Abstract
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 inE 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 theE 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 theE 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 β4 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 β4 nAChR subunits.
Footnotes
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Send reprint requests to: Dennis B. McKay, Ph.D., Division of Pharmacology, The Ohio State University College of Pharmacy, 500 West 12th Ave., Columbus, OH 43210.
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↵1 This work was supported by grants from the Bremer Foundation (Columbus, OH) and the American Heart Association (Ohio Affiliate) and by Grant NS29746 (R.T.B.) from the National Institutes of Health. Support from the Student Achievement in Research and Scholarship Program of the Ohio State University is also appreciated.
- Abbreviations:
- α-bgt
- α-bungarotoxin
- brACh
- bromoacetylcholine
- DMEM
- Dulbecco’s modified Eagle medium
- nAChR
- nicotinic acetylcholine receptor
- NE
- norepinephrine
- PCR
- polymerase chain reaction
- PSS
- physiological saline solution
- RT
- reverse transcription
- SSPE
- standard saline/phosphate/EDTA
- TM
- transmembrane domain
- Received September 17, 1996.
- Accepted January 6, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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