715
Development 109, 7L5-722 (1990)
Printed in Great Britain © T h e Company of Biologists Limited 1990
Ammonia promotes accumulation of intracellular cAMP in differentiating
amoebae of Dictyostelium discoideum
BRUCE B. RILEY and STEPHEN L. BARCLAY*
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI53706 USA
*To whom reprint requests should be addressed
Summary
We used sporogenous mutants of Dictyostelium discoideum to investigate the mechanism(s) by which exogenous
NH4CI and high ambient pH promote spore formation
during in vitro differentiation. The level of NH4CI
required to optimize spore formation is correlated inversely with pH, indicating that NH3 rather than NH 4 + is
the active species. The spore-promoting activity of high
ambient pH (without exogenous NH4CI) was eliminated
by the addition of an NH^-scavenging cocktail, suggesting that high pH promotes spore differentiation by
increasing the ratio of NH 3 :NH4 + secreted into the
medium by developing cells. High ammonia levels and
high pH stimulated precocious accumulation of intracellular cAMP in both sporogenous and wild-type cells.
In both treatments, peak cAMP levels equaled or
exceeded control levels and were maintained for longer
periods than in control cells. In contrast, ammonia
strongly inhibited accumulation of extracellular cAMP
without increasing the rate of extracellular cAMP hydrolysis, indicating that ammonia promotes accumulation of intracellular cAMP by inhibiting cAMP secretion. These results are consistent with previous
observations that factors that raise intracellular cAMP
levels increase spore formation. Lowering intracellular
cAMP levels with caffeine or progesterone inhibited
spore formation, but simultaneous exposure to these
drugs and optimal concentrations of NH4CI restored
both cAMP accumulation and spore formation to normal levels. These data suggest that ammonia, which is a
natural Dictyostelium morphogen, favors spore formation by promoting accumulation or maintenance of high
intracellular cAMP levels.
Introduction
completion of stalk cell differentiation and induces
several genes that are expressed only in prestalk and
stalk cells (Kopachik et al. 1983; Jermyn et al. 1987). In
contrast, maintaining high levels of extracellular cAMP
and inhibiting DIF accumulation favors spore differentiation (Ishida, 1980; Riley and Barclay, 1986; Berks
and Kay, 1988).
Extracellular cAMP functions by binding to cell
surface receptors that are analogous and homologous to
mammalian G-protein regulated hormone receptors
(Gomer et al. 1986; Oyama and Blumberg, 1986;
Haribabu and Dottin, 1986; Klein et al. 1988). Binding
causes rapid accumulation of several intracellular
second messengers including Ca2+ ions, inositol triphosphate, cAMP and cGMP (Newell et al. 1987), any
of which could regulate the choice between stalk cell
and spore differentiation.
In previous studies, we used caffeine and progesterone, which inhibit accumulation of intracellular cAMP
by different mechanisms (Brenner and Thorns, 1984;
Klein and Brachet, 1975), to investigate the role of
intracellular cAMP in differentiation. Both drugs pre-
Mechanisms that regulate differentiation of Dictyostelium discoideum amoebae during multicellular development can be conveniently studied under in vitro
culture conditions that permit differentiation of single
cells. Monolayers of wild-type V12M2 cells differentiate as stalk cells if supplied with cAMP, and monolayers of sporogenous derivatives of V12M2 form both
stalk cells and spores (Town el al. 1976; Kay et al. 1978;
Kay, 1982). The dependence of stalk cell formation on
cell density led to the identification of another essential
morphogen, differentiation inducing factor (DIF)
(Town et al. 1976; Morris et al. 1987). It is now widely
held that the choice between stalk cell and spore
differentiation is regulated in part by levels of cAMP
and DIF. Extracellular cAMP is required to initiate
development but inhibits terminal stalk cell differentiation (Sobolewski et al. 1983; Berks and Kay, 1988).
Developing cells degrade exogenous cAMP and secrete
DIF, which antagonizes extracellular cAMP late in
development (Wang et al. 1986). DIF is essential for the
Key words: Dictyostelium discoideum, spore differentiation,
ammonia, extracellular pH, intracellular cAMP.
716
B. B. Riley and S. L. Barclay
vent spore and increase stalk cell formation during in
vitro differentiation of sporogenous mutants (Riley and
Barclay, 1986). These results are not due to pleiotropic
effects on other second messenger systems because
simultaneous exposure to 8-Br-cAMP, a membranepermeable cAMP analog with little affinity for the cell
surface cAMP receptor (Van Haastert and Kein, 1983),
completely restores spore formation to caffeine- and
progesterone-treated cultures (Riley etal. 1989). In the
absence of caffeine and progesterone, conditions that
increase rates of endogenous cAMP synthesis and
accumulation increase spore and decrease stalk cell
formation during standard development on agar as well
as during in vitro differentiation (Riley et al. 1989).
From these data, we proposed that intracellular cAMP
levels regulate cell fate, with high levels promoting
spore and/or inhibiting stalk cell differentiation. This
conclusion was supported in a separate series of experiments (Kay, 1989).
Previous studies showed that high ambient pH and
ammonia promote spore formation during in vitro
differentiation of sporogenous mutants (Gross et al.
1981; Gross etal. 1983). Do these environmental factors
affect cell fate by promoting intracellular cAMP accumulation or is another mechanism involved? To
address this, we examined the effects of high pH and
ammonia on cell fate and accumulation of intracellular
cAMP in cultures of wild-type strain V12M2 and a
sporogenous derivative, HB200. Our results support
the hypothesis that high intracellular cAMP levels
promote spore and/or inhibit stalk cell formation in
cultures exposed to ammonia or high pH.
Materials and methods
Strains and culture conditions
In all experiments, we used wild-type D. discoideum strain
V12M2 or a spontaneous sporogenous derivative, HB200.
Amoebae were grown as previously described (Riley et al.
1989). For in vitro differentiation, washed amoebae were
distributed on 6 cm tissues culture dishes at SxlO^cellscm"2
and submerged in 2.5ml of KM (10mM KC1, 5mM MgCb,
200^gml~1 streptomycin sulfate, and either 10mM MES,
pH6.2 or 10 mni Hepes, pH7.5) containing lmM cAMP.
Under these conditions, cells terminally differentiate within
12-24 h as highly vacuolated stalk cells or phase-bright spores.
This occurs without formation of large cell aggregates,
although loose clumps of 10-20 cells often form by 6h. For
submerged aggregation, cells were distributed at
2.5xlO^cellscm~2 and submerged in KM without exogenous
cAMP to promote aggregation. In this case, cells form large
cohesive aggregates (up to 1000 cells), but roughly half of
these cells fail to complete differentiation. The remainder
differentiate asynchronously as stalk cells or spores after 2-4
days. Despite these differences, early development (through
8h) follows the same time course during in vitro differentiation and submerged aggregation as judged by accumulation
of cellular phosphodiesterase activity: Levels peak at 6h and
decline by 8h (Riley, unpublished data).
Measurement of cAMP
Intracellular and extracellular cAMP levels were measured by
radio immune assay as previously described (Riley et al.
1989).
Measurement of phosphodiesterase activity
Phosphodiesterase assays were performed according to
Boudreau and Drummond (1975), with minor modifications
noted in the legend for Fig. 5.
Results
Effects of extracellular ammonia and high pH on cell
fate
We repeated and extended earlier studies (Gross et al.
1981; Gross et al. 1983) showing that increasing extracellular pH or ammonia levels during in vitro differentiation increases spore formation and decreases stalk
cell formation in sporogenous mutants. Fig. 1 shows
that, in the absence of added NH4C1, nearly two times
more HB200 cells formed spores at pH7.5 than at
pH6.2. The effects of exogenous NH4CI on cell fate
varied with ambient pH. The ratio of NH3:NH4+ is 20
times higher at pH7.5 than at pH6.2 and 15-20 times
more NH4CI had to be added at pH6.2 to optimize
spore formation (Fig. 1). These data indicate that NH3
rather than NH 4 + is the active species in promoting
spore formation. The increase in HB200 spore production in response to ammonia or high pH was highly
reproducible and similar in magnitude to that observed
previously with other sporogenous strains (Gross et al.
1981; Gross etal. 1983). Spore formation increased to as
much as 80 % when cAMP hydrolysis and accumulation
of DIF (a prestalk morphogen) were inhibited by
plating cells at lower cell densities (not shown).
We were puzzled that continuous exposure to NH4CI
concentrations exceeding lmM at pH7.5 or 15mM at
pH6.2 reduced both stalk cell and spore formation.
However, high ammonia levels delay early develop-
NH Cl Concentration (mM)
4
Fig. 1. Effect of raising pH and/or NH3 levels on cell fate.
Vegetative HB200 cells were plated for in vitro
differentiation at pH6.2 (triangles) or pH7.5 (squares) with
varying NH4CI concentrations as indicated. Stalk cells
(open figures), spores (closed figures) and amoebae (not
shown) were scored with an inverted phase-contrast
microscope after 24 h at 22 °C. Data are means and standard
deviations of 2 independent experiments.
Ammonia promotes cAMP accumulation
ment (see later) which could allow cells to prematurely
degrade exogenous cAMP. This could inhibit spore
formation because terminal differentiation of spores is
sensitive to extracellular cAMP concentrations (Ishida,
1980; Riley and Barclay, 1986; Berks and Kay, 1988).
Fig. 2A shows that adding NH4CI (up to 20ITIM) to
cultures at pH7.5 after 10 h of differentiation bypassed
the early delay and promoted spore formation. Furthermore, continuous exposure to 20mM NH4C1 at pH7.5
promoted spore differentiation if extracellular cAMP
concentrations were increased (Fig. 2B). Thus, high
ammonia levels promote spore formation if the developmental delay associated with ammonia is bypassed or
if high exogenous cAMP levels are maintained.
Distinguishing the effects of NH3 and pH on
differentiation
Raising the pH of the media could affect cell fate
directly by a pH sensitive mechanism. Alternatively,
high pH might act indirectly by increasing the ratio of
NH3:NH4+ secreted into the medium. From published
rates of ammonia secretion (Schindler and Sussman,
1977; Aeckerle et al. 1985), average concentrations of
NH 3 +NH4 + could approach 100 ^M during the course
of in vitro differentiation, and concentrations within
loose cell clumps could be much higher. Such ammonia
levels are probably too low to affect cell fate at pH6.2
but might be adequate at pH7.5 because the
NH 3 :NH 4 + ratio is 20 times higher. If pH acts only to
raise the NH 3 :NH 4 + ratio, then raising pH should not
affect cell fate if secreted ammonia is completely
removed.
We removed ammonia enzymatically by adding a
cocktail containing glutamate dehydrogenase, alpha-
111
ketoglutarate and NADPH (Schindler and Sussman,
1977). To prevent exhaustion of the cocktail, and
because the spore-promoting activity of ammonia is
strongest after 10 h of in vitro differentiation (see
below), the effect of enzymatic ammonia depletion was
tested at this time (Fig. 3). Increasing the pH from 6.2
to 7.5 nearly doubled spore formation in control cultures that received no cocktail. Cultures at pH7.5 that
received incomplete cocktails (missing one or more
reagents) formed the same number of spores as cultures
at pH7.5 that received no cocktail. In contrast, cultures
at pH7.5 that received complete cocktail formed the
same number of spores as cultures at pH6.2 that
received either complete cocktail or no cocktail at all.
These results were not due to greater accumulation of
enzymatic byproduct (glutamate) at pH7.5 since 10 mM
glutamate by itself had no effect on cell fate (not
shown). Thus, high pH alone cannot enhance spore
formation because enzymatic removal of ammonia
abolished the spore-promoting activity of high pH. This
supports the hypothesis that high pH increases spore
formation by increasing the NH3:NH4+ ratio.
The effects of changing levels of exogenous ammonia
or intracellular cAMP late in development
To determine when ammonia or high pH are most
effective in promoting spore formation, HB200 cells
were allowed to differentiate in vitro for 10 h under one
set of conditions (defined by pH and NH4CI levels) after
which the media were changed and cells were allowed
to complete differentiation under another set of conditions. 10 h corresponds to a relatively late stage of
development because HB200 cells differentiate quite
rapidly. Transcript levels of the prespore-specific gene
D19, which encodes a cell surface glycoprotein of
60n
10
15
20
NH Cl Concentration (mM)
0
5
10
15
20
25
cAMP Concentration (mM)
Fig. 2. (A) Effect on cell fate of adding high NH3 levels
late during differentiation. Vegetative HB200 cells were
allowed to differentiate in vitro at pH7.5 without
exogenous NH4Cl for 10 h after which NH4C1 was added to
final concentrations ranging from 1 to 20mM as indicated.
(B) Effect on cell fate of increasing exogenous cAMP levels
during continuous differentiation in the presence of high
NH3 levels. HB200 cells were plated for in vitro
differentiation at pH7.5 with 20 mM NH4CI and cAMP
concentrations ranging from 1 to 25 mM as indicated. Stalk
cells (open figures), spores (closed figures) and amoebae
(not shown) were scored as described in Fig. 1 legend.
10
?o
30
Percent Spore Formation
Fig. 3. Effect of enzymatic removal of NH3 on cell fate.
Vegetative HB200 cells were plated for in vitro
differentiation at pH6.2 or pH7.5. After 10 h, media in test
cultures were replaced with fresh KM containing 1 mM
cAMP and one or more of the following as indicated: (a),
lOmM alpha-ketoglutarate; (e), 0.5 units glutamate
dehydrogenase; (n) 0.15 mM NADPH. At the same time,
media in control cultures were replaced with KM containing
1 mM cAMP only. Stalk cells, spores, and amoebae were
scored as described in Fig. 1 legend. Data are means and
standard deviations of spore percentages in 3 independent
experiments.
718
B. B. Riley and S. L. Barclay
unknown function (Early et al. 1988), are maximal by
8h and are almost undetectable by 12 h, the time when
the first mature spores appear (not shown). Adding
15 mM NH4CI (pH 6.2) or increasing pH from 6.2 to 7.5
after 10 h increased spore formation to the same extent
as continuous exposure to high pH or ammonia
(Table 1, compare line 1 with lines 2, 3, 5, and 6). In
contrast, spore formation did not increase over the
control if cells were exposed for the first 10 h of
differentiation to either pH7.5 or to 15 mM NH4CI at
pH6.2 and then shifted back to the control conditions
of pH6.2 with no exogenous NH4CI (Table 1, compare
line 1 with lines 4 and 7). Thus, exposure to high pH or
ammonia after 10 h of differentiation is completely
effective in promoting spore formation, but early exposure is neither necessary nor sufficient for optimal
spore formation.
Because previous work showed that spore formation
correlates with elevated intracellular cAMP levels
(Kay, 1989; Riley et al. 1989), we performed similar
experiments with drugs that raise or lower intracellular
cAMP levels (Table 1). 8-Br-cAMP is a membranepermeable cAMP analogue that has high affinity for
intracellular targets of cAMP, such as the regulatory
subunit of cAMP-dependent protein kinase, but has
very low affinity for the cell surface cAMP receptor
(DeWit etal. 1984; Van Haastert and Kein, 1983). This
allows 8-Br-cAMP to enter cells and mimic endogenous
cAMP without stimulating other second messenger
systems via the cell surface receptor. Adding 1 mM
8-Br-cAMP after 10 h was as effective in promoting
spore formation as continuous exposure to 8-Br-cAMP
(Table 1, lines 8 and 9). However, spore formation did
not increase over the control if 8-Br-cAMP was added
for the first 10 h and then removed (compare lines 1 and
10). Thus, the effects of adding or removing
8-Br-cAMP after 10 h were the same as adding or
removing NH3, suggesting that these agents might
operate by a common mechanism.
In contrast, treating cells with caffeine or progesterone, drugs that lower intracellular cAMP levels (Brenner and Thorns, 1984; Klein and Brachet, 1975; Riley et
al. 1989) gave quite different results. Adding 5mM
caffeine or 20 piM progesterone after 10 h completely
inhibited spore differentiation and promoted stalk cell
differentiation (Table 1, lines 11 and 14). Moreover, the
effects of caffeine and progesterone on cell fate persisted even after the drugs were removed. Spore formation was inhibited and nearly all cells differentiated as
stalk cells when caffeine or progesterone were added
for the first 10 h and then removed (Table 1, lines 13 and
16). These results are not due to retention of caffeine or
progesterone inside cells after washing because the
effects of these drugs on cAMP synthesis and aggregation are rapidly reversible (Brenner and Thorns,
1984; Klein and Brachet, 1975). Instead, our results
may reflect earlier observations that prestalk differentiation is only slowly reversible (Raper, 1940; Bonner, 1949).
In summary, these data show that (1) drugs that
increase (8-Br-cAMP) or decrease (caffeine and progesterone) intracellular cAMP levels effectively alter
cell fate when added late in development, (2) adding
caffeine or progesterone early in development induces
stable changes in cell fate while raising cAMP levels
early has no lasting effects, and (3) high pH and
ammonia promote spore formation by a mechanism
that is consistent with elevation, but not reduction, of
intracellular cAMP levels.
Effects of ammonia and high pH on accumulation of
intracellular cAMP
To establish whether a link exists between extracellular
NH3 and intracellular cAMP, we determined the effects
Table 1. Effects of changing developmental conditions after 10 h of in vitro differentiation
Developmental conditions
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Percent cell-type±s.D.
0-10 h
After 10 h
Spore
Stalk cell
Amoeb;
Control
Control
pH7.5
pH7.5
Control
NH3
NH3
Control
8-Br-cAMP
8-Br-cAMP
Control
Caf
Caf
Control
Prog
Prog
Control
pH7.5
pH7.5
Control
NH3
NH3
Control
8-Br-cAMP
8-Br-cAMP
Control
Caf
Caf
Control
Prog
Prog
Control
23±2
42±3
39±1
21±3
46±4
47±4
25±0
49±1
5O±2
25±2
0±0
72±3
54±4
54±1
73±2
5O±3
48±0
69±5
49±0
49±1
66±2
94±5
96±3
98±2
91±1
96±5
97±2
5±1
0±0
0±0
4±1
7±2
6±1
4±1
5±2
6±5
2±1
9±1
6±4
3±3
9±1
4±4
2±2
HB2O0 cells were plated form vitro differentiation at pH6.2 with 15 mM NH4C1 (NH3), lmM 8-Br-cAMP (8-Br-cAMP), 5mM caffeine
(Caf), 20/<M progesterone (Prog), or with no drug (control), or at pH7.5 with no drug (pH7.5). After lOh, media were changed as
indicated. Data represent means and standard deviations of two independent experiments.
Ammonia promotes cAMP accumulation
of high pH and ammonia on cAMP accumulation
during 'submerged aggregation', culture conditions that
permit reliable measurements of both intracellular and
secreted cAMP levels (see Methods to distinguish from
'in vitro differentiation'). Under these conditions, cells
in control cultures began streaming by 4.5 h and completed aggregation by 6h. Adding 15mM NH4C1 to
cultures at pH6.2 or raising the pH from 6.2 to 7.5
delayed the onset of aggregation by 1 h. Adding 15 mM
NH4CI to cultures at pH7.5 delayed aggregation by
3-4h. However, once initiated, aggregation proceeded
normally in cultures exposed to ammonia and/or high
pH (not shown).
Fig. 4A shows intracellular cAMP levels during submerged aggregation of HB200 cells. Cells cultured at
pH6.2 and pH7.5 had identical levels of intracellular
cAMP at 6h of development. However, cAMP accumulated more quickly and was maintained at maximal levels through 8h of development at pH7.5, while
cAMP levels declined sharply by 8 h in control cultures.
50-,
o
E
Q.
0
2
4
6
Hours Development
Fig. 4. Effect of high pH and NH3 levels on accumulation
of intracellular cAMP in strains HB200 (A) and V12M2
(B), and extracellular cAMP in HB200 cultures (C).
Vegetative amoebae were plated for submerged aggregation
(see Methods) at pH6.2 (closed triangles), pH7.5 (open
triangles), pH6.2 with 15 mM NH4C1 (closed squares), or
pH7.5 with 15 mM NH4CI (open squares). At the indicated
times, cAMP samples were taken and measured in
duplicate by radioimmune assay. Data are means and
standard deviations of 2 or more independent experiments.
719
Still more striking was the finding that, in cells cultured
with 15 mM NH4CI at pH6.2 or pH7.5, intracellular
cAMP were nearly maximal at 2h. These levels equaled
or exceeded the 6h peak in control cells and were
maintained through 8 h of development. Identical results were obtained with V12M2 cells (Fig. 4B). Thus,
increasing ammonia levels, either by increasing the pH
or by direct addition of NH4CI, stimulated precocious
accumulation and prolonged maintenance of high intracellular cAMP levels in both wild-type and sporogenous
amoebae. This could explain the role of NH3 as a
prespore morphogen because elevating intracellular
cAMP levels is sufficient to promote spore formation
(Kay, 1989; Riley et al. 1989).
Ammonia might promote accumulation of intracellular cAMP by altering rates of cAMP synthesis, degradation or secretion. However, we were unable to detect
any effects of ammonia on specific activities of cellular
phosphodiesterase (PDE) or adenylate cyclase activities in cells developed for 2h, the time when ammoniatreated cells first achieved maximal intracellular cAMP
levels. In fact, enzyme activities in cells developed for
2h (with or without ammonia) were not significantly
higher than in vegetative cells (not shown). In contrast,
ammonia did affect the amount of cAMP secreted into
the medium. Cultures exposed to 15 mM NH4CI at
pH7.5 accumulated only 1/10 as much cAMP in the
extracellular medium as control cultures at pH6.2
(Fig. 4C). Intermediate levels of extracellular cAMP
accumulated in cultures at pH7.5 with no exogenous
NH4CI and at pH6.2 with 15mM NH4CI (Fig. 4C).
Decreased levels of extracellular cAMP accumulation
were not due to increased rates of cAMP hydrolysis
because secreted PDE activities were the same in
ammonia-treated and control cultures (Fig. 5). These
data indicate that ammonia raises intracellular cAMP
levels by inhibiting cAMP secretion. This mechanism
explains the apparent discrepancy between our results
and those of previous studies (see Discussion) and
could also explain why ammonia delays aggregation by
several hours.
Ammonia antagonizes the effects of caffeine and
progesterone
HB200 amoebae terminally differentiate as stalk cells
when caffeine or progesterone are used to lower intracellular cAMP levels (Table 1). However, simultaneous
exposure to 8-Br-cAMP completely reverses inhibition
of spore formation in cultures exposed to caffeine or
progesterone (Riley et al. 1989). This change in cell fate
is thought to result from diffusion of 8-Br-cAMP into
cells where it binds to intracellular targets of cAMP,
such as cAMP-dependent protein kinase, for which it
has high affinity (DeWit et al. 1984). Table 2 shows that
ammonia also restores spore formation to caffeine- and
progesterone-treated cultures. Spore formation was
inhibited and nearly all cells differentiated as stalk cells
when cultured with 2.5 mM caffeine or IOJJM progesterone, but spore inhibition was completely reversed if
15 mM NH4CI was also added. It is possible that
ammonia bypasses the effects of caffeine and progester-
720
B. B. Riley and S. L. Barclay
•5
2
4
6
Hours Devalopment
40-
2
8
4
6
Hours Development
Fig. 5. Effect of NH3 on accumulation of secreted
phosphodiesterase. Vegetative HB200 cells were plated for
submerged aggregation at pH6.2 (circles) or at pH7.5 with
15 mM NH4CI (squares). At the indicated times,
extracellular media were drawn off, centrifuged to remove
cells, and dialyzed at 4°C against 50 mM Tris, pH7.6, and
5mM MgCl2. Dialysis was performed for 24 h, during which
the buffer was changed twice. 80/d of dialyzed media were
mixed with assay cocktail to give 200 ;d containing 20 mM
Tris, pH7.6, 2mM MgCl2, 100HM CAMP, and 0.13^M 3H-
cAMP (31.2Cimmol~l), and incubated at 37°C for 20min.
1 unit of phosphodiesterase degrades 1 pmole cAMPmin"1.
The data shown are means and standard deviations of two
independent experiments.
Table 2. Ammonia reverses inhibition of spore
formation in the presence of caffeine or progesterone
Percent cell type±s.D.
Developmental
conditions
Spore
Stalk cell
Amoeba
Control
NH3
Caf
Prog
Caf+NH3
Prog+NHa
24±3
52±5
3±1
2±1
32±4
20±2
74±4
39±8
90±l
97±1
57±3
73±1
2±2
9±4
7±2
l±0
7±1
HB200 cells were plated for in vitro differentiation at pH 6.2 in
the presence of 15 mM NR,C1 (NH3), 2.5 mM caffeine (Caf), 10/uu
progesterone (Prog), or with no drugs (Control). Higher
concentrations of caffeine (5ITIM) or progesterone (20 /an) were
toxic if ammonia was also present. Data are means and standard
deviations of two independent experiments.
one by inhibiting cAMP secretion and thereby raising
intracellular cAMP levels. (Basal rates of cAMP synthesis persist in the presence of caffeine and progesterone.) Alternatively, ammonia might directly activate
targets of intracellular cAMP, such as cAMP-dependent protein kinase, or another second messenger
system that is dominant over changes in intracellular
cAMP.
We cannot test the effects of ammonia on intracellular cAMP accumulation during in vitro differentiation
because high levels of exogenous cAMP (1 mM) preclude reliable measurement of intracellular cAMP
levels. However, ammonia reversed the effects of
Fig. 6. Effect of simultaneous treatment with ammonia and
caffeine or progesterone on accumulation of intracellular
cAMP. Vegetative HB200 cells were plated for submerged
aggregation at pH6.2 with 2.5 mM caffeine (closed squares),
10 jiM progesterone (closed circles), 15 mM NH3C1 (open
triangles), 2.5 mM caffeine and 15 mM NH4CI (open
squares), 10 ^M progesterone and 15 mM NH4CI (open
circles), or with no drugs (closed triangles). At the
indicated times, cAMP samples were taken and measured
in duplicate by radio immune assay. Data are means and
standard deviations of two experiments.
caffeine and progesterone on intracellular cAMP accumulation during submerged aggregation. Fig. 6
shows that 2.5 mM caffeine and 10 /JM progesterone
reduced internal cAMP levels unless 15 mM NH4CI was
also present, in which case normal cAMP levels accumulated. These data support the hypothesis that
ammonia reversed the effects of caffeine and progesterone on cell fate by restoring normal intracellular cAMP
levels.
Discussion
In agreement with studies of other sporogenous strains
(Gross et al. 1981; Gross et al. 1983), we have shown
that high concentrations of NH4CI or high pH enhance
spore formation by HB200 cells during in vitro differentiation. While others have speculated that both agents
exert this effect by a common mechanism (Sussman,
1982), we provide the first direct evidence that exogenous NH4CI and high pH function by increasing the
concentration of NH3 in the medium. High pH has no
separate role. In addition, we have shown that concentrations of ammonia that promote spore formation lead
to precocious accumulation and maintenance of high
intracellular cAMP levels in HB200 and V12M2 cells.
This is probably the mechanism by which ammonia
promotes spore formation because other conditions
that raise intracellular cAMP levels also promote spore
formation (Kay, 1989; Riley et al. 1989).
Ammonia raises intracellular cAMP levels by inhibiting cAMP secretion. This explains how ammonia permits cells with low adenylate cyclase activities (early in
development or in the presence of caffeine) to accumulate or maintain high intracellular cAMP levels (Figs 4
Ammonia promotes cAMP accumulation 111
and 6). Inhibition of cAMP secretion also explains the
ability of ammonia to antagonize the effects of progesterone (Table 2 and Fig. 6), a drug that normally
stimulates cAMP secretion (Klein and Brachet, 1975).
The mechanism of cAMP secretion in Dictyostelium is
unknown, but differential regulation of this function
might be important during development because prestalk cells of migrating slugs secrete much more cAMP
than do prespore cells (Bonner and Slifkin, 1949). This
raises the interesting possibility that ammonia induces
differentiation of prespore cells in migrating slugs by
inhibiting cAMP secretion and thereby raising intracellular cAMP levels.
Stimulation of intracellular cAMP accumulation by
ammonia would not have been expected based on
earlier studies showing that 15 mM NH4C1 at pH7.2
inhibits cAMP synthesis by aggregation-competent cells
of wild-type strain NC4 (Schindler and Sussman, 1979;
Williams et al. 1984). However, the previous studies
examined the short term effects of NH4CI on cAMP
synthesis by aggregating cells, whereas we looked at
accumulation, not the rate of synthesis, in cells cultured
from the time of starvation to 8 h of development. As
we have shown, ammonia can increase accumulation of
intracellular cAMP even when adenylate cyclase activity is low. Moreover, it is possible that adenylate
cyclase of Dictyostelium is only transiently sensitive to
changes in pH or ammonia concentrations and that,
after a period of adjustment, high rates of synthesis
return. Indeed, Khachatrian et al. (1987) showed that
preincubating Dictyostelium membranes with very high
levels of NH4SO4 (>100ITIM) increases activation of
adenylate cyclase by inhibiting an inhibitory G protein.
Low rates of cAMP degradation could also play a
part in maintaining high cAMP levels in ammoniatreated cells. We have recently shown that expression of
the Dictyostelium cAMP phosphodiesterase gene and
accumulation of enzyme activity are negatively regulated by high intracellular cAMP levels: treatment with
ammonia or 8-Br-cAMP reduces cellular PDE activity
by nearly half (B. B. Riley and S. L. Barclay, unpublished data). This suggests that, as ammonia raises
intracellular cAMP levels, reduction of cellular PDE
activity helps to maintain high cAMP levels.
These results and other recent studies suggest that
intracellular cAMP regulates Dictyostelium development, perhaps by more than one mechanism. Overexpression of the regulatory subunit of cAMP-dependent protein kinase disrupts development at a stage
prior to aggregation (Simon et al. 1989; Firtel and
Chapman, 1990). This block presumably results from
low kinase activity. Another cAMP-binding protein,
CABP1, may function to transduce the cAMP signal
from the cell membrane into the nucleus (Kay et al.
1987) and has been implicated in regulation of the rate
of development (Tsang et al. 1987). It is not yet clear
what cell functions these proteins regulate or how they
affect cell fate. However, this study and others (Riley
and Barclay, 1986; Riley et al. 1989; Kay, 1989) clearly
show that cell fate correlates with intracellular cAMP
levels.
Genes that respond to changes in intracellular cAMP
levels should be useful in elucidating the mechanisms by
which cAMP affects development. Use of drugs like
ammonia, 8-Br-cAMP, caffeine, and progesterone to
alter intracellular cAMP levels during in vitro differentiation could be a powerful system for detecting such
genes and studying their expression.
This research was supported by N1H grant GM35432
awarded to S. L. Barclay.
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{Accepted 22 March 1990)