Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils.
Aggregation of human platelets induced by a variety of agonists was inhibited by 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dionel (U-73122) (IC50 values 1-5 microM), but not by the close analog 1-[6-[[17 beta-3-methoxyestra- 1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343) in which pyrrolidinedione was substituted for pyrroledione. Inhibition by U-73122 was not mediated by an increase in intracellular cyclic AMP. In contrast, the production of inositol 1,4,5-trisphosphate (IP3) and the subsequent rapid increase in cytosolic Ca++ induced by either thrombin or the thromboxane-mimetic, (5Z,9 alpha, 11 alpha, 13E, 15S) 15-hydroxy-11,9-(epoxymethano)prosta- 5,13,-dien-1-oic acid (U-46619), was inhibited by U-73122 but not by U-73343. Reduction of IP3 levels appeared to reflect an inhibition of IP3 production because the hydrolysis of phosphatidyl[3H]inositol and phosphatidyl[3H]inositol 4,5-bisphosphate catalyzed by a soluble fraction from platelets was inhibited by U-73122 (Ki = 9 and 40 microM, respectively). In addition, U-73122 inhibited thromboxane B2 production induced by collagen but not that supported by exogenously added arachidonic acid, suggesting that U-73122 also inhibited receptor-coupled mobilization of arachidonic acid. After preincubation of platelets with [3H]arachidonic acid, the loss of [3H]phosphatidylinositol and accumulation of [3H]phosphatidic acid induced by thrombin was attenuated by U-73122. U-73122 did not inhibit the activities of phospholipases A2 purified either from porcine pancreas or from the venoms of Crotalus adamanteus and Naja naja. Although U-73122 inhibited neither the conversion of exogenous arachidonic acid to thromboxane B2 nor the binding of the thromboxane receptor antagonist [1S-[1 alpha, 2 beta (5Z), 3 beta, 4 alpha]]-7-[3-[[2- [2-[(phenylamino)-carbonyl]- hydrazino]methyl]-7-oxabicyclo [2.2.1]-hept-2-yl-5-heptenoic acid to platelet membranes, it was an effective inhibitor of arachidonic acid-induced aggregation of platelets. These data are consistent with the observed inhibition by U-73122 of platelet activation by the thromboxane receptor agonist, U-46619, via a mechanism that involves inhibition of a phospholipase C-dependent component(s) of signal transduction. U-73122, but not U-73343, inhibited also N-formyl-methionyl-leucyl-phenylalanine-induced aggregation of human polymorphonuclear neutrophils (PMN) and the associated production of IP3 and diacyglycerol. Diradylglycerol produced in PMN stimulated with N-formyl- methionyl-leucyl-phenylalanine was 74 +/- 7% saponifiable and inhibited by U-73122 (Ki = 2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)
Bleasdale
J E
JE
Department of Lipids Research, Upjohn Company, Kalamazoo, Michigan.
Thakur
N R
NR
Gremban
R S
RS
Bundy
G L
GL
Fitzpatrick
F A
FA
Smith
R J
RJ
Bunting
S
S
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology*; D011982:Receptors, Prostaglandin / Q000187:drug effects*; D017482:Receptors, Thromboxane; D013329:Structure-Activity Relationship; D013928:Thromboxane A2 / Q000096:biosynthesis; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D001095:Arachidonic Acids; D004075:Diglycerides; D004963:Estrenes; D007295:Inositol Phosphates; D010975:Platelet Aggregation Inhibitors; D011760:Pyrrolidinones; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D016718:Arachidonic Acid; D013928:Thromboxane A2; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010738:Type C Phospholipases; D002118:Calcium