Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production.
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 GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS 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.
Ronco
A M
AM
Unidad de Hormonas y Receptores, INTA, Universidad de Chile, Casilla 138-11, Santiago, Chile. amronco@uec.inta.uchile.cl
Moraga
P F
PF
Llanos
M N
MN
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008555:Melitten / Q000494:pharmacology; D037342:Pertussis Toxin; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D011796:Quinacrine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013268:Stimulation, Chemical; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000098:Acetophenones; D006063:Chorionic Gonadotropin; D004791:Enzyme Inhibitors; D010566:Virulence Factors, Bordetella; D008555:Melitten; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D037342:Pertussis Toxin; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; D011796:Quinacrine; C006821:4-bromophenacyl bromide