Effect of methylprednisolone sodium succinate on hypoxic heart muscle.

The ability of 30 mg/litre methylprednisolone sodium succinate (MPSS) to modify the effects of hypoxia on isolated Langendorff-perfused rat hearts was investigated. When perfused under hypoxic conditions (pO2 less than 0.8 kPa[6 mmHg]) these hearts lose intracellular enzymes, including creatine phosphokinase (CPK) and succinic dehydrogenase (SDH). The size of the extracellular space is enhanced, the cells gain Na+ and Ca2+ and lose K+, and the endogenous stores of ATP and CP are depleted. Initially the resistance to flow in the coronary circulation falls but after 75 min of hypoxic perfusion it increases so that coronary flow is reduced. MPSS failed to prevent hypoxic muscle from either gaining Na+ and Ca2+ or losing K+. It did, however, delay the release of CPK and SDH from the hypoxic muscle, prolong the phase of increased coronary flow, and decrease the rate of depletion of the energy-rich phosphate stores. MPSS potentiated the hypoxic-induced gain in Ca2+. Whilst the effects of MPSS on coronary flow and tissue Ca2+ were probably due to the steroid part of the complex, the other changes, including the protection of the ATP and CP stores and the delayed enzyme release, were probably due to the presence of the sodium succinate.

Calcium accumulating and ATPase activity of cardiac sarcoplasmic reticulum before and after birth.

The possibility that the cardiac SR undergoes developmental changes at about the time of birth, and that these changes affect its ability to accumulate Ca2+ and to hydrolyse ATP has been studied. SR-rich microsomal fractions were prepared from heart muscle excised from foetal guinea pigs and rabbits 1 day before their anticipated date of birth, and from 1 day old and adult animals. For control purposes microsomes were also prepared from the relevant maternal stock animals. One day before birth the cardiac microsomes of the foetal but not of the maternal animals exhibited a decreased ability to accumulate Ca2+ by uptake but not by the binding process, and a decreased ability to hydrolyse ATP. This reduction in ATPase activity involved both the Ca2+-dependent and the Ca2+-independent ATPase enzymes. One day after birth the Ca2+-accumulating activity of the neonatal microsomes had increased, that of the rabbit via an increase in Ca2+ uptake and that of the guinea pig by an increase in Ca2+ binding. These changes were accompanied by an increase in the activity of the Ca2+-dependent ATPase. The results are interpreted to mean that the cardiac SR changes at about the time of birth, and that although the pattern of these changes may be species specific they result in an increase in the Ca2+-accumulating activity of the SR.

A protective effect of verapamil on hypoxic heart muscle.

Hypoxic-induced damage of rabbit heart muscle has been quantitated in terms of the release of intracellular enzymes (including creating phosphokinase, CPK) into the extracellular space, a gain in tissue Na+ and Ca2+, a loss of tissue K+, the depletion of the adenosine triphosphate (ATP) and creatine phosphate (CP) reserves, and ultrastructural damage. This ultrastructural damage incolves the disruption of the plasmalemma, swelling and distortion of the mitochondria, disruption of the myofilaments, and the development of contraction bands. In isolated Langendorff-perfused rabbit hearts perfused under either aerobie (pO2 greater than 80.0 kPa [600 mm Hg]) or hypoxie (pO2 less than 0.80 kPa [6 mmHg]) conditions, and either with or without glucose substrate, 0.5-1.0 mg/litre dl verapamil reduced the amount of ultrastructural damage caused by hypoxie perfusion. Verapamil (0.5-1.0 mg/litre) also reduced the rate at which the hypoxie muscle gained Na+ and lost K+; it reduced the rate at which the endogenous stores of ATP and CP were depleted and, provided that the extracellular phase contained Ca2+, it decreased the rate at which CPK appeared in the coronary effluent. Verapamil failed to prevent the hypoxie muscle from gaining Ca2+. These results are discussed in terms of a possible protective effect of dl verapamil on hypoxie heart muscle.

Beta-adrenoceptor antagonists and the release of creatine phosphokinase from hypoxic heart muscle.

The ability of several beta-adrenoceptor antagonists with partial agonist activity (dl-oxprenolol, dl-acebutolol and dl-practolol) to attenuate the release of CPK that occurs during hypoxia (pO2 less than 0.8 kPa [6 MMHg]) has been studied and compared with the protection provided by dl-propranolol. dl-propranolol attenuated the hypoxic-induced release of CPK. The activity resided in the l isomer. dl-oxprenolol, acebutolol, and practolol were either less effective than propranolol in preventing CPK release, or they exacerbated the release. The protective effect of dl-propranolol extended to the hypoxic hyperthyroid heart but not to hearts that were perfused under aerobic (pO2 greater than 80 kPa [600 mmHg]) Ca2+-free conditions.

Pharmacological protection of mitochondrial function in hypoxic heart muscle: Effect of verapamil, propranolol, and methylprednisolone.

Experiments were undertaken to determine if drugs (verapamil, propranolol, and methylprednisolone sodium saccinate) that protect the fine ultrastructure of heart muscle against damage caused by hypoxia, protect mitochondrial function. Mitochondrial function was assessed in terms of oxidative phosphorylating and Ca2 +-accumulating activities. Isolated rabbit hearts were used, and hypoxic conditions induced by reducing the perfusate PO2 from 80.8 to 0.80 kPa (600 to 6 mmHg). The drugs were either added at the start of the hypoxic perfusion or (verapamil and propranolol) the rabbits were pretreated with them. Verapamil, propranolol and, to a lesser extent, methylprednisolone sodium succinate, provided protection evidenced by the maintainance of near normal mitochondrial oxidative phosphorylating and Ca2 +-accumulating activities after 60 min hypoxic perfusion. When added directly to mitochondria isolated from hypoxic-perfused muscle, the drugs had no effect.

The effect of beta-adrenoceptor and Ca2+ antagonist drugs on the hypoxia-induced increased in resting tension.

Using isolated, Langendorff-perfused, electrically-paced guinea-pig hearts, we have investigated the rise in resting tension that occurs when mammalian heart muscle becomes hypoxic. Substrate-depletion, tachycardia, hyperthyroidism, and inotropic interventions (ouabain, isoprenaline, and beta-receptor antagonists at concentrations which increase inotropic state) enhanced the rate of development of this increase in resting tension. 3.86 mumol.litre-1 propranolol, 0.22 to 2.20 mumol.litre-1 verapamil or removing Ca2+ from the extracellular phase at the start of the hypoxic episode prevented (or delayed) the rise in resting tension. Adding these same agents or removing Ca2+ from the extracellular phase after the hypoxia-induced rise in resting tension had started to develop failed to prevent its progression. These results provide some support for an hypothesis that the hypoxia-induced increase in resting tension is independent of an enhanced Ca2+ influx.

Protective effect of methylprednisolone sodium succinate on the ultrastructure and resting tension of hypoxic heart muscle.

Experiments were undertaken to further investigate the protective effect of methylprednisolone sodium succinate on hypoxic heart muscle. Hypoxia was induced in isolated Langendorff perfused rat and rabbit hearts by gassing the perfusate with 95% N2 + 5% CO2. The hypoxia-induced damage was quantitated in terms of an altered ultrastructure and increased resting tension. When added at the start of the hypoxic episode 6 X 10(-5) mol.litre-1 methylprednisolone sodium succinate protected the fine ultrastructure of the heart, and delayed the increase in resting tension. This protective effect could not be accounted for in terms of the sodium succinate moiety.

Vascular injury: mechanisms and manifestations.

Abnormalities in regulatory mechanisms for calcium handling play a key role in cell death and tissue necrosis. In the cardiovascular system this applies to the vasculature and the myocardium alike. In the aged population, where hypertension is a known risk factor, manifestations of vascular injury include atherogenesis and stroke. The newly developed dihydropyridine-based calcium antagonist amlodipine was used in investigations to determine whether calcium antagonists with sustained activity, in addition to lowering blood pressure, slow the development of atherogenesis in rabbits receiving high cholesterol diets, or reduce mortality in stroke-prone hypertensive rats. To establish whether this drug protects the vasculature against excessive atheroma formation in the presence of high cholesterol intake, rabbits were given 2% cholesterol in addition to their normal food intake and either 0, 1, or 5 mg/kg/day amlodipine orally for either 8 or 12 weeks. One day after the conclusion of the treatment protocol, the thoracic aorta was excised, assayed for calcium or cholesterol concentrations, and stained to identify sudanophilic-positive lesions. Amlodipine caused a time- and dose-dependent reduction in lesion formation, calcium overload, and cholesterol level. In the second series of experiments, amlodipine (5 mg/kg/day) was added to the diets of stroke-prone hypertensive rats. Treatment was initiated at age 5 weeks and continued for 30 weeks. During the treatment period, systolic blood pressure was reduced in the amlodipine-treated rats (166 +/- 9 mm Hg) versus those treated with placebo (248 +/- 12 mm Hg) (p less than 0.001). A significant reduction in mortality was observed in the amlodipine-treated rats (p less than 0.001), with 93% surviving versus only 26% in the placebo group at the end of the 30-week treatment period. Concomitantly, cardiac hypertrophy was attenuated in the treated group compared with the placebo group (heart-to-body weight ratios of 4.5 +/- 0.01 vs 5.8 +/- 0.6, respectively [p less than 0.01]). These results extend the evidence that calcium antagonists provide vascular protection in animal models. This finding may become increasingly important in the management of an aging hypertensive population.

Amlodipine pretreatment and the ischemic heart.

Amlodipine is a long-acting dihydropyridine-based Ca2+ channel blocker, developed for use on a once-daily basis. Experiments using hearts from amlodipine-pretreated rats were undertaken to further test the hypothesis that Ca2+ channel blockers can be used as prophylactic therapy to reduce the severity of the mechanical and biochemical consequences of ischemia and reperfusion. Amlodipine was given intravenously, 0.25 mg/kg, 5 hours before excising the hearts. Ischemia (global) was induced at 37 degrees C for 10, 30 or 60 minutes, and was followed by reperfusion. Protection was quantitated in terms of functional recovery, adenosine triphosphate and creatine phosphate retention, tissue acidosis and Ca2+ gain. The results show that amlodipine pretreatment supplied protection, provided that the ischemic episode did not exceed 30 minutes. The protection resulted in improved recovery of peak developed tension on reperfusion, reduced Ca2+ gain, retention of tissue adenosine triphosphate and creatine phosphate, and reduced acidosis.

Protecting the vasculature: an eye toward the future.

Although calcium antagonists were originally developed for use in the management of patients with angina pectoris, they are now used in the management of other cardiovascular disorders, including hypertension. More recently, the calcium antagonists have been under investigation for their potential protective role in atherosclerosis. Coupled with these new possibilities for therapeutic use are the development of new, long-acting, tissue-specific calcium antagonists. Amlodipine belongs to this group, and although it is a dihydropyridine-based calcium antagonist, its pharmacologic profile differs from that of other dihydropyridine-based calcium antagonists. Differences include: different pH optimum for receptor binding, different rates of association and dissociation, and differences in allosteric interaction with the diltiazem and verapamil binding sites. Amlodipine, when given orally to rabbits receiving a high-cholesterol diet, reduces atheroma formation. Evidence of its ability to protect the vasculature is provided by its ability to significantly increase (p less than 0.001) survival in stroke-prone hypertensive rats.

Reperfusion-induced calcium gain after ischemia.

Reperfusion-induced calcium gain provides a marker of irreversible injury, but whether the cells gain calcium because of irreversible injury caused by the ischemic episode, or whether it is the reperfusion-induced calcium gain that triggers the irreversible injury has yet to be established. Using isolated rat hearts made ischemic for either 30 or 60 minutes, and reperfusing with Krebs-Henseleit buffer or Krebs-Henseleit buffer containing either 2,3-butanedione monoxime (to inhibit contractile activity) or 2,4-dinitrophenol or nitrogen-gassed substrate-free Krebs-Henseleit buffer (to inhibit oxidative phosphorylation), the effect of reperfusion was monitored in terms of calcium gain and ultrastructural changes including loss of sarcolemmal integrity. The results establish that the routes of calcium entry during postischemic reperfusion are complex. The calcium gain can occur in the absence of mitochondrial oxidative phosphorylation and is modulated by interventions introduced at the moment of reperfusion which affect the contractile state. There are at least 2 routes of calcium entry: contraction-dependent and contraction independent. The former is probably associated with the development of sarcolemmal discontinuities. The results also establish that when sarcolemmal integrity has been destroyed, the cells can gain excess calcium under conditions that prevent mitochondrial calcium uptake. Accordingly, the mitochondria cannot be the only intracellular organelles that accumulate calcium under these conditions. Additional studies are needed to identify the other sites of calcium binding under conditions of adenosine triphosphate deprivation.

Fundamental mechanisms of action of calcium antagonists in myocardial ischemia.

The mammalian myocardium exhibits a spectrum of damage during an ischemic episode. After relatively short periods of ischemia the damage is reversible, but with longer periods of ischemia the number of cells that are lethally injured increases. When coronary flow is restored the lethally injured cells become overloaded with Ca++ and fail to regenerate adenosine triphosphate. The calcium antagonists provide protection under these circumstances, but only if used prophylactically. When added only upon reperfusion the calcium antagonists slow, but do not inhibit, the excessive gain in Ca++ that occurs during postischemic reperfusion. Nicotine, in a concentration equivalent to that found in the plasma of smokers (0.15 microgram/ml), exacerbates the reperfusion-induced Ca++ gain. Treatment with the long-acting calcium antagonist, anipamil, on a once-daily basis attenuates the reperfusion-induced Ca++ gain in spontaneously hypertensive rats and its exacerbation by nicotine in Sprague Dawley rats. The prolonged oral administration of at least 1 calcium antagonist, verapamil (50 mg/kg body weight/day), causes a significant (p less than 0.001) depletion of left ventricular norepinephrine.

Comparative partial agonist activity of -adrenoceptor antagonists.

Inotropic dose-response curves were constructed for a series of beta-adrenoceptor antagonists, based on the inotropic responses of isolated dog trabecular muscles stimulated to contract at a regular rate. Propranolol exerted only a negative inotropic effect but K01366, LB46, oxprenolol and practolol all evoked a positive inotropic response, whether or not catecholamine-depleted muscles were used. The order of potency for this positive inotropic activity was K01366>LB46>oxprenolol>practolol. The duration of the positive inotropic response to the antagonists was more prolonged than that due to isoprenaline. Propranolol significantly reduced the positive inotropic response to K01366, LB46, oxprenolol and practolol.

The Ca2+ -antagonist and binding properties of the phenylalkylamine, anipamil.

1. Isolated, Langendorff-perfused rat hearts, isolated membranes, and pharmacological and receptor binding techniques were used to study the properties of the newly developed verapamil derivative, anipamil. 2. When added acutely to isolated, spontaneously beating or electrically paced hearts, anipamil (0.01-0.15 microM) exerted a dose-dependent negative inotropic effect which developed slowly and persisted after 60 min washout. 3. When added acutely (0.05-0.1 microM) to isolated hearts, or when given intravenously (2 mg kg-1 body weight 1 h before the animals were killed), anipamil displaced the dose-response curves for the positive inotropic effect of (0.10-3.0 mM) Ca2+ and (10-50 nM) Bay K 8644 to the right. 4. When added to freshly isolated cardiac membranes, 0.1 microM anipamil increased the dissociation constant (KD) of the phenylalkylamine (-)-[3H]-desmethoxyverapamil ((-)-[3H]-D888) from 1.22 +/- 0.2 to 2.91 +/- 0.46 nM, without any significant change in density (Bmax; control: 163 +/- 17; anipamil: 117 +/- 20 fmol mg-1 protein). Bound (-)-[3H]-D888 was displaceable by (-)-D888 (Ki 1.7 +/- 0.4 nM) greater than (-)-D600 (Ki 12 +/- 0.5 nM) greater than verapamil (Ki 55 +/- 11 nM) greater than (+)-D600 (Ki 108 +/- 12.2) greater than anipamil (Ki 471 +/- 52 nM). 5. In cardiac membranes isolated from rats pretreated with anipamil (2 mg kg-1 i.v.) 1h before they were killed, the KD of (-)-[3H]-D888 binding was increased (P less than 0.05) from 1.59 +/- 0.18 to 3.28 +/-0.65 nM with no significant change in density, compared to the placebo-treated (control) rats. 6. These results establish that anipamil interacts in a competitive manner with the phenylalkylamine binding sites in cardiac membranes, and that it resembles other Ca2+ antagonists in displacing the dose-response curve for the positive inotropic effect of Ca2+ to the right. The results also show that although anipamil binds tightly to the cardiac membranes, it binds to the (-)-[3H]-D888 recognition sites less potently than (-)-D888, (-)-D600 or verapamil.

[3H]-verapamil binding to rat cardiac sarcolemmal membrane fragments; an effect of ischaemia.

The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained.

The effect of verapamil on the Ca2+-transporting and Ca2+-ATPase activity of isolated cardiac sarcolemmal preparations.

1 The effect of (+/-)-, (+)- and (-)-verapamil on the Ca2+-binding, Ca2+-transporting activity, and Ca2+-dependent adenosine triphosphatase (ATPase) activity of isolated cardiac sarcolemmal preparations was studied. Enzymatic treatment was used to establish the nature of the sites facilitating [14C]-(+/-)-verapamil binding. 2 (+/-)-Verapamil 1 microM inhibited the passive binding of 45Ca2+. The (+/-)- and (-)-isomers were equiactive. 3 (+/-)-Verapamil 1 microM inhibited the ATP-dependent transport of 45Ca2+ and the associated activation of the Ca2+-sensitive ATPase. The activity resided in the (-)-isomer. 4 Lineweaver-Burk plots for the initial rates of ATP-dependent transport showed that the inhibition induced by the (-)-isomer was accompanied by a reduced Km and Vmax. 5 Enzymatic removal of N-acetyl neuraminic acid and galactose residues increased [14C]-(+/-)-verapamil binding; removal of N-acetylglucosamine and treatment with phospholipase C and trypsin decreased the binding. 6 These results have been interpreted to mean that (-)-verapamil interferes with the ATP-dependent Ca2+-transporting properties of the sarcolemma, and that this effect is accompanied by an altered activity of the intrinsic Ca2+-sensitive ATPase. N-acetylneuramic acid and galactose residues do not provide binding sites for verapamil at the cell surface.

The effect of chemical sympathectomy on mitochondrial function in the ischaemic and reperfused myocardium.

1 Isolated rabbit hearts were perfused aerobically for 120 min, made ischaemic for 90 min, or made ischaemic for 90 min and then reperfused for 30 min. 2 Some rabbits were pretreated with 6-hydroxydopamine (6-OHDA), given as three separate intravenous doses of 30, 20 and 20 mg/kg, 20 to 48 h before they were killed; others (controls) received saline according to the same regime. 3 Mitochondria were harvested from left ventricular homogenates and their function assessed by measuring state 3O2 consumption (state 3 QO2), respiratory control index (RCI), phosphate: oxygen ratio (ADP:O), Ca2+ content, and ATP-producing activity. In other experiments peak left ventricular developed tension was recorded. 4 In hearts from saline-treated animals, mitochondrial state 3 QO2, RCI and ATP producing activities were reduced after global ischaemia, with or without reperfusion. There was a small gain in mitochondrial Ca2+ after ischaemia, and a large gain upon reperfusion. 5 6-OHDA pretreatment provided some protection against the effects of ischaemia and reperfusion on mitochondrial function and on peak developed tension. 6 It was concluded that chemical sympathectomy with 6-OHDA does not duplicate the effect of prolonged beta-adrenoceptor blockade in protecting mitochondrial function against the deleterious effects of ischaemia and reperfusion.

Streptozotocin-induced diabetes reduces the density of [125I]-endothelin-binding sites in rat cardiac membranes.

The effect of acute, streptozotocin-induced diabetes on the affinity (KD), density (Bmax) and selectivity of specific, high affinity binding sites for [125I]-endothelin [( 125I]-ET) in rat cardiac membrane fragments was determined. Three days after a single i.v. bolus dose of streptozotocin (60 mg kg-1), the density of [125I]ET binding sites was reduced (P less than 0.01) without changes in affinity or selectivity.

The failure of endothelin to displace bound, radioactively-labelled, calcium antagonists (PN 200/110, D888 and diltiazem).

The effect of endothelin, a potent vasoconstrictor polypeptide, on three types of calcium antagonist binding sites was examined, in rat cardiac membrane fragments. Endothelin 10 nM affected neither the affinity nor density of dihydropyridine binding sites. At concentrations of 10(-12)-10(-7) M, endothelin failed to displace bound (+)-[3H]-PN 200/110, (-)-[3H]-D888 and (+)-cis-[3H]-diltiazem. These results suggest that the calcium antagonist binding sites associated with L-type calcium channels are not the primary site of action of endothelin.

Effect of prolonged beta-adrenoceptor blockade on heart weight and ultrastructure in young rabbits.

1 Young rabbits were treated for 30 days or 6 weeks with propranolol or oxprenolol to provide dose-levels similar to those used clinically. 2 Relative to litter matched controls the treated rabbits exhibited a reduced rate of growth. 3 Morphometric studies showed that prolonged beta-adrenoceptor blockade had no effect on the myofibrillar volume as a proportion of cell volume. 4 Prolonged beta-blockade increased the proportion of the extracellular space occupied by vascular tissue.