Beta-adrenergic receptors and intracellular signalling pathway in stunned and non-ischemic regions of pig myocardium.

The beta-adrenergic pathway may have a role in the pathophysiology of ischemic syndromes characterised by reversible left ventricular dysfunction, such as myocardial stunning and other clinical conditions of unstable angina or coronary spasms, or chronic reversible left ventricular dysfunction, which might be a consequence of repeated events of short-term ischemia ("repetitive stunning"). A partial-to-total occlusion of the left anterior descending coronary artery in pigs was used to induce short periods of ischemia (total ischemic time 12 +/- 2 min). Hypokinesis and dyskinesis of the myocardium were considered signs of myocardial dysfunction. We found a maintained function of the beta-adrenergic signalling system. Density and affinity of beta-adrenergic receptors were not different in stunned and non-ischemic regions, nor were cyclic AMP and cyclic GMP intracellular contents and ratio, nor well as the ratio of stimulatory/inhibitory G protein a subunits. Our findings are in agreement with a maintained beta-adrenergic signalling system in the pathophysiology of chronic reversible left ventricular dysfunction.

Reduction of oxidative stress by carvedilol: role in maintenance of ischaemic myocardium viability.

OBJECTIVES: To differentiate the impact of the beta-blocking and the anti-oxidant activity of carvedilol in maintaining myocardium viability. METHODS: Isolated rabbit hearts, subjected to aerobic perfusion, or low-flow ischaemia followed by reperfusion, were treated with two doses of carvedilol, one dose (2.0 microM) with marked negative inotropic effect due to beta-blockage and the other (0.1 microM) with no beta-blockage nor negative inotropism. Carvedilol was compared with two doses of propranolol, 1.0 - without - and 5.0 microM - with negative inotropic effect. Anti-oxidant activity was measured as the capacity to counteract the occurrence of oxidative stress and myocardium viability as recovery of left ventricular function on reperfusion, membrane damage and energetic status. RESULTS: Carvedilol counteracted the ischemia and reperfusion induced oxidative stress: myocardial content of reduced glutathione, protein and non-protein sulfhydryl groups after ischaemia and particularly after reperfusion, was higher in hearts treated with carvedilol, while the myocardial content of oxidised glutathione was significantly reduced (0.30+/-0.03 and 0.21+/-0.02 vs. 0.39+/-0.03 nmol/mg prot, both P<0.01, in 0.1 and 2.0 microM). At the same time, carvedilol improved myocardium viability independently from its beta-blocking effect. On the contrary, propranolol maintained viability only at the higher dose, although to a lesser extent than carvedilol. This suggests that the effects of propranolol are dependent on energy saving due to negative inotropism. The extra-protection observed with carvedilol at both doses is likely due to its anti-oxidant effect. CONCLUSIONS: Our data show that the anti-oxidant activity of carvedilol is relevant for the maintenance of myocardium viability.

New insights on myocardial pyridine nucleotides and thiol redox state in ischemia and reperfusion damage.

OBJECTIVE: to investigate the changes of pyridine nucleotides and thiol redox state in cardiac tissue following ischemia and reperfusion. NADH/NAD and NADPH/NADP redox couples were specifically studied and the influence of NADPH availability on cellular thiol redox was also investigated. METHODS: isolated rabbit hearts were Langendorff perfused and subjected to a protocol of ischemia and reperfusion. An improved technique for extraction and selective quantitation of pyridine nucleotides was applied. RESULTS: ischemia and reperfusion induced an increase in diastolic pressure, limited recovery in developed pressure and loss of creatine phosphokinase. Creatine phosphate and ATP were decreased by ischemia and only partially recovered during reperfusion. NADH was increased (from 0. 36+/-0.04 to 1.96+/-0.15 micromol/g dry wt. in ischemia, P<0.001), whereas NADPH decreased during ischemia (from 0.78+/-0.04 to 0. 50+/-0.06 micromol/g dry wt., P<0.01) and reperfusion (0.45+/-0.03 micromol/g dry wt.). Furthermore, we observed: (a) release of reduced (GSH) and oxidised glutathione (GSSG) during reperfusion; (b) decreased content of reduced sulfhydryl groups during ischemia and reperfusion (GSH: from 10.02+/-0.76 to 7.11+/-0.81 nmol/mg protein, P<0.05, and to 5.48+/-0.57 nmol/mg protein; protein-SH: from 280.42+/-12.16 to 135.11+/-17.00 nmol/mg protein, P<0.001, and to 190.21+/-11.98 nmol/mg protein); (c) increased content in GSSG during reperfusion (from 0.17+/-0.02 to 0.36+/-0.02 nmol/mg protein, P<0.001); (d) increased content in mixed disulphides during ischemia (from 6.14+/-0.13 to 8.31+/-0.44 nmol/mg protein, P<0.01) and reperfusion (to 9.87+/-0.82 nmol/mg protein, P<0.01). CONCLUSIONS: under severe low-flow ischemia, myocardial NADPH levels can decrease despite the accumulation of NADH. The reduced myocardial capacity to maintain NADPH/NADP redox potential can result in thiol redox state changes. These abnormalities may have important consequences on cellular function and viability.

Role of A2A receptor in the modulation of myocardial reperfusion damage.

Adenosine protects myocardium from ischemia and reperfusion damage; however, the mechanism of action is still under discussion. We investigated whether (a) adenosine protects isolated crystalloid-perfused rabbit heart from ischemia/ reperfusion injury; (b) this action is receptor mediated and what receptor subtypes are involved, and (c) this action is dependent on an enhanced nitric oxide production. Our results showed a cardioprotective effect of adenosine (10(-4) M), of nonselective adenosine-receptor agonist 5'-N-ethyl-carboxamidoadenosine (NECA; 5 x 10(-6) M), and of A2A agonists CGS 21680 (10(-8) and 10(-6) M), 2-hexynylNECA (10(-7) M). On the contrary, A1 agonist CCPA (10(-8) and 10(-6) M) does not provide any protection. The effect has been achieved in terms of significant reduction in contracture development during reperfusion [diastolic pressure was 46.8 +/- 7.1 mm Hg (p < 0.01); 46.1 +/- 7.8 mm Hg (p < 0.01); 46.9 +/- 5.5 mm Hg (p < 0.01); and 59.3 +/- 6.7 mm Hg (p < 0.05) with 10(-4) M adenosine, 5 x 10(-6) M NECA, 10(-6) M CGS 21680, and 10(-7) M 2-hexynylNECA, respectively, versus 77.6 +/- 5.0 mm Hg in control]; reduced creatine phosphokinase release (13.5 +/- 1.6, 22.2 +/- 7.9, 14.2 +/- 3.3, and 14.1 +/- 4.5 U/gww in treated hearts vs. 34.6 +/- 7.2 U/gww in controls; p < 0.05); improved energy metabolism [adenosine triphosphate (ATP) content is 9.9 +/- 0.5, 10.4 +/- 0.6, 9.8 +/- 0.5, and 10.5 +/- 0.5 micromol/gdw in treated hearts vs. 7.6 +/- 0.2 micromol/gdw; p < 0.05]. Moreover, our data indirectly show a functional presence of A2A receptors on cardiomyocytes as the protection is A2A mediated and exerted only during reperfusion, although in the absence of blood and coronary flow changes. These activities appear independent of nitric oxide pathways, as adenosine and 2-hexynylNECA effects are not affected by the presence of a nitric oxide-synthase inhibitor (10(-4) M L-NNA).

Changes in oxidative stress and cellular redox potential during myocardial storage for transplantation: experimental studies.

BACKGROUND: Cardioplegic solutions assure only a sub-optimal myocardial protection during prolonged storage for transplantation. The ultimate cause of myocardial damage during storage is unknown, but oxygen free radicals might be involved. We evaluated the occurrence of oxidative stress and changes in cellular redox potential after different periods of hypothermic storage. METHODS: Langendorff-perfused rabbit hearts were subjected to a protocol mimicking each stage of a cardiac transplantation procedure: explantation, storage and reperfusion. Three periods of storage were considered: Group A = 5 hours, Group B = 15 hours, and Group C = 24 hours. Oxidative stress was determined in terms of myocardial content and release of reduced (GSH) and oxidized (GSSG) glutathione, and cellular redox potential as oxidized and reduced pyridine nucleotides ratio (NAD/NADH). Data on mechanical function, cellular integrity and myocardial energetic status were collected. RESULTS: At the end of reperfusion, despite the different timings of storage, recovery of left ventricular developed pressure (46.1+/-7.0, 54.7+/-6.7, and 45.7+/-7.4% of the baseline pre-ischaemic value), energy charge (0.81+/-0.02, 0.81+/-0.02, and 0.77+/-0.01) and NAD/NADH ratio (8.87+/-1.08, 9.39+/-1.72, and 10.26+/-1.98) were similar in all groups (A, B and C). On the contrary, the rise in left ventricular resting pressure (LVRP) and GSH/GSSG ratio were significantly different between Group C, and Groups A and B (p<0.0001, analyzed by Generalized Estimating Equations model for repeated measures, and p<0.05, respectively). CONCLUSIONS: The pathophysiology of myocardial damage during hypothermic storage cannot be considered as a normothermic ischaemic injury and parameters other than energetic metabolism, such as thiolic redox state, are more predictive of functional recovery upon reperfusion.

Modification of the gating of the cardiac sarcoplasmic reticulum Ca(2+)-release channel by H2O2 and dithiothreitol.

The effect of hydrogen peroxide (H2O2) on the sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel has been investigated under voltage-clamp conditions after incorporation of native membrane vesicles into planar phospholipid bilayers. In the presence of micromolar activating calcium concentrations on the cytosolic side of the membrane, H2O2 (3-5 mM) increased open probability of the channels. H2O2 did not affect the conductance of the channel or the response to activating compounds, such as ATP and caffeine. H2O2 did not alter the inhibitory response to magnesium or the modification of channels by ryanodine. At subactivating calcium concentrations (approximately 45 pM) on the cytosolic side of the membrane, 5 mM H2O2 was still able to open the channel. Analysis of single-channel open and closed lifetimes suggested that H2O2 had a direct effect on the gating mechanism of the channel. Open probability of the SR Ca(2+)-release channel is reduced by millimolar concentrations of dithiothreitol, a sulfhydryl-protecting compound, in a concentration-dependent manner. In conclusion, it is probable that H2O2 activates the SR Ca(2+)-release channel via an oxidation of cysteine thiol groups in the channel protein.

How do calcium antagonists differ in clinical practice?

The majority of calcium antagonists used clinically belong to three distinct chemical classes: the phenylalkylamines, the dihydropyridines, and the benzothiazepines. In recent years their mode of action has been unravelled, their limitations recognized, and their efficacy and use in the management of patients with a broad spectrum of cardiovascular and other disorders defined. It is clear, however, that these drugs are not all alike, providing an explanation for their differing effects. The final therapeutic effect in humans depends on the mechanisms of action at the molecular level, the tissue selectivity, and the hemodynamic changes of each agent. All these aspects are examined in detail in this article. Concepts that are highlighted are as follows: (a) Molecular biology has allowed recognition of the polypeptide components of the alpha 1 subunit of the L-type Ca2+ channel and the finding of peptide segments covalently labelled by all three classes of drugs. (b) The location of these segments within the peptides is different: Binding sites for dihydropyridines are located externally, whereas those for verapamil and diltiazem are located internally, in the cytosolic part of the membrane. (c) Dihydropyridine binding is voltage dependent. This explains the selectivity of this class of drugs for vascular smooth muscle, which is more depolarized than cardiac muscle. (d) Phenylalkylamines and benzothiazepines reach their receptors at the internal surface of the sarcolemma through the channel lumen. Their binding is facilitated by the repetitive depolarization of atrioventricular and cardiac tissue, a phenomenon described as use dependence. This explains why these drugs are not highly selective, but equipotent for the myocardium, the atrioventricular conducting tissue, and the vasculature. (e) Dihydropyridines act through selective vasodilatation and may increase heart rate and contractility via a reflex mechanism. On the contrary, phenylalkylamines and diltiazem act through a combination of effects, including reduction of afterload, heart rate, and contractility. When taken together, all these differences distinguish the preferential clinical utilization of one of these compounds for a given cardiovascular pathology.

Effect of angiotensin converting enzyme inhibition with quinaprilat on the ischaemic and reperfused myocardium.

We assessed whether the local inhibition of myocardial converting enzyme by quinaprilat and captopril reduces the functional and metabolic damage caused by ischaemia and reperfusion. Quinaprilat and captopril were either subcutaneously injected (0.3 mg/kg once daily for 5-6 days) in the rabbit before isolation of the heart or delivered to the isolated hearts in the perfusate (10(-6) M) 60 min before ischaemia. Cardiac protection was evaluated in terms of left ventricular pressure recovery during reperfusion, creatine phosphokinase (CPK) release, mitochondrial function, ATP and CP tissue contents, calcium homeostasis and the occurrence of oxidative stress, established by measuring content and release of reduced and oxidized glutathione. Both drugs exerted cardioprotection. Optimal myocardial preservation is achieved when quinaprilat is prophylactically administered to the rabbit. Recovery of developed pressure on reperfusion improved from 11.3 +/- 2.7 (S.E.) to 25.4 +/- 5.4 mmHg, P < 0.01 and the release of CPK was reduced from 665.8 +/- 101.4 to 231.8 +/- 81.4 mU/min/g wet wt, P < 0.01. Peak of noradrenaline release was also attenuated, from 5.253 ng/min/g wet wt to 1.764 ng/min/g wet wt. The accumulation of tissue and mitochondrial calcium was reduced from 52.3 +/- 7.5 and 44.1 +/- 5.6 to 20.5 +/- 3.2 and 27.3 +/- 4.6 nmol/kg dry wt, respectively, P < 0.01. This resulted in significant (P < 0.01) improvement of left ventricular diastolic dysfunction during ischaemia and reperfusion and in a preservation of all indices of mitochondrial function, allowing a higher recovery of ATP and CP after reperfusion (from 4.1 +/- 0.5 and 5.2 +/- 0.5 to 11.1 +/- 1.1 and 24.8 +/- 1.0 mumol/g dry wt, respectively, P < 0.01). Reperfusion-induced myocardial accumulation and release of oxidized glutathione were reduced from 0.301 +/- 0.056 and 0.318 +/- 0.083 to 0.138 +/- 0.025 nmol/mg protein and 0.076 +/- 0.012 nmol/min/g wet wt, respectively, P < 0.01. Similar results were obtained when quinaprilat was administered to the isolated heart. These data suggest that the cardioprotective effect of quinaprilat is independent from haemodynamic changes or direct reduction of toxicity due to oxygen free-radicals but it is likely to be related to a reduction in the release of noradrenaline, maintenance of high energy phosphates and membrane integrity.

[Congestive heart failure: from cardiac muscle to skeletal muscle]. / Insufficienza cardiaca congestizia: dal muscolo cardiaco al muscolo scheletrico.

It is well established that in patients with chronic heart failure, exercise capacity and clinical symptoms such as fatigue or dyspnea correlate poorly with the extent of left ventricular dysfunction. The increase in cardiac output caused by vasodilators, cannot be translated immediately into increased exercise capacity and peak oxygen consumption in patients with chronic heart failure. These observations have prompted the hypothesis that in chronic heart failure intrinsic abnormalities of skeletal muscle emerge that prevent acute improvement in peak VO2 and blood lactate accumulation. Studies using nuclear magnetic resonance demonstrate abnormal skeletal muscle metabolism during exercise, even in the absence of reduced flow or under ischaemic conditions. Histological examination of skeletal muscle reveals a variable extent of atrophy, increased interstitial cellularity and increase in type IIb fibres. Ultrastructural analysis shows abnormalities indicative of depressed oxidative capacity. Biochemical analysis of skeletal muscle biopsies demonstrates reduced activity of enzymes involved in aerobic metabolism and free fatty acid accumulation. These data indicate morphological, biochemical and metabolic alterations of skeletal muscle that should contribute significantly to the reduced muscle strength and rapid fatigue in patients with chronic heart failure. It has also been speculated that a generalized myopathy may occur in a subset of patients with dilated cardiomyopathy. These findings have clinical implications. Prolonged immobilization of patients with chronic heart failure was often suggested, is not practised anymore. Physical training in chronic heart failure has been shown to improve skeletal muscle function, exercise capacity and clinical symptoms in small controlled trials. Pharmacological treatment might be targeted for skeletal muscle disorders in patients with heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of lacidipine on ischaemic and reperfused isolated rabbit hearts.

Lacidipine is a new developed dihydropyridine calcium-antagonist, showing a slow onset and long lasting-selective activity. To assess whether the administration of lacidipine protects the myocardium in a dose-dependent manner against ischaemia and reperfusion, isolated rabbit heart were infused with three different concentrations of lacidipine: 10(-10); 10(-9); 10(-8) M. Diastolic and developed pressures were monitored; coronary effluent was collected and assayed for CPK activity and for noradrenaline concentration; mitochondria were harvested and assayed for respiratory activity, ATP production and calcium content and tissue concentration of ATP, creatine phosphate (CP) and calcium were determined. Occurrence of oxidative stress during ischaemia and reperfusion was also monitored in terms of tissue content and release of reduced (GSH) and oxidized (GSSG) glutathione. Treatment with lacidipine at 10(-10) and 10(-9) M had no effects on the hearts when perfused under aerobic condition, whilst the higher dose reduced developed pressure of 36%. The ischaemic-induced deterioration of mitochondrial function was attenuated. On reperfusion treated hearts recovered better than the untreated hearts with respect to left ventricular performance, replenishment of ATP and CP stores and mitochondrial function. The reperfusion-induced tissue and mitochondrial calcium overload, release of CPK and of noradrenaline and oxidative stress were also significantly reduced. The effects of lacidipine were dose-dependent. The lower concentration (10(-10) M) failed to modify ischaemic and reperfusion damage. The dose of 10(-9) M was cardioprotective, but the best effect was found at 10(-8) M. It is concluded that lacidipine infusion provides a dose dependent protection of the heart against ischaemia and reperfusion. Because this protection occurred also at 10(-9) M, in the absence of negative inotropic effect during normoxia and of a coronary dilatory effect during ischaemia, it cannot be attributed to an energy sparing effect or to improvement of oxygen delivery. From our data we can envisage two other major mechanism: -1) membrane protection -2) reduction of oxygen toxicity. The ATP sparing effect occurring at 10(-8) M is likely to be responsable for the further protection.

The effects of L-arginine mono(2-mercaptoethanesulfonate) on the ischemic and reperfused heart.

We evaluated the effectiveness of L-arginine mono(2-mercaptoethanesulfonate) (argimesna) to limit the extent of myocardial damage resulting from 60 minutes of severe ischemia followed by 30 minutes of reperfusion in the Langendorff-perfused rabbit heart. Argimesna is a sulfhydryl group containing molecule which has no effect on glutathione status or on the total thiol pool. The effects of 10(-6) M argimesna were compared with those of 10(-6) M L-arginine and of 10(-6) M sodium salt of 2-mercaptoethanesulfonate (mesna). Pretreatment of the hearts with 10(-6) M argimesna resulted in marked myocardial protection, measured in terms of improved recovery of developed pressure (p less than 0.01), reduced release of creatine kinase (p less than 0.01), maintenance of mitochondrial function and increased stores of ATP on reperfusion (p less than 0.01). On reperfusion less oxidative stress developed, as indicated by less accumulation of oxidized glutathione (p less than 0.01). These effects were specific for argimesna; no significant protection could be found for mesna and L-arginine. The beneficial effects of argimesna could not be explained by hemodynamic differences or effects on anaerobic metabolism. Neither is it likely that argimesna acts as a free radical scavenger at the concentrations employed. The protection may be achieved by maintenance of -SH groups during ischemia and reperfusion.

Role of timing of administration in the cardioprotective effect of fructose-1,6-bisphosphate.

We administered fructose-1,6-bisphosphate (FDP), 1 mM, to isolated and perfused rabbit hearts submitted, after 90 minutes of equilibration, to an ischemic period (60 minutes at a coronary flow of 0.17 ml/min/g), followed by a period of reperfusion (30 minutes at a coronary flow of 3.6 ml/min/g). FDP was delivered at different times following the experimental protocol: 60 minutes before ischemia and for the entire experiment; 60 minutes before and during ischemia, but not at reperfusion; at the onset of ischemia and during reperfusion; and only during reperfusion. The FDP cardioprotective effect was evaluated in terms of recovery of left ventricular pressure developed during reperfusion, creatine phosphokinase (CPK) and noradrenaline release, mitochondrial function (expressed as yield, RCI, QO2, ADP/O), ATP and creatine phosphate (CP) tissue contents, calcium homeostasis, and by measuring oxidative stress in terms of reduced and oxidized glutathione release and tissue contents. Our data show that the cytoprotective action of FDP is closely related to the time of administration. Optimal myocardial preservation was achieved when it was present prior to ischemia and during reperfusion. When given at the time of ischemia or only on reperfusion, FDP does not exert cardioprotection. The data suggest that the FDP cardioprotective effect is related to improvement of energy metabolism.

Protection of the ischemic myocardium by the converting-enzyme inhibitor zofenopril: insight into its mechanism of action.

We assessed whether local inhibition of myocardial converting enzyme by captopril and zofenopril reduces the functional and metabolic damage caused by ischemia and reperfusion. First we investigated the effects of zofenopril and captopril on the mechanical function, cellular redox state, and norepinephrine (NE) content of isolated and aerobically perfused rabbit hearts. Both drugs failed to modify the myocardial redox state. At concentrations > 10(-6) M, zofenopril, but not captopril, caused a reduction in myocardial NE content. At 10(-4) M, both drugs caused a reduction in developed pressure and an increase in diastolic pressure and release of creatine phosphokinase (CPK). Second we investigated their effects on ischemic and reperfused myocardium. Both drugs exerted a cardioprotection; zofenopril was always more potent than captopril. Recovery of developed pressure on reperfusion improved, and peak release of NE was reduced, as was release of CPK. Calcium homeostasis and mitochondrial function were maintained. Captopril had no effect on occurrence of oxidative stress during reperfusion, whereas zofenopril reduced it. In hearts treated with the converting enzyme inhibitors, peak release of NE was correlated to mitochondrial calcium content, production of ATP, and recovery of mechanical function on reperfusion. These data suggest that the cardioprotective effect of zofenopril and captopril is independent of hemodynamic changes or reduction of the toxicity of oxygen free radicals and that it could be related to a reduction in release of NE.

Role of timing of administration in the cardioprotective effect of iloprost, a stable prostacyclin mimetic.

We administered iloprost, a stable prostacyclin mimetic, 27 nM, to isolated and perfused rabbit hearts submitted, after 60 min of equilibration, to an ischaemic period (60 min at a coronary flow of 1 ml/min) followed by a period of reperfusion (30 min at a coronary flow of 25 ml/min). Iloprost was delivered at different times during the experimental protocol: 60 min before ischaemia, at the onset and after 30 min of ischaemia and only during reperfusion. The iloprost cardioprotective effect was evaluated in terms of recovery of left ventricular pressure developed during reperfusion, creatine phosphokinase (CPK) and noradrenaline release, mitochondrial function (expressed as yield, RCI (respiratory control index), QO2, ADP/O), ATP and creatine phosphate (CP) tissue contents, calcium homeostasis and by measuring several parameters of oxidative stress: reduced and oxidized glutathione release and tissue contents, Mn and Cu-Zn superoxide dismutase activities; glutathione reductase and peroxidase activities. Our data show that the cytoprotective action of iloprost is closely related to the time of administration. Optimal myocardial preservation was achieved when it was given before or at the onset of ischaemia. Iloprost administration 30 min after the onset of ischaemia was still beneficial, although to a lesser extent. Iloprost lost its protective effect when given only on reperfusion. The data suggest that the iloprost cardioprotective effect is related to maintainance of membrane integrity.

Effect of propionyl-L-carnitine on mechanical function of isolated rabbit heart.

We studied the acute and chronic effects of propionyl-L-carnitine (PLC) on mechanical function of isolated rabbit heart. Propionyl-L-carnitine was either directly delivered in the perfusate (10(-9) to 10(-3) M) or intraperitoneally injected (250 mg/kg) for 10 days to the animals. When added acutely, propionyl-L-carnitine had no effect on inotropism, heart rate, or coronary perfusion pressure. When added chronically, propionyl-L-carnitine induced a positive inotropic effect, with no changes in heart rate or in coronary perfusion pressure, and it ameliorated the pressure-volume relationship. This effect of propionyl-L-carnitine was independent of the calcium concentration of the perfusion medium, but it was correlated with an increase in the myocardial content of propionyl-L-carnitine. The effect was not apparent after 5 days of treatment, although the tissue content of propionyl-L-carnitine remained unchanged. These data suggest that propionyl-L-carnitine, when given chronically, exerts a positive inotropic effect.

Effects of anipamil on myocardial sarcolemmal and mitochondrial calcium transport, comparison with verapamil and nifedipine.

The calcium antagonists anipamil, verapamil and nifedipine inhibited, dose dependently, passive and ATP-driven 45Ca2(+)-uptake in purified rabbit ventricular sarcolemmal vesicles exposed to a wide range of free calcium concentration (from 0 to 200 microM). The IC50 values for passive binding were virtually identical for all calcium antagonists and the inhibition was relatively independent of the amount of free calcium employed. On the contrary, the order of potency for inhibition of the ATP-driven calcium uptake was: anipamil greater than verapamil greater than nifedipine. The inhibition of nifedipine, at free calcium concentrations lower than 80 microM, was preceded by a slight stimulation. The inhibitory effects of anipamil and verapamil, but not those of nifedipine, on the ATP-driven calcium uptake were more evident with increasing external calcium concentration. Verapamil and nifedipine failed to modify the initial rate of mitochondrial calcium transport either in the presence or in the absence of ADP; on the contrary, anipamil induced a dose-dependent inhibition of mitochondrial calcium transport. The inhibition occurred over the whole range of calcium concentrations tested, independent of the presence of ADP. The effects of anipamil, but not those of verapamil and nifedipine, on sarcolemmal and mitochondrial calcium transport were long lasting and survived membrane isolation.

Effect of D-600 on ischemic and reperfused rabbit myocardium: relation with timing and modality of administration.

In this study we have investigated the possibility that D-600, a phenylalkylamine calcium antagonist, protects the isolated rabbit heart against ischemia and reperfusion-induced damage. D-600 was either subcutaneously injected (2mg/kg, twice daily for 5 to 6 days) in the rabbit before isolation of the heart, or delivered to the isolated hearts in the perfusate (10(-7) M), either at the onset of ischemia and during reperfusion, or only during post-ischemic reperfusion. Ischemia (90 min) was induced by reducing coronary flow from 25 to 1 ml/min, followed by 30 min of reperfusion. Myocardial damage was determined in terms of mechanical function, release of creatine phosphokinase (CPK) and noradrenaline, mitochondrial function, calcium homeostasis, and endogenous stores of ATP and creatine phosphate (CP). Administration of D-600 to the rabbits or to the isolated hearts at the time of ischemia exerted protection. There are four groups of evidence in support of this conclusion: 1) the rise in diastolic pressure during ischemia was diminished with greater recovery of developed pressure during reperfusion; 2) CPK and noradrenaline release during reperfusion were reduced; 3) the oxygen consumption and ATP generating capacities of mitochondria were better maintained; and 4) associated with this preservation of mitochondrial function was the maintenance of near normal calcium homeostasis and of endogenous ATP and CP stores. The two different modalities of administration did not produce substantially different results. When administered to the isolated hearts after the ischemic period, D-600 failed to improve mechanical recovery and release of endogenous substances. However, it reduced mitochondrial calcium overload and improved ATP production. The mechanism of the protective effect of D-600 seems to be multiple: energy-sparing effect, reduction of the toxicity mediated by endogenous catecholamines, and direct inhibition of mitochondrial calcium transport.

Protective effects of gallopamil against ischemia and reperfusion damage.

To establish if the administration of gallopamil, a derivative of verapamil, protects heart muscle against the deleterious effect of ischemia and subsequent reperfusion, rabbits were injected subcutaneously twice daily with 2 mg/kg of Gallopamil for 5-6 days. The hearts were isolated and perfused with aerobic Krebs-Henseleit buffer solution by the Langendorff method. The hearts were paced (180 b/min) and wall temperature was controlled. Ischemia was induced by reducing coronary flow from 25 ml/min to 1 ml/min for 90 min and then the hearts were reperfused for 30 min. At the end of either the ischemic period or reperfusion, the hearts were assayed for ATP, CP, and calcium. Others were homogenized, their mitochondria harvested and monitored for oxidative phosphorylating and ATP generating activity as well as calcium content and uptake. The mechanical function of the hearts and noradrenaline release was also measured. Hearts that were made ischemic gained calcium, their endogenous stores of ATP and CP were depleted, their mitochondria had reduced RCI and state 3 respiration and increased calcium concentrations. During reperfusion tissue and mitochondrial calcium was significantly increased, the capacity of mitochondria to use oxygen for state 3 respiration was further impaired and their ATP generating capacity reduced. Diastolic pressure increased and there was no recovery of developed pressure and important noradrenaline release. Pretreatment with gallopamil protected the mitochondria against the ischemically induced changes in RCI, state 3 respiration. There was also a less marked rise in tissue and mitochondrial calcium and a reduced increase of diastolic pressure. Gallopamil also diminished the effect of reperfusion on the calcium accumulating activity of mitochondria and on the decline in the ATP generating and oxygen utilizing capacity of the mitochondria. The tissue levels of ATP and CP were better maintained, and noradrenaline release was reduced, the systolic pressure generating capacity was enhanced by the treatment with gallopamil. These results are discussed in accordance with the hypothesis that this drug protects heart muscle against the deleterious effects of ischemia and reperfusion by ensuring that sufficient ATP remains available to maintain homeostasis with respect to calcium.