Intracellular Buffering Capacity Research Articles

Scaling of anaerobic metabolism during exercise in the estuarine crocodile (Crocodylus porosus)

The allometry of anaerobic metabolism was examined in wild estuarine crocodiles (Crocodylus porosus) exercised to exhaustion during capture, with the aim of identifying factors underlying the higher anaerobic capacity but possibly lower anaerobic scope in larger animals. Exercise time to exhaustion, and concentrations of lactate and protons in blood and tail muscle, increased with increasing body mass. Muscle glycogen, both before and after exercise, also showed positive allometric scaling. Muscle intracellular pH buffering capacity scaled negatively with body mass. The effects of pH on the activities of muscle phosphorylase and phosphofructokinase were independent of body mass. It is proposed that anaerobic capacity may be limited by the size of muscle glycogen reserves in smaller crocodiles and by glycolytic inhibition at the lower pH values encountered in larger crocodiles. Mean anaerobic scope, and phosphorylase activity in tail muscle, decreased with increasing body mass, while lactate dehydrogenase activity was independent of body mass. Negative allometric scaling of anaerobic scope in crocodiles may relate to changes in predator-prey and social interactions that involve reduced dependence on frequent bursts of anaerobic muscle work with increasing body mass.

A comparison of metabolic stress during air exposure in two species of New Zealand abalone, Haliotis iris and Haliotis australis: implications for the handling and shipping of live animals

Anaerobic metabolism in New Zealand blackfoot and yellowfoot abalone, Haliotis iris and Haliotis australis, is supported by specific pyruvate reductase enzymes and a moderate intracellular pH buffering capacity. Lactate and tauropine increased, while ATP and energy charge decreased in both species after 24 h air exposure. Responses were most marked in yellowfoot abalone, though allometric analysis revealed the simple explanation that the difference was due to size alone. Thus, small abalone accumulate higher metabolite concentrations and have a significantly lowered energetic status compared with larger specimens similarly exposed to air. These stress responses, which may ultimately affect both meat quality and survival during transport, are evaluated in the context of proposals to culture “cocktail” sized abalone.

Cytoplasmic calcium buffer capacity determined with Nitr-5 and DM-nitrophen

We have examined intracellular calcium buffer capacity of cytoplasm from the giant axon of the marine invertebrate Myxicola infundibulum by photolytically releasing calcium from ‘caged’ compounds, while monitoring free calcium, [Ca 2+], with Ca-sensing electrodes. In cytoplasm containing intact organelles, two features of the [Ca 2+] response were seen upon light exposure: an initial spike from basal [Ca 2+], followed by a slower phase recovery. Both the amplitude of the spike in [Ca 2+] and the recovery were reduced by removal of MgATP. If organelles were removed from the cytoplasm, light exposure caused only a step-like change in [Ca 2+] with no recovery.Apparent buffer capacities (Δ bound Ca/Δ free Ca) were unaffected by changing pH from 7.0 to 7.5; however, raising basal free calcium above 3 μM significantly reduced this parameter. The buffer capacity measured after the initial spike varied by as much as an order of magnitude from one giant axon to another but averaged −50 in the absence and ∼100 in the presence of 1 mM MgATP for [Ca 2+] below 3 μM.

Proton-Lactate Cotransport in the Apical Membrane of Frog Retinal Pigment Epithelium

We studied lactate- and pyruvate-dependent proton transport across the apical membrane of frog RPE. The epithelium was mounted in a modified Ussing-chamber that allowed measurement of transepithelial potential and resistance while intracellular pH was measured with either intracellular microelectrodes or a pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). To estimate the rate of lactate influx from the change in intracellular pH, we used the NH 4 pulse technique to measure intracellular buffering capacity and its dependence on intracellular pH. We found that the buffering capacity was 16 mm at pH i = 7·28, and that it increased as intracellular pH decreased. Intracellular pH was monitored with the tissue bathed in nominally HCO 3-free (Hepes buffered) Ringer. The perfusate on the apical side of the epithelium was then changed to a Ringer that contained between 5 and 100 mM lactate or pyruvate. When 10-100 mM lactate or pyruvate was added to the apical bath the cells acidified by 0·05-0·:50 pH units. For each of these acidifications, the initial acid influx into the RPE cells was calculated from the intracellular buffering capacity and the initial rate of intracellular acidification. These influxes were plotted as functions of the concentrations of lactate or pyruvate and this relationship was analysed using Michaelis-Menten kinetics. The K m values were: 33 ± 5 mM for lactate and 9 ± 3 mM for pyruvate. There were no differences in the rates of acid influx caused by L- or D-lactate. The rates of acidification caused by 50 mM apical L-lactate were reversibly reduced by 56% after apical administration of probenecid (2 mM), and irreversibly reduced by 63% after apical administration of the SH-reagent mersalyl acid (2 mM). These results indicate the presence of a proton-lactate cotransport system in the apical membrane of the frog RPE.

Regulation of intracellular pH in crypt cells from rabbit distal colon

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end crypts of Lieberkühn were isolated by microdissection, and intracellular pH (pHi) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein. In the absence of HCO(3-)-CO2 and presence of Na+, resting pHi was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 min after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A1, pHi in cells at the bottom of the crypts was significantly reduced, whereas pHi in cells at the top of the crypts remained unchanged. The intrinsic buffering capacity of cells from the middle to the top portion of crypts was significantly higher in the pHi range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH(4+)-NH3 pulse amounted to 245 +/- 53 microM/s (n = 73/7) at pHi 7.1 and was largely Na+ dependent and ethylisopropylamiloride sensitive. The Na(+)-independent recovery of pHi after an acid load was insensitive to Sch-28080 and bafilomycin A1. In conclusion, pHi in colonic crypt cells is regulated through Na+/H+ exchange activity in the absence of HCO3-. In addition, intracellular buffering capacity varied with the position along the crypt axis, whereas Na+/H+ exchange activity and pHi did not.

Hepatocellular Na+/H+ exchange is activated at transcriptional and posttranscriptional levels in rat biliary cirrhosis

Background/Aims: Rat hepatocyte Na+/H+ exchange is activated in vitro by growth factors and in vivo following partial hepatectomy. This study explored by which mechanism(s) it is activated in a cirrhosis model characterized by chronic stimulation of hepatocyte proliferation. Methods: Rat hepatocytes were isolated 4 weeks after bile duct ligation or sham operation. Intracellular pH (pHi) was fluorimetrically determined, and plasma membranes and messenger RNA (mRNA) were prepared from isolated hepatocytes by standard methods. Results: Resting pHi was higher in bile duct-ligated than in control rats (7.42 ± 0.03 vs. 7.06 ± 0.04; P < 0.001). Although plasma membrane lipid composition and intracellular buffering capacity were similar, initial Na+/H+ exchange-mediated rates of pHi recovery following acid loading were higher in bile duct-ligated than in control rats (0.098 ± 0.011 vs. 0.055 ± 0.005 pH units/min; P < 0.05). The antiporter's set point was shifted ∼0.3 pH units towards more alkaline values and its steady-state mRNA levels were doubled after bile duct ligation. Conclusions: Hepatocellular Na+/H+ exchange is transcriptionally and posttranscriptionally activated in rat biliary cirrhosis further supporting a relationship between hepatocyte proliferation and Na+/H+ exchange activation.

Vulnerability to excitotoxic stimuli of cultured rat hippocampal neurons containing the calcium-binding proteins calretinin and calbindin D 28K

Rat embryonic hippocampal neurons cultured on astrocyte feeder-layers were sensitive to different excitotoxic stimuli after 10–12 DIV. Almost all neurons (∼ 95%) died within 20 h after a transient exposure for 10 min to 50 μM glutamate, a continuous exposure to either 25 μM MMDA or 250 μM kainate or after a 15-min deprivation of glucose and oxygen. Dizocilpine at 10 μM protected neurons against the glutamate- and NMDA-mediated toxicity as well as against 30 min glucose and oxygen deprivation. However, it failed to protect against kainate toxicity and prolonged glucose/oxygen deprivation (60 min). An additional treatment with CNQX (100 μM) protected neurons even under the latter two conditions. This indicates that the vast majority of neurons was sensitive to different excitotoxic stimuli acting through different types of glutamate receptors leading to calcium overload of the cells which might be the common denominator of triggering cell death under these conditions. Expression of calcium-binding proteins, such as calbindin D 28K or calretinin, might increase the intracellular calcium buffer capacity of neurons, thus, rendering them more resistant to calcium overload. Therefore, we analyzed whether neurons expressing these calcium-binding proteins would survive these toxic stimuli. Indeed, a small population of the neurons (3–5%) survived, including a subpopulation of calretinin-positive but not calbindin D 28K-positive neurons. This implies that the expression of calcium-binding proteins per se does not render neurons more resistant towards these excitotoxic stimuli. Moreover, most of the surviving calretinin-positive neurons showed morphological damage as indicated by loss of neurites. When cytotoxicity due to calcium overload was induced by an exposure of the cells to the calcium ionophore 4-bromo-A23187 rather than by activation of glutamate receptors, calretinin-positive cells were found not to be significantly more resistant than the vast majority of neurons. This may indicate that the lower sensitivity of a subpopulation of calretinin-positive neurons to excitotoxic stimuli may be due to a lower expression of gluatmate receptors.

Hepatocellular Na+/H+ exchange is activated early, transiently and at a posttranscriptional level during rat liver regeneration

In hepatocytes in vitro, Na+/H+ exchange, an important regulator of intracellular pH, is activated by epidermal growth factor, but its activity during liver regeneration in vivo is unknown. We therefore compared activity and regulation of Na+/H+ exchange in hepatocytes isolated after two-thirds partial hepatectomy or sham surgery, respectively, by measuring intracellular pH (fluorimetry) and steady state Na+/H+ exchange mRNA levels (Northern blotting). Resting intracellular pH increased from 7.06 +/- 0.02 to 7.12 +/- 0.02 (p < 0.05) 2 hr but not 20 hr after partial hepatectomy. Na+/H+ exchange-mediated rates of intracellular pH recovery from an acid load increased from 0.075 +/- 0.018 to 0.151 +/- 0.018 pH units/min (p < 0.05) 2 hr but not 20 hr after partial hepatectomy. Because intracellular buffering capacity was not affected, this reflects increased Na+/H+ exchange activity. The inverse relationship between Na+/H+ exchange activity and intracellular pH was shifted by about 0.1 pH units toward more alkaline pH values 2 hr but not 20 hr after partial hepatectomy, whereas steady-state Na+/H+ exchange mRNA levels remained unchanged. In conclusion, hepatocellular Na+/H+ exchange is activated early, transiently and at a posttranscriptional level during liver regeneration induced in the rat by partial hepatectomy.

Polarized distribution of Na(+)-H+ antiport activity in rat alveolar epithelial cells

In this study, we investigated the polarized distribution of Na(+)-H+ antiport activity in alveolar epithelial cell monolayers. Rat alveolar type II pneumocytes were grown on detachable tissue culture-treated Nuclepore filters. The membrane filters, with their adherent intact alveolar epithelial cell monolayers, were mounted in a cuvette designed to contain two fluid compartments separated by the monolayer. Cells were loaded with the pH-sensitive dye 2',7'-biscarboxyethyl-5,6-carboxylfluorescein and intracellular pH (pHi) measured spectrofluorometrically. Monolayers were studied at ambient temperature on days 3-4 in culture, coincident with the development of high tissue resistance (RT > or = 2000 omega.cm2). Cells were incubated in HCO(3-)-free Na+ buffer [(in mM) 140 NaCl, 6 HEPES, pH 7.4] and acidified by NH3 prepulse. Rates of realkalinization (JH+) were calculated as the product of the initial rate of recovery (dpHi/dt) and the intracellular buffer capacity (beta i). Under control conditions, recovery occurred with an initial JH+ of 28.4 mM/min. When 100 microM dimethylamiloride (DMA), an amiloride analogue with enhanced specificity for inhibiting the Na(+)-H+ antiporter, was present in the basolateral fluid, recovery was inhibited by > 90%. Conversely, when the monolayers were acidified in Na+ buffer containing DMA (100 microM) in the apical fluid, acidification and recovery were identical to control. Recovery from acidification was inhibited by basolateral DMA with a one-half maximal inhibitory concentration (IC50) of 100 nm and by basolateral amiloride with an IC50 of 10 microns. Recovery was completely inhibited by omission of Na+ from the basolateral fluid, but omission of Na+ from apical fluid had no effect. We conclude that Na(+)-H+ antiport activity is located exclusively on the basolateral surface of these alveolar epithelial cell monolayers, where it most likely represents the high-amiloride affinity isoform of the Na(+)-H+ antiporter, NHE-1. The Na(+)-H+ antiporter, asymmetrically distributed to the basolateral surface of the polarized alveolar epithelium, contributes to intracellular homeostasis in alveolar pneumocytes and may also play a role in signal transduction in these cells.

Regulation of intracellular pH in cardiac muscle during cell shrinkage and swelling in anisosmolar solutions.

The effect on intracellular pH (pHi) of exposure to solutions of progressively increasing osmolarity from 418 to 620 mosM and to hyposmolar solutions (240 mosM) was examined in guinea pig ventricular muscle using ion-selective microelectrodes. Exposure of tissue to 418 mosM Tyrode solution (100 mM sucrose added) produced an intracellular alkalosis of approximately 0.1 U, whereas exposure to 620 mosM solution (300 mM sucrose added) caused an intracellular acidosis of approximately 0.1 U. The maximal rate of recovery of pHi from acidosis induced by an NH4Cl prepulse increased progressively as extracellular osmolarity was raised from 310 to 620 mosM. This suggests that the acidosis observed at steady state in 620 mosM solution is not due to inhibition of the Na(+)-H+ exchanger. In the presence of 10 microM ryanodine, exposure to 620 mosM solution produced a sustained intracellular alkalosis. We suggest that the decrease in pHi during exposure to 620 mosM solution is due, at least in part, to the acidifying influence of Ca2+ release from the sarcoplasmic reticulum. This decrease in pHi is expected to contribute to the negative inotrop reported in studies of cardiac contractility in markedly hyperosmolar solutions. There was no change in pHi when tissue was exposed to hyposmolar solution. However, the maximal rate of recovery of pHi from acidosis was slower in hyposmolar than in isosmolar solution, despite a concomitant decrease in the intracellular buffer capacity. This suggests that osmotic cell swelling results in inhibition of the sarcolemmal Na(+)-H+ exchanger.

Intracellular pH recovery during respiratory acidosis in perfused hearts.

Na(+)-H+ exchange and Na(+)-dependent HCO3- influx both contribute to recovery of intracellular pH (pHi) after an acidosis induced by using the NH4Cl prepulse technique in mammalian and avian cardiac tissue. We have investigated the relative contributions of these mechanisms to pHi recovery during respiratory acidosis in the Langendorff-perfused ferret heart with and without correction of extracellular pH (pHo). pHi was measured from the chemical shift of the exogenous 31P nuclear magnetic resonance pH indicator 2-deoxy-D-glucose 6-phosphate. Intrinsic intracellular buffering capacity, calculated from the change in intracellular HCO3- concentration after a change in CO2, was reduced from approximately 33 (no inhibitors of acid extrusion present) to 19 +/- 5 mM when H+ extrusion during the acid loading phase was inhibited. During respiratory acidosis (pHo approximately 6.95), the proton efflux rate (JH) calculated at pHi 6.85 was 0.30 +/- 0.04 mmol.l-1.min-1 (n = 9). When pHo was corrected by increasing external HCO3- concentration to 60 mM during respiratory acidosis (pHo approximately 7.33), JH was 1.11 +/- 0.11 mmol.l-1.min-1 (n = 7), and when pHo was partially corrected by the addition of 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to the perfusion solution (pHo approximately 7.1), JH was 0.64 +/- 0.08 mmol.l-1.min-1 (n = 6). In all three groups Na(+)-H+ exchange and HCO3- influx each contributed approximately 50% to acid-equivalent efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Na(+)-H+ antiporter activity in relation to membrane fatty acid composition and cell proliferation.

Na(+)-H+ exchange activity was studied in a human breast cancer cell line. At steady state, the intracellular pH (pHi) of the cells was 7.23 +/- 0.01, and intracellular buffering capacity (beta i) was 44 +/- 4 mM/pH unit. pHi was controlled by a Na(+)-H+ antiporter that was reversible, electroneutral, inhibited by 5-(N-methyl-N-isobutyl)amiloride, and dependent on extracellular Na+. The exchanger function depended on internal H+ concentration, according to an allosteric activation mechanism obeying the model of Hill. The exchanger was inactive at pHi > or = 7.22, and its maximal activity was reached at pHi < 6.60. The exchanger was stimulated by osmotic shrinking but was unaffected by growth factors (epidermal growth factor, insulin-like growth factor I) or by serum. When cells were grown in a medium supplemented with linoleic or alpha-linolenic acids, large quantities of the additional fatty acid accumulated in membranes, saturated fatty acids increased, and monounsaturated fatty acids decreased. These changes reduced cell proliferation but had no effect on the steady-state value of pHi, on beta i, or on the kinetic parameters of the Na(+)-H+ exchanger. Therefore, in this system, cell proliferation is not directly related to the activation of the Na(+)-H+ exchanger.

Sublytic C5b-9 on oligodendrocytes reduces myelin protein mRNAs, induces oncogenes and cdc2 kinase activity

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

Calcium entry through nicotinic receptor channels and calcium channels in cultured rat superior cervical ganglion cells.

1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.

Regulation of intracellular pH in the perfused heart by external HCO3- and Na(+)-H+ exchange

Both Na(+)-dependent HCO3- influx and the Na(+)-H+ antiport have been shown to contribute to recovery from intracellular acidosis in avian and mammalian cardiac tissue. We have investigated the participation of these mechanisms in the recovery of intracellular pH (pHi) after an acid load in the Langendorff-perfused ferret heart. pHi was measured from the phosphorus-31 nuclear magnetic resonance chemical shift of 2-deoxy-D-glucose 6-phosphate. Basal pHi was higher in HCO(3-)-buffered solution (7.05 +/- 0.01; n = 8) than in nominally HCO(3-)-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution (6.98 +/- 0.02; n = 9). Addition of 5-(N-ethyl-N-isopropyl)amiloride (EIPA) caused a significant fall in pHi in HEPES solution (6.91 +/- 0.02; n = 5) but not in HCO3- solution (7.02 +/- 0.02; n = 5). Intrinsic intracellular buffering capacity in 0 Na(+)-HEPES solution was 37 +/- 2 mmol/l (n = 4), and additional buffering due to HCO(3-)-CO2 was approximately 13 mmol/l in HCO3- solution. After an intracellular acidosis induced by an NH4Cl prepulse, the proton efflux rate (JH) at pHi 6.90 was 0.5 +/- 0.2 nmol.l-1.min-1 (n = 14) in HEPES solution and 1.2 +/- 0.4 mmol.l-1.min-1 (n = 13) in HCO3- solution. The addition of 1 microM EIPA effectively blocked proton efflux in HEPES solution (JH < 0.1 mmol.l-1.min-1; n = 8), whereas it slowed pHi recovery in HCO3- solution (JH = 0.6 +/- 0.2 mmol.l-1.min-1; n = 9). There was no recovery of pHi in Na(+)-free HCO3- solution (JH < 0.1 mmol.l-1.min-1; n = 3). The Na(+)-H+ antiport and a mechanism requiring both external Na+ and HCO3- each contribute approximately 50% to proton efflux at pHi 6.90 during the recovery from intracellular acidosis in the isolated perfused mammalian heart.

Studies of Intracellular pH Regulation in Cardiac Myocytes From the Marine Bivalve Mollusk, Mercenaria campechiensis.

Myocytes were isolated from the ventricle of the marine clam Mercenaria campechiensis by enzymatic dispersion procedures. Intracellular pH (pHi) was measured via fluorescence imaging techniques using an inverted microscope interfaced with a high sensitivity television camera. Myocyte pHi was similar to values observed in other molluscan muscles measured by weak acid distribution and nuclear magnetic resonance (NMR) techniques. Myocytes displayed a good capacity for defending pHi against changes in extracellular pH (pHe) as the pHi remained unchanged in the pHe range of 7.1 to 8.0, but gradually declined at lower pHe values. Myocytes had a relatively high non-bicarbonate intracellular buffering capacity. Further, these cells showed recovery from imposed acid loads. This recovery was accelerated by increasing HCO3- concentrations, was not dependent on external Na+ and was blocked by a stilbene transport inhibitor, suggesting that a HCO3-:CI- transporter plays a central role in regulation of pHi. Collectively, these data show that ventricular myocytes of M. campechiensis have a relatively high capacity for dealing with potential metabolic proton loads associated with environmental anaerobiosis.

Ammonium chloride-induced acidification in renal TALH SVE.1 cells monitored by 31P-NMR

The aim of this study was to investigate the effect of NH 4 + on the intracellular pH in TALH SVE.1 cells derived from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. These cells are specialized to perform NH 4 + transport in vivo. Intracellular pH was monitored by 31P-NMR. The steady state intracellular pH (pH i) under standard conditions was 7.24 ± 0.04 ( n = 46). Exposure to NH 4Cl resulted in an initial intracellular acidification of the TALH SVE.1 cells, followed by a recovery to the initial steady-state pH i value. The NH 4 +-induced acidification followed saturation kinetics up to 20 mM NH 4Cl (ΔpH max= 0.2 pH units). Half-maximal acidification was observed at 0.6 mmol/1. The intracellular acidification due to NH 2Cl exposure was completely inhibited by 0.1 mM of the diuretic bumetanide, an inhibitor of the Na +/K +/2Cl − cotransporter. The effect of bumetanide was dose-dependent and a K i value of 8 · 10 −7 M was calculated. NH 4 + influx via K + channels or the (Na + + K +)ATPase could not be detected. pH i recovery to the initial value was caused mainly by amiloride-sensitive Na +/H + exchange and to a lesser extent by an amiloride-insensitive system, which was not studied in detail. In the presence of bumetanide, pulses of high concentrations of NH 4Cl induced small intracellular alkalinizations. From these experiments, an intrinsic buffer capacity (β i) in TALH SVE.1 cells of 26 ± 3 mM × pH −1 (pH i = 7.65) was determined. It could also be shown that the TALH SVE.1 cells exhibit maximal ‘functional buffer capability’ between pH out 6.9 and 7.3. Within these limits the cells can maintain their intracellular pH at a constant level, even though the extracellular pH changes. These data strongly suggest that the Na +/K +/2Cl − cotransporter is the main site of NH 4 + entry into rabbit thick ascending limb cells in culture. A high intracellular buffer capacity and potent acid extrusion mechanism cooperate in counteracting the intracellular acidification caused by NH 4 + influx into the cell.

Alterations in Na(+)-H+ exchange in mesenteric arteries from spontaneously hypertensive rats.

Na(+)-H+ exchange was examined in segments of the superior mesenteric artery from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal pH values were identical in the two strains (SHR = 7.18 +/- 0.02, WKY = 7.15 +/- 0.01) and exposure to 20-50 mM NH4Cl produced comparable changes in pH in both strains (tissues acidified to pH 6.8). The recovery of intracellular pH (pH(i)) from acid loading in bicarbonate-free buffer was dependent on extracellular Na+, inhibited by dimethyl amiloride, and thus was presumably mediated by Na(+)-H+ exchange. Exchange activity was markedly increased in SHR (0.308 vs. 0.182 pH U/min), and the increased activity could not be explained by differences in intracellular buffer capacity or in the Na+ affinity of the transporter. The rate of Na(+)-H+ exchange was more steeply dependent on pH(i) in SHR vessels Hill coefficient = 6.6 in SHR vs. 4.1 in WKY), suggesting an alteration in the transport protein. Young SHR (4-6 wk) exhibited kinetic characteristics resembling those of 12- to 14-wk-old WKY rats. Exposure to the Ca2+ ionophore bromo-A23187 significantly increased the Hill coefficient of WKY arteries but had no effect on vessels from SHR. Exposure of vessels from either strain to the phorbol ester phorbol 12-myristate 13 acetate (PMA) had no effect on Na(+)-H+ exchange.

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

Na +/H + exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pH i) was 7.13±0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity β i was determined using the NH 4Cl-pulse method, yielding a β i value of 85 ± 12 mM/pH unit. The relationship between extracellular Na + and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent K m value of 44±26 mM and an V max value of 0.3±0.01 pH unit/min. The agonist-induced activation of Na +/H + exchange was investigated in cells acidified with nigericin. Addition of 12- O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 μM amiloride, by substituting extracellular Na + with equimolar concentrations of choline +, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca 2+, or depleating intracellular Ca 2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na +/H + exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.

Intracellular Buffering Capacity of Platelets is Decreased in Microalbuminuric, but not in Normoalbuminuric, Non-Insulin-Dependent Diabetic Subjects

Intracellular Buffering Capacity of Platelets is Decreased in Microalbuminuric, but not in Normoalbuminuric, Non-Insulin-Dependent Diabetic Subjects