EVIDENCE FOR SPARE NICOTINIC ACETYLCHOLINE-RECEPTORS AND A BETA-4 SUBUNIT IN BOVINE ADRENAL CHROMAFFIN CELLS - STUDIES USING BROMOACETYLCHOLINE, EPIBATIDINE, CYTISINE AND MAB35

Relatively little is known about the type and number of nicotinic acet ylcholine receptors (nAChRs) that mediate secretion from adrenal chrom affin 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 c atecholamine release was reduced (IC50 similar to 0.3 mu M) This effec t was slowly reversible. Submaximal concentrations of brACh caused shi fts to the right in concentration-response curves of approximately 4-f old, as well as decreases in E-max values for the agonists nicotine an d epibatidine. Cytisine is a nAChR agonist (EC50 similar to 46 mu M) t hat was somewhat less efficacious than nicotine (E-max similar to 85% of 10 mu M nicotine) in adrenal chromaffin cells. Submaximal concentra tions 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-me diated secretion, produced a time-dependent shift to the right in agon ist concentration-response curves and a reduction in E-max for nicotin e and epibatidine. mAb35 treatment produced only a reduction in the E- max value of cytisine. Finally, we cloned and sequenced a reverse tran scription-polymerase chain reaction product from bovine adrenal chroma ffin RNA that shares a high degree of homology with beta 4 nAChR subun its. Northern analysis provided evidence for the presence of this tran script in chromaffin cell cultures. Together, these studies support th e presence of a nAChR reserve in adrenal chromaffin cells that is down -regulated by mAb35. These studies also support the presence of more t han one nAChR population mediating secretion and the presence of beta 4 nAChR subunits.