Evidence that M3 muscarinic receptors in rat parotid gland couple to two second messenger systems

Y. Dai, I. S. Ambudkar, V. J. Horn, C. K. Yeh, E. E. Kousvelari, S. J. Wall, M. Li, R. P. Yasuda, B. B. Wolfe, B. J. Baum

Research output: Contribution to journalArticle

107 Scopus citations

Abstract

The binding affinities of muscarinic antagonists were compared with their abilities to block carbachol (CCh)-mediated stimulation of Ca2+ mobilization and inhibition of isoproterenol-elicited adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat parotid cells. The binding of [3H]quinuclidinyl benzilate (QNB) to membranes was inhibited by antagonists with the following potencies (dissociation constant, nM): atropine (1.1) ~ 4-diphenylacetoxy-N-methylpiperidine methbromide (4-DAMP) (1.6) >> pirenzepine (136) > 11-[[2-[(diethylamino)methyl-1-piperidinyl]-acetyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116) (5,293). AF-DX 116 blocked Ca2+ mobilization and inhibition of cAMP accumulation with low affinities [inhibitory concentration at 50% (IC50) = 3150 and 6,528 nM, respectively], whereas 4-DAMP blocked these responses with considerably higher affinities (IC50 = 4.3 and 11.4 nM, respectively). Schild plots of 4-DAMP and AF-DX 116 antagonism of CCh-stimulated inositol trisphosphate accumulation showed inhibitor constant (K(i)) values of 0.85 and 1,585 nM, respectively, whereas Schild plots of 4-DAMP, AF-DX 116, and methoctramine antagonism of CCh-induced inhibition of cAMP accumulation showed K(i) values of 1.3, 1,585, and 2,754 nM, respectively. Preincubation of cells with 0.1 mM 3-isobutyl-1-methylxanthine did not prevent the capacity of CCh to inhibit cAMP accumulation. Pertussis toxin blocked the CCh-elicited and G(i)-mediated inhibition of cAMP formation. Northern blot analysis showed the presence of mRNA for the M3, but not for the M2, subtype in parotid gland. An immunochemical procedure using m1-m5 specific antibodies was performed in parotid membranes and showed that the m3 receptor accounts for 93% of precipitable receptors. These data suggest that M3 receptors in the rat parotid are coupled to both the stimulation of Ca2+ mobilization and the inhibition of cAMP accumulation.

Original languageEnglish (US)
Pages (from-to)C1063-C1073
JournalAmerican Journal of Physiology - Cell Physiology
Volume261
Issue number6 30-6
StatePublished - Dec 1 1991
Externally publishedYes

Keywords

  • Adenosine 3',5'-cyclic monophosphate inhibition
  • Antimuscarinic receptor antibodies
  • Cytosolic calcium mobilization
  • Immunochemistry
  • Inositol trisphosphate formation
  • Muscarinic receptor subtypes
  • Northern blots
  • Receptor coupling
  • [H]quinuclidinyl benzilate binding

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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