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In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding.
Proc Natl Acad Sci U S A. 1995 Aug 01; 92(16):7242-6.PN

Abstract

Agonists stimulate guanylyl 5'-[gamma-[35S]thio]-triphosphate (GTP[gamma-35S]) binding to receptor-coupled guanine nucleotide binding protein (G proteins) in cell membranes as revealed in the presence of excess GDP. We now report that this reaction can be used to neuroanatomically localize receptor-activated G proteins in brain sections by in vitro autoradiography of GTP[gamma-35S] binding. Using the mu opioid-selective peptide [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) as an agonist in rat brain sections and isolated thalamic membranes, agonist stimulation of GTP[gamma-35S] binding required the presence of excess GDP (1-2 mM GDP in sections vs. 10-30 microM GDP in membranes) to decrease basal G-protein activity and reveal agonist-stimulated GTP[gamma-35S] binding. Similar concentrations of DAMGO were required to stimulate GTP[gamma-35S] binding in sections and membranes. To demonstrate the general applicability of the technique, agonist-stimulated GTP[gamma-35S] binding in tissue sections was assessed with agonists for the mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyric acid type B (baclofen) receptors. For opioid and cannabinoid receptors, agonist stimulation of GTP[gamma-35S] binding was blocked by incubation with agonists in the presence of the appropriate antagonists (naloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrating that the effect was specifically receptor mediated. The anatomical distribution of agonist-stimulated GTP[gamma-35S] binding qualitatively paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that variations in coupling efficiency may exist between different receptors in various brain regions. This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins by specific receptors.

Authors+Show Affiliations

Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

7638174

Citation

Sim, L J., et al. "In Vitro Autoradiography of Receptor-activated G Proteins in Rat Brain By Agonist-stimulated Guanylyl 5'-[gamma-[35S]thio]-triphosphate Binding." Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 16, 1995, pp. 7242-6.
Sim LJ, Selley DE, Childers SR. In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding. Proc Natl Acad Sci U S A. 1995;92(16):7242-6.
Sim, L. J., Selley, D. E., & Childers, S. R. (1995). In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding. Proceedings of the National Academy of Sciences of the United States of America, 92(16), 7242-6.
Sim LJ, Selley DE, Childers SR. In Vitro Autoradiography of Receptor-activated G Proteins in Rat Brain By Agonist-stimulated Guanylyl 5'-[gamma-[35S]thio]-triphosphate Binding. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7242-6. PubMed PMID: 7638174.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding. AU - Sim,L J, AU - Selley,D E, AU - Childers,S R, PY - 1995/8/1/pubmed PY - 1995/8/1/medline PY - 1995/8/1/entrez SP - 7242 EP - 6 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc Natl Acad Sci U S A VL - 92 IS - 16 N2 - Agonists stimulate guanylyl 5'-[gamma-[35S]thio]-triphosphate (GTP[gamma-35S]) binding to receptor-coupled guanine nucleotide binding protein (G proteins) in cell membranes as revealed in the presence of excess GDP. We now report that this reaction can be used to neuroanatomically localize receptor-activated G proteins in brain sections by in vitro autoradiography of GTP[gamma-35S] binding. Using the mu opioid-selective peptide [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) as an agonist in rat brain sections and isolated thalamic membranes, agonist stimulation of GTP[gamma-35S] binding required the presence of excess GDP (1-2 mM GDP in sections vs. 10-30 microM GDP in membranes) to decrease basal G-protein activity and reveal agonist-stimulated GTP[gamma-35S] binding. Similar concentrations of DAMGO were required to stimulate GTP[gamma-35S] binding in sections and membranes. To demonstrate the general applicability of the technique, agonist-stimulated GTP[gamma-35S] binding in tissue sections was assessed with agonists for the mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyric acid type B (baclofen) receptors. For opioid and cannabinoid receptors, agonist stimulation of GTP[gamma-35S] binding was blocked by incubation with agonists in the presence of the appropriate antagonists (naloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrating that the effect was specifically receptor mediated. The anatomical distribution of agonist-stimulated GTP[gamma-35S] binding qualitatively paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that variations in coupling efficiency may exist between different receptors in various brain regions. This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins by specific receptors. SN - 0027-8424 UR - https://www.unboundmedicine.com/medline/citation/7638174/In_vitro_autoradiography_of_receptor_activated_G_proteins_in_rat_brain_by_agonist_stimulated_guanylyl_5'_[gamma_[35S]thio]_triphosphate_binding_ L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=7638174 DB - PRIME DP - Unbound Medicine ER -