Surface and intracellular nicotinic receptors expressed in intact adrenal chromaffin cells: direct measurements using [3H]epibatidine.
The presence and importance of assembled, intracellular neuronal nicotinic acetylcholine receptors (nAChRs) has not been established in native systems. In these studies [3H]epibatidine binding techniques were used to characterize surface and intracellular sites expressed in intact bovine adrenal chromaffin cells in culture. Permeant (300 microM nicotine) and impermeant (5 mM carbachol) cholinergic agents were used to define specific [3H]epibatidine binding to total (surface and intracellular) sites and surface sites, respectively. Intracellular [3H]epibatidine binding sites were characterized after eliminating surface binding sites via alkylation. Equilibrium binding to all sites was reached within 30 min at room temperature. Homologous (epibatidine) competition experiments on total (surface and intracellular) binding sites demonstrated a significant fraction of the high affinity sites were localized to intracellular compartments. Saturation binding assays to surface and intracellular sites revealed K(d) values of 1.9+/-1.1 and 3.6+/-1.9 nM, respectively. These binding studies document the existence of a significant population of high affinity, intracellular binding sites in native neuronal cells and support their characterization as assembled, alpha3beta4* nAChRs. Although the intracellular nAChRs represent approximately 70% of the total, high-affinity nAChRs expressed in cultured chromaffin cells, they do not appear to be involved in functional recovery after nAChR down-regulation.
Division of Pharmacology, The Ohio State University College of Pharmacy, 500 West 12th Avenue, Columbus 43210, USA.
R Benjamin
RB
Free
Dennis B
DB
McKay
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000478:Alkylation; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002395:Catecholamines / Q000096:biosynthesis; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation / Q000502:physiology; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D018613:Microscopy, Confocal; D018722:Nicotinic Agonists; D011725:Pyridines; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D011978:Receptors, Nicotinic / Q000096:biosynthesis*
D000903:Antibiotics, Antineoplastic; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002395:Catecholamines; D018722:Nicotinic Agonists; D011725:Pyridines; D011956:Receptors, Cell Surface; D011978:Receptors, Nicotinic; C403530:nicotinic receptor alpha3beta4; D003609:Dactinomycin; C082748:epibatidine