Acidification of the interior of Ehrlich ascites tumor cells by nigericin inhibits DNA synthesis

In Ehrlich ascites carcinoma cells, acidification of the cytoplasm down to pH 6.2-6.3 arrests DNA synthesis. Such acidification can be obtained by decreasing the pH outside the cell or, alternatively, by addition of a micromolar concentration of the K+/H+ antiporter nigericin. Thus, nigericin may be regarded as a new type of cytostatic, the effect of which is mediated by alteration of the intracellular pH.     

Neuronal intracellular pH directly mediates nitric oxide-induced programmed cell death.

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH(i)) regulation during nitric oxide (NO)-induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 microM), 3-morpholinosydnonimine (300 microM), or 6-(2-hyrdroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex anamine (300 microM). The pH(i) was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH(i) 7.36 +/- 0.01 to approximately 7.00 (p <.0001). Within 45 min, a biphasic alkaline response was evident, with pH(i) reaching 7.40 +/- 0.02, that was persistent for a 6-h period. To mimic the effect of NO-induced acidification, neurons were acid-loaded with ammonium ions to yield a pH(i) of 7.09 +/- 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 +/- 2% (24 h) and DNA fragmentation increased to 58 +/- 8% (24 h) (p <.0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH(i) of 7.60 +/- 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO-induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

Acidification induces Bax translocation to the mitochondria and promotes ultraviolet light-induced apoptosis

It has been suggested that Bax translocation to the mitochondria is related to apoptosis, and that cytosol acidification contributes to apoptosis events. However, the mechanisms remain obscure. We investigated the effect of acidification on Bax translocation and on ultraviolet (UV) light-induced apoptosis. The Bax translocation assay in vitro showed that Bax translocated to the mitochondria at pH 6.5, whereas no Bax translocation was observed at pH 7.4. VHDBB cells expressing the GFP-Bax fusion protein were treated for 12 h with a pH 6.5 DMEM medium, nigericin (5 μg/ml) and UV light (50 J/cm²), separately or in combination, and Bax translocation to the mitochondria was determined by SDS-PAGE and Western blot, and apoptotic cell death was detected by flow cytometry. The results showed that some of the Bax translocated to the mitochondria in the cells treated with the normal medium, nigericin and UV in combination, whereas all of the Bax translocated to the mitochondria in the cells treated with the pH 6.5 medium, nigericin and UV in combination. In VHDBB cells treated for 12 h with nigericin, UV alone, and UV and nigericin in combination, the respective rates of apoptotic cell death were 25.08%, 33.25% and 52.88%. In cells treated with pH 6.5 medium and nigericin, pH 6.5 medium and UV, and pH 6.5 medium, nigericin and UV in combination, the respective rates of apoptotic cell death increased to 37.19%, 41.42% and 89.44%. Our results indicated that acidification induces Bax translocation from the cytosol to the mitochondria, and promotes UV lightmediated apoptosis. This suggests that there is a possibility of improving cancer treatment by combining acidification with irradiation or chemotherapeutic drugs.     

Resistance to alloxan of tumoral insulin-producing cells

Rat pancreatic islets and insulin-producing cells of the RINm5F line were incubated for 5 min at 7 or 23 degrees C in media containing 3H2O and either L-[1-14C]glucose or [2-14C]alloxan. In the islets the intracellular distribution space of [2-14C]alloxan represented, at 7 and 23 degrees C respectively, 11.4 +/- 1.0 and 25.5 +/- 2.3% of the intracellular 3H2O space. In the RINm5F cells, the distribution space of [2-14C]alloxan failed to be affected by the ambient temperature and represented, after correction for extracellular contamination, no more than 5.2 +/- 0.5% of the intracellular 3H2O space. Preincubation for 30 min at 7 degrees C in the presence of alloxan (10 mM) failed to affect subsequent D-[U-14C]glucose oxidation in the tumoral cells, whilst causing a 70% inhibition of glucose oxidation in the islets. It is proposed that RINm5F cells are resistant to the cytotoxic action of alloxan, this being attributable, in part at least, to poor uptake of the diabetogenic agent.     

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.     

Leucine transport-induced activation of the Na+/H+ exchanger in human peripheral lymphocytes

Adjustment of amino-acid-induced cytoplasmic pH decrease by the Na+/H+ exchange system in human lymphocytes has been studied using a fluorometric technique to monitor the intracellular pH change. When the interior of lymphocytes is acidified by addition of nigericin to medium, cytoplasmic pH is immediately corrected toward its resting value. This recovery of the cytoplasmic pH depends on extracellular Na+ and is inhibited by amiloride. A temporary (less than 2 min) decrease in the cytoplasmic pH, followed by a slow recovery phase, was observed in incubation with 1.0 mM leucine in Na+-containing medium. This leucine-dependent decrease of cytoplasmic pH persisted longer when amiloride was added to the medium. Cytoplasmic pH recovery from the leucine-induced acidification depends on external Na+ concentration. Amiloride-sensitive Na+/H+ exchanger was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in the lymphocytes and preincubation of the cells with TPA partially prevented the leucine-induced cytoplasmic acidification. We conclude that human peripheral lymphocytes are provided with an amino acid-H+ cotransport system, which is cooperatively coupled to the amiloride-sensitive Na+/H+ exchanger to correct the cytoplasmic pH anomaly.     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Uncoupling protein-2 participates in cellular defense against oxidative stress in clonal beta-cells

The role of uncoupling protein-2 (UCP-2) in beta-cells is presently unclear. We have tested the notion that UCP-2 participates in beta-cell defense against oxidants. Expression of the UCP-2 gene in clonal beta-cells (INS-1) was decreased by 45% after 48 h of culture with vitamin E and selenite. When INS-1 cells were exposed to 200 microM H(2)O(2) for 5 min, the cell viability (MTT assay) decreased to 85 +/- 1, 61 +/- 1, 40 +/- 2, and 39 +/- 2% of control when measured respectively 30 min, 2 h, 6 h, and 16 h after H(2)O(2) exposure. At corresponding time points UCP-2 mRNA levels were 1.01 +/- 0.09, 1.53 +/- 0.15 (P < 0.05), 1.44 +/- 0.18 (P = 0.06), and 1.12 +/- 0.09 fold of control, i.e., transiently increased. We next tested whether overexpression of UCP-2 could enhance resistance of beta-cells toward H(2)O(2) toxicity. A cotransfection method using EGFP as a suitable marker and a human cDNA UCP-2 construct was used for transient overexpression of UCP-2. Transfected cells expressed the gene about 30-fold more than normal cells. After exposure to H(2)O(2) (200 micrometer, 5 min), the survival of UCP-2 overexpressing cells was measured 30-45 min later by flow cytometry. Survival was 13 +/- 0.05% higher than control (EGFP only) cells, P < 0.004 for difference. The results indicate that oxidative stress induces UCP-2 expression in beta-cells, and that UCP-2 serves a role in beta-cell defense against oxidative stress.     

Nigericin-induced death of an acidophilic bacterium.

At an external pH of 3.5, nigericin (which catalyses an electroneutral H+/K+ exchange) abolished the transmembrane proton gradient (delta pH) of Bacillus acidocaldarius, causing a rapid acidification of the cytoplasm from approximately pH 6.0 to pH 3.5. A pronounced loss of viability and fine-structural changes rapidly followed treatment with nigericin. A marked decline in respiration and an even more rapid decrease in cytoplasmic ATP were observed. Activity of at least one cytoplasmic enzyme decreased more slowly. There was no generalized loss in the integrity of the cytoplasmic membrane, as assayed by permeability to inulin or Na+ or by release of ultraviolet light-absorbing compounds. The loss of viability upon treatment with carbonyl cyanide m-chlorophenylhydrazone was similar to what observed with nigericin, so proton influx alone, rather than together with K+ efflux, was probably involved in the death of the organism. Moreover, acidification of the cytoplasm rather than abolition of the delta pH was the lethal event, since no loss of viability was observed when the delta pH was abolished by elevation of the external pH.     

Evidence that Na+/H+ exchange regulates angiotensin II-stimulated diacylglycerol accumulation in vascular smooth muscle cells

Angiotensin II stimulation of vascular smooth muscle cells results in initial, rapid diacylglycerol (DG) formation from the polyphosphoinositides accompanied by intracellular acidification, as well as a more sustained DG accumulation which is accompanied by a prolonged intracellular alkalinization. To determine whether intracellular pH (pHi) modulates DG accumulation, NH4Cl and potassium acetate were used to alter pHi and DG formation was measured. NH4Cl (10 mM) increased pHi from 7.15 +/- 0.05 to 7.34 +/- 0.02 pH units and markedly enhanced the sustained (5 min), but not the initial (15 s), phase of DG formation in response to 100 nM angiotensin II (65 +/- 13% increase). Conversely, intracellular acidification with Na+-free buffer and potassium acetate (20 mM) decreased pHi to 6.93 +/- 0.08 and reduced subsequent angiotensin II-induced sustained DG formation by 82 +/- 9%. In intact cells, inhibition of angiotensin II-stimulated alkalinization by incubation in Na+-free buffer or by addition of the Na+/H+ exchange inhibitor dimethylamiloride (10 microM) decreased the ability of the cell to sustain DG formation, suggesting that active Na+/H+ exchange is necessary for continued DG formation. Thus, it seems that sustained, angiotensin II-induced diacylglycerol accumulation is regulated by intracellular alkalinization secondary to Na+/H+ exchange in cultured vascular smooth muscle cells.     

The Na+/H+ exchanger in immature and mature granulocytic HL-60 cells. Property changes induced by intracellular acidification and cell maturation

We have compared the properties of the Na+/H+ exchanger in two cell populations: growing promyelocytic HL-60 cells (immature) and HL-60 cells induced to mature into granulocytes by dimethyl sulfoxide. The exchanger was activated by intracellular acidification from pH 7.25 to pH 5.5. In both immature and mature granulocytic cells, this type of activation resulted in the expected increase in Vmax for Na+ uptake but also in an increase in KmNa. Maximum acidification caused an increase in Vmax of approximately 10-fold in both types of cells. The increase in KmNa was influenced by cell maturation. In immature cells, the KmNa was higher than in mature cells at all pH values tested, and this difference increased with acidification. Maximum acidification increased the KmNa from 15 +/- 4 to 124 +/- 17 mM in immature cells and from 10 +/- 3 to 43 +/- 20 mM in mature cells. Intracellular pH also influenced the pattern of inhibition of 22Na uptake by dimethylamiloride, a specific inhibitor of the exchanger. At intracellular pH 7.0 dimethylamiloride inhibition was mostly competitive in immature and competitive in mature cells. At lower intracellular pH, 5.9, the inhibition was mixed in both types of cells. Thus, the properties of the exchanger in granulocytic cells are influenced by the cell maturation stage and the intracellular pH.     

Ysp2 mediates death of yeast induced by amiodarone or intracellular acidification

Recently we have found that the drug amiodarone induces apoptosis in yeast, which is mediated by reactive oxygen species (ROS). Here we have used this finding as a tool to screen for genes involved in the death program. We have described a novel mitochondrial protein, Ysp2, acting in the amiodarone-induced death cascade. After amiodarone addition both the control and amiodarone-resistant ysp2-deleted cells formed ROS, but the mutant (unlike the control) did not undergo the mitochondrial thread-to-grain transition. To test whether the action of Ysp2 is amiodarone-specific we tried to induce PCD by other agents. We have found that acetic acid-induced PCD also depends on Ysp2. We also demonstrate that, like acetic acid, propionic acid or nigericin triggered intracellular acidification causing ROS-dependent death. We suggest that intracellular acidification results in the protonation of superoxide anion (O2-*) to form HO2, one of the most aggressive ROS, which in turn induces Ysp2-mediated PCD.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].     

NAD(P)H,a primary target of 1O2 in mitochondria of intact cells

Direct reaction of NAD(P)H with oxidants like singlet oxygen ((1)O(2)) has not yet been demonstrated in biological systems. We therefore chose different rhodamine derivatives (tetramethylrhodamine methyl ester, TMRM; 2',4',5',7'-tetrabromorhodamine 123 bromide; and rhodamine 123; Rho 123) to selectively generate singlet oxygen within the NAD(P)H-rich mitochondrial matrix of cultured hepatocytes. In a cell-free system, photoactivation of all of these dyes led to the formation of (1)O(2), which readily oxidized NAD(P)H to NAD(P)(+). In hepatocytes loaded with the various dyes only TMRM and Rho 123 proved suited to generating (1)O(2) within the mitochondrial matrix space. Photoactivation of the intracellular dyes (TMRM for 5-10 s, Rho 123 for 60 s) led to a significant (29.6 +/- 8.2 and 30.2 +/- 5.2%) and rapid decrease in mitochondrial NAD(P)H fluorescence followed by a slow increase. Prolonged photoactivation (> or =15 s) of TMRM-loaded cells resulted in even stronger NAD(P)H oxidation, the rapid onset of mitochondrial permeability transition, and apoptotic cell death. These results demonstrate that NAD(P)H is the primary target for (1)O(2) in hepatocyte mitochondria. Thus NAD(P)H may operate directly as an intracellular antioxidant, as long as it is regenerated. At cell-injurious concentrations of the oxidant, however, NAD(P)H depletion may be the event that triggers cell death.     

Contribution of anion transporters to the acidosis-induced swelling and intracellular acidification of glial cells

This study examines the contribution of anion transporters to the swelling and intracellular acidification of glial cells from an extracellular lactacidosis, a condition well-known to accompany cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were exposed to lactacidosis in physiological or anion-depleted media, and different anion transport inhibitors were applied. Changes in cell volume and intracellular pH (pH(i)) were simultaneously quantified by flow cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell volume to 125.1 +/- 2.5% of baseline within 60 min, whereas the pH(i) dropped from the physiological value of 7.13 +/- 0.05 to 6.32 +/- 0.03. Suspension in Cl(-)-free or HCO(3)(-)/CO(2)-free media or application of anion transport inhibitors [0.1 mM bumetanide or 0.5 mM 4, 4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS)] did not affect cell volume during baseline conditions but significantly reduced cell swelling from lactacidosis. In addition, the Cl(-)-free or HCO(3)(-)/CO(2)-free media and DIDS attenuated intracellular acidosis on extracellular acidification. From these findings it is concluded that besides the known activation of the Na(+)/H(+) exchanger, activation of the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and the Na(+)-K(+)-Cl(-) cotransporter contributes to acidosis-induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.     

Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C.

Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.     

Elevated cAMP level attenuates 2-deoxy-d-ribose-induced oxidative damage in pancreatic beta-cells

Glucose toxicity to pancreatic beta-cells is defined as irreversible beta-cell damage, including apoptosis, caused by chronic exposure to high glucose levels in type 2 diabetes. Oxidative stress is an important mechanism for glucose toxicity to pancreatic beta-cells. Reducing sugars produce reactive oxygen species through autoxidation and protein glycosylation. 2-Deoxy-d-ribose (dRib) is a reducing sugar with high reactivity. We investigated whether cAMP-stimulating agents could protect beta-cells from dRib-induced oxidative damage. HIT-T15 cells were cultured with various concentrations of dRib for 24 h. We measured cell survival, intracellular cAMP and H2O2 levels, and apoptosis. dRib decreased cell survival in a dose- and time-dependent manner and markedly increased intracellular H2O2 levels and apoptosis. N-Acetyl-l-cysteine decreased dRib-induced rises in intracellular H2O2 and apoptosis to control levels. Forskolin, IBMX, and dbcAMP markedly elevated intracellular cAMP levels and significantly attenuated dRib-induced cytotoxicity and apoptosis, but had no influence on the dRib-induced rise in intracellular H2O2 levels. These results demonstrate that dRib produced oxidative stress and apoptosis in pancreatic beta-cells and that elevated intracellular cAMP levels reduced dRib-induced damage, independent of reactive oxygen species metabolism.     

Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation

We investigated the role of the endoplasmic reticulum (ER) stress response in intracellular Ca2+ regulation, MAPK activation, and cytoprotection in LLC-PK1 renal epithelial cells in an attempt to identify the mechanisms of protection afforded by ER stress. Cells preconditioned with trans-4,5-dihydroxy-1,2-dithiane, tunicamycin, thapsigargin, or A23187 expressed ER stress proteins and were resistant to subsequent H2O2-induced cell injury. In addition, ER stress preconditioning prevented the increase in intracellular Ca2+ concentration that normally follows H2O2 exposure. Stable transfection of cells with antisense RNA targeted against GRP78 (pkASgrp78 cells) prevented GRP78 induction, disabled the ER stress response, sensitized cells to H2O2-induced injury, and prevented the development of tolerance to H2O2 that normally occurs with preconditioning. ERK and JNK were transiently (30-60 min) phosphorylated in response to H2O2. ER stress-preconditioned cells had more ERK and less JNK phosphorylation than control cells in response to H2O2 exposure. Preincubation with a specific inhibitor of JNK activation or adenoviral infection with a construct that encodes constitutively active MEK1, the upstream activator of ERKs, also protected cells against H2O2 toxicity. In contrast, the pkASgrp78 cells had less ERK and more JNK phosphorylation upon H2O2 exposure. Expression of constitutively active ERK also conferred protection on native as well as pkAS-grp78 cells. These results indicate that GRP78 plays an important role in the ER stress response and cytoprotection. ER stress preconditioning attenuates H2O2-induced cell injury in LLC-PK1 cells by preventing an increase in intracellular Ca2+ concentration, potentiating ERK activation, and decreasing JNK activation. Thus, the ER stress response modulates the balance between ERK and JNK signaling pathways to prevent cell death after oxidative injury. Furthermore, ERK activation is an important downstream effector mechanism for cellular protection by ER stress.