In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding.

Abstract

Agonists stimulate guanylyl 5'-[y-[35S] (23,24). More recently, this technique has been used in isolated membranes to measure the activation of G proteins by specific receptors (25,26). These results demonstrated that significant agonist stimulation of GTP [.y-35S] binding occurred in membranes only in the presence of relatively large concentrations (3-10 ,uM) of GDP. Such concentrations of GDP were necessary to inactivate G proteins, so that stimulation of GTP[_y-35S] binding over basal levels could be observed when agonists were added to membrane preparations. We now report the development of an in vitro anatomical technique by which receptor-activated G proteins can be identified autoradiographically by using GTP[y-35S] binding to tissue sections. The present study compares the autoradiographic distribution of GTP[y35S] binding after stimulation by agonists for three different receptors, which numerous studies have identified as typical members of the G-protein-coupled receptor superfamily: ,u opioid (27), cannabinoid (28), and type B y-aminobutyric acid receptor (GABAB) (29).

Upregulation of CB1 receptors and agonist-stimulated [35S]GTPγS binding in the prefrontal cortex of depressed suicide victims

“…27 The results of the present study suggest greater CB 1 receptor-stimulated [ 35 31 This is borne out from direct comparison between the efficacy of cannabinoids and opiates in which opiates signaling was found to be 20-fold more efficient than cannabinoid signaling. 32 Therefore, it is speculated that the CB 1 receptor signaling functions as a subtle, fine-tuning mechanism for cells. The high density of receptors makes the CB 1 receptor highly sensitive to agonists; however, the poor coupling efficiency ensures that overactivation of the system will not occur.…”

Adenylyl cyclase type 5 (AC5) is an essential mediator of morphine action

“…Opioid-stimulated [␥- 35 S]GTP binding assay was performed as described in ref. 46. Striatal membranes (10 g) were incubated at 30°C for 1 h in assay buffer (50 mM Tris⅐HCl, pH 7.4͞3 mM MgCl 2 ͞0.2 mM EGTA͞100 mM NaCl) with 10 M GDP, 0.05 nM [␥- 35 S]GTP, and 10 M DAMGO.…”

Inverse Agonists and Serotonergic Transmission From Recombinant, Human Serotonin (5-HT)1B Receptors to G-Protein Coupling and Function in Corticolimbic Structures in vivo

“…In the substantia nigra, employing a procedure based on that detailed by Sim et al (1995), a pronounced stimula- In contrast to the present study, in which the sections were extensively washed to ensure removal of endogenous 5-HT, Stanton et al (1997) (Dupuis et al 1998). …”

“…First, we examine binding of the GTP analogue, [ Thomas et al 1995). Second, we discuss the binding of [ 35 S]-GTP ␥ S to native rat brain h5-HT 1B receptors visualized by quantitative autoradiography (Sim et al 1995). Third, we consider the modulation of extracellular 5-HT levels as determined by dialysis of the FCX of freely moving rats ).…”