Chronic Exposure of NG 108‐15 Cells to Opiate Agonists Does Not Alter the Amount of the Guanine Nucleotide‐Binding Proteins Gi and GO

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Abstract
We have characterized the pertussis toxin substrate in NG 108–15 cell membranes using site‐specific antisera and ADP‐ribosylation. Cell membranes contain two pertussis toxin‐sensitive guanine nucleotide‐binding protein α‐subunits (Gα) whose Rf values in gel electrophoresis coincide with those of Gαo and Gαi2. The total quantity of Gi and Go im‐munoreactivity amounted to 24.3 ± 2.8 pmol/mg, whereas only 1.5 ± 0.2 pmol/mg are capable of undergoing ADP‐ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D‐Ala2,D‐Leu2]‐enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP‐ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP‐ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down‐regulation and desensitization of the γ‐opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between α‐and β‐subunits of Gi and/or Go.