Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.

PURPOSE: This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism. METHODS: Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels of Trp53, Oct-4, and Cdx2 were monitored by quantitative polymerase chain reactions (qPCRs). The effect of Trp53 on the expression of Oct-4 and Cdx2 was assessed by depleting Trp53 using Trp53 siRNA. RESULTS: Aneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels of Oct-4 and Cdx2. Adding 1 mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels of Oct-4 and Cdx2 mRNA. Trp53 RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, and Trp53 siRNA levels reduced the levels of Trp53 and increased levels of Oct-4 and Cdx2 mRNA by > twofold. CONCLUSION: These studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels of Trp53 mRNA that suppresses Oct-4 and Cdx2 expression.

Slender and stumpy bloodstream forms of Trypanosoma brucei display a differential response to extracellular acidic and proteolytic stress.

Natural infections of mammals with African trypanosomes, such as Trypanosoma brucei, are generally pleomorphic, the population consisting of different forms, termed slender and stumpy forms, that vary in number as the parasitaemia develops. We show that the differentiation of slender into stumpy forms is characterized by the acquisition by the parasite of the ability to regulate its internal pH, even in the face of a large, inwardly directed gradient of H+, as well as a tolerance towards external proteolytic stress. These adaptations effectively abbrogate cellular stress-activated signalling pathways involving adenylate cyclase and glycosylphosphoinositol-specific phospholipase-C mediated release of the surface coat. Although in metabolic terms stumpy forms of the parasite are considered to be preadapted to life in the arthropod vector, these data clearly demonstrate that these forms also possess additional cellular adaptations designed to deal with the immediate and potentially harmful changes in the extracellular environment that occur upon ingestion of a bloodmeal by the tsetse fly vector.

Tissue and intracellular pH in normal periarticular soft tissue and during different phases of antigen induced arthritis in the rat.

OBJECTIVE: To measure intracellular and tissue pH in periarticular soft tissue during different phases of antigen induced arthritis in the rat. METHODS: pH was calculated using the following values: (1) the distribution of [14C]-dimethyl-oxazolidinedione; (2) the total tissue water and the extracellular space water volume, which was measured as [14C]-sucrose distribution in nephrectomized rats. Experiments were performed during both maximal inflammation (Day 3) and the restorative phase (Day 14). RESULTS: In all animals both tissue (pHt) and intracellular (pHi) pH were lower in arthritic joints than in the contralateral control. Mean pHt in control joints was 7.37+/-0.03. In arthritic rats it was 7.30+/-0.05 on Day 3 after challenge and 7.27+/-0.03 on Day 14. The pHi ranges were 6.86-7.81 for controls, 6.65-7.28 for arthritis Day 3, and 5.66-6.91 for arthritis Day 14. CONCLUSION: In this model there is a reduction in pH in the periarticular tissue of arthritic joints. The magnitude is, however, relatively small and the pannus tissue is not uniquely acidic in comparison with other compartments. There does not seem to be a correlation between pH and changes in metabolic balance, pannus formation, or healing.

The effects of organic solvents on trimethadione n-demethylation in rats.

Many organic solvents are frequently used as support solvents to dissolve chemicals in the study concerning drug metabolism mediated by cytochrome P450. However, some organic solvents used as the support solvents affect the chemical's metabolism. It has been reported that some organic solvents are metabolized by CYP2E1 or inhibit its enzymatic reaction. In this study we investigated the effects of organic solvents, such as acetonitrile (AN), dimethylsulfoxide (DMSO), ethanol (EtOH), methanol (MeOH), polyethylene glycol (PEG) and propylene glycol (PG) on TMO (trimethadione) metabolism, which is mainly mediated by CYP2E1 in the rat. In the in vivo study, male SD rats were pretreated with an organic solvent intraperitoneally at a dosage of 0.5, 1 or 2 mmol/kg 1 hour before TMO administration orally at the dose of 4 mg/kg. After 2 hours, serum concentrations of TMO and DMO were determined by gas chromatography/flame detection (CG/FTD) and the serum DMO/TMO ratio was employed for assessment of the metabolic capacity of TMO. In the in vitro study, hepatic microsomal fraction was used as an enzyme source of TMO N-demethylase and enzyme activities were determined by the production of DMO. Pretreatment with DMSO and PG decreased the DMO/TMO ratio in a dose-related manner in vivo study. Furthermore, in vitro study TMO N-demethylase activity was inhibited by DMSO, EtOH and PG with different potency in a concentration related manner. However, no remarkable effects were observed by AN or PEG both in vivo and in vitro study. These results indicated that there are variations in the inhibitory effects of these organic solvents on CYP2E1-mediated metabolism and AN and PEG will be useful solvents to dissolve chemicals in the metabolic study mainly mediated by CYP2E1.

In vivo uptake of [3H]nimodipine in focal cerebral ischemia: modulation by hyperglycemia.

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 +/- 0.11 in normoglycemic rats (n = 12) and 6.57 +/- 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 +/- 0.45) than hyperglycemic (1.40 +/- 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 +/- 22 mL/100 g in normoglycemic rats (n = 8) and 33 +/- 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.

Intracellular pH monkey embryos at various stages of organogenesis estimated by dimethadione distribution.

Previous experiments using the transplacental distribution of 14C-DMO (5,5-dimethyloxazolidine-2,4-dione or commonly known as dimethadione) have demonstrated that the pH of rat embryos and fluids progressively decreases during organogenesis. The aim of the present experiments was to similarly evaluate pH changes during organogenesis in the cynomolgus monkey, which is a model for human embryogenesis. Using DMO quantitated by gas chromatography-mass spectrometry as opposed to the counting of radiolabelled compound, cynomolgus monkey embryos were determined to undergo a similar decrease in embryonic pHi over an approximately comparable period of development (Days 24-36 of gestation). The ratio of DMO in chorionic fluid to DMO in maternal plasma in the cynomolgus monkey also displayed a decrease with advancing gestational age indicative of a pH decrease. The DMO transplacental distribution was found to be significantly slower in the cynomolgus monkey than that in rodents. The present investigation indicates that the magnitude of the reduction of pH in embryonic cells and in extra-embryonic fluids over a period of organogenesis in the cynomolgus monkey is similar to the reduction detected in rodent embryos and fluids over a comparable developmental period, but the relative gradient between maternal blood pH and embryonic intracellular pH is different. The difference in the pH gradient between the two species may lead to differential transplacental distribution of exogenous and endogenous substances.

Influence of partial hepatectomy in dogs on trimethadione metabolism and microsomal monooxygenases.

1. The recovery of trimethadione (TMO) metabolism and its association with liver weight and the activity of TMO N-demethylase have been reported in rat following partial (68%) hepatectomy. In the present study, we examined the effect of liver regeneration on hepatic P450 isozymes and TMO metabolism in dog. 2. The ratio of dimethadione (DMO), being the only TMO metabolite, to TMO at 2 h after i.v. injection of TMO (4 mg/kg) fell to 80% of that in the preoperative animals by 24 h after hepatectomy. The DMO/TMO ratio gradually recovered from days 7 to 14, and by day 21 after hepatectomy it had increased to about 25%. At 28 days post-hepatectomy the ratio had returned to preoperative levels. 3. The activity of benzphetamine N-demethylase, TMO N-demethylase, p-nitro-anisole O-demethylase and aniline hydroxylase increased 3 days post-hepatectomy, exhibiting levels 4.77, 3.45, 1.51 and 1.91 times greater respectively than that of the preoperative liver in the same animal. Two weeks post-hepatectomy these activities had returned to normal. The activity of the 16 beta- and 2 beta-hydroxylation of testosterone was unchanged. However, the activity of 6 beta-hydroxylase decreased 7 days post-hepatectomy, while 16 alpha-hydroxylation had increased at 3 and 7 days post-hepatectomy compared with controls. 4. The changes in liver weight were nearly restored to preoperative levels 7 days post-hepatectomy. 5. Although the P450 content was unchanged from days 1 to 7 post-hepatectomy, it had decreased by 30% at day 14 and by 20% at day 28. The P4502B11 content 3, 7 and 14 days post-hepatectomy had increased 8, 10 and 2 times respectively, while the P4503A12 content at 7 and 14 days decreased by 30 approximately 50% compared with that of the pre-operative liver. 6. The data presented above do not reveal any relationship between P4502B11 induction and liver regeneration. The reason for such a change is unknown, therefore further investigation needs to be carried out.

Effect of perfusate pH on the influx of 5-5'-dimethyl-oxazolidine-2,4-dione and dissociation of epidermal growth factor from the cell-surface receptor: the existence of the proton diffusion barrier in the Disse space.

The influx clearance (PSinf.MID) of the weak acid 5,5'-dimethyl-oxazolidine-2,4-dione (DMO) was determined by the multiple indicator dilution method with the isolated perfused rat liver under various perfusate pH conditions, ranging from 6.4 to 7.6. Although the pH partition theory predicted an increase in influx clearance of ten times in proportion to the change in the unionized fraction of DMO, there was no measurable change in this value. The effect of medium pH on the steady-state cell/medium concentration ratio (C/M) ratio of DMO was also investigated using isolated hepatocytes. The C/M ratio increased while medium pH decreased, but this change was less marked than predicted by the pH partition theory. Finally the pH dependency of the dissociation rate constant (koff) of epidermal growth factor from its receptor was also investigated using both isolated rat hepatocytes and the perfused rat liver. When the extracellular pH was changed from 6.4 to 5.6, the koff value of isolated hepatocytes increased 44 times, while that of the perfused rat liver increased only 9 times. Therefore, the effect of changing the extracellular pH on pH-dependent dissociation of epidermal growth factor from its cell-surface receptor was less in the perfused liver than in isolated hepatocytes. These findings, in addition to the well-known existence of the Na(+)-H+ exchanger on the sinusoidal membrane and the possible existence of the unstirred water layer in the Disse space, seem to suggest the existence of the proton diffusion barrier in the rat liver, which remains stronger in the perfused liver than in isolated hepatocytes.

The electrochemical proton gradient in the bloodstream form of Trypanosoma brucei is dependent on the temperature.

The membrane potential and pH gradient over the plasma membrane of the protozoan parasite Trypanosoma brucei were measured with radioactive indicators in combination with the silicone oil centrifugation technique over a range of temperatures. At 37 degrees C a small membrane potential and pH gradient of similar magnitude, but of opposite polarity, were measured. The resulting electrochemical proton gradient was almost zero. However, when the temperature was lowered from 37 degrees C to 22 degrees C, the internal pH was kept constant independent of the external pH and a membrane potential of between -100 and -150 mV was measured, depending on the external pH. Measurements at various temperatures between 15 degrees C and 37 degrees C revealed that above 26 degrees C the membrane potential collapsed and that this collapse correlated with a sudden increase in membrane fluidity. The uptake of 2-deoxy-D-glucose and of pyruvate, which are both mediated by facilitated diffusion carriers in the plasma membrane of the trypanosome, were also affected by this sudden increase in fluidity of the membrane. The overall rate of the conversion of glucose into its metabolites, which is independent of the plasma membrane, varied only gradually. We conclude (i) that major changes occur in the plasma membrane of T. brucei around 26 degrees C, that affect all membrane related processes; (ii) that the electrochemical proton gradient plays a minor role in the energy metabolism of T. brucei when it resides in the bloodstream of the mammalian host at 37 degrees C; and (iii) that below 26 degrees C an electrochemical proton gradient is maintained over the plasma membrane.

Effect of dimethadione derived from repeated oral administration of trimethadione on pancreatic secretion in dogs.

The effect of the weak organic acid of dimethadione (DMO) on secretin-stimulated pancreatic secretion was studied with repeated oral administration of trimethadione (TMO), the precursor of DMO, to dogs at a dose of 10 to 160mg/kg/day for a period of 14 days. The bicarbonate concentration in pancreatic juice at a steady state decreased significantly, reflecting a close correlation with the dose of TMO and DMO concentrations in plasma and pancreatic juice. The maximal decrement from the control of cases of no TMO administration was 18.8 mEq/l (12.1% of the control level). The chloride concentration in pancreatic juice showed a reciprocal relation to the bicarbonate concentration. The sum of both anion concentration was constant, irrespective of the dose of TMO. The average carbon dioxide tension of pancreatic juice in all doses of TMO was lower than that of the control, but differences were not statistically significant. The pH, flow rate, sodium and potassium concentrations in pancreatic juice at a steady state did not differ significantly in relation to the dose of TMO. These findings suggest that repeated oral administration of TMO cause a significant decrease in bicarbonate concentration in pancreatic juice, resulting probably from the buffer action of bicarbonate on protons provided from the undissociated form of DMO.

Evidence for poliovirus-induced cytoplasmic alkalinization in HeLa cells.

During the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that Na+/K+ ATPase activation is a consequence of Na+/H+ antiporter activation. Increased uptake of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione by infected cells around 2 h after infection suggested cytoplasmic alkalinization equivalent to pH 7.7 during the biosynthetic phase of viral replication. Consistent with the involvement of Na+/H+ antiporter activation in this phenomenon, it was found to be [Na+]-dependent and inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). However, the pH increase was not associated with an increase in amiloride-sensitive Na+ uptake by infected cells predicted by this mechanism. By contrast, the alkalinization could be abolished with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), implicating an anion-exchange mechanism, such as Cl-/HCO3- exchange, in this process. In addition to abolishing virus-induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular pH with nigericin in the incubation medium revealed that maximum viral replication required a pH of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the pH increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful pH increase in cells infected with a lytic virus.

Interactions of micromolar concentrations of fluoride with Streptococcus rattus FA-1.

Inhibition of the metabolic activities of bacteria by trace amounts of fluoride is manifested phenomenologically as changes in the pH gradient and/or the electrical potential between the cellular interior and the surrounding medium. These data were obtained from the intracellular/extracellular distribution of radioactivity labelled fluoride (18F), 5,5-dimethyloxazolidine-2,4-dione (14C), and tetraphenylphosphonium chloride (14C). When taken up from acidic media, trace concentrations of fluoride (1-100 microM) reduce the intracellular/extracellular pH gradient and affect the electrical potential across the cell membrane. The chromatographic fractionation of fluoride-charged bacterial homogenates showed that fluoride is attached to many proteins of the cytoplasm, the cell membrane, and to nonproteinaceous components of the cell wall. Lysozyme treatment synergistically affects the vulnerability of the bacteria to micromolar concentrations of fluoride.

Pancreatic excretion of dimethadione and trimethadione by repeated oral administration of trimethadione in dogs.

To examine pancreatic excretion of dimethadione (DMO), a weak organic acid, as well as of its precursor trimethadione (TMO), TMO was given orally to dogs with pancreatic fistulae at a dose of 10-160 mg/kg/day over a period of 14 days. Blood samples were taken once a day during the administration of TMO and for 7 days after discontinuation of the drug. On the 15th day, pancreatic juice was collected under stimulation by secretin (2 Crick-Haper-Raper units/kg/h). DMO concentration in plasma reached a maximal plateau around the 10th day after starting TMO administration, and depended directly on the dose of TMO. Pancreatic excretion of DMO at a steady state closely depended on both the dose of TMO and the DMO concentration in plasma. The pancreatic juice/plasma concentration ratio for DMO exceeded 1.0 at a steady rate and decreased with the increased flow rate. Pancreatic DMO clearance (DMO output/DMO concentration in plasma) increased, depending on the flow rate, the bicarbonate concentration, and pH of pancreatic juice. Pancreatic excretion of TMO was zero or extremely low.

Glucose metabolism and acidosis in the metabolic penumbra of rat brain.

The characterization of tissue acid-base status related to the penumbral zone of increased glucose consumption surrounding a focal cerebral ischemic lesion may suggest therapeutic techniques to maximize tissue survivability from stoke. We measured local cerebral metabolic rate for glucose (1 CMRglc) and an index of brain tissue pH (pHt) concurrently and characterized their interaction in a model of focal cerebral ischemia in rats in a double-label autoradiographic study, using [14C]2-deoxyglucose and [14C]dimethyloxazolidinedione. Computer-assisted digitization and analysis permitted the simultaneous quantification of the two variables on a pixel-by-pixel basis in the same brain slices. Hemispheres ipsilateral to intravascular tamponade-induced middle cerebral artery occlusion showed areas of normal, depressed, and elevated glucose metabolic rate (as defined by an interhemispheric asymmetry index) after 2 hr of ischemia. Regions of increased 1 CMRglc showed moderate acidosis (6.87 +/- 0.05), while regions of normal glucose metabolic rate showed normal pHt (pH +/- SD = 6.98 +/- 0.05) and regions of decreased 1 CMRglc showed severe acidosis (6.69 +/- 0.11). A repeated-measures analysis of variance found these values to differ from each other at the P less than 0.0005 significance level. The finding of moderate acidosis coupled with increased 1 CRMglc in the metabolic penumbra suggests that the excess protons may result from the anaerobic dissociation of ATP synthesis and hydrolysis.

Decreasing pH of rat embryos and fluids estimated by transplacental distribution of DMO.

Utilizing the transplacental distribution of a weak acid, 5,5-dimethyloxazolidine-2,4-dione (DMO), we have measured the pH of cells within the rat embryo in vivo on days 11.5-14 of gestation. This is a period of rapid organogenesis in this species when the cells of many organ systems begin to change from a proliferative mode into a differentiated state. We found that intracellular pH of the day 11.5 rat embryo is 7.47 +/- 0.03 and decreases steadily to day 14 at which time it reaches 7.11 +/- 0.03. Because there is a concomitant fall in proliferative rate over this span of development, we suggest this correlation to be additional evidence of an association between proliferation and alkalinization of the cell interior. A number of other compartments including embryo plasma, amniotic fluid, exocoelomic fluid, and yolk sac have a decreasing concentration of DMO as development advances, indicative of a steadily declining pH. These changes could have developmental and pharmacokinetic implications.

The metabolism and excretion of trimethadione in patients with percutaneous transhepatic biliary drainage and renal dysfunction.

The metabolism and excretion of trimethadione (TMO) following an oral dose of 4 mg/kg has been examined in patients with percutaneous transhepatic biliary drainage (PTBD) and renal dysfunction. Biliary excretion as the total amount of TMO and its metabolite, dimethadione (DMO) was 2.0% of the dose during 0 to 48 h after TMO administration in patients with PTBD. Total urinary excretion (0-48 h) was 2.8% and 3.0% of the dose in healthy volunteers and patients with renal dysfunction, respectively. The serum DMO/TMO ratio at 4 h after oral dosing in patients of PTBD and renal dysfunction was not significantly changed in comparison with the ratio reported previously in healthy volunteers. The elimination half-life of TMO was also not altered in patients with PTBD in comparison with that reported previously in volunteers. These results suggest that metabolism and urinary and biliary excretion of TMO are not changed in patients with PTBD and renal dysfunction.

Inhibition of trimethadione and dimethadione teratogenicity by the cyclooxygenase inhibitor acetylsalicylic acid: a unifying hypothesis for the teratologic effects of hydantoin anticonvulsants and structurally related compounds.

Teratogenicity of the anticonvulsant phenytoin may be due in part to its bioactivation by prostaglandin synthetase, forming a reactive free radical intermediate. We examined whether teratogenicity of the structurally similar oxazolidinedione anticonvulsants, trimethadione and its N-demethylated metabolite dimethadione, could be inhibited by the prostaglandin synthetase inhibitor acetylsalicylic acid (ASA). Trimethadione, 700 or 1000 mg/kg intraperitoneally (ip), was given to pregnant CD-1 mice during (Gestational Days 12 and 13) or before (Days 11 and 12) the critical period of susceptibility to phenytoin-induced fetal cleft palates. Dimethadione was given similarly on Days 11 and 12, or 12 and 13, in a dose (900 mg/kg ip) that was equimolar to 1000 mg/kg of trimethadione. ASA, 10 or 1 mg/kg ip, was given 2 hr before trimethadione or dimethadione on Days 11 and 12, and before trimethadione on Day 11 only. Dams were killed on Day 19 and fetuses were examined for anomalies. Either dose of trimethadione given on Days 12 and 13 was negligibly teratogenic, as evidenced by a non-dose-related, 1.1% mean incidence of fetal cleft palates. However, when given earlier on Days 11 and 12, trimethadione 1000 mg/kg caused an 8.9% incidence of cleft palates (p less than 0.05). Similarly, dimethadione caused a 3.9-fold higher incidence of cleft palates when given earlier on Days 11 and 12 (17.3-34.9%) than on Days 12 and 13 (4.4%) (p less than 0.05). At equimolar doses, dimethadione caused a 1.9- to 3.9-fold higher incidence of cleft palates compared to trimethadione (p less than 0.05), suggesting that dimethadione may be the proximate teratogen. Either dose of ASA given on both days before trimethadione totally prevented cleft palates, and ASA 10 mg/kg given only on Day 11 reduced the incidence of trimethadione-induced cleft palates to 1.1% (p less than 0.05). ASA reduced the incidence of cleft palates caused by dimethadione given on Days 11 and 12 from 34.9 to 20.3% (p less than 0.05). These results suggest that the teratogenic potential of trimethadione may depend at least in part upon its prior N-demethylation to dimethadione, which then can be bioactivated by prostaglandin synthetase to a teratogenic reactive intermediate, possibly involving a free radical located in the oxazolidinedione ring. This would provide a unifying hypothesis for the teratogenicity of hydantoins, as well as structurally related teratogens like trimethadione, which lack the molecular configuration necessary for the formation of a teratogenic arene oxide intermediate.

pH homeostasis in Leishmania donovani amastigotes and promastigotes.

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment.