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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.

H. Stunff,Lien Dokhac,Simone Harbon

Published 2000 in Journal of Pharmacology and Experimental Therapeutics

ABSTRACT

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.

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