Detection of cannabinoid CB1, adenosine A1, muscarinic acetylcholine, and GABA(B) receptor-dependent G protein activity in transducin-deactivated membranes and autoradiography sections of rat retina.
1. Several G-protein-coupled receptors (GPCRs) have been localized to various layers of the vertebrate retina, using autoradiographic and immunohistochemical techniques, but the functional data concerning G protein activation are limited. Here, we establish optimized assay conditions to detect receptor-dependent G protein activity in membranes and tissue sections of the rat retina. 2. Agonist-stimulated [35S]GTPgammaS-binding responses were characterized for the Gi/o-linked adenosine A1, cannabinoid CB1, m2/m4 muscarinic acetylcholine, and GABA(B) receptors. Initial assumption was that G protein activity under "basal conditions" is high due to enrichment and activity of rhodopsin and transducin in this tissue. 3. We found that pretreatment of retina membranes with hydroxylamine (10 mM), a rhodopsin-inactivating drug, substantially (up to 60%) reduced basal G protein activity, thereby improving signal-to-noise ratio to detect agonist-stimulated G protein activation for all studied receptors. [35S]GTPgammaS autoradiography revealed that hydroxylamine specifically reduced basal binding in the transducin-enriched photoreceptor layer. In contrast, hydroxylamine did not affect GPCR signaling in brain membranes, indicating specific action on retinal transducin. 4. For all studied receptors, [35S]GTPgammaS autoradiography allowed localization of G protein activity to different retinal layers, with the bulk of signal detected in the ganglion cell layer. Strongest responses were observed for adenosine and muscarinic receptor agonists. Additional G protein activity was detected in the inner plexiform layer. 5. Responses to all tested agonists were reversed in the presence of appropriate receptor-selective antagonists, indicating receptor-mediated G protein activation.
Savinainen
Juha R
JR
Department of Physiology, University of Kuopio, Kuopio, Finland.
Laitinen
Jarmo T
JT
D000241:Adenosine / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D018756:GABA Antagonists / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019811:Hydroxylamine / Q000494:pharmacology; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D043682:Receptor, Adenosine A1 / Q000187:drug effects / Q000378:metabolism; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000378:metabolism*; D018080:Receptors, GABA-B / Q000187:drug effects / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism; D012160:Retina / Q000187:drug effects / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013462:Sulfur Radioisotopes; D015293:Transducin / Q000187:drug effects / Q000378:metabolism*
D018756:GABA Antagonists; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043682:Receptor, Adenosine A1; D043884:Receptor, Cannabinoid, CB1; D043562:Receptors, G-Protein-Coupled; D018080:Receptors, GABA-B; D011976:Receptors, Muscarinic; D013462:Sulfur Radioisotopes; D019811:Hydroxylamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D015293:Transducin; D000241:Adenosine