Journal of General Microbiology (1984), 130, 549-556.
Printed in Great Britain
549
Variation of Intracellular Cyclic AMP and Cyclic GMP Following
Chemical Stimulation in Relation to Contractility in
Physarum polycephdum
By TATSUO AKITAYA, T A T S U M I HIROSE, T E T S U O U E D A * A N D
YONOSUKE KOBATAKE
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan
(Received 20 September I983 ;revised 14 November 1983)
The plasmodium of Physarum polycephalum reacts to external stimuli tactically. Positive and
negative taxes correspond to relaxation and contraction, respectively. Variations of intracellular
CAMPand cGMP concentrations in response to chemical stimuli were examined in relation to
the regulation of contractility. Concentrations of the two cyclic nucleotides oscillated, with a
gradual shift, for some time after stimulation.The period of oscillation was 4-5 min, with phases
being the same in response to repellents, but different when attractants were tested. Therefore,
changes in the accumulation of the nucleotides summed over 15min were taken as a
quantitative measure of the external stimuli. Attractants (glucose, 2-deoxyglucose, galactose,
maltose) induced decreases both in CAMPand in cGMP concentration, the latter being larger
than the former. Repellents (KCl, CaCl,, MgCl,, sucrose)induced decreases both in CAMPand
in cGMP concentration, the former being larger than the latter. Variations of the intracellular
CAMPconcentration for repellents and those of cGMP concentration for attractants, took place
at similar concentrationsof stimulants as variations of contraction and relaxation, respectively.
Microinjection of cAMP and cGMP into the plasmodia1 strands induced contraction, cGMP
being about 10 times as effective as CAMP. The results indicate that both CAMPand cGMP
regulate the ability to contract, not antagonistically, but cooperatively, in the sensory
transduction of the Physarum plasmodium.
INTRODUCTION
The plasmodium of Physarum polycephalum reacts to chemical stimuli tactically (Knowles &
Carlile, 1978; Chet et al., 1977; Kincaid & Mansour, 1978). Positive and negative taxes
correspond to relaxation and contraction of the cell, respectively (Ueda et al., 1975, 1976; Hato
et al., 1976). What chemicals mediate the sensory signal and finally regulate the ability to
contract? It has been found that intracellular ATP concentration is increased by repellents
(Hirose et al., 1980a), and that intracellular pH is decreased by attractants (Hirose et al., 1982).
Although these findings are consistent with the regulation mechanism of an actomyosin system,
other important factors such as cyclic nucleotides are still left unstudied. Cyclic nucleotides play
a role in the chemotactic transduction of, for example, leucocytes (Schiffmann, 1982), and the
cellular slime mould Dictyostelium discoideum (Gerisch, 1982), but in Physarum involvement of
CAMPis only hinted at by the observation that inhibitors of adenyiate phosphodiesteraseact as
attractants (Kincaid & Mansour, 1979). Cyclic nucleotides may participate in regulating
contraction of the actomyosin system, but their role is still controversial in smooth muscle
(Goldberg & Haddox, 1977) and non-muscle cells.
Here we examine changes in intracellular cAMP and cGMP concentrations after chemical
stimulation and also changes in contractile activity produced by injecting these cyclic
nucleotides into the cell. Our results show that CAMPand cGMP have similar regulatory effects
on contractility, their increase and decrease corresponding to contraction and relaxation,
respectively.
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550
T. AKITAYA A N D OTHERS
METHODS
Organism. The true slime mould fhysanun plycephalm was cultured by the method of Camp (1936). Before
experimental use, the plasmodia were allowed to differentiate into tips and strands by migrating on wet filter
paper overnight without feeding. The contraction-relaxation cycle is synchronousthroughout the whole organism
(Grenbecki & Cieslawska, 1978).If the organism is separatedinto pieces, contractileactivityof each piece follows
a similar time course, at least for a while, when monitored optically (Hirose et al., 19806).
Measurements of intracellular cAMP and cGMP concentrations afier chemical stimulation. Experimental
procedures for the determination of CAMP and cGMP concentrationsafter chemical stimulationwere as follows.
The frontal region of a plasmodium migrating on a wet filter paper (Whatman no. 1,30 x 30 cm) was cut into
strips(5 x 10 mm each), which were dipped in water for 20 min (Petri dish, 9 cm wide, 1 cm deep) and then in the
solution to be tested. At 1 min intervalseach strip was immersed in isopmtane (cooled by liquid N2),transferred
to 1 ml icecooled 1 M-HCl, and homogenized after scraping the plasmodium from the filter paper. The
homogenate was washed twice with 0.5 ml 1 M-HCleach time, and centrifuged (17000g, 10 min). The pellet was
used for protein assay, and the supernatant for cAMP and cGMP assays. cAMP and cGMP were assayed
radioimmunologically by using commercial kits (Yamasa assay kit, Yamasa Shop K. K., Choshi 288, Japan)
which are based on the method developed by Honma er al. (1977). Protein was determinedby the Lowry method.
Concentrationsof CAMP and cGMP before stimulation were 3.5-40 pmol (mg protein)- and 1-4-7-8pmol (mg
protein)-', respectively. Experiments were done twice, giving similar time courses. The temperature was 22 f
1 "C.
Measurements of changes in contractile activity accompanying injection of cyclic nucleotides. Given amounts of
cyclic nucleotides were dissolved in a basal solution containing 30 mM-KCl, 3 m-MgCl,, 0.2 ~ M - A T P2, x
lo-' wCaZ+with 10 mra-Ca/EGTA buffer, 10 m-Tris/HCl, pH 7-1. After measuring the contractile activity of
a plasmodium for a while, flowing plasma sol in the plasmodial vein was pushed away and replaced with the test
solution. Changes in contractileactivity of the plasmodium were monitored tensiometrically before and after the
injection, as shown in Fig. 5. Injection of the basal solution had little effect on the tension generation.Detailsof the
tensiometric method in combination with injection procedure were described by Ueda & Giitz von Olenhusen
(1 978).
RESULTS
Changes in cAMP concentration after chemical stimulation
Variations in the concentration of CAMP after stimulation with sugars and salts are shown in
Fig. 1. Low concentrations of glucose, KC1 and CaC12 brought about only small changes in
cAMP concentration. At high concentrations, glucose induced a decrease in cAMP
concentration. 2-Deoxyglucose, a non-metabolizable analogue of glucose, also induced a
decrease in cAMP concentration. Galactose and maltose induced large oscillatory variations of
cAMP concentration; averaged over the experimental period there was a tendency for the
concentration to decrease slightly. Repellents such as sucrose and KCl induced an oscillatory
increase in the cAMP concentration. In response to CaC12, the concentration of cAMP
increased gradually, with oscillation, and stayed at an elevated level. In all cases examined,
attractants (glucose, 2-deoxyglucose, galactose, maltose) induced a decrease in cAMP
concentration, while repellents (sucrose, KCl, CaC12) caused an increase in cAMP
concentration.
Changes in cGMP concentration afier chemical stimulation
Time courses for changes in the concentration of cGMP after chemical stimulation are shown
in Fig. 2. The attractant sugars glucose, 2-deoxyglucose, galactose and maltose induced a
decrease in cGMP concentration; occasional oscillatory variations were seen. The repellents
sucrose, KCl and CaC12 induced an increase in cGMP concentration, with oscillation.
Thus, cAMP and cGMP decrease and increase similarly in response to attractants and
repellents.
Correlation bet ween oscillations of CAMP and cGMP concentrations
Continuous chemical stimulation induced oscillatory changes in cAMP and cGMP
concentrations (Figs 1 and 2). The two oscillations were related to each other. Phases of the
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Role of cAMP and cGMP in Physarum chemotaxis
55 I
1.0
0.5
1*o
I
E
2.0
(c) Galactose
1.5
1.0
4
Oa5
t
(d) Maltose
1.5
1.0
0.5
0-5
5
10
1
15
Time (min)
5
10
15
Fig. 1. Time courses of the changes in intracellular cAMP concentration after chemical stimulation.
Stimulations are with: (a)glucose, 0.1 mM ( 0 )and 10 mM (0);(b)2-deoxyglucose, 10 m ~(c); galactose,
10 mM; (d)maltose, 30 mM; (e) sucrose, 100 mM; (f)KCl, 10 r
mi( 0 )and 30 m~ (0);(g) CaC12, 1 mM
( 0 )and 10 m (0).cAMP concentrations in pmol (mg protein)-’ units at time zero (represented by
‘1.O’on theordinates)were: 5*4(O)and12(0)in(a);40in(b); 3.7in(c);6.2in(d); 10-1in(e); 12.6(*)
and 9.2 ( 0 )in (f);3.5 ( 0 )and 32.5 ( 0 )in (g).
oscillation in the concentrations of the two cyclic nucleotides are compared in Fig. 3, where the
time axes are displaced in order to overlap the two oscillations. The concentration of cAMP
oscillated in advance of cGMP on stimulation with attractant sugars, e.g. maltose (Fig. 3b) and
galactose, while cAMP and cGMP concentrations oscillated in phase for repellents, e.g. MgClz
(Fig. 3 a ) and sucrose. These results indicate that temporal order (oscillation) develops under
continuous stimulation and that the oscillatory systems of cAMP and cGMP are coupled, the
mode of coupling being varied by the chemical environment.
Quantitative evaluation of the non-linear response of cyclic nucleotides to chemical stimuli
As seen in Figs 1 and 2, responses of intracellular cAMP and cGMP to chemical stimuli are
neither necessarily stationary, nor simply transient, nor simply oscillatory. All these responses
are here quantified by taking an integral over time t after stimulation at time 0:
P
=
Ji(C - Co)/Co d t
where Co is the concentration before stimulation. For experimental convenience t was taken as
15 min. The values of P for cAMP and cGMP in response to various chemicals are summarized
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552
-
T. AKITAYA AND OTHERS
[a) Glucose
I
-
(e) Sucrose
(b) 2-Deoxyglucose
2*o
1.5
(c) Galactose
w
-
1.0
0.5
1
LO r n M
lornM
( d ) Maltose
2.0
1.5
1.0
5
10
15
0*5
5
10
15
Time (min)
Fig. 2. Time courses of the changes in intracellular cGMP concentrations after chemical stimulation.
Stimulationsare with (a)glucose, 0.1 m~ (0)and 10 m~ (0);(6)2deoxyglucose, 10 m ~(c); galactose,
10 mu; (d)maltose, 30 m;(e) sucnrse, 100 my; (f)KCI, 10 m~ (0)and 30 m~ (0);
(g) C a Q , 1 m~
(0)and 10 m~ (0).
cGMP concentrations in pmol (mg protein)-' units at time zero (represented by
'l-o'on the ordinates)were: 2.6 (0)and 7.5 (0)in (a);7.8 in (b);1.4 in (c); 1.6 in (4;
3-7 in (e); 4.3 (0)
and 2.9 (0)in (f);1.4 (0)and 7.5 (0)in (g).
in Table 1. The concentrationsof both cAMP and cGMP decreased in response to attractants
and increased in response to repellents. Generally, the decrease in cGMP concentration was
larger than that in cAMP concentration, while the increase in cAMP concentration was larger
than that in cGMP concentration.
Concentration depndence of the response
In Fig. 4 changes in the accumulation of CAMP and of cGMP and in tension are plotted as a
function of the concentration of stimulus chemicals. The response of cGMP to repellents
occurred at about ten times lower concentrationsthan that of CAMP. Conversely, in response to
attractants, decrease in cGMP concentration occurred at about ten times lower concentrations
than those required for a decrease in cAMP concentration (compare a with b in Fig. 4). Increase
in cAMP concentration and decrease in cGMP concentration occurred at similar concentrations of stimulants, as did contraction and relaxation, respectively (compare Q and b with c in
Fig. 4). This suggests that repellents are mediated by CAMP,and attractants by cGMP at low
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Role of CAMP and cGMP in Physarum chemotaxis
5
I s
553
15
10
I
I
15
10
10
15
Time (min)
Fig. 3. Phase relationship between cAMP and cGMP oscillations. (a) Following stimulation with
30 mM MgCl,, intracellular CAMP and cGMP concentrations oscillatejust in phase. cAMP and cGMP
concentrations at time zero were 5.2 and 2.5 pmol (mg protein)- l , respectively. (b) Following
stimulation with 30 mM maltose, cAMP oscillates about 1 min in advance of the cGMP oscillation (the
time axis for cGMP is displaced so that the two oscillations overlap). The period of oscillation is
4-5 min. The data are taken from Figs 1 and 2. 0 , CAMP; 0 , cGMP.
5
Table 1. Changes in intracellular concentrations of CAMP and cGMP following chemical
stimulation
Chemical
Attractants
Glucose
2-Deox yglucose
Galactose
Maltose
Repellents
Sucrose
KCl
Concn
(mM)
0.1
1
10
10
10
30
c-~-,
P value*
cAMP
0.2
-0.5
-3.5
-10.7
-1.2
-2.5
30
100
3
10
30
30
0.1
1
10
0.5
4.1
0.5
2.0
7.1
2.8
0.3
2.1
8-6
cGMP
-0.1
-3.3
-5.6
-5.8
-6.8
-5.9
0.1
2.0
0.2
0.3
4.8
6.0
0.1
0.2
9-0
* P, the change in intracellular cAMP or cGMP concentration,
is defined by P = so(C - C,)/C, dt where
t = 15 min and C, is the value before stimulation. The values are averages of two experiments, and the standard
deviation is within 20%.
concentrations. At high concentrations both nucleotides act cooperatively to induce contraction
or relaxation.
Effects of injected cAMP and cGMP on contractility
Effects of intracellular cAMP and cGMP concentration on contractility were studied by
injecting these cyclic nucleotides into the plasmodia1 strands (Fig. 5). Low concentrations of
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554
T. AKITAYA AND OTHERS
-4
-3
-2
-I
log (Stimulant concn. M)
Fig. 4. Dependence of the responses on the concentration of chemical stimuli. (a) Changes in
intracellular cAMP concentration; (b)changes in intracellular cGMP concentration; (c) changes in
tension generation (data for c taken from Ueda et al., 1976). 0,CaCl,; 0,KCI; A, sucrose; 0 ,
glucose.
(a)
cGMP
cAMP
10
c
--8
I
I
-.7
-6
I
-5
I
-4
log (Cyclic nucleotide concn. %I)
Fig. 5. Effects of injected cAMP and cGMP on the contractile activity in the plasmodia1 strands. (a)
Time courses of tension generation before and after the injection. Injection was performed at the time
indicated by arrows. (b) Dependence of contraction on the concentration of injected cAMP ( 0 )and
cGMP (0).Vertical bars indicate standard deviations based on 5-7 experiments.
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Role of cAMP and cGMP in Physarum chemotaxis
555
cAMP and cGMP had little effect on contractility. At high concentrations, both cyclic
nucleotides elicited a transient contraction. These effects were quantified by taking the integral,
S, over this transient time, as shown by the shaded area in the upper trace of Fig. 5. The lower
trace shows the dependence of the contraction on the concentration of injected cAMP and
cGMP. Cyclic GMP is about 10 times as effective in eliciting contraction in the plasmodium of
P . polycephalum.
DISCUSSION
There is a hypothesis that cAMP and cGMP regulate antagonistically such cellular functions
as receptor potential in synapses, contractility, cell movement, etc. (Kupfermann, 1980;
Goldberg & Haddox, 1977; Estensen et al., 1973). However, this does not always hold. In smooth
muscle a rise in cAMP concentration corresponds to contraction, but an elevation in cGMP
concentration does not always correlate with relaxation (Diamond, 1983). In the cellular slime
mould Dictyostelium discoideum, an increase both in cAMP and in cGMP concentration is
correlated with a decrease in light scattering, i.e. presumably with contraction (Gerisch et al.,
1979). Our chemical analysis and injection study argue against the antagonistic action, but
suggest concerted action of the two cyclic nucleotides in regulating the contractility in the
Physarum plasmodium.
cAMP and cGMP oscillate with a period similar to that of contractile activity (Figs 1 and 2).
Thus, it is tempting to assume that these cyclic nucleotides also oscillate in association with the
contraction-relaxation cycle. If so, we have several chemical oscillations of such mediators as
ATP (Yoshimoto et a!., 1981a), Ca2 (Yoshimoto et al., 1981b, 1982), pH (Yoshimoto et al.,
1981c ; Kamiya, 1981) and cyclic nucleotides in relation to rhythmic contraction. How and why
are these oscillations coupled to each other and organized to evolve such a regular rhythmic
contraction in the Physarum plasmodium? This point has yet to be clarified, although
suggestions have been made based on a feedback system either among calcium-cyclic AMP
control loops (Rapp & Berridge, 1977) or among the contractile apparatus, the calcium
regulatory system and the energy metabolism (Korohoda et al., 1983).
+
This work was partially supported by a Grant in Aid for Scientific Research from the Ministry of Education,
Science and Culture, Japan.
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