SK&F 96365, a novel inhibitor of receptor-mediated calcium entry

Abstract

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)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&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&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&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&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&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&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&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&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.

Inhibition of lipopolysaccharide‐induced cyclooxygenase‐2, tumor necrosis factor‐α and [Ca2+]i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195

“…However, SKF96365 had no effects in blocking the LPS-induced entry of Ca 2+ indicating that SOC were not involved. Although SKF96365 shows non-selective actions in tissue (Merritt et al 1990), this compound (at 50 lM) inhibits SOC in a variety of cells including microglia. In terms of the intracellular component of the LPS response, the endotoxin, added following application of ATP in Ca 2+ -free solution, was still able to elicit an increase in [Ca 2+ ] i .…”

Role of endothelial Ca2+ stores in the regulation of hydraulic conductivity of Rana microvessels in vivo

“…To exclude the effect of Ni 2ϩ on other mechanisms of Ca 2ϩ regulation a specific cation channel blocker, 100 M SKF (17,28), was used instead of Ni 2ϩ . Vessels were perfused with TG and SKF for 20 min once a baseline with 1% BSA had been established.…”

Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium

“…Barium is not a substrate for Ca# + -ATPase [39], and cannot easily be extruded from the cells or sequestered. However, barium entry was inhibited by SKF96356, an inhibitor of store-operated calcium entry [40]. The above results thus exclude a direct inhibitory effect of thimerosal on the calcium entry pathway.…”

“…In Figure 4, it is clearly seen that no difference in the entry of barium could be detected between control cells and cells treated with 100 µM thimerosal. For comparison, the calcium-entry inhibitor SKF96365 [40] was tested. This compound almost totally inhibited barium entry (Figure 4).…”