A GppNHp‐insensitivity factor modulates the activation of β‐adrenoceptor‐coupled Gsprotein in rat cortex and cerebellum

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Abstract
Abstract—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 β‐adrenoceptors ((βAR) in cultured rat brain astrocytes. In contrast, a proportion of βAR 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 Gsproteins and not present in a functional form in cultured astrocytes. Here we assessed the effect of this factor on the βAR‐mediated activation of Gsproteins. 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 Gsproteins was monitored by the binding of another non‐hydrolysable GTP‐analog, guanylyl 5′‐[γ‐[35S]thio]‐triphosphate ([35S]GTPγS). The βAR‐Gsprotein 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 Gsproteins 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.