ammonia promotes accumulation of intracellular camp in ... · ammonia on cell fate and accumulation...

Development 109, 7L5-722 (1990)Printed in Great Britain © T h e Company of Biologists Limited 1990

715

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 discoid-eum to investigate the mechanism(s) by which exogenousNH4CI and high ambient pH promote spore formationduring in vitro differentiation. The level of NH4CIrequired to optimize spore formation is correlated inver-sely with pH, indicating that NH3 rather than NH4

+ isthe active species. The spore-promoting activity of highambient pH (without exogenous NH4CI) was eliminatedby the addition of an NH^-scavenging cocktail, sugges-ting that high pH promotes spore differentiation byincreasing the ratio of NH3:NH4+ secreted into themedium by developing cells. High ammonia levels andhigh pH stimulated precocious accumulation of intra-cellular cAMP in both sporogenous and wild-type cells.In both treatments, peak cAMP levels equaled orexceeded control levels and were maintained for longerperiods than in control cells. In contrast, ammoniastrongly inhibited accumulation of extracellular cAMP

without increasing the rate of extracellular cAMP hy-drolysis, indicating that ammonia promotes accumu-lation of intracellular cAMP by inhibiting cAMP se-cretion. These results are consistent with previousobservations that factors that raise intracellular cAMPlevels increase spore formation. Lowering intracellularcAMP levels with caffeine or progesterone inhibitedspore formation, but simultaneous exposure to thesedrugs and optimal concentrations of NH4CI restoredboth cAMP accumulation and spore formation to nor-mal levels. These data suggest that ammonia, which is anatural Dictyostelium morphogen, favors spore forma-tion by promoting accumulation or maintenance of highintracellular cAMP levels.

Key words: Dictyostelium discoideum, spore differentiation,ammonia, extracellular pH, intracellular cAMP.

Introduction

Mechanisms that regulate differentiation of Dictyo-stelium discoideum amoebae during multicellular devel-opment can be conveniently studied under in vitroculture conditions that permit differentiation of singlecells. Monolayers of wild-type V12M2 cells differen-tiate as stalk cells if supplied with cAMP, and mono-layers of sporogenous derivatives of V12M2 form bothstalk cells and spores (Town el al. 1976; Kay et al. 1978;Kay, 1982). The dependence of stalk cell formation oncell density led to the identification of another essentialmorphogen, differentiation inducing factor (DIF)(Town et al. 1976; Morris et al. 1987). It is now widelyheld that the choice between stalk cell and sporedifferentiation is regulated in part by levels of cAMPand DIF. Extracellular cAMP is required to initiatedevelopment but inhibits terminal stalk cell differen-tiation (Sobolewski et al. 1983; Berks and Kay, 1988).Developing cells degrade exogenous cAMP and secreteDIF, which antagonizes extracellular cAMP late indevelopment (Wang et al. 1986). DIF is essential for the

completion of stalk cell differentiation and inducesseveral genes that are expressed only in prestalk andstalk cells (Kopachik et al. 1983; Jermyn et al. 1987). Incontrast, maintaining high levels of extracellular cAMPand inhibiting DIF accumulation favors spore differen-tiation (Ishida, 1980; Riley and Barclay, 1986; Berksand Kay, 1988).

Extracellular cAMP functions by binding to cellsurface receptors that are analogous and homologous tomammalian G-protein regulated hormone receptors(Gomer et al. 1986; Oyama and Blumberg, 1986;Haribabu and Dottin, 1986; Klein et al. 1988). Bindingcauses rapid accumulation of several intracellularsecond messengers including Ca2+ ions, inositol tri-phosphate, cAMP and cGMP (Newell et al. 1987), anyof which could regulate the choice between stalk celland spore differentiation.

In previous studies, we used caffeine and progester-one, which inhibit accumulation of intracellular cAMPby different mechanisms (Brenner and Thorns, 1984;Klein and Brachet, 1975), to investigate the role ofintracellular cAMP in differentiation. Both drugs pre-

716 B. B. Riley and S. L. Barclay

vent spore and increase stalk cell formation during invitro differentiation of sporogenous mutants (Riley andBarclay, 1986). These results are not due to pleiotropiceffects on other second messenger systems becausesimultaneous exposure to 8-Br-cAMP, a membrane-permeable cAMP analog with little affinity for the cellsurface cAMP receptor (Van Haastert and Kein, 1983),completely restores spore formation to caffeine- andprogesterone-treated cultures (Riley etal. 1989). In theabsence of caffeine and progesterone, conditions thatincrease rates of endogenous cAMP synthesis andaccumulation increase spore and decrease stalk cellformation during standard development on agar as wellas during in vitro differentiation (Riley et al. 1989).From these data, we proposed that intracellular cAMPlevels regulate cell fate, with high levels promotingspore and/or inhibiting stalk cell differentiation. Thisconclusion was supported in a separate series of exper-iments (Kay, 1989).

Previous studies showed that high ambient pH andammonia promote spore formation during in vitrodifferentiation of sporogenous mutants (Gross et al.1981; Gross etal. 1983). Do these environmental factorsaffect cell fate by promoting intracellular cAMP ac-cumulation or is another mechanism involved? Toaddress this, we examined the effects of high pH andammonia on cell fate and accumulation of intracellularcAMP in cultures of wild-type strain V12M2 and asporogenous derivative, HB200. Our results supportthe hypothesis that high intracellular cAMP levelspromote spore and/or inhibit stalk cell formation incultures exposed to ammonia or high pH.

Materials and methods

Strains and culture conditionsIn all experiments, we used wild-type D. discoideum strainV12M2 or a spontaneous sporogenous derivative, HB200.Amoebae were grown as previously described (Riley et al.1989). For in vitro differentiation, washed amoebae weredistributed 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 within12-24 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. Forsubmerged aggregation, cells were distributed at2.5xlO^cellscm~2 and submerged in KM without exogenouscAMP to promote aggregation. In this case, cells form largecohesive aggregates (up to 1000 cells), but roughly half ofthese cells fail to complete differentiation. The remainderdifferentiate asynchronously as stalk cells or spores after 2-4days. Despite these differences, early development (through8h) follows the same time course during in vitro differen-tiation and submerged aggregation as judged by accumulationof cellular phosphodiesterase activity: Levels peak at 6h anddecline by 8h (Riley, unpublished data).

Measurement of cAMPIntracellular and extracellular cAMP levels were measured by

radio immune assay as previously described (Riley et al.1989).

Measurement of phosphodiesterase activityPhosphodiesterase assays were performed according toBoudreau and Drummond (1975), with minor modificationsnoted in the legend for Fig. 5.

Results

Effects of extracellular ammonia and high pH on cellfateWe repeated and extended earlier studies (Gross et al.1981; Gross et al. 1983) showing that increasing extra-cellular pH or ammonia levels during in vitro differen-tiation increases spore formation and decreases stalkcell formation in sporogenous mutants. Fig. 1 showsthat, in the absence of added NH4C1, nearly two timesmore HB200 cells formed spores at pH7.5 than atpH6.2. The effects of exogenous NH4CI on cell fatevaried with ambient pH. The ratio of NH3:NH4+ is 20times higher at pH7.5 than at pH6.2 and 15-20 timesmore NH4CI had to be added at pH6.2 to optimizespore formation (Fig. 1). These data indicate that NH3rather than NH4

+ is the active species in promotingspore formation. The increase in HB200 spore pro-duction in response to ammonia or high pH was highlyreproducible and similar in magnitude to that observedpreviously with other sporogenous strains (Gross et al.1981; Gross etal. 1983). Spore formation increased to asmuch as 80 % when cAMP hydrolysis and accumulationof DIF (a prestalk morphogen) were inhibited byplating cells at lower cell densities (not shown).

We were puzzled that continuous exposure to NH4CIconcentrations exceeding lmM at pH7.5 or 15mM atpH6.2 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 vitrodifferentiation at pH6.2 (triangles) or pH7.5 (squares) withvarying NH4CI concentrations as indicated. Stalk cells(open figures), spores (closed figures) and amoebae (notshown) were scored with an inverted phase-contrastmicroscope after 24 h at 22 °C. Data are means and standarddeviations of 2 independent experiments.

Ammonia promotes cAMP accumulation 111

ment (see later) which could allow cells to prematurelydegrade exogenous cAMP. This could inhibit sporeformation because terminal differentiation of spores issensitive to extracellular cAMP concentrations (Ishida,1980; Riley and Barclay, 1986; Berks and Kay, 1988).Fig. 2A shows that adding NH4CI (up to 20ITIM) tocultures at pH7.5 after 10 h of differentiation bypassedthe early delay and promoted spore formation. Further-more, continuous exposure to 20mM NH4C1 at pH7.5promoted spore differentiation if extracellular cAMPconcentrations were increased (Fig. 2B). Thus, highammonia levels promote spore formation if the devel-opmental delay associated with ammonia is bypassed orif high exogenous cAMP levels are maintained.

Distinguishing the effects of NH3 and pH ondifferentiationRaising the pH of the media could affect cell fatedirectly by a pH sensitive mechanism. Alternatively,high pH might act indirectly by increasing the ratio ofNH3:NH4+ secreted into the medium. From publishedrates of ammonia secretion (Schindler and Sussman,1977; Aeckerle et al. 1985), average concentrations ofNH3+NH4+ could approach 100 ̂ M during the courseof in vitro differentiation, and concentrations withinloose cell clumps could be much higher. Such ammonialevels are probably too low to affect cell fate at pH6.2but might be adequate at pH7.5 because theNH3:NH4

+ ratio is 20 times higher. If pH acts only toraise the NH3:NH4

+ ratio, then raising pH should notaffect cell fate if secreted ammonia is completelyremoved.

We removed ammonia enzymatically by adding acocktail containing glutamate dehydrogenase, alpha-

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 levelslate during differentiation. Vegetative HB200 cells wereallowed to differentiate in vitro at pH7.5 withoutexogenous NH4Cl for 10 h after which NH4C1 was added tofinal concentrations ranging from 1 to 20mM as indicated.(B) Effect on cell fate of increasing exogenous cAMP levelsduring continuous differentiation in the presence of highNH3 levels. HB200 cells were plated for in vitrodifferentiation at pH7.5 with 20 mM NH4CI and cAMPconcentrations ranging from 1 to 25 mM as indicated. Stalkcells (open figures), spores (closed figures) and amoebae(not shown) were scored as described in Fig. 1 legend.

ketoglutarate and NADPH (Schindler and Sussman,1977). To prevent exhaustion of the cocktail, andbecause the spore-promoting activity of ammonia isstrongest after 10 h of in vitro differentiation (seebelow), the effect of enzymatic ammonia depletion wastested at this time (Fig. 3). Increasing the pH from 6.2to 7.5 nearly doubled spore formation in control cul-tures that received no cocktail. Cultures at pH7.5 thatreceived incomplete cocktails (missing one or morereagents) formed the same number of spores as culturesat pH7.5 that received no cocktail. In contrast, culturesat pH7.5 that received complete cocktail formed thesame number of spores as cultures at pH6.2 thatreceived either complete cocktail or no cocktail at all.These results were not due to greater accumulation ofenzymatic byproduct (glutamate) at pH7.5 since 10 mMglutamate by itself had no effect on cell fate (notshown). Thus, high pH alone cannot enhance sporeformation because enzymatic removal of ammoniaabolished the spore-promoting activity of high pH. Thissupports the hypothesis that high pH increases sporeformation by increasing the NH3:NH4+ ratio.

The effects of changing levels of exogenous ammoniaor intracellular cAMP late in developmentTo determine when ammonia or high pH are mosteffective in promoting spore formation, HB200 cellswere allowed to differentiate in vitro for 10 h under oneset of conditions (defined by pH and NH4CI levels) afterwhich the media were changed and cells were allowedto complete differentiation under another set of con-ditions. 10 h corresponds to a relatively late stage ofdevelopment because HB200 cells differentiate quiterapidly. Transcript levels of the prespore-specific geneD19, which encodes a cell surface glycoprotein of

10 ?o 30

Percent Spore Formation

Fig. 3. Effect of enzymatic removal of NH3 on cell fate.Vegetative HB200 cells were plated for in vitrodifferentiation at pH6.2 or pH7.5. After 10 h, media in testcultures were replaced with fresh KM containing 1 mMcAMP and one or more of the following as indicated: (a),lOmM alpha-ketoglutarate; (e), 0.5 units glutamatedehydrogenase; (n) 0.15 mM NADPH. At the same time,media in control cultures were replaced with KM containing1 mM cAMP only. Stalk cells, spores, and amoebae werescored as described in Fig. 1 legend. Data are means andstandard deviations of spore percentages in 3 independentexperiments.


unknown function (Early et al. 1988), are maximal by8h and are almost undetectable by 12 h, the time whenthe first mature spores appear (not shown). Adding15 mM NH4CI (pH 6.2) or increasing pH from 6.2 to 7.5after 10 h increased spore formation to the same extentas continuous exposure to high pH or ammonia(Table 1, compare line 1 with lines 2, 3, 5, and 6). Incontrast, spore formation did not increase over thecontrol if cells were exposed for the first 10 h ofdifferentiation to either pH7.5 or to 15 mM NH4CI atpH6.2 and then shifted back to the control conditionsof pH6.2 with no exogenous NH4CI (Table 1, compareline 1 with lines 4 and 7). Thus, exposure to high pH orammonia after 10 h of differentiation is completelyeffective in promoting spore formation, but early ex-posure is neither necessary nor sufficient for optimalspore formation.

Because previous work showed that spore formationcorrelates with elevated intracellular cAMP levels(Kay, 1989; Riley et al. 1989), we performed similarexperiments with drugs that raise or lower intracellularcAMP levels (Table 1). 8-Br-cAMP is a membrane-permeable cAMP analogue that has high affinity forintracellular targets of cAMP, such as the regulatorysubunit of cAMP-dependent protein kinase, but hasvery low affinity for the cell surface cAMP receptor(DeWit etal. 1984; Van Haastert and Kein, 1983). Thisallows 8-Br-cAMP to enter cells and mimic endogenouscAMP without stimulating other second messengersystems via the cell surface receptor. Adding 1 mM8-Br-cAMP after 10 h was as effective in promotingspore formation as continuous exposure to 8-Br-cAMP(Table 1, lines 8 and 9). However, spore formation didnot increase over the control if 8-Br-cAMP was addedfor the first 10 h and then removed (compare lines 1 and10). Thus, the effects of adding or removing8-Br-cAMP after 10 h were the same as adding or

removing NH3, suggesting that these agents mightoperate by a common mechanism.

In contrast, treating cells with caffeine or progester-one, drugs that lower intracellular cAMP levels (Bren-ner and Thorns, 1984; Klein and Brachet, 1975; Riley etal. 1989) gave quite different results. Adding 5mMcaffeine or 20 piM progesterone after 10 h completelyinhibited spore differentiation and promoted stalk celldifferentiation (Table 1, lines 11 and 14). Moreover, theeffects of caffeine and progesterone on cell fate per-sisted even after the drugs were removed. Spore forma-tion was inhibited and nearly all cells differentiated asstalk cells when caffeine or progesterone were addedfor the first 10 h and then removed (Table 1, lines 13 and16). These results are not due to retention of caffeine orprogesterone inside cells after washing because theeffects of these drugs on cAMP synthesis and aggre-gation are rapidly reversible (Brenner and Thorns,1984; Klein and Brachet, 1975). Instead, our resultsmay reflect earlier observations that prestalk differ-entiation is only slowly reversible (Raper, 1940; Bon-ner, 1949).

In summary, these data show that (1) drugs thatincrease (8-Br-cAMP) or decrease (caffeine and pro-gesterone) intracellular cAMP levels effectively altercell fate when added late in development, (2) addingcaffeine or progesterone early in development inducesstable changes in cell fate while raising cAMP levelsearly has no lasting effects, and (3) high pH andammonia promote spore formation by a mechanismthat is consistent with elevation, but not reduction, ofintracellular cAMP levels.

Effects of ammonia and high pH on accumulation ofintracellular cAMPTo establish whether a link exists between extracellularNH3 and intracellular cAMP, we determined the effects

Table 1. Effects of changing developmental conditions after 10 h of in vitro differentiation

Developmental conditions Percent cell-type±s.D.

1

2345678910111213141516

0-10 h

ControlControlpH7.5pH7.5Control

NH3NH3

Control8-Br-cAMP8-Br-cAMP

ControlCafCaf

ControlProgProg

After 10 h

ControlpH7.5pH7.5Control

NH3NH3

Control8-Br-cAMP8-Br-cAMP

ControlCafCaf

ControlProgProg

Control

Spore

23±242±339±121±346±447±425±049±15O±225±20±0

0±00±0

Stalk cell

72±354±454±173±25O±348±069±549±049±166±294±596±398±291±196±597±2

Amoeb;

5±1

4±17±26±14±15±26±52±1

9±16±43±3

9±14±42±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 asindicated. Data represent means and standard deviations of two independent experiments.


of high pH and ammonia on cAMP accumulationduring 'submerged aggregation', culture conditions thatpermit reliable measurements of both intracellular andsecreted cAMP levels (see Methods to distinguish from'in vitro differentiation'). Under these conditions, cellsin control cultures began streaming by 4.5 h and com-pleted aggregation by 6h. Adding 15mM NH4C1 tocultures at pH6.2 or raising the pH from 6.2 to 7.5delayed the onset of aggregation by 1 h. Adding 15 mMNH4CI to cultures at pH7.5 delayed aggregation by3-4h. However, once initiated, aggregation proceedednormally in cultures exposed to ammonia and/or highpH (not shown).

Fig. 4A shows intracellular cAMP levels during sub-merged aggregation of HB200 cells. Cells cultured atpH6.2 and pH7.5 had identical levels of intracellularcAMP at 6h of development. However, cAMP ac-cumulated more quickly and was maintained at maxi-mal levels through 8h of development at pH7.5, whilecAMP 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 accumulationof 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 (opentriangles), pH6.2 with 15 mM NH4C1 (closed squares), orpH7.5 with 15 mM NH4CI (open squares). At the indicatedtimes, cAMP samples were taken and measured induplicate by radioimmune assay. Data are means andstandard deviations of 2 or more independent experiments.

Still more striking was the finding that, in cells culturedwith 15 mM NH4CI at pH6.2 or pH7.5, intracellularcAMP were nearly maximal at 2h. These levels equaledor exceeded the 6h peak in control cells and weremaintained through 8 h of development. Identical re-sults were obtained with V12M2 cells (Fig. 4B). Thus,increasing ammonia levels, either by increasing the pHor by direct addition of NH4CI, stimulated precociousaccumulation and prolonged maintenance of high intra-cellular cAMP levels in both wild-type and sporogenousamoebae. This could explain the role of NH3 as aprespore morphogen because elevating intracellularcAMP levels is sufficient to promote spore formation(Kay, 1989; Riley et al. 1989).

Ammonia might promote accumulation of intracellu-lar cAMP by altering rates of cAMP synthesis, degra-dation or secretion. However, we were unable to detectany effects of ammonia on specific activities of cellularphosphodiesterase (PDE) or adenylate cyclase activi-ties in cells developed for 2h, the time when ammonia-treated cells first achieved maximal intracellular cAMPlevels. In fact, enzyme activities in cells developed for2h (with or without ammonia) were not significantlyhigher than in vegetative cells (not shown). In contrast,ammonia did affect the amount of cAMP secreted intothe medium. Cultures exposed to 15 mM NH4CI atpH7.5 accumulated only 1/10 as much cAMP in theextracellular medium as control cultures at pH6.2(Fig. 4C). Intermediate levels of extracellular cAMPaccumulated in cultures at pH7.5 with no exogenousNH4CI and at pH6.2 with 15mM NH4CI (Fig. 4C).Decreased levels of extracellular cAMP accumulationwere not due to increased rates of cAMP hydrolysisbecause secreted PDE activities were the same inammonia-treated and control cultures (Fig. 5). Thesedata indicate that ammonia raises intracellular cAMPlevels by inhibiting cAMP secretion. This mechanismexplains the apparent discrepancy between our resultsand those of previous studies (see Discussion) andcould also explain why ammonia delays aggregation byseveral hours.

Ammonia antagonizes the effects of caffeine andprogesteroneHB200 amoebae terminally differentiate as stalk cellswhen caffeine or progesterone are used to lower intra-cellular cAMP levels (Table 1). However, simultaneousexposure to 8-Br-cAMP completely reverses inhibitionof spore formation in cultures exposed to caffeine orprogesterone (Riley et al. 1989). This change in cell fateis thought to result from diffusion of 8-Br-cAMP intocells where it binds to intracellular targets of cAMP,such as cAMP-dependent protein kinase, for which ithas high affinity (DeWit et al. 1984). Table 2 shows thatammonia also restores spore formation to caffeine- andprogesterone-treated cultures. Spore formation wasinhibited and nearly all cells differentiated as stalk cellswhen cultured with 2.5 mM caffeine or IOJJM progester-one, but spore inhibition was completely reversed if15 mM NH4CI was also added. It is possible thatammonia bypasses the effects of caffeine and progester-


2 4 6 8Hours Devalopment

Fig. 5. Effect of NH3 on accumulation of secretedphosphodiesterase. Vegetative HB200 cells were plated forsubmerged aggregation at pH6.2 (circles) or at pH7.5 with15 mM NH4CI (squares). At the indicated times,extracellular media were drawn off, centrifuged to removecells, and dialyzed at 4°C against 50 mM Tris, pH7.6, and5mM MgCl2. Dialysis was performed for 24 h, during whichthe buffer was changed twice. 80/d of dialyzed media weremixed with assay cocktail to give 200 ;d containing 20 mMTris, 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 twoindependent experiments.

Table 2. Ammonia reverses inhibition of sporeformation in the presence of caffeine or progesterone

Developmentalconditions

Percent cell type±s.D.

Spore Stalk cell Amoeba

ControlNH3CafProgCaf+NH3Prog+NHa

24±352±53±12±1

32±420±2

74±439±890±l97±157±373±1

2±29±47±2l±0

7±1

HB200 cells were plated for in vitro differentiation at pH 6.2 inthe presence of 15 mM NR,C1 (NH3), 2.5 mM caffeine (Caf), 10/uuprogesterone (Prog), or with no drugs (Control). Higherconcentrations of caffeine (5ITIM) or progesterone (20 /an) weretoxic if ammonia was also present. Data are means and standarddeviations of two independent experiments.

one by inhibiting cAMP secretion and thereby raisingintracellular cAMP levels. (Basal rates of cAMP syn-thesis persist in the presence of caffeine and progester-one.) Alternatively, ammonia might directly activatetargets of intracellular cAMP, such as cAMP-depen-dent protein kinase, or another second messengersystem that is dominant over changes in intracellularcAMP.

We cannot test the effects of ammonia on intracellu-lar cAMP accumulation during in vitro differentiationbecause high levels of exogenous cAMP (1 mM) pre-clude reliable measurement of intracellular cAMPlevels. However, ammonia reversed the effects of

•5 40-

2 4 6

Hours Development

Fig. 6. Effect of simultaneous treatment with ammonia andcaffeine or progesterone on accumulation of intracellularcAMP. Vegetative HB200 cells were plated for submergedaggregation at pH6.2 with 2.5 mM caffeine (closed squares),10 jiM progesterone (closed circles), 15 mM NH3C1 (opentriangles), 2.5 mM caffeine and 15 mM NH4CI (opensquares), 10 ̂ M progesterone and 15 mM NH4CI (opencircles), or with no drugs (closed triangles). At theindicated times, cAMP samples were taken and measuredin duplicate by radio immune assay. Data are means andstandard deviations of two experiments.

caffeine and progesterone on intracellular cAMP ac-cumulation during submerged aggregation. Fig. 6shows that 2.5 mM caffeine and 10 /JM progesteronereduced internal cAMP levels unless 15 mM NH4CI wasalso present, in which case normal cAMP levels ac-cumulated. These data support the hypothesis thatammonia reversed the effects of caffeine and progester-one on cell fate by restoring normal intracellular cAMPlevels.

Discussion

In agreement with studies of other sporogenous strains(Gross et al. 1981; Gross et al. 1983), we have shownthat high concentrations of NH4CI or high pH enhancespore formation by HB200 cells during in vitro differen-tiation. While others have speculated that both agentsexert this effect by a common mechanism (Sussman,1982), we provide the first direct evidence that exogen-ous NH4CI and high pH function by increasing theconcentration of NH3 in the medium. High pH has noseparate role. In addition, we have shown that concen-trations of ammonia that promote spore formation leadto precocious accumulation and maintenance of highintracellular cAMP levels in HB200 and V12M2 cells.This is probably the mechanism by which ammoniapromotes spore formation because other conditionsthat raise intracellular cAMP levels also promote sporeformation (Kay, 1989; Riley et al. 1989).

Ammonia raises intracellular cAMP levels by inhibit-ing cAMP secretion. This explains how ammonia per-mits cells with low adenylate cyclase activities (early indevelopment or in the presence of caffeine) to accumu-late or maintain high intracellular cAMP levels (Figs 4


and 6). Inhibition of cAMP secretion also explains theability of ammonia to antagonize the effects of pro-gesterone (Table 2 and Fig. 6), a drug that normallystimulates cAMP secretion (Klein and Brachet, 1975).The mechanism of cAMP secretion in Dictyostelium isunknown, but differential regulation of this functionmight be important during development because pre-stalk cells of migrating slugs secrete much more cAMPthan do prespore cells (Bonner and Slifkin, 1949). Thisraises the interesting possibility that ammonia inducesdifferentiation of prespore cells in migrating slugs byinhibiting cAMP secretion and thereby raising intra-cellular cAMP levels.

Stimulation of intracellular cAMP accumulation byammonia would not have been expected based onearlier studies showing that 15 mM NH4C1 at pH7.2inhibits cAMP synthesis by aggregation-competent cellsof wild-type strain NC4 (Schindler and Sussman, 1979;Williams et al. 1984). However, the previous studiesexamined the short term effects of NH4CI on cAMPsynthesis by aggregating cells, whereas we looked ataccumulation, not the rate of synthesis, in cells culturedfrom the time of starvation to 8 h of development. Aswe have shown, ammonia can increase accumulation ofintracellular cAMP even when adenylate cyclase ac-tivity is low. Moreover, it is possible that adenylatecyclase of Dictyostelium is only transiently sensitive tochanges in pH or ammonia concentrations and that,after a period of adjustment, high rates of synthesisreturn. Indeed, Khachatrian et al. (1987) showed thatpreincubating Dictyostelium membranes with very highlevels of NH4SO4 (>100ITIM) increases activation ofadenylate cyclase by inhibiting an inhibitory G protein.

Low rates of cAMP degradation could also play apart in maintaining high cAMP levels in ammonia-treated cells. We have recently shown that expression ofthe Dictyostelium cAMP phosphodiesterase gene andaccumulation of enzyme activity are negatively regu-lated by high intracellular cAMP levels: treatment withammonia or 8-Br-cAMP reduces cellular PDE activityby nearly half (B. B. Riley and S. L. Barclay, unpub-lished data). This suggests that, as ammonia raisesintracellular cAMP levels, reduction of cellular PDEactivity helps to maintain high cAMP levels.

These results and other recent studies suggest thatintracellular cAMP regulates Dictyostelium develop-ment, perhaps by more than one mechanism. Over-expression of the regulatory subunit of cAMP-depen-dent protein kinase disrupts development at a stageprior to aggregation (Simon et al. 1989; Firtel andChapman, 1990). This block presumably results fromlow kinase activity. Another cAMP-binding protein,CABP1, may function to transduce the cAMP signalfrom the cell membrane into the nucleus (Kay et al.1987) and has been implicated in regulation of the rateof development (Tsang et al. 1987). It is not yet clearwhat cell functions these proteins regulate or how theyaffect cell fate. However, this study and others (Rileyand Barclay, 1986; Riley et al. 1989; Kay, 1989) clearlyshow that cell fate correlates with intracellular cAMPlevels.

Genes that respond to changes in intracellular cAMPlevels should be useful in elucidating the mechanisms bywhich cAMP affects development. Use of drugs likeammonia, 8-Br-cAMP, caffeine, and progesterone toalter intracellular cAMP levels during in vitro differen-tiation could be a powerful system for detecting suchgenes and studying their expression.

This research was supported by N1H grant GM35432awarded to S. L. Barclay.

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{Accepted 22 March 1990)

ammonia promotes accumulation of intracellular camp in ... · ammonia on cell fate and accumulation...

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