Influence of phorbol esters on stimulation-induced noradrenaline release and contractile force of isolated guinea-pig atria

Phorbol-12,13-dibutyrate (PDB) and phorbol-12-myristate-13-acetate (PMA) increased the field-stimulation-induced efflux of radioactivity from guinea-pig atria preloaded with 7-[³H]-noradrenaline. The efflux was more than doubled by 10^–7 mol/l of either compound. Phorbol-12-myristate-13-acetate-4-O-methylether (PME), which has no effect on the protein kinase C (PKC), did not modify the stimulation-induced efflux of radioactivity, while a slight reduction was seen with 70 M of the PKC inhibitor polymyxin B. In the presence of polymyxin B, the effects of PMA and PDB were greatly attenuated. In addition, PDB had a concentration-dependent negative inotropic effect (EC₅₀ 7,0 × 10^–10 mol/l). Pretreatment with polymyxin B shifted the concentration-response curve for PDB to the right (EC₅₀ 4,6 × 10^–9 mol/l). No negative inotropic effect was seen with PMA or PME. The results suggest that all effects of the phorbol esters were mediated by a stimulation of PKC. The different lipophilicity of PMA and PDB or a different influence on the various isozymes of PKC may account for the diverging postjunctional effects of the two compounds.     

Effects of the divalent cation ionophore ionomycin on the performance of isolated guinea-pig atria

Summary In isolated electrically driven left and in spontancously beating right guinea-pig atria, the calcium ionophore ionomycin produced a concentration-dependent positive inotropic and chronotropic effect with a threshold near 10^–7 mol/l and a pD₂ of 6.31±0.09 and 5.94±0.07, respectively. At low [Ca²⁺]_o (0.5 mmol/l), the positive inotropic effect of ionomycin (3×10^–6 mol/l) was strongly attenuated by ryanodine and nifedipine, and slightly attenuated by pindolol and mepyramine; atropine had no effect. The positive chronotropic effect of ionomycin was slightly reduced by cimetidine or pindolol, whereas atropine, nifedipine, and ryanodine showed no inhibitory activity. The oxygen consumption of resting left atria was significantly enhanced by addition of ionomycin.It is concluded that the action of ionomycin involves at least the following mechanisms: I) release of Ca²⁺ from sarcoplasmic reticulum, II) influx of Ca²⁺ from the extracellular space, and having little significance, III) release of catecholamines and histamine from sympathetic nerve endings and tissue mast cells. However, additional mechanisms of action of ionomycin cannot be excluded.Preliminary results were presented at the 29th Spring Meeting of the Deutsche Gesellschaft für Pharmakologie und Toxikologie, March 8–11, 1988, Mainz, FRG     

Analysis of the inotropic: chronotropic selectivity of dobutamine and dopamine in anaethetised dogs and guinea-pig isolated atria.

The previously reported in vivo inotropic selectivity of dobutamine and dopamine compared with isoprenaline has been demonstrated in anaesthetised bivagotamised open chest dogs. Responses of heart rate, right ventricular tension, left ventricular dP/dtmax, and blood pressure were measured. Compared with isoprenaline, dobutamine and dopamine were 1.8 and 2.4 times more active in increasing cardiac contractility than rate. The inotropic selectivity of dopamine but not that of dobutamine was abolished by pretreatment of dogs with syrosingopine. In guinea-pig isolated atria, isoprenaline, dobutamine, and dopamine were all slightly rate selective although this was less for dopamine. In atria incubated with phenoxybenzamine from guinea-pigs pretreated with reserpine only the dopamine dose-response curves were displaced to the right indicating a considerable indirect sympathomimetic component. The in vivo inotropic selectivity of dopamine could therefore be explained on the basis of this indirect activity being manifested at lower doses in the myocardium where the stores of catecholamines are more abundant than at the sinoatrial node. However, it is concluded that the inotropic selectivity of dobutamine seen in vivo is not due to indirect activity, reflex effects, or to a difference in the beta-adrenoceptors mediating the rate and tension responses of the heart. Possible alternative explanations are discussed.     

黄芪及其活性成分对离体豚鼠心脏的作用

目的 探讨黄芪及其活性成分黄芪总黄酮、黄芪总皂苷、黄芪多糖对豚鼠离体心脏心肌缺血再灌注损伤的影响.方法 将36只豚鼠的离体心脏分为正常对照组(对照组)、缺血再灌注组(模型组)、黄芪组(AST组)、黄芪总黄酮组(TFA组)、黄芪总皂苷组(TSA组)和黄芪多糖组(APS组)6组.检测豚鼠离体心脏的心率、冠状动脉流量、心肌梗死面积、灌流液中乳酸脱氢酶(LDH)和肌酸激酶(CK)的活性、心肌组织中丙二醛(MDA)含量和超氧化物歧化酶(SOD)的活性等.结果 再灌注期各时间点模型组心率均较对照组相应的时间点低(P ＜0.05或0.01),AST组和TFA组则均较模型组高(P ＜0.05或0.01),TSA组和APS组心率没有明显改善.再灌注5、10、15、20、25、30、40、50、60 min时模型组冠状动脉流量均较对照组相应时间点低(P均＜0.01).再灌注期AST组冠状动脉流量均高于模型组(P＜0.05或0.01).再灌注5、10、25、30、40 min时TFA组冠状动脉流量均高于模型组(P ＜0.05或0.01).TSA组和APS组冠状动脉流量没有明显改善.再灌注15、25、40、60 min时模型组灌流液中LDH活力均高于对照组(P均＜0.01).再灌注15、25、40、60 min时AST组灌流液中LDH活力均低于模型组(P均＜0.01).再灌注5、15、25、40、60 min时TFA组灌流液中LDH活力均低于模型组(P＜0.05或0.01).再灌注5 min时APS组灌流液中LDH活力显著低于模型组(P＜0.05).再灌注后各时间点TSA组灌流液中LDH活力与模型组比较差异均无统计学意义.再灌注15、60 min时模型组灌流液中CK活力均高于对照组(P均＜0.01).再灌注15 min时AST组和TFA组灌流液中CK活性均低于模型组(P均＜0.01).再灌注60 min时模型组灌流液中CK活力显著高于对照组(P＜0.01).AST组与TFA组灌流液中CK活力分别均低于模型组(P值分别为0.008和0.015).模型组缺血再灌注心肌组织中MDA含量为(5.95±0.91) μmol/g蛋白高于对照组的(3.62±0.58) μmol/g蛋白(P＜0.01).AST组和TFA组心肌组织中MDA含量分别为(4.52 ±0.47)和(4.47±0.78)μmol/g蛋白,均低于模型组(P分别＜0.01和0.05).模型组缺血再灌注心肌组织中SOD活力为(98.64±12.49) U/mg蛋白低于对照组的(153.55±13.25) U/mg蛋白(P＜0.01).AST组和TFA组心肌组织中SOD活力分别为(130.73 ±14.85)和(124.38±13.35) U/mg蛋白,均高于模型组(P均＜0.01).模型组心肌梗死面积百分比为(18.9±2.27)％高于对照组的(1.60±0.33)％(P＜0.01).AST组和TFA组心肌梗死面积百分比分别为(11.9±2.03)％和(13.31±1.17)％,均低于模型组(P均＜0.01).结论 黄芪及其有效成分黄芪总黄酮对豚鼠离体心脏心肌缺血再灌注损伤有保护作用,其机制可能与抗氧化应激有关.     

Accumulation and washout of glycerol in isolated rabbit atria

Continuously stimulated rabbit atria were equilibrated in Ringer-Locke solution, transfered to Ringer-Locke solution containing 400mm glycerol, and returned to Ringer-Locke solution. Accumulation and washout of glycerol were determined using tritium labeled glycerol. During exposure to the hypertonic media, glycerol was observed to accumulate in atria at a half time of approximately 12 min. Return of atria to Ringer-Locke solution resulted in glycerol washout at a half time of approximately 16 min. Data indicated that only about 80% of the total tissue water is accessible to glycerol, and that the remaining 20% becomes accessible to glycerol when CuSO₄ is added to the hypertonic media. A permanent decrease in wet tissue weight was seen as a result of glycerol treatment.     

Cardiovascular effects of digitalis on intact dogs and isolated cross-perfused atria

The chronotropic effects of digoxin and deslanoside were studied in canine atria cross-perfused with heparinized arterial blood from donor dogs. Intravenous injections of either drug (100 micrograms/Kg) into the donor dog produced bradycardia followed by ventricular tachyarrhythmia, with or without hypertension, in the donor dog. A significant increase in the developed tension was observed in the isolated atria, with or without slight sinus acceleration. These effects continued over 150 min after the injection. Digoxin (200 micrograms/Kg, i.v.) caused an immediate bradycardia followed by ventricular tachycardia. In addition, ventricular fibrillation occurred in 3 out of 5 donor dogs within 20 min of the drug administration. In isolated atria, there was a marked increase in the developed tension, usually with a little sinus tachycardia. Deslanoside (200 micrograms/Kg, i.v.) caused almost the same response patterns as digoxin. However, this dose of deslanoside caused ventricular fibrillation in all 6 experiments. Drug concentrations in the donor's arterial blood decreased rapidly for 15-20 min and then decreased slowly in all experiments. It is concluded that digoxin and deslanoside have no significant direct accelerating action on the SA node in doses which produced marked increases in the developed tension; only extremely high doses cause a direct, slight sinus acceleration.     

Comparison between the cardiac effects induced by muzolimine and furosemide in guinea-pig atria

Summary Muzolimine (10–500 M) induced a concentration-dependent reduction of both the contractile force and frequency in spontaneously beating atria and in electrically driven left atrium from reserpine-treated guinea pigs. This negative inotropic response was unaffected by the addition of atropine to the perfusion fluid, and it was highly sensitive to changes in external Ca²⁺ concentration. Both in spontaneously beating and in electrically driven atrium, muzolimine (50–400 M) antagonized, in an apparently competitive manner, the increase in contractile force induced by cumulative addition of CaCl₂ (0.68–9.59 mM) to the bathing fluid. Muzolimine (50–100 M) reduced the inotropic response to low (5–30 nM), but not high (50–100 nM) concentrations of Bay K 8644, a calcium-channel agonist. The inotropic effects of 8-phenyltheophylline and of ouabain were antagonized by muzolimine (10–100 M) in a noncompetitive manner, while the response to noradrenaline was not altered. Similar to muzolimine, verapamil at a concentration suitable to block calcium channels inhibited, in a noncompetitive way, the inotropic effect induced by 8-phenyltheophylline and by ouabain without altering the contractile response to noradrenaline. Furosemide (10 and 100 M) did not influence the contractile force or the frequency of spontaneously beating atria, nor the inotropic effect induced by CaCl₂, 8-phenyltheophylline, ouabain, or noradrenaline. These results indicate that the influence of muzolimine on guinea-pig atria originates from an inhibition of Ca²⁺ influx into cardiac cells and that furosemide does not mimic the effect of muzolimine at this level.     

Effects of quinidine, procainamide and disopyramide on automaticity and cyclic AMP content of guinea-pig atria

Atrial automaticity can be importantly influenced by fluctuations in intracellular cAMP. The purpose of this study was to determine the effects of quinidine, procainamide and disopyramide (atrial antiarrhythmic drugs) on spontaneous rate and cAMP content of guinea-pig atria. Superfusion of quinidine, procainamide, and disopyramide to isolated tissues at concentrations of 10^?7m to 10^?5m produced concentration-related reductions in spontaneous atrial rate and cAMP content. Lidocaine (a membrane-active drug with no atrial antiarrhythmic effects) did not alter atrial rate or cAMP content. When atrial tissues were pretreated with 8-Bromo cAMP (3 × 10^?4m) to maintain tissue cAMP levels, the negative chronotropic effects of quinidine, procainamide, and disopyramide were attenuated. All three drugs produced a similar magnitude of atrial rate slowing when administered alone or in the presence of 8-Bromo 5′ AMP (3 × 10^?4m). These studies demonstrate that the negative chronotropic effects of quinidine, procainamide, and disopyramide are paralleled by reductions in atrial cAMP content. These results suggest that certain antiarrhythmic drugs may influence electrical impulse formation in part through alterations in the adenylate cyclase - cAMP system.     

Effects of carbocromene on performance and oxidation of FFA and glucose in isolated atria

Conclusions Carbocromene inhibits only inhypoxic injured atria the palmitate oxidation (–32%), whereas glucose oxidation-rate (+17%) increased at the incubation with the combination of these 2 substrates. In thenormoxic controls, however, the competition of substrates in the biological oxidation after the positive inotropic effect of C. was dependent on the V_max-and K_m-values of the oxidation-rate of each substrate as well as its concentration.If carbocromene inhibits the palmitate oxidation in the stage of its penetration through the cell membrane or in the mitochondrial membrane or by other interference in the hypoxic injured atria cannot be decided yet.

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Wirkungen von Carbocromen auf Funktion und Oxidation von FFA und Glukose in isolierten Herzvorhöfen

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Paper, presented at the Erwin Riesch Symposium, Tübingen, April 3–7, 1979

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With 1 figure     

Direct effect of ACTH on renin release in isolated perfused guinea-pig kidneys with adrenal glands.

To investigate the direct effect of corticotropin (ACTH) on the renin-angiotensin-aldosterone system, isolated guinea-pig kidneys with adrenal glands were perfused with various doses of ACTH (0.1-1000 micrograms/l) and 0.3 mmol/l of dibutyryl cyclic AMP (cAMP) through each cannula inserted into the abdominal aorta and the inferior caval vein. Perfusate renin activity was increased in a dose-dependent manner by the addition of ACTH in a range of 0.1-1000 micrograms/l, and reached a plateau at 20 min with each dose. The perfusate cAMP level was dose-dependently increased with 10-1000 micrograms/l of ACTH. Perfusate renin activity was also markedly increased by the addition of dibutyryl cAMP. The same effects of ACTH on renin and cAMP secretions were observed in the kidney perfusion model from which the adrenal glands were excluded. Aldosterone secretion failed to respond to 0.1 micrograms/l of ACTH, and was increased by higher concentrations (1-1000 micrograms/l) in the same experiments. These results demonstrate that ACTH has a direct effect on renal renin release in a physiological concentration (0.1 micrograms/l), and that the action of ACTH is probably mediated by cAMP. The sensitivity of renin release to ACTH stimulation is no less than that of aldosterone secretion during ACTH infusion, so it is possible that ACTH is an important stimulator of the renin-angiotensin system.     

The effects of aging on responses to verapamil in isolated right atria

Potential senescence-related effects of verapamil were studied in spontaneously beating isometrically contracting right atria from 12 mature and 9 senescent male Fischer 344 rats. Verapamil 0-300 mcg/L prolonged spontaneous cycle length in all atria but produced significantly greater prolongation of spontaneous cycle length in the senescent vs mature right atria (p less than .03). In addition, cessation of spontaneous activity occurred more frequently and at lower concentrations in senescent vs mature atria. Recovery times from pacing at 3.5 pps and 5.0 pps also showed greater prolongation in response to verapamil in the senescent atria when compared to the mature right atria (p less than .004 and p less than .002). In contrast, the percent reduction of developed force produced by verapamil during pacing at 3.5 and 5.0 pps did not differ between senescent and mature atria.     

Effects of dopamine on sinoatrial conduction in isolated, blood-perfused dog atria

Dopamine, administered at a constant infusion rate of 1-2 micrograms/min into the cannulated sinus node artery of the isolated dog atrium, decreased sinus cycle length (SCL) from 630 +/- 19 to 501 +/- 22 msec (mean +/- SEM, 38 trials in 12 atria). However, on sinoatrial conduction time (SACT) estimated by a constant atrial pacing method, dopamine produced 2 types of response (shortening and lengthening) with sinus tachycardia. In 24 trials in 11 atria, the drug decreased SACT from 86 +/- 8 to 56 +/- 4 msec, and in 14 trials in 6 atria it increased SACT from 67 +/- 7 to 101 +/- 9 msec. In general, the effects of dopamine on SACT were dependent on the control levels of SCL: dopamine caused a reduction of SACT at small levels of SCL and a prolongation at large levels. At a control sinus rate of 120 beats/min, dopamine usually shortened SACT. Dopamine-induced shortening of SACT was blocked by a beta-adrenoceptor blocker, propranolol, and an uptake blocker, imipramine, but not by a dopaminergic inhibitor, sulpiride. Furthermore, dopamine-induced lengthening of SACT tended to be suppressed by propranolol, but not by sulpiride. It is concluded that the dopamine-induced changes in SACT are mediated via beta-adrenergic mechanism and partially due to a tyramine-like action.     

Calcium and potassium currents in cells from adult and aged canine right atria

BACKGROUND: Action potential (AP) contours vary considerably between normal adult and aged right atrial fibers. The ionic bases for these differences remain unknown. Therefore we studied Ca(2+) and K(+) currents in cells from adult and aged canine right atria (RA). METHODS AND RESULTS: We used whole cell patch clamp recording techniques to measure L-type Ca(2+) currents (I(CaL)) with either Ca(2+) or Ba(2+) (3 mM) as the charge carrier, and both the transient outward (I(to)) and sustained potassium currents (I(sus)) in cells dispersed from normal adult (Adult, 2-5 years) and older dogs (Aged, >8 years). There is a significant reduction in peak I(CaL) (47%) and I(BaL) (43%) in Aged cells, yet differences in I(BaL) disappear with maximal beta adrenergic stimulation (isoproterenol, 1 microM). Composite I(to) and I(sus) densities were significantly increased in the Aged versus Adult cell group (by 31 and 27% at +50 mV, respectively). I(to) decay during a maintained depolarization was slowed in Aged cells. Furthermore, I(to) steady-state inactivation curve was shifted positively in Aged cells. Finally, composite I(to) and I(sus) currents of Aged cells were more sensitive to tetraethylammonium chloride (TEA), a specific inhibitor of some types of K(+) currents. In the presence of TEA (5 mM), I(to) in Aged cells was significantly greater than that in Adult cells. CONCLUSIONS: Ionic currents differ in Aged versus Adult right atrial cells, such that a reduced Ca(2+) current and augmented outward currents could contribute significantly to the altered AP contour of the Aged RA cell. Adrenergic stimulation appears to restore Ba(2+) currents in Aged cells. Finally, an augmented TEA sensitive current plays a role in changes of I(sus) in Aged right atrial cells.     

Cardiac effects of piretanide and furosemide on intact anesthetized dogs and on isolated atria

The effects of piretanide and furosemide on systemic arterial blood pressure and heart rate were examined in the anesthetized dog and the effects on atrial rate and contractile force were assessed in isolated atrial muscle perfused with heparinized arterial blood from a donor dog. When piretanide was administered intravenously to intact dogs, the depressor and bradycardic responses were produced dose-dependently. There were no significant simultaneous chronotropic or inotropic changes in the isolated atrium. On the other hand, furosemide (1-3 mg/kg) did not induce significant changes in either systemic blood pressure or heart rate in the intact dog. The atrial rate and developed tension were also not affected in the isolated atrium. A potent beta-adrenoceptor blocking agent, propranolol (1 mg/kg i.v.), consistently produced a significant depressor response and a profound negative chronotropic effect in the intact dogs; significant negative chronotropic and inotropic effects were also observed in the isolated atrium. When large doses of piretanide and furosemide were injected intraarterially into the sinus node artery of the isolated atrium, atropine-insensitive negative chronotropic and inotropic effects were induced dose-dependently. The potency of the negative chronotropic effect of piretanide was slightly greater than that of furosemide, but the negative inotropic effect of piretanide was slightly smaller than that of furosemide. These data indicate that piretanide has a depressor effect without significant cardiac influences. However, a high dose of piretanide has negative chronotropic and inotropic effects. These effects were not observed with the doses of furosemide (1-3 mg/kg) employed in this study.     

Direct effects of catecholamines and tyramine on sinoatrial conduction in isolated and blood-perfused dog atria

We have investigated the effects of catecholamines (dopamine, norepinephrine, epinephrine and isoproterenol) and tyramine on sinoatrial conduction time (SACT), sinus cycle length (SCL) and developed tension (DT) in isolated atrial muscle, using an isolated and blood-perfused dog atrial preparation, perfused with heparinized blood from the carotid artery of the anesthetized donor dog. Each substance was continuously administered intraarterially into the cannulated sinus node artery. They had dose-dependent positive chronotropic and inotropic effects. Each produced not only a shortening but a prolongation of SACT. In experiments in which only a shortening occurred, the order of the potency for inducing the shortening of the SACT was isoproterenol greater than norepinephrine = epinephrine greater than dopamine greater than tyramine. The ratio of doses required to produce roughly a 15-25% shortening of the SACT was 1: 10: 10: 100: 300, respectively. The isoproterenol-induced shortening of the SACT was inhibited by treatment with propranolol. Tyramine-induced shortening was inhibited by imipramine. From these results, it is suggested that sympathomimetic amines induce a shortening of SACT through adrenergic beta-receptors and also readily induce a pacemaker shift in the SA nodal area.