©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
cGMP Accumulation Induced by Hypertonic Stress in Dictyostelium discoideum(*)

(Received for publication, July 27, 1995; and in revised form, December 15, 1995)

Masakazu Oyama

From the Department of Life Science, Faculty of Science, Himeji Institute of Technology, Shosha 2167, Himeji, Hyogo 671-22, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The change in extracellular osmolarity from 0.07 osM to 0.38 osM caused rapid cell shrinkage and loss of pseudopodes in Dictyostelium discoideum amoebae and induced elevation of total (cellular + extracellular) cGMP with a 2.5-min lag. cGMP accumulation reached a peak at 10-15 min after the change, and then the total cGMP gradually decreased. cGMP first accumulated intracellularly and was then secreted. A roughly identical osmotic concentration was required for the accumulation when the effect of KCl and glucose was tested. The non-osmolytes, formamide and ethanol, did not induce the accumulation. We concluded that hypertonic stress induces cGMP accumulation in D. discoideum amoebae.

The hypertonic stress-induced accumulation of cGMP was observed in a streamer F mutant (NP368) that lacks cGMP-specific phosphodiesterase. While Dictyostelium cells also have nonspecific phosphodiesterases that degrade both cGMP and cAMP, hypertonic stress induced only a small increase in cAMP in wild type and streamer F cells. These results suggest that hypertonic stress-induced accumulation of cGMP is due to the activation of guanylate cyclase rather than the inhibition of phosphodiesterases.

Binding of folic acid to the specific receptors on the cell surface induces a rapid transient accumulation of cGMP that reaches a peak at 10 s. When cells were stimulated by folic acid after the addition of 0.31 M glucose, rapid transient cGMP accumulation was observed immediately after the stimulation by folic acid and prolonged cGMP accumulation was induced 2-3 min after the addition of glucose irrespective of the timing of folic acid stimulation. These results suggest that the hypertonic stress-induced and the receptor-mediated accumulation proceed independently of one another. 2,3-Dimercapto-1-propanol, a thiol-reducing reagent, induces prolonged cGMP accumulation similar to hypertonic stress. However, the hypertonic stress-induced cGMP accumulation was enhanced by EDTA and was not suppressed by folic acid and cAMP. These characteristics are distinct from the reducing reagent-induced accumulation that is suppressed by EDTA, folic acid, and cAMP. These findings show that hypertonic stress has a unique effect on the activation of guanylate cyclase.


INTRODUCTION

Amoebae of the cellular slime mold, Dictyostelium discoideum, grow as single cells feeding on bacteria. The growing amoebae move chemotactically toward folic acid which is secreted by various bacteria(1) . This chemotaxis is mediated by cell surface folate receptors (2, 3) and is thought to be a mechanism for finding food bacteria. Starvation induces expression of cAMP receptors on the cell surface (4, 5, 6) and Dictyostelium amoebae begin to show chemotactic movement to cAMP(7) . They aggregate by chemotaxis to cAMP secreted by the amoebae themselves and form a multicellular organism. Through a complex morphogenetic process, the cells within the organism finally differentiate into spores and stalk cells. Extracellular cAMP plays important roles not only in the aggregation but also in cell differentiation during the multicellular stage of development in this organism(8, 9, 10, 30, 43) .

Binding of folic acid or cAMP to the specific receptors on the cell surface induces complex intracellular signals. These intracellular signals cooperatively induce chemotaxis and differentiation. GTP-binding proteins(11) , ERK2(12) , guanylate cyclase(13, 14) , adenylate cyclase (15, 16, 17) and inositol phosphate turnover(18, 19, 20) play a role in signal transduction. Among them, guanylate cyclase is thought to be regulated by both an activation signal and an adaptation signal. These two intracellular signals induce a transient activation of this enzyme and a transient accumulation of cGMP.

Living organisms have a defense mechanism to cope with changes in extracellular osmotic conditions. It has been known that hyperosmotic conditions suppress the receptor-mediated activation of adenylate cyclase (21, 22) and induce prespore-specific enzyme(24) , spore formation(23) , and the phosphorylation of proteins (25) in this organism. Since conditions, which specifically modify the receptor-mediated signals, are useful for the investigation of intracellular signal transduction, we further studied the effect of hypertonic stress on signal transduction. We report here that hypertonic stress induces cGMP accumulation in Dictyostelium cells.


MATERIALS AND METHODS

Dictyostelium discoideum wild type (NC4) and a streamer F mutant (NP368), which lacks cGMP-phosphodiesterase(29) , were grown in shaking culture in phosphate buffer with Escherichia coli B/r as a food source. When aggregating cells were needed, the cells were developed on a nitrocellulose filter. Details have been described previously(26, 27) .

The cells were washed with a salt solution (30 mM NaCl, 30 mM KCl) and resuspended in 20 mM phosphate buffer (pH 6.4) containing 10 mM KCl (PBK) at 1.0-1.5 times 10^7 cells/ml. When the effects of EDTA were to be tested (Fig. 6), the cells were washed with the salt solution containing 2 mM EDTA and then washed with the EDTA-free salt solution. The cell suspension in PBK was shaken at 22 °C for at least 1 h (vegetative cells) or 0.5 h (aggregating cells). Then PBK containing 3 times concentrated additives was added. When cells were stimulated by 2,3-dimercapto-1-propanol (Kanto Chemical), folic acid (Nakarai Chemicals), 2`-deoxyadenosine cyclic 3`:5`-monophosphate (dcAMP, Sigma) or cAMP (Sigma) alone, a 50-200 times concentrated solution was added to the cell suspension instead of the 3 times concentrated one. The final concentrations of additives are indicated in the text. An aliquot of the cell suspension was removed to a tube containing HClO(4). The HClO(4) was neutralized by the addition of KHCO(3). cAMP or cGMP in the supernatant was assayed using the Amersham isotope dilution assay kit.


Figure 6: Effect of EDTA on the hypertonic stress-induced and the thiol-reducing reagent-induced accumulation of cGMP. Vegetative amoebae washed with the salt solution containing 2 mM EDTA were cultured with shaking in PBK containing 1 mg/ml K252a with (closed symbols) or without (open symbols) 2 mM EDTA. They were stimulated by 6 mM dimercaptopropanol (A) or 0.31 M glucose (B). The cell suspensions were sampled for total cGMP assay at the indicated times.



When cellular and extracellular cGMP were assayed, a 200-µl cell suspension was sampled at the indicated times and centrifuged at 12,000 rpm for 10 s. Part of the supernatant was removed and mixed with HClO(4) 25 s after the indicated times and then the remaining supernatant was removed using an aspirator and the cell pellet was dissolved in HClO(4) solution 50 s after the indicated times. cGMP in each sample was determined as extracellular and cellular cGMP. The cell suspension was sampled 10 s before the indicated times for total cGMP assay.

When the cell volume was measured, vegetative cells were cultured with shaking in PBK for 1 h and then glucose (final 0.31 M) in PBK or PBK alone (control) was added as described above. Ten to twenty min after the addition, the cell suspension (10 ml) was removed into a tube containing 2 ml of 20 mg/ml blue dextran with an average molecular weight of 2,000,000 (Sigma) dissolved in PBK (control) or PBK containing 0.31 M glucose. The cells were mixed and then centrifuged at 3000 rpm for 1 min. The supernatant was removed using an aspirator. The total weight (test tube plus cell pellet) was measured using a balance (Sartorius R200D) and then the cell pellet was dissolved in 2 ml of PBK. The cell suspension was centrifuged at 12,000 rpm for 15 s. The absorbance of the supernatant at 620 nm (A) was measured using a spectrophotometer (Hitachi U1100). The same treatment without blue dextran was also performed, and the A of the supernatant was measured as the background. The wet cell weight was estimated by substraction of the dry weight of the tube from the total weight. The actual cell weight was estimated by substraction of the extracellular liquid which was estimated by the absorbance of blue dextran from the wet cell weight.

All results described in this report were reproduced in at least two separate experiments.


RESULTS

Cell Shrinkage Induced by Hypertonic Stress

Vegetative Dictyostelium amoebae were washed and cultured with shaking in PBK. The osmotic strength of PBK was about 0.07 osM. The addition of a glucose solution dissolved in PBK (final 0.31 M glucose in PBK) induced rapid cell shrinkage. The cells almost completely lost their pseudopodes, became round in shape and the cell surface began to ripple probably due to excess plasma membrane about 1 min after the addition. The cell volume in a centrifuged cell pellet was estimated using blue dextran as an indicator of the volume of the extracellular medium (Table 1). The cell volume decreased 45% under the hypertonic conditions (Table 1). The shrunken cell shape did not change after a 1-h culture under the hypertonic conditions.



cGMP Accumulation Induced by Hypertonic Stress

Adenylate cyclase and guanylate cyclase are major components of intracellular signal transduction in this organism. Since it has been reported that hypertonic conditions suppress the receptor-mediated activation of adenylate cyclase, we studied the effects of hypertonic stress on cGMP metabolism (Fig. 1). Total cGMP began to increase after a 2.5-min lag and reached a peak at 10-15 min. While cGMP then gradually decreased, the total cGMP under the hypertonic conditions was still higher than that in the control 1 h after the addition (Fig. 1).


Figure 1: Time course of the hypertonic stress-induced accumulation of cGMP. Vegetative amoebae were cultured with shaking in PBK (about 0.07 osM) for more than 1 h and then a half volume of glucose dissolved in PBK was added at zero time. The final concentration of glucose was 0.31 M and the final osmotic concentration of the culture medium was about 0.38 osM. The cell suspension was sampled at the indicated times for cGMP assay. The mean and standard deviation of 5 separate experiments are shown.



Sugars, amino acids, glycerol, and inorganic salts induced a similar accumulation (Table 2). While inorganic salts were less effective than the organic osmolytes, the time course of cGMP accumulation induced by 0.15 M KCl was roughly identical to that induced by 0.31 M glucose (data not shown). Furthermore, equivalent osmotic concentration (two times more glucose than KCl in molar concentration) was required for the induction of cGMP accumulation (Fig. 2). In contrast to osmolytes, non-osmolytes, such as ethanol and formamide, did not induce the accumulation. Additionally, glucose could induce the accumulation in the presence of these non-osmolytes (Table 2). These results suggest that hypertonic conditions rather than a specific effect of additives induce cGMP accumulation.




Figure 2: Concentrations of glucose and KCl required for the induction of cGMP accumulation. Glucose (closed symbols) or KCl (open symbols) solution dissolved in PBK was added to vegetative amoebae cultured with shaking in PBK. Their final concentrations are indicated. The cell suspensions were sampled for cGMP assay 10 min after the addition. Normalized results are shown. Two symbols (circles and triangles) show two separate experiments.



Distribution of Accumulated cGMP

Cellular cGMP increased and nearly reached a peak 3 min after the addition of glucose. While the elevated cGMP decreased gradually, cellular cGMP was higher than control for more than 30 min (Fig. 3). Extracellular cGMP began to increase shortly after the increase in cellular cGMP. The sum of cellular and extracellular cGMP was close to the total cGMP assayed directly (Fig. 3). These results show that cGMP first accumulated intracellularly and that the accumulated cGMP was secreted quickly. The function of extracellular cGMP is still unknown.


Figure 3: Time course of the hypertonic stress-induced cellular, extracellular, and total cGMP accumulation. Vegetative amoebae were cultured with shaking in PBK. Glucose (final 0.31 M) in PBK was added at zero time. The cell suspension was sampled 10 s before the indicated times for assay of total cGMP (circles). The cell suspension was sampled at the indicated times and centrifuged. Part of the supernatant was sampled 25 s after the indicated times for extracellular cGMP assay (triangles). The remaining supernatant was removed and the cell pellet was dissolved in perchloric acid for cellular cGMP assay (squares) 50 s after the indicated times. The sum of the cellular and extracellular cGMPs is also shown (crosses).



Hypertonic Stress Induces Activation of Guanylate Cyclase

Both the inhibition of degradation and the activation of synthesis could induce the accumulation of cGMP. Dictyostelium has two types of degradation enzymes, cGMP-specific phosphodiesterase and a nonspecific phosphodiesterase which degrades both cAMP and cGMP equally. It has been reported that the streamer F mutant (NP368) lacks the cGMP-specific phosphodiesterase (29) . This mutant accumulated cGMP in response to hypertonic stress (Fig. 4). We confirmed that the folic acid-induced cGMP accumulation is prolonged in this mutant (data not shown). Furthermore, hypertonic conditions induced only a small increase in cAMP in both wild type cells (vegetative cells and aggregating cells) and vegetative streamer F cells (data not shown). These results suggest that the accumulation of cGMP induced by hypertonic conditions is due to the activation of guanylate cyclase rather than the inhibition of cGMP-specific and nonspecific phosphodiesterases.


Figure 4: Time course of the hypertonic stress-induced accumulation of cGMP in streamer F mutant cells. Glucose (final 0.31 M) in PBK (closed symbols) or PBK alone (open symbols) was added to vegetative streamer F (NP368) cells cultured with shaking in PBK at zero time. The cell suspensions were sampled for total cGMP assay at the indicated times.



Relation of Hypertonic Stress and Folic Acid

Vegetative cells have cell surface receptors for folic acid that are involved in chemotaxis(2, 3) . The binding of folic acid to the receptors induces a rapid transient accumulation of cGMP that reaches a peak at 10 s (13, 14) (Fig. 5). Since the pattern of cGMP accumulation induced by folic acid and hypertonic stress is clearly different, we compared the effects of folic acid and hypertonic stress. The addition of both folic acid and hypertonic shock to cells induced the rapid transient accumulation of total cGMP identical to that observed when folic acid was added alone (Fig. 5). This was then followed by the prolonged elevation of total cGMP identical to that observed with hypertonic stress alone both over time and in amount (data not shown). Since folic acid is degraded rapidly, we further tested their relationship. When folic acid was added after glucose addition, rapid cGMP accumulation was observed for the first 10 s with a peak that was slightly lower than that induced by folic acid alone (Fig. 5). cGMP accumulation induced by hypertonic stress was observed 2-3 min after the addition of glucose, irrespective of the timing of folic acid stimulation (Fig. 5). These results suggest that the adaptation signal induced by folic acid does not interfere with the accumulation of cGMP induced by hypertonic conditions and that hypertonic conditions do not affect the folic acid-induced activation and adaptation of guanylate cyclase.


Figure 5: Relation between the hypertonic stress-induced and the folic acid-induced cGMP accumulation. Vegetative amoebae were cultured with shaking in PBK and then folic acid alone (200 µM, open circles), glucose alone (final 0.31 M, crosses), or glucose + folic acid in PBK (closed circles) was added. The cells treated with glucose (crosses) were stimulated by 50 times concentrated folic acid solution (final 200 µM) 0.5 min (open triangles), 1 min (closed triangles), 1.5 min (open squares), 2 min (closed squares), 2.5 min (open diamonds), or 3 min (closed diamonds) after the addition of glucose. The cell suspensions were sampled for total cGMP assay at the indicated times.



Relation of Hypertonic Stress and Reducing Reagent

We compared the effect of hypertonic stress with that of a thiol-reducing reagent, 2,3-dimercapto-1-propanol, which induces continuous cGMP accumulation (27) (Fig. 6). We tested the effect of K252a, a protein kinase inhibitor, and EDTA on the hypertonic stress-induced accumulation of cGMP, since K252a is an enhancer (27) and EDTA is a potential inhibitor (28) of the reducing reagent-induced accumulation of cGMP. We found that K252a enhanced the hypertonic stress-induced accumulation of cGMP (data not shown). EDTA enhanced the hypertonic stress-induced accumulation of cGMP (Fig. 6B) but inhibited the reducing reagent-induced one (Fig. 6A). Therefore, although both hypertonic stress and reducing reagent induce the continuous (or prolonged) accumulation of cGMP (Fig. 6), these two inducers act through different mechanisms.

Effects of Hypertonic Stress in Aggregating Cells

Hypertonic stress induced cGMP accumulation in aggregating cells as well as in vegetative cells with a time course identical to that in vegetative cells except for a temporary decrease between 5 and 10 min after the osmotic shock (Fig. 7). The amount of cGMP accumulated in aggregating cells was lower than that in vegetative cells (Fig. 7).


Figure 7: Time course of cGMP accumulation induced by cAMP, hypertonic stress, or cAMP + hypertonic stress. Aggregating cells developed on filter were collected and cultured with shaking in PBK. Glucose alone (0.31 M, circle), cAMP alone (100 µM, triangle), or the mixture of them (square) was added at zero time. The cell suspensions were sampled for assay of total cGMP at the indicated times.



Aggregating cells have cAMP receptors. cAMP induces a rapid transient cGMP accumulation similar to that induced by folic acid in vegetative cells. cAMP + glucose induced rapid cGMP accumulation identical to the one induced by cAMP alone (Fig. 7, details of the results are not shown). Since we used a high concentration of cAMP (100 µM), the cAMP receptors were continuously saturated by cAMP for more than 10 min(26) . Glucose alone and cAMP + glucose induced the continuous cGMP accumulation, which began at the same time (2.5 min after the addition, Fig. 7). cAMP + glucose induced much more cGMP accumulation than glucose alone (Fig. 7), probably due to competitive protection of secreted cGMP against phosphodiesterase by 100 µM cAMP.

cAMP induces the activation of adenylate cyclase as well as that of guanylate cyclase(15, 16, 17) . It is known that the cAMP-induced activation of adenylate cyclase is suppressed by hypertonic stress(21, 22) . We tested the relation between the activation of guanylate cyclase and the inhibition of adenylate cyclase by hypertonic stress. The concentration of glucose required for the half maximum effect was 0.15 M for the inhibition and 0.27 M for the activation (Fig. 8). The inhibition began just after the addition of glucose. dcAMP stimulation 1 min after the osmotic shock did not induce cAMP accumulation (data not shown). Since the cell suspension was sampled for cAMP assay 2 min after the stimulation by dcAMP, the inhibition was completed within 3 min after the addition of glucose. Therefore, the inhibition had been completed at the beginning of cGMP accumulation.


Figure 8: Osmolarity required for cGMP accumulation and the inhibition of the receptor-mediated activation of adenylate cyclase. Aggregating cells developed on filters were collected and cultured with shaking in PBK. The indicated concentrations of glucose was added, and the cell suspensions were stimulated by 10 µM dcAMP 2 min after the glucose addition. The cell suspension was sampled 2 min after the dcAMP stimulation for total cAMP assay (closed symbols). The cell suspension stimulated by glucose alone was sampled 10 min after the glucose addition for assay of total cGMP (open symbols). Normalized results are shown. Two symbols (circles and triangles) show two separate experiments.




DISCUSSION

We found that cGMP accumulates under hypertonic conditions in D. discoideum. We also found that the hypertonic stress-induced accumulation is observed in a mutant lacking cGMP-specific phosphodiesterase (29) and that cAMP accumulation only marginally occurs under hypertonic conditions. These results suggest that hypertonic stress induces the accumulation of cGMP through the activation of guanylate cyclase rather than the inhibition of phosphodiesterases.

The hypertonic stress-induced accumulation of total cGMP reaches a peak at 10-15 min, which is distinct from the receptor-mediated activation of guanylate cyclase that lasts 10 s(13, 14) . A thiol-reducing reagent induces continuous (or prolonged) cGMP accumulation similar to hypertonic stress(27) . However, the reducing reagent-induced accumulation is inhibited by EDTA, while the hypertonic stress-induced one is enhanced by EDTA (Fig. 6). Furthermore, the hypertonic stress-induced accumulation is suppressed by neither folic acid (Fig. 5) nor cAMP (Fig. 7). This is clearly different from the reducing reagent-induced accumulation which is suppressed by cAMP and folic acid (27) . These facts show that hypertonic stress has a unique effect on the activation of guanylate cyclase and suggest that hypertonic stress induces or mimics the intracellular activation signal after a point where the adaptation signal interferes with the activation signal or that the hypertonic stress-induced activation is independent of the receptor-mediated signals.

It has been reported that various substances, such as caffeine(33) , adenosine(33) , concanavalin A(34, 35, 40, 41) , and K252a(27, 36) , which inhibit the cAMP-induced activation of adenylate cyclase, enhance the receptor-mediated activation of guanylate cyclase. Hypertonic stress is also known to suppress the cAMP-induced activation of adenylate cyclase(21, 22) . However, hypertonic stress is exceptional, since other inhibitors do not induce the activation of guanylate cyclase by themselves. We found that the inhibition by hypertonic stress of the cAMP-induced activation of adenylate cyclase occurs at a lower concentration and faster than the hypertonic stress-induced activation of guanylate cyclase (Fig. 8). These reports and our findings in this report suggest that the mechanism required for the inhibition of the cAMP-induced activation of adenylate cyclase may be related to (but not sufficient for) the activation of guanylate cyclase.

A change in osmotic conditions is a common stress for all organisms. Mechanisms to cope with this stress are observed in various organisms. Dictyostelium amoebae have such mechanisms, since it has been reported that cell volume is regulated constantly under different osmotic environments in this organism(37) . The cGMP accumulation reported here may be a part of such a mechanism. It has been reported that cGMP is involved in the regulation of the association of myosin II with the cytoskeleton in this organism(31, 32, 42) . cGMP may act to adjust the size of the cytoskeleton to the reduced cell volume. Involvement of mitogen-activated protein kinase cascade in response to osmotic stress has been reported in the yeast, Sacchromyces cerevisiae(38, 39) . It has been reported in Dictyostelium that the mitogen-activated protein kinase-related protein is involved in the receptor-mediated signal transduction (12) and that protein phosphorylation is induced by hypertonic stress(25) . An interesting question is whether these phosphorylated proteins mediate responses to hypertonic stress and whether the mitogen-activated protein kinase cascade plays a role in the hypertonic stress-induced responses in Dictyostelium.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


ACKNOWLEDGEMENTS

I thank Drs. K. Okamoto and Y. Yamada for the supply of NP368 and Dr. D. Blumberg for her advice to this manuscript. I also thank Dr. K. Kubota for encouragement.


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