tITHSO:IIAN SC1 NCE INFORMATION EXCtiAPlCE
    f                    U.S. DEPARTblEiiT OF       PROJECT EiUt:!SiR
?OJECT NUMBER Do NOT use this space)  HEALTH EDUCAT 108 AND WELFARE
                    PdBLlC HEALTk SERVICE
                                  NOTICE OF
                          INTRAMURAL RESEARCH PftOJECT   201 HL 00015-01 LBG

`El? I GO COVEREO
July 1, 1975 through June 30, 1976
ITLE OF PROJECT (80 characters or less)

Regulation of cyclic nucleotide biosynthesis by neurotransmitters and opiates

AMES, LABORATORY AND INSTITUTE AFFILlATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AH0 ALL OTHER
`RGFESSIONAL PERSONNEL ENGAGED ON THE PROJECT


PI:    Marshall Nirenberg    Chief, Lab. of Biochem. Genetics   LBG NKLI
      Steven L. Sabol      Research Associate            -LBG NHLI









DOPERATING UNITS (if any)


None

AB/BRANCH
Laboratory of Biochemical Genetics
;ECT I ON
Section on Molecular Biology
NSTITUTE AND LOCATION
NHLI, NIH, Bethesda, Maryland 20014
`OTAL MANY EARS:         PROFESS I ON AL:           OTHER:
     2.0                1.5                   .5
MMARY DF WORK 200 words or less - underline keywords)

   The role of the cyclic nucleotides adenosine 3'5' monophosphate and
guanosine 3'5' monophosphate in synaptic transmission is under study using
cultured cells of neural origin. The topics of 4-c
                             A.lbLerest d-uring the current
year have been the following:  1) The receptor-mediated inhibition of adenylate
cyclase activity by alpha-adrenergic agents in neuroblastoma x glioma hybrid
cells; 2) Demonstration of a compensatory induction of adenylate cyclase
activity in cells treated for one or more days with alpha-adrenergic agents, as
had been previously demonstrated by others for opiates, and study of the
mechanism of this compensatory induction; 3) The effect of opiates and alpha-
adrenergic agents on guanosine 3'5' monophosphate levels in the hybrid cells;
4) Search for a cultured-cell system which synthesizes a peptide with opiate-
like properties.









PHS-6020
(12-75)


201 HL 00015-01 LBG

Project Description:

  Objectives:  Previous work in this laboratory demonstrated that opiates,
a-adrenergic agonists, and muscarinic cholinergic agonists lower cyclic AMP
(adenosine 3'5' monophosphate) levels by receptor-mediated mechanisms in the
neuroblastoma x glioma hybrid cell line NG108-15. Also demonstrated was the
inhibition of adenylate cyclase by opiates and a compensatory induction of
adenylate cyclase activity (measured in the absence of opiates) in cells grown
in the presence of opiates.  One aspect of the present project attempts to
demonstrate and analyze similar effects of adrenergic agonists on adenylate
cyclase in cell-free systems.  Another aspect is to relate these findings to
the action of the enkephalins, recently discovered endogenous opiate-like
peptides.

  Major Findings:  (1) Adrenergic agents, like opiates, inhibit adenylate
cyclase activity in NG108-15 homogenates.  The order of agonist potencies is
consistent with an alpha receptor, and the effect is blocked by alpha receptor
antagonists.

  (2) Cultivation of NG108-15 with norepinephrine for 1 to 4 days results,
as in the case with opiates, in an increase in adenylate cyclase activity
which compensates for the inhibition of this enzyme by norepinephrine.  Cells
then appear tolerant to norepinephrine, but are dependent upon norepinephrine
for maintenance of normal CAMP levels.  Withdrawal of norepinephrine or the
addition of an alpha-adrenergic antagonist results in an increase in cellular
cyclic AMP levels to abnormally high values.

  Significance to Biomedical Research:  The results show that cells exposed
to a-receptor activators for several days become tolerant to and dependent
upon these compounds and that tolerance and dependence are established by the
mechanism of dual regulation of adenylate cyclase.

  Proposed Course:  It is intended to continue the investigation of the
induction of adenylate cyclase activity by cyclase inhibitors, including the
possible mediation of cyclase activators.  Also the relationship among the
opiate, adrenergic and cholinergic receptors in NG108-15 should be studied.
Final1y.a search for endogenous "endorphin" synthesis in cell lines of neural
origin is being initiated, with the goal of developing a tissue culture system
for the study of the biosynthesis of this peptide or peptides.

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