Evidence that M3 muscarinic receptors in rat parotid gland couple to two second messenger systems.
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) approximately 4-diphenylacetoxy-N-methylpiperidine methbromide (4-DAMP) (1.6) much greater than pirenzepine (136) greater than 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 (Ki) 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 Ki 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 Gi-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.
Dai
Y S
YS
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Ambudkar
I S
IS
Horn
V J
VJ
Yeh
C K
CK
Kousvelari
E E
EE
Wall
S J
SJ
Li
M
M
Yasuda
R P
RP
Wolfe
B B
BB
Baum
B J
BJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D018727:Muscarinic Antagonists; D010306:Parotid Gland / Q000378:metabolism*; D011233:Precipitin Tests; D011813:Quinuclidinyl Benzilate / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D015290:Second Messenger Systems
D018727:Muscarinic Antagonists; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D011813:Quinuclidinyl Benzilate; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D000242:Cyclic AMP; D007545:Isoproterenol; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine