Vol. 281, Issue 2, 905-913, 1997

Evidence for Spare Nicotinic Acetylcholine Receptors and a 4 Subunit in Bovine Adrenal Chromaffin Cells: Studies using Bromoacetylcholine, Epibatidine, Cytisine and mAb35¹

Bryan W. Wenger, Darrell L. Bryant, R. Thomas Boyd and Dennis B. McKay

Division of Pharmacology, College of Pharmacy (B.W.W., D.L.B., D.B.M.), and Department of Pharmacology, College of Medicine (R.T.B.), The Ohio State University, Columbus, Ohio

Relatively little is known about the type and number of nicotinic acetylcholine receptors (nAChRs) that mediate secretion from adrenal chromaffin cells. In these studies, we investigated nAChR reserve pools and their modulation using bromoacetylcholine (brACh) and the anti-nAChR antibody mAb35. By using brACh under acetylating conditions, adrenal catecholamine release was reduced (IC₅₀, ~0.3 µM). This effect was slowly reversible. Submaximal concentrations of brACh caused shifts to the right in concentration-response curves of approximately 4-fold, as well as decreases in E_max values for the agonists nicotine and epibatidine. Cytisine is a nAChR agonist (EC₅₀, ~46 µM) that was somewhat less efficacious than nicotine (E_max, ~85% of 10 µM nicotine) in adrenal chromaffin cells. Submaximal concentrations of brACh caused a small shift to the right in the concentration-response curves for the agonist cytisine, as well as a decrease in the E_max value. mAb35, which causes a slowly developing loss of nAChR-mediated secretion, produced a time-dependent shift to the right in agonist concentration-response curves and a reduction in E_max for nicotine and epibatidine. mAb35 treatment produced only a reduction in the E_max value of cytisine. Finally, we cloned and sequenced a reverse transcription-polymerase chain reaction product from bovine adrenal chromaffin RNA that shares a high degree of homology with 4 nAChR subunits. Northern analysis provided evidence for the presence of this transcript in chromaffin cell cultures. Together, these studies support the presence of a nAChR reserve in adrenal chromaffin cells that is down-regulated by mAb35. These studies also support the presence of more than one nAChR population mediating secretion and the presence of 4 nAChR subunits.