A GppNHp-insensitivity factor modulates the activation of beta-adrenoceptor-coupled Gs protein in rat cortex and cerebellum.
The effect known as the GTP-shift refers to the complete conversion of receptors from the high- to the low agonist-affinity state in the presence of an excess of GTP or one of its analogs. 5'-Guanylylimidodiphosphate (GppNHp) was able to fully suppress the high (-)-isoproterenol-affinity of beta-adrenoceptors (beta AR) in cultured rat brain astrocytes. In contrast, a proportion of beta AR in rat cortex and cerebellum synaptosomes was found to be insensitive to this GTP analog. This GppNHp-insensitivity was due to a membrane-associated factor, presumably interacting with Gs proteins and not present in a functional form in cultured astrocytes. Here we assessed the effect of this factor on the beta AR-mediated activation of Gs proteins. The removal of the GppNHp-insensitivity factor from the synaptosomes was achieved using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a mild detergent. The activation of Gs proteins was monitored by the binding of another non-hydrolysable GTP-analog, guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTP gamma S). The beta AR-Gs protein coupling was at least twofold less efficient in synaptosomes relative to cultured astrocytes. The CHAPS treatment induced a twofold increase in the coupling efficiency in cortex and cerebellum synaptosomes, but had no effect in cultured astrocytes. It undoubtedly indicated the inhibitory effect of the GppNHp-insensitivity factor on the activation of Gs proteins in the synaptosomes. Using CHAPS-soluble material extracted from synaptosomes, it was possible to reconstitute the GppNHp-insensitivity of CHAPS-treated membranes or even to induce it in cultured astrocytes. This effect correlated with the amount of CHAPS-soluble material according to a sigmoid curve, but it was abolished by the heat of CHAPS-soluble material. Successful crossed reconstitutions of the GppNHp-insensitivity suggest that the GppNHp-insensitivity factor is the same regardless of its originating area, and that it might play a general role in the central nervous system. Further investigations should help to identify the GppNHp-insensitivity factor.
Garnier
V
V
Département de Pharmacologie, Faculté de Médecine de Paris XII, Créteil, France.
Zini
R
R
Tillement
J P
JP
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D002793:Cholic Acids / Q000494:pharmacology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D011412:Propanolamines / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D002793:Cholic Acids; D011412:Propanolamines; D011943:Receptors, Adrenergic, beta; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011433:Propranolol; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C033394:CGP 12177