Decrease In ATP Content Research Articles

Effect of Ca2+-independent Mechanisms on the Hypoxic Relaxation of Guinea-pig Tracheal Rings

Hypoxia induces bronchodilation in vivo and in vitro, but the mechanisms are still unclear. To evaluate whether an extra- or intracellular free Ca2+ion is involved in the mechanisms of hypoxic relaxation, we simultaneously measured cytosolic Ca2+levels and tensions in both intact and denuded guinea-pig tracheal strips precontracted with histamine (100 μM), and assessed the effect of hypoxia on guinea-pig tracheal rings precontracted with okadaic acid (10 μM) and calyculin-A (0.1≈10 μM) under an extracellular Ca2+-free state. The exposure of tracheal rings to hypoxia induced an immediate decrease of tracheal tension without decrease in intracellular free Ca2+levels. In the presence of okadaic acid but not calyculin-A, hypoxic air exposure caused significant transient reductions in tracheal tone. Further, thapsigargin (5 μM or 10 μM) did not affect hypoxic bronchodilation, suggesting that the release of intracellular Ca2+does not take a role in hypoxic bronchodilation. Hypoxic dilation decreased ATP content in epithelium-intact rings but not epithelium-denuded rings, indicating a relationship between hypoxic dilation and change of adenine nucleotide in epithelium-intact rings. Our findings indicate that the epithelium dependent mechanisms of hypoxic relaxation of guinea pig tracheal rings preconstricted with histamine may not be related to the mobilization of extra and intra-cellular Ca2+.

Blunted effect of parathyroid hormone on adenosine 3′,5′-cyclic monophosphate production is derived from ATP depletion in proximal convoluted tubules of hypophosphatemic mice

Background. To clarify the pathophysiological features associated with phosphate depletion in hypophosphatemic (Hyp) mice, we examined the effect of extracellular phosphate on parathyroid hormone (PTH)-stimulated adenosine 3′,5′-cyclic monophosphate (cAMP) production in the proximal convoluted tubules (PCT). Methods. PTH-Stimulated cAMP production and ATP content were determined by radioimmunoassay and luciferin-luciferase chemiluminescence methods, respectively. Results. The level of cAMP stimulated by PTH at an extracellular phosphate concentration of 0.78 mM was lower in the Hyp mice than in normal mice. The PTH-stimulated cAMP production in the Hyp mice was increased when the extracellular phosphate concentration was raised. In contrast, an increase in extracellular phosphate did not affect the PTH-stimulated cAMP production in the normal mice. Although the ATP content of the PCT was not different between the normal and Hyp mice immediately after microdissection, after 60 min of incubation, it had decreased to a greater extent in Hyp mice than in the normal animals. Raising the extracellular phosphate concentration from 0.78 to 2.3 mM prevented the decrease in ATP content in Hyp mice, and the intracellular ATP content then became comparable to that in the normal control. Conclusions. These results suggest that ATP content in the PCT tended to be decreased in Hyp mice by a decreased phosphate supply and that the blunted effect of PTH on cAMP production in these mice is due to ATP depletion.

Rasagiline, a novel monoamine oxidase-B inhibitor with neuroprotective effects under ischemic conditions in PC12 cells

Rasagiline (N-propargyl-1R-aminoindan) is a novel, potent, selective, and irreversible inhibitor of monoamine oxidase B (MAO-B), currently in Phase III trials for the treatment of Parkinson disease (PD). Rasagiline was shown to have neuroprotective properties in various in vitro and in vivo models independently of MAO-B inhibition. Recently, we developed an in vitro oxygen-glucose deprivation (OGD) model of time-course dependent neuronal cell death in nerve growth factor (NGF)-differentiated PC12 cultures. OGD was accompanied by activation of the arachidonic acid cascade and a decrease in ATP content, changes typical of ischemic conditions. OGD for 3 h followed by reoxygenation for 18 h caused about 30–40% cell death. These conditions are suitable for testing the effect of potential neuroprotective compounds on neuronal cell death. Rasagiline markedly reduced OGD-induced cell death independently of MAO-B inhibition, reducing OGD-induced cell death even when added after the OGD insult. The compound also inhibited OGD-induced prostaglandin E2 (PGE2) release in a dose-dependent manner. At present, selegiline remains the only drug approved for PD therapy based on MAO-B inhibition. However, in contrast to selegiline, rasagiline is not metabolized to amphetamine-like products, which cause adverse side effects and neuronal cell death. Therefore, rasagiline, whose neuroprotective properties are uncomplicated by the production of neurotoxic metabolites, may have an advantage over selegiline in the treatment of PD. Drug Dev. Res. 50:285–290, 2000. © 2000 Wiley-Liss, Inc.

Progressive loss of myocardial ATP due to a loss of total purines during the development of heart failure in dogs: a compensatory role for the parallel loss of creatine.

Whether myocardial ATP content falls in heart failure is a long-standing and controversial issue. The mechanism(s) to explain any decrease in ATP content during heart failure have not been identified. Cardiac dysfunction, heart failure, and a prolonged steady state of heart failure were induced by chronic right ventricular pacing for 1 to 2 weeks, 3 to 4 weeks, and 7 to 9 weeks in dogs. Cardiac function and myocardial O(2) consumption (Mf1.gif" BORDER="0">O(2)) were measured with the dogs in the conscious state. ATP, total purine, and creatine were measured in biopsy specimens obtained at each stage. ATP and the total purine pool progressively fell at rates of 0.12 and 0.15 nmol. mg protein(-1). d(-1), despite an increase in Mf1.gif" BORDER="0">O(2). The rate of loss of creatine was 1.06 nmol. mg protein(-1). d(-1), 7 times faster than the depletion of total purine. (1) ATP contents progressively decreased during heart failure as a result of a loss of the total purine pool. The loss of purines may be due to inhibition of de novo purine synthesis. (2) Loss of creatine is an early marker of heart failure and may serve as a compensatory mechanism minimizing the reduction of the total purine pool in the failing heart.

Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A→G mutation in mitochondrial DNA

The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A→G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A→G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A→G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A→G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.

Rasagiline, a monoamine oxidase-B inhibitor, protects NGF-differentiated PC12 cells against oxygen-glucose deprivation

In our in vitro model, rasagiline a selective irreversible monoamine oxidase-B (MAO-B) inhibitor, protected nerve growth factor (NGF)-differentiated PC12 cells from cell death under oxygen and glucose deprivation (OGD). The severity of the OGD insult, as expressed by cell death, was time-dependent. Exposure of the cells to OGD for 3 hr followed by 18 hr of reoxygenation caused about 30-40% cell death. Under these conditions, the neuroprotective effect of rasagiline was dose-dependent: rasagiline reducing OGD-induced cell death by 68% and 80% at 100 nM and 1 microM, respectively. The neuroprotective effect of rasagiline was also observed when added after the OGD insult (55% reduction in cell death). Under rasagiline treatment, there was a lesser decrease in ATP content in cultures exposed to OGD compared with that in untreated cultures. OGD followed by reoxygenation resulted in a several fold increase in PGE(2) release into the extracellular medium. Rasagiline (100 nM-1 microM) markedly inhibited OGD-induced PGE(2) release. Clorgyline, a monoamine oxidase-A (MAO-A) inhibitor, did not protect NGF-differentiated PC12 cells against OGD-induced cell death. As NGF-differentiated PC12 cells contain exclusively MAO type A, these data suggest that the neuroprotective effect of rasagiline under OGD conditions is independent of MAO inhibition.

Increase in external glutamate and NMDA receptor activation contribute to H2O2-induced neuronal apoptosis.

The present study aims to investigate the role of extracellular glutamate and NMDA receptor stimulation in the neuronal death induced by a transient exposure to H2O2 of cultured neurons originating from mouse cerebral cortex. Most of the neuronal loss following a transient exposure to H2O2 of cortical neurons results from an apoptotic process involving a secondary stimulation of NMDA receptors, which occurs after H2O2 washout. Indeed, (a) the neurotoxic effect of H2O2 was strongly reduced by antagonists of NMDA receptors, (b) the neurotoxic effect of H2O2 was enhanced in the absence of Mg2+, (c) the protective effect of MK-801 progressively decayed when it was applied with increasing delay time after H2O2 exposure, and (d), finally, the extracellular concentration of glutamate was increased after H2O2 exposure. The major part of H2O2-induced neurotoxicity is mediated by the formation of hydroxyl radicals, which might be involved in (a) the delayed accumulation of extracellular glutamate and NMDA receptor activation and (b) the poly(ADP-ribose) polymerase activation and the related NAD content decrease. The combination of these two mechanisms could lead to both an increase in ATP consumption and a decrease of ATP synthesis. The resulting large decrease in ATP content might be finally responsible for the neuronal death.

Sodium Palmitate Induces Partial Mitochondrial Uncoupling and Reactive Oxygen Species in Rat Pancreatic Isletsin Vitro1

The aim of the present investigation was to study whether prolonged exposure of isolated rat islets to the long chain fatty acid sodium palmitate leads to uncoupling of respiration. It was found that culture of islets in the presence of palmitate abolished glucose-sensitive insulin release and decreased insulin contents. This was paralleled by decreased ATP contents, increased respiration, and decreased islet cell mitochondrial membrane potential. Using electron microscopy, an increase in the beta-cell mitochondrial volume in islets exposed to palmitate was observed. The addition of the uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, at a concentration that decreased mitochondrial membrane potential to a similar extent as palmitate, diminished the glucose-induced insulin release. In addition, islet generation of reactive oxygen species, but not of nitric oxide, was increased in response to a long-term palmitate exposure. It is concluded that long-term exposure to a long chain fatty acid induces partial uncoupling of beta-cell oxidative phosphorylation and that this may contribute to the loss of glucose-sensitive insulin release.

Effect of Dietary Fat on Chronic Ethanol‐Induced Oxidative Stress in Hepatocytes

Although oxidative stress and deficits in hepatic energy metabolism have been implicated as important factors in the initiation of alcoholic liver disease, their relative contribution to ethanol-induced cell death is not known. The purpose of this study was to examine the effects of chronic ethanol administration on hepatocyte reactive oxygen species (ROS) generation, energy state, and viability, as well as the effect of dietary fat on these parameters. Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol, with fat as either 12% (low fat) or 35% (high fat) of total calories. Pair-fed controls received liquid diets in which maltose-dextrin was substituted for ethanol calories. The fluorescent probe 2',7'-dichlorofluorescin diacetate was used to detect ROS, lactate dehydrogenase leakage was used to assess viability, and ATP levels were used as a measure of the energy state. The effect of chronic ethanol feeding on these parameters was determined by incubating hepatocytes under a 5% oxygen-containing atmosphere or an atmosphere < or = 1% oxygen for 60 min. In general, chronic ethanol feeding stimulated ROS production and decreased ATP concentrations, which were associated with decreased viability in hepatocytes isolated from rats fed either high- or low-fat, ethanol-containing diets, compared to the corresponding controls. Incubation under an atmosphere < or = 1% oxygen and/or ethanol (10 mM) augmented these effects in both high- and low-fat control and ethanol-fed hepatocytes. The addition of antimycin to the incubations increased ROS production, decreased ATP concentrations, and accelerated loss of hepatocyte viability. Viability loss under all conditions used in this study was correlated with decreases in cellular ATP. Comparisons of incubations performed under the two oxygenation conditions revealed that viability loss was inversely associated with ROS production, which indicates that ATP loss and not ROS production was a better predictor of loss in cell integrity. This study also demonstrates that the level of dietary fat has only minor effects on generation of ROS and the cellular energy state. In contrast, ethanol consumption had significant effects on generation of ROS, energy state, and hepatocyte viability.

Homogeneous Bioluminescence Assay for Galactosuria: Interference and Kinetic Analysis

Elevated galactose concentration in urine is an important clinical symptom of galactosemia and other metabolic disorders. A quantitative assay for galactose using firefly luciferase bioluminescence is presented. The assay couples the galactokinase and firefly luciferase reactions. A higher concentration of galactose present in the sample produces a faster decrease in ATP concentration, which is monitored by firefly luciferase bioluminescence. The kinetic assay is modeled and analyzed. The interference between the two reactions, the interference of certain sugars and other components in the urine, the specificity, and the optimal pH for galactokinase were studied. Calibration curves were constructed and compared with a conventional spectrophotometric assay for galactose. The bioluminescence assay is relatively fast and specific for galactose with a linear range from 1 to 20 mM galactose. The effect of other galactose metabolites (galactonate and galactitol) has also been studied.

Acyl phosphatase activity of NO-inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH): a potential mechanism for uncoupling glycolysis from ATP generation in NO-producing cells

Treatment of glyceraldehyde-3-phosphate - dehydrogenase (GA (GAPDH) with the NO donors S-nitrosoglutathione, 3-morpholinosydnonimine or diethylamine NONOate (diethylamine diazeniumdiolate) in vitro, inhibited its dehydrogenase activity and induced its acyl phosphatase activity. NO-producing cells, in turn, exhibited reduced GAPDH activity, increased glycolysis, and decreased ATP content, synthesis and turnover. These cellular alterations could be explained by the uncoupling of glycolytic flux from substrate level phosphorylation by the acyl phosphatase activity of NO-modified GAPDH.

An enhancement of nitric oxide production regulates energy metabolism in rat hepatocytes after a partial hepatectomy

Background/Aims: Infection after a liver resection often results in hepatic failure. Nitric oxide is one of the candidates which has been suspected to cause cellular dysfunction during infection in the liver. We have previously reported that the inflammatory cytokine interleukin-1β (IL-1β) induced the expression of the inducible nitric oxide synthase gene in primary cultured rat hepatocytes. We hypothesized that an enhancement of nitric oxide production after the resection was implicated in a change in liver energy metabolism, thus resulting in liver dysfunction. Methods: In this study, we performed a 70% hepatectomy or a sham operation in rats, and then isolated hepatocytes from the remnant liver by collagenase perfusion. The cultured hepatocytes were treated with cytokines including IL-1β. The effects on nitric oxide induction, the ATP content and ketone body ratio (acetoacetate/β-hydroxybutyrate) were then compared between the partial hepatectomized (PH) and shamoperated (control) rats. Results: IL-1β augmented the induction of nitric oxide production two-fold in hepatocytes from the PH rats as compared to the control rats. IL-1β markedly decreased the ATP content in the PH rats, although IL-1β also decreased the ATP content in the control rats, but to a lesser extent. IL-1β also decreased the ketone body ratio in both groups. The addition of l-arginine further stimulated the inhibition of the ATP levels and the ketone body ratio concomitantly with increased nitric oxide production in the PH rats. N G-monomethyl- l-arginine, an inhibitor of nitric oxide synthase, abolished the effects of IL-1β on the ATP levels and ketone body ratio, as well as on the nitric oxide production. Conclusions: These results demonstrate that the decreased ATP content observed in PH rats resulted from an increase in nitric oxide production. The decrease in ketone body ratio indicates that nitric oxide-induced mitochondrial dysfunction contributes significantly to ATP attenuation in hepatocytes. Therefore, the regulation of nitric oxide induction may be crucial for preventing liver failure after a hepatic resection.

The effect of α-phenyl- N-tert-butyl nitrone on bioenergetic state in substantia nigra following flurothyl-induced status epilepticus in rats

Status epilepticus (SE), i.e. ongoing seizures of more than 30 min duration, gives rise to bilateral pan-necrotic lesions of the substantia nigra, pars reticulata (SNPR). These are known to be preceded by an initial increase, followed by a depression of metabolic rate, and by failure of the bioenergetic state, suggesting mitochondrial dysfunction. We have previously shown that the spin trap α-phenyl- N-tert-butyl nitrone (PBN) prevents the lesions caused by 45 min of SE from occurring, in spite of ongoing seizure activity. In this article, we demonstrate that PBN, given 30 min before seizure induction, reduces or prevents the decrease in ATP concentration and adenylate energy charge, without significantly reducing the amount of lactate accumulated, or the decrease in intracellular pH (pH i). The results suggest that the spin trap nitrone preserves the structural and functional integrity of SNPR neurons by protecting the mitochondria against oxidative damage.

Suppression of Calcium Oscillation by Tri-n-Butyltin Chloride in Cultured Rat Hepatocytes

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on cytoplasmic free calcium ion concentration ([Ca2+]i) were investigated in primary cultured rat hepatocytes. A high concentration (4.0 μM) of TBT increased resting levels of [Ca2+]iand then induced cell blebs resulting in cell death within 2 h. The increase in [Ca2+]i, but not the cell death, depended on the presence of extracellular Ca2+, suggesting that the increase in [Ca2+]iis not critical for the cytotoxicity of TBT. A low concentration (0.1 μM) of TBT did not have any toxic effect (decrease in ATP content, decrease in viability, and shape change) on cultured hepatocytes and did not change [Ca2+]i. However, the calcium responses induced by phenylephrine, [Arg8]-vasopressin, and ATP were suppressed in the cells pretreated with 0.1 μM TBT for 30 min. The suppression was not observed in the cells pretreated with 0.1 μM TBT for only 1 min. Pretreatment with 0.1 μM TBT for 30 min had no effect on the inositol 1,4,5-triphosphate content or its increase in response to hormonal stimulation. These results suggest that TBT suppresses hormone-induced calcium responses at nontoxic low concentrations.

Metabolic depression in animals: physiological perspectives and biochemical generalizations

Metabolic depression in animals: physiological perspectives and biochemical generalizations

Effect of alcohol on energy storage of primary astrocytes and C6-glioma cells in vitro.

The present experiments were conducted to investigate the direct effects of ethanol on the energy metabolism of astrocytes and C6-glioma cells. Primary astrocytes were prepared from cerebral cortices of neonatal rats, and C6-glioma cells were purchased from American Type Culture Collection (ATCC). These cells were exposed to different concentrations of alcohol (100 mM, 200 mM, and 300 mM) for 15 minutes and 24 hours. The amount of ATP and PCr was measured by the method of Lowry and Passonneau (1972). Following 15 minutes treatment with different doses of ethanol the amount of ATP and PCr increased, in both cell types. Only the increase of ATP concentration with varying doses of ethanol (100 mM, 200 mM, and 300 mM) was statistically significant. Following 24 hours treatment of astrocytes with different doses of ethanol the concentration of ATP and PCr decreased. The decrease in concentration of ATP was significant with all three doses of ethanol, but the decrease of PCr concentration was only statistically significant with 300 mM ethanol. Following 24 hours treatment of C6-glioma cells to varying doses of ethanol, the concentration of PCr and ATP decreased. The decrease of PCr was statistically significant with all three doses of ethanol and the decrease of ATP concentration was only significant with 300 mM ethanol.

The Molecular Energetics of the Failing Heart from Animal Models―Small Animal Models

Small animal models of heart failure have been widely used. Availability of transgenic mice with perturbations of the molecular pathways known to be altered in heart failure will substantially increase the number of small animal models for heart failure research. This chapter summarizes the alterations in myocardial energetics observed in failing hearts of small animal models using the combined tools of 31P NMR spectroscopy and biochemical assays. These include: early depletion of total creatine and phosphocreatine followed by decreased creatine kinase activity; both contribute to decreased capacity and velocity of the creatine kinase reaction resulting in decreased energy reserve during the development of heart failure; decreased ATP content at end-stage failing hearts; and a switch in substrate utilization with increased preference for glucose in hypertrophied and failing hearts. Evidence presented here suggest that these abnormalities contribute to failure to recruit contractile reserve, increased susceptibility for diastolic dysfunction and impaired calcium homeostasis in hypertrophied and failing hearts.

Peroxynitrite affects Ca 2+ transport in Trypanosoma cruzi

Macrophages play an important role against Trypanosoma cruzi infection, via superoxide, nitric oxide, and peroxynitrite production. Peroxynitrite has been shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. Calcium is involved in many vital functions of the parasites, being its intracellular concentration governed by several transport systems, involving mitochondrial and non-mitochondrial compartments. In this paper, we report the effect of peroxynitrite on the calcium uptake systems, as studied by digitonin-permeabilized trypanosomes in the presence of arsenazo III. Peroxynitrite, at biologically relevant concentrations produced within phagosomes (250–750 μM), inhibited calcium uptake in a dose-dependent manner. Peroxynitrite decreased the mitochondrial membrane potential obtained in the presence of tetramethyl- p-phenylenediamine (TMPD)/ascorbate. In addition, a decrease of the non-mitochondrial Ca 2+-uptake, concomitant with the inactivation of a Ca 2+-dependent ATPase activity, was observed. HPLC analyses of the cellular adenine nucleotide pool showed a time-dependent decrease of ATP content and energy charge of the parasite; however this drop in ATP levels was significantly delayed with respect to decrease of the ATP-dependent Ca 2+-transport. We conclude that the disruption of calcium homeostasis by peroxynitrite may contribute to the observed cytotoxic effects of macrophages against T. cruzi.

Reversible association between the V1 and V0 domains of yeast vacuolar H+-ATPase is an unconventional glucose-induced effect.

The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore. Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.

Mechanisms through which high glucose concentration raises [Ca2+]i in renal proximal tubular cells

The basal levels of cytosolic calcium ([Ca2+]i) of renal proximal tubular cells of rats with streptozotocin-induced diabetes are elevated. It is possible that this phenomenon is mediated by the hyperglycemia, which may cause both increased calcium influx into and/or decreased calcium efflux out of these cells. We examined whether high glucose concentration in vitro causes acute rise in [Ca2+]i of freshly isolated renal proximal tubular cells and explored the pathways that are involved in such an event. There were dose and time dependent increments in [Ca2+]i of renal proximal tubular cells exposed to high concentrations of glucose. A similar effect was observed with equimolar concentrations of mannitol or choline chloride but not urea. A substantial part of the rise in [Ca2+]i was inhibited when the media contained verapamil, nifedipine, amlodipine or ryanodine and when the cells were placed in a calcium free media. Inhibitors of G protein(s) (GDPbetaS or pertussis toxin), inhibitors of cAMP-protein kinase A pathway (RpcAMP or H-89), inhibitors of protein kinase C (staurosporine or calphostin) and inhibitor of Na+-H+ exchanger (HOE 694) blocked the rise in a dose dependent manner. High glucose concentration also caused a decrease in ATP content of these cells and a reduction in the Vmax of their Ca2+ATPase. The results are consistent with the formulation that the osmotic activity (cell shrinkage) of the high glucose concentration may activate a stretch receptor with subsequent stimulation of various cellular pathways including G protein(s), cAMP-protein kinase A and phospholipase C systems and calcium channels. Activation of these cellular pathways permits both calcium influx into renal tubular cells and mobilization of calcium from their intracellular stores. Further, a decrease in calcium efflux secondary to the reduction in the Vmax of Ca2+ ATPase may occur. It is possible that the rise in [Ca2+]i is critical for the stimulation of the events that lead to restoration of cell volume to normal.