A ROLE OF DIACYLGLYCEROL KINASE IN STIMULUS-SECRET I ON COUPLING OF HUMAN PLATELETS -DISSOCIATION OF SEROTONIN SECRETION FROM Ca²⁺ MOBILIZATION-

Diacylglycerol (DG) kinase catalyzes the reaction: DG + ATP phosphatidic acid + ADP and it is widely distributed in animal tissues. The enzyme seems to play a pivotal role in removing a second messenger, DG, which activates protein kinase C. DG kinase inhibitor, R 59 022 (6-[2-[4-[(4-fluorophenyl)pheny1- methylene] -1-piperidinyl] ethyl] -7-methyl-5H-thiazolo [3,2-a] -pirimidin-5-one) has recently been developed. In order to gain further insight into the role of DG in the secretory response, effects of the DG kinase inhibitor on secretory responses and Ca²⁺ mobilization were investigated in human platelets.

The addition of the DG kinase inhibitor (10 μM) potentiated thrombin-induced accumulation of [³H]radioactivity of DG in platelets loaded with [³H] arachidonate. Thrombin-induced release of [³H] arachidonic acid and its metabolites was not affected by the inhibitor. The inhibitor did not cause significant secretion of [¹⁴C] serotonin by itself. However, the pretreatment with this agent potentiated the level of secretion in thrombin-stimulated platelets. When l-oleoyl-2-acetylglycerol(OAG) was added to [³²pjpi-iabeled platelets in the presence of the DG kinase inhibitor, the formation of [³²P] l-oleoyl-2-acetylphosphatidic acid was greatly prevented. The pretreatment with the inhibitor also potentiated OAG-induced serotonin secretion. With the view that Ca²⁺ is thought to be another important second messenger, we investigated the effect of the DG kinase inhibitor on Ca²⁺ mobilization. Two types of Ca2+ indicators, Quin2 and aequorin were used to measure cytosolic free Ca²⁺ concentration ([Ca²⁺]i). The inhibitor alone did not affect [Ca²⁺]i. Interestingly, thrombin-induced increase in [Ca²⁺]i was suppressed by the pretreatment with this agent both in the Quin2-loaded and aequorin-loaded platelets.

These results indicate that diacylglycerol kinase may operate as an attenuator in the signal transduction system involving protein kinase C and that Ca2+ mobilization may not be tightly coupled to serotonin secretion.