Inhibition of human platelet secretion and of Ca2+, calmodulin-dependent protein phosphorylation by the antiallergic agent GMCHA.

Calcium ion-dependent interaction with purified calmodulin (CaM), of a potent inhibitor of histamine release from mast cells, trans-4-guanidinomethylcyclohexanecarboxylic acid p-tertbutylphenylester (GMCHA), was investigated using 5-(dimethylamino)-1-naphthalenesulfonyl-calmodulin (dansyl-CaM). GMCHA undergoes a fluorescence increase with the Ca2+-dansyl-CaM complex but there is no significant change in the dansyl-CaM fluorescence with GMCHA, up to a 10 microM in the absence of calcium ion. This suggests that binding of GMCHA to CaM is Ca2+-dependent on the apparent Kd is approximately 1 microM. GMCHA suppressed the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS), in the presence of the Ca2+-CaM complex, with an IC50 value of 3 microM. [3H]N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) bound to the purified CaM was displaced, in a concentration dependent manner by GMCHA, the Ki value of GMCHA against the binding of W-7 to CaM was 2.3 microM, and there was a selective inhibition of the Ca2+-CaM induced activation of enzymes such as myosin light chain kinase. Increasing the CaM concentration in the presence of Ca2+ overcame the GMCHA-induced inhibition of myosin light chain kinase activation, with a Ki value of 2.7 microM. GMCHA at these concentrations is effective in inhibiting the release of histamine from mast cells. Moreover, this compound suppresses platelet secretion and relaxes vascular strips, at concentrations similar to those seen with the CaM interacting action and characteristic of CaM antagonists such as W-7. GMCHA also inhibits the Ca2+, CaM-dependent myosin light chain phosphorylation of human platelets. These results suggest that GMCHA, a potent inhibitor of histamine release from mast cells, suppresses platelet secretion, relaxes vascular smooth muscle and inhibits Ca2+, CaM-dependent protein phosphorylation, all at similar concentrations.