Regulation of cardiac AMP-specific 5'-nucleotidase during ischemia mediates ATP resynthesis on reflow

The ability toresynthesize ATP during recovery from ischemia is limited tothe size of endogenous pool of adenine nucleotides. CytosolicAMP-specific 5'-nucleotidase (5'-NT) plays a key role inATP degradation and hence the capacity for ATP resynthesis. We havesuggested (J. Clin. Invest. 93:40-49, 1994) that intracellular acidosis [intracellular pH(pH_i)] is a potentinhibitor of 5'-NT under in vivo conditions. To test thishypothesis further, we used the hyperthyroid rat heart because we couldalter pH_i during ischemiaand determine the consequences of lowerpH_i on AMPaccumulation (by chemical assay) and ATP resynthesis (by³¹P nuclear magnetic resonancespectroscopy) during reperfusion. Global no-flow ischemiacaused pH_i to decrease from 7.1 under well-oxygenated control perfusion to 6.7. We found thatdecreasing pH_i further from pH 6.7 to 6.4 leads to increased accumulation (30%) of AMP duringischemia and to a 2.5-fold increase in ATP resynthesis duringreperfusion. Analysis of all known substrates, products, activators,and inhibitors of the 5'-NT suggests that 5'-NT isactivated primarily by Mg²⁺ andADP and is inhibited by H⁺. Thusthese observations provide evidence for a salutary effect ofintracellular acidosis on preserving the AMP pool due to inhibition of5'-NT and suggest a novel role ofH⁺ in protecting ischemic tissue.

     

Glucose activates H(+)-ATPase in kidney epithelial cells

The vacuolar H⁺-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H⁺-ATPase remain largely unknown. The present study examines the effect of glucose on H⁺-ATPase activity in the pig kidney epithelial cell line LLC-PK₁. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH₃/NH₄⁺ prepulse, and rates of intracellular pH (pH_i) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K⁺ free to eliminate Na⁺/H⁺ exchange and H⁺-K⁺-ATPase activity. After NH₃/NH₄⁺-induced acidification, LLC-PK₁ cells had a significant pH_i recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pH_i recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pH_i recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H⁺-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A     

Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH_i). pH_i was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH_i in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H₂O₂ decreased pH_i by 0.19 ± 0.007, 0.20 ± 0.015, 0.15 ± 0.013, and 0.08 ± 0.002 pH unit, respectively. Hypercapnia decreased pH_i by 0.26 ± 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH_i, causing a 0.42 ± 0.03 (n = 21) pH unit acidification. CT slowed pH_i recovery mediated by Na⁺/H⁺ exchange (NHE) from NH₄Cl-induced acidification by 53% (n = 20) in -buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH_i. These results indicate that oxidative stress 1) decreases pH_i in some SC neurons, 2) together with hypercapnia has an additive effect on pH_i, 3) partially inhibits NHE, and 4) directly affects excitability of CO₂/H⁺-chemosensitive SC neurons independently of pH_i changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction. hyperoxic hyperventilation; O₂ toxicity; pH regulation; brain stem; reactive oxygen species     

Effect of intracellular pH on depolarization-evoked calcium influx in human sperm

Human sperm are endowed with putative voltage-dependent calcium channels (VDCC) that produce measurable increases in intracellular calcium concentration ([Ca²⁺]_i) in response to membrane depolarization with potassium. These channels are blocked by nickel, inactivate in 1–2 min in calcium-deprived medium, and are remarkably stimulated by NH₄Cl, suggesting a role for intracellular pH (pH_i). In a previous work, we showed that calcium permeability through these channels increases approximately onefold during in vitro "capacitation," a calcium-dependent process that sperm require to fertilize eggs. In this work, we have determined the pH_i dependence of sperm VDCC. Simultaneous depolarization and pH_i alkalinization with NH₄Cl induced an [Ca²⁺]_i increase that depended on the amount of NH₄Cl added. VDCC stimulation as a function of pH_i showed a sigmoid curve in the 6.6–7.2 pH_i range, with a half-maximum stimulation at pH 7.00. At higher pH_i (7.3), a further stimulation occurred. Calcium release from internal stores did not contribute to the stimulating effect of pH_i because the [Ca²⁺]_i increase induced by progesterone, which opens a calcium permeability pathway that does not involve gating of VDCC, was unaffected by ammonium. The ratio of pH_i-stimulated-to-nonstimulated calcium influx was nearly constant at different test depolarization values. Likewise, depolarization-induced calcium influx in pH_i-stimulated and nonstimulated cells was equally blocked by nickel. In our capacitating conditions pH_i increased 0.11 pH units, suggesting that the calcium influx stimulation observed during sperm capacitation might be partially caused by pH_i alkalinization. Additionally, a calcium permeability pathway triggered exclusively by pH_i alkalinization was detected. mammalian sperm; capacitation; intracellular calcium     

Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes

Malignantgliomas exhibit alkaline intracellular pH (pH_i) and acidicextracellular pH (pH_e) compared with nontransformedastrocytes, despite increased metabolic H⁺ production. Theacidic pH_e limits the availability ofHCO₃, thereby reducing both passiveand dynamic HCO₃-dependent buffering.This implies that gliomas are dependent upon dynamic HCO₃-independent H⁺buffering pathways such as the type 1 Na⁺/H⁺exchanger (NHE1). In this study, four rapidly proliferating gliomas exhibited significantly more alkaline steady-state pH_i(pH_i = 7.31-7.48) than normal astrocytes(pH_i = 6.98), and increased rates of recovery fromacidification, under nominallyCO₂/HCO₃-free conditions.Inhibition of NHE1 in the absence ofCO₂/HCO₃ resulted inpronounced acidification of gliomas, whereas normal astrocytes wereunaffected. When suspended inCO₂/HCO₃ medium astrocytepH_i increased, yet glioma pH_i unexpectedlyacidified, suggesting the presence of anHCO₃-dependent acid loadingpathway. Nucleotide sequencing of NHE1 cDNA from the gliomasdemonstrated that genetic alterations were not responsible for thisaltered NHE1 function. The data suggest that NHE1 activity issignificantly elevated in gliomas and may provide a useful target forthe development of tumor-selective therapies.

     

Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes

The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia–reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4 h and reoxygenated at pH 7.4. Chelatable Fe²⁺, ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe²⁺. Desferal and starch–desferal 1 h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch–desferal, and the inhibitor of the mitochondrial Ca²⁺ uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch–desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe²⁺ during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.     

pH heterogeneity at intracellular and extracellular plasma membrane sites in HT29-C1 cell monolayers

In the colonic mucosa, short-chain fatty acids changeintracellular pH (pH_i) and extracellular pH(pH_e). In this report, confocal microscopy anddual-emission ratio imaging of carboxyseminaphthorhodofluor-1 were usedfor direct evaluation of pH_i and pH_e in asimple model epithelium, HT29-C1 cells. Live cell imaging along theapical-to-basal axis of filter-grown cells allowed simultaneousmeasurement of pH in the aqueous environment near the apical membrane,the lateral membrane, and the basal membrane. Subapical cytoplasmreported the largest changes in pH_i after isosmoticaddition of 130 mM propionate or 30 mM NH₄Cl. In restingcells and cells with an imposed acid load, lateral membranes hadpH_i values intermediate between the relatively acidicsubapical region (pH 6.3-6.9) and the relatively alkaline basalpole of the cells (pH 7.4-7.1). Transcellular pH_igradients were diminished or eliminated during an induced alkalineload. Propionate differentially altered pH_e near the apicalmembrane, in lateral intracellular spaces between adjacent cells, andnear the basal membrane. Luminal or serosal propionate causedalkalinization of the cis compartment (where propionate wasadded) but acidification of the trans compartment only inresponse to luminal propionate. Addition of NH₄Cl produced qualitatively opposite pH_e excursions. The microscopicvalues of pH_i and pH_e can explain a portion ofthe selective activation of polarized Na/H exchangers observed inHT29-C1 cells in the presence of transepithelial propionate gradients.

     

Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0

During maturation of oocytes,Cl conductance (G_Cl) oscillatesand intracellular pH (pH_i) increases. ElevatingpH_i permits the protein synthesis essential to maturation.To examine whether changes in G_Cl andpH_i are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespH_i, decreases intracellular Cl concentration([Cl]_i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pH_ichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pH_i. In contrast, hyperpolarization elevates pH_i. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pH_iand decreases [Cl]_i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpH_i elevation, although the decrease in[Cl]_i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pH_i and decreases[Cl]_i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte G_Cl may exert an effect onpH_i via shrinkage, thus inducing meiotic maturation.

     

Skeletal muscle chemoreflex and pHi in exercise ventilatory control

Oelberg, David A., Allison B. Evans, Mirko I. Hrovat, PaulP. Pappagianopoulos, Samuel Patz, and David M. Systrom. Skeletal muscle chemoreflex and pH_i inexercise ventilatory control. J. Appl.Physiol. 84(2): 676-682, 1998.To determinewhether skeletal muscle hydrogen ion mediates ventilatory drive inhumans during exercise, 12 healthy subjects performed three bouts ofisotonic submaximal quadriceps exercise on each of 2 days in a 1.5-Tmagnet for ³¹P-magnetic resonancespectroscopy(³¹P-MRS). Bilaterallower extremity positive pressure cuffs were inflated to 45 Torr duringexercise (BLPP_ex) or recovery(BLPP_rec) in a randomized orderto accentuate a muscle chemoreflex. Simultaneous measurements were madeof breath-by-breath expired gases and minute ventilation, arterializedvenous blood, and by ³¹P-MRS ofthe vastus medialis, acquired from the average of 12 radio-frequencypulses at a repetition time of 2.5 s. WithBLPP_ex, end-exercise minuteventilation was higher (53.3 ± 3.8 vs. 37.3 ± 2.2 l/min;P < 0.0001), arterializedPCO₂ lower (33 ± 1 vs. 36 ± 1 Torr; P = 0.0009), and quadricepsintracellular pH (pH_i) more acid (6.44 ± 0.07 vs. 6.62 ± 0.07; P = 0.004), compared withBLPP_rec. Bloodlactate was modestly increased withBLPP_ex but without a change inarterialized pH. For each subject, pH_i was linearly relatedto minute ventilation during exercise but not to arterialized pH. Thesedata suggest that skeletal muscle hydrogen ion contributes to theexercise ventilatory response.

     

Effect of reactive oxygen species on NH4+ permeation in Xenopus laevis oocytes

To investigate theeffects of reactive oxygen species (ROS) on NHpermeation in Xenopus laevis oocytes, we used intracellulardouble-barreled microelectrodes to monitor the changes in membranepotential (V_m) and intracellular pH(pH_i) induced by a 20 mM NH₄Cl-containingsolution. Under control conditions, NH₄Cl exposure induceda large membrane depolarization (to V_m = 4.0 ± 1.5 mV; n = 21) and intracellularacidification [reaching a change in pH_i(pH_i) of 0.59 ± 0.06 pH units in 12 min]; theinitial rate of cell acidification (dpH_i/dt) was0.06 ± 0.01 pH units/min. Incubation of the oocytes in thepresence of H₂O₂ or -amyloid protein had nomarked effect on the NH₄Cl-induced pH_i. Bycontrast, in the presence of photoactivated rose bengal (RB),tert-butyl-hydroxyperoxide (t-BHP), orxanthine/xanthine oxidase (X/XO), the same experimental maneuverinduced significantly greater pH_i anddpH_i/dt. These increases in pH_iand dpH_i/dt were prevented by the ROS scavengershistidine and desferrioxamine, suggesting involvement of the reactivespecies ¹gO₂ and ·OH. Using thevoltage-clamp technique to identify the mechanism underlying theROS-measured effects, we found that RB induced a large increase in theoocyte membrane conductance (G_m). ThisRB-induced G_m increase was prevented by 1 mMdiphenylamine-2-carboxylate (DPC) and by a low Na⁺concentration in the bath. We conclude that RB, t-BHP, andX/XO enhance NH influx into the oocyte via activationof a DPC-sensitive nonselective cation conductance pathway.

     

The role of intracellular pH in cell growth arrest induced by ATP

In this study, we investigated ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained intracellular pH (pH_i) decrease from 7.41 to 7.11. Inhibition of Ca²⁺ influx, lowering extracellular Ca²⁺, and buffering cytoplasmic Ca²⁺ inhibited ATP-induced acidification, thereby demonstrating that acidification is a consequence of Ca²⁺ entry. We show that ATP induced reuptake of Ca²⁺ by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced after the dissipation of the mitochondrial proton gradient by rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, after inhibition of Ca²⁺ uptake into the mitochondria by ruthenium red, and after inhibition of the F₀F₁-ATPase with oligomycin. ATP-induced acidification was not induced by either stimulation of the Cl^–/HCO₃^– exchanger or inhibition of the Na⁺/H⁺ exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable Ca²⁺ from the endoplasmic reticulum, assessed by measuring change in cytosolic Ca²⁺ induced by thapsigargin or ATP in a Ca²⁺-free medium. This latter finding reveals cross talk between pH_i and Ca²⁺ homeostasis in which the Ca²⁺-induced intracellular acidification can in turn regulate the amount of Ca²⁺ that can be released from the endoplasmic reticulum. Furthermore, pH_i decrease was capable of reducing cell growth. Taken together, our results suggest that ATP-induced acidification in DU-145 cells results from specific effect of mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP. prostate; cancer; acidification     

V-type H+-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration

We investigated for the presence of avacuolar-type H⁺-ATPase (V-ATPase) in the human eccrinesweat duct (SD). With the use of immunocytochemistry, ananti-V- ATPase antibody showed a strong staining at the apicalmembrane and a weaker one in the cytoplasm. Cold preservation followedby rewarming did not alter this staining pattern. With the use of thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein onisolated and perfused straight SD under HCO-free conditions and in the absence of Na⁺, proton extrusion wasdetermined from the recovery rate of intracellular pH(dpH_i/dt) following an acid load. Oligomycin (25 µM), an inhibitor of F-type ATPases, decreaseddpH_i/dt by 88 ± 6%, suggesting a role foran ATP-dependent process involved in pH_i recovery.Moreover, dpH_i/dt was inhibited at 95 ± 3% by 100 nM luminal concanamycin A, a specific inhibitor ofV-ATPases, whereas 10 µM bafilomycin A₁, another specificinhibitor of V-ATPases, was required to decrease dpH_i/dt by 73%. These results strongly suggestthat a V-ATPase is involved in proton secretion in the human eccrine SD.

     

Effect of osmolarity on LDL binding and internalization in hepatocytes

The present study has been performed to elucidate a possiblerole of cell volume in low-density lipoprotein (LDL) binding andinternalization (LDL_b+i). Asshown previously, increase of extracellular osmolarity (OSMe) andK⁺ depletion, both known to shrinkcells, interfere with the formation of clathrin-coated pits and thuswith LDL_b+i. On the other hand,alterations of cell volume have been shown to modify lysosomal pH,which is a determinant of LDL_b+i.LDL_b+i have been estimated fromheparin-releasable (binding) or heparin-insensitive (internalization)uptake of ¹²⁵I-labeled LDL. OSMewas modified by alterations of extracellular concentrations of ions,glucose, urea, or raffinose. When OSMe was altered by varying NaClconcentrations, LDL_b+i decreased (by 0.5 ± 0.1%/mM) with increasing OSMe andLDL_b+i increased (by 1.2 ± 0.1%/mM) with decreasing OSMe, an effect mainly due to alteredaffinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 µg/ml at 219, 293, and 435 mosM, respectively. A 25%increase of OSMe increased cytosolic (by 0.46 ± 0.03) and decreasedlysosomal (by 0.14 ± 0.02) pH. Conversely, a 25% decrease of OSMedecreased cytosolic (by 0.28 ± 0.02) and increased lysosomal (by0.17 ± 0.02) pH. Partial replacement of extracellularNa⁺ withK⁺ had little effect onLDL_b+i, although it swelledhepatocytes and increased lysosomal and cytosolic pH. Hypertonicglucose, urea, or raffinose did not exert similar effects despite ashrinking effect of hypertonic raffinose. Monensin, which completelydissipates lysosomal acidity, virtually abolishedLDL_b+i. In conclusion, theobservations reveal a significant effect of ionic strength onLDL_b+i. The effect is, however,not likely to be mediated by alterations of cell volume or alterationsof lysosomal pH.

     

Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons

To determine the role ofmembrane transporters in intracellular pH (pH_i) regulationunder conditions of low microenvironmental O₂, we monitoredpH_i in isolated single CA1 neurons using the fluorescentindicator carboxyseminaphthorhodafluor-1 and confocal microscopy. Aftertotal O₂ deprivation or anoxia (PO₂ 0 Torr), a large increase in pH_i was seen in CA1neurons in HEPES buffer, but a drop in pH_i, albeit small,was observed in the presence of HCO. Ionicsubstitution and pharmacological experiments showed that the largeanoxia-induced pH_i increase in HEPES buffer was totallyNa⁺ dependent and was blocked by HOE-694, stronglysuggesting the activation of the Na⁺/H⁺exchanger (NHE). Also, this pH_i increase in HEPES bufferwas significantly smaller in Na⁺/H⁺ exchangerisoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons,demonstrating that NHE1 is responsible for part of the pH_iincrease following anoxia. Both chelerythrine and H-89 partly blocked,and H-7 totally eliminated, this anoxia-induced pH_iincrease in the absence of HCO. We conclude that1) O₂ deprivation activatesNa⁺/H⁺ exchange by enhancing protein kinaseactivity and 2) membrane proteins, such as NHE, activelyparticipate in regulating pH_i during low-O₂states in neurons.

     

Somatic vs. dendritic responses to hypercapnia in chemosensitive locus coeruleus neurons from neonatal rats

Cardiorespiratory control is mediated in part by central chemosensitive neurons that respond to increased CO₂ (hypercapnia). Activation of these neurons is thought to involve hypercapnia-induced decreases in intracellular pH (pH_i). All previous measurements of hypercapnia-induced pH_i changes in chemosensitive neurons have been obtained from the soma, but chemosensitive signaling could be initiated in the dendrites of these neurons. In this study, membrane potential (V_m) and pH_i were measured simultaneously in chemosensitive locus coeruleus (LC) neurons from neonatal rat brain stem slices using whole cell pipettes and the pH-sensitive fluorescent dye pyranine. We measured pH_i from the soma as well as from primary dendrites to a distance 160 µm from the edge of the soma. Hypercapnia [15% CO₂, external pH (pH_o) 7.00; control, 5% CO₂, pH_o 7.45] resulted in an acidification of similar magnitude in dendrites and soma (0.26 pH unit), but acidification was faster in the more distal regions of the dendrites. Neither the dendrites nor the soma exhibited pH_i recovery during hypercapnia-induced acidification; but both regions contained pH-regulating transporters, because they exhibited pH_i recovery from an NH₄Cl prepulse-induced acidification (at constant pH_o 7.45). Exposure of a portion of the dendrites to hypercapnic solution did not increase the firing rate, but exposing the soma to hypercapnic solution resulted in a near-maximal increase in firing rate. These data show that while the pH_i response to hypercapnia is similar in the dendrites and soma, somatic exposure to hypercapnia plays a major role in the activation of chemosensitive LC neurons from neonatal rats. acid; brain stem; intracellular pH; pyranine; respiratory control; whole cell     

Reactive oxygen species, but not Ca2+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion

We investigated the role of pH, reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+ imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+ increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+ overloading again occurred late after reperfusion. This late Ca2+ overloading was blocked by MPT inhibition. Intramitochondrial Ca2+ chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+ overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.     

Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH-dependent cell death after reperfusion

Reperfusion of ischemic tissue can precipitate cell death. Much of this cell killing is related to the return of physiological pH after the tissue acidosis of ischemia. The mitochondrial permeability transition (MPT) is a key mechanism contributing to this pH-dependent reperfusion injury in hepatocytes, myocytes, and other cell types. When ATP depletion occurs after the MPT, necrotic cell death ensues. If ATP levels are maintained, at least in part, the MPT initiates apoptosis caused by mitochondrial swelling and release of cytochrome c and other proapoptotic factors. Cyclosporin A and acidotic pH inhibit opening of permeability transition pores and protect cells against oxidative stress and ischemia/reperfusion injury, whereas Ca²⁺, mitochondrial reactive oxygen species, and pH above 7 promote mitochondrial inner membrane permeabilization. Reperfusion with nitric oxide (NO) donors also blocks the MPT via a guanylyl cyclase and protein kinase G-dependent signaling pathway, which in turn prevents reperfusion-induced cell killing. In isolated mitochondria, a combination of cGMP, cytosolic extract, and ATP blocks the Ca²⁺-induced MPT, an effect that is reversed by protein kinase G inhibition. Thus, NO prevents pH-dependent cell killing after ischemia/reperfusion by a guanylyl cyclase/cGMP/protein kinase G signaling cascade that blocks the MPT.     

Cytoprotection of rat hepatocytes by desipramine in a model of simulated ischemia/reperfusion

We investigated the cytoprotective effect of desipramine (DMI) during in vitro simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 μM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Extracellular Cl(-) modulates shrinkage-induced activation of Na(+)/H(+) exchanger in rat mesangial cells

To examine theeffect of hyperosmolality on Na⁺/H⁺ exchanger(NHE) activity in mesangial cells (MCs), we used apH-sensitive dye,2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pH_i) in a single MC from ratglomeruli. All the experiments were performed inCO₂/HCO₃-free HEPESsolutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH₂O) treated with mannitol caused cellalkalinization. The hyperosmolality-induced cell alkalinization wasinhibited by 100 µM ethylisopropylamiloride, a specific NHEinhibitor, and was dependent on extracellular Na⁺. Thehyperosmolality shifted the Na⁺-dependent acid extrusionrate vs. pH_i by 0.15-0.3 pH units in thealkaline direction. Removal of extracellular Cl byreplacement with gluconate completely abolished the rate of cellalkalinization induced by hyperosmolality and inhibited the Na⁺-dependent acid extrusion rate, whereas, under isosmoticconditions, it caused no effect on Na⁺-dependentpH_i recovery rate or Na⁺-dependent acidextrusion rate. The Cl-dependent cell alkalinizationrate under hyperosmotic conditions was partially inhibited bypretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS,and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acidextrusion rate via NHE is greater under hyperosmotic conditions thanunder isosmotic conditions at a wide range of pH_i,3) the NHE activation under hyperosmotic conditions, but notunder isosmotic conditions, requires extracellularCl, and 4) theCl-dependent NHE activation under hyperosmoticconditions partly occurs via Cl channel andmicrotubule-dependent processes.