Acid Extrusion Rate Research Articles

Graphene oxide promoted chromium uptake by zebrafish embryos with multiple effects: Adsorption, bioenergetic flux and metabolism

The increasing production and application of graphene oxide (GO, a popular carbon nanomaterial), makes their release into aqueous environment inevitably. The capability of GO to enhance the toxicity of background contaminants has been widely concerned. However, the effect of GO on heavy metal accumulation in fish embryos remains unclear. Here, we show that GO-promoted chromium (Cr) uptake by zebrafish embryos with multiple effects. The adsorption accelerated the aggregation and settlement of Cr6+-adsorbed GO and decreased the Cr6+ concentration in the upper water, which enhanced the interaction of chorions and contaminants (Cr6+, GO and Cr6+-adsorbed GO). In the presence of GO, the Cr content in chorions and intra-chorion embryos was increased by four times and 57% respectively, compared to that of the single Cr6+ exposure. Furthermore, GO+Cr6+ increased the oxygen consumption rates, embryonic acid extrusion rates and ATP production, induced more serious oxidative stress, and disturbed amino acid metabolism, fatty acid metabolism and TCA cycle. These findings provide new insights into the effect of GO on heavy metal bioaccumulation and toxicity during embryogenesis.

Modulation of acid extrusion and acid loading in neurons and astrocytes by RPTPγ and RPTPζ

Receptor protein tyrosine phosphatases γ and ζ (RPTPγ & RPTPζ) are transmembrane signaling proteins that, as monomers, catalyze dephosphorylation of tyrosine residues on cytosolic targets. RPTPγ and RPTPζ are unusual in having an extracellular carbonic-anhydrase–like domain (CALD). Carbonic anhydrases (CAs) catalyze the reaction, CO₂ + H₂O ⇌ HCO3– + H⁺, but CALDs are enzymatically inactive. Previous studies on renal proximal tubules (PTs) strongly suggest that RPTPγ is a dual extracellular CO₂/HCO₃⁻ sensor. RPTPγ—expressed on the PT basal (i.e., blood-side) membrane—is essential for modulating acid-base transport in response to changes in basolateral [CO₂] and [HCO₃⁻]. RPTPγ and RPTPζ are also present in the central nervous system (CNS), including mouse hippocampus (HC). RPTPγ is expressed primarily in neurons and RPTPζ, in astrocytes. Here, we investigate whether RPTPγ and RPTPζ in brain, like RPTPγ in kidney, play roles in sensing extracellular [CO₂]ₒ and [HCO₃⁻]ₒ and altering acid-base transport. We hypothesize that activation/termination of the intracellular phosphatase activity of these RPTPs regulates initial steps in signaling cascades that defend cells against intracellular pH (pHi) decreases triggered by metabolic acidosis (MAc, caused by ↓ [HCO₃⁻]ₒ) or respiratory acidosis (RAc, caused by ↑ [CO₂]ₒ). We propose that the RPTPs respond to ↑[CO₂]ₒ or ↓[HCO₃⁻]ₒ by promoting a shift from dimers to monomers, thereby dis-inhibiting the activity of the intracellular phosphatase domains. The ultimate result of the RPTP dis-inhibition would be increased acid-extrusion rate (JE) or decreasedacid-loading rate (JL), both of which would minimize the fall of pHi during a first exposure to MAc or RAc, and may promote adaptation during a second exposure. Immunocytochemistry of mixed HC neuron-astrocyte cultures from wild-type (WT) mice using a primary antibody against the RPTPγ fibronectin III domain shows that RPTPγ colocalizes with MAP2 in HC neurons but not with GFAP in HC astrocytes . RT-PCR cloning from mixed HC cultures identifies specific RPTPγ and RPTPζ variants. Presumably it is the neurons that express two identified RPTPγ variants: the full-length protein, encoded by all 30 ptprg exons (NM_008981), and a second newly-detected variant that corresponds to the hypothetical assembly X1 (XM_006517956). The HC co-cultures also express six RPTPζ variants: Three RPTPζ variants (V) have previously been validated in mice: VR3 (NM_001081306), VR4 (NM_001311064), and VR5 (NM_001361349). Three others were previously only reported as hypothetical assemblies: X2 (XM_006505013), X3 (XM_006505014), and X4 (XM_006505015). To assess the influence of RPTPζ on pHi homeostasis, we loaded cells with the pH-sensitive dye BCECF, digitally monitored dye fluorescence, and computed pHi of cells subjected to acid-base disturbances. In MAc-MAc protocols, the effects of the knockout (KO) of RPTPζ are unremarkable in both neurons and astrocytes. In RAc-RAc protocols, the KO has unremarkable effects on neurons. However, in astrocytes the RPTPζ-KO markedly increases both the rates and the magnitudes of pHi decrease during both RAc pulses. Thus, RPTPζ may stabilize pHi in astrocytes subjected to RAc.

Simultaneous monitoring of oxygen consumption and acidification rates of a single zebrafish embryo during embryonic development within a microfluidic device

A microfluidic device with a light modulation system was developed to simultaneously measure the oxygen consumption rate (OCR) and acid extrusion rate (AER) of a single zebrafish embryo during embryonic development. The device combines two components: an array of acrylic microwells containing two sensing layers as the dual luminescent sensor for oxygen (O2) and acid (pH) detection, and a microfluidic module with pneumatically actuated glass lids to controllably seal the microwells. The continuous blue LED and modulated UV LED lights were simultaneously used to excite the dual luminescent sensor, with the emission detected by a single photodetector. The detection signals were then split into DC and AC components to measure the time variations in fluorescence intensity and phosphorescence lifetime for pH and O2 detection, respectively. We have successfully measured the OCR and AER of a single developing zebrafish embryo inside a sealed microwell from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage of 48 hpf. We also demonstrated the measurement of the OCR and AER of a single 48 hpf zebrafish that experienced acute hypoxia by using our device to monitor the transition between aerobic and anaerobic metabolism. We observed that the AER began to significantly increase, while the OCR rapidly decreased after 20 min of hypoxia, indicating the time point of transition where the non-mitochondrial metabolism subsequently dominated the energy production. Our proposed methodology provides the potential for studying the bioenergetic metabolism in a developing organism that relates mitochondrial physiology and disease.

Inflammation reduces the activity of the NaHCO3 cotransporter, NBCe1, in the surface cells of the proximal colon of IL10 knockout mice (908.1)

The impact of inflammation on intestinal secretion is contentious. Here the effect of inflammation on activity of the NaHCO3 cotransporter, NBCe1, in the proximal colon was investigated. Interleukin10‐knockout (IL10‐/‐) mice infected with Helicobacter typhlonius, which develop colitis, were used as a model of inflammation, and uninfected, asymptomatic IL10‐/‐ mice served as controls. NBCe1 activity was measured as the 5‐(N‐ethyl‐N‐isopropyl) amiloride‐insensitive (EIPA, 20µM), 4,4'‐diisothiocyano‐2,2'‐stilbenedisulfonic acid‐sensitive (DIDS, 500µM), rate of intracellular pH recovery and acid extrusion (JH+) following acid loading of isolated crypts, while NBCe1 expression was measured by Western blotting and immunohistochemistry. In control tissues NBCe1 immunoreactivity was restricted to the surface cells and upper third of the crypts. Consistent with this there was little evidence of NBCe1 activity in the lower half of the crypts, with or without forskolin (10µM) stimulation. In contrast, in the surface cells, while NBCe1 was inactive under basal conditions (JH+EIPA=16.4±1.8mM.min‐1; JH+EIPA+DIDS=18.7±1.8 mM.min‐1), its activity was stimulated by forskolin (after forskolin JH+EIPA=24.5±2.7 mM.min‐1 and JH+EIPA+DIDS=13.6±1.1mM.min‐1). In inflamed tissues, total NBCe1 expression was reduced (P<0.001, n=6), there was no NBCe1 immunoreactivity in the surface cells and upper crypts, and forskolin did not stimulate NBCe1 activity in the surface cells. This suggests inflammation reduces colonic HCO3‐ secretion, which may affect luminal pH regulation and growth of luminal bacteria, possibly contributing to the dysbiosis seen in patients with inflammatory bowel disease, as well as modifying mucus hydration.Grant Funding Source: Supported by The Otago Medical Research Foundation

Sensor monitoring of organotypic human tumor short term cultures may reveal profiles of metabolism and chemosensitivity

Event Abstract Back to Event Sensor monitoring of organotypic human tumor short term cultures may reveal profiles of metabolism and chemosensitivity Martin Brischwein1*, Regina Kleinhans1, Marina Haas1 and Bernhard Wolf1 1 Technische Universitaet Muenchen, Germany Background and aim. There is an urgent clinical demand for improved stratification of cancer patients and allocation into distinct therapeutic regimens. In this proof-of-principle study we analyse the feasibilty of a sensor-based profiling of metabolism and chemosensitivity on an organotypic human breast cancer model preserving the native, heterogeneous character of the tumor tissue. Methods. The approach is based on surgically explanted human breast cancer tissue samples which were subjected to careful slicing preparation (Kleinhans, 2012). These organotypic slices in a size of ≈ 4 µl were subsequently cultured for 3-5 days in an automated assay platform (Wolf, 2013). This platform analyzes the pH and dissolved oxygen values with optochemical sensors in the close vicinity of the tumor slices. Acid extrusion and oxygen consumption rates are determined before, during and after treatments with pharmaceutical compounds. Results . A number of 23 surgical samples was analysed. After the introduction of a careful vibratome slicing protocol the success rate of generating viable short term tumor cultures was generally good. The optimum slice thickness was found to be at 200-250 µm. Both a recording of pre-treatment metabolic profiles (i.e. ratios of extracellular acidification rates and oxygen consumption rates) and assessments of chemoresponses to drugs such as chloracetaldehyde and doxorubicine is feasible. Additionally, a computational reaction-diffusion modelling (Grundl, 2013) allows an estimation of pH and dissolved oxygen values in temporal and spatial resolution for an improved control about physico-chemical conditions of the cellular microenvironment. Conclusions / Discussion. A non-interventional, prospective clinical study should be initiated on the basis of this proof-of-principle research. In such a study, the microphysiometric, sensor-based approach should be complemented by data from histopathological, metabolomic and genomic profiling.

Slc26a11, a chloride transporter, localizes with the vacuolar H+-ATPase of A-intercalated cells of the kidney

Chloride has an important role in regulating vacuolar H(+)-ATPase activity across specialized cellular and intracellular membranes. In the kidney, vacuolar H(+)-ATPase is expressed on the apical membrane of acid-secreting A-type intercalated cells in the collecting duct where it has an essential role in acid secretion and systemic acid base homeostasis. Here, we report the identification of a chloride transporter, which co-localizes with and regulates the activity of plasma membrane H(+)-ATPase in the kidney collecting duct. Immunoblotting and immunofluorescent labeling identified Slc26a11 (∼72 kDa), expressed in a subset of cells in the collecting duct. On the basis of double-immunofluorescent labeling with AQP2 and identical co-localization with H(+)-ATPase, cells expressing Slc26a11 were deemed to be distinct from principal cells and were found to be intercalated cells. Functional studies in transiently transfected COS7 cells indicated that Slc26a11 (designated as kidney brain anion transporter (KBAT)) can transport chloride and increase the rate of acid extrusion by means of H(+)-ATPase. Thus, Slc26a11 is a partner of vacuolar H(+)-ATPase facilitating acid secretion in the collecting duct.

Platelet-activating factor stimulates sodium-hydrogen exchange in ventricular myocytes

Sodium-hydrogen exchanger (NHE), the principal sarcolemmal acid extruder in ventricular myocytes, is stimulated by a variety of autocrine/paracrine factors and contributes to myocardial injury and arrhythmias during ischemia-reperfusion. Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent proinflammatory phospholipid that is released in the heart in response to oxidative stress and promotes myocardial ischemia-reperfusion injury. PAF stimulates NHE in neutrophils and platelets, but its effect on cardiac NHE (NHE1) is unresolved. We utilized quiescent guinea pig ventricular myocytes bathed in bicarbonate-free solutions and epifluorescence to measure intracellular pH (pH(i)). Methylcarbamyl-PAF (C-PAF; 200 nM), a metabolically stable analog of PAF, significantly increased steady-state pH(i). The alkalosis was completely blocked by the NHE inhibitor, cariporide, and by sodium-free bathing solutions, indicating it was mediated by NHE activation. C-PAF also significantly increased the rate of acid extrusion induced by intracellular acidosis. The ability of C-PAF to increase steady-state pH(i) was completely blocked by the PAF receptor inhibitor WEB 2086 (10 μM), indicating the PAF receptor is required. A MEK inhibitor (PD98059; 25 μM) also completely blocked the rise in pH(i) induced by C-PAF, suggesting participation of the MAP kinase signaling cascade downstream of the PAF receptor. Inhibition of PKC with GF109203X (1 μM) and chelerythrine (2 μM) did not significantly affect the alkalosis induced by C-PAF. In summary, these results provide evidence that PAF stimulates cardiac NHE1, the effect occurs via the PAF receptor, and signal relay requires participation of the MAP kinase cascade.

ATP Dependence of Na+-Driven Cl–HCO3 Exchange in Squid Axons

Squid giant axons recover from acid loads by activating a Na(+)-driven Cl-HCO(3) exchanger. We internally dialyzed axons to an intracellular pH (pH( i )) of 6.7, halted dialysis and monitored the pH(i) recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (J(H)) from the rate of pH(i) increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP](i), finding that Michaelis-Menten kinetics describes J (H) vs. [ATP](i), with an apparent V(max) of 15.6 pmole cm(-2 )s(-1) and K (m) of 124 microM. In series 2, we examined ATP gamma S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP beta S and GDP beta S to determine if any, by themselves, could support transport. Only ATP gamma S (8 mM) supported acid extrusion; ATP gamma S also supported the HCO (3)(-) -dependent (36)Cl efflux expected of a Na(+)-driven Cl-HCO(3) exchanger. Finally, in series 3, we asked whether any nucleotide could alter J (H) in the presence of a background [ATP](i) of approximately 230 microM (control J (H) = 11.7 pmol cm(-2 )s(-1)). We found J (H) was decreased modestly by 8 mM AMP-PNP (J (H) = 8.0 pmol cm(-2 )s(-1)) but increased modestly by 1 mM ADP beta S (J (H) = 16.0 pmol cm(-2 )s(-1)). We suggest that ATP gamma S leads to stable phosphorylation of the transporter or an essential activator.

Effects of acute hypoxia on intracellular-pH regulation in astrocytes cultured from rat hippocampus

We used the pH-sensitive dye BCECF to evaluate the effect of acute (5–10 min) hypoxia (∼ 3% O 2) on the regulation of intracellular pH (pH i) in astrocyte populations cultured from rat hippocampus. For cells in the nominal absence of CO 2/HCO 3 − at an extracellular pH of 7.40 (37 °C), acute hypoxia caused a small (0.05) decrease in steady-state pH i, but increased the pH i recovery rate from an acid load during all but the late phase of the pH i recovery. During such pH i recoveries, the total acid extrusion rate ( φ E, the product of dpH i/d t and proton buffering power) decreased with increasing pH i. Hypoxia alkali shifted the plot of φ E vs. pH i; over the upper ∼ 85% of the φ E range, this shift was 0.15–0.30. Hypoxia also stimulated the pH i recovery rate from an alkali load. Under normoxic conditions, switching the extracellular buffer to 5% CO 2/22 mM HCO − 3 also alkali shifted the φ E–pH i plot (upper ~ 85%) by 0.4–0.5. Superimposing hypoxia on CO 2/HCO − 3 further alkali shifted the φ E–pH i plot (upper ∼ 85% of the φ E range) by 0.05–0.15. The SITS-insensitive component of φ E was alkali shifted by 0.20–0.30, whereas the SITS-sensitive component of φ E was depressed in the low pH i range. Thus, in the nominal absence of CO 2/HCO 3 −, acute hypoxia has little effect on steady-state pH i but stimulates acid extrusion and acid loading, whereas in the presence of CO 2/HCO − 3, hypoxia stimulates the SITS-insensitive but inhibits the SITS-sensitive acid extrusion.

Mechanisms for nongenomic and genomic effects of aldosterone on Na+/H+ exchange in vascular smooth muscle cells

We have reported that exposure of vascular smooth muscle cells (VSMCs) to aldosterone for 3 and 24 h activated Na+/H+ exchange (NHE) via nongenomic and genomic mechanisms, respectively. The present study determined whether aldosterone-induced nongenomic and genomic NHE activation depends on the number of transporters, the turnover rate of a single transporter, and/or the change in intracellular pH (pHi) sensitivity of the transporter, and whether aldosterone-induced NHE activation is inhibited by the selective mineralocorticoid receptor (MR) antagonist (eplerenone). Using a fluorescent dye, we assessed NHE activity by Na-dependent acid extrusion rates (JH) after an acid load in the absence of CO2/HCO3- in VSMCs treated with aldosterone. Treatment with aldosterone for 3 and 24 h increased JH at the wide pHi range, and shifted the JH versus pHi in the alkaline direction. Without affecting the apparent Km for external Na+, the Vmax increased in VSMCs treated with aldosterone for 3 and 24 h. Both eplerenone and spironolactone inhibited only aldosterone-induced genomic NHE activation, but the IC50 of eplerenone was smaller than that of spironolactone. We demonstrated that: (1) both nongenomic and genomic stimulatory effects of aldosterone on NHE activity in VSMCs occur by an increase in the number of NHEs and the alkaline shift in pHi sensitivity of the NHE; (2) only the aldosterone-induced genomic NHE activation occurs via MR; and (3) both eplerenone and spironolactone inhibit the aldosterone-induced genomic NHE activation, but eplerenone is more effective than spironolactone, based on the IC50 value in VSMCs.

Regulation of intracellular pH

The approach that most animal cells employ to regulate intracellular pH (pH(i)) is not too different conceptually from the way a sophisticated system might regulate the temperature of a house. Just as the heat capacity (C) of a house minimizes sudden temperature (T) shifts caused by acute cold and heat loads, the buffering power (beta) of a cell minimizes sudden pH(i) shifts caused by acute acid and alkali loads. However, increasing C (or beta) only minimizes T (or pH(i)) changes; it does not eliminate the changes, return T (or pH(i)) to normal, or shift steady-state T (or pH(i)). Whereas a house may have a furnace to raise T, a cell generally has more than one acid-extruding transporter (which exports acid and/or imports alkali) to raise pH(i). Whereas an air conditioner lowers T, a cell generally has more than one acid-loading transporter to lower pH(i). Just as a house might respond to graded decreases (or increases) in T by producing graded increases in heat (or cold) output, cells respond to graded decreases (or increases) in pH(i) with graded increases (or decreases) in acid-extrusion (or acid-loading) rate. Steady-state T (or pH(i)) can change only in response to a change in chronic cold (or acid) loading or chronic heat (or alkali) loading as produced, for example, by a change in environmental T (or pH) or a change in the kinetics of the furnace (or acid extrudes) or air conditioner (or acid loaders). Finally, just as a temperature-control system might benefit from environmental sensors that provide clues about cold and heat loading, at least some cells seem to have extracellular CO(2) or extracellular HCO(3)(-) sensors that modulate acid-base transport.

Hyperosmotic urea activates basolateral NHE in proximal tubule from P-gp null and wild-type mice.

Using the pH-sensitive fluorescent dye BCECF, we compared the effects of hyperosmotic urea on basolateral Na(+)/H(+) exchange (NHE) with those of hyperosmotic mannitol in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type (WT) mice. All the experiments were performed in CO(2)/HCO-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH(2)O by adding mannitol or urea. NHE activity was assessed by the Na(+)-dependent acid extrusion rate (J(H)) after an acid load with NH(4)Cl prepulse. In WT mice, hyperosmotic mannitol had no effect on J(H) at over the entire range of intracellular pH (pH(i)) studied (6.20-6.90), whereas in KO mice it increased J(H) at a pH(i) range of 6.20-6.45. In contrast, in both WT and KO mice, hyperosmotic urea increased J(H) at a pH(i) range of 6.20-6.90. In KO mice, J(H) in a hyperosmotic urea solution were similar to those in a hyperosmotic mannitol solution at a pH(i) range of 6.20-6.40 but were greater than in a hyperosmotic mannitol solution at a pH(i) range of 6.45-6.90. In WT mice, hyperosmotic urea caused an increase in V(max) without changing K(m) for peritubular Na(+). Staurosporine (the PKC inhibitor) inhibited hyperosmotic mannitol-induced NHE activation in KO mice, whereas it had no effect on hyperosmotic urea-induced NHE activation in WT or KO mice. Genistein (the tyrosine kinase inhibitor) inhibited hyperosmotic urea-induced NHE activation in WT and KO mice, whereas it caused no effect on hyperosmotic mannitol-induced NHE activation in KO mice. We conclude that hyperosmotic urea activates basolateral NHE via tyrosine kinase in tubules from both WT and KO mice, whereas hyperosmotic mannitol activates it via PKC only in tubules from KO mice.

Continuous Measurement of Glucose Utilization in Heart Myoblasts

Understanding quantitative aspects of cell energy metabolism and how it is influenced by environment is central to biology, medicine, and biotechnology. Most methods used for measuring metabolic fluxes associated with energy metabolism require considerable personnel effort or high maintenance instrumentation. The microphysiometer is a commercially available instrument that measures acid extrusion rates, which are commonly used for drug screening. With the addition of oxygen sensors, the instrument can also be used to measure cell oxygen consumption rates and thereby calculate glycolytic fluxes. In the work described here, oxygen consumption and acid extrusion rates were used to measure glucose utilization by the H9c2 rat heart myoblast cell line and these results are compared with fluxes measured with a radiometric assay. Both assays were used to investigate changes in H9c2 energy metabolism due to cell stimulation with carbachol and insulin. The results demonstrate the utility of the microphysiometer method for measuring both transient and sustained changes in partitioning of glucose utilization between glycolysis and oxidation in live cells.

The cystic fibrosis transmembrane conductance regulator is not a base transporter in isolated duodenal epithelial cells.

Duodenal epithelial bicarbonate secretion has previously been shown to be greatly impaired in mice deficient of the cystic fibrosis transmembrane conductance regulator (CFTR). It has been proposed that transmembranal bicarbonate transport occurs through the CFTR channel itself. In the present study, the transport of acid/base equivalents across the plasma membrane of proximal duodenal epithelial cells from CFTR deficient mice was compared with that of cells from normal littermates. Mixed epithelial cells from both villi and crypts were isolated from proximal duodenum and intracellular pH was assessed by cuvette-based fluorescence spectrometry using the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The steady state intracellular pH, the acid extrusion rate and the alkaline extrusion rate were unaffected by CFTR deficiency in the presence of CO(2)/HCO(-)(3). Forskolin had no effect on acid extrusion or alkaline extrusion rates. In control experiments without CO(2)/HCO(-)(3), the intrinsic buffering capacities, the steady state intracellular pH and the acid extrusion rates were equivalent in the cells from CFTR deficient mice and normal littermates. The results are consistent with a model where acid/base transport is almost exclusively mediated by the previously described transporters in the murine duodenum (i.e. Na+/H+ exchange, Cl(-)/HCO(-)(3). exchange and Na+:HCO(-)(3). cotransport). There were no evidence for significant CFTR dependent HCO(-)(3). transport in proximal duodenal epithelial cells of mixed villus and crypt origin.

Hyperosmotic mannitol activates basolateral NHE in proximal tubule from P-glycoprotein null mice.

Using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of hyperosmotic mannitol on basolateral Na(+)/H(+) exchange (NHE) activity in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type mice (WT). All experiments were performed in CO(2)/HCO-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH(2)O by the addition of mannitol. NHE activity was assessed by Na(+)-dependent acid extrusion rates (J(H)) after an acid load with NH(4)Cl prepulse. Under isosmotic conditions, J(H) values at a wide intracellular pH (pH(i)) range of 6.20-6.90 were not different between the two groups. In WT mice, hyperosmotic mannitol had no effect on J(H) at the wide pH(i) range. In contrast, in KO mice, hyperosmotic mannitol increased J(H) at a pH(i) range of 6.20-6.45 and shifted the J(H)-pH(i) relationship by 0.15 pH units in the alkaline direction. In KO mice, hyperosmotic mannitol caused an increase in maximal velocity without changing the Michaelis-Menten constant for peritubular Na(+). Exposure of cells from WT mice to the hyperosmotic mannitol solution including the P-gp inhibitor cyclosporin A increased J(H) (at pH(i) 6.30) to an extent similar to that in cells from KO mice exposed to hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C inhibited the hyperosmotic mannitol-induced increase in J(H). The stimulatory effect of hyperosmotic mannitol on J(H) was mimicked by addition to the isosmotic control solution, including phorbol 12-myristate 13-acetate (PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA increased J(H). We conclude that, in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via protein kinase C, whereas in the presence of P-gp activity, it does not.

Effect of eccentric contraction-induced injury on force and intracellular pH in rat skeletal muscles.

The effect of eccentric contraction on force generation and intracellular pH (pH(i)) regulation was investigated in rat soleus muscle. Eccentric muscle damage was induced by stretching muscle bundles by 30% of the optimal length for a series of 10 tetani. After eccentric contractions, there was reduction in force at all stimulation frequencies and a greater reduction in relative force at low-stimulus frequencies. There was also a shift of optimal length to longer lengths. pH(i) was measured with a pH-sensitive probe, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein AM. pH(i) regulation was studied by inducing an acute acid load with the removal of 20-40 mM ammonium chloride, and the rate of pH(i) recovery was monitored. The acid extrusion rate was obtained by multiplying the rate of pH(i) recovery by the buffering power. The resting pH(i) after eccentric contractions was more acidic, and the rate of recovery from acid load post-eccentric contractions was slower than that from postisometric controls. This is further supported by the slower acid extrusion rate. Amiloride slowed the recovery from an acid load in control experiments. Because the Na(+)/H(+) exchanger is the dominant mechanism for the recovery of pH(i), this suggests that the impairment in the ability of the muscle to regulate pH(i) after eccentric contractions is caused by decreased activity of the Na(+)/H(+) exchanger.

Elevated lysosomal pH in neuronal ceroid lipofuscinoses (NCLs).

We report here the intracellular (pHi) and lysosomal pH in fibroblasts of six forms of neuronal ceroid lipofuscinoses (NCLs). Acid extrusion rate and pH(i) values were measured by the membrane-permeant acetoxymethyl ester of the indicator dye, 2',7'-bis(carboxyethyl)-5-(and-6)-carboxy-fluorescein (BCECF) and lysosomal pH by a spectrofluorometric assay utilizing a novel acidotropic probe, Lysosensor yellow/blue. Intracellular pH was normal in all NCLs. Elevated lysosomal pH was detected in all NCL forms except CLN2 and CLN8. Elevated pH most probably disturbs the catalytic activity of lysosomes and is one important factor in explaining accumulation of ceroid and lipofuscin-like autofluorescent lipopigments characteristic of NCLs. Using the novel spectrofluorometric assay introduced in this study provides a fast and repeatable technique to measure intralysosomal pH from cell suspensions.

Modulation of Na(+) x H(+) exchange by osmotic shock in isolated bovinearticular chondrocytes.

The effects of hyperosmotic shock on intracellular pH (pHi) have been characterized in bovine articular chondrocytes. Osmotic shock is one of a variety of physicochemical stimuli experienced by chondrocytes upon cartilage loading. Cells were isolated from their extracellular matrix, and loaded with the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Hyperosmotic shocks were imposed by addition of KCl or sucrose to the extracellular medium. For cells at steady-state pHi, resuspension in hyperosmotic solutions elicited an alkalinization, which was significantly inhibited by removal of extracellular Na+ ions, or treatment with amiloride (1 mM) or HOE-694 (10 microM), both inhibitors of Na+ x H+ exchange. For cells acidified by ammonium rebound, recovery of pHi towards resting levels was significantly stimulated by exposure to hyperosmotic solutions, and the effect was again attenuated by inhibition of Na+ x H+ exchange. Determination of the rate of acid extrusion at different levels of acidification indicated that the affinity of acid extrusion systems for H+ ions was increased by hypertonic shock. The response to hyperosmotic media could be abolished by treatment of chondrocytes with the non-specific kinase inhibitor staurosporine (10 nM), while the phosphatase inhibitor okadaic acid (1 mM) was able to augment recovery rates to values similar to those measured under hyperosmotic conditions. The osmotic sensitivity of recovery was unaffected by exposure to the protein kinase C inhibitor calphostin C, but was abolished in cells treated with ML-7, a specific inhibitor of myosin light chain kinase. These results confirm that - as for other components of mechanical load - increased osmolarity can modulate the activity of Na+ x H+ exchange, in this case by altered patterns of phosphorylation of transporter-associated myosin. The changes of pHi which will result dictate in part the rate of cartilage macromolecule synthesis.

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

To examine the effect of hyperosmolality on Na(+)/H(+) exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pH(i)) in a single MC from rat glomeruli. All the experiments were performed in CO(2)/HCO(-)(3)-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH(2)O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 microM ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular Na(+). The hyperosmolality shifted the Na(+)-dependent acid extrusion rate vs. pH(i) by 0.15-0.3 pH units in the alkaline direction. Removal of extracellular Cl(-) by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the Na(+)-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on Na(+)-dependent pH(i) recovery rate or Na(+)-dependent acid extrusion rate. The Cl(-)-dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment 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 acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of pH(i), 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular Cl(-), and 4) the Cl(-)-dependent NHE activation under hyperosmotic conditions partly occurs via Cl(-) channel and microtubule-dependent processes.

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.

Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units.