Changes In pHi Research Articles

Relationships Between Calcium and pH in the Regulation of the Slow Afterhyperpolarization in Cultured Rat Hippocampal Neurons

The Ca(2+)-dependent slow afterhyperpolarization (AHP) is an important determinant of neuronal excitability. Although it is established that modest changes in extracellular pH (pH(o)) modulate the slow AHP, the relative contributions of changes in the priming Ca(2+) signal and intracellular pH (pH(i)) to this effect remain poorly defined. To gain a better understanding of the modulation of the slow AHP by changes in pH(o), we performed simultaneous recordings of intracellular free calcium concentration ([Ca(2+)](i)), pH(i), and the slow AHP in cultured rat hippocampal neurons coloaded with the Ca(2+)- and pH-sensitive fluorophores fura-2 and SNARF-5F, respectively, and whole cell patch-clamped using the perforated patch technique. Decreasing pH(o) from 7.2 to 6.5 lowered pH(i), reduced the magnitude of depolarization-evoked [Ca(2+)](i) transients, and inhibited the subsequent slow AHP; opposite effects were observed when pH(o) was increased from 7.2 to 7.5. Although decreases and increases in pH(i) (at a constant pH(o)) reduced and augmented, respectively, the slow AHP in the absence of marked changes in preceding [Ca(2+)](i) transients, the inhibition of the slow AHP by decreases in pH(o) was correlated with low pH(o)-dependent reductions in [Ca(2+)](i) transients rather than the decreases in pH(i) that accompanied the decreases in pH(o). In contrast, high pH(o)-induced increases in the slow AHP were correlated with the accompanying increases in pH(i) rather than high pH(o)-dependent increases in [Ca(2+)](i) transients. The results indicate that changes in pH(o) modulate the slow AHP in a manner that depends on the direction of the pH(o) change and substantiate a role for changes in pH(i) in modulating the slow AHP during changes in pH(o).

Assessment of splanchnic perfusion by gastric tonometry in patients with acute hypovolemic burn shock

Objective To compare the changes in pHi and intramucosal–arterial CO 2-gap with invasive haemodynamic and global perfusion measurements during hypovolemic burn shock and to evaluate the sensitivity of these parameters as an early predictor of mortality in patients with extensive burns. Design Prospective, controlled, clinical study. Setting An eight-bed intensive burn care unit in a university-affiliated hospital. Patients Fifty severely burned patients with TBSA burned >25% BSA. Methods During the first 48 h after burn, gastric intramucosal CO 2 was measured every 8 h using automated air tonometry. pHi and intramucosal–arterial CO 2-gap were calculated. Simultaneously invasive haemodynamic data were registered by the transpulmonary thermodilution technique, using the mean of triplicate injections. The intramucosal–arterial CO 2-gradient and pHi were compared with haemodynamic and global perfusion data by regression analysis. Mean pHi and CO 2-gap values at 8 and 24 h after injury were compared between survivors and non-survivors to evaluate the prognostic significance of this parameter. Results Regression analyses revealed no or a negligible correlation between intramucosal and haemodynamic or perfusion data, even during the critical low flow–high resistance phase of resuscitation. Mean pHi and PCO 2-gap at 8 and 24 h did not differ significantly between survivors and non-survivors. Conclusion Gastric tonometry is a poor indicator of splanchnic perfusion in patients with burn shock, even when all precautions are taken to prevent methodological errors. The intramucosal–arterial PCO 2-gap and pHi do not distinguish survivors from non-survivors. Therefore, gastric tonometry does not seem to improve the ability to anticipate and avert regional anaerobic metabolism during burn shock and its routine use in these patients cannot be recommended.

Effect of Human Carbonic Anhydrase II on the Activity of the Human Electrogenic Na/HCO3 Cotransporter NBCe1-A in Xenopus Oocytes

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (I(NBC)) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I(NBC) or the initial rate at which the intracellular pH fell (dpH(i)/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I(NBC) or dpH(i)/dt (Day 4). Because dpH(i)/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I(NBC) slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5' end of NBCe1-A and CA II at the 3' end (EGFP-e1-CAII). We measured I(NBC) or dpH(i)/dt (days 3-4), exposed oocytes to EZA, and measured I(NBC) or dpH(i)/dt (Day 3-4). dpH(i)/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity.

Lactic acid accumulation is an advantage/disadvantage during muscle activity

Lactic acid accumulation is an advantage/disadvantage during muscle activity

Mechanism of iodide transport in the rabbit cortical collecting duct

Pendrin, an anion exchanger known to participate in iodide transport in the apical membrane of follicular cells of the thyroid gland, has recently been shown to exist in the apical membrane of the beta- and gamma-intercalated (beta/gamma-IC) cells of the cortical collecting duct (CCD). We examined mechanisms of iodide transport in the CCD. Rabbit CCD was perfused in vitro, and lumen-to-bath flux coefficients for both (125)I(-) (K(I (lb))) and (36)Cl(-) (K(Cl (lb))) were measured simultaneously. The intracellular pH (pHi) of beta/gamma-IC cells in the perfused CCD was measured by microscopic fluorometory, by loading 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxy methylester (BCECF-AM), a fluorescent marker for pHi. The effects on pHi of the replacement of NaCl with Na cyclamate, NaI, or NaBr in the lumen or bath were observed. K(I (lb)) was comparable to or slightly higher than K(Cl (lb)). Both iodide and chloride in the lumen caused self- and cross-inhibitions to both fluxes. The addition of 5-nitro-2-(-3-phenylpropylamino)-benzoate (NPPB), a Cl(-) channel inhibitor, to the bath significantly reduced K(Cl (lb)), but not K(I (lb)). Replacement of luminal fluid NaCl with Na cyclamate, NaI, or NaBr caused alkalization of pHi, no change in pHi, and slight acidification of pHi, respectively. Replacement of bath NaCl with Na cyclamate, NaI, or NaBr caused alkalization, alkalization, and acidification of pHi, respectively. Luminal NaI prevented the acidification of pHi caused by bath Na cyclamate. The data are consistent with the model that iodide is transported via the Cl(-)/HCO(3) (-) exchanger in the apical membrane of beta/gamma-IC cells and exits the basolateral membrane via an electroneutral transporter that is distinct from the Cl(-) channel. We could not, however, identify which type of beta/gamma-IC cell was mainly responsible.

The Na+/H+ exchanger NHE1 in stress-induced signal transduction: implications for cell proliferation and cell death

The ubiquitous plasma membrane Na+/H+ exchanger NHE1 is highly conserved across vertebrate species and is extensively characterized as a major membrane transport mechanism in the regulation of cellular pH and volume. In recent years, the understanding of the role of NHE1 in regulating cell function has expanded from one of a household protein involved in ion homeostasis to that of a multifaceted regulator and/or modulator of a wide variety of cell functions. NHE1 plays pivotal roles in response to a number of important physiological stress conditions which, in addition to cell shrinkage and acidification, include hypoxia and mechanical stimuli, such as cell stretch. It has recently become apparent that NHE1-mediated modulation of, e.g., cell migration, morphology, proliferation, and death results not only from NHE1-mediated changes in pHi, cell volume, and/or [Na+]i, but also from direct protein-protein interactions with, e.g., ezrin/radixin/moesin (ERM) proteins and regulation of cellular signaling events, including the activity of mitogen-activated protein kinases (MAPKs) and Akt/protein kinase B (PKB). The aim of this review is to present and discuss new findings implicating NHE1 activation as a central signaling event activated by stress conditions and modulating cell proliferation and death. The pathophysiological importance of NHE1 in modulating the balance between cell proliferation and cell death in cancer and in ischemia/severe hypoxia will also be briefly addressed.

Role of Na + /Ca 2+ exchange in maintenance of elevated pulmonary arterial smooth muscle cell (PASMC) intracellular Ca 2+ concentration ([Ca 2+ ] i ) and intracellular pH (pHi) during chronic hypoxia (CH)

In the lung, exposure to CH may result in pulmonary hypertension. Development of this condition is due to increased muscularity and contraction of the pulmonary arteries. These processes are associated with an increase in both pHi and [Ca2+]i. We previously demonstrated that the alkaline shift in pHi in PASMCs isolated from chronically hypoxic rats was partially dependent on the increase in [Ca2+]i. In this study, we examined the role of Na+/Ca2+ exchange (NCX) and Na+/H+ exchange (NHE) in mediating the elevated basal [Ca2+]i and Ca2+-dependent changes in pHi in PASMCs isolated from rats exposed to CH (3weeks, 10% O2). Ratiometric fluorescent microscopy was used to measure PASMC pHi and [Ca2+]i with the pH-sensitive dye, BCECF-AM, and the Ca2+-sensitive dye, Fura-2-AM. At baseline, both bepridil, a general Na+/Ca2+ exchange inhibitor, and KB-R7943, an inhibitor selective for reverse mode NCX (Ca2+ entry), caused a small but significant decrease in resting [Ca2+]i. In the presence of KB-R7943, the changes in pHi induced by increasing [Ca2+]i with KCl or decreasing [Ca2+]i with Ca2+-free extracellular solution or NiCl2 were markedly reduced. Exposure to ethyl isopropyl amiloride (EIPA), a NHE inhibitor, decreased resting pHi. In the presence of EIPA, no further changes in pHi were observed in response to changing [Ca2+]i. These results suggest that during CH, Na+ efflux through reverse mode NCX facilitated increased activation of NHE activity and an alkaline shift in pHi, while Ca2+ entry contributed to regulation of increased basal [Ca2+]i.

Effect of intracellular pH clamping on the response to hypercapnia of locus coeruleus (LC) neurons

Central chemosensitive neurons increase their firing rate (FR) upon exposure to hypercapnia. Currently, pHi is believed to be the major signal in the chemosensitive (CS) response; however, a new model has been proposed suggesting that multiple factors (such as Ca++, CO2, pHi, and pHo) contribute to the CS response. While there is evidence for a significant role of pHi in the CS response, we hypothesize that hypercapnic acidosis (HA) can still increase FR even with no change in pHi. To test this hypothesis, we developed a method to clamp pHi using rapid diffusion of the weak acid, acetic acid, through the cell membrane according to the relationship: pHi = pHo + log ([C2H3O2] i/[C2H3O2] o). Pontine brainstem slices (300 μm) were cut from neonatal rat pups (P4-P16). FR of LC neurons was measured with whole cell patch pipettes, pHi was measured by fluorescence imaging using the dye pyranine, and neurons were loaded from the patch pipette with 50 mM acetate. Extracellular acetate was varied to set pHi at desired values using the weak acid diffusion technique. We eliminated pHi decreases (0.07 ± 0.06 pH unit, p>0.05, n=8) of LC neurons upon exposure to HA (15% CO2, pHo 7.0) compared to the normal response (0.25 ± 0.03 pH unit, p<0.05, n=20). Despite pHi clamping, HA induced an increase in FR (CS index = 144 ± 14%, n=7) similar to controls (140 ± 5%, n=17). While a decrease in pHi is sufficient to elicit the chemosensitive response, these data indicate that intracellular acidification is not required. A chemosensitive response to hypercapnia without a change in pHi strongly supports the multiple factors model of chemosensitivity. [NIH grants RO1 HL56683 and F32 HL80877].

Sodium and Epithelial Sodium Channels Participate in the Regulation of the Capacitation-associated Hyperpolarization in Mouse Sperm

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Fluorescent measurement of the intracellular pH during sporulation of Saccharomyces cerevisiae.

This work reports the intracellular pH (pHi) dynamics of Saccharomyces cerevisiae cells in sporulation medium. Cells loaded with the pH-sensitive dye carboxy-seminaphthorhodafluor-1 (C.SNARF-1) exhibited an alkalization of the pHi following the extracellular pH during sporulation in the absence of buffer and almost no change in pHi or delta pH when sporulation was carried out in buffered medium. The results indicate that the pH gradient does not appear to be directly involved in the regulation of acetate uptake during sporulation. However, the alkalization of pHi by eliciting a decrease in metabolic fluxes could account for a lower demand for acetate.

Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.

Conformational Preference and Cis−Trans Isomerization of 4(R)-Substituted Proline Residues

We report here the conformational preference and prolyl cis-trans isomerization of 4(R)-substituted proline dipeptides, N-acetyl-N'-methylamides of 4(R)-hydroxy-L-proline and 4(R)-fluoro-L-proline (Ac-Hyp-NHMe and Ac-Flp-NHMe, respectively), studied at the HF/6-31+G(d), B3LYP/6-31+G(d), and B3LYP/6-311++G(d,p) levels of theory. The 4(R)-substitution by electron-withdrawing groups did not result in significant changes in backbone torsion angles as well as endocyclic torsion angles of the prolyl ring. However, the small changes in backbone torsion angles phi and psi and the decrease of bond lengths r(Cbeta-Cgamma) or r(Cgamma-Cdelta) appear to induce the increase of the relative stability of the trans up-puckered conformation and to alter the relative stabilities of transition states for prolyl cis-trans isomerization. Solvation free energies of local minima and transition states in chloroform and water were calculated using the conductor-like polarizable continuum model at the HF/6-31+G(d) level of theory. The population of trans up-puckered conformations increases in the order Ac-Pro-NHMe < Ac-Hyp-NHMe < Ac-Flp-NHMe in chloroform and water. The increase in population for trans up-puckered conformations in solution is attributed to the increase in population for the polyproline-II-like conformations with up puckering. The barriers DeltaGct++ to prolyl cis-to-trans isomerization for Ac-Hyp-NHMe and Ac-Flp-NHMe increase as the solvent polarity increases, as seen for Ac-Pro-NHMe. In particular, it was identified that the cis-trans isomerization proceeds through the clockwise rotation about the prolyl peptide bond for Ac-Hyp-NHMe and Ac-Flp-NHMe in chloroform and water, as seen for Ac-Pro-NHMe.

Functional characterization of Cl-/HCO3- exchange in villous cells of the mouse ileum

At least three kinds of Cl(-)/HCO(3)(-) exchangers, SLC26A3, SLC26A6 and AE2, have been demonstrated to be expressed in the intestinal epithelial cell. To examine the functional expression of these exchangers in the native enterocyte, we studied the Cl(-)/HCO(3)(-)- exchange activity in isolated villi from the mouse ileum by microfluorometric intracellular pH (pH(i)) measurement. The pH(i) value increased upon Cl(-) removal when the villus was superfused with an HCO(3)(-)/CO(2)-buffered solution, while the response was blunted when superfused with an HCO(3)(-)/CO(2)-free, Hepes-buffered solution. The recovery of pH(i) value induced by Cl(-) re-addition (after initial Cl(-) removal) was totally or partially mimicked by the addition of Br(-), I(-), F(-), NO(3)(-), or SO(4)(2-) (in the absence of Cl(-)). The increase in pH(i) value induced by Cl(-) removal was partially inhibited in the presence of DIDS (30 muM), tenidap (10 muM), niflumic acid (30 muM) or NPPB (30 muM). Increasing the K(+) concentration from 5 mM to 60 mM in the superfusion solution induced a reversible increase in pH(i) value under the HCO(3)(-)/CO(2)-buffered condition, while it had hardly any effect on pH(i) under the Hepesbuffered condition. The K(+)-induced pH(i) changes were partially suppressed by removing Cl(-) from the superfusion solution. These results, together with the reported findings of mouse slc26a3, slc26a6 and AE2 in heterologously expressed systems, suggest the possibility that these three exchangers may all be functionally expressed in mouse ileal villous cells.

Shank2 Associates with and Regulates Na+/H+ Exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.

Transport Activity of MCT1 Expressed in Xenopus Oocytes Is Increased by Interaction with Carbonic Anhydrase

Injection of carbonic anhydrase isoform II (CA) into Xenopus frog oocytes increased the rate of H+ flux via the rat monocarboxylate transporter isoform 1 (MCT1) expressed in the oocytes. MCT1 activity was assessed by changes of intracellular H+ concentration measured by pH-selective microelectrodes during application of lactate. CA-induced augmentation of the rate of H+ flux mediated by MCT1 was not inhibited by ethoxyzolamide (10 microM) and did not depend on the presence of added CO2/HCO3- but was suppressed by injection of an antibody against CA. Deleting the C terminus of the MCT1 greatly reduced its transport rate and removed transport facilitation by CA. Injected CA accelerated the CO2/HCO3(-)-induced acidification severalfold, which was blocked by ethoxyzolamide and was independent of MCT1 expression. Mass spectrometry confirmed activity of CA as injected into the frog oocytes. With pulldown assays we demonstrated a specific binding of CA to MCT1 that was not attributed to the C terminus of MCT1. Our results suggest that CA enhances MCT1 transport activity, independent of its enzymatic reaction center, presumably by binding to MCT1.

Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains

The effects of NaCl stress on cell area and intracellular pH (pHi) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30-35%), it caused different pHi changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pHi homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pKa (pK(a,i)) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pK(a,i) close to its pHi homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pK(a,i) to its pHi homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pHi were mainly due to hyperosmotic stress and not ionic stress.

Effects of Alcohol on Intracellular pH Regulators and Electromechanical Parameters in Human Myocardium

Disturbances in intracellular pH (pHi) of the heart can trigger major changes in the strength and rhythm of the heartbeat. It is well known that two extruders, Na+/H+ exchange (NHE) and Na+/HCO3- symporter (NHS), and a monocarboxylic acid transporter (MCT) are involved in acid-equivalent extruding in the human heart. Drinking alcohol has been proven to affect blood pressure and heart contractility and, sometimes, causes cardiac arrhythmia. To assess the effects of alcohol on pHi regulators and electromechanical parameters, various concentrations of alcohol were superfused into human myocardium in the present study. Human atrial myocardium was obtained from hearts of patients undergoing corrective cardiac surgery. Institutional rules for the protection of human subjects were observed. In the whole study, pHi was measured by an epifluorescent, ratiometric microspectrofluorimetry technique with the dye BCECF, while electrophysiological experiments were performed by traditional micropipette. NHE and NHS activities were measured after pHi recovery from intracellular acidosis induced by NH4Cl prepulse, while MCT activity was measured by a lactate adding/removing technique. In pHi experiments, we demonstrated that alcohol could induce a biphasic, concentration-dependent (30-1000 mM) pHi change (i.e., alkalosis after acidosis) in human atrium in HEPES-buffered Tyrode solution. To a smaller extent, similar results were found when the superfusate was replaced by HCO3- -buffered Tyrode solution. NHE activity was increased by a moderate concentration of alcohol (30 mM), while it was inhibited in a concentration-dependent manner by higher concentrations of alcohol (>100 mM). On the contrary, 30-1000 mM alcohol increased the activity of NHS in a concentration-dependent manner. Surprisingly, MCT activity was not affected by alcohol. In electromechanical experiments, we found that alcohol (30-1000 mM) had a notable concentration-dependent inhibitory effect on the contractile force, while higher concentrations of alcohol (>100 mM) decreased the action potential amplitude, upstroke velocity, duration of repolarization, and force of contractions in a concentration-dependent way. All these alcohol-induced pHi changes and electromechanical inhibitions were reversible. To our knowledge, this study provides the first evidence that alcohol can affect pHi in human myocardial tissue by changing the activity of acid extruders (i.e., NHE and NHS).

Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell-matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin alpha2beta1. Cells reached their maximum motility at pHe approximately 7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell-matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin alpha2beta1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell-matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition).

Apport de la spectroscopie RMN à l'évaluation du traumatisme crânien

Nuclear magnetic spectroscopy (MRS) is a useful method for noninvasively studying intracerebral metabolism. Proton MRS can identify markers of the neuronal viability (N-acetyl-aspartate, NAA), of the metabolism of cellular membranes (choline), of the cellular energy metabolism (creatine, lactate). In Phosphorus MRS, the peaks most readily identified are involved in the high-energy cellular metabolism (ATP, phosphocreatine, inorganic phosphate), and intracellular pH (pHi) can be determined using this method. MRS has been used in experimental models of traumatic brain injury (TBI), primarily to study the cellular metabolism and the relation between biochemical and histological changes after trauma. In trauma patients, significant changes in NAA, choline and pHi were found in both grey and white matter comparing with controls, and these alterations correlated with injury severity. Correlations have been reported between these biochimical changes (reduction in NAA, increase in choline) measured at 1 to 6 months after TBI and the clinical outcome of the patients. However, there are methodological issues which still impede to recommend MRS as a tool for predicting neurological outcome in the clinical setting.

Photosynthesis and photoinhibition in two xerophytic shrubs during drought

Seasonal changes in water relations, net photosynthetic rate (P(N)), and fluorescence of chlorophyll (Chl) a of two perennial C(3) deciduous shrubs, Ipomoea carnea and Jatropha gossypifolia, growing in a thorn scrub in Venezuela were studied in order to establish the possible occurrence of photoinhibition during dry season and determine whether changes in photochemical activity of photosystem 2 (PS2) may explain variations of PN in these species. Leaf water potential (psi) decreased from -0.2 to -2.1 MPa during drought in both species. The PN decreased with y in T carnea and J. gossypifolia by 64 and 74%, respectively. Carboxylation efficiency (CE) decreased by more than 50 and 70% in I. carnea and J gossypifolia, respectively. In I. carnea, relative stomatal limitation (L(s)) increased by 17% and mesophyll limitation (L(m)) by 65% during drought, while in J. gossypifolia Ls decreased by 27% and L. increased by 51%. Drought caused a reduction in quantum yield of PS2 (theta(PS2)) in both species. Drought affected the capacity of energy dissipation of leaves, judging from the changes in the photochemical (q(P)) and non-photochemical quenching (NPQ) coefficients. Photo-inhibition during drought in I. carnea and J gossypifolia was evidenced in the field by a drop in the maximum quantum yield of PS2 (F(v)/F(m)) below 0.8 and also by non-coordinated changes in phi(PS2) and quantum yield of non-photochemical excitation quenching (Y(n)). Total soluble protein content on an area basis increased with psi but the ribulose-1,5-bisphosphate carboxylase/oxygenase content remained unchanged. A reduction of total Chl content with drought was observed. Hence in the species studied photoinhibition occurred, which imposed an important limitation on carbon assimilation during drought.