SMITHSONIAN SCI NCE INFORMATION EXCHANGE
       00 NOT use this space)      U.S. DEPARTMENT OF       PROJECT NUMBER                  I

PROJECT NUMBER                  HEALTH EDUCATION AND WELFARE
                     P6BLlC HEALTk SERVICE
                                 NOTICE OF
                          INTRAWRAL RESEARCH PROJECT   Zol HL 00012-02 LBG

1                                   I                         I-
PERT 00 COVERED

July 1, 1975 through June 30, 1976
TITLE OF PROJECT (80 characters or less)

Muscarinic Acetylcholine Receptors of Cultured Cell Lines

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:   Marshall Nirenberg       Chief, Lab. of Biochemical

                             Genetics                LBG NHLI
OTHER:  William Klein            Postdoctoral Fellow        LBG NHLI
      Orest Hurko             Staff Fellow              LBG NHLI
   Wolfgang Burgermeister     Guest Worker               LCNIAMOD
    Bernhard Witkop        Chief, Laboratory of
                       Chemistry               LC NIAMDD

COOPERATING UNITS (if any)

Laboratory.of Chemistry, NIAMDD

LAB/BRANCH
Laboratory of Biochemical Genetics
SECT I ON

    on vv
INSTITUTE AND LOCATION

NHLI, NIH, Bethesda, Maryl_and 20014
TOTAL MANYEARS:       1 PROFESSIONAL:

/OTHER:

   2.5       I    2.5      I
SUMMARY OF WORK (200 words or less - underline keywords)
  Our work on muscarinic acetylcholine receptors focuses on two fundamental
questions:

  (1) How is binding of acetylcholine to receptors converted into an intra-
cellular response?

  (2) What factors regulate receptor concentrations in the membrane?

  A binding assay employing tritiated quinuclidinyl benzilate permits us to
investigate properties of receptor binding sites and to measure receptor levels
in cultured neuroblastoma and hybrid cells under systematically varied conditions.

PHS-6040
(12-75)


ZOl HL 00012-02 LBG

Project Description:

  Objectives:  The objectives of our work have been (1) to characterize the
nature of ligand interactions with muscarinic receptors, and (2) to assess
various factors likely to affect receptor concentrations.  Although most
methodology involved has been straightforward, our experiments required the
custom labeling and purification of muscarinic ligands as commercially availa-
ble materials were unsuitable due to low specific activity.

Major Findings:  The principal findings are:

  (1) Excitatory and inhibitory muscarinic receptors for ligands which do
'not differ pharmacologically.

  (2) The affinity of receptors for acetylcholine is affected by the ionic
composition of the medium.

  (3) Antagonists and agonists differ strikingly in the way that they inter-
act with the receptor.  Antagonist binding to receptor follows the law of mass
action whereas agonist binding does not.  When analyzed on Hill plots, agonist
interactfons with receptor exhibit negative cooperativity that may be related
to receptor desensitization.

  (4) The receptor concentration is greatly reduced by exposure of cultured
cells to carbamylcholfne or other receptor activators.  The [receptor*agonist]
complex is degraded more rapidly than the free receptor.

  (5) Muscarinic excitatory and inhibitory acetylcholine receptors have been
solubilized and have been characterized partially.

  Significance to Biomedical Research:  This work with a model system suggests
that, in the normal nervous system, receptor levels, which ultimately influence
the pattern of behavior of neural nets, are regulated by synaptic transmission.
Some of the events which result from the interaction of acetylcholine with
muscarinic acetylcholine receptors have been defined.  Two regulatory processes
were found which depend upon acetylcholine.  One regulates receptor activity,
the other regulates receptor concentration.

Proposed Course:  Further elucidation of QNB binding will be pursued.