Proc. Naf. kad. Sci. USA
Vol. 72, No. 9, pp. 3472-3476, September 1975
Biochemistry

Receptor-mediated shifts in cGMP and CAMP levels in
neuroblastoma cells

(acetylcholine receptor/prostaglandin El receptor/adenosine receptor/synapse/neurotransmitter)

HIROSHI MATSUZAWA AND MARSHALL NIRENBERG
Laboratory of Biochemical Genetics, National Heart and Lung Institute. National Institutes of Health, Bethesda, !kuyland 20011
Contributed by Marshall Nirenberg, July 10,19X

ABSTRACT   3':5'-cCMP levels of neuroblastoma NlE-115

cells increase as much as 2OO-fold upon activation of muscar-
inic acetvlcholine recenters. resulting in intracellular cGMP

                .
concentritions >600 pmol/k of pro;ein. The cells also have
receptors for adenosine whlc mediate an increase in 3':5'-
        3
cAaP levels. Unexpectedly, prostaglandin El was found to
increase the concentrations of both cCMP and CAMP. Car-
bamylcholine, adenosine, and PGEl were added to cells sep-
arately and in pairs to determine the effect of one compound
on cell responses to another. Reciprocal inhibition, unilateral
inhibition, additive, and nonadditive responses were ob
served with respect to cCMP and CAMP levels when differ-
ent pairs of receptors were activated simultaneously-

The response of neurons to transmitters frequently can be
modified by other species of (transmitter.receptor] interac-
tions; thus, two types of synaptic stimuli may potentiate or
inhibit one another. The pharmacology and electrophysiolo-
gy of such phenomena have been studied extensively but
much remains to be learned about the mechanisms which
couple the activities of different species of receptors. In part,
the difficulty in defining the reactions associated with re-
ceptor activation stems from the heterogeneity of neural
tissues with regard to cell type. One approach to this prob-
lem is to define receptor-mediated responses of relatively
homogeneous, clonal neuroblastoma cells which have excit-
able membranes and respond to chemical and electrical
stimuli.

In this report the effects of activators of inhibitory mus-
carinic acetylcholine receptors, adenosine receptors, and
prostaglandin El (PGEl) receptors (l-5) on 3':5'-cGMP and
3':5'-CAMP levels of neuroblastoma cells are described. Mus-
carinic acetylcholine receptors frequently mediate slow,
prolonged responses which may be excitatory or inhibitory;
in contrast to the nicotinic acetylcholine receptor, which in
higher organisms mediates fast excitatory responses. Activa-
tion of the inhibitory or excitatory muscarinic acetylcholine
receptors of cardiac (6, 7) and smooth muscle (7) results in
an increase in cGMP concentration. We find that cGMP lev-
els of neuroblastoma NlE-115 cells are elevated as much as
200-fold upon activation of muscarinic acetylcholine recep-
tors. When different pairs of receptors were activated simul-
taneously, reciprocal inhibition, unilateral inhibition, addi-
tive, and nonadditive responses were observed with respect
to cGMP and CAMP levels.

      MATERIALS AND METHODS
Chemicals were obtained from the following sources: car-
bamylcholine chloride, atropine sulfate, and d-tubocurarine

Abbreviations: cGMP, guanosine  3'6'~cyclic monophosphate;
CAMP, adenosine 3':5'-cyclic monophosphate; PGE,, prostaglandin
El; IBMX, 3-isobutyl-l-methyl-xanthine; Hepes, N-2-hydroxy-
ethylpiperazine-N'-2-ethanesulfonic acid; DMEM, Dulbecco-Vogt
modification of Eagle's minimal essential medium.

chloride from Sigma; N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic acid (Hepes), Calbiochem; S-isobutyl-l-
methyl-xanthine (IBMX) from Aldrich Chem. Co.; PGE1, a
gift from Dr. John Pike, the Upjohn Co.; adenosine, Nutri-
tional Biochem. Corp.; CAMP, SchwarzjMann; cGMP, JEM
Research Products; (G-3H]cAMP (22.1 Ci/mmol), New En-
gland Nuclear; [8-3H]cGMP (15 Ci/mmol), Amersham/
Searle Corp.; CAMP-dependent protein kinase, Sigma;
cGMP antiserum and succinyl-cGMP `=I-tyrosine methyl
ester, Collaborative Research, Inc.; fetal bovine serum, Colo-
rado Serum Co.; and DMEM (the Dulbecco-Vogt modifica-
tion of Eagle's minimal essential medium), GIBCO, Cat. no.
H-21. Other chemicals were of reagent grade purity.

Cell Culture. The.following cell lines were used: mouse
neuroblastoma C-1300 clones NlE-115 and N18 (8). mouse
neuroblastoma X mouse L cell hybrid clone NLlF (9), and
rat glioma clone CGBU-1 (10). Neuroblastoma cells were
grown in 100 mm petri dishes (Falcon, 55 cm" surface area)
in 15 ml of 90% DMEM-10% fetal-bovine serum (340
mOsm/kg) in a humidified atmosphere of 10% COZ-90% air
at 37". Hybrid cells were grown in the same medium sup-
plemented with 0.1 mM hypoxanthine, 1 PM aminopterin,
and 16 PM thymidine (HAT). Cells usually were grown to
confluency (1 to 3 X I@ cells, 0.1 to 0.3 mg of protein per
cm2) prior to use.

Incubation of Cells. Each plate was washed three times
with 7 ml portions of solution A (DMEM minus h'aHC03
with 25 mM Hepes adjusted to pH 7.4 with NaOH and to
340 mOsm/kg with NaCl). Unless stated otherwise, 10 ml of
solution A supplemented with 0.5 mM IBMX were added to
each plate and cells were equilibrated for 30 min at 37". Re-
actions were initiated by the addition of 0.1 ml of a solution
of the compound to be tested dissolved in 0.17 M NaCl, or
0.1 ml of a PGE] solution (1 mh4 PGE,, 0.153 M NaCI, and
10% ethanol). The final concentration of ethanol in the petri
dish, O.l%, did not alter cGMP or CAMP levels of cells. Cells
were incubated at 37" for the times specified;then the me-
dium was removed by aspiration (discarded unless specified
otherwise) and 4 ml of cold 5% trichloroacetic acid was
added to the monolayer to terminate reactions and extract
nucleotides. A solution (0.05 ml) containing 10,000 cpm of
[3H]cGMP (0.67 pmol) and/or [3H]cAMP (0.45 pmol) was
added to each dish and cells were detached by scraping. The
suspension was transferred to a tube, the dish was washed
with 1 ml of cold 5% trichloroacetic acid, and the combineti
suspension and wash was centrifuged for 15 min at 20,000 X
g at 3".

Purification of cGMP and CAMP. A rapid method was
devised for separating cGMP from CAMP and removing tri-
chloroacetic acid and endogenous interfering compounds
from cyclic nucleotide fractions. The supernatant solution of
the deproteinized sample in 5% trichloroacetic acid was ap-
plied to an ion exchange column (0.7 X 8 cm) of AG50W-Wd

3472


Biochemistry: Matsuzawa and Nirenberg

(200-400 mesh, hydrogen form, Bio-Rad) previously washed
and equilibrated with H20. The column was washed with 2
ml of Hz0 (eluates discarded) and then with 2 additional ml
of HzO. The latter eluate (2.0 ml) which contains cGMP was
collected on a neutral alumina (WN-3, Sigma) column (0.7
X 3 cm) previously washed with 20 ml of 200 mM sodium
acetate, pH 6.2, and then with 10 ml of 5 mM sodium ace-
tate, pH 6.2. The eluate was discarded, and the column was
washed with 3 ml of 5 mM sodium acetate buffer, pH 6.2,
and then with 1 ml of 200 mM sodium acetate buffer, pH
6.2 (eluates discarded). Then cGMP was eiuted with 2 ml of
200 mM sodium acetate, pH 6.2, and portions of the eluate
were assayed for cGMP concentration and for recovery. The
average recovery of cGMP was about 50%; <l% of the
CAMP of the sample was present in the cGMP fraction.
Cyclic AMP fractions were obtained as follows: after
cGMP was eluted from the AGSOW-X4 column, the column
was washed with 2 ml of Hz0 (eluate discarded) and CAMP
was eluted with 3 additional ml of H20. Portions of the el-
uate were assayed for CAMP concentration and recovery.
The CAMP fraction contained <0.2% of the cGMP of the
sample prior to fractionation. The average recovery of
[3H]cAMP was 70%.

Each AGSOW-X4 column was regenerated by washing
with 8 ml of 1 N HCl and, prior to use, with 30 ml of water.
Assay of cGMP and CAMP. The cGMP concentrations of
purified alumina column eluates were determined by the ra-
dioimmune method of Steiner et al. (11) modified as fol-
lows: each 0.5 ml reaction mixture contained 200 mM (in
some experiments 140 mM) sodium acetate, pH 6.2 (solution
B); 17 fmol of succinyl-cGMP 1251-tyrosine methyl ester
(lO,OOO-20,000 cpm); cGMP antibody; and sample (O-20
pmol of cGMP). After incubation (14-18 hr. 3") each reac-
tion mixture was diluted with 2 ml of solution B at 3"; 10
min later the sample was passed through a Millipore filter
(0.45 pm pore size, 25 mm diameter, HAWP 02500) pre-
viously rinsed with solution B and washed three times with
cold solution B (4 ml each wash). Each filter was placed in a
scintillation vial with 1 ml of 2-methoxyethanol; after filters
dissolved (0.5-I hr) I5 ml of Aquasol (New England Nucle-
ar) were added. Radioactivity was determined 12-24 hr
later in a Beckman liquid scintillation counter (`H plus `"C
isoset).

Cyclic AMP concentrations of samples purified as de-
scribed above were determined by the method of Gilman
(12). Each cGMP or CAMP sample was assayed at three or
four concentrations. Each value shown corresponds to 100%
recovery of cyclic nucleotide and is the mean of duplicate
dishes. Protein was determined by a modification of the
method of Lowry et al. (13).

RESULTS

A rapid method was devised for separating cGMP from
CAMP and removing trichloroacetic acid and endogenous
factors which interfere with the cGMP assay. The proce-
dure, based upon the Materials and Methods section, is
based upon the methods of Salomon, Londos, and Rodbell
(14) and White and Zenser (15); cGMP and CAMP are recov-
ered in separate fractions in high yield and in sufficiently
high concentration to assay directly without lyophilization.
One hundred samples can be purified in 2-3 hr.

The effect of carbamylcholine, a relatively stable analog
of acetvlcholine, upon intracellular cGMP levels of neuro-
blastoma and other cell lines in the absence of a 3':5'-cyclic
nucleotide phosphodiesterase inhibitor is shown in Fig. 1.
Cyclic GMP levels of neuroblastoma clones N18 and NlE-

Proc. Nat. Acad. Sci. USA 72 (1975)   3473

600

5
E
a200
             NIE-115

MINUTES

FIG. 1.  Effect of 1 mM carbamylcholine on intracellular levels
of cGMP in neuroblastoma clones NlE-115 and N18, neuroblasto-
ma x L cell hybrid clone NLlF, and glioma clone CGBU-1. Cul-
tures were confluent; the average protein content was: 11.5, 16.4,
7.6 and 6.9 rng of nrotein ner 100 mm oetri dish for NlE-115. N18.
NLlF, and c6Bu-1, resiectively. A `3':5'-cyclic nucleotide `phos:
phodiesterase inhibitor was not present.

II5 rose 40- and 2IO-fold, respectively, upon incubation
with carbamylcholine for 15 sec. and then fell rapidly to
normal levels (50% decrease in cGMP at 45 set). Carbamyl-
choline also elicited a 6-fold increase in the cGMP concen-
tration of the neuroblastoma X L cell hybrid, NL-lF, but
had no effect upon cGMP levels of rat glioma clone CGBU-1.
Thus, two types of cell lines were found with respect to ace-
tylcholine-receptor-mediated  reactions  which  increase
cGMP levels: cell lines which are sensitive to carbamylcho-
line and a line which is insensitive.

The effect of IBMX, a 3':5'-cyclic nucleotide phosphodies-
terase inhibitor, upon the rate of disappearance of intracel-
lular cGMP after stimulation with carbamylcholine is shown
in Fig. 2A. In the absence of IBMX, maximum cGMP levels
were attained 15 set after the addition of carbamylcholine,
and then rapidly decreased (50% decrease at 45 set; Figs. 1
and 2A). In the presence of 0.5 mM IBMX, maximum cGMP
levels were attained at 30 set and cGMP levels then de-
creased at a slower rate (50% decrease at 3 min).

The effect of IBMX upon carbamylcholine dependent
cGMP accumulation also was studied with NlE-115 cells
adapted to grow in suspension culture. The assay was modi-
fied so that both intra- and extracellular cGMP was deter-
mined. As shown in Fig. 2B, carbamylcholine elevated
cGMP levels of suspension cells and in the presence of IBMX
cGMP levels remained constant during the 10 min incuba-
tion period. These results suggest that part of the cGMP
formed by cells may be excreted into the medium. Other
3':5'-cyclic nucleolide phosphodiesterase inhibitors such as
4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone  (Ro-20-
1724) and papaverine were not as effective as IBMX in
maintaining cGMP levels of N18 cells.

The relation between carbamylcholine concentration and
cGMP accumulation in NlE-115 cells is shown in Fig. 3.
The maximum increase in cGMP concentration was ob-
tained with lo-" M carbamylcholine; 50% of the maximum
increase in cGMP concentration was evoked by 10mJ M car-
bamylcholine.

The effects of atropine and d-tubocurarine, selective in-
hibitors of muscarinic and nicotinic acetylcholine receptors,


3474  Biochemistry: Matsuzawa and Nirenberg



         1000   ' ' ' ' I / ' ' ' '
     !i-@

I k      MONOLAYER CELLS    4

    SUSPENSION CELLS AND MEDIUM

200 7

SUSPENSION CELLS AND MEDIUM

5ttc   1 1   1 1   ' '   1 1   ' '   ' '   ' '   ' '  11
                       '
0 I  2 2  3 3  4 4  5 5  6 6  7 7  8 8  9 9  IO
                       IO
        MINUTES
        MINUTES

FIG. 2.  Effect of 1 mM carbamylcholine in the presence of 0.5
mM IBMX, a 3':5'-cyclic nucleotide phosphodiesterase inhibitor,
on cGMP levels of neuroblastoma NIE-115 cells. (A) Cells were
grown for 9 days to confluence; the average cellular protein was
12.2 mg/lOO mm dish. The broken line corresponds to the data for
NIE-I15 shown in Fig. 1 (cells were incubated in the absence of
IBMX). (B) Cells were grown in suspension culture in DMEM
with 10 mM NaHCO:s, 25 mM Hepes, and 2% fetal bovine serum
(pH 7.3, 340 mOsm/kg). Cells were washed and 1 ml portions (4 X
10s cells) were preincubated in the presence of 0.5 mM IBMX for
15 min with gentle shaking. Two experiments are shown. Cells
were incubated for 3 min or less (A) with 1 mM carbamylcholine;
or (A) without this compound. In another experiment (I) cells
were incubated for 0 to 10 min with 1 mM carbamylcholine. In
both experiments reactions were terminated by the addition of 4
ml of cold 6% trichloroacetic acid to each tube. Hence the com-
bined intra- and extracellular cGMP concentration was deter-
mined.

respectively, upon carbamylcholine-dependent accumula-
tion of cGMP in NlE-115 cells are shown in Fig. 4. Atro-
pine, at 1 X lo-' M, inhibited the carbamylcholine-depen-
dent increase in cGMP concentration by 50%; 97% inhibi-
tion was obtained with 1 X 10m6 M atropine. In contrast, d-
tubocurarine did not inhibit appreciably even at 1 X 10e4
M; in fact, in other experiments d-tubocurarine potentiated
the carbamylcholine-dependent elevation of cGMP. The in-
hibition by atropine suggests that the increase in cGMP elic-
ited by carbamylcholine is mediated by the muscarinic ace-
tylcholine receptors.

The effects of PGEl upon CAMP and cGMP levels of
NlE-115 cells are shown in Fig. 5. Cyclic AMP levels were
found to increase markedly upon the addition of 10 PM
PGEl. Unexpectedly, the level of cGMP also increased 6-
fold in the presence of PGEl. The maximum concentration
of CAMP attained in this experiment was g-fold higher than
that of cGMP; however, in other experiments, the concen-
tration of cGMP dependent on PGEl increased to 250 pmol

Proc. Nat. Acad. Sci. USA 72 (1975)

MCXARlTY

FIG. 3 (left).  Relation between carbamylcholine concentration
and cGMP levels of neuroblastoma NIE-115 cells. Cultures were
grown for 9 days to confluence; the average cellular protein was
13.5 m&00 mm dish. Cells were incubated for 30 set in the pres-
ence of the indicated concentrations of carbamylcholine and
IBMX.

WC. 4 (right).  Effect of atropine and d-tubocurarine concen-
tration on the carbamylcholine dependent increase in cGMP level
of NIE-115 cells. Cultures were grown for 9 days to confluence; the
average cellular protein was 14.6 mg/lOO mm dish. Cells were incu-
bated for 30 set in the presence of 1 mM carbamylcholine and the
indicated concentrations of atropine or d-tubocurarine.

of cGMP per mg of protein, approximately 60% of the con-
centration attained by CAMP in the presence of PGEl. In
other experiments not shown here, the increase in cGMP
concentration elicited by PGEl was shown to be insensitive
to atropine. Thus, the effect of PGEl upon cGMP is not me-
diated by the muscarinic acetylcholine receptor. The maxi-
mum cGMP concentration was achieved 30 set after the ad-
dition of PGEl; cGMP levels decreased during subsequent
incubation. In contrast, the maximum CAMP level was
achieved 4 min after the addition of PGEi.

The effects of carbamylcholine. PGE1, and adenosine
upon cGMP and CAMP levels of NlE-115 cells plotted on a
logarithmic scale to illustrate inhibitory as well as stimulato-
ry effects are shown in Fig. 6A and B, respectively. In the
presence of carbamylcholine or PGE,, cGMP levels in-
creased and reached maximum levels within 1 min. The
rates of disappearance of cGMP during the subsequent incu-
bation period were linear. In contrast, the basal level of
cGMP was reduced approximately 50% in the presence of
0.1 mM adenosine. As shown in Fig. 6B, CAMP levels in-
creased markedly in the presence of PGEl or adenosine, but
decreased 30% in the presence of carbamylcholine.

Various combinations of carbamylcholine, PGE1, and
adenosine were added simultaneously to determine the ef-
fect of one compound upon cell responses lo another (Fig.

MINUTES

FIG. 5. Effect of PGEl on intracellular levels of cGMP and
CAMP in neuroblastoma NlE-115 cells. Cultures were grown for c'
days; the average cellular protein was 6.8 mg/lOO mm dish. Closed
and open circles correspond to cGMP levels with and without 1"
FM PGEI, respectively; closed and open triangles correspond tc'
CAMP levels with and without 10 FM PGE,, respectively.


Biochemistry: Matsuzawa and Nirenberg             Proc. Nat. Acad. Sci. USA 72 (197.5)   3475

cGMP   ?ED    CAMP

w-  CONTROL   Cl I              -I

MINUTES

FIG. 6.  Effect of 1 mM carbamylcholine (carbachol), 0.1 mM
adenosine, or 10 MM PGEt on intracellular levels of cGMP (A) and
CAMP (B) of neuroblastoma NlE-115 cells. Culture conditions
were identical to those described in the legend to Fig. 5.

7). The effect of exposing cells to both carbamylcholine and
adenosine upon cGMP and CAMP levels is shown in Fig. 7A
and B, respectively. Adenosine inhibited the carbamylcho-
line-dependent increase in cGMP concentration 25 and 60%
when incubations were 30 and 60 set, respectively.

In Fig. 7B, the effect of carbamylcholine upon the adeno-
sine-dependent increase in CAMP concentration is shown.
Carbamylcholine did not inhibit the adenosine-stimulated
increase in CAMP during the first 30 set of incubation even
though the maximum increase in cGMP concentration
evoked by carbamylcholine was achieved at 30 sec. How-
ever, during further incubation, carbamylcholine inhibited
almost completely the adenosine-dependent elevation in
CAMP concentration.

Carbamylcholine and PCEr added separately increased
cCMP levels as indicated by the broken lines in Fig. 7C. The
effects of carbamylcholine and PGEi added simultaneously
to cultures were additive or more than additive. However,
carbamylcholine inhibited the PGEi-induced increase in
CAMP concentration, particularly during the latter part of
the incubation period (Fig. 7D). Thus, carbamylcholine may
have a delayed inhibitory effect upon both adenosine- and
PGEi-dependent increases in CAMP concentrations. Atro-
pine completely blocked the inhibitory effect of carbamvl-
choline upon adenosine- or PGEr-dependent increases *in
CAMP levels (data not shown).

The effect of activating receptors for adenosine and PGEl
separately and simultaneously upon cGMP and CAMP levels
is shown in Fig. 7E and F, respectively. Adenosine inhibited
slightly the rise in cGMP levels elicited by PGEr. The results
show that the increases in CAMP levels elicited either by
adenosine or PGEi are not additive. In other experiments,
the sequence of addition of carbamylcholine and either
adenosine or PGEi was varied, but the results did not differ
significantly from those found when two compounds were
added simultaneously.

DISCUSSION

Two types of cells were found with respect to muscarinic
acetylcholine receptor activity: cells sensitive to carbamyl-
choline and cells insensitive to this compound. The degree of
response also varied widely from one clone to another. Acti-
vation of the muscarinic acetylcholine receptors of neuro-

I      ADENOSINE +PGE,  ,I' p A+P  300

MINUTES

FIG. 7.  Effect of various combinations of 1 mM carbamylcho-
line, 0.1 mM adenosine, and 10 JLM PGE, on intracellular levels of
cGMP (panels A, C, and E) and CAMP (panels B, D, and F) of neu-
roblastoma NlE-115 cells. Culture conditions were identical to
those described in the legend to Fig. 5. The broken lines corre-
spond to the effect of each compound added separately (data
shown in Fig. 6); C, A, and P represent carbamylchoiine, adeno-
sine, and PGEr, respectively.

blastoma NlE-115 cells evoked up to a 2OO-fold increase in
cGMP concentration, yielding levels in excess of 600 pmol of
cGMP per mg of protein. These cells have high tyrosine hy-
droxylase activity (8), generate action potentials in response
to electrical stimuli (8), have neurosecretory vesicles, and
also possess PGEi receptors and adenosine receptors which
upon activation lead to elevated CAMP levels (2, 3).

Adenosine, carbamylcholine, and PGEr were added to
cells separately and in pairs to determine the effect of one
compound upon cell responses to another. The results, sum-
marized in Table 1, show that in the presence of adenosine
CAMP levels increase and cGMP levels decrease; whereas, in

Table 1.  Summary of receptor-mediated shifts in cGMP
    and CAMP levels in neuroblastoma cells

cGMP

CAMP

      Addition                percent

None                        100        100
Adenosine                      40        500
Carbamylcholine               1500         70
PGE,                         500       1250
Adenosine + carbamylcholine        650        250
PGE, + carbamylcholine           2050       1000
PGE, + adenosine                300       1100

100% corresponds to 7.5 pmol of cGMP/mg of protein or 27.5
pmol of cAMP/mg of protein. The data are from the experiments
shown in Figs. 6 and 7.


3476  Biochemistry: Matsuzawa and Nirenberg
the presence of carbamylcholine, cGMP levels increase and
CAMP levels decrease. In addition to the inverse relationship
between cGMP and CAMP concentrations, a novel phenom-
enon was observed; that is, in the presence of PGEl the lev-
els of both cGMP and CAMP increase. The increase in cGMP
elicited by carbamylcholine was not inhibited by PGEl in
spite of the fact that PGEl elicits a marked increase in
CAMP levels. Prostaglandin-El- and carbamylcholine-de-
pendent increases in cGMP were fully additive, and in some
experiments, were greater than additive. In contrast, the ele-
vated CAMP concentrations induced either by PGEl or by
adenosine were not additive when both compounds were
added simultaneously to cells.

We conclude that activation of a species of muscarinic
acetylcholine receptor inhibits adenosine- and PGEl-recep-
tor-mediated reactions which elevate CAMP levels; whereas,
activation of adenosine receptors inhibits reactions mediated
by muscarinic acetylcholine receptors and PGEl receptors
which elevate cGMP levels. In contrast to these results, acti-
vation of PGEl receptors did not affect acetylcholine- or
adenosine-receptor-mediated reactions which affect cGMP
or CAMP levels. Thus, reciprocal inhibition, unilateral inhi-
bition, additive, and nonadditive responses were observed
when different pairs of receptors were activated simulta-
neously.

Relatively little is known at the molecular level about the
mechanisms which couple one receptor with another. The
observed phenomena are obviously complex and may reflect
changes in membrane permeability, metabolism of cyclic
nucleotides, and so forth. Other examples of reciprocal cou-
pling of cGMP and CAMP levels and possible mechanisms of
coupling have been discussed by Goldberg et al. (22, 23).
Blume et al. (5) have reported that acetylcholine inhibits
the increase in CAMP levels induced by PGEl or adenosine
in mouse neuroblastoma cells. Acetylcholine also has been
shown to inhibit the PGEl-dependent elevations of CAMP
levels, the inhibition mediated by excitatory muscarinic ace-
tylcholine receptors in neuroblastoma X glioma hybrid cells
(16). Acetylcholine and carbamylcholine also have been re-
ported to stimulate mouse neuroblastoma adenylate cyclase
activity (17).

The inhibitory effect of carbamylcholine on reactions me-
diated by other species of receptors shows that some NlE-
115 cells with muscarinic acetylcholine receptors also have
adenosine or PGEl receptors. Some cells also have both
adenosine receptors and PGEl receptors. Although NlE-115
is a clonal cell line, single cell analysis by electrophysiologic
methods reveals two acetylcholine receptor phenotypes; cells
sensitive and cells insensitive to carbamylcholine. All NlE-
115 cells sensitive to acetylcholine or carbamylcholine exhib-
it hyperpolarizing responses to these compounds, frequently
-25 mV in magnitude and lo-15 set in duration. Hence,
NlE-115 cells have inhibitory muscarinic acetylcholine re-
ceptors.

The activity of PGEl in eliciting increases in both cGMP
and CAMP levels should be considered in context with the
different actions of PGEl on various tissues which have been
reported. For example, PGEl elevates CAMP levels in some
tissues and reduces hormone-dependent elevation of CAMP
levels in others (18), activates adenylate cyclase activity in

Proc. Nat. Acad. Sci. USA 72 (1975)

homogenates (19), including those prepared from neuroblas-
toma x glioma hvbrid cells (20), and inhibits the vasopres-
sin-dependent activation of adenylate cyclase (21).
Prostaglandin El elicits a transitory, 30 sec. increase in
cGMP accumulation and a prolonged increase in CAMP ac-
cumulation. The difference in the duration of cGMP and
CAMP accumulations may reflect the activities of different
enzvmes which catalyze the synthesis and perhaps the hy-
droiysis of the nucleotides, or alternatively, may indicate the
presence of two species of PGEl receptor, one mediating an
increase in CAMP concentration, the other, an increase in
cGMP. Preliminary results which support the latter hypoth-
esis will be described elsewhere.

We thank Doyle Illullinax, Deborah Carper, and Linda Lee for
their excellent assistance, and Mary Ellen Miller for typing the
manuscript.

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21


22

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