Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production

Abstract

We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose-and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A 2 (PLA 2 ) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [ 14 C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180 12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA 2 inhibitors, decreased the hormone-stimulated AA release to 85 9 and 70 24% respectively. Conversely, melittin, a PLA 2 stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTP S, a non-hydrolyzable analog of GTP. Results demonstrate that GTP S was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G i family was involved in the hCG-dependent release of AA. Cells treated with PLA 2 inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of PLA 2 and G proteins in the release of AA from hCG-stimulated Leydig cells, and under particular conditions, regulation of cAMP production by this fatty acid in these cells.

The involvement of epoxygenase metabolites of arachidonic acid in cAMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression

“…It is widely accepted that the LH stimulation of Leydig cells induces the formation of the second messenger cAMP, which activates protein kinase A (PKA), and leads to the phosphorylation of several transcription factors that regulate StAR gene transcription (Reinhart et al 1999). Previous studies also indicated that LH and cAMP induce arachidonic acid (AA) release (Cooke et al 1991, Moraga et al 1997, Finkielstein et al 1998, Ronco et al 2002, Wang et al 2002. After the release of AA, its metabolic derivatives transduce signals to the nucleus to regulate StAR gene expression and steroidogenesis (Cooke 1999, Wang et al 2000.…”

Cyclooxygenases in Rat Leydig Cells: Effects of Luteinizing Hormone and Aging

“…In turn, arachidonic acid and/or its metabolites have been proposed to be involved in modulating the acute effects of LH on steroidogenesis (12)(13)(14)(15)(16). Arachidonic acid can be metabolized in vivo either to prostaglandins by cyclooxygenases (COX1, COX2) or into 5-hydroxyeicosatetraenoic acid and 5-hydro-peroxyeicosatetraenoic acid by 5-lipoxygenase.…”

Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid

“…Both OTR concentrations and PKC activity were higher in the non-pregnant horns of unilaterally pregnant ewes on day 16 after oestrus than they were in the pregnant horns of the same sheep (Abayasekara et al 1995), which is consistent with oxytocin-induced production of PKC activators such as diacylglycerol or arachidonic acid. Control of arachidonic acid release from cells through endocrine or immune stimulation is consistent with paracrine or autocrine functions of fatty acids in other tissues (Zheng et al 1999, Ronco et al 2002.…”