title: Muscarinic receptors and second-messenger responses of neurons in primary culture.
abstract: The coupling of muscarinic receptors to second messenger responses was investigated in primary cultures of neurons from the fetal mouse brain. Neurons were maintained in monolayer culture, in serum-free medium; immunocytochemical studies found these cultures to be nearly exclusively neuronal. In striatal cultures, [3H]N-methylscopolamine (NMS) bound specifically and with high affinity (Kd = 70 pM) to a homogeneous population of receptors on intact neurons (320 fmol/mg cellular protein). Displacement of the binding of [3H]NMS by pirenzepine indicated the presence of heterogeneous sites (81% high affinity sites, Kh = 51 nM, K1 = 1.5 microM); AF-DX 116 showed the opposite selectivity (15% high affinity sites, Kh = 56 nM, K1 = 1.3 microM). The dopamine agonist SKF-38393 (1 microM) enhanced the accumulation of cyclic adenosine monophosphate (AMP) in these cultures 2.5-fold; addition of carbachol reduced cyclic AMP levels by 30% (EC50, 1.7 microM). In the presence of 1 mM lithium, carbachol stimulated the accumulation of inositol monophosphate 5-fold (EC50, 61 microM). Both responses were antagonized by pirenzepine (apparent Ki of 23 nM for the phosphoinositide response and 200 nM for the cyclic AMP response) and AF-DX 116 (apparent Ki 540 nM and 160 nM, respectively). In binding studies on brainstem cultures, AF-DX 116 indicated the presence of two sites of approximately equal abundance (Kh = 170 nM, K1 = 2.9 microM); data for pirenzepine were adequately fit by a one-site model (Kd = 630 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Psychiatry, University of Vermont College of Medicine, Burlington 05405.
authors.forename: J
authors.initials: J
authors.lastname: Ellis
authors.forename: J H
authors.initials: JH
authors.lastname: Huyler
authors.forename: D E
authors.initials: DE
authors.lastname: Kemp
authors.forename: S
authors.initials: S
authors.lastname: Weiss
mesh_terms: D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D018727:Muscarinic Antagonists; D009474:Neurons / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D010095:Oxotremorine / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012601:Scopolamine / Q000378:metabolism; D015290:Second Messenger Systems
chemical_list: D007295:Inositol Phosphates; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D010095:Oxotremorine; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D012601:Scopolamine; D000242:Cyclic AMP