulpecula). Erythromycin (25–200 g/kg) and motilin (25–200 ng/kg) were administered as graded doses by close intraarterial injection. Trans-sphincteric flow was measured as inflow and outflow. Both motilin and erythromycin decreased trans-sphincteric inflow (both P<0.0001) and outflow (P<0.0001 and P=0.0017 respectively) in a dose-dependent manner. The highest dose of each agent abolished trans-sphincteric flow. These agents increased sphincter of Oddi phasic contraction frequency and basal pressure up to 2 and 3 fold respectively (P<0.05). The amplitude of the sphincter of Oddi phasic contractions were not influenced in any consistent fashion by either agent. The durations of the responses (trans-sphincteric inflow) elicited by erythromycin and motilin were dose dependent (P = 0.0225 and P = 0.0001 respectively). The actions of erythromycin (200 g/kg) or motilin (100 ng/kg) on trans-sphincteric flow and sphincter of Oddi motility were not influenced by neural blockade with tetrodotoxin. These findings support the hypothesis that erythromycin acts as a motilin agonist and both substances increase the resistance to flow through the sphincter of Oddi by raising the basal pressure and frequency of contractions.Correspondence to G. T P. Saccone at the above address     

Pharmacological characterization of the alpha adrenoceptors of the dog basilar artery

Summary The effects of alpha adrenoceptor agonists and antagonists on the postsynaptic alpha receptors were examined in the dog basilar, mesenteric and renal arteries and the type of alpha adrenoceptors present was characterized. In the basilar artery, noradrenaline, clonidine and phenylephrine produced almost the same maximal contraction, the pD₂ values being 6.51±0.11, 5.49±0.16 and 5.65±0.13, respectively. Yohimbine (1–3×10^–8 M) inhibited the contractile responses to noradrenaline and clonidine competitively and the response to phenylephrine noncompetitively. Corynanthine (10^–6 M) had no effect on such contractile responses. In the mesenteric and renal arteries, the maximal responses to noradrenaline and phenylephrine were markedly greater than those to clonidine. Yohimbine (10^–7–10^–5 M) and corynanthine (10^–7–10^–5 M) both antagonized noradrenaline competitively in these vessels. In the basilar, mesenteric and renal arteries preloaded with ³H-noradrenaline, ³H-efflux induced by electrical transmural stimulation was attenuated by clonidine (10^–10–10^–7 M), while phenylephrine (10^–10–10^–8 M) was without effect. Yohimbine at considerably lower concentrations than corynanthine increased the ³H-efflux clicited by electrical stimulation. These results indicate that presynaptic and postsynaptic alpha receptors of the dog basilar artery are largely alpha₂ in contrast to those of peripheral arteries.This work was supported by a Grant-in-Aid for Co-operative Research (No. 00437006), by a Grant-in-Aid for Special Project Research (No. 56220016) and by a Grant-in-Aid for Encouragement of Young Scientists (No. 577106, 56770118) from the Ministry of Education, Science and Culture, Japan     

The role of intracellular calcium stores in motilin induced contractions of the longitudinal muscle of the rabbit duodenum

Summary The contraction of longitudinal muscle strips of the rabbit duodenum in response to motilin and acetylcholine was investigated in normal and high K⁺-solutions in the presence and absence of external calcium, in order to demonstrate the existence of pharmaco-mechanical coupling for motilin and to examine whether the peptide mobilizes calcium from an intracellular store. In depolarized smooth muscle (140 mM K⁺), motilin (3.2×10⁹ –1×10^–7 M) and acetylcholine (1×10^–5 M) were still capable of causing a considerable, transient, concentration-dependent contraction in the presence of Ca²⁺. The extra-contraction to motilin was not blocked by tetrodotoxin (1 g/ml) nor by atropine (10^–7 M), but acetylcholine (10^–5 M) was blocked by atropine. Verapamil (10^–7 M) could selectively block the K⁺ contraction without affecting the extra agonist contraction. Nitroprusside was ineffective up to 10^–4 M in high K⁺-solutions, but in normal Hepes-buffer it caused a concentration-dependent rightward shift of the concentration-response curve of motilin and acetylcholine contractions. In a calcium-depleted medium, high K⁺-depolarized muscle strips were still responsive to motilin and acetylcholine, but higher concentrations (10^–6 M) were needed than in the presence of calcium and the contractions reached only 57 +- 11% and 74 +- 9% respectively of the maximal contraction in 1.2 mM Ca²⁺ containing solutions. The response to motilin (10^–6 M) was not only smaller than that to acetylcholine (10^–5 M), it also faded more rapidly with time. The response to one agonist could not be repeated except by using a higher concentration of the same or the other agonist, and the magnitude of this second response depended upon the dose used in the first one. We conclude that pharmaco-mechanical coupling exists for motilin and that this peptide is able to elicit contractions by mobilization of calcium from an intracellular store. This store overlaps with the one used by acetylcholine. Our experiments also reinforce the hypothesis that in the rabbit motilin exerts a direct action upon smooth muscle cells.     

The role of extracellular Ca2+ in carbachol-induced tonic contraction of the pig detrusor smooth muscle

The role of extracellular Ca²⁺ in the toniccontractile response to muscarinic receptor stimulation was investigated in isolated detrusor smooth muscle from the pig urinary bladder.Carbachol (10^–8 –10^–5 M) produced a concentration-dependent contractile response in isolated pig detrusor smooth muscle strips consisting of an initial phasic component followed by a tonic component. During the plateau of the tonic contractions induced by carbachol at the submaximal concentration of 10^–6 M, the inhibiting effects of atropine, EGTA, nifedipine (a voltage-dependent calcium channel antagonist), H-7 [a protein kinase C (PKC) inhibitor] and YM 934 (a potassium channel opener) on the contractions were evaluated. Atropine (10^–10 –3 × 10^–8 M) concentration-dependently inhibited the tonic contractions induced by carbachol. In the same experimental conditions, EGTA (4 mM) and nifedipine (10^–9 –3 × 10^–7 M) depressed the tonic contractions in a concentration-dependent manner as did H-7 (10^–5 –3 × 10^–5 M) and YM934 (10^–8 -10^–6 M). However, H-7 (10^–5-3 × 10^–5 M) and YM934 (10^–6 M) were very weak in inhibiting the contractions induced by KCl (50 mM) in isolated pig detrusor smooth muscle strips.These results suggest that the tonic-contractile response induced by carbachol in pig detrusor smooth muscle strips is dependent mainly on depolarization of the cell membranes and an influx of extracellular Ca²⁺, and also suggest that this depolarizing response may be due to inactivation of ATP-sensitive potassium channels through muscarinic activation of PKC. Correspondence to: W. Uchida at the above address     

Ca2+ dependence of motilide-induced contractions in rabbit duodenal muscle strips in vitro

Summary Recent studies suggested that certain erythromycin A (EM-A) derivatives are motilin receptor agonists. As proposed by Itoh they may be called motilides. We have investigated the Ca²⁺-dependence of contractions induced by two potent motilides, ME-34 [de(N-methyl) 8,9-anhydroeryhtromycin A 6,9-hemiacetal] and EM-523 [de(N-methyl)-N-ethyl-8,9-anhydro-erythromycin A 6,9-hemiacetal], in duodenal tissues and compared the results with those previously obtained with motilin.Isometric and isotonic contractile responses of isolated longitudinal muscle sheets from the rabbit duodenum were tested under normal, Ca²⁺-free and depolarizing conditions. Prior to stimulation with motilides, the maximal response to acetylcholine was recorded and all responses were always expressed as a percentage of this response. Both motilides induced contractions in normally polarized tissue, with an EC₅₀ of 26 ± 5 nM for ME-34 (n = 7), and 27 ± 5 nM for EM-5231 (n = 16) and maximal responses of respectively 88 ± 4% and 80 ± 3%. Like motilin, both compounds induced an extra-contraction in depolarized tissues. The EM-523 response in 140 mM K⁺under isotonic conditions was 84 ± 3% (n = 5) at 10^–5 M, with an EC₅₀ that was shifted to 65 ± 18 nM. Similar figures were obtained for ME-34. When Ca²⁺ was added to Ca²⁺-depleted strips, half-maximal Ca²⁺ values (in mM) were 1.10 ± 0.11 (n = 9) for EM-523 and 1.13 ± 0.12 (n = 3) for ME-34, as compared with 1.12 ± 0.13 (n = 7) for motilin and 2.8 ± 1.1 (n = 9) for K⁺. Both ME-34 and EM-523 also induced a transient contraction in Ca⁺-free solutions under isometric conditions. The response to EM-523 (5 × 10^–6 M) was 49 ± 15% (n = 4) after 3 min. A maximal EM-523 -stimulation reduced a subsequent ACh response by 78 ± 7%, whereas EM-523 and ME-34 could not induce a contraction after ACh.We conclude that motilides depend upon external Ca²⁺ to a similar extent to motilin. Like motilin, they are also able to mobilize intracellular Ca Z + stores. Our results support the hypothesis that motilides act on motilin receptors. Send offprint requests to T. L. Peeters at the above address     

Pre- and postjunctional actions of endothelin in the rat iris sphincter preparation

Effects of endothelins (ETs) were studied in the rat iris sphincter preparation. Three peptides (ET1, ET-2 and ET-3) caused contractile responses, and the rank order of agonist potency was: ET-1 = ET-2 > ET-3. The concentration-response curve to ET-1 was shifted to the right by the ET_A receptor antagonist cyclo [d-Asp-l-Pro-d-Val-l-Leu-d-Trp] (BQ-123: 10^–7 M), the pA₂ value of which was 7.41 ± 0.09 (n = 4).ET-1 and ET-3, at the concentration of 10^–9 M, potentiated cholinergic contractions evoked by electrical field stimulation (5 and 20 Hz) without affecting the postjunctional sensitivity to carbachol. This potentiating effect was not influenced by BQ-123 (10^–6 M). The ET-evoked percentage increase in the stimulation-induced contraction observed at 5 Hz was significantly greater than that at 20 Hz. A release of immunoreactive ET was detected when the preparation was stimulated at 20 Hz (1.81 ± 0.36 pg/sphincter n = 6). ET release evoked by 20 Hz stimulation was completely abolished by tetrodotoxin (10^–7 M).In conclusion, ET interacts with two different receptor types, ET_A and non-ET_A receptors (probably ET_B) which exist post- and presynaptically at cholinergic neuroeffector junctions of the rat iris preparation. Stimulation of ET_A receptor results in a direct muscle contraction and non-ET_A receptor activation facilitates the acetylcholine output from cholinergic nerve endings. It is suggested that ET released from a tetrodotoxin-sensitive site is involved in the modulation of acetylcholine release in the rat iris sphincter preparation. Correspondence to: I. Takayanagi at the above address     

Evidence for the involvement of calcitonin gene-related peptide in the epithelium-dependent contraction of guinea-pig trachea in response to capsaicin

Summary Capsaicin (10^–9 to 10^–5 M) contracted guinea-pig tracheal strips. Epithelium-containing tracheal strips developed a maximum active tension which was significantly higher than that observed in epithelium-free strips. Anti-CGRP (calcitonin gene-related peptide) serum blocked the epithelium-dependent potentiation of the capsaicin-induced contraction in the intact tracheal strips, without affecting the response of the epithelium-free strips. This result suggests the occurrence of an epithelium-dependent release of CGRP. This same serum markedly reduced the contraction induced by exogenous rat CGRP in both intact and epithelium-free tracheal strips. In epithelium-free tracheal strips, capsaicin-induced contraction was abolished by spantide (10^–6 and 10^–5 M), a substance P antagonist, but, in intact tracheal strips, spantide did not abolish the capsaicin-induced contraction, showing that both CGRP and substance P release are directly induced by capsaicin. Moreover, the contractile responses to rat CGRP of intact tracheal strips from guinea pig suggest that CGRP itself might be able to release a contracting factor from the airway epithelium. Therefore, CGRP originating from the airway epithelium may play a major role in the control of airway smooth muscle tone.Send offprint request to E. Tschirhart at the above address     

Does motilin stimulate the gastrointestinal motility of the pig? In vitro study using smooth muscle strips and dispersed muscle cells

To clarify the physiological role of motilin in the pig gastrointestinal (GI) tract, effect of Leu¹³-porcine motilin (LMT) on the contractility of GI smooth muscle was investigated in studies using isolated muscle strips and dispersed muscle cells. LMT produced no contraction in either longitudinal muscle (LM) or circular muscle (CM) of the stomach (fundus, corpus, antrum), duodenum, ileum and colon even at 1 μM. Pretreatment with LMT (1 nM-1 μM) did not potentiate the contractile response to acetylcholine (ACh) in each muscle strip. Dispersed cells from the duodenum responded to ACh in a concentration-dependent manner (EC₅₀= 10 pM), but not to LMT even at a high concentration (10 μM). Electrical field stimulation (EFS) caused a frequency-dependent (0.2–10 Hz) contraction of the duodenal LM that was almost completely inhibited by atropine or tetrodotoxin. EFS caused the relaxation of duodenal CM in a frequency-dependent manner (0.1–10 Hz). This relaxation was not inhibited by atropine, propranolol, phentolamine or guanethidine, indicating the involvement of noncholinergic, nonadrenergic (NCNA) nerves. N^G-nitro l-arginine methylester (l-NAME, 100 μM) attenuated the EFS-induced relaxation and the inhibition at low frequency was larger than that at high frequency. l-Arginine prevented the inhibition by l-NAME but d-arginine did not. LMT (1 nM-1 μM) had no influence on EFS-induced cholinergic contraction of LM and EFS-induced NCNA relaxation of CM layer. The present in vitro studies indicate that motilin is ineffective in producing contraction and in modulating the autonomic neuroeffector transmission of the pig GI smooth muscle, and suggest that pig GI smooth muscle lacks functional motilin receptors.     

Positive inotropic effects of the calcium channel activator Bay K 8644 on guinea-pig and human isolated myocardium

Summary 1. The positive inotropic effects of the dihydropyridine calcium activator Bay K 8644 were studied in guinea-pig isolated contracting myocardium and human papillary muscle strips obtained from patients undergoing mitral valve replacement or cardiac transplantation. 2. Bay K 8644 produced a slowly developing, concentration-dependent positive inotropic response in all cardiac tissues studied. In guinea-pig papillary muscle, the increase in force of contraction was half-maximal at 3.9 × 10^–8 mol/l and the maximal inotropic effect was comparable to that obtained with ouabain, dobutamine or calcium. The guinea-pig left atrium (EC₅₀, 2.1 × 10^–7 mol/l) was fivefold less sensitive than the papillary muscle. 3. The maximal inotropic response to dihydroouabain was significantly increased after preincubation with Bay K 8644 (1 × 10^–6 mol/l) in papillary muscles from both guinea-pig and human. In guinea-pig papillary muscles, the maximal inotropic response to dobutamine was not changed by preincubation with Bay K 8644 whereas in human papillary muscle strips, Bay K 8644 increased the inotropic response to dobutamine. 4. Bay K 8644 increased force of contraction (EC₅₀, 4 × 10^–8 mol/l) in human papillary muscle strips from patients undergoing mitral valve replacement. However, the maximal inotropic response to Bay K 8644 was reduced to 32 ± 4.4% that of calcium (15 mmol/l) measured in the same muscle strips. 5. A further reduction in maximal inotropic response to Bay K 8644 to 13 ± 1.2% that of calcium (15 mmol/l) with no change in potency was measured in human papillary muscle strips taken from terminally failing hearts of cardiac transplant recipients. 6. There was a significant correlation between the preoperative left ventricular ejection fraction and the maximal inotropic response to Bay K 8644 in isolated human papillary muscle strips. 7. These results suggest that Bay K 8644 affects excitation-contraction coupling of cardiac muscle so as to increase the maximal inotropic effect of the digitalis glycosides. Further, the inotropic response of human myocardial tissue to calcium channel activator Bay K 8644 may be reduced in states of pathological heart function.The human heart papillary muscles were provided by Prof. E. Kreuzer, Prof. B. Kemkes, Dr. C. Weinhold and their colleagues, Herzchirurgische Klinik der Universität, Klinikum Grosshadern, D-8000 München 70, Federal Republic of Germany Send offprint requests to E. Erdmann     

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.     

Precontraction-induced contractile response of isolated canine portal vein to alpha-2 adrenoceptor agonists

Summary The responses of isolated canine portal veins, in either transverse or longitudinal strip, to alpha-2 adrenoceptor agonists were examined. B-HT920, a selective alpha-2 adrenoceptor agonist, did not elicit an appreciable response in the transverse strips of the portal vein under resting tone (0.5 g/mm width). When the preparation was partially precontracted with phenylephrine, prostaglandin F₂, KCl, Bay K 8644, acetylcholine, 5-hydroxytryptamine, histamine, or A23187, B-HT920 evoked concentration-dependent contractile responses (3 × 10^–9-10^–6 M). Maximum contractions, which depended on the precontraction levels and the precontracting substances, ranged from 10–35% of those evoked by norepinephrine 10^–5 M. Similar precontraction-dependent contractile responses were obtained in the longitudinal strips. Concentration-response curves of B-HT920 in the transverse strips precontracted with prostaglandin F₂ were shifted by yohimbine to the right, but not by prazosin. Schild analysis yielded a slope of unity and a pA₂ of 8.79. Clonidine also showed a similar precontraction-dependent contractile response. The lower part of the concentration-response curve of clonidine was shifted by yohimbine. These results may be explained by the presence of postsynaptic alpha-2 adrenoceptors in canine portal vein, which mediate a contractile response under certain conditions. Send offprint requests to T. Furuta at the above address     

P2Y受体介导大鼠胃体环行肌收缩反应的药理学特点

观察大鼠离体胃体环行肌和胃底环行肌不同的药理学特征，分析核苷及核苷酸类物质诱发胃体环行肌收缩反应的作用特点和受体机制。制备大鼠离体胃体环行肌和胃底环行肌标本，利用受体药理学技术观察药物诱发的收缩反应。在胃体环行肌KCl所致收缩反应与胃底环行肌无显著性差别；但是，CCh收缩胃体环行肌的EC₅₀值 [(0.45 ± 0.15) μmol·L⁻¹] 显著高于胃底环行肌 [(0.20 ± 0.09) μmol·L⁻¹, P < 0.01]。5-HT和His收缩两种标本的EC₅₀值无显著差异 (P > 0.05); 但是, 在胃体环行肌5-HT和His产生收缩反应的E_max值 [(0.81 ± 0.26) 和 (0.88 ± 0.27) g] 显著小于胃底环行肌 [(2.67 ± 0.61) 和 (1.90 ± 0.68) g, P < 0.01]。在预收缩胃体环行肌，ATP (0.1～3 000 μmol·L⁻¹) 诱发浓度依赖性收缩反应，未见舒张反应；在预收缩胃底环行肌标本，同浓度ATP诱发先舒张后收缩的双相反应，并呈浓度依赖性。ATP、UTP、ADP、2-MeSATP和α, β-MeATP浓度依赖性诱发大鼠胃体环行肌收缩反应，2-MeSATP的EC₅₀值为 (7.2 ± 5.2) nmol·L⁻¹比Ach [(3.47 ± 1.20) μmol·L⁻¹] 低500倍；各药物产生收缩反应的效价序列为：2-MeSATP>>ADP>ATP=UTP>α, β-MeATP>>腺苷。酚妥拉明、普萘洛尔、阿托品及河豚毒素不影响ATP和UTP诱发的胃体环行肌收缩反应。研究结果表明, 大鼠胃体环行肌的药理学特征明显不同于胃底环行肌; 核苷酸类物质通过某种特殊的P2Y受体介导胃体环行肌收缩反应，是调节胃体环行肌收缩功能的重要介质。
     

Characteristics of histamine tachyphylaxis in canine tracheal smooth muscle

Summary In isolated canine tracheal smooth muscle, repeated administrations of histamine result in a rapid reduction in contractile response to about 15% of the initial contraction (tachyphylaxis). Development of this tachyphylaxis is specific inasmuch as: 1) it does not develop to acetylcholine (10^–6 M or 10^–4 M), or serotonin (10^–5 M); and 2) maximally developed histamine tachyphylaxis is not associated with a parallel reduction in response to acetylcholine. Pretreatment with propranolol (10^–5 M) or phentolamine (10^–4 M) does not prevent tachyphylaxis: however, pretreatment with atropine (10^–4 M) does prevent tachyphylaxis in about 50% of the animals tested.Tachyphylaxis to histamine can be reversed in a dose- and time-dependent fashion with prostaglandin synthesis inhibiting agents. The order of potency obtained with such compounds (indomethacin > mefenamic acid > oxyphenbutazone > acetylsalicylic acid) is consistent with potencies for inhibition of prostaglandin synthesis found in the literature. Also, in indomethacin pretreated strips in which tachyphylaxis to histamine was prevented, exogenous addition of PGE₂ (1.42×10^–10 M to 2.84×10^–9 M) and PGA₂ in a high concentration (2.9×10^–9 M) are capable of selectively reducing the response to histamine without an effect on acetylcholine-induced contractions. These data suggest that the mechanism of histamine tachyphylaxis in the canine tracheal smooth muscle preparation involves prostaglandin synthesis.This report is part of a dissertation to be presented by W. H. A. to the University of South Florida College of Medicine in partial fulfillment of the requirements for a Doctor of Philosophy degree     

Comparison of in vitro effects of cicletanine,its enantiomers and major metabolite on rat thoracic aorta

Summary The effects of cicletanine (((±)-cic)), its optical isomers (BN-50417,(+)-cic); (BN-50418,(–)-cic) and major metabolite (BN-50699,cic-Met) on active tension were investigated in rat thoracic aortas. (±)-cic, (+)-cic, (–)-cic shifted the K⁺-concentration response curve to the right and depressed the maximum contractile response. Cic-Met was devoid of inhibitory effect on K⁺-induced contractions. (–)-Pinacidil had a far more potent inhibitory effect on K+-induced contractions than the cicletanine enantiomers and shifted the K⁺-concentration response curve to the right without affecting the maximum contractile response. (±)-Cic and nifedipine caused a concentration-related inhibition of Ca²⁺-induced contractions. Nifedipine was far more potent than (±)-cic in this respect. The slope of the Schild plot for nifedipine was not different from unity contrary to the significantly different slope for (±)-cic.(±)-Cic, (+)-cic, (–)-cic and cic-Met (3 × 10^–5 M to 3 × 10^–4 M) caused a concentration-related relaxation of noradrenaline (NA), serotonin (5-HT) and prostaglandin F₂, (PGF₂)-induced contractions. In NA-precontracted preparations (–)-cic (10^–4M–3×10^–4M) had a stronger relaxant effect than (+)-cic. Cic-Met was a weaker antagonist of NA-induced contractions than (+) and (–)cic. The enantiomers were far less potent relaxing NA-induced contractions than phentolamine. (+)-Cic, (–)-cic and cic-Met had a similar relaxant effect on 5-HT-induced contractions. The drugs were far less potent in relaxing 5-HT-contracted aortas than ketanserin. (+)-Cic, (–)-cic and cic-Met had an equally concentration-dependent relaxant effect on rat aortas precontracted with PGF₂.The results showed that cicletanine, its enantiomers and major metabolite all caused relaxation of rat aortas in vitro. The mechanism of vasodilator action of cicletanine is complex, its action on K⁺- and Ca²⁺-induced contraction differing quantitatively as well as qualitatively from those of pinacidil and nifedipine. The results indicate that cicletanine stereoselectivity is of minor importance and cannot explain its complex mechanism of action. Correspondence to E.O. Mikkelsen at the above address     

CONTRACTILE ACTION OF GALANIN ANALOGUES ON RAT ISOLATED GASTRIC FUNDUS STRIPS IS MODIFIED BY TACHYPHYLAXIS TO SUBSTANCE P

This study was undertaken to characterize the interaction of porcine galanin (Gal) and some of its analogues with their receptors on rat gastric fundus muscle strips.Gal, galantide (M15) and Gal(1–14)-[Abu⁸]SCY-I evoked concentration-dependent contractions of gastric smooth muscle strips. Reproducible effects were observed in concentrations of 1–300, 3–1000 and 100–3000n , respectively. Specific EC₅₀for the contractile effect equalled 13, 70 and 187n .Hill's coefficient for Gal is 1.03 indicating an interaction of one Gal molecule with one receptor, fulfilling the criteria of classical receptor theory. For M15 and Gal(1–14)-[Abu⁸]SCY-I Hill's coefficients are different from 1, namely 0.73 and 1.56, pointing out that the principle of interaction of one drug molecule with one receptor may not apply. The contraction induced by 300n of Gal was not significantly modified by tachyphylaxis to substance P (SP). On the contrary the introduction of tachyphylaxis to SP decreased the contractile effects of M15 and Gal(1–14)-[Abu⁸]SCY-I by about 57.7±3% and 39.6±5%, respectively. The findings suggest that contractile actions of M15 and Gal(1–14)-[Abu⁸]SCY-I are probably not only due to their agonist activities at Gal receptors but may result from a subsequent stimulation of receptors for SP or release of endogenous SP.     

Compared effects of calcium entry blockers on calcium-induced tension in rat isolated cerebral and peripheral resistance vessels

Summary The effects of the calcium entry blockers verapamil (V), diltiazem (D), nifedipine (NF) and nicardipine (NC) have been studied on calcium concentration-effect curves elicited in depolarized (K⁺, 40 mmol/l) and in serotonin-exposed (6 mol/l) rat middle cerebral arteries (RMCA) in order to compare the relative potencies of the blockers against these two calcium channel activating mechanisms. In control conditions, Ca²⁺ sensitivity expressed as pD₂ and maximal active wall tension (AWT) were not significantly different in depolarized and in 5-HT-exposed vessels: pD₂: 3.39 ±0.08 vs 3.50 ± 0.06 and AWT: 0.93 ± 0.15 mN · mm^–1 vs 0.90 ± 0.16 mN · mm^–1 respectively. V, D, NF and NC displaced Ca²⁺ control curves to the right and depressed the maximum contractile response in the two experimental conditions, which suggests a noncompetitive type of antagonism. All the blockers were more potent inhibitors of Ca²⁺-induced contractions in depolarized than in serotonin-exposed middle cerebral arteries. The IC₅₀ values (concentration of blockers producing a 50% inhibition of maximal control contractile response) were (nmol/l) : V = 20, D = 120, NF = 0.4, NC = 1 and V = 400, D = 10000, NF = 20, NC = 7 in depolarized and serotonin-exposed arteries respectively. From these IC₅₀ values, the relative order of potency of the CEB's was not the same in the two experimental conditions suggesting that while serotonin and K⁺ both promote the entry of Ca²⁺ into vascular smooth muscle cells of RMCA, they either activate a different gating mechanism associated with a single common channel or perhaps distinct channels. Comparison of the results obtained in this study for depolarized rat middle cerebral arteries with those previously obtained in depolarized rat mesenteric resistance arteries (RMRA) revealed that while Ca²⁺-induced contractile responses were inhibited in a similar non-competitive manner by the four CEB's, the respective IC₅₀ values showed that potencies and rank of relative potency of the blockers were different in the two types of vessels. D and NC were equally potent in both preparations (IC₅₀ ratio = 2.5 and 3 respectively) but RMCA were more sensitive to V and NF than RMRA (IC₅₀ ratio = 6.5 and 11 respectively). These results are discussed and it is proposed that regional differencies in the conformation and/or the activation of the voltage-gated Ca²⁺ channels may exist in different vascular beds. Send offprint requests to J. L. Freston     

Direct and indirect actions of dopamine on tracheal smooth muscle

Summary The effects of dopamine (DA) were studied on guinea-pig isolated tracheal chains. At a low concentration (10^–6M) DA occasionally produced a small contraction; this was followed by a dose-dependent relaxation 3×10^–6–3×10^–3 M).On a molar basis, DA was about 40 times less potent than noradrenaline (NA) in relaxing tracheal chains, and about 2700 times less potent than isoprenaline (ISO). The maximum degree of relaxation obtained with each drug was the same.Pretreatment of the guinea-pig with reserpine (5 mg/kg) resulted in a 3-fold shift of the DA curve to the right without concomitantly affecting the ISO doseresponse curve. Reserpine completely abolished the relaxant effects of tyramine, but a small contractile response remained.Desipramine (DMI), at a concentration of 10^–5 M, caused a 4-fold shift of the DA curve to th right. The same concentration of DMI resulted in a shift to the left of the NA dose-response curve by about 8-fold. Benztropine (10^–5 M) and haloperidol (10^–5 M and 3×10^–5 M) did not affect the DA dose-response curve.The DA-induced relaxation was inhibited by propranolol (10^–8–10^–6 M) in a dose-dependent manner. The higher concentrations of propranolol (10^–7 and 10^–6 M) unmasked the contractile effect of DA. In the presence of propranolol, phentolamine (10^–5 M) abolished the contractile effect of DA.It is concluded that DA has both direct and indirect actions on guinea-pig isolated tracheal smooth muscle. The relaxant effects of DA are predominantly due to a direct action on smooth muscle -adrenoceptors, with a component due to release of NA from adrenergic nerves. The contractile effects, which under normal conditions are masked by the relaxant effect of DA, are mediated by functional -adrenoceptors. There is no evidence for either specific dopaminergic nerves, uptake mechanisms or receptors in guinea-pig trachealis muscle.     

Low concentrations of UD-CG 212 enhance myocyte contractility by an increase in calcium responsiveness in the presence of inorganic phosphate

Recent data show that UD-CG 212 in nanomolar concentrations increases myofibrillar Ca⁺⁺ responsiveness of chemically skinned cardiac preparations in the presence of elevated inorganic phosphate. We studied the effects of UD-CG 212 on cell shortening of intact myocytes and in addition measured the intracellular calcium transients with the aid of INDO-1 fluorescence in the presence of 5 mM inorganic phosphate.The validity of our experimental system was first tested with the calcium channel opener Bay k 8644. Bay k 8644 at 10^–8 M did not significantly influence myocyte shortening ( + 13.9 ± 4.6%, n = 9) but at 10^–7 M and 10^–6 M significantly increased contraction by 40.1 +- 13.6%and52.5 ± 17.0% respectively. Bay k 8644 at 10^–8 M increased the INDO-1 fluorescence ratio by 17.3 ± 4.7% (P < 0.01; n = 9), and at 10^–7 M by 21.5 + 4.3% (P < 0.01; n = 9), whereas 10^–6 M Bay k 8644 had no significant effect on peak INDO-1 ratio. However, 10^–7and 10^–6 M Bay k 8644 accelerated and broadened the calcium transients.Cell shortening of guinea pig ventricular myocytes electrically stimulated at 1 Hz was significantly increased by UD-CG 212 (10^–9-10^–7 M) and isoprena line(3 × 10^–8 M). An increase of 37.0 ± 14.0% (P < 0.05; n = 9) was observed at 10^–9 M UD-CG 212, 90.5±18.2% (P<0.05; n=9) at 10^–8 M UD-CG 212, 164.0 ± 34.9% (P < 0.05; n = 9) at 10^–7 M UD-CG 212, and 258.2 ± 67.4% (P < 0.05; n = 9) at 3 × 10-8 M isoprenaline. Peak INDO-1 fluorescence ratios were not significantly (P > 0.05) influenced after addition of 10^–9 M and 10^–8 M UD-CG 212, but significantly increased by 19.4 ± 4.9%(P < 0.05; n = 9) at 10^–7 MUD-CG 212 and by 81.1 ± 11.1% (P < 0.05; n = 9) at 3 x 10^–8 M isoprenaline.In conclusion, UD-CG 212 (10^–9 - 10^–7 M) Concentration-dependently increased myocyte shortening in the presence of 5 mM inorganic phosphate. Low concentrations of 10^–9 and 10^–8 M UD-CG 212 increased myocyte contractility without altering the peak INDO-1 fluorescence ratio whereas 10^–7 M UD-CG 212 and 3 × 10^–8 M isoprenaline increased cell shortening as well as peak INDO-1 fluorescence ratio. These data suggest that low concentrations of UD-CG 212 increase myocyte contractility by enhancing myofibrillar calcium responsiveness whereas higher concentrations elevate intracellular calcium probably via increased intracellular CAMP brought about by phosphodiesterase inhibition.     

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     

Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha1-adrenoceptors in the rabbit ventricular myocardium

Summary The influence of the alpha_lb-adrenoceptor-selective antagonist chlorethylclonidine on the alpha₁-adrenergic positive inotropic effect and the phosphoinositide hydrolysis induced by phenylephrine was investigated in the rabbit ventricular myocardium. Pretreatment of membrane fractions derived from the rabbit ventricular muscle with 10^–5 mol/l chlorethylclonidine decreased the specific binding of [³H]prazosin (at a saturating concentration of 10^–9 mol/l) from the control value of 11.27±0.48 to 4.18±1.87 fmol/mg protein. The inhibition by adrenaline of the binding of [³H]prazosin (slope factor and affinity) was not affected by chlorethylclonidine. The positive inotropic effect of phenylephrine (in the presence of 3 × 10^–7 mol/l bupranolol) was inhibited by chlorethylclonidine in a concentration-dependent manner (10^–7–10^–5 mol/l) and abolished by 10^–5 mol/l chlorethylclonidine. The concentration of chlorethylclonidine to inhibit the phenylephrine-induced maximum response to 50% was 2.4 × 10^–6 mol/l. The accumulation of [³H]inositol monophosphate and [³H]inositol trisphosphate induced by 10^–5 mol/l phenylephrine was inhibited by chlorethylclonidine in the same concentration range. These findings indicate that the myocardial alpha₁-adrenoceptors mediating a positive inotropic effect in the rabbit ventricular myocardium may belong to the chlorethylclonidine-sensitive alpha_1b-subtype, and that the subcellular mechanism of action involve phosphoinositide hydrolysis. Send offprint requests to M. Endoh at the above address