SK-AND-F-96365, A NOVEL INHIBITOR OF RECEPTOR-MEDIATED CALCIUM ENTRY

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK and F 96365 (1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2+-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK and F 96365 in platelets stimulated with ADP or thrombin was 8.5 μM or 11.7 μM respectively; these concentrations of SK and F 96365 did not affect internal Ca2+ release. Similar effects of SK and F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK and F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK and F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK and F 96365; however, the ATP-gated Ca2+-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK and F 96365. Thus SK and F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK and F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK and F 96365 with an IC50 of 15.9 μM, which is similar to the IC50 of 8-12 μM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK and F 96365. SK and F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK and F 96365 is not as potent (IC50 around 10 μM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.