Laboratory project: "Regulation of Adenylate Cyclase by Alpha-Adrenergic Receptors"
Summary of work on this project as indicated in the report: "The role of the cyclic nucleotides adenosine 3':5'
monophosphate (cyclic AMP) and guanosine 3':5' monophosphate (cyclic GMP) in synaptic transmission is under study
using cultured cells of neural origin. The topics of interest during the current year have been the following: 1) The alpha-receptor-mediated
inhibition of adenylate cyclase activity by norepinephrine in neuroblastoma x glioma hybrid cells; and 2) characterization
of a compensatory increase in adeylate cyclase activity in cells treated for 10 hours or longer with norepinephrine and study
of the mechanism of this increase, which results in cell tolerance to and dependence on norepinephrine with respect to cyclic
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1977-09 (September 1977)
Nirenberg, Marshall W.
Sabol, Steven L.
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
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From Neuroblastoma to Homeobox Genes, 1976-1992
Annual Report of the Laboratory of Biochemical Genetics, July 1, 1976 - September 1, 1977 (September 1977)
Box Number: 13
Folder Number: 35
Project Number: Z01 HL 00015-02 LBG
Period Covered: July 1, 1976 through September 30, 1977
Title of Project: Regulation of adenylate cyclase by alpha-adrenergic receptors
Names, Laboratory and Institute Affiliations, and Titles of Principal Investigators and All Other Professional Personnel Engaged
on the Project:
PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI
OTHER: Steven L. Sabol, Research Associate, LBG NHLBI
Saburo Ayukawa, Visiting Associate, LBG NHLBI
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section on Molecular Biology
Institute and Location: NHLBI, NIH, Bethesda, Maryland 20014
Summary of Work:
The role of the cyclic nucleotides adenosine 3':5' monophosphate (cyclic AMP) and guanosine 3':5' monophosphate
(cyclic GMP) in synaptic transmission is under study using cultured cells of neural origin. The topics of interest during
the current year have been the following: 1) The alpha-receptor-mediated inhibition of adenylate cyclase activity by norepinephrine
in neuroblastoma x glioma hybrid cells; and 2) characterization of a compensatory increase of adenylate cyclase activity in
cells treated for 10 hours or longer with norepinephrine and study of the mechanism of this increase, which results in cell
tolerance to and dependence upon norepinephrine with respect to cyclic AMP synthesis.
Objectives: Alpha-receptor activators such as norepinephrine rapidly lower cAMP levels of NG108-15 cells by inhibiting adenylate
cyclase activity. Furthermore, prolonged exposure of cells to alpha receptor agonists results in an increase in adenylate
cyclase activity which compensates for the inhibition. Similar rapid inhibitions and compensatory increases elicited by opiate
and muscarinic cholinergic receptor agonists have been recently observed by others. During the past year, attempts have been
made to characterize these phenomena further and to elucidate the regulatory mechanisms.
Major Findings: NG108-15 hybrid cells possess alpha-adrengeric receptors which in concert with receptor activators inhibit
adenylate cyclase. Cells were cultured in the presence of norepinephrine for 0-48 hours, then the effects of withdrawal of
norepinephrine either by replacing the medium or by the addition of a receptor antagonist was tested. Withdrawal of norepinephrine
resulted in a 9-fold increase in cAMP levels of intact cells. Adenylate cyclase activity also increased but to a lesser extent.
Studies on the specificity of receptor antagonists showed that both the inhibition of adenylate cyclase by norepinephrine
and the subsequent increase in adenylate cyclase activity are mediated by alpha-receptors. These and other results show that
dual regulation of adenylate cyclase is a general phenomenon and that cells can become dependent upon norepinephrine, acetylcholine,
or opiates. The cells develop an apparent tolerance to these compounds but in fact remain sensitive to the compound used.
NG108-15 alpha-receptors were characterized by studying the specific binding of [3H]-dihydroergocryptine and other ligands
to the receptors. The specificity of the binding sites for ligands resembles that of alpha-receptors. The binding of the
ligand to the membrane preparation is a saturable process. The average NG108-15 cell possesses 60,000 alpha-receptors.
Significance to Biomedical Research: 1. The fact that dual regulation of NG108-15 adenylate cyclase has been observed now
with three classes of inhibitors each mediated by a different species of receptor, suggests that dual regulation may be a
general phenomenon. 2. Norepinephrine released at adrenergic synapses may regulate cAMP levels in post-synaptic or pre-synaptic
cells by the mechanism discussed here. Such regulation may modulate the cell's responsiveness to ligands for other species
of receptors which activate adenylate cyclase and thus may affect information transfer in the nervous system.
Proposed Course: The potencies of alpha-receptor activators and antagonists with respect to inhibition of adenylate cyclase
will be compared with the effects of ligand binding to NG108-15 alpha-receptors. The mechanism of coupling inhibition of
adenylate cyclase with a subsequent compensatory increase in enzyme activity will be studied further.
Effects of alpha-adrenergic activators and antagonists on [3H] dihydroergocryptine binding will be determined to define the
specificity of the alpha-receptor and the kinetics of binding. The regulation of receptor concentration will be studied.
1. Archer, Ellen G., Breakefield, Xandra O. and Sharata, Mary N.: Transport of tyrosine, phenylalanine, trytophan and glycine
in neuroblastoma clones. J. Neurochem. 28: 127-135, 1977.