Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

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     

Effects of the potassium channel openers KRN4884 and levcromakalim on the contraction of rat aorta induced by A23187, compared with nifedipine

We examined the different vasodilatory effects of the K⁺ channel openers levcromakalim and 5-amino-N2-[2-(2-chlorophenyl)ethyl]-N-cyano-3-pyridinecarboxamidine (KRN4884), and the Ca²⁺ channel blocker nifedipine in the rat aorta. KRN4884 (10^–10-10^–5 M) and nifedipine (10^–10–10^–5 M) produced concentration-dependent relaxation in the rat aorta precontracted by 25 mM KCl. The K⁺ channel blocker glibenclamide (1 M) inhibited the relaxation induced by KRN4884 but did not influence nifedipine-induced relaxation. KRN 4884 had almost no effect on contraction induced by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl. These results indicate that KRN4884 is a K+ channel opener. We investigated the relaxant effects of KRN4884 (10^–10-10^–5 M), levcromakalim (10^–9-10^–5 M) and nifedipine (10^–9-10^–5 M) on A23187 (1 M)-induced contraction. KRN4884 and levcromakalim had a potent relaxant effect but nifedipine only a weak effect on the smooth muscle contracted by A23187. Glibenclamide (1 M) inhibited the relaxation induced by KRN4884 and levcromakalim, but did not influence the nifedipine-induced relaxation. KRN 4884 (1 M) produced a larger relaxation of A23187-induced contraction but had little effect on the increase in intracellular [Ca²⁺] induced by A23187. These results suggest that KRN4884 is a specific K⁺ channel opener and its vasodilating mechanisms involve not only deactivation of Ca²⁺ channels but also a decrease in the Ca²⁺ sensitivity of contractile elements.     

Use of ion channel blockers in studying the regulation of skeletal muscle contractions

Summary Effects of K⁺- and Cl^–-channel blockers on the muscle contraction of mouse diaphragm in response to direct electrical muscle stimulation were studied. K⁺-channel blockers (0.1–1 mmol/1 4-aminopyridine, 0.4–1.2 mmol/l uranyl nitrate and 2-30 mmol/l tetraethylammonium chloride) and a Cl^–-channel blocker (0.01–0.03 mmol/1 9-anthracene carboxylic acid) increased the contractile amplitudes in a limited extent not to exceed over 50% of control. However, the sequential applications of two different channel blockers at a rather low concentration markedly increased the contractile responses mostly over 300% of control except the combination of 4-aminopyridine and uranyl nitrate. It appears that two K⁺-channel blockers synergistically exerted their effects rather than additionally in the regulation of muscle contractions. Investigation on the possible mechanism of the synergistic action of K⁺-channel blockers suggested that prolongation of action potential durations was in a linear correlation with the increased contractions. On the other hand, the contractile potentiation induced by combination of K⁺- and Cl^–-channel blockers was attributed to the production of repetitive action potential firings (150±12 Hz) upon a single electrical stimulation. Similar to Cl^–-channel blocker, low Cl^– as well as low Ca²⁺ enchanced K⁺-channel blockers in producing contractile potentiation accompanied with stimulus-bound repetitive discharges. Tetrodotoxin at a concentration of 0.03 mol/l which did not affect the twitches evoked by electrical stimulations completely inhibited the contractile potentiation induced by the combined application of K⁺- and Cl^–-channel blockers. It was believed that these studies on the contractions of mouse diaphragm carried out in the physiological salt solution provided a better approach in exploring the possible functions of K⁺- and Cl^–-channels in the regulation of skeletal muscle contractions. Moreover, because of the different K⁺-channels inhibited by these blockers (4-aminopyridine and uranyl nitrate majorly on delayed rectifier K⁺-channel and tetraethylammonium ion on Ca²⁺-activated and ATP-sensitive K⁺-channel), it was concluded that the different types of K⁺-channels as well as Cl^–-channels exert their effects in a synergistic manner on the regulation of the skeletal muscle contractions.     

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     

Two phases of the prostaglandin F2α-induced contraction in guinea-pig taenia coli involve different Ca2+ channels

Summary Properties of the contraction produced by PGF₂ in the guinea-pig taenia coli were compared to those produced by ACh. Prostaglandin (PG) F₂ (3 × 10^–7 M) and acetylcholine (ACh, 10^–5 M) induced an initial transient contraction (phasic contraction) and a subsequent late contraction (tonic contraction). Both phasic and tonic contractions produced by PGF₂ or ACh were abolished in Ca²⁺-free Krebs solution containing 0.5 mM EGTA. The tonic contractions caused by PGF₂ and ACh were markedly suppressed by -[3-[[2-(3,4-dimethoxy-phenyl)-ethyl]methylamino]-propyl]-3,4,5-trimethoxy--(1-methylethyl) benzeneacetonitrile hydrochloride (D600, > 10^–7 M) as well as nifedipine (5 × 10^–9 M), a Ca²⁺-antagonist. However, the phasic contraction produced by PGF₂, but not by ACh, was greatly inhibited by Mn²⁺ (> 10^–4 M). Furthermore, the phasic contraction caused by PGF₂ was abolished in 18 mM K⁺ Krebs solution with D600 (2 × 10^–7 M), whereas that induced by ACh and the tonic contractions produced by PGF₂ as well as by ACh were unaffected in this high K⁺ solution without D600. Membrane potentials of the tissue in normal (K⁺, 5.9 mM) and 18 mM K⁺ Krebs solution containing D600 were about –55 mV and –43 mV, respectively. In a fluorescence study which used Fura-2 an intracellular free Ca²⁺ indicator in the presence of D600, PGF₂ and ACh increased fluorescence intensity in the tissue, which coupled with the magnitude of contractions. Both the enhanced fluorescence intensity and tension development evoked by PGF₂, but not by ACh, were markedly decreased in 18 mM K⁺ Krebs solution. It is, thus, suggested that the phasic and tonic contractions evoked by PGF₂, unlike those by ACh, may be mediated via certain different Ca²⁺ channels from extracellular Ca²⁺-source.Send offprint requests to S. Usune at the above address     

Presence of muscarinic inhibitory and absence of nicotinic excitatory receptors at the terminal sympathetic nerves of chicken hearts

Summary Nicotine (2×10^–4 M) or acetylcholine (5.5×10^–4 M) in the presence of 3×10^–6 M atropine did not increase the rate or amplitude of contraction in isolated atria or ventricular strips of the chicken heart; both drugs also did not cause an output of noradrenaline or adrenaline and did not evoke antidromic discharges in the right sympathetic nerves of isolated perfused chicken hearts. In contrast, high K⁺-solutions evoked an output of noradrenaline and adrenaline and caused a burst of antidromic discharges. Dimethylphenylpiperazine (DMPP; 3.1×10^–4 M), by a tyramine-like action, elicited a small output of noradrenaline and increased rate and amplitude of contraction, but did not evoke antidromic discharges. The noradrenaline output caused by DMPP was not reduced by lowering the extracellular Ca²⁺ concentration from 1.8 to 0.1125 mM.-Acetylcholine (10^–5 and 10^–4 M) inhibited the noradrenaline and adrenaline outputs evoked by electrical stimulation of the right sympathetic nerves (3 Hz, 1 ms, 30 V); the inhibition was blocked by 3×10^–6 M atropine. —It is concluded that the terminal parts of sympathetic nerves of the chicken heart posses muscarinic inhibitory receptors but lack nicotinic excitatory receptors. Thus prejunctional nicotinic receptors are not an integral part of the terminal sympathetic neurone otherwise they would be present at this neurone in all species.This work was part of the M. D. thesis of U. Engel     

Effect of calcium antagonists (ω-conotoxin GVIA,verapamil, gallopamil,diltiazem) on bronchial smooth muscle contractions induced by soman

Summary The effect of the calcium antagonists -conotoxin GVIA, verapamil, gallopamil and diltiazem was investigated on in vitro bronchial smooth muscle contraction in the rat induced by the nerve agent soman. Soman inhibits the acetylcholinesterase activity irreversibly. The effect of the calcium channel antagonists on contractions induced by electrical field stimulation and carbachol was also investigated, in order to elucidate the mechanism by which calcium antagonists inhibit the soman induced contraction.-Conotoxin GVIA reduced the bronchial smooth muscle contraction induced by electrical field stimulation with an almost complete inhibition at approximately 1.0× 10^–6 M. The soman induced contraction was only inhibited by 15% at a concentration of 3.0× 10^–6 M -conotoxin GVIA. The organic calcium antagonists verapamil, gallopamil and diltiazem reduced both electrically and soman induced smooth muscle contraction. Complete inhibition of the contractions induced by soman was achieved at 1.4x 10^–4 M for verapamil and gallopamil, while diltiazem inhibited the contraction to 7% of control at 1.4 × 10^–4 M. Verapamil, gallopamil and diltiazem increased the EC₅₀ for carbachol significantly, while co-conotoxin GVIA had no effect. None of the calcium antagonists had any effect on the maximal contraction induced by carbachol. Verapamil, gallopamil and diltiazem blocked, however, sub-maximal contractions induced by carbachol (10^–7 -10^–5 M) resulting in a right-shift of the dose response curve.The results show that co-conotoxin GVIA inhibits the calcium-dependent release of acetylcholine which causes contraction of airway smooth muscle, while it has no effect on smooth muscle contraction induced by soman. Gallopamil, verapamil and diltiazem inhibit the contraction of bronchial smooth muscle following stimulation of postjunctional muscarinic receptors resulting from inhibition of the acetylcholinesterase activity by soman. Gallopamil and verapamil inhibit the contraction more potently than diltiazem. Correspondence to P. Aas at the above address     

Functional role of sodium pump in human placental arteries

Summary Ouabain (10^–7 to 10^–4 M) elicited concentration-dependent contractile responses in human placental arteries. The contractions were reduced by 10^–4 M amiloride and Ca²⁺-free medium, but not affected by 10^–6 M nifedipine or 10^–6 M Bay-K-8644, which markedly reduced or potentiated 75 mM K⁺-induced contractions, respectively. After contracting the vessels with 10^–6 M prostaglandin F₂ in a K⁺-free medium, the subsequent addition of 7.5 mM K⁺ induced a marked relaxation, which was blocked by 10^–6 M ouabain. This glycoside (10^–8 to 10^–4 M) also produced a concentration-dependent reduction of ⁸⁶Rb⁺ uptake. Scatchard analysis of the [³H]-ouabain binding to membrane fractions from human placental arteries suggests a single class of binding sites with a KD of 88.3 nM and a B_max of 345 fmol/mg. 5-Hydroxytryptamine (5-HT; 10^–9 to 10^–5 M) caused concentration-dependent contractions. Single concentrations produced transient responses composed of an initial contraction, followed by a slow fall in tension. Ouabain (10^–8 to 10^–6 M), K⁺-free medium or the reduction of bath temperature (28°C) did not modify contractions but inhibited the relaxant phase of the response. 5-HT (10^–8 to 10^–6 M) increased both total and ouabain-insensitive ⁸⁶Rb⁺ uptake, but the difference between them was not modified. These data indicate that: (1) human placental arteries possess an important sodium pump activity, inhibition or stimulation of which markedly alters vascular tone, (2) ouabain-evoked contractions are produced by Ca²⁺ entry mainly through Na⁺-Ca²⁺ exchange, secondary to intracellular Na⁺ accumulation, (3) the relaxant component of 5-HT response is dependent on the activity of the sodium pump, (4) the activation of Na⁺,K⁺-ATPase activity by this amine is not apparently due to direct effect, and (5) the inhibition of the sodium pump can cause long lasting increases of placental vascular resistance in the presence of physiological concentrations of 5-HT. Send offprint requests to J. Marin at the above address     

Field stimulation-induced responses of circular smooth muscle from guinea-pig stomach

Summary Field stimulation of circular smooth muscle of guinea-pig stomach from the regions of the cardia and fundus caused contraction responses at low stimulation frequencies (0.25–1 Hz) with relaxation at higher frequencies (1–10 Hz), whilst tissues of the body and antrum responded with contraction throughout the frequency range. Atropine (10^–9–10^–8 M) antagonised the contraction responses of all tissues, with relaxation developing at higher concentrations (except for antral tissue). In contrast, metoclopramide (10^–8–10^–6 M) caused modest (cardia, fundus) or marked (body, antrum) enhancement of contractions to field stimulation, whilst domperidone (10^–8–10^–7 M), haloperidol (10^–8–10^–6 M), prazosin, propranolol and methysergide (10^–8–10^–6 M) failed to modify the contraction responses. However, whilst yohimbine and guanethidine failed to modify the contractions of the cardia, fundus and body tissues, those of the antral preparations were antagonised by nanomolar concentrations of yohimbine and by guanethidine (10^–6–5×10^–5 M). To optimise the relaxation responses for study, atropine was included in the physiological solution. Relaxation to field stimulation of preparations from the body and cardia, but not the fundus, was antagonised by reserpine pretreatment (5 mg/kg i.p., 24h), addition of guanethidine (10^–5–10^–4 M), phentolamine, prazosin or propranolol (10^–7–10^–6 M) (the effects of prazosin and propranolol being additive). Higher concentrations of haloperidol and domperidone antagonised the relaxation responses of the body preparations only. Metoclopramide, yohimbine and methysergide (10^–8–10^–6 M) were ineffective. Thus, it is concluded that the contractile effects of the 4 stomach areas to field stimulation reflects a major cholinergic involvement, with an additional ₂-adrenoceptor contractile component in antral tissue. Relaxation responses of cardia and body tissue involve ₂- and -adrenoceptors plus a further, unidentified, non-adrenergic component; the latter represents the total relaxation response of the fundic preparation.     

Characteristics and possible mechanisms of low - Na+ induced contractions in rat aorta

The influence of reducing external Na⁺ concentration ([Na⁺]^ex) upon vascular smooth muscle contractility was investigated using the rat isolated aorta. NaCl from the physiological saline solution (PSS) was replaced with either choline-Cl, sucrose, or LiCl to give the following [Na⁺]_ex (mM): 115, 85, 55, and 25 (115NaPSS to 25NaPSS). Small reductions in [Na⁺]_ex (115NaPSS) induced a biphasic contraction, comparable in amplitude with the control one induced by phenylephrine 10^–6 M. Elimination of the endogenous catecholamine participation using either phentolamine 10^–5 M or guanethidine 3.10^–6 M similarly reduces these contractions to 25% (sucrose replacement). A similar relaxing effect was obtained with D600 10^–5 M, an antagonist of the voltage operated Ca²⁺ channels (25–30% residual tension for all the substitutes). Large reductions in [Na⁺]_ex (25NaPSS) induced contractions comparable in amplitude and shape, but less sensitive to phentolamine and guanethidine (residual tension 65–75 %, sucrose replacement) and insensitive to D600 (all the substitutes). The Na⁺/K⁺ ATPase inhibitor ouabain (10^–4 M) elicited slowly developing contractions, the amplitude being 115% of the phenylephrine 10^–6 M control.Phenylephrine further contracted the 115NaPSS precontracted preparations, but was significantly less effective in 25NaPSS, although the precontraction levels were similar for the same substitute used. The amplitude of the superimposed phenylephrine contractions exhibited [Na⁺]_ex dependence. Phenylephrine 10^–6 M failed to further contract the ouabain 10^–4 M precontracted rings.We conclude that relatively small reductions in [Na⁺]_ex are able to induce contractions of rat aorta primarily through release of endogenous catecholamines, probably through neural Na⁺/Ca²⁺ exchange. Larger reductions in [Na⁺]_ex appear to cause contraction through muscular Na⁺/Ca²⁺ exchange.     

Effect of moderate cooling on contractile responses in mouse vas deferens and its relation to calcium

Isolated mouse vas deferens preparations were used to study the effect of temperature on noradrenaline-induced contractions. Preparations were suspended in the organ bath containing Krebs-Henseleit solution for isometric tension recording. Contractile responses to noradrenaline were investigated in the mouse vas deferens after moderate cooling from 37 to 26 or 22° C. A significant increase of the phasic contractions to noradrenaline was observed at 26 or 22°C compared with responses obtained at 37° C (about 12.3 and 35.6% increase at 26 and 22° C, respectively). The secondary noradrenaline-induced sustained contraction was also significantly enhanced after moderate cooling to 26° C. The potentiation of noradrenaline-induced contraction at 26° C remained in a Ca²⁺-free EGTA (1 mM)-containing solution. However, sustained contraction was suppressed after removal of the calcium from the medium at 37 and 26°C. Contraction to caffeine was significantly enhanced at 22° C compared with 37°C. By contrast, barium chloride-induced contraction of the vas deferens was markedly decreased after moderate cooling to 22° C. In the presence of ouabain (0.1 mM), the noradrenaline-induced peak contraction was significantly increased at 37°C. However, potentiation of the noradrenaline response at 22° C was unaffected by the Na⁺/K⁺ pump inhibitor. Noradrenaline-induced peak contractions were depressed in the presence of vanadate (1 mM) and cyclopiazonic acid (10 M), two Ca²⁺-ATPase inhibitors, at 37° C and also at 22° C. These results suggest that temperature-induced hyperreactivity is partly due to an increase of the amount of calcium released from intracellular stores. The inhibition of the Na⁺/K⁺ pump due to cooling may participate in this effect whereas Ca²⁺-ATPase inhibition does not seem to be involved.     

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     

A target K+ channel for the LP-805-induced hyperpolarization in smooth muscle cells of the rabbit portal vein

Summary The resting membrane potential of smooth muscle cells of the rabbit portal vein was –51.2 mV. LP-805 (8-tert-butyl-6,7-dihydropyrrolo[3,2-e] 5-methylpyrazolo [1,5-a] pyrimidine-3-carbonitrile) hyperpolarized the membrane to –62.3 mV (10 M) and inhibited the burst spike discharges as measured using the microelectrode method. In dispersed smooth muscle cells, LP-805 (10 M) generated an outward-current with a maximum amplitude of 68 pA at a holding potential of –40 mV in experiments using the voltage-clamp procedure. The reversal potential of the outward current evoked by LP-805 was –82 mV and this value was close to the equilibrium potential for K⁺ (–80 mV) in the present ionic conditions, suggesting that LP-805 activated the K⁺ channel. Generation of both the hyperpolarization and the outward c urrent by LP-805 was inhibited by glibenclamide ( 1 M). Using the cell-attached and cell-free patch-clamp (in the presence of GDP) procedures, the maxi-K⁺ channel current (150 pS) could be recorded in the absence of LP-805; application of LP-805 additionally opened a small conductance K⁺ channel current (15 pS) without change in the activity of the maxi-K⁺ channel. The maxi-K⁺ channel was sensitive to charybdotoxin (0.1 M) and to intracellular Ca²⁺ ([Ca²⁺]_i) concentration. The 15 pS channel was insensitive to [Ca²⁺]_i and charybdotoxin, but sensitive to intracellular ATP concentration. Glibenclamide (> 1 M) inhibited the 15 pS K⁺ channel activated by LP-805. These actions of LP-805 on the maxi-K⁺ and 15 pS K⁺ channels are the same as those previously observed for nicorandil and pinacidil. Thus, LP-805 is a K⁺ channel opener with a chemical structure different from those of the known openers. Correspondence to M. Kamouchi at the above address     

Relaxation Patterns of Human Gastric Corporal Smooth Muscle by Cyclic Nucleotides Producing Agents

To elucidate the mechanism of cyclic nucleotides, such as adenosine 3'',5''-cyclic monophosphate (cAMP) and guanosine 3'',5'' -cyclic monophosphate (cGMP), in the regulation of human gastric motility, we examined the effects of forskolin (FSK), isoproterenol (ISO) and sodium nitroprusside (SNP) on the spontaneous, high K⁺ and acetylcholine (ACh)-induced contractions of corporal circular smooth muscle in human stomach. Gastric circular smooth muscle showed regular spontaneous contraction, and FSK, ISO and SNP inhibited its phasic contraction and basal tone in a concentration-dependent manner. High K⁺ (50 mM) produced sustained tonic contraction, and ACh (10 µM) produced initial transient contraction followed by later sustained tonic contraction with superimposed phasic contractions. FSK, ISO and SNP inhibited high K⁺-induced tonic contraction and also ACh-induced phasic and tonic contraction in a reversible manner. Nifedipine (1 µM), inhibitor of voltage-dependent L-type calcium current (VDCC_L), almost abolished ACh-induced phasic contractions. These findings suggest that FSK, ISO and SNP, which are known cyclic nucleotide stimulators, inhibit smooth muscle contraction in human stomach partly via inhibition of VDCC_L.     

Coenzyme Q10 attenuates cyanide-activation of the ATP-sensitive K+ channel current in single cardiac myocytes of the guinea-pig

Summary The effect of coenzyme Q₁₀ (CoQ₁₀) on the cyanide (CN^–)-induced ATP-sensitive K⁺ channel current (K_ATP) was examined in single atrial myocytes, using the patch clamp technique. Superfusion of the cells with a CN^–/low glucose bathing solution induced an outward current in the whole-cell clamp condition. Glibenclamide (1 M) abolished this current, indicating that the current was carried through the K_ATP channel. After steady-state activation by CN^–, pinacidil (a K_ATP channel opener, 300 M) failed to further increase the current. In cell-attached patches, CN^–, when applied to the bath, induced bursting openings of an 80 pS channel (the K_ATP channel). In cells preincubated for 30 min in a solution containing CoQ₁₀ (100 g/ml), CN^–-activation of the K_ATP channel was markedly attenuated both at the whole cell and at the single channel level. At the steady-state effect of CN^– in CoQ₁₀-treated cells, pinacidil (300 M) activated the current to the maximum level achieved by CN^– in the control cells. These results suggest that CoQ₁₀ reduces in the CN^–-induced KATP current not by affecting the channel itself but by preventing depletion of intracellular ATP caused by CN^–. Send offprint requests to Y. Kurachi at Mayo Foundation     

Effects of phthalazinol on smooth muscle cells of the porcine coronary artery and on neuromuscular junction on the guinea-pig vas deferens

Summary In smooth muscle cells of the porcine coronary artery, phthalazinol (10^–5 M) did not modify the membrane potential and the membrane resistance. At a concentration of 10^–4 M or higher, it hyperpolarized the membrane, reduced the membrane resistance and enhanced the rectifying property of the membrane. At the concentration of 10^–5 M, phthalazinol raised the threshold for the induction of a contraction and suppressed nonselectively the amplitude of the contraction evoked by application of high [K]₀, acetylcholine or electrical depolarization of the membrane. Phthalazinol (10^–5–10^–4 M) did not modify the membrane properties of smooth muscle cells from the guinea-pig vas deferens. However, it suppressed the amplitude of the excitatory junction potentials and the facilitation phenomenon produced by repetitive stimulation at various rates. The action potential recorded from the adrenergic nerves was not affected in the presence of phthalazinol (10^–5 and 10^–4 M). The mean amplitude of the miniature excitatory junction potentials (m.e.j.p.s.) was not affected by treatment with phthalazinol (10^–5–10^–4 M), but the rate of which of m.e.j.p.s. appeared was reduced by phthalazinol (>5×10^–5 M). These results indicate that the vasodilator property of phthalazinol may result from a suppression of Ca-mobilization in both the smooth muscle cells and the adrenergic nerve terminals. The former affects the mechanical response directly and the latter leads to an inhibition of noradrenaline release.     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Die verschiedene Empfindlichkeit der Na+ + K+-aktivierten ATPase des Herz- und Skeletmuskels gegen k-Strophanthin

Zusammenfassung 1. Membran- und Vesikelfraktionen des Skeletmuskels enthalten ebenso wie die des Herzmuskels eine durch Na⁺ + K⁺-aktivierbare und durch k-Strophanthin hemmbare ATPase.2. Im Gegensatz zum Herzmuskel findet sich in Membranfraktionen des Skeletmuskels eine maximale Aktivität der Na⁺ + K⁺-stimulierten ATPase nicht nur bei hohen Na⁺- und niedrigen K⁺-Konzentrationen, sondern auch bei der Umkehr des Ionenverhältnisses, d. h. bei hohen K⁺- und niedrigen Na⁺-Konzentrationen.3. Untersuchungen über die Wirksamkeit des Strophanthins an Membranfraktionen des Herz- und Skeletmuskels von Kaninchen, Meerschweinchen und Ratten ergaben deutliche artspezifische Empfindlichkeitsunterschiede: Eine halbmaximale Hemmung der Na⁺ + K⁺-aktivierten ATPase des Herz- bzw. Skeletmuskels fand sich an Kaninchen bei 0,7 bzw. 700 M/l, an Meerschweinchen bei 1,0 bzw. 500 M/l und an Ratten bei 50 bzw. 300 M/l. Die Na⁺ + K⁺-aktivierte Membran-ATPase des Skeletmuskels ist demnach bei Kaninchen 1000 mal, bei Meerschweinchen 500 mal und bei Ratten sechsmal unempfindlicher als diejenige des Herzmuskels. Die Na⁺ + K⁺-aktivierte Membran-ATPase des Rattenherzens ist gegen k-Strophanthin 50–70 mal unempfindlicher als die des Meerschweinchen-bzw. Kaninchenherzens.4. Die Empfindlichkeit der Vesikelfraktion des Herz- und Skeletmuskels des Kaninchens gegenüber Strophanthin entspricht derjenigen der Membranfraktion. Der Zusatz von Na-Azid vermindert die Empfindlichkeit der Na⁺ + K⁺-aktivierten Vesikel-ATPase des Skeletmuskels gegen Strophanthin und hat zur Folge, daß niedrige Strophanthinkonzentrationen (10^–6–10^–8) eine Aktivierung verursachen.

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Summary 1. Membrane and vesicle fractions of skeletal muscle contain like those of cardiac muscle a Na⁺ + K⁺ activated ATPase which is inhibited by k-strophanthine.2. Contrary to the cardiac muscle there is a maximum of activity of the Na⁺ + K⁺ stimulated ATPase in the membrane fraction of the skeletal muscle not only at high Na⁺ and low K⁺ concentrations, but also at the reversal of the ion relations, that is at high K⁺ and low Na⁺ concentrations.3. Investigations of the effectiveness of strophanthine on the membrane fractions of cardiac and skeletal muscle of rabbits, guinea-pigs and rats resulted in a significant species specifity in sensitiveness: a half maximal inhibition of the Na⁺ + K⁺ activated ATPase of the heart or skeletal muscle was found in rabbits at 0,7 or 700 M/l, in guinea-pigs at 1,0 or 500 M/l and in rats at 50 or 300 M/l respectively. The Na⁺ and K⁺ activated membrane ATPase of the skeletal muscle is accordingly in rabbits 1000 times, in guinea-pigs 500 times but in rats only 6 times less sensitive than that of the cardiac muscle. The Na⁺ and K⁺ activated membrane ATPase of the rat hearts is 50–70 times less sensitive to strophanthine than that of guinea-pig or rabbit hearts.4. The sensitiveness of the vesicle fraction of heart- and skeletal muscle of the rabbit to strophanthine corresponds to that of the membrane fraction. Addition of sodiumacide diminishes the sensitiveness of the Na⁺ and K⁺ activated vesicle fraction and results in an activation by low strophanthine concentrations (10^–6–10^–8).

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Herrn Professor Dr. O. Schaumann zum 75. Geburtstag gewidmet.

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Die Ergebnisse wurden mit Ausnahme der Befunde an Vesikelfraktionen auf der 28. Tagung der Deutschen Pharmakologischen Gesellschaft am 8. Oktober 1964 in Bad Nauheim vorgetragen [Dransfeld, H., u. K. Greeff: Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 250, 244 (1965)].     

Effects of propofol on responses of rat isolated renal arteriole to vasoactive agents

Rat renal arterial rings were suspended in organ chambers for isometric tension recording. The effects of propofol on the resting tone, KCl-, norepinephrine (NE)-, serotonin- and thromboxane A₂ analog U46619-induced contractions were observed. The relaxation responses to propofol on KCl-, NE- and U46619-induced contractions were assessed in the absence or presence of cyclooxygenase inhibitor, nitric oxide synthetase inhibitor or specific K⁺ channel inhibitors. Propofol did not significantly affect the resting tone, but inhibited the contractions induced by KCl-, NE-, serotonin- and U46619. Propofol (1–100 µM) concentration-dependently relaxed 60 mM KCl-, 10 µM NE-, and 1 µM U46619-induced contractions with the values of RC₅₀ (concentration to decline the precontraction by 50%) being 18.9 µM, 70.6 µM and 12.7 µM, respectively. Propofol-induced relaxation was attenuated by indomethacin, but not by either N^G nitro-l-arginine methyl ester (L-NAME) or any K⁺ channel specific inhibitors used. The vasorelaxations induced by acetylcholine, sodium nitroprusside and amrinone were not affected by the presence of propofol. The present results indicate that propofol antagonizes provoked contractions of the arteriole and suggest that inhibition of extracellular Ca²⁺ influx and synthesis of vasodilator prostanoid may be involved in propofol-induced relaxation of the arteriole.