J. S. Dillon

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 10−12‐10−7m, 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.

An effect of ischemia on myocardial dihydropyridine binding sites

The [3H]nitrendipine binding activity of sarcolemmal fragments isolated from aerobically perfused or ischemic rat hearts was studied. After 90 min aerobic perfusion, two populations of binding sites were detected--high affinity sites with KD of 0.24 +/- 0.04 nM and Bmax 313 +/- 110 fmol/mg protein, and low affinity sites with KD of 47.6 +/- 8.7 nM and Bmax 12.4 +/- 1.88 pmol/mg protein. Sixty minutes global ischemia significantly reduced the KD of the low (15.8 +/- 2.9 nM, P less than 0.03) but not of the high (0.22 +/- 0.05 nM) affinity sites. Under these same conditions the Bmax of both the high (82.4 +/- 14.5 fmol/mg protein, P less than 0.03) and low (6.1 +/- 1.7 pmol/mg protein, P less than 0.01) affinity binding sites was reduced but the sites retained their selectivity, with nifedipine displacing bound [3H]nitrendipine more potently than D600. Bay K 8644, when added upon reperfusion, promoted a dose-related increase in Ca2+ entry which was reduced by nifedipine, indicating that dihydropyridine binding sites can be activated after 60 min ischemia.

Dihydropyridine binding sites in aerobically perfused, ischemic, and reperfused rat hearts: effect of temperature and time.

The time course of normothermic (37°C) and hypothermie (22°C) global ischemia, and of post-ischemic reperfusion on the affinity (KD), selectivity, and density (Bmax) of the high-affinity 1,4 dihydropyridine (DHP) calcium channel antagonist binding sites were studied in isolated rat heart membranes, using (+)[3H]PN 200–110. Short periods (10 or 20 min) of ischemia at either 37°C or 22°C altered neither the KD nor Bmax of these binding sites as compared with aerobic controls. By contrast, longer periods of normothermic ischemia (30 or 60 min) caused a reduction (p < 0.05) in Bmax (36% after 30 min and 32.7% after 60 min) without change of KD. This reduction in Bmax was reversed by 15-min reperfusion at 37°C after 30 but not 60-min ischemia. There was no effect of ischemia (up to 60 min) on the density of DHP binding sites under hypothermie conditions. Irrespective of the experimental conditions the selectivity of the binding sites was maintained, with (+)PN 200–100 > (−)Bay K8644 > (−)PN 200–110 = (+)Bay K8644 ≫ (−)D600 in displacing (+)-[3H]PN 200–110. and D-cis diltiazem stimulating the binding. These results show that the ischemia-induced change in Bmax is specific, reversible, and time- and temperature-dependent.

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

Summary: 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 β1-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 < 0.001), dopamine (43%, p < 0.02), and 3.4-dihydroxyphenylethyleneglycol (30%, p < 0.02). Cardiac membranes prepared from rats maintained on standard (control) diet contained a single population of high affinity β1-adrenoceptor binding sites, with an affinity (Kd) of 0.24 ± 0.02 n/W and a density (Bmax) of 35.5 ± 19.5 fmol/mg protein. Including verapamil (dissolved in either ethanol or water) in the diet for ≤ 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 < 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 β1-adrenoceptor density during 6 weeks therapy.

CALCIUM ANTAGONISTS AND HYPERTENSION

1. Calcium antagonists, including verapamil, are now used widely in the management of patients with hypertension.

Effect of age and of hypertrophy on cardiac Ca2+ antagonist binding sites

We explored the effect of age and of hypertrophy on Ca2+ antagonist binding site density (Bmax), affinity (Kd), and selectivity in cardiac membranes harvested from the hearts of young adult (9-week-old) and older (25-week-old) Sprague Dawley (SD) rats, Wistar Kyoto rats (WKY), and spontaneously hypertensive rats (SHR). Radioligand binding studies with (+)[3H]PN200–110 failed to show a significant difference between the Bmax obtained for the cardiac membranes of 9-week-old SD, WKY, or SHR. Similarly, at 25 weeks, the Bmax values were the same for each group, but in each group the Bmax tended to increase with age. The Kd and selectivity were unchanged. For (−)[3H]D888 binding, the Kd values changed with age, but there was no hypertension or hypertrophy-linked increase in Bmax In the SD and SHR series, but not in the WKY, there was a tendency for the Bmax to increase with age. We interpreted these results to mean that age may contribute to the different Kd and Bmax values described for cardiac membranes from 25-week-old WKY and SHR.

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.

Calcium Channels and the Heart

Calcium ions play a key role in muscle contraction. In the resting state the cytosolic Ca2+ is maintained at a concentration of around 10-7 M, but upon excitation this level increases by a factor of 100 or more. In cardiac muscle this increase involves an influx of Ca2+ through the voltage-sensitive Ca2+ -selective channels which traverse the sarcolemma and a secondary release of intracellularily stored Ca2+ (Fabiato and Fabiato 1979). The structure, biochemistry, ionic selectivity and voltage regulation of these trans-sarcolemmal channels have been the subject of previous papers in this symposium, as has their isolation and reconstitution.