Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range

The proportion of prestalk and prespore cells in Dictyostelium discoideum slugs is often cited as an example of "almost perfect" regulation. The pattern is similar over a very wide range of cell number; furthermore, removal of either of the cell types leads to compensatory transdifferentiation. Several studies of Dictyostelium fruiting bodies, however, have suggested that proportioning in Dictyostelium differs systematically from true constancy. We have confirmed this in the slug stage using a short-lived beta-galactosidase as a reporter of the prestalk specific ecmA gene expression: the prestalk proportion decreases from 24+/-5% in slugs of 10(3) cells to 10+/-3% when 10(5) cells are present. Regeneration experiments suggest that this difference is not due to a modulation of the proportioning set-point by size, as one might have expected; instead there appears to be a regulatory "tolerance zone" at all sizes. After amputation of the whole posterior region, transdifferentiation stops after the fraction of prestalk has been reduced from 100% to 28+/-20%, well above the initial value of 10+/-3%, while after anterior removal the transdifferentiation endpoint is about 10%. Most strikingly, we find no regulation at all after partial amputations of the prespore region. It seems that any prestalk proportion is stable between a approximately 10% lower threshold and a approximately 30% upper threshold. To explain this, we propose a regulation mechanism based on a negative feedback plus cell type bistability. In both intact and regenerating slugs we find that the slug morphology is regulated so that the length-to-width ratio of the anterior region is constant.     

Dual control of the intracellular pH in aortic smooth muscle cells by a cAMP-sensitive HCO3-/Cl- antiporter and a protein kinase C-sensitive Na+/H+ antiporter

Two mechanisms are involved in the regulation of the intracellular pH (pHi) of aortic smooth muscle cells: the Na+/H+ antiporter and a Na+-independent HCO3-/Cl- antiporter. The Na+/H+ antiporter acts as a cell alkalinizing mechanism. It is activated by vasopressin and by phorbol esters when cells are incubated in the presence of bicarbonate but is not affected in the absence of bicarbonate. The HCO3-/Cl- antiporter acts as a cell acidifying mechanism. Agents such as forskolin, 8-Br-cAMP, and isoproterenol which raise intracellular cAMP levels inhibit the HCO3-/Cl- antiporter by shifting its pHi dependence in the alkaline direction. Thus, within the same cell type, different hormones control pHi variations by acting on different pHi regulating systems. An increase in pHi can be achieved either by a stimulation of a cell alkalinizing mechanism or by inhibition of a cell acidifying mechanism. A change of the activity of one pHi regulating mechanism modifies the responsiveness of the other to regulatory agents. Bicarbonate turns on the HCO3-/Cl- antiporter, decreases pHi and allows its regulation by protein kinase C through the Na+/H+ antiporter. Inhibition of the HCO3-/Cl- antiporter by cAMP increases the pHi and switches off the protein kinase C-mediated regulation.     

Characterization of Na+/H+ exchange in a rabbit corneal epithelial cell line (SIRC)

Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.     

Analysis of proportion regulation in slugs of Dictyostelium discoideum using a monoclonal antibody and a FACS-IV

A quantitative assay for estimating the proportion of prespore cells in D. discoideum slugs was established by labelling disaggregated slug cells with a prespore specific monoclonal antibody and analysing the cell population with a FACS-IV. The method is validated using a wild-type strain and its stalky mutant. "Wild-type" strains have different proportions of prespore cells and it is demonstrated that slugs of some strains have an increased percentage of prespore cells when migrated in the dark compared to the light and in the presence of EGTA. The technique is rapid and will make possible genetic analysis of proportion regulation in D. discoideum.     

Induction of Cell Differentiation of Isolated Cells in Dictyostelium discoideum by Low Extracellular pH

In Dictyostelium discoideum , the formation of multicellular masses is necessary for cell differentiation. However, the present study shows that amoebae of strain V12M2 efficiently differentiate to prespore or stalk cells under submerged incubation in a simple medium containing cAMP and salts without cell contact, only if the pH of the medium is maintained at acidic values; differentiation scarcely occurs in the neutral pH range. The optimum pH values for prespore and stalk cell differentiation are 5.1 and 4.5, respectively. In addition to the extracellular pH, Mg ions and the concentration of cAMP also affect the choice of the differentiation pathway. The time courses of differentiation of both cell types under optimum conditions are also presented.     

Cell-type Conversion at Low Temperature in Dictyostelium discoideum

The effects of low temperature (5°C) on cell-type conversion in whole slugs of Dictyostelium discoideum and their anterior prestalk- and posterior prespore-isolates were examined immunohistochemically and electronmicroscopically. When slugs were incubated for nine days at 5°C, the proportion of cells containing spore-antigens increased from about 75% to 85%. More important, the proportion of prespore and spore cells increased from about 3% to 40% in anterior prestalk isolates incubated at 5°C for 12 days, but no cell-type conversion from prespore to prestalk cells occurred in posterior prespore isolates. Therefore, the mechanism regulating the proportions of cells that operates at 21 °C does not operate at low temperature. The cells with full competence for stalk differentiation could change into stalk cells even at 5°C, because a short stalk was always formed when early culminants were transferred to low temperature. The effects of low temperature on several sequential steps of cell differentiation are discussed on the basis of these findings. The ultrastructural characteristics during the process of cell-type conversion are also described.     

Control of cell type proportioning in Dictyostelium discoideum by differentiation-inducing factor as determined by in situ hybridization

We have determined the proportions of the prespore and prestalk regions in Dictyostelium discoideum slugs by in situ hybridization with a large number of prespore- and prestalk-specific genes. Microarrays were used to discover genes expressed in a cell type-specific manner. Fifty-four prespore-specific genes were verified by in situ hybridization, including 18 that had been previously shown to be cell type specific. The 36 new genes more than doubles the number of available prespore markers. At the slug stage, the prespore genes hybridized to cells uniformly in the posterior 80% of wild-type slugs but hybridized to the posterior 90% of slugs lacking the secreted alkylphenone differentiation-inducing factor 1 (DIF-1). There was a compensatory twofold decrease in prestalk cells in DIF-less slugs. Removal of prespore cells resulted in cell type conversion in both wild-type and DIF-less anterior fragments. Thus, DIF-1 appears to act in concert with other processes to establish cell type proportions.     

CHANGES OF THE PRESPORE SPECIFIC STRUCTURE DURING DEDIFFERENTIATION AND CELL TYPE CONVERSION OF A SLIME MOLD CELL

In the slug of the cellular slime mold, Dictyostelium discoideum , are differentiated the anterior prestalk cells and the posterior prespore cells, whose differentiation is characterized by formation of the prespore specific vacuole (PSV). The ultrastructural changes of the PSV were investigated during dedifferentiation of a prespore cell disaggregated from a slug and also during conversion of the cell type, caused by fragmentation of a slug, between the prespore and the prestalk cells.
During the dedifferentiation, the PSV first lost its lining membrane which subsequently congregated, together with the inner filamentous material, to form some electron dense granules. Finally, the vacuole membrane was punctured, and the granules were released into cytoplasm. During conversion of the prespore to the prestalk cell, the PSV was degraded through the same process as in dedifferentiation, but the degradation proceeded much more synchronously in a converting cell. When a prestalk fragment was isolated from a slug, formation of the PSV was detected in no cell until 2 hr of incubation. After a lag, the PSV was formed in a converting cell through the process which is not a simple reversal of its degrading process.     

Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell

Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol. The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin. The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells. Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units. The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-[phenyl-methoxy]-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity. In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units. This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion. In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA). This compound also blocked changes in pHi due to changes in extracellular Na+. Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane. The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation. In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively. Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached. Thereafter, acidification continued without further change in pHi. To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl-. DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90%. Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

A 31P-NMR study on the recovery of intracellular pH in LLC-PK1/Cl4 cells from intracellular alkalinization

The regulation of intracellular pH (pHi) in a renal epithelial cell line, LLC-PK1/Cl4, during re-acidification from an alkaline load was studied by 31P-NMR. Intracellular alkalinization was induced by 10 mM ammonium glucuronate or by preloading with and subsequent removal of 20% CO2; the rate of re-acidification was found to be 0.047 pH units/min and 0.053 pH units/min, respectively. This rate of re-acidification was inhibited by 83% if Cl- was removed from the extracellular medium. A similar inhibition was found in the presence of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (76% inhibition) and 1 mM bumetanide (81% inhibition). No change in recovery was found after removing sodium from the extracellular medium, indicating that LLC-PK1/Cl4 cells recover from an intracellular alkaline load by a Cl-/HCO3- exchanger, which is SITS- and bumetanide-sensitive and has no requirement for sodium. In addition, the steady-state pHi in Cl4 cells was monitored by 31P-NMR. Removal of Cl- from the extracellular medium introduced an increase in pHi by 0.33 pH units, whereas 1 mM SITS and 1 mM bumetanide caused an increase in pHi by 0.14 or 0.13 pH units. In the presence of 1 mM amiloride, an inhibitor of the Na+/H+ exchanger, the steady-state pHi did not change significantly. These results indicate that at pHo 7.4 the steady-state intracellular pH of LLC-PK1/Cl4 cells strongly depends on the activity of the Cl-/HCO3- exchanger. Under the same conditions the activity of the Na+/H+ exchanger seems to be negligible.     

Differential Cell Cohesiveness Expressed by Prespore and Prestalk Cells of Dictyostelium discoideum

Pseudoplasmodia of Dictyostelium discoideum at the culmination stage were separated into two cell populations by sedimentation in a discontinuous renografin gradient. The two lighter fractions (I and II) had enzymatic activities characteristic of the anterior prestalk cells, while the heaviest fraction (III) showed enzyme activities characteristic of the posterior prespore cells. Cell-cell adhesion among prespore cells is much more resistant to EDTA dissociation than 10-h cells and prestalk cells. Fab fragments prepared from antibodies directed against a specific cell surface glycoprotein gp150 were more effective in dissociating prespore cells than prestalk cells. In addition, prespore cells contained an approximately 2-fold higher concentration of the endogenous carbohydrate binding protein discoidin-I than prestalk cells. These differences may account for the differential cohesiveness of these two cell populations and provide a basis for cell recognition and cell sorting at the slug stage.     

Transient increase in intracellular pH during Dictyostelium differentiation

The intracellular pH (pHi) of Dictyostelium discoideum amebae has been determined using the pH-dependent fluorescence of intracellularly trapped fluorescein (Thomas, J. A., R. N. Buschbaum, A. Zimiak, and E. Racker, Biochemistry, 18:2210-2218). The pHi of cells measured 45-60 min after initiation of differentiation was between 6.2 and 6.3. At approximately 2 h into differentiation cells underwent a transient intracellular alkalinization during which the pHi rose to 7.13 (+/- 0.3, n = 4), after which the pHi returned to approximately the original value (6.2-6.4). Cells that were removed from growth medium but were incubated in differentiation medium containing 3% dextrose did not exhibit this transient increase in pHi. The alkalinization event can also be prevented from occurring by differentiation in Na+-free solutions or by the addition of amiloride to sodium-containing buffer solutions, suggesting that the alkalinization is sodium dependent. When the alkalinization was prevented by amiloride treatment, cells did not progress normally into differentiation. This increase in pHi was initiated by the cells 2 h after removal from nutrient medium and it could be inhibited by several treatments that had been observed to delay the differentiation program, suggesting that it plays a major role in the initiation of the developmental program of this organism.     

Day-night variations in blood and intracellular pH in a lizard,Dipsosaurus dorsalis

Blood pH, PCO2 and PO2 of Dipsosaurus dorsalis were measured during the day and at night. Lizards at constant body temperature (25, 37 degrees C) and lizards experiencing diurnal changes in body temperature similar to those in nature were studied. In lizards at constant body temperatures, blood pH was about 0.1 unit less and blood PCO2 was 4-7 Torr higher at night compared to day. Similar patterns were seen in lizards on natural thermal cycles. Intracellular pH (pHi) of skeletal muscle, esophagus and liver was about 0.2 units lower at night than day but myocardial pHi was unchanged. Reduction in breathing frequency, and thus a relative hypercapnia from hypoventilation was consistent with the nocturnal acidification of the blood and intracellular compartments. Nocturnal acidification (CO2 retention) corresponds to periods of minimum metabolism. The possible impacts of diurnal shifts in hydrogen ion concentration on energy metabolism and metabolic regulation are discussed.     

Development of thermotolerance and changes in intracellular pH in CHO cells heated at 45.0 degrees C at pH 6.6

Chinese hamster ovary (CHO) cells were given short heat pulses (5 to 20 min) at 45.0 degrees C and incubated at 37 degrees C for up to 20 h under either pH 7.3 or 6.6 conditions. Thermotolerance developed under both pH conditions, but at a slower rate in the pH 6.6 medium. Intracellular pH (pHi) was measured with the dye, 1,4-diacetoxy-2,3-dicyanobenzene, combined with flow cytometry. Time-dependent changes in the intracellular pH occurred under either pH condition. CHO cells incubated under normal pH conditions had a transient increase in the pHi. This pHi elevation was followed by a rapid intracellular acidification of approximately 0.15 to 0.25 pH units. The timing of both the increases and decreases in the pHi was dependent on the magnitude of the initial heat dose. With heat doses less than or equal to 10 min, the pHi returned to normal unheated levels after the acidification phase. Although cells incubated under low pH (6.6) conditions showed similar pHi alterations, differences in the kinetics were measured. The intracellular pH increased immediately after heating. In addition, when intracellular acidification occurred, the rate of acidification was significantly reduced. With heat doses longer than 5 min under the low pH conditions, the pHi did not return to normal unheated levels.     

Intracellular pH changes of cultured bovine aortic endothelial cells in response to ATP addition

The changes of the intracellular pH (pHi) of cultured bovine aortic endothelial cells were fluorometrically monitored using 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). A biphasic pHi change was observed by addition of ATP: an initial acidification followed by an alkalinization of about 0.2 pH unit above the resting level of pHi 7.23. The alkalinization was dependent on [Na+]o and [H+]o, and was inhibited by 5-(N,N-hexamethylene)amiloride, indicating that the alkalinization is mediated by the Na+/H+ exchanger. The 50% effective concentration of ATP was about 1.4 microM. ADP similarly induced pHi changes, whereas AMP and adenosine were inactive. The pHi changes induced by ATP were dependent on the extracellular Ca2+, and the addition of calcium ionophore A23187 induced similar pHi changes. The results indicate that ATP activates the Na+/H+ exchanger in cultured bovine aortic endothelial cells and the activation is mediated by the P2-purinergic receptor and is dependent on the extracellular Ca2+.     

Flow cytometric measurement of intracellular pH in B16 tumors: intercell variance and effects of pretreatment with glucose

Flow cytometry was used to measure cytoplasmic pH (pHi) of B16 melanoma cells taken from tumor-bearing animals. We used a ratiometric method to allow measurements on an individual cell basis which were independent of cellular content of the pH indicator BCECF. In order to "freeze" any intercell variance which may have existed within the tumor mass, tumors were mechanically disaggregated in bicarbonate-free medium containing 0.5 mM amiloride at 4 degrees C and loaded with BCECF in choline chloride-based Earle's solution at 37 degrees C. Studies using cells grown in vitro showed that this protocol prevented acid load recovery during the 30-min period typically required between tumor excision and pHi measurement. A calibration curve was obtained by resuspending BCECF-stained cells in a range of buffers containing the proton ionophore nigericin. The range of values for individual cells was estimated by comparing the coefficient of variation of the test sample with that obtained when nigericin was used to reduce all cells to the pHi of the calibration buffer. The average value for mean tumor cell pH was 7.32 +/- 0.05 SD. Pretreatment of animals with intraperitoneal glucose for one hour resulted in an average for mean pHi of 7.17 +/- 0.17 SD. Mean coefficient of variation was 8.7%, and in the presence of nigericin, 8.1%. These values indicate a variance in measured pHi of approximately +/- 0.4 pH units, but most of this results from experimental error rather than true intercell pHi variance. The method used here is capable of detecting reduction in mean tumour pHi caused by ip glucose, but incapable of precise estimation of individual cell values. Despite these uncertainties, the results suggest that the range of pHi within B 16 tumors is small.     

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.     

Role of Cell Sorting in Pattern Formation in Dictyostelium discoideum

To examine the relationship between cell sorting and cell differentiation in the development of Dictyostelium discoideum , labeled cells grown in the absence of glucose [G(–) cells] and unlabeled cells grown in its presence [G(+) cells] were mixed and either allowed to undergo normal morphogenesis or cultured under submerged conditions. Changes in the distributions within a cell aggregate of labeled cells and cells stained with the conjugated antispore serum (prespore cells) were followed on the same sections by the methods of autoradiography and immunohistochemistry. In normal morphogenesis, differentiation of prespore cells apparently initiated and proceeded coincident with sorting out between G(+) and G(–) cells, during formation of a standing slug. By contrast, within an aggregate formed under submerged conditions, prespore cells began to differentiate long before G(+) and G(–) cells were sorted out, indicating that the cell sorting is not a prerequisite for the cell differentiation. The sorting out, however, brought about an accumulation of prespore cells in a hemisphere, thus producing a prestalk-prespore pattern within the aggregate.