Xin Hua Gu

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.

Alpha1 adrenoceptors in the ischaemic and reperfused myocardium

The [3H]-prazosin binding activity of membranes isolated from aerobically perfused, ischaemic and reperfused cat and rat hearts was investigated. Alpha 1 adrenoceptors in membranes from aerobically perfused cat hearts had a KD of 0.363 +/- 0.067 nM and a Bmax of 68.8 +/- 7.0 fmol/mg protein; 30 min normothermic global ischaemia caused an increase (P less than 0.01) in density (Bmax, 111.4 +/- 9.3 fmol/mg protein), without any change in affinity (KD 0.430 +/- 0.069 nM). Post-ischaemic reperfusion (15 min) caused the Bmax to return to control values, with no change in KD. Membranes isolated from aerobically perfused rat hearts contained a single population of high affinity alpha 1 adrenoceptor binding sites, with a KD of 0.092 +/- 0.02 nM and a Bmax of 43.02 +/- 2.49 fmol/mg protein. Neither global nor low-flow (0.1 ml/min) ischaemia for either 30 or 60 min, nor post-ischaemic reperfusion, caused any change in either the affinity or density of these binding sites. In both species, and under all the above experimental conditions, the selectivity of the alpha 1 adrenoceptor binding sites was maintained, with phentolamine much greater than rauwolscine in displacing bound [3H]-prazosin. These results show that the ability of ischaemia to increase alpha 1 adrenoceptor density in cardiac membranes is species specific, and that it can occur as a direct (as opposed to a reflex) response to ischaemia.

Cyclosporine increases endothelin-1 binding site density in cardiac cell membranes

When used in high concentrations, the immunosuppressive agent cyclosporine A has toxic side effects, including cardiac fibrosis and calcification. Cyclosporine-treated mice were used to investigate whether a cyclosporine dose regime which produces cardiac fibrosis and calcification alters the density of the specific binding sites for the endothelial-derived vasoconstrictor polypeptide, endothelin-1. After twenty-one days of treatment the endothelin-1 binding site density in cardiac cell membranes was increased, without any change in selectivity. This increase in endothelin-1 binding site density could contribute to the cytotoxic effects of cyclosporine.

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.

(-)[3H]amlodipine binding to rat cardiac membranes.

Amlodipine is a newly developed long-acting dihydropyridine-based calcium antagonist. To characterize the binding properties of this compound, saturation binding studies were undertaken, using (-)[3H]amlodipine and rat cardiac membrane fragments. (-)[3H]Amlodipine bound to a single population of high-affinity binding sites with a KD of 1.68 +/- 0.12 nM, a Bmax of 0.34 +/- 0.08 pmol/mg protein, and a Hill coefficient approaching unity. Binding required up to 5 h to reach asymptote, and was pH- and temperature-sensitive. The specific binding was totally inhibited by (-) amlodipine and (-) D600 (IC50 values of 9.20 +/- 5.56 and 6.58 +/- 6.57 nM, respectively) and only partially inhibited by (+) PN 200-110, (-) Bay K 8644, (+) D600, and d-cis diltiazem (IC50 values of 60 +/- 10, 160 +/- 20, 250 +/- 40, and 200 +/- 30 nM, respectively). These results indicate that in addition to its ability to bind to the dihydropyridine and benzothiazepine recognition sites in rat cardiac membrane fragments, (-)[3H]amlodipine also binds strongly to the recognition sites for the phenylalkylamine-based calcium antagonists. The results also show that the inhibition of (-)[3H]amlodipine binding by D600 is stereospecific with (-) greater than (+)D600. Dissociation of bound (-)[3H]amlodipine was slowed under acidotic (pH 6.0) and accelerated under alkalotic (pH 10.0) conditions.

The inhibitory effect of [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P on endothelin-1 binding sites in rat cardiac membranes

The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments.

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.

Effect of amlodipine pretreatment on ischaemia-reperfusion-induced increase in cardiac endothelin-1 binding site density.

Summary: Endothelin-1 (ET-1) may be implicated in the pathophysiology of myocardial ischaemia. To determine whether the long-acting calcium antagonist amlodipine attenuates the ischaemia- and reperfusion-induced increase in cardiac ET-1 binding sites, hearts from rats pretreated with amlodipine (0.25 or 0.5 mg/kg) 2 or 5 h before they were killed were made ischaemic for 20 or 40 min, reper-fused, and subfractionated. Twenty- and 40-min ischaemia caused a time-dependent increase in ET-1 binding site density (Bmax) identified with [125I]ET-1. Amlodipine pretreatment attenuated this increase in a time- and dose-dependent manner. 0.25 and 0.5 mg/kg amlodipine also suppressed the reperfusion-induced increase in [125I]ET-1 binding site density, even when the 0.5-mg/kg pretreatment series reperfusion was administered after 40-min ischaemia.

Sarafotoxin S6c is a relatively weak displacer of specifically bound 125I-endothelin.

Sarafotoxin S6a, S6b and S6c are chemically related vasoconstrictor polypeptides obtained from the venom of the snake, Atractaspis engaddensis. Each contains twenty one amino acid residues, two intrachain cysteine linkages and a long hydrophobic tail. Structurally these polypeptides resemble endothelin. Binding studies with 125I-endothelin showed that 125I-endothelin bound to rat ventricular membranes is totally displaceable by sarafotoxin S6b and endothelin, with IC50 values of 0.21 and 0.16 nM, respectively. Sarafotoxin S6c, which differs from sarafotoxin S6b in containing threonine instead of serine at residue 2, arginine instead of lysine at residue 4, and glutamic acid instead of lysine at residue 9, only weakly displaced bound 125I-endothelin (IC50, 854 nM). These results indicate that the ability of the sarafotoxins to interact with the endothelin binding site is not solely dependent on the long hydrophobic tail or the cysteine linkages.

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.