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.

Inhibitory effect of calcium antagonists on the depletion of cardiac norepinephrine during postischemic reperfusion.

We have investigated whether Ca2+ antagonists of the dihydropyridine type (nifedipine, nisoldipine, and nitrendipine) attenuate or abolish the ischemia-reperfusion-induced depletion of the cardiac stores of norepinephrine (NE). The experiments were performed using isolated, Langendorff-perfused rat hearts. Ischemia (global) was induced for 15 or 60 min at 37 degrees C and was followed by normothermic reperfusion. Left ventricular NE content was assayed by high-performance liquid chromatography with electrochemical detection. The continued presence of nifedipine (0.03 microM), nisoldipine (0.03 microM), or nitrendipine (0.03 microM) before and after the ischemic episode abolished the loss of NE caused by 15 min but not 60 min of ischemia and reperfusion. Nifedipine, 0.03 microM, but not diltiazem, 0.24-1.22 microM, or verapamil, 0.22-1.09 microM, attenuated (p less than 0.05) NE depletion when added only on reperfusion.

An inhibitory effect of verapamil and diltiazem on the release of noradrenaline from ischaemic and reperfused hearts.

We have investigated whether Ca2+ antagonists reduce the amount of noradrenaline lost from the myocardium during periods of ischaemia and reperfusion. Hearts obtained from adult, male normotensive Sprague Dawley, Wistar Kyoto and spontaneously hypertensive rats were perfused in the Langendorff mode at 37 degrees C before being made globally ischaemic for either 15, 30 or 60 min. Some of the hearts were reperfused, and in some ECG records were made. 90-min normothermic aerobic perfusion failed to cause a significant change in left ventricular noradrenaline content. In Sprague Dawley and spontaneously hypertensive, but not Wistar Kyoto rats, 15 min ischaemia followed by 1 min reperfusion caused a significant (P less than 0.05) loss of noradrenaline. Extending the ischemic episode to 60 min resulted in a further loss of noradrenaline (P less than 0.005) in the Sprague Dawley, Wistar Kyoto and spontaneously hypertensive hearts and this loss was exacerbated upon reperfusion. Neither dl verapamil (2.5 X 10(-8) to 1.2 X 10(-6) mol/l) nor diltiazem (0.25 to 1.25 X 10(-6) mol/l) caused any change in the noradrenaline content of the aerobically perfused hearts. (1.25 X 10(-1) to 1.2 X 10(-1) mol/l) verapamil abolished the release of noradrenaline caused by 15 min ischaemia and reduced the release caused by 60 min ischaemia and 15 min reperfusion. The dose-response curve for verapamil was bell-shaped and the activity resided in the l form. Diltiazem (1.25 X 10(-6) mol/l but not 2.5 X 10(-1) mol/l) also abolished the loss of noradrenaline caused by short periods of ischaemia and reperfusion.

Calcium antagonists and the ischaemic myocardium.

We have investigated whether Ca2+ antagonists reduce the amount of noradrenaline (NA) lost from the myocardium during periods of ischaemia and reperfusion. Hearts obtained from adult, male, normotensive Sprague-Dawley rats were perfused at 37 degrees C before being made globally ischaemic for 15 or 60 min and assayed for catecholamines. Some of these hearts were reperfused. Aerobic perfusion for 90 min failed to cause a significant loss of left ventricular NA. Ischaemia for 15 min followed by reperfusion for 15 min, and ischaemia for 60 min with or without reperfusion caused a significant loss of NA. The loss of NA caused by 15 min ischaemia and reperfusion was abolished or attenuated by verapamil, nifedipine, nitrendipine, nisoldipine and diltiazem.

Calcium antagonists and hypertension.

1. Calcium antagonists, including verapamil, are now used widely in the management of patients with hypertension. 2. Six weeks of chronic therapy with verapamil (50 mg/kg per day, orally) to produce a plasma level of 80-100 ng/ml in Sprague-Dawley rats depletes cardiac noradrenaline (NA) without apparently causing beta 1 adrenoceptor 'up' regulation. 3. The effect of verapamil on cardiac NA is rapidly reversed upon verapamil withdrawal. 4. Chronic therapy with nisoldipine (100 mg/kg per day, orally) had no effect on cardiac NA. 5. Verapamil (50 mg/kg per day, orally) and nisoldipine (100 mg/kg per day, orally) therapy for 6 weeks prevented the time-dependent increase in systolic blood pressure in SHR rats. 6. Binding studies with (-)[3H]-D888 (desmethoxyverapamil) indicated that the affinity of the phenylalkylamine binding sites is higher in hearts of SHR relative to hearts from age-matched (25 weeks) WKY and SD, without any change in density.

Chronic calcium antagonist therapy: some unexpected results.

The calcium antagonists protect the myocardium against the deleterious effects of ischemia and postischemic reperfusion provided that they are used prophylactically. This requires chronic therapy. Experiments were undertaken to establish whether chronic verapamil therapy alters the cardiac noradrenaline reserves or provokes a change in beta 1 adrenoceptor density. Sprague-Dawley (SD) rats were fed a diet containing placebo, or placebo plus dl verapamil (V) to provide plasma V levels of around 100 ng/ml. After 6 weeks of therapy the hearts were excised and assayed for noradrenaline (NA), adrenaline (A), and dopamine (DA) using high performance liquid chromatography. In addition, cardiac membranes were isolated in the presence of Tris, 10 mM MgCl2 and 9 microM phenylmethylsulfoxylfluoride and assayed for beta 1 adrenoceptor density (Bmax) and affinity (KD), using [3H]dihydroalprenolol as the ligand. Three days of therapy reduced left ventricular NA by 45%. Asymptote was reached within 11 days, when the NA content has decreased (p less than 0.001) to only 0.9 +/- 0.1 mu/g dry wt, mean +/- SEM, n = 6. The tissue level of DA was also reduced from 0.14 +/- 0.02 to 0.08 +/- 0.01 microgram/g dry weight (p less than 0.02). Further treatment for up to 6 weeks caused no further change in NA, A, or DA. Even after 6 weeks of therapy the density (35.5 +/- 1.9 before and 31.2 +/- 2.3 fmol/mg protein after therapy) and affinity (0.24 +/- 0.02 and 0.21 +/- 0.02 nM) of the beta 1 adrenoceptors were unchanged. These results show that although chronic verapamil therapy depletes the cardiac reserves of NA, beta-adrenoceptor density remains constant.

Effect of chronic verapamil therapy on cardiac norepinephrine and beta-adrenoceptor density.

Experiments were undertaken to establish whether the chronic administration of verapamil, to provide a therapeutically relevant plasma level, results in a loss of cardiac norepinephrine (NE) and, if so, whether this is accompanied by a change in cardiac beta 1-adrenoceptor density, affinity, or selectivity. Adult male Sprague-Dawley rats were used. Verapamil was mixed with the food to provide a daily verapamil intake of 50 mg/kg body weight. This yielded a plasma level of 80-110 ng/ml and caused a significant but reversible decrease in left ventricular NE (77%, p less than 0.001), dopamine (43%, p less than 0.02), and 3,4-dihydroxyphenylethyleneglycol (30%, p less than 0.02). Cardiac membranes prepared from rats maintained on standard (control) diet contained a single population of high affinity beta 1-adrenoceptor binding sites, with an affinity (Kd) of 0.24 +/- 0.02 nM and a density (Bmax) of 35.5 +/- 19.5 fmol/mg protein. Including verapamil (dissolved in either ethanol or water) in the diet for less than or equal to 6 weeks had no effect on the Kd, Bmax, or selectivity of these sites. However, the ethanol containing, but not the ethanol-free, placebo diet caused a small (p less than 0.05) reduction in Bmax after 6 but not after 2 weeks treatment. These results show that the chronic oral administration of verapamil to provide a clinically relevant plasma level depletes the cardiac stores of NE by 77% without causing an increase in beta 1-adrenoceptor density during 6 weeks therapy.

The protective effect of prazosin on the ischaemic and reperfused myocardium.

Experiments were undertaken to establish whether prazosin prevents isolated hearts from gaining excess Ca2+ during post-ischaemic reperfusion, to determine whether this effect is dose-dependent, if it is accompanied by a change in the energy-rich phosphate reserves, and whether prazosin is effective when added only upon reperfusion. Isolated, spontaneously beating rat hearts were used. The ischaemic episodes ranged from 15 to 60 min, and prazosin (0.01 to 10 micro mol/1) was added both before inducing ischaemia and upon reperfusion. When 0.01 to 1 micro mol/1 prazosin was present before and after the ischaemic episode the reperfusion-induced gain in Ca2+ was attenuated, but not abolished. Pretreatment with 0.01 to 1 micro mol/1 prazosin slowed the ischaemic-induced rise in resting tension, enhanced mechanical recovery after 30 but not 60 min ischaemia, and exerted a dose-dependent slowing effect on the ischaemia-induced depletion of ATP and CP, with 1 micro mol/1 being the optimal dose. Adding 0.01 to 1 micro mol/1 prazosin at the time of reperfusion neither prevented excess Ca2+ accumulation upon reperfusion nor did it exert an energy-sparing effect. 5 to 10 micro mol/1 prazosin did not attenuate the reperfusion-induced gain in Ca2+, irrespective of whether it was added before or only at the time of reperfusion. These results show that the dose-response curve for the inhibitory effect of prazosin on Ca2+ overload is complex, and that adding prazosin coincident with the reperfusion of isolated ischaemic hearts does not attenuate Ca2+ gain.