Regulation of guanine nucleotide turnover on Gi/Go by agonist-stimulated and spontaneously active muscarinic receptors in cardiac membranes.
Muscarinic receptor regulation of guanine nucleotide turnover on Gi/Go proteins in ventricular sarcolemma was investigated. In the absence of a muscarinic receptor (MR) agonist, GTP bound to background sites with a Kapp value of 60 nM and a Bmax of 50 pmol/mg. The addition of the MR agonist, carbachol, further increased GTP binding by 50 pmol/mg to sites with the same Kapp value of 60 nM. Pertussis toxin treatment reduced GTP binding to carbachol-regulated and background binding sites, thus identifying both sites as Gi/Go. The identity of the carbachol-regulated GTP binding sites was further confirmed by demonstrating that carbachol stimulated GTP binding and inhibited adenylyl cyclase with an EC50 value of 200 nM. Background and carbachol-regulated guanine nucleotide binding sites bound GDP with a Kapp value of 150 nM. However, maximal background GDP binding was 50 pmol/mg, whereas maximal carbachol-regulated GDP binding was only 12-15 pmol/mg. In sarcolemma preloaded with [3H]GDP, carbachol-regulated [3H]GDP release was strictly dependent on the presence of guanine nucleotides. The Kapp values for GTP and GDP to support carbachol-regulated [3H]GDP release were 60 nM and 150 nM, respectively. Guanosine 5'-O-(3-thiotriphosphate) (GDPbetaS) facilitated carbachol-regulated [3H]GDP release with a Kapp value of 2 microM. However, GTP was two times more efficacious than GDP or GDPbetaS in facilitating carbachol-regulated [3H]GDP release. Mn2+ also stimulated [3H]GDP release from carbachol-regulated sites by a mechanism not requiring guanine nucleotides. These studies indicate that two pools of muscarinic receptors, carbachol regulated and spontaneously active, regulate guanine nucleotide turnover on pertussis toxin sensitive Gi/Go. These studies further suggest that guanine nucleotide binding provides the signal to stimulate GDP release from receptor activated Gi/Go proteins. A quaternary mechanism involving G-protein interactions may be necessary to promote guanine nucleotide exchange on Gi/Go.
Quist
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E
Department of Pharmacology, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107, USA.
Satumtira
N
N
Vasan
R
R
D037361:Adenylate Cyclase Toxin; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002217:Carbachol / Q000378:metabolism / Q000494:pharmacology; D004285:Dogs; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism* / Q000502:physiology; D006153:Guanosine Diphosphate / Q000378:metabolism; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology*; D009206:Myocardium / Q000378:metabolism*; D037342:Pertussis Toxin; D011485:Protein Binding / Q000187:drug effects; D011976:Receptors, Muscarinic / Q000378:metabolism / Q000502:physiology*; D012508:Sarcolemma / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000378:metabolism / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D006150:Guanine Nucleotides; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D002217:Carbachol; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go