Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by betagamma subunits of heterotrimeric G-proteins.

Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629-637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5'-[gamma-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6((2-13))] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1((2-17)) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF(-)(4) and this inhibition was counteracted by the fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1 (495-689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein betagamma-subunit scavengers. It is concluded that G-protein subunits betagamma are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins.

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.

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.

ETA receptors mediate activation of phospholipases C and D in rat myometrium.

In estrogen-treated rat myometrium, endothelin-1 (ET-1) activated both the phospholipase C (PLC) which degrades PtdInsP2, resulting in an increased accumulation of inositol phosphates, and the phospholipase D pathway (PLD) as evidenced in the presence of butanol by an increased production of phosphatidylbutanol (PBut). Both ET-1 effects displayed similar concentration dependencies (EC50 50 nM) and were mediated by ET(A) receptors in that they were antagonized by BQ123 and were elicited by ET-3 with a rank order of potency ET-1 >> ET-3. Bombesin, another activator of the PLC/PtdInsP2 pathway, also increased PBut accumulation. Enhanced production of PBut could also be observed with the Ca2+ ionophore ionomycin and the phorbol ester PMA, an activator of protein kinase C, suggesting a potential contribution of the PLC/PtdInsP2 pathway in ET-1 induced PLD activity.

Myosin light chain phosphorylation in intact rat uterine smooth muscle. Role of calcium and cyclic AMP.

Myometrial strips from oestrogen-primed rat uterus were exposed to various treatments, isometric contraction was measured, and the extent of myosin light chain phosphorylation determined after rapid freezing in liquid nitrogen. Two-dimensional electrophoresis revealed five spots having the same molecular weight as the light chain, with isoelectric points comprised between 5.15 and 4.95. Two of these spots (pI 5.09 and 5.00) were not present in pure uterine myosin, whether prepared from incubated or nonincubated tissue; they do not represent light chain isoforms or electrophoresis artefacts but rather degradation products appearing during the treatment. Two spots (pI 5.15 and 5.06) were identified as the nonphosphorylated and the phosphorylated forms of the light chain. The fifth minor spot (pI 4.95) may represent a diphosphorylated myosin species. Strips incubated in a normal Ca2+-medium 0.8 mM) exhibited basal contractions and an incorporation of 0.2 mol phosphate per mol light chain. Removal of Ca2+ resulted in almost complete dephosphorylation, coincident with a total relaxation of the muscle. Exposure of the myometrium to carbachol caused tetanic contractions with an increase to 0.5 mol phosphate per mol light chain. Isoproterenol, a beta-adrenergic agonist elevated intracellular cyclic AMP and induced uterine relaxation. Addition of isoproternol to a resting myometrium caused a slight but significant decrease in phosphorylation; its addition prior to carbachol markedly prevented the increase in myosin phosphorylation normally induced by the cholinergic effector. Forskolin (1 microM) increased intracellular cyclic AMP, caused relaxation and a concomitant decrease in basal myosin phosphorylation. Prostaglandin E2-induced elevation in intracellular cyclic AMP was however accompanied by an increase in contraction together with an increase in light chain phosphorylation. The data imply that light chain phosphorylation-dephosphorylation, regulated by Ca2+-dependent mechanisms, is essential for both uterine contraction and relaxation but question the role of cyclic AMP in exclusively mediating relaxation and myosin dephosphorylation in intact myometrium.

Differential role of microtubules in the control of prostaglandin E2 and beta-adrenergic stimulation of cyclic AMP accumulation in the rat myometrium.

A possible modulatory role of microtubules was investigated for the beta-adrenergic and prostaglandin E2 (PGE2)-induced cyclic AMP accumulation in the estrogen-treated rat myometrium. Colchicine, vinblastine and nocodazole, three different antitubulin drugs, enhanced cyclic AMP accumulation caused by PGE2. The effect of inhibitors was dose-(0.1-5 microM) and time-dependent, increased maximal responses without changing EC50 for PGE2, did not occur with trimethyl-colchicinic acid, which does not bind to tubulin, and was totally prevented in tissues pretreated with taxol, an agent that enhances polymerization and stabilization of microtubules. Concomitantly, colchicine reduced the rate and extent of PGE2-induced refractoriness in terms of cyclic AMP. In contrast, the antitubulin drugs failed to affect the rise in cyclic AMP evoked by isoproterenol and cholera toxin but enhanced the response to prostacyclin (PGI2), which is assumed to share common receptors with PGE2. Colchicine and vinblastine also failed to alter the ability of the beta-adrenergic agonist to provoke a cyclic AMP refractory state. Stimulations induced by all effectors were totally insensitive to cytochalasin B. The data suggest a relation between the constraints associated with the microtubules and/or membrane tubulin of the myometrium and the efficacy of PGE2 and PGI2 (but not the beta-adrenergic agonist or cholera toxin) to interact with the cyclic AMP forming system.