Modulation of Synapse Formation by
Cyclic Adenosine Monophosphate

  M. Nirenberg, S. Wilson, H. Higashida, A. Rotter
K. Krueger, N. Busis, R. Ray, J. G. Kenimer, M. Adler

How neurons in the developing ner-
vous system form synapses and distin-
guish appropriate from inappropriate
synapses remains one of the central,
unsolved problems in neurobiology. In
1963, Sperry (I) proposed the chemoaf-
finity hypothesis; namely, that neurons
bear positional labels (that is, molecular
addresses) that are recognized by com-
plementary molecules on the synaptic
target cells and thereby determine the
specificity of neuronal connections. He
also suggested that two gradients of mol-
ecules on retina neurons at right angles
to one another, which interact with com-
plementary molecules on the target neu-
rons in the tectum, might be a mecha-
nism for matching synaptic connections
and reproducing a point-to-point map of
the retina in the tectum. If synapse rec-
ognition molecules exist, monoclonal

antibody technology should be a power-
ful tool for their detection. Many investi-
gators are now using this approach.

Other mechanisms such as regulation
of gene expression by environmental fac-
tors such as hormones, neuromodula-
tars, transynaptic communication, or
molecules secreted by neighboring or
other cells surely play important roles in
the assembly of synaptic circuits. For
example, Le Douarin (2) and Patterson
(3) and their colleagues have shown that
during development neurons from the
neural crest can express either the gene
for tyrosine hydroxylase, which cata-
lyzes the first step in the pathway for
norepinephrine synthesis, or the gene for
choline acetyltransferase, which cata-
lyzes the synthesis of acetylcholine, de-
pending on the presence of an extracellu-
lar macromolecule, purified by Weber

(4), which is secreted by other cells, or
the extent of depolarization of the neu-
ron. In addition, Mudge (5) has shown
that the expression of somatostatin. a
peptide transmitter or neuromodulator.
by dorsal root ganglia sensory neurons is
dependent on molecules secreted b}
nonneural cells. Raff et al. (6) also have
shown that fetal calf serum markedly
influences the differentiation pathway
expressed by glial cells in the central
nervous system.

Edelman and his colleagues (7) discov-
ered a neuronal glycoprotein rich in sial-
ic acid residues, termed N-CAM (neural
cell adhesion molecule), that mediates
intercellular adhesion in the absence of
Ca*+ and probably plays an important
role in the development of the nervous
system by conserving the topographic
relationships between individual neuron5
or axons (or both) in a set of neurons~
even though axons may migrate lo@
distances before synapsing. Molecule5
that mediate Ca*+-dependent intercell"-
lar adhesion (8) and factors that promote
retina cell adhesion, such as cognin c9'.
and ligand and agglutinin (IO), also ha"'
been described, but little is known aho"'
their function in the nervous system-
Other mechanisms such as contact guiJ.
ante, chemotaxis, cell survival factop'
guidance of neurites by gha (II). sn'

. .

The authors are or have been membe;;$*~
Laboratory of Biochemical Genetics:
Heart. Lung, and Blood Institute. NatIona' r:
tutes of Health. Building 36. Room 1C-06. Beth
Maryland 20205.


,lection for synchronous or sequential
;p,mission across two or more synap-
3j that innervate a neuron may also play
,,portant roles in synaptogenesis.
' \Ke have used monoclonal antibodies
,d cultured cell systems to study syn-
,pje formation and plasticity. Some
,,,,dies with retina cells are discussed
nrst, and then studies on the plasticity of
,,.,,apses formed by clonal neuroblasto-
,&hybrid cells with striated muscle
,.clls are reviewed.

A dorsal-ventral gradient of protein in
,t,ina. Trisler et al. (12) obtained a
monoclonal antibody that recognizes a
~1 membrane protein distributed in a
large dorsal-ventral topographic gradient
,,, chick retina (Fig. 1). The concentra-
tion of antigen detected at the dorsal
margin was at least 35fold higher than
[hat found at the ventral margin of the
retina, and the concentration of antigen
detected varied continuously add loga-
rithmically with the logarithm of distance
&ng the circumference of the retina
from ventral to dorsal poles of the gradi-
ent. Thus, the protein defines a bilateral-
ly symmetrical, dorsal-ventral axis of the
retina and can be used as a marker of cell
position in the retina with respect to the
dorsal-ventral axis. The antigen, termed
TOP (toponimic), was detected on all
cells examined in dorsal and middle reti-
na, but more TOP was detected on cells
from dorsal retina than on cells from
middle retina.

The TOP antigen was solubilized and
purified by antibody-agarose column
chromatography and sodium dodecyl
sulfate (SDS)-polyacrylamide gel elec-
trophoresis. A single band of protein was
obtained with a molecular weight (M,) of
approximately 47,000 (13). TOP was de-
tected in optic cups of 4%hour chick
embryos (14), and evidence for a gradi-
ent of TOP was found in 4-day embryo
retinas. A gradient therefore is generated
as neurons are generated in the retina
and the gradient is maintained through-
out embryonic development and in the
adult. Neurons first appear in the central
Portion of retina and then are added in
concentric, ever widening rings. Thus,
central retina is the oldest portion of the
retina and peripheral retina is the young-
est. How a dorsal-ventral gradient is
generated as the retina forms and is
Perpetuated is not known.
TOP was detected, in order of de-
creasing concentration, in retina, cere-
brum, and thalamus; little or no antigen
was found in other parts of the nervous
sYstem or in other tissues. Gradients of
TOP were found in chicken, turkey,
duck, and quail retina, but the antigen
was not detected in rat, Xenopus laevis,
AQfla pipiens. or goldfish retina.

The antigenicity of TOP is destroyed  monoclonal antibodies Fe specific for a
by trypsin; however, cells dissociated  single class of cells in retina such as
with trypsin from dorsal, middle, or ven-  photoreceptors,  horizontal neurons,
tral retina, cultured separately or com-  Miiller cells, or ganglion neurons, or for
bined in various proportions, continue to  a family of cells such as those in the
synthesize the antigen and accumulate  inner nuclear layer of retina. Another
the amount of TOP that would be expect-  monoclonal antibody, A2B5 (18), recog-
ed with cells from the corresponding nizes unidentified gangliosides with sial-

Summary. Synapses between neuroblastoma-hybrid cells and myotubesexhibit a
high degree of plasticity. Increase of cyclic adenosine monophosphate (AMP) ,levels
of the hybrid cells for several days results in the appearance of functional voltage-
sensitive Ca2' channels, which are requjred for evoked secretion of acetylcholine.
The results show that cyclic AMP regulates synaptogenesis by regulating the

                         2+
expression of voltage-sensitive Ca  channels, and suggest that cyclic AMP affects
posttranslational modifications of some glycoproteins and cellular levels of certain
proteins.

region in the intact retina in ovo. Thus,
the number of antigen molecules detect-
ed on retina cells after 10 days in culttire
depends on the prior position of the cells
in the intact retina.

These results suggest that the retina is
composed of a gradient of cells that
express different amounts of TOP, de-
pending on the position of the ceils in
retina along the dorsal-ventral axis of the
retina. The function of TOP is not
known. Monoclonal antibodies that rec-
ognize an anterior-posterior gradient of
molecules in retina were looked for, but
were not found (15). However, the dem-
onstration that TOP is a cell membrane
protein and is expressed on the basis of
cell position in the retina, rather than cell
type, suggests that TOP may play a role
in the specification of positional informa-
tion in the retina. We are trying to clone
complementary DNA (cDNA) corre-
sponding to TOP messenger RNA
(mRNA) to use to define the amino acid
sequence of TOP and to explore the
mechanism of regulating TOP expres-
sion.

Other monoclonal antibodies to reti-
na. Grunwald et al. (16) showed that
antibody 13H9 recognizes cell mem-
brane protein detected on most or all
cells in retina; however, antigen was not
detected on neurons or glia in other parts
of the nervous system. It is of interest to
determine whether the protein specifies
a compartment of cells; that is, functions
as a cell adhesion molecule that enables
retina cells to adhere preferentially to
one another rather than to other cells.
Three monoclonal antibodies recognize
antigens that are restricted to the outer
synaptic layer of retina (113F4, 92A2,
and 18B8); another antibody (16G6) rec-
ognizes antigen in both the inner and
outer synaptic layers of retina. Antibody
18B8 binds to glycoproteins and uniden-
tified species of gangliosides (f 7). Other

ic acid residues and glycoproteins (17)
that are markers of neurons and some
glia (6, 18).

Cultured retina ceils. Chick retina
contains abundant nicotinic and musca-
rinic acetylcholine receptors that mostly
are distributed in layers within the inner
synaptic layer of retina (19). Cultured
neurons dissociated from chick embryo
retina also express choline acetyltrans-
ferase and acetylcholine receptors, and
the neurons form approximately as many
synapses in vitro (1.5 X lo9 synapses
per milligram of protein) as they do in
ovo, as judged by electron microscopy
(20).

The specificity of synapse formation
by retina neurons was examined by co-
culturing dissociated chick embryo or rat
retina neurons with inappropriate synap-
tic partner cells such as striated muscle
cells that possess many nicotinic acetyl-
choline receptors. Retina neurons form
functional synapses with most striated
muscle cells in 90 minutes, but these
synapses are transient and slowly disap-
pear over a period of 5 to 10 days (21-
23). Cholinergic neurons that are able to
synapse with myotubes first appear in
chick retina on day 6 of embryonic de-
velopment, are most abundant on day 8
and comprise approximately 8 percent of
the retina cell population, atid lose the
ability to form synapses with.myotubes
by day 16 of embryonic development
(23). However, synapses between retina
neurons increase during the culture peri-
od and remain abundant after all synap-
ses between retina neurons and muscle
cells terminate.

Two processes contribute to the turn-
over of retina neuron synapses with
myotubes. First, .retina neurons are able
to form synapses with striated muscle
cells only for a short time during devel-
opment (23); and second, synapses be-
tween retina neurons and myotubes ter-


minate because retina neurons preferen-
tially adhere to other retina cells rather
than to myotubes (21).

Preparations of neurons from chick
embryo spinal cord, which presumably
contain motor neurons that normally in-
nervate striated muscle cells, also form
synapses with cultured muscle cells, but
the number of synapses remains con-
stant during subsequent culture (22).
Therefore, spinal cord neurons either
form stable, long-lived synapses w&h
muscle cells or attain a steady sf%t'e
wherein the rate of synapse formation is
equal to the rate of synapse termination.
These results show that inappropriate
synapses between retina neurons and
myotubes form rapidly and are terminat-
ed slowly, that synapses formed by cho-
linergic neurons from retina and spinal
cord turn over at different rates, and that
differences in synapse turnover rates of
two populations of synapses can result in
the selective retention of one population
and the loss of the other.

Clonal Neuroblastoma Cell Lines

Adult neurons do not divide; however,
the establishment of clonal lines of neu-
roblastoma cells from a transplantable
mouse neuroblastoma tumor (C- 1300) of
spontaneous origin provided a source of
relatively homogeneous populations of
dividing cells of neural origin (24). Char-
acterization of these (24) and other (25)
clonal lines of C-1300 neuroblastoma
`showed that the cells have excitable
membranes (26) and other neural proper-
ties, and that the expression of genes for
neural properties is inherited and thus
can be perpetuated. Clonally inherited
differences in phenotype also were
found; for example, some neuroblastoma
cell lines synthesize acetylcholine (25),
others catecholamines; but most do not
synthesize these compounds.

Cells from neuroblastoma lines that
synthesize acetylcholine were cocul-
tured with striated muscle cells, which
possess abundant nicotinic acetylcholine

Table I. Cell line phenotypes [see (32, 34, 35)].

Cell    ACh'
lines   forma-
(No.)    tion

K+-Dependent

45Ca2'   [3H]ACh
uptake    release

  Vesicles


Small   Large
clear   dense
       core

ACh
receptor
aggregation
protein

Synapse

:       +      +++      +++      +       +         +        +++
        +        +        +        +       +         +         +
2       +                -        +       +         +         -
5       +       ++        -        +       +         +       - or +
3       +       ++        2       +                        - or +
9 -                -                                 -

`Acetylcholine (ACh).

2     4     6     8                 5     10           50    100
  Retina section                        Maximal distance t%)

Perpendicular.

Fig. 1. Geometry of the TOP gradient in 14-day chicken embryo retina (12). (A) Specifically
bound `2sI-labeled F(ab'), (pmole per milligram of protein) is shown on the ordinates in (A) and
(B) and within the appropriate segment of retina tested. (B) The circumference of the retina is
14.5 mm which corresponds to 100 percent on the abscissa. (A) Strips of retina from
ventroanterior (0 percent) to dorsoposterior (100 percent) retina margins were removed, and
each was cut into nine segments and assayed for TOP. (0) Strips of retina from anterior (0
percent) to posterior (100 percent) margins of the retina perpendicular to the choroid fissure
were prepared and assayed as above; (0) data from panel A.

7%

receptors, or with cardiac muscle cells
that have muscarinic acetylcholine re-
ceptors. However, for several years we,
and others, failed to detect synapses. We
thought that these cell lines might not
express ah genes for proteins that might
be required for synaptic communication,
and therefore we fused- neuroblastoms
cells with other cells and generated many
somatic hybrid cell lines (26). Hybrid cell
lines were found that express new neural
properties not detected with parental
cells (27, 28); with other hybrid cell lines
some neural properties were extin-
guished. Eventually five cell lines were
found that synthesize acetylcholine and
form many synapses with cultured myo-
tubes (32, 33). The early attempts to
form synapses with neuroblastoma cells
failed for two reasons. (i) The extent of
neural maturation and ability of cells to
form synapses are regulated and are
highly sensitive to environmental condi-
tions, making it necessary to find condi-
tions that yield populations of "differen-
tiated" cells. (ii) Most, but not all. of the
cholinergic neuroblastoma cell lines that
were tested do, indeed, lack reactions
that are required for synapse formation.
Empirically, we found that populations
of neuroblastoma or hybrid cells can be
shifted from a poorly differentiated, syn-
apse incompetent state, to a well-differ-
entiated, synapse competent state, by
increasing intracellular levels of cyclic
adenosine monophosphate (AMP) for
days. Selection for nondividing ceils also
yields well-differentiated populations of
cells.

In Fig. 2 are shown photomicrographs
of cells from four of the five cell lines
that form many synapses with striated
muscle cells. The NBrlO-A and NBr20-A
cells originated by fusion of mouse neu-
roblastoma N18TG-2 (26) with clonal
BRL30-E rat liver cells, NCB-20 cells
(29) resulted from fusion of N18TG-2
cells with fetal Chinese hamster brain
cells, and NG108-15 (30) resulted from
fusion of N18TG-2 with C6BU-I (28) rar
glioma cells. Few neurites or synapse'
were found when cells were in the W-
rithmic phase of growth. However, es.
posure of cells for 7 days to I mM dib".
tyryl cyclic AMP, which promotes nrur.
ite extension, and to 1 percent (rather
than 5 percent) fetal bovine s;lzs'
which reduces neurite retraction. .   '
cells with neurites that can be more thnn
2 mm in length (31).

Other cell lines have high concen"'
tions of acetylcholine, adhere Wei' "
myotubes, but do not form sYz:z,;
(32). A summary of phenotypes
lines with or without synaptic defects "
shown in Table 1. The NBrlO-A. NBBr3"


A, FJCB-20, NG108-15, and NS-26 neu-
&astoma cells (25) form many synap-
,gs with cultured myotubes (32,33), syn-
,hzsize acetylcholine (32), have function-
.,I "oItage-sensitive CaZf channels (34),
have small clear vesicles approximately
4 nm in diameter and large dense-core
,,,sicIes 180 nm in diameter (41), and
+se acetylcholine into the medium
Joj a protein that stimulates the aggrega-
I,on of nicotinic acetylcholine receptors
on myotube plasma membranes (35).
Cells from three lines take up Ca'+ ions
jlo~Iy (34) and secrete little acetylcho-
line when depolarized by 80 mM K'
ions, and form few synapses with muscle
ce~~~, Cells from two lines lack functional
,oltage-sensitive Ca2+ channels (34) and
~0 not form synapses. Cells from five
lines take up Ca2+ when depolarized by
K- ions but do not respond by secreting
more acetylcholine (32), and few or no
synapses were found. These cells lack a
ca?+-dependent acetylcholine secretion
reaction (or reactions); however, acetyl-
choline is secreted into the medium in
the basal, unstimulated state. Cells from
[hree lines have small clear vesicles but
lack large dense-core vesicles and func-
tional protein that induces nicotinic ace-
tylcholine receptor aggregation on myo-
tube membranes (35), and form few or no
synapses. Nine additional cell lines have
little or no choline acetyltransferase ac-
tivity, and therefore they synthesize lit-
tle or no acetylcholine (32) and do not
form functional synapses with striated
muscle cells.

Regulation of synaptogenesis. Thus
far. we have identified 12 species of
receptors that are expressed by NGlOS-
IS cells, including receptors for prosta-
glandin E, (PGE,) (36,37), prostaglandin
Fz (PGF2) (36). adenosine (38), Met-
enkephalin (36), alpha-Zadrenergic re-
ceptors (39), depolarizing muscarinic
acetylcholine receptors (40), serotonin
and LSD receptors (29), and receptors
for bradykinin, neurotensin, angiotensin
II. and somatastatin (32), and have de-
fined cell responses to the ligands for
these receptors. Some receptors, such as
those for PGE,, mediate activation of
adenylate cyclase; other receptors such
as Met-enkephalin receptors, muscarinic
depolarizing acetylcholine receptors,
and alpha-2-adrenergic receptors medi-
ate inhibition of adenylate cyclase.
Increase of cyclic AMP in neuroblas-
toma or hybrid .cells for 5 to 7 days,
obtained either by treating cells with
PGE, to increase the endogenous rate of
Cyclic AMP synthesis or by inhibition of
Cyclic  nucleotide phosphodiesterase
with dibutyryl cyclic AMP, or theophyl-
line. resulted in increases in the percent-

Table 2. Effect of culture conditions on synap-
togenesis and acetylcholine secretion by
NGlOS-15 cells. Each value is the mean of
values obtained from more than 75 myotubes.
[Data from (32)]

Culture conditions

Myo-   Syn-
tubes   aptic
with     re-
syn-   sponse
apses    fre-
(%) quency*

Control
1 mM dibutyryl cyclic
AMP
1 mM theophylline
10 m PGE,
lOpMPGE, + 1mM
theophylline

IS     0.7
55     14

64     10
63     II
98     32

*The number per minute per myotube.

age of myotubes tested that were inner-
vated and the rate of spontaneous secre-
tion of acetylcholine from NG108-15
cells at synapses (32) (Table 2). Presum-
ably, each depolarizing response of a
myotube to acetylcholine is due to the
spontaneous secretion of acetyicholine
from a single NG108-15 vesicle. NG108-
15 cells and myotubes were cocultured
and treated for 5 to 7 days with the
compounds shown; then myotubes were
assayed for synapses by intracellular mi-
croelectrode recording. Treatment of
cells with 1 mM dibutyryl cyclic AMP,
1 mM theophylline, or 10 PM PGE, in-
creased the percentage of muscle ceils
tested that were innervated from 15 to

approximately 60 percent and increased
14- to 20-fold the frequency of spontane-
ous synaptic responses of myotubes (the
miniature end-plate potential frequency).
Treatment of cells with 10 PM PGE, and
1 mM theophylline resulted in innerva-
tion of 98 percent of the myotubes tested
and increased the frequency of synaptic
responses of myotubes 45-fold. No im-
mediate effect of these compounds on
the cell membrane potential or rate of
acetylcholine secretion was detected.
Half-maximal ingreases in synapses and
rate of sfiontaneous acetylcholine secre-
tion at synapses were observed when
cellular cyclic AMP levels were in-
creased for 1 to 2 days; maximal in-
creases were obtained when cells were
treated for 3 to 5 days (32).

In other experiments, NGIOS-15 cells
were incubated with PGE,, theophylline,
dibutyryl cyclic AMP, or PGE, and theo-
phylline for 5 to 7 days: then the com-
pounds were withdrawn and cells were
incubated for an additional 4 to 14 days
to determine whether the effects on syn-
apses and acetylcholine secretion were
reversible. On withdrawal of the com-
pounds, synapses and acetylcholine se-
cretion gradually returned to control val-
ues in 7 to 11 days (32). Thus, the effects
of the compounds on synapses are ex-
pressed slowly and are long-lived.

Cyclic AMP levels of NG108-15 cells
increase markedly in the presence of 10
PM PGE, and 1 mM theophylline and

Fig. 2. Neuroblastoma hybnd cells from lines that form many synapses with cultured myotubes
were treated for 7 days with 1 r&f dibutyryl cyclic AMP and the concentration of fetal bovine
serum was reduced from 5 to 1 percent between day 5 and day 7. The bar in the lower right hand
panel corresponds to 50 km in each panel. [Data from (32)]

797


remain higher than those of control cells
for seven or more days. `Intracellular
acetylcholine in NG108-15 cells also in-
creases eight- and threefold when cells
are treated for 3 days with PGE, and
theophylline or dibutyryl cyclic AMP,
respectively (32). NGlOS-15 cells treated
with dibutyryl cyclic AMP (41) or PGE,
and theophylline (32) for five or more
days contain many large dense-core vesi-
cles and small clear vesicles, whereas
control cells contain few vesicles. The
cyclic AMP-dependent increase in intra-
cellular acetylcholine is due, at least in
part, to an increase in the number of
acetylcholine storage vesicles in cells.
Depolarization of NG108-15 or NBrlO-
A cells with 80 mM K+ ions, in place of
80 mM Na+ ions, has no effect on the
rate of acetylcholine secretion bjl un-
treated NG108-15 or NBrlO-A cells.
However, cells gradually are shifted
from an unresponsive to a responsive
state with respect to depolarization-de-
pendent secretion of acetylcholine when
treated for 5 to 7 days with 1 mM dibu-
tyryl cyclic AMP or 10 pM PGE, and
1 mM theophylline. Half-maximal and
maximal increases in acetylcholine se-
cretion due to cell depolarization were
obtained when NG108-15 cells were
treated with 1 mM dibutyryl cyclic AMP
for 2 and 5 days, respectively (42).
Depolarization of nerve terminals is
known to activate voltage-sensitive Ca2+
channels; Ca2+ ions then flow into the
cytoplasm of axon terminals and in-
crease the rate of secretion of transmitter
at the synapse. We therefore examined
the effect of prolonged elevation of cy-
clic AMP levels of NBrlO-A or NGlOS-
15 cells on voltage-sensitive Ca*+ chan-
nel activity. Four kinds of assays were
used (34). 45Ca2+ flux, net uptake of
Ca*' by cells was measured with a Ca*+
specific electrode, Ca2' fluxes were de-
termined in the presence of murexide by
a spectrophotometric assay with a
stopped-flow apparatus, and Ca" action
potentials of cells were assayed by intra-
cellular microelectrode recording. We
found by each method of assay that
logarithmically dividing cpntrol cells
have little or no voltage-sensitive Ca*+
channel activity; however, prolonged el-
evation of cellular cyclic AMP activation
of adenylate cyclase of cells with PGE, ,
or by inhibition of cyclic nucleotide
phosphodiesterase with dibutyryl cyclic
AMP or theophylline, gradually results
in the acquisition of functional voltage-
sensitive Ca2+ channels by cells. Assay
of Ca*' action potentials elicited by elec-
trical stimulation of single cells with in-
tracellular   microelectric  recording
showed that most untreated NG108-15 or
NBrlO-A cells lack functional voltage-

sensitive Ca*+ channels. However, Ca"  with PGE, and theophylline or dibutyryl
action potentials were found in 100 per-  cyclic AMP for 2 and 4 days, respective-
cent of the cells tested that had been ly.
treated for four more days with dibutyryl   Relatively weak voltage-sensitive
cyclic AMP.            Ca2+ channel activity appears in untreat.
As shown in Fig. 3, 45Ca2+ uptake by  ed NBrlO-A cells when cells form co,,-
logarithmically dividing, control NBrlO-  fluent monolayers. Thus, cell concentra.
A ceils is not affected by depolarization  tion or adhesive interactions between
of cells with 80 mM K+. However, cells  cells also regulates the expression of
that had been treated for 7 days with 10  voltage-sensitive Ca'+ to some extent.
pM PGE, and 1 mM theophylline or   Nitrendipine and other dihydropyc.
with 1 mM dibutyryl cyclic AMP re-  dine derivatives inhibit voltage-sensitive
spond to depolarization by 80 mM K+
with a rapid influx of 4'Ca2+ via voltage- Ca2+ channels of smooth muscle (43),
                   striated muscle (44), and cardiac muscle

sensitive Ca*+ channels (34). Depolar- (45), and specific binding sites for 3H-
ization-dependent 45Ca2+ uptake is in-  labeled nitrendipine have been found in
hibited completely by 1 X 10e4M D-600  these tissues and in brain (46). The ni-
(half-maximal inhibition was obtained  trendipine receptors are thought to be
with 9 X lo-`M D-600), an alkaloid part of the voltage-sensitive Ca2+ chan-
known to inhibit voltage-sensitive Ca2+  nel complex, perhaps functioning as reg
channels and slow Na+ channels. 45Ca2+  ulators of channel activity.
uptake also is inhibited by La3+, Co'+,   Kongsamut and Miiller have shown
and Ni*+ ions.            that 45Ca2+ uptake by NG108-I5 cells
Exposure of NG108-15 cells to PGE,  mediated by voltage-sensitive  Ca2+
increases cellular cyclic AMP levels  channels is inhibited by nitrendipine
within seconds: however, no immediate  (47). We have confirmed this and find
effects of PGE,, PGE, and theophylline,  that NBrlO-A cells are inhibited half-
or dibutyryl cyclic AMP on voltage-sen-  maximally by 3 nM nitrendipine. A sin-
sitive Ca2+ channel activity were detect-  gle class of specific binding sites for 3H-
ed. Half-maximal and maximal voltage-  labeled nitrendipine was found in mem-
sensitive Ca2' channel activity were ex-  branes from NBrlO-A cells that had been
pressed by cells that had been treated  treated with PGE, and theophylline with
                    a dissociation constant, estimated by
                 Scatchard analysis, of 2 x lO-"M,
                    which is similar to values reported for
             POE, o theophylline
                 o  other tissues (43, 4546). The maximum
                   number of specific nitrendipine binding
                    sites was estimated to be 61 fmole per
                    milligram of NBrlO-A membrane pro-
                    tein, which is equivalent to approximate-
                   ly 16,000 specific sites for nitrendipine
                  per cell. In contrast, few or no specific
                 binding sites for 3H-labeled nitrendipine
                       were detected in membranes from un-
                  treated, logarithmically dividing NBrlO-
                   A cells. These results show that cyclic
                 AMP regulates the number of specific
                nitrendipine receptors per cell. Specific
                 binding sites for 3H-labeled nitrendipine
                Control cells       also were not detected in membranes
                prepared from two lines of hybrid cells
    0     2     4    8     0    10
            Time (minuted       (SB21B-1 and SB37-B) that lack func-
                 tional voltage-sensitive Ca2+ channels
Fig. 3. The effect of culture conditions on the
expression of functional voltage-sensitive  and do not synapse with muscle cells.
Ca'+ channels of NBrlO-A cells. Uptake of  Cyclic AMP increases the probabilitY
"Cat' due to activation of voltage-sensitive  of opening Ca*+ channels of cardiac
Ca" channels of untreated logarithmically  muscle cells (48); however, responses t0
dividing control NBrlO-A cells, cells cultured  cyclic AMP are rapid and thus differ
for 6 days with 1 mit4 dibutyryl cyclic AMP,
or 10 @I4 PGE, and 1 m&I theophylline. The  from the slow effects found with NBrlO-

cells were depolarized with 80 mM K' (in  A cells.
place of 80 ITIM Na+). Values for +Za'+   The molecular weights of nitrendipine
binding to cells or uptake at 5.4 m&I K',  receptors in intact membranes of smooth
which were not inhibited by 100 m D-600
and were not mediated by voltage-sensitive  muscle (49), transverse tubule mefl
Ca" channels, were subtracted from the val- branes of skeletal muscle, and cerebral
ues shown. Uptake of Ca*' dependent on cell  cortex synaptic membranes (44) Were
depolarization was completely inhibited by  estimated by radiation inactivation tar@'
100 p& D-600. [Data from (34)]     analysis to be 278,000, 210,000, and


210,000, respectively. Available infor-
mation suggests that the nitrendipine re-
ceptor complex is a glycoprotein with N-
acetylglucosamine or sialic acid residues
(or both) (50). Nitrendipine receptors of
smooth and cardiac muscle were report-
ed to be covalently labeled with a radio-
active affinity label analog of nitrendi-
pine,  3H-labeled 2,6-dimethyl-3,5-di-
carbomethoxy - 4 - (2 - isothiocyanato-
phenyl)- 1,4-diiydropyridine; labeled pro-
tein then was solubilized and fractionat-
ed. A peak of labeled protein with a
molecular weight of 45,000 was identi-
fied (49). These results suggest that the
molecular weight of voltage-sensitive
Ca*+ channel in membranes is 210,000 to
278,000, that each channel is composed
of two or more subunits, and that one
subunit is a protein with a molecular
weight of 45,000, which binds nitrendi-
pine.
NG108-15 cells that had been grown
with or without 10 m PGE, were incu-
bated with ["Slmethionine to label the
protein; the 35S-labeled glycoproteins
then were solubilized and fractionated
by wheat germ agglutinin-, ricin-, or len-
til-lectin column chromatography and by
two-dimensional gel  electrophoresis
(51). Elevation of cellular cyclic AMP
levels resulted in the disappearance of
some 3'S-labeled glycoproteins, the ap-
pearance of new 35S-labeled glycopro-
teins with different molecular weights,
changes in the apparent abundance of
some 35S-labeled glycoproteins, as well
as changes in the isoelectric points of
other 35S-labeled glycoproteins. A "S-
labeled glycoprotein with a molecular
weight of approximately 45,000 was elut-
ed from wheat germ agglutinin-sepha-
rose with N-acetylglucosamine was ob-
tained from cells with high cyclic AMP
levels, but was not detected in untreated
cells. Twelve "S-glycoproteins were de-
tected that were expressed by NG108-15
cells with high cyclic AMP levels but not
by control cells, and many other )$-
labeled glycoproteins were obtained
from PGE,-treated cells with radioactiv-
ities 2.5- to IO-fold higher than those of
control cells. These results extend previ-
ous reports of differentiation-specific
changes in neuroblastoma proteins (52).

Exposure of neuroblastoma or hybrid
cells to dibutyryl cyclic AMP alters the
levels of some species of polysomal
mRNA (53). Polysomal polyadenylated
(~01~ A+) RNA from "undifferentiated"
and  "differentiated" neuroblastoma
cells were compared; many species of
Polysomal poly A' RNA were found in
RNA from undifferentiated cells, but not
differentiated cells (54, 55), and con-
versely, many species of poly A+ RNA
were expressed by differentiated neuro-

blastoma cells that were not expressed
by undifferentiated cells (55).

In prokaryotic cells (cyclic AMP * ca-
tabolite activator protein) complexes
bind to certain sites on DNA and thereby
regulate the initiation of transcription of
certain genes. Cyclic AMP also regulates
the levels of some species of mRNA and
protein in eukaryotic cells (56), but rela-
tively little is known about the mecha-
nisms of regulation. Cyclic AMP mark-
edly increases the expression of many
neural properties in neuroblastoma or
hybrid cells, such as voltage-sensitive
channels for Ca2', Naf, and KC, and
also Ca*+-dependent K' channels, neur-
ite extension, vesicles, synapses, acetyl-
cholinesterase, and with some cell lines,
choline acetyltransferase, or tyrosine hy-
droxylase activities. We find that cyclic
AMP regulateg synaptogenesis, at least
in part, by regulating the expression of
voltage-sensitive Ca*+ channels, which
are required for stimulus-dependent se-
cretion of transmitter at synapses. The
results suggest that cyclic AMP affects
posttranslational modifications of some
species of glycoprotein. Appropriate
cloned cDNA probes are needed to de-
termine whether cyclic AMP affects the
levels of some species of mRNA and to
define further the cyclic AMP-depen-
dent mechanisms that affect synapse for-
mation and plasticity.

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