Laboratory project: "Receptor Mediated Regulation of Adenylate Cyclase"
Summary of work for this project as indicated on the report: "Receptor-mediated activation and inhibition of adenylate
cyclase of neuroblastoma x glioma hybrid cells and other cell lines were studied."
Item is a photocopy.
Number of Image Pages:
3 (240,432 Bytes)
1979-09 (September 1979)
Nirenberg, Marshall W.
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, October 1, 1978 - September 30, 1979 (September 1979)
Period Covered: October 1, 1978 - September 30, 1979
Title of Project: Receptor Mediated Regulation of Adenylate Cyclase.
Names, Laboratory and Institute affiliations, and titles of principal investigations and all other professional personnel
engaged on the project:
PI: Marshall Nirenberg, Chief, LBG, LBG NHLBI
OTHERS: Douglas Wilkening, PRAT Fellow, LBG, NHLBI
John MacDermot, Visiting Fellow, LBG, NHLBI
Saburo Ayukawa, Visiting Fellow, LBG, NHLBI
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section on Molecular Biology
Institute and Location: NHLI, NIH, Bethesda, Maryland 20205
Total Man Years: 3.5
Summary of Work:
Receptor-mediated activation and inhibition of adenylate cyclase of neuroblastoma x glioma hybrid cells and other cell lines
Major Findings: The inhibition of adenylate cyclase by morphine and the gradual increase in adenylate cyclase activity that
results when NG108-15 cells are incubated for 12 or more hours in the presence of morphine was previously proposed as a model
for the analgesic action of opiates and for the phenomena of opiate dependence and tolerance. We now find that linoleic acid
or serum lipids are required for the morphine-dependent increase in adenylate cyclase activity, but not for inhibition of
the enzyme. Similar results were obtained with norepinephrine which activates alpha-receptors of NG108-15 cells. In this
model system, therefore, the inhibition of NG108-15 adenylate cyclase by morphine or norepinephrine can be dissociated from
the acquisition of dependence upon opiates or norepinephrine.
Ten mu-M morphine of norepinephrine completely inhibit the activation of adenylate cyclase by Ca2+ ions, but inhibit basal
or PGE1-activated adenylate cyclase by no more than 55 percent in NG108-15 homogenates. The extent of inhibition of adenylate
cyclase by morphine or norepinephrine thus is a function of the Ca2+ ion concentration and the proportion of adenylate cyclase
molecules that are activated by Ca2+ ions.
Activation of serotonin receptors of NG108-15 or NCB-20 hybrid cells by serotonin results in cell depolarization, action potentials,
and secretion of acetylcholine into the medium. These responses desensitize in less than 15 sec and are not inhibited or
mimicked by LSD. Serotonin also stimulates adenylate cyclase activity of NCB-20 hybrid cells, but this effect of serotonin
does not desensitize. Eadie-Scatchard analysis suggests a bimolecular interaction and reveals no evidence of receptor heterogeneity.
The Hill interaction coefficient is 1.0, indicating independent, noncooperative reactions. LSD activates adenylate cyclase
(Kact = 12 nM) and also inhibits the activation of the enzyme by serotonin (Ki - 10 nM). In addition, mianserin and cyproheptadine
inhibit serotonin activation of adenylate cyclase (Ki = 43 nM and 95 nM, respectively) and LSD activation of adenylate cyclaae
(Ki - 100 nM and 64 nM, respectively). These results show that serotonin and LSD interact during activation of adenylate
Binding sites for [3H]LSD were detected in NCB-20 homogenates; the KDapp was 36 nM, the Hill coefficient was 1.0, and the
receptor concentration was 385 fmol/mg of protein. [3H]LSD was displaced by serotonin (Ki = 110-180 nM). These results
agree well with those found to be mediated by a serotonin receptor responsive to LSD that mediates activation of adenylate
cyclase. Two binding sites for [3H]serotonin were detected in NCB-20 homogenates [KDapp = 200 nM and 3750 nM] and serotonin-LSD
interactions also were detected.
We conclude that NCB-20 hybrid cells possess two species of serotonin receptors, one coupled to activation of adenylate cyclase,
the other to cell depolarization and acetylcholine release; that activation of adenylate cyclase does not affect the rate
of acetylcholine release, and, conversely, that serotonin-dependent cell depolarization does not affect intracellular levels
of cAMP or cGMP in the hybrid cells tested.
Significance to Biomedical Research:
The results suggest that fatty acids may be required for cellular acquisition of opiate dependence and tolerance and that
the analgesic action of morphine may be uncoupled from the acquisition of morphine dependence and tolerance.
1. McDermot, J., Higashida, H., Wilson, S. P., Matsuzawa, H., Minna, J. and Nirenberg, M. Adenylate Cyclase and Acetylcholine
Release Regulated By Separate Serotonin Receptors Of Somatic Cell Hybrids, Proc. Natl. Acad. Sci. USA 76, 1135-1139 (1979).
2. Wilkening, D., and Nirenberg, M. A Lipid Requirement For Acquisition Of Opiate Or Epinephrine Dependence By Neuroblastoma
x Glioma Hybrid Cells, J. Neurochem., In Press.
3. Wilkening, D., Sabol, S. L., and Nirenberg, M. Control of Opiate Receptor-Adenylate Cyclase Interactions By Calcium Ions
and Guanosine-5'-Triphosphate, Brain Res., In Press.
4. Sabol, S. L., and Nirenberg, M. Regulation of Adenylate Cyclase Of Neuroblastoma x Glioma Hybrid Cells By alpha-Receptors,
I. Inhibition Of Adenylate Cyclase Mediated By alpha-Receptors, J. Biol. Chem. 254, 1913-1920 (1979).
5. Sabol, S. L., and Nirenberg, M. Regulation Of Adenylate Cyclase Of Neuroblastoma x Glioma Hybrid Cells By alpha-Adrenergic
Receptors. II. Long-lived Increase Of Adenylate Cyclase Activity Mediated By alpha-Receptors. J. Biol. Chem. 254, 1921-1926