[Gamma-35S]GTP autoradiography allows region-specific detection of muscarinic receptor-dependent G-protein activation in the chick optic tectum.
A recently introduced technique of [gamma-35S]GTP autoradiography was used to localize and characterize muscarinic receptor-dependent activation of G-proteins in tissue sections of the chick optic tectum, a brain region with relatively high expression of G-protein-coupled receptors for the neurotransmitter acetylcholine. Within the highly stratified tectal structure, the bulk of muscarinic receptor-mediated [gamma-35S]GTP signal was localized to the stratum griseum et fibrosum superficiale with considerably lower binding responses in other tectal layers. Quantitative comparison of [gamma-35S]GTP binding responses in tectal sections and membranes revealed a close match between the two tissue preparations for the response elicited by the cholinergic agonist carbachol, its dose-dependent reversal with the non-selective muscarinic antagonist atropine, its approximately 100-fold sensitivity towards blockade with M1-type (pirenzepine) over M2-type (gallamine) muscarinic antagonists, as well as absolute requirement for micromolar concentrations of GDP (EC50 approximately 10 microM) for the receptor-mediated [gamma-35S]GTP response. The pharmacological profile is consistent with that of cm4, a recently cloned chicken homolog of the mammalian m4 muscarinic acetylcholine receptor. Moreover, the strict GDP-dependence of the binding response suggest activation of Gi/o, the inhibitory class of G-proteins. These data provide the first functional characterization of the chick tectal muscarinic receptors. A close match between [gamma-35S]GTP responses in membranes and tissue sections strongly suggest that [gamma-35S]GTP autoradiography offers great potential for studies on G-protein-mediated signaling, with particular use within anatomically restricted regions that are not readily approached with more conventional techniques. It is anticipated that [gamma-35S]GTP autoradiography should greatly facilitate studies on signaling capacity of an individual receptor subtype whilst in its native cellular environment.
Kurkinen
K M
KM
Department of Physiology, University of Kuopio, Finland.
Koistinaho
J
J
Laitinen
J T
JT
D000818:Animals; D000831:Animals, Newborn; D001345:Autoradiography; D002217:Carbachol / Q000494:pharmacology; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000502:physiology*; D013462:Sulfur Radioisotopes; D013477:Superior Colliculi / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution
D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D013462:Sulfur Radioisotopes; D006153:Guanosine Diphosphate; D010890:Pirenzepine; D006160:Guanosine Triphosphate; D002217:Carbachol; D019204:GTP-Binding Proteins