G protein expression and second messenger formation in human granulosa cells.
The expression of heterotrimeric (alpha beta gamma subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express alpha s, alpha i3 alpha i1,2, alpha q,11 and beta subunits. Three antibodies against alpha o failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of G alpha s. The alpha 2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a Gi-type protein, most likely G alpha i2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of G alpha q,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating Gi-mediated phospholipase C activation (by either alpha i or beta gamma subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of G alpha s:G alpha i activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both Gq,11 and Gi pathways.
López Bernal
A
A
Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford, UK.
Bellinger
J
J
Marshall
J M
JM
Phaneuf
S
S
Europe-Finner
G N
GN
Asbóth
G
G
Barlow
D H
DH
D002118:Calcium / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003000:Clonidine / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010121:Oxytocin / Q000494:pharmacology; D037342:Pertussis Toxin; D015290:Second Messenger Systems; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D006063:Chorionic Gonadotropin; D010566:Virulence Factors, Bordetella; D010121:Oxytocin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015232:Dinoprostone; D003000:Clonidine; D002118:Calcium