Human red‐blood‐cell Ca2+‐antagonist binding sites

Abstract

The human red blood cell ghost Ca²⁺-antagonist binding sites were characterized with (±)-[³H]nimodipine. The labelled 1,4-dihydropyridine bound in a non-cooperative, reversible manner with a K_d of 52 nM at 25°C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4-dihydropyridines. In contrast to the 1,4-dihydropyridine-selective receptors on Ca²⁺ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (−) enantiomer of the benzoxadiazol 1,4-dihydropyridine (PN 200–110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [³H]nimodipine-labelled sites also interacted with the inorganic Ca²⁺-antagonist La³⁺ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine-type Ca²⁺-antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d- or l-cis-diltiazem were without effect. Nucleosides (adenosine ∼ inosine > cytidine) were inhibitory at the nimodipine-labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [³H]nimodipine binding was irreversibly lost. (+)-Nimodipine > (−)-nimodipine inhibited [¹⁴C]adenosine transport into human red blood cells. A good correlation between IC₅₀ values for inhibition of [³H]nimodipine binding and IC₅₀ values for inhibition of [¹⁴C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [³H]nimodipine binding sites. It is concluded that the Ca²⁺-antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.