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

Abstract Calcium ion-dependent interaction with purified calmodulin (CaM), of a potent inhibitor of histamine release from mast cells, tran s-4-guanidinomethylcyclohexanecarboxylic acid p -tertbutylphenylester (GMCHA), was investigated using 5-(dimethylamino)-1-naphthalenesulfonyl-calmodulin (dansyl-CaM). GMCHA undergoes a fluorescence increase with the Ca 2+ -dansyl-CaM complex but there is no significant change in the dansyl-CaM fluorescence with GMCHA, up to a 10 μM in the absence of calcium ion. This suggests that binding of GMCHA to CaM is Ca 2+ -dependent on the apparent K d is approximately 1 μM. GMCHA suppressed the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS), in the presence of the Ca 2+ -CaM complex, with an ic 50 value of 3μM. [ 3 H ]N-(6- aminohexyl )-5- chloro -1- naphthalenesulfonamide (W-7) bound to the purified CaM was displaced, in a concentration dependent manner by GMCHA, the K i value of GMCHA against the binding of W-7 to CaM was 2.3 μM, and there was a selective inhibition of the Ca 2+ -CaM induced activation of enzymes such as myosin light chain kinase. Increasing the CaM concentration in the presence of Ca 2+ overcame the GMCHA-induced inhibition of myosin light chain kinase activation, with a K i , value of 2.7 μM. 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 Ca 2+ , 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 Ca 2+ , CaM-dependent protein phosphorylation, all at similar concentrations.