Summary
To characterize the properties of nicotinic acetylcholine receptors (nAChRs) in autonomic ganglia, we examined l-[3H]nicotine binding to membrane fraction prepared from cultured bovine adrenal chromaffin cells, using a modified filtration method. Binding of l-[3H]nicotine to non-treated glass fiber filters interfered with the detection of specific binding to the membrane fraction. Presoaking glass fiber filters in 3% or higher concentrations of polyethyleneimine (PEI) solution (sixty times higher than earlier used concentration) for at least 5 h could reduce the binding of l-[3H]nicotine to the filters to the background level. Specific l-[3H]nicotine binding to the membrane fraction was detected only when the membrane fraction was prepared in Ca2+- and Mg2+ (EDTA, EGTA and protease inhibitors were added)-free buffer. Specific binding of l-[3H]nicotine was saturable and reversible. Both computer program and Scatchard analysis revealed a single class of high affinity binding sites with an average Kd of 8.9 nM and a Bmax of 42.5 fmol/mg protein. The Hill coefficient was 0.98. In inhibition studies, both cholinergic agonists (carbachol and l-nicotine) and ganglionic agonists (lobeline and 1,1-dimethyl-4-phenylpiperazinium iodide) were much effective in inhibiting l-[3H]nicotine binding, whereas both neuromuscular blocking (α-bungarotoxin and d-tubocurarine) and ganglionic blocking agents were less effective. These results suggest that high affinity nicotinic binding sites on adrenal chromaffin cells are nAChRs of the ganglion-type, which have properties different from nAChRs on the neuromuscular junction but similar to nAChRs in the brain.
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References
Abood LG, Lowy K, Tometsko A, MacNeil M (1979) Evidence for a noncholinergic site for nicotine's action in brain: psychopharmacological, electrophysiological, and receptor binding studies. Arch Int Pharmacodyn 237:213–229
Ascher P, Large WA, Rang HP (1979) Studies on the mechanism of action of acetylcholine antagonists on rat parasympathetic ganglion cells. J Physiol (Lond) 295:139–170
Boulter J, Evans K, Goldman D, Martin G, Treco D, Heinemann S, Patrick J (1986) Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor α-subunit. Nature 319:368–374
Brown DA (1979) Neurotoxins and the ganglionic (C6) type of nicotinic receptor. In: Ceccarelli B, Clementi F (eds) Advances in cytopharmacology, vol. 3: Neurotoxins: tools in neurobiology. Raven Press, New York, pp 225–230
Chiappinelli VA (1985) Actions of snake venom toxins on neuronal nicotinic receptors and other neuronal receptors. Pharmac Ther 31:1–32
Coloquhoun D, Ogden DC, Mathie A (1987) Nicotinic acetylcholine receptors of nerve and muscle: functional aspects. Trends Pharmacol Sci 8:465–472
Costa LG, Murphy SD (1983) [3H]nicotine binding in rat brain: Alteration after chronic acetylcholinesterase inhibition. J Pharmacol Exp Ther 226:392–397
Deneris ES, Connolly J, Boulter J, Wada E, Wada K, Swanson LW, Patrick J, Heinemann S (1988) Primary structure and expression of β2: A novel subunit of neuronal nicotinic acetylcholine receptors. Neuron 1:45–54
Fenwick EM, Fajdiga PB, Howe NBS, Livett BG (1978) Functional and morphological characterization of isolated bovine adrenal medullary cells. J Cell Biol 76:12–30
Goldman D, Simmons D, Swanson LW, Patrick J, Heinemann S (1986) Mapping of brain areas expressing RNA homologous to two different acetylcholine receptor α-subunit cDNAs. Proc Natl Acad Sci USA 83:4076–4080
Goldman D, Deneris E, Luyten W, Kochhar A, Patrick J, Heinemann S (1987) Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system. Cell 48:965–973
Guroff G (1964) A neutral, calcium-activated proteinase from the soluble fraction of rat brain. J Biol Chem 239:149–155
Larsson C, Nordberg A (1985) Comparative analysis of nicotine-like receptor-ligand interactions in rodent brain homogenate. J Neurochem 45:24–31
Lee K, Miwa S, Koshimura K, Hasegawa H, Hamahata K, Fujiwara M (1990) Effects of hypoxia on the catecholamine release, Ca2+ uptake and cytosolic free Ca2+ concentration in cultured bovine adrenal chromaffin cells. J Neurochem 55:1131–1137
Lippiello PM, Fernandes KG (1986) The binding of l-[3H]nicotine to a single class of high affinity sites in rat brain membranes. Mol Pharmacol 29:448–454
Lippiello PM, Fernandes KG (1988) Identification of putative high affinity nicotine receptors on cultured cortical neurons. J Pharmacol Exp Ther 246:409–416
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Marks MJ, Collins A (1982) Characterization of nicotine binding in mouse brain and comparison with binding of α-bungarotoxin and quinuclidinyl benzilate. Mol Pharmacol 22:554–564
Martino-Barrows AM, Kellar KJ (1987) [3H]Acetylcholine and [3H](−)nicotine label the same recognition site in rat brain. Mol Pharmacol 31:169–174
Mellgren RL (1980) Canine cardiac calcium-dependent proteases: Resolution of two forms with different requirements for calcium. FEBS Lett 109:129–133
Mishina M, Takai T, Imoto K, Noda M, Takahashi T, Numa S (1986) Molecular distribution between fetal and adult forms of muscle acetylcholine receptor. Nature 321:406–411
Nef P, Oneyser C, Alliod C, Couturier S, Ballivet M (1988) Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors. EMBO J 7:595–601
Nordberg A, Adem A, Hardy J, Winblad B (1988) Change in nicotinic receptor subtypes in temporal cortex of Alzheimer brains. Neurosci Lett 86:317–321
Paton WDM, Zaimis EJ (1949) The pharmacological actions of polymethylene-bis-trimethylammonium salts. Br J Pharmacol 4:381–400
Reavill C, Jenner P, Kumar R, Stolerman IP (1988) High affinity binding of [3H](−)-nicotine to rat brain membranes and its inhibition by analogues of nicotine. Neuropharmacology 27:235–241
Romano C, Goldstein A (1980) Stereospecific nicotine receptors on rat brain membranes. Science 210:647–650
Shimohama S, Taniguchi T, Fujiwara M, Kameyama M (1985) Biochemical characterization of the nicotinic cholinergic receptors in human brain: Binding of (−)-[3H]nicotine. J Neurochem 45:604–610
Slotkin TH, Orband-Miller L, Queen KL (1987) Development of [3H]nicotine binding sites in brain regions of rats exposed to nicotine prenatally via maternal injections or infusions. J Pharmacol Exp Ther 242:232–237
Tallarida RJ, Murray B (1987a) Quantal dose-response: Probits. In: Tallarida RJ, Murray B (eds) Pharmacologic calculations with computer program. Springer, Berlin Heidelberg New York, pp 31–35
Tallarida RJ, Murray B (1987b) Dissociation constant III: Perturbation methods (rate constants in the drug-receptor reaction). In: Tallarida RJ, Murray B (eds) Pharmacologic calculations with computer programs. Springer, Berlin Heidelberg New York, pp 50–53
Volle RL (1980) Nicotinic ganglion-stimulating agents. In: Kharkevich DA (ed) Pharmacology of ganglionic transmission. Springer, Berlin Heidelberg New York, pp 281–312
Wada K, Ballivet M, Boulter J, Connolly J, Wada E, Deneris ES, Swanson LW, Heinemann S, Patrick J (1988) Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science 240:330–334
Zaimis E, Head S (1976) Depolarizing neuromuscular blocking drugs. In: Zaimis E (ed) Neuromuscular junction. Springer, Berlin Heidelberg New York, pp 365–420
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Lee, K., Miwa, S., Koshimura, K. et al. Characterization of nicotinic acetylcholine receptors on cultured bovine adrenal chromaffin cells using modified l-[3H]nicotine binding assay. Naunyn-Schmiedeberg's Arch Pharmacol 345, 363–369 (1992). https://doi.org/10.1007/BF00176611
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DOI: https://doi.org/10.1007/BF00176611