Guinea-pig Ventricular Strips Research Articles

Pharmacological Profile of LAS 31180, a New Inotropic/Vasodilator Quinolone Derivative

LAS 31180 (3-methylsulphonylamino-1-methyl-4(1H)-quinolone, CAS 137338-43-3) was found to have positive inotropic and vasodilator properties through its inhibitory action on type 3 phosphodiesterase. The inotropic effects of LAS 31180 were demonstrated in vitro both in isolated guinea-pig ventricular strips (EC50 of 1.2 mumol/l) and in isolated guinea-pig working hearts. In conscious chronically instrumented Beagle dogs, LAS 31180 administered either by intravenous or oral route, showed a dose-dependent, long-lasting positive inotropic effect with minimal effects on heart rate. In hypertensive Beagle dogs LAS 31180 elicited a potent and long-lasting fall in blood pressure.

Electrophysiological effects of dofetilide in an in vitro model of "border zone" between normal and ischemic/reperfused myocardium.

To evaluate both class III activity and antiarrhythmic action of dofetilide at the level of the "border zone," we investigated its electrophysiological effects on guinea pig ventricular strips submitted partly to normoxia (normal zone, NZ) and partly to simulated severe ischemia, then reperfusion (altered zone, AZ). Because of the differential class III effects of dofetilide in normal and ischemic regions, the dispersion of the action potential duration at 90% repolarization (APD(90)) between NZ and AZ was reduced by 5 nmol/L of drug during early ischemia (at 10 minutes, APD(90) NZ/APD(90) AZ was 1.68+/-0.22 versus 2.82+/-0.17 in control, P<0.05), whereas 50 nmol/L dofetilide worsened it during late ischemia (at 30 minutes, APD(90) NZ/APD(90) AZ was 4.62+/-0.76 versus 2.57+/-0.21 in control, P<0.05). Concomitantly, dofetilide at 5, 10, and 50 nmol/L abolished the early extrastimulus (ES)-induced arrhythmias, and at 10 and 50 nmol/L, it significantly enhanced the incidence of late spontaneous repetitive responses (in 86% and 75% of preparations treated with 10 and 50 nmol/L, respectively, versus 25% in control, P<0.05). During reperfusion, dofetilide at 5, 10, and 50 nmol/L exhibited concentration-dependent class III effects, as it did in the NZ, and did not modify the incidence of spontaneous arrhythmias. Dofetilide 5 nmol/L decreased APD(90) dispersion between NZ and AZ and reduced the early ES-induced arrhythmias. However, dofetilide 50 nmol/L increased APD(90) dispersion, and at 10 and 50 nmol/L, it increased the late spontaneous arrhythmias.

Cicletanine prevents the excitation–conduction blocks induced by terfenadine in ischemic myocardium

Terfenadine, a histamine H 1 receptor antagonist, has been associated with clinical ventricular arrhythmias and in vitro excitation–conduction blocks, whereas anti-ischemic and antiarrhythmic effects have been shown with cicletanine, a prostacyclin generation stimulator. We aimed at determining in vitro if cicletanine can protect the ischemic myocardium from excitation–conduction blocks and specifically those induced by terfenadine. In a double-chamber bath, isolated guinea pig ventricular strips were partly exposed to normoxia and partly to ischemic, then reperfused, conditions, in the presence of 10 μM terfenadine, 10 μM indomethacin (prostacyclin generation blocker) or the solvent (dimethylsulfoxide 1:100, control) randomly allocated, and thus either in the absence ( n=20) or presence ( n=21) of 10 μM cicletanine during the total protocol duration. The multivariate Cox's model was used to predict the excitation–conduction block events and to assess the estimated survival of preparations (excitation–conduction block-free rate). Cicletanine protected the preparations (relative risk=0.08, t=−3.28) from the ischemia-induced excitation–conduction blocks (estimated survival=0.83 versus 0.30 in control), and this effect was abolished by indomethacin (estimated survival=0.35). Terfenadine enhanced 3.58-fold the risk of occurrence of excitation–conduction blocks during ischemia ( t=2.10) and this effect was inhibited by cicletanine pretreatment (estimated survival=0.40 versus 0.10 in untreated preparations). In conclusion, these in vitro findings have provided evidence for (1) protective effects of cicletanine against ischemia-induced excitation–conduction blocks, possibly related to its stimulating activity on local prostacyclin generation, and (2) efficacy of cicletanine to prevent excitation–conduction blocks induced by terfenadine in ischemic cardiac tissue.

KATP channels and 'border zone' arrhythmias: role of the repolarization dispersion between normal and ischaemic ventricular regions.

1. In order to investigate the role of KATP channel activation and repolarization dispersion on the 'border zone' arrhythmias induced by ischaemia-reperfusion, the effects of glibenclamide and bimakalim, agents modifying action potential (AP) duration, were studied in an in vitro model of myocardial 'border zone'. 2. The electrophysiological effects of 10 microM glibenclamide and 1 microM bimakalim (n=8 each), respectively KATP channel blocker and activator, were investigated on guinea-pig ventricular strips submitted partly to normal conditions (normal zone, NZ) and partly to simulated ischaemic then reperfused conditions (altered zone, AZ). 3. By preventing the ischaemia-induced AP shortening (P<0.0001), glibenclamide reduced the dispersion of AP duration 90% (APD90) between NZ and AZ (P<0.0001), and concomitantly inhibited the 'border zone' arrhythmias induced by an extrastimulus (ES), their absence being significantly related to the lessened APD90 dispersion (chi2=8.28, P<0.01). 4. Bimakalim, which also reduced the APD90 dispersion (P<0.005) due to differential AP shortening in normal and ischaemic tissues, decreased the incidence of myocardial conduction blocks (25% of preparations versus 83% in control, n=12, P<0.05) and favoured 'border zone' spontaneous arrhythmias (75% of preparations versus 25% in control, P<0.05). 5. During reperfusion, unlike bimakalim, glibenclamide inhibited the ES-induced arrhythmias and reduced the incidence of the spontaneous ones (12% of preparations versus 92% in control, P<0.05), this latter effect being significantly related (chi2=6.13, P<0.02) to the lessened ischaemia-induced AP shortening in the presence of glibenclamide (P<0.0001). 6. These results suggest that KATP blockade may protect the ischaemic-reperfused myocardium from 'border zone' arrhythmias concomitantly with a reduction of APD90 dispersion between normal and ischaemic regions. Conversely, KATP channel activation may modify the incidence of conduction blocks and exacerbate the ischaemia-induced 'border zone' arrhythmias.

Proarrhythmic effects of DL- and D-sotalol on the "border zone" between normal and ischemic regions of isolated ventricular myocardium and antiarrhythmic effects on reperfusion.

Considering the Survival With ORal D-sotalol (SWORD) study results, in which mortality was higher in patients treated by the pure class III agent D-sotalol, we tested DL- and D-sotalol (5 and 10 microM) in an in vitro model of "border zone" arrhythmias. Isolated guinea-pig ventricular strips were partly exposed to normoxia ("Normal Zone," NZ) and partly to modified Tyrode's solution ("Ischemic Zone," IZ) for 15 or 30 min ("ischemia"), followed by return to normoxia for 30 min ("reperfusion"). Resting membrane potential, action potential (AP) amplitude, and maximal upstroke velocity of AP were not significantly modified. DL- And D-sotalol, 5 and 10 microM, lengthened AP duration 90% (APD90) in NZ (p < 0.05), whereas these drugs were unable to prevent ischemia-induced APD shortening. By using the accelerated failure time Weibull's model, and a large number of reference experiments to control random variability of analyzed covariates, DL- and D-sotalol increased significantly the incidence of spontaneous arrhythmias during ischemia (chi2 = 24.79; p = 0.0367): 83 (5 microM D- and DL-sotalol), 86, and 62% (10 microM D- and DL-sotalol, respectively) versus 32% of controls. During reperfusion, 10 microM DL-sotalol prevented the occurrence of spontaneous arrhythmias (chi2 = 46.74; p = 0.0001) similar to what seen with the beta-blocking agent propranolol (10 microM). These data, providing evidence for proarrhythmic effects of DL- and D-sotalol on border-zone arrhythmias, concomitant with differential class III actions on NZ versus IZ, might be considered for understanding the SWORD study results.

PACAP induces bradycardia in guinea-pig heart by stimulation of atrial cholinergic neurones.

Based on previous studies which indicated that pituitary adenylate cyclase activating peptide (PACAP) acts as a positive inotropic and chronotropic substance in different species via the cAMP signal transduction pathway, the objective of the present work was to investigate cAMP-regulated myocardial key proteins in response to PACAP in isolated ventricular cells of the guinea pig. Surprisingly, the two molecular forms of PACAP, PACAP(1-27) and PACAP(1-38), showed no effect on intracellular cAMP-levels, L-type Ca2+ channel current or phosphorylation of troponin inhibitor (TnI) and phospholamban (PLB). Additionally, inotropy of isolated guinea-pig ventricular strips was not affected by the neuropeptide. However, in isolated spontaneously beating guinea-pig atria, PACAP(1-27) and PACAP(1-38), but not VIP induced severe bradycardia in a dose-dependent manner. This effect could be prevented by preincubation with the PACAP receptor antagonist PACAP(6-38), by atropine and by omega-conotoxin, a blocker of neuronal N-type Ca2+ channels. PACAP stimulates release of [3H]-labelled acetylcholine. Only preparations showing an increase in [3H]acetylcholine release developed bradycardia, indicating a causal relationship between both phenomena. It was concluded that PACAP exerts no influence on guinea-pig ventricular tissue, but induces negative chronotropic effects in isolated guinea-pig atria by stimulation of acetylcholine release from parasympathetic neurons via PACAP type 1 receptors.

Effects of 2-methyl-1,4-naphthoquinone (menadione) on myocardial contractility and cardiac sarcoplasmic reticulum Ca-ATPase

(1) In electrically driven guinea-pig left atria, menadione (2-methyl-1,4-naphthoquinone) (1 to 20 mumol/l) and menadione sodium bisulfite (30 to 200 mumol/l) produced marked positive inotropic effects. Endogenously released catecholamines and histamine contributed to 80-85% of the effect, the residual 15-20% appearing as a direct effect. (2) In electrically driven guinea-pig ventricular strips, low micromolar concentrations of menadione (0.05 to 0.3 mumol/l) exerted a catecholamine-mediated small positive inotropic effect. (3) In both myocardial preparations, the increase in force of contraction was followed by a non-reversible rise of resting force. In its effects on cardiac contractility menadione resembled the thiol group blocking agent p-chloromercuribenzoate and H2O2. Pretreatment of atria with glutathione prevented the increase in resting force, while dithiothreitol only slightly delayed it. By contrast, the pretreatment with the NAD(P)H-quinone reductase (DT-diaphorase) inhibitor, dicumarol, markedly increased the rate of appearance of the toxic effect of menadione. (4) Among enzymatic and transport systems involved in the onset and control of cardiac contractility, sarcoplasmic reticulum Ca-ATPase was significantly inhibited by menadione after a long contact time. The inhibition was concentration-dependent and persistent, and was antagonized by addition of glutathione. (5) On the basis of these results, the increase in resting force caused by menadione appears to be related to an impairment of the thiol groups of proteins (Ca-ATPase), presumably caused by the drug per se.

Indirect evidence for a role of phosphatidylinositol turnover in the cardiac response to H1-receptor stimulation.

The influence of lithium on the positive inotropic effect of the H1-agonist 2-pyridyl-ethylamine (PEA) and of the H2-receptor agonist 4-methylhistamine was studied in isolated guinea-pig ventricular strips electrically stimulated at 1 Hz. Lithium (1-10 mM) was devoid of any effect on cardiac contraction; the positive inotropic effect of 4-methylhistamine was unaffected in the presence of 10 mM lithium. On the other hand, lithium (1-10 mM) dose-dependently shifted the dose-inotropic effect curve for PEA to the right; an antagonistic effect, qualitatively similar to that of lithium, was induced by the myoinositol antagonist 2-2'-anhydro-2-C-hydroxymethyl-myoinositol, at a concentration of 100 microM. Moreover the antagonistic effect of the higher lithium concentration (10 mM) was almost completely prevented in preparations superfused with 10 mM myoinositol. Since it is known that lithium is able to reduce the cellular availability of myoinositol by an interference with the phosphatidylinositol (PI) cycle, these results suggest that the H1-receptor-mediated increase in contractility may be linked to an increased turnover of PI, while the H2-receptor-mediated one is not.

Barium cardiotoxicity: Relationship between ultrastructural damage and mechanical effects

The ultrastructural damage in guinea-pig ventricular strips caused by barium was analysed. At a concentration of 1 mmol/litre, barium chloride caused a dramatic increase in the developed tension associated with the onset of automaticity. The ultrastructural analysis demonstrated that barium caused notable and consistent alterations which affected most myocyte components. Various degenerative aspects were observed in mitochondria and in the contractile apparatus. Glycogen deposits were completely depleted. Preparations driven at 4 Hz (i.e. the rate of spontaneous firing of barium-treated preparations) showed moderate ultrastructural alterations, thus demonstrating that the increase in the rate of beating is not the only determinant of the observed damage. These results suggest that the myocardial toxicity of barium is due not only to the well-known modifications in membrane permeability, but possibly also to alterations in cell function.

Differential temperature-dependency of electrophysiological and inotropic actions of nifedipine, verapamil and cinnarizine in K +-depolarized ventricular myocardium

1. 1. Temperature dependency (in the range 27–37°C) of inotropic and electrophysiological effects of equieffective (EC 30 at 37°C) concentrations of nifedipine, verapamil and cinnarizine was assessed in potassium depolarized isoprenaline-reactivated guinea-pig ventricular strips. 2. 2. Lowering temperature greatly enhanced nifedipine inhibition of (a) maximal rate of depolarization ( V max) of slow action potentials and (b) amplitude of contractions. 3. 3. Electrophysiological and inotropic actions of verapamil was virtually unaffected by temperature changes. 4. 4. Negative inotropic action of cinnarizine was greater at 37°C than at lower temperature. At 37°C, but not at 32°C, cinnarizine reduced V max of slow action potentials.

Proarrhythmic effects of an oxygen-derived free radical generating system on action potentials recorded from guinea pig ventricular myocardium: a possible cause of reperfusion-induced arrhythmias.

Standard microelectrode techniques were used to study the effects of a free radical generating system on action potentials recorded from guinea pig ventricular myocardium. Free radicals were generated by mixing xanthine oxidase (0.02-0.04 mu/ml) with the superfusate-modified Locke's solution containing purine 2.3 mM. The system was validated by demonstrating that it could reduce cytochrome C at a rate of 15.9 +/- 1.5 mol/l/min. This rate was decreased to 3.0 +/- 0.3 (p less than 0.001) in the presence of superoxide dismutase (12 mg/100 ml), and the reaction was absent if xanthine oxidase and purine were premixed for 60 minutes prior to adding cytochrome C. Superfusion of guinea pig ventricular strips with the free radical generating system (20-30 minutes) resulted in a highly significant reduction in resting potential from -79.3 +/- 1.8 mV to -70.9 +/- 1.4 mV (p less than 0.0001, n = 6) and in action potential amplitude from 110.9 +/- 2.2 mV to 101.7 +/- 4.0 mV (p less than 0.0001). There was an accompanying fall in maximum rate of depolarization (Vmax) from 254.1 +/- 17.7 V/sec to 207.1 +/- 18.6 V/sec (p less than 0.01) and no significant change in action potential duration. These changes were accompanied by spontaneous activity in 3 of 6 preparations and reversed after 20-30 minutes washing in Locke's solution. They were largely abolished by adding superoxide dismutase (12 mg/100 ml) to the superfusate and completely absent if the xanthine oxidase and purine were premixed for 60 minutes before superfusing the myocardium. We conclude that the phenomena observed may contribute to the genesis of reperfusion arrhythmias.

Inotropic activity of orthanilic and L-cysteic acid on isolated guinea-pig ventricular strips.

The positive inotropic effect of taurine in low calcium medium has been demonstrated extensively in guinea-pig (1,2,4,6,9), and chick heart (11). This effect of taurine is dose- and calcium-concentration dependent (2,6,8), and is not suppressed by the addition of propranolol (4,11), cimetidine or indomethacin (8). Furthermore, taurine antagonizes the negative inotropic effect of D-600 (7) in guinea-pig ventricular strips. More recently it has been demonstrated that two taurine-related compounds, orthanilic acid (2-aminobenzenesulfonic acid) and L-cysteic acid (Fig. 1), exert a positive inotropic effect on guinea-pig ventricular strips incubated in a medium containing 0.9 mM CaCl2 (4). This effect is dose-dependent and occurs over the same concentration range as the inotropic action of taurine (5–20 mM). It is also similar with respect to the time course (4). Both compounds are characterized by primary amino and sulfonic groups bound to adjacent carbons (Fig. 1). Chain length, sulfonic and primary amino groups are a structural requirement for taurine-like inotropic activity (4). The present study investigates other properties of these two compounds in guinea-pig ventricular strips.

On the presence of opioid receptors in guinea-pig ventricular tissue

1. 1. The cardiac response to sympathetic nerve stimulation, induced by trains of field pulses, was studied in isolated guinea-pig ventricular strips. 2. 2. Dynorphin-(1-13) and [ d-Ala2, d-Leu5]enkephalinamide, but not morphine, reduced, in a dose-dependent manner, the cardiac sympathetic response. 3. 3. The effect of the two opioid peptides was antagonized by naloxone. 4. 4. The opioid agonists did not affect the response to exogenous noradrenaline. 5. 5. Neither naloxone nor a mixture of peptidase inhibitors modified the cardiac response to sympathetic nerve stimulation.

Positive inotropic effect of some taurine-related compounds on guinea-pig ventricular strips perfused with low calcium medium

Taurine exerts a positive inotropic effect at a low calcium concentration. Of the compounds chemically related to taurine only L-cysteic acid and 2-aminobenzenesulfonic acid mimic taurine action. Their effect is concentration-dependent and not linked to the restoration of taurine tissue concentration in guinea-pig ventricular strips. Our data demonstrate that: (1) carbon chain length between amino and sulfonic groups is a crucial factor in the development of activity. (2) Substitution of the sulfonic group with other acid functions such as the substitution of the primary amino group or the introduction of the -COOH group onto the ß-carbon brings about a lack of activity.

Functional and binding evidence of taurine inhibition of α-adrenoceptor effects on guinea-pig ventricle

The effect of taurine on alpha- and beta-mediated positive inotropic effects was investigated in guinea-pig ventricular strips. Loading with taurine dose-dependently antagonized the phenylephrine-induced positive inotropic effect while it was ineffective on beta-mediated positive inotropic effect. The superfusion with a taurine-free medium determined a loss of intracellular taurine, while the loading with 20 mM taurine prevented this depletion. Preincubation of cardiac membranes with different concentrations of taurine (1 to 20 mM) decreased 3H-prazosin binding, and the saturation curve obtained after preincubation of cardiac membranes with 20 mM taurine showed that taurine had a more marked effect on the number of binding sites. In both experimental models, beta-alanine did not mimic any taurine effects, suggesting that taurine actions might be specific.

Effect of temperature on isoprenaline- and barium-induced slow action potentials in guinea-pig ventricular strips

1. 1. The effect of variation in temperature (37-32 and 27°C) on electrical and mechanical activity of depolarized and isoprenaline- or barium-reactivated guinea pig ventricular strips was studied. 2. 2. Lowering the temperature brings a marked prolongation of isoprenaline-induced slow action potentials. In addition the maximal rate of depolarization was strongly reduced at lower temperatures. These effects were observed at an extracellular Ca 2+ concentration of either 0.9 or 2.5 mM. The accompanying mechanical activities was significantly increased by reduction in temperature. 3. 3. Barium-induced slow action potentials were similarly affected by temperature variations. 4. 4. These observations suggest that hypothermia exert a sort of calcium antagonistic action probably coupled to a reduction of repolarizing outward potassium currents.

Positive inotropic effect of Mg 2+ in K +-depolarized isoprenaline-reactivated guinea-pig ventricular strip

1. 1. In K +-depolarized isoprenaline-reactivated guinea-pig ventricular strips increase in external Mg 2+ concentration produced a biphasic inotropic effect i.e. at low concentrations (2.5–7.5 mM) a slight negative inotropic effect was observed, while at higher Mg 2+ concentrations (10–17.5 mM) a strong positive inotropic effect become predominant. 2. 2. Variation in extracellular K + concentration (17–22 and 25 mM) determined a concentration related reduction in Mg 2+-induced positive inotropic effect. 3. 3. Increase in extracellular Ca 2+ concentration (2.5–3.6 and 5.4 mM) prevented the development of Mg 2+-induced negative inotropic effect but did not affect the magnitude of the positive inotropic effect. 4. 4. Mg 2+-induced positive inotropic effect was unaffected by TTX (3 mg/l) and even enhanced by nifedipine (65 nM). 5. 5. Caffeine (0.2–15 mM) antagonized in a concentration dependent manner the positive inotropic effect of Mg 2+. On the other hand in the presence of Mg 2+ (15 mM) caffeine (0.2 mM) was unable to exert its positive inotropic effect. 6. 6. Mg 2+ (15 mM) determined a “Ca 2+ entry blocking”—like modifications of electrophysiological parameters of slow action potentials elicited by isoprenaline in K +-depolarized preparations. Such effects of Mg 2+ were also present during the positive inotropic effect. 7. 7. The possible mechanisms responsible for these complex inotropic effects of Mg 2+ are discussed.

Extra- and intracellular lanthanum: modified calcium distribution, inward currents and contractility in guinea pig ventricular preparations

In guinea pig ventricular strips and isolated cells, 0.1 mM LaCl3 blocks contractility and shortens the action potential (AP) in less than 2 min ("early La-effect"). After 30 min, it prolongs the APs which trigger slow contractions ("late La-effect"). These results confirm earlier reports. X-ray microprobe analysis shows that La initially displaces only a small fraction of that Ca which is superficially bound to the sarcolemma. But, since this Ca is completely removed by Ca-free solutions within 2 min, we suggest that La blocks contractility not by displacing superficial Ca but by blocking the Ca inward current iCa. Blocking of iCa is analyzed with voltage clamp experiments. It is not La-specific, and can also be observed with other calcium channel blockers as well. When iCa has been blocked, the membrane can still generate 100-200 ms long plateaus via the sodium inward current iNa. During the late La-effect, the cells internalize La. Intracellular La is detected by x-ray microprobe analysis in cryosections of frozen muscles and as La-precipitates in EM images from freeze substituted preparations. Simultaneously, the cytosol gains Na and Ca, but the plasmalemmal and sarcoplasmic reticulum (SR) membranes are no longer occupied by Ca but by La. The late La-effect on the prolongation of the AP is La-specific. In the absence of extracellular La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional inward current which is insensitive to Ca-removal, is inactivated by holding potentials of -40 mV, and is TTX-sensitive. We suggest that the current flows through a fraction of original Na-channels that is modified by i.c. La with respect to inactivation and selectivity. We attribute the late re-occurrence of contractility to activator Ca entering from the bath. Ca-entry might be mediated via enhanced Na/Ca-exchange whose rate is increased by the i.c. Na-load. In addition, Ca may enter through the La-modified Na-channels due to their impaired selectivity. Since i.c. La is known to interfere with the Ca-sequestration by the SR, it is expected to impair relaxation.

Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue.

An estimation of the actual Na/K-ATPase transport activity in intact cardiac cells was made by measuring the binding of [3H]-ouabain to rat and guinea-pig ventricular strips. At the low [3H]-ouabain concentration of 1 nM equilibrium binding was hardly obtained after an incubation time of five hours. Different procedures known to alter the sodium load of the cardiac preparations influenced [3H]-ouabain binding: the sodium ionophore monensin enhanced [3H]-ouabain binding, the local anaesthetic dibucaine and a reduction of external sodium ion concentration diminished [3H]-ouabain binding; [3H]-ouabain binding was similarly affected by these procedures in the rat and guinea-pig. Since [3H]-ouabain binding occurred predominantly at the high-affinity binding sites of rat myocardium under the applied experimental conditions, it was concluded that these binding sites represent Na/K-ATPase molecules involved in sodium ion transport.

Inotropic effect of taurine in guinea-pig ventricular strips

At 0.9 mM CaCl 2 taurine prevented the negative inotropic effect due to low calcium concentration in guinea-pig ventricular strips but not in rat strips. This taurine effect was dose-dependent. The presence (in the perfusion medium) of β-alanine, an inhibitor of taurine transport, antagonized the effect of taurine suggesting that the action of taurine was correlated to its transport. Neither propranolol, cimetidine nor indomethacin affected the response to taurine in guinea-pig ventricular strips.