Winifred G. Nayler

Protective effect of pretreatment with verapamil, nifedipine and propranolol on mitochondrial function in the ischemic and reperfused myocardium

Abstract To establish whether the prophylactic use of verapamil, nifedipine or propranolol protects heart muscle against the deleterious effects of global ischemia and reperfusion, rabbits were injected subcutaneously twice daily with 2.0 mg/kg of one of these drugs for 4 to 5 days. The hearts were then isolated, paced and either perfused aerobically, made totally ischemic for 90 minutes or made ischemic for 90 minutes and then reperfused. At the end of this time some of the hearts were assayed for adenosine triphosphate (ATP), creatine phosphate (CP) and calcium (Ca ++ ). Other hearts were homogenized, the mitochondria harvested and monitored for oxidative phosphorylating and ATP-generating capacity and Ca ++ content. The effect of Ca ++ on the ATP-generatlng capacity of cardiac mitochondria was also determined. Hearts that were made ischemic gained Ca ++ . The endogenous stores of ATP and CP were depleted; the mitochondria accumulated Ca ++ and the oxidative phosphorylating activity (respiratory control index and oxygen quotient) was impaired. During reperfusion, tissue and mitochondrial Ca ++ was substantially increased, the capacity of the mitochondria to use O 2 for state III respiration was further impaired and the ATP-generating capacity reduced. Resting tension increased and there was only a small recovery of active tension generation. Hearts from the rabbits treated with propranolol, verapamil or nifedipine were protected against ischemia, and ischemia with reperfusion-induced decline in the ATP-generating and O 2 -utillzing capacity of the mitochondria. There was also a less marked increase in tissue and mitochondrial Ca ++ and the systolic tension-generating capacity of the hearts was better maintained.

Hypoxia and calcium

Abstract During hypoxia peak developed tension falls rapidly, resting tension inceeases slowly and recovery is incomplete if the hypoxia is sufficiently prolonged for resting tension to become greatly elevated. We propose that the initial fall of peak developed tension is due to a failure of excitation-contraction coupling at a time when high energy phosphate stores are only partially depleted. The rise of resting tension appears to reflect a deficiency of ATP necessary for the maintenance of intracellular calcium homeostasis. The removal of extracellular calcium, the addition of verapamil, and the measurement of calcium influx, indicate that the initial rise in resting tension is not due to an inward movement of calcium from the extracellular phase. Mechanical recovery after a period of hypoxia is jeopardized when there has been a substantial net gain of calcium.

A protective effect of a mild acidosis on hypoxic heart muscle.

Isolated spontaneously beating and electrically-paced rabbit hearts were perfused aerobically and then with hypoxic buffer (PO2 < 25 mmHg) at pH 7.4, 6.9 and 6.6, the required pH being established by varying the CO2 content of the gas mixture bubbled through the perfusion buffer. After the required period of hypoxic perfusion the hearts were either reoxygenated with buffer solution at pH 7.4 or they were assayed for ATP and creatine phosphate or their mitochondria were harvested and assayed for QO2, respiratory control index and Ca2+-accumulating activity. Resting and peak developed tension were monitored continuously. n nHearts made hypoxic at pH 7.4 exhibited reduced ATP and creatine phosphate content. The respiratory activity of their mitochondria was low and their Ca2+-accumulating activity high relative to that obtained for hearts made hypoxic at pH 6.9. n nThere was a greater recovery of tension development during reoxygenation of hearts made hypoxic at pH 6.8, relative to recovery obtained for the pH 7.4 hypoxic hearts. Perfusion of pH 6.9 also resulted in a relatively smaller rise in resting tension relative to that found during hypoxic perfusion at 7.4. n nThese results have been interpreted to mean that a mild respiratory acidosis has a protective effect on hypoxic heart muscle. This protective effect was not evident if the pH of the hypoxic buffer was decreased further, to 6.6.

An effect of ouabain on the superficially-located stores of calcium in cardiac muscle cells.

The effect of ouabain on the amount of Ca2+ (detected as 45Ca2+) displaceable by La3+ from 45Ca2+-labelled heart muscle was studied. The doses of ouabain used were restricted to those which evoke a positive inotropic response without contracture. La3+ had a negative inotropic effect. Inotropically-active doses of ouabain increased the amount of Ca2+ displaced by La3+. These results are cited as evidence of a membrane-located site of action for ouabain.

Calcium efflux from cardiac sarcoplasmic reticulum: effects of calcium and magnesium.

Abstract The dependence of the rate of calcium efflux from cardiac sarcoplasmic reticulum on the concentration of ionized calcium in the medium has been investigated. A high concentration of ionized calcium outside the sarcoplasmic reticulum stimulates calcium efflux. Stimulation of calcium efflux from cardiac sarcoplasmic reticulum by high levels of ionized calcium outside the membranes is inhibited by magnesium. This inhibition can be overcome at very high concentrations of calcium in the medium. This resembles the magnesium inhibition of calcium triggered calcium release from skinned cardiac muscle fibres, which can also be overcome by increasing the concentration of calcium in the medium. Ouabain at 10−6 m and verapamil at 10−5 m have no effet on the stimulation of calcium efflux by calcium.

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 μm inhibited the passive binding of 45Ca2+. The (+)‐ and (‐)‐isomers were equiactive. 3 (±)‐Verapamil 1 μm 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‐acetylneuraminic acid and galactose residues do not provide binding sites for verapamil at the cell surface.

Review of preclinical data of calcium channel blockers and atherosclerosis.

A variety of animal models have been used to determine whether calcium channel blockers exert an inhibitory effect on atherosclerotic lesion formation. These models include the cholesterol-fed rabbit, in which the lesions resemble the fatty-streak stage of atherosclerotic lesion development in humans. Diet-induced atherosclerosis in monkeys is also used and, in this case, the lesions resemble those found in humans, both in pathology and distribution. Other models involve mechanical injury superimposed on cholesterol feeding. Cellular and subcellular preparations are being used to investigate the mechanisms involved in the antiatherosclerotic activity of the calcium channel blockers. The ability of calcium channel blockers to slow atherosclerotic lesion formation is a class effect that is independent of their blood pressure-lowering effect, and occurs without any significant change in the plasma lipid profile. It is accompanied by a reduction in vessel wall cholesterol and calcium and is maintained over prolonged periods of treatment. The mechanisms that may be involved include inhibition of smooth muscle cell proliferation and migration, slowed platelet aggregation, restructuring of cholesterol-enriched cell membranes, enhanced gene expression for low-density lipoprotein receptor protein, inhibition of growth factor release, slowed calcium uptake, and restoration of endothelium-dependent relaxation.

The calcium accumulating activity of subcellular fractions isolated from rat and guinea pig heart muscle

Abstract Microsomal fractions prepared from rat ventricular muscle accumulated 23.15 ± 3.6 and 27.8 ± 2.4 nmol Ca2+/mg microsomal protein when incubated for 2 and 5 min respectively in an oxalate-free incubation medium, and 263.2 ± 26.0 and 327.3 ± 48.6 nmol Ca2+/mg microsomal protein when incubated for 2 and 5 min respectively in the presence of oxalate. These values for Ca2+ binding (absence of oxalate) and uptake (presence of oxalate) are significantly (P In the absence of Ca2+ the rat microsomal fractions hydrolysed ATP at a rate which was significantly faster than that exhibited by the guinea pig microsomes. Doses of 1 and 5 m m caffeine increased the peak tension developed by guinea pig and decreased the peak tension developed by rat papillary muscles. These same doses of caffeine prolonged the time required to develop peak tension in both species, and showed the rate of Ca2+ uptake. Doses of 1 and 5 m m caffeine slowed the rate at which the rat but not the guinea pig microsomes bound Ca2+ (in the absence of oxalate). These results are interpreted to mean that there is a species variation in the Ca2+-accumulating and ATPase activity of rat and guinea pig microsomal fractions.

ATP-induced stimulation of calcium binding to cardiac sarcolemma

Abstract Sarcolemma vesicles isolated from rabbit ventricular muscle showed a high degree of purity when assessed in terms of calcium binding and marker enzyme activity. Calcium binding to the sarcolemma increased as the free calcium concentration of the medium increased. ATP-independent (passive) and ATP-dependent (active) calcium binding activities were observed. The active binding was associated with an increase in the calcium stimulated ATPase activity of the preparation. A Lineweaver-Burke plot of the ATP-dependent calcium binding data was linear over the whole range of calcium concentrations (20–200 μM) investigated.

Two orientations of isolated cardiac sarcolemmal vesicles separated by affinity chromatography

Abstract Isolated cardiac sarcolemmal vesicles showing a high degree of purity were separated into two populations of opposite orientation using affinity chromatography. Separations employed the carbohydrate binding characteristics of a wheat germ lectin-Sepharose 6MB column and the location of carbohydrate residues exclusively on the extracellular face of the sarcolemma. The calcium-handling capacities of the two different orientations of vesicle were investigated. Inside-out vesicles showed very low passive binding but high ATP-dependent or active transport. In contrast, right-side-out vesicles showed significant passive binding and very little active transport. These results are discussed in terms of the calcium-regulating mechanisms of the cardiac muscle cell.