Activation of phospholipase D by endothelin-1 in rat myometrium. Role of calcium and protein kinase C.

In rat myometrium labeled with [3H]myristic acid, endothelin (ET)-1 via ET(A) receptors stimulated, in the presence of 0.3% butanol, the formation of [3H]phosphatidylbutanol ([3H]PBut) as a result of phospholipase D activity. Fluoroaluminates increased [3H]PBut generation, which indicated that a heterotrimeric G protein was involved. The ET-1 effect was insensitive to pertussis toxin and was rapidly desensitized. The calcium ionophore ionomycin as well as 4beta-phorbol 12-myristate-13-acetate and 4beta-phorbol 12,13-dibutyrate also stimulated [3H]P-But production. Protein kinase C (PKC) inhibition, particularly with Ro-31-8220, and down-regulation of PKC by 4beta-phorbol 12-myristate-13-acetate, abrogated 4beta-phorbol 12,13-dibutyrate responses but partially reduced (50%) ET-1 and ionomycin stimulatory effects. [3H]PBut production induced by ionomycin depended on Ca++ influx, whereas that induced by 4beta-phorbol 12,13-dibutyrate did not. Decrease of extracellular Ca++ partially reduced (60%) ET-1 stimulation that was additionally attenuated (75%) by chelerythrine, a PKC inhibitor. The data indicate that in myometrium, phospholipase D was activated by PKC and Ca++, which both contribute at least partially to ET-1-mediated phospholipase D activation.