Adenosine inhibition of the hormonal response in the Sertoli cell is reversed by pertussis toxin.
The rat Sertoli cell in culture expresses A1 inhibitory adenosine receptors. In this study, we have used pertussis toxin as a tool to characterize the mechanism of action of adenosine on these cells. Cells were preincubated for 18-24 h with pertussis toxin, and the responses to FSH and to the adenosine analog phenylisopropyladenosine (PIA) were measured by assaying cAMP accumulation. The effect of toxin on adenosine receptors was also evaluated by measuring binding of the adenosine agonist cyclohexyladenosine (CHA). The total number of specific CHA-binding sites was reduced 60-70% in membranes prepared from cells cultured for 24 h in the presence of pertussis toxin; the binding sites remaining after treatment displayed no apparent change in affinity for [3H]CHA. The effect of guanine nucleotides on CHA binding was also reduced after toxin pretreatment, but not abolished. PIA inhibited FSH-stimulated cAMP accumulation by 70-80%. Maximal inhibition was observed at a concentration of 10 nM PIA, and the ED50 of the dose-response curve was 1 nM. Pretreatment of the Sertoli cell with pertussis toxin completely blocked the PIA inhibition. The pertussis toxin effect was time and dose dependent. Reversal of the inhibition was observed after 6 h of treatment with a maximal dose of toxin (100 ng/ml). The dose of toxin producing a half-maximal effect was 10-30 ng/ml. In addition to this blockade of purine nucleotide inhibitory effects, exposure of the Sertoli cell to pertussis toxin concentrations ranging from 1-400 ng/ml consistently led to a potentiation of the FSH response measured as cAMP accumulation. In cell-free preparations (crude particulate fraction of the Sertoli cells, or sucrose gradient-purified plasma membranes), pertussis toxin catalyzed the incorporation of [32P]ADP ribose into a polypeptide with a molecular mass of 40-41 K. This peptide had electrophoretic mobility similar to that of a partially purified guanine nucleotide-binding protein (Gi). These data indicate that adenosine A1 inhibitory receptors are coupled to an inhibitory component (Gi) of adenylate cyclase. In the Sertoli cell, inhibitory and stimulatory signals interact in a bimodal regulation of adenylate cyclase and intracellular cAMP.
Monaco
L
L
Institute of Histology and General Embryology, University of Rome, Italy.
DeManno
D A
DA
Martin
M W
MW
Conti
M
M
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D037342:Pertussis Toxin; D010660:Phenylisopropyladenosine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011983:Receptors, Purinergic / Q000187:drug effects / Q000378:metabolism; D012708:Sertoli Cells / Q000187:drug effects / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D010455:Peptides; D011983:Receptors, Purinergic; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D010660:Phenylisopropyladenosine; D006165:Guanylyl Imidodiphosphate; C027513:N(6)-cyclohexyladenosine; D005640:Follicle Stimulating Hormone; D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000241:Adenosine