Segment-dependent activation of muscarinic acetylcholine receptor-mediated [35S]Guanosine-5'-O-(gamma-thiotriphosphate) binding in airway tissue membranes.
Muscarinic acetylcholine receptor (mAChR)-mediated guanine nucleotide-binding regulatory protein (G protein) activation and the functional interaction between receptors and the respective G proteins were investigated using an agonist-induced [(35)S]guanosine-5'-O-(gamma-thiotriphosphate) ([(35)S]GTPgammaS)-binding approach in membranes of 3 native equine airway segments (trachea, bronchus and lung), which differ tremendously in mAChR density and subtype distribution; especially subtypes that couple negatively to adenylyl cyclase through G(i/0) proteins, i.e. M(2) receptors. The assay was initially optimized by determining the influence of incubation time, guanosine 5'-diphosphate (GDP), MgCl(2) and NaCl on basal and agonist-stimulated [(35)S]GTPgammaS binding. In standard assays, the presence of 10 mumol/l GDP, 10 mmol/l MgCl(2) and 200 mmol/l NaCl increased carbachol-induced specific [(35)S]GTPgammaS binding in a segment- and receptor-density-dependent manner. Hereby, mAChR agonists stimulated [(35)S]GTPgammaS binding with a rank order of potency (oxotremorine M > carbachol > acetylcholine), and in a specific segment- and receptor-density-dependent manner (trachea > bronchus > lung). The increase in the specific [(35)S]GTPgammaS binding was potently inhibited by the mAChR antagonist atropine. Pertussis toxin and N-ethylmaleimide as G(i/0) protein ADP-ribosylating and alkylating agents reduced basal and agonist-stimulated [(35)S]GTPgammaS binding. The mAChR-stimulated [(35)S]GTPgammaS binding serves as a useful method for investigating the functional interaction between mAChRs and their respective G proteins in native airway tissue membranes of equines.
Hajek
Peter
P
Institute of Pharmacology, Pharmacy and Toxicology, Leipzig University, Leipzig, Germany. gabraham@rz.uni-leipzig.de
Ungemach
Fritz Rupert
FR
Abraham
Getu
G
D000818:Animals; D001980:Bronchi / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D004347:Drug Interactions; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006736:Horses; D066298:In Vitro Techniques; D008168:Lung / Q000378:metabolism; D015636:Magnesium Chloride / Q000008:administration & dosage / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000187:drug effects*; D012137:Respiratory System / Q000187:drug effects / Q000378:metabolism*; D012965:Sodium Chloride / Q000008:administration & dosage / Q000494:pharmacology; D014018:Tissue Distribution; D014132:Trachea / Q000378:metabolism
D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D015636:Magnesium Chloride; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012965:Sodium Chloride; D002217:Carbachol