The roles of protein kinase C and tyrosine kinases in mediating endothelin-1-stimulated phospholipase D activity in rat myometrium: differential inhibition by ceramides and cyclic AMP

The aim of the present study was to investigate the mechanisms that regulate the activation of phospholipase D (PLD) by endothelin (ET)-1 in rat myometrium. We previously reported that ET-1 exerted part ( approximately 50%) of its effect via protein kinase C (PKC) activation. We now show that in addition to ET-1 and 4beta-phorbol-12,13-dibutyrate (PDBu), pervanadate also stimulated PLD activity. Stimulation by pervanadate was not affected by the PKC inhibitor Ro-31-8220 but was abolished by protein tyrosine kinase (PTK) inhibitors genistein and tyrphostin-47. Genistein partially reduced (52%) ET-1 stimulation, which was further attenuated (96%) by Ro-31-8220, indicating that PTKs may account for the PKC-independent arm of ET-1-stimulated PLD activity. Cell-permeable ceramides reduced ( approximately 50%) the activation of PLD by ET-1 and PDBu but not that by pervanadate. Inhibition was also achieved by sphingomyelinase but not with sphingosine. Inhibition by genistein and D-erythro-N-hexanoyl-sphingosine was additive, whereas inhibition by Ro-31-8220 and D-erythro-N-hexanoyl-sphingosine was not, indicating that ceramide affected the PKC-dependent process involved in PLD activation by ET-1. Forskolin, as well as dibutyryl-cAMP and iloprost, attenuated (approximately 50%) the activation of PLD by ET-1 and pervanadate but not that by PDBu. Inhibition by forskolin was prevented by H-89, an inhibitor of protein kinase A. Inhibition by forskolin and ceramide was additive, whereas inhibition by genistein and forskolin was not, indicating that the cAMP/protein kinase A cascade affected the PTK-dependent process involved in PLD activation by ET-1. The data illustrate a cross-talk between separate signaling pathways, resulting in positive and negative regulation of PLD in rat myometrium.