Influence of extracellular pH and perfusion rate on Na+/H+ exchange in cultured opossum kidney cells

Studies were undertaken in cultured opossum kidney (OK) cells to determine whether the rate of H⁺ secretion by apical membrane Na⁺/H⁺ exchange is modulated by changes in extracellular pH or perfusion rate. H⁺ secretion was assessed in single cells by measuring the rate of Na⁺-dependent intracellular pH recovery after NH₄Cl loading, using the pH-sensitive fluorescent dye, 27-bis(carboxyethyl)-5, 6-carboxyfluorescein, in monolayers mounted to allow independent perfusion of the apical and basolateral surfaces. At constant intracellular pH, Na⁺-dependent H⁺ secretion was found to be inversely related to extracellular H⁺ activity, and directly related to the perfusate flow rate. Inhibition of H⁺ secretion by perfusate acidity occurred immediately and was greater when perfusate Na⁺ was reduced, consistent with H⁺ competition with Na⁺ for binding to the transporter. By contrast, the effect of the perfusion rate was a delayed response, requiring 20 min of exposure, and was independent of perfusate Na⁺ concentration. The results indicate that both extracellular pH and the perfusion rate modulate H⁺ secretion by OK cells, and that the two effects are independent.     

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone,cyclic AMP and phorbol esters

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na⁺/phosphate co-transport and Na⁺/H⁺ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na⁺/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na⁺/H⁺ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca²⁺ concentration do not induce inhibition of Na⁺/H⁺ exchange; PTH-dependent inhibition of Na⁺/H⁺ exchange is not prevented by ionomycin or by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (Ca²⁺ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40–50% inhibition of Na⁺/H⁺ exchange. An analysis of intracellular pH sensitivity of Na⁺/H⁺ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced V _max of Na⁺/H⁺ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na⁺/H⁺ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulphonic acid - EDTA ethylenediaminetetraacetic acid - FCS fetal calf serum - PTH parathyroid hormone - Tris 2-amino-2-hydroxyme-thylpropane-1,3-diol - BCECF 2,7-bis(2-carboxyethyl)-5,6-carboxyfluorescein - P_i inorganic phosphate - pH_i intracellular pH - Ca_i cytosolic free Ca²⁺ - BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - EGTA ethylene glycol-bis(-amino-ethylether)-N,N,N,N-tetraacetic acid - IP₃ inositol 1,4,5-trisphos-phate - DAG 1,2-diacylglycerol     

Functional adaptation to high PCO2 of apically and basolaterally located Na+/H+ exchange activities in cultured renal cell lines

Cultured renal epithelial cells grown on filter support were examined for functional adaptation of Na⁺/H⁺ exchange activities to respiratory acidaemia, which was mimicked by increasing PCO₂ from 5% to 10% during 24 h or 48 h of cell culture. We have selected proximal tubular cell lines with either dual location of Na⁺/H⁺ exchange activities (MCT cells, RKPC-2 cells), apical location of Na⁺/H⁺ exchange activity (OK/ WOK cells) or a basolateral location of Na⁺/H⁺ exchange activities (LLC-PK₁/clone 4 cells, MDCK cells). Na⁺/H⁺ exchange activity was determined microspectrofluorometrically (using BCECF) in the absence of CO₂/HCO ₃ ^– . Respiratory acidaemia specifically increased apical Na⁺/H⁺ exchange activity (previously classified as amiloride-resistant) in MCT cells, in RKPC-2 cells and in WOK cells; it stimulated basolateral Na⁺/H⁺ exchange activity (previously shown to be amiloride-sensitive) in RKPC-2 cells, in LLC-PK₁/clone 4 cells and in MDCK cells, but did not affect basolateral Na⁺/H⁺ exchange activity in MCT cells. In MCT and in RKPC-2 cells the effect of high PCO₂ on apical Na⁺/H⁺ exchange was prevented by inhibition of protein kinase C. In RKPC-2 cells, activation of basolateral Na⁺/H⁺ exchange by high PCO₂ occurred also when protein kinase C was inhibited. In conclusion, these studies demonstrate stimulation of apical Na⁺/H⁺ exchange, but differential regulation of basolateral Na⁺/H⁺ exchange activities in response to a high-PCO₂-induced acid environment. Protein kinase C activation might be involved in mediating the effect of acidaemia on stimulation of apical Na⁺/H⁺ exchange activity (MCT and RKPC-2 cells).     

Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells

In recent studies, there has been a re-evaluation of the polarity of Na⁺/H⁺ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na⁺/H⁺ exchange of MDCK cell monolayers grown on permeant filter supports; pH_i was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10–20 min to an alkalization of pH_i ( 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na⁺/H⁺ exchange activity (Na⁺-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Preexposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na⁺/H⁺ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na⁺/H⁺ exchange activity. An inhibitory effect of aldosterone was observed if Na⁺/H⁺ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%–30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na⁺ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na⁺/H⁺ exchange activity under conditions of low Na⁺, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na⁺ concentrations.     

pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

The colon carcinoma cell line Caco-2 contains an H+/K+-ATPase that contributes to intracellular pH regulation

The presence of an H⁺/K⁺-ATPase and its contribution to the regulation of intracellular pH (pH_i) was investigated in Caco-2 cells. The H⁺/K⁺-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H⁺/K⁺-ATPase. Cell pH was determined using the pH-sensitive dye 2,7-bis(carboxyethyl)-carboxyfruorescein. Control pH_i, measured in HCO ₃ ^– -free medium, was 7.62±0.03 (n=27) when cells were cultured for 14 days and decreased to 7.40±0.03 (n=18) after 35 days in culture. Recovery of pH_i following a NH ₄ ⁺ /NH₃ pulse could be reduced by either 100 M SCH 28080 or 1 mM amiloride, or by removing extracellular Na⁺. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H⁺/K⁺-ATPase and a Na⁺/H⁺ exchanger in regulating pH_i. Recovery rates at pH_i 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na⁺/H⁺ exchange to the recovery. Recovery of pH_i was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H⁺-ATPase, since bafilomycin A₁ had no effect on pH_i recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pH_i, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na⁺/H⁺ exchanger to pH_i regulation is decreased.     

Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO 3 − /Cl− exchange

We have estimated the changes in cytosolic pH (pH_i) that occur when human platelets are stimulated by thrombin. Changes in pH_i were estimated (i) from the H⁺ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H⁺ efflux that averaged 7.7±1.6 mol/10¹¹ platelets (means±SD) leading to an increase in pH_i, from 7.05±0.04 to 7.45±0.05. Both H⁺ efflux and pH_i changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4-acetamido 4-isothiostilbene-2,2-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO ₃ ^– /Cl^– exchange. Removal of HCO ₃ ^– or Cl^– from the suspending buffer had no effect on the extent of the rise in pH_i. After blockade of Na⁺/H⁺ exchange by 100 M ethylisopropylamiloride (EIPA), thrombin induced a decrease in pH_i the rate of which averaged 0.39 unit/min in HCO ₃ ^– -containing medium, and 0.57 unit/min in HCO ₃ ^– -free medium. The cytosolic buffer capacity for H⁺ was determined by the nigericin/ NH₄Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO ₃ ^– , but only 17.2 mmol/(1xpH) in HCO ₃ ^– -free buffer. The total amount of H⁺ transferred by Na⁺/H⁺ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO ₃ ^– and to 14 mmol/l platelet cytosol in the presence of HCO ₃ ^– , and is in good agreement with the estimated amount of Na⁺ uptake by ADP-stimulated platelets. We conclude that net extrusion of H⁺ from stimulated platelets is predominantly mediated by Na⁺/H⁺ exchange without an apparent contribution of HCO ₃ ^– /Cl^– exchange.     

Na+-HCO3 − cotransporter and intracellular pH regulation in chicken enterocytes

The current studies examine the presence of the Na⁺-HCO₃ ^– cotransporter in chicken enterocytes and its role in cytosolic pH (pH_i) regulation. The pH-sensitive dye 2,7-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pH_i. Under resting conditions, pH_i was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO₃ ^–. Removal of external Na⁺ decreased pH_i and readdition of Na⁺ rapidly increased pH_i towards the control values. These Na⁺-dependent changes were greater in HCO ₃ ^– than in HEPES-buffered solutions. In HCO ₃ ^– - free solutions the Na⁺-dependent changes in pH_i were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4-diisothiocyanatostilbene disulphonic acid (H₂-DIDS). In the presence of HCO ₃ ^– , the Na⁺-induced changes in pH_i were sensitive to both EIPA and H₂-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na⁺ and HCO ₃ ^– were present. This pH_i recovery, which was EIPA resistant, and dependent on Na⁺ and HCO ₃ ^– , was inhibited by H₂-DIDS and occurred at equal rates in both Cl^–-containing and Cl^–-free solutions. Kinetic analysis of the rate of HCO ₃ ^– - and Na⁺- dependent pH_i recovery from an acid load as a function of the Na⁺ concentration revealed first-order kinetics with a Michaelis constant, K _m, of 11 mmol/l Na⁺. It is concluded that in HCO₃ ^/– buffered solutions both the Na⁺/H⁺ exchanger and the Na⁺-HCO₃ ^– cotransporter participate in setting the resting pH_i in isolated chicken enterocytes and help the recovery from acid loads.     

Altered cell volume regulation in ras oncogene expressing NIH fibroblasts

Expression of the Ha-ras oncogene has been reported to stimulate the dimethylamiloride sensitive Na⁺/H⁺ exchanger and Na⁺, K⁺, 2Cl^– cotransport, both transport systems which are involved in cell volume regulation. The present study has been performed to test for an influence of ras oncogene expression on cell volume regulation in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras). As controls served NIH 3T3 fibroblasts not expressing the ras oncogene (– ras). In isotonic extracellular fluid, the cell volume of + ras cells (2.70±0.08 pl) is significantly greater than the cell volume of –ras cells (2.04±0.10 pl). Both, + ras and – ras cells exhibit a regulatory cell volume increase in hypertonic extracellular fluid and a regulatory cell volume decrease in hypotonic extracellular fluid. The regulatory cell volume decrease is inhibited by 1 mmol/l quinidine and barium, the regulatory cell volume increase is inhibited in – ras and + ras cells by dimethyl-amiloride (100 mol/l) and, only in + ras cells, by furosemide (100 mol/l) and bumetanide (10 mol/l). In conclusion, expression of the ras oncogene leads to a shift of the set point for cell volume regulation to greater cell volumes, which may contribute to the activation of the Na⁺/H⁺ exchanger and Na⁺, K⁺, 2Cl^– cotransport.     

Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy

The properties of pH and calcium homeostasis have been investigated in single bovine lactotrophs by the use of the fluorescent indicators 2,7-bis(carboxyethyl)-carboxyfluorescein (BCECF) and fura-2 respectively. A method of simultaneous recording from both dyes loaded in the same cell was used. Despite slight crosstalk between the two dyes, physiologically relevant information about the interrelationship between pH and calcium homeostasis was obtained. Three types of interactions were recorded. First, an increase in calcium due to the discharge of intracellular stores by thyrotropin-releasing hormone resulted in no change in cytosolic pH. Secondly, alkalinization by the addition of a weak base, NH₄Cl, induced a large transient (around 1000 nM) and a small (a few tens of nanomoles per liter) sustained increase in cytosolic calcium. The former is partly due to release from agonist-sensitive stores. Thirdly, upon the removal of NH₄Cl the cytoplasm became acidic, which induced a release of calcium from intracellular stores in some cells. In addition we demonstrate that the recovery from acid load is sensitive to extracellular Na⁺, suggesting the presence of Na⁺/H⁺ exchange mechanisms in bovine lactotrophs. Interestingly we have also found that, at rest, removal of Na⁺ from the bathing medium results in a decrease in resting [Ca²⁺]_i, paralleled by a reduction in pH_i. This suggests a role for Na⁺/H⁺ exchange in determining resting [Ca²⁺]_i.     

Coupling between proximal tubular transport processes

Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (-methyl-d-glycoside), H⁺ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (c) of these substances. In the case of calcium the electrochemical potential differencec +zFc_i /RT) was the criterion employed. The rate of isotonic Na⁺-absorption (J_Na) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4isothiocyanatostilbene 2,2-disulfonic acid) was observed in rats.Ouabain (1 mM) applied peritubularly incompletely inhibited J_Na (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, -methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H⁺ (glycodiazine) transport and PAH transport was only slightly affected.When SITS (1 mM) was applied from both sides of the cell it inhibited H⁺ (glycodiazine) transport by 72% and reduced J_Na by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H⁺ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied.When the HCO ₃ ^– buffer or physically related buffers were omitted from the perfusate the absorption of Na⁺ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected.The data are consistent with the hypothesis that the active transport processes of histidine, -methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na⁺-Ca²⁺ countertransport located at the contraluminal cell side. The residual Na⁺ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H⁺ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO ₃ ^– from the cell and secondary to that, the luminal H⁺-Na⁺ exchange and consequently the Na⁺ reabsorption. In the absence of HCO ₃ ^– buffer in the perfusates the luminal H⁺-Na⁺ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onP _i reabsorption observed under these conditions might be explained by possible variations in intracellular pH.     

HCO 3 − -dependent ACh-activated Na+ influx in sheep parotid secretory endpieces

In the present study we used the Na⁺-sensitive fluorescent dye SBFI and optical measurement of endpiece volume to investigate the transport of Na⁺ in sheep parotid secretory cells. Sheep parotid endpiece cells bathed in a HCO ₃ ^– -free Cl^–-rich solution had a resting intracellular Na⁺ concentration ([Na⁺]_i) of 17±2 mmol/l (n=39). Exposure of the cells to a 2-min pulse of acetylcholine (ACh) (3×10^–7 mol/l) in a HCO ₃ ^– -free bathing solution produced no change in [Na⁺]_i or in cell volume. Changing from a Cl^–-containing HCO ₃ ^– -free bath solution to a Cl^– solution containing 25 mmol/l HCO ₃ ^– caused the endpieces to swell by 8±2 % (n=11) and the [Na⁺]_i to increase by 10±2 mmol/l (n=14). Subsequent exposure of the cells to ACh led to shrinkage of the cells by 12±2 % from the volume in the HCO ₃ ^– -containing solution prior to ACh exposure, with the maximum decrease occurring after 29±7 s (n=9). This shrinkage was accompanied by a rapid and transient increase in [Na⁺]_i, the [Na⁺]_i reaching a peak at 70±5 mmol/l above the unstimulated level (n=9). Substitution of gluconate for Cl^– did not significantly alter the effects of HCO ₃ ^– on unstimulated [Na⁺]_i or endpiece volume, nor did it significantly inhibit the effects of ACh on these two parameters when HCO ₃ ^– was present. Addition of 200 mol/l dihydrogen-4,4-diisothiocyanatostilbene-2,2-disulfonic acid (H₂-DIDS) to the gluconate/HCO ₃ ^– solution significantly reduced the peak of the ACh-induced increase in [Na⁺]_i to 34±10 mmol/l (n=4), but did not have any significant effect on the magnitude of the ACh-induced shrinkage. At 500 mol/l, H₂-DIDS abolished the ACh-induced increase in [Na⁺]_i and also significantly reduced the shrinkage due to ACh. Finally, we found that the rate of endpiece shrinkage following ACh stimulation did not depend on the presence of Cl^–.We interpret these results as indicating that sheep parotid secretory cells do not contain significant Na⁺-K⁺-2Cl^– co-transport activity and do not actively accumulate Cl^–. Rather, the mechanism of spontaneous basal secretion by these cells, in the presence of extracellular HCO ₃ ^– , is based on the accumulation of HCO ₃ ^– by the Na⁺-H⁺ exchanger. During ACh stimulation, the concentration of HCO ₃ ^– in the cytosol is also maintained by the operation of a H₂-DIDS-sensitive Na⁺-HCO ₃ ^– co-transporter. HCO ₃ ^– efflux across the apical membrane occurs via a HCO ₃ ^– conductance pathway rather than by the coupled operation of a Cl^– channel and a Cl^–-HCO ₃ ^– exchanger.     

Inhibitory action of norepinephrine on sodium transport in vascular smooth muscle cells in culture

Cultured vascular smooth muscle cells from porcine aortas incubated in Na⁺-free medium rapidly release their intracellular Na⁺ contents (Na_i) (23±4% of baseline after 60 min incubation, mean ± SEM of 18 experiments). Total Na_i release was inhibited by 35–40% after addition of ouabain and by 60–70% after addition of ouabain + bumetanide. Norepinephrine inhibited ouabain and bumetanide-sensitives Na⁺ efflux with an IC₅₀ of about 10^–9–10^–8 M. Addition of the alpha-adrenergic agonist phenylephrine (10 M) to the cells mimicked the inhibitory action of norepinephrine on Na_i release. Conversely, the beta-adrenergic agonist isoproterenol was without effect on Na_i release. Simultaneous addition of 10 M norepinephrine and the alpha-adrenergic antagonist phentolamine prevented any effect of norepinephrine on the rate of Na_i decline. In A-10 cultured vascular smooth muscle cells, the alpha-adrenergic agonist phenylephrine (10 M) inhibited 40.0±8.1% of ouabain-sensitive Rb⁺ influx and 70.7±6.9% of bumetanide-sensitive Rb⁺ influx (mean ± SEM of three experiments). 50% inhibition of bumetanide-sensitive Rb⁺ influx was obtained with about 5×10^–7 M of phenylephrine. Our results show that in vascular smooth muscle cells a [Na⁺, K⁺, Cl^–]-cotransport system is able to catalyze outward Na⁺ movements (in Na⁺-free media) of a similar order of magnitude to those of the Na⁺, K⁺ pump and that alpha-adrenergic stimulation markedly inhibits Na⁺ efflux (and Rb⁺ influx) through these two transport systems.     

Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules

The purpose of this study was to investigate intracytoplasmic pH (pH_i) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pH_i was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pH_i values, determined in bicarbonatefree medium (extracellular pH: 7.40), were 7.25±0.02 (n=23) and 7.17±0.04 (n=30) for cultured PCT and DCTb respecitively. After the acid-loading procedure, cultured proximal cells recovered their pH_i by means of the classic Na⁺/H⁺ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pH_i recovery was mediated by a Na⁺/H⁺ exchange present in the basolateral side. Moreover, at physiological initial pH_i values, chloride removal from the apical solution caused the pH_i to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pH_i recovery was induced in sodium-free media by 15 mM HCO ₃ ^– in the presence of an outward chloride gradient. This pH_i change was completely abolished by 4,4-diisothiocyanostilbene 2,2-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na⁺-independent Cl^–/HCO ₃ ^– exchanger.     

Abnormal erythrocyte sodium leak in a subset of essential hypertensive patients

Summary We measured the ouabain- and bumetanide-resistant Na⁺ efflux in Mg²⁺-sucrose medium (passive Na⁺ leak) in erythrocytes from 30 normotensive controls and 72 essential hypertensive patients. The mean values (±SEM) of the rate constant of Na⁺ leak (k_pNa) were not significantly different between normotensives and hypertensives. Nevertheless, using the 95% confidence limits of the k_pNa (in 10^–3.h^–1) in the normotensive group as a cut-off point, 7 (9.7%) essential hypertensives exhibited increased values (58.96±10.12) when compared with the other 65 patients (23.86±0.74). revealing increased passive Na⁺ permeability in the former (leak + hypertensives). Na⁺ fluxes depending on the Na⁺-K⁺ pump, outward Na⁺-K⁺ cotransport, and Na⁺-Li⁺ countertransport were also measured in fresh erythrocytes from the same 72 patients. Three of them (4.2%) exhibited decreased values of ouabain-sensitive Na⁺ efflux and 6 (8.3%) of bumetanide-sensitive Na⁺ efflux, while 8 patients (11.1%) showed increased values of Li⁺-stimulated Na⁺ efflux and, finally, 48 patients (59.7%) did not present any evident abnormality in these Na⁺ transport systems. No differences were observed between leak + hypertensives and the remaining 65 patients when both basal erythrocyte Na⁺ content and clinical parameters of hypertension were compared. However, Na⁺ efflux depending on the outward Na⁺-K⁺ cotransport was significantly higher in the leak + hypertensive subset (299.43±43.18 vs 181.52±10.76 µmol.(l cells.h)^–1;P=0.0078), suggesting a compensatory phenomenon. Enhancement of Na⁺ permeability detected in 3% to 16% of essential hypertensives may be implicated in the pathogenesis of primary hypertension.Abbreviations ATPase adenosine triphosphatase - Dcat difference between the external Na⁺ concentration after incubation at 37° C and at zero time - k_pNa rate constant of passive Na⁺ leak - Leak + hypertensive essential hypertensive patient with abnormally high erythrocyte Na⁺ leak - MOPS 4-morpholinopropanesulfonic acid - OBR ouabain- and bumetanide-resistant - PRA plasma renin activity - sPRA plasma renin activity stimulated after furosemide infusion - SEM standard error of the mean Supported in part by Grant 87/1078 of the Fondo de Investigaciones Sanitarias de la Seguridad Social and Grant PA85/0168 of the Comisión Asesora de Investigación Científica y Técnica     

Short-term and long-term stimulation of Na+−H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney

The effect of unilateral nephrectomy on Na⁺–H⁺ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70–75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted toV _max, whereas theK _m for Na⁺ was similar in control and compensatory growing kidney. The increase of Na⁺–H⁺ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na⁺–H⁺ exchange. The late, but not the initial stimulations of Na⁺–H⁺ exchange were associated with elevated activities of cortical (Na⁺+K⁺)-ATPase indicating that changes in antiporter activity precede those in the (Na⁺+K⁺)-pump. The early stimulation of Na⁺–H⁺ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na⁺–H⁺ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na⁺–H⁺ exchanger may be regulated by phosphorylation-dephosphorylation events. In support, the concentration of a well known protein kinase C activator, 1,2-diacylglycerol, in the cortical tissue of the remnant kidney increased up to 100% within 5 min following unilateral nephrectomy and preceded the increase in Na⁺–H⁺ exchange. The early stimulation of Na⁺–H⁺ exchange may be a trigger in initiating the kidney growth.     

Electrical properties of cultured renal tubular cells (OK) grown in confluent monolayers

OK cells grown to confluent monolayers were investigated by microelectrode techniques and microinjection. Cell membrane potential difference (PD_m) in bi-carbonate-free solution is –61.8±0.6 mV (n=208), cell membrane resistance (R_m) amounts to 1.4±0.2k · cm² (n=8). The apparent transference number for potassium (t_K ⁺) is 71±3% (n=28) and can be reduced by 3 mmol/l BaCl₂ to 7.5±4.0%; (n=8). In the presence of extracellular CO₂ and HCO ₃ ^– (pH 7.4) the cells acidify by 0.34±0.05 pH units (n=12). This leads to a depolarization of PD_m by 8.4±1.8 mV (n=8), an increase in R_m by 49±10% (n= 10), and a reduction of K⁺-conductance to 63±5% (n= 13). Intracellular acidification by the NH₄Cl-prepulse technique also inhibits K⁺-conductance and depolarizes the membrane. Recovery from an intracellular acid load is reflected by cell membrane repolarization. This recovery can be inhibited by amiloride (10^–3 mol/l). Na⁺- and Cl^–-conductances could not be detected.The transepithelial resistance (R _te) of OK cell monolayers 1 day after plating is 41±6 ·cm² and decreases with time after plating. Intercellular communication (electrical or dye coupling) was not observed.Conclusions: 1. The membrane potential of OK cells is largely determined by a pH-sensitive, barium-blockable K⁺-conductance. 2. Amiloride-blockable Na⁺/H⁺-exchange is reflected by membrane potential changes via this K⁺-conductance. 3. Monolayers of OK cells are electrically leaky.Parts of this study were presented at the 66th meeting of the Deutsche Physiologische Gesellschaft, Würzburg, September 1988 [Pflügers Arch 412 (Suppl 1):R55].     

Oscillations: a key event in transformed renal epithelial cells

Summary Intracellular pH (pH_i) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pH_i may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form foci in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca²⁺ flux activates plasma membrane K⁺ channels and Na⁺/H⁺ exchange. This leads to periodic changes of membrane voltage and pH_i at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.Abbreviations CICR calcium-induced calcium release - IP₃a inositol triphosphate - MDCK Madin-Darby canine kidney - pH_ia intracellular pH Dedicated to our friend and teacher Prof. Dr. Gerhard Giebisch in gratitude for his long standing support     

Intracellular pH regulation in cultured rat astrocytes in CO2/HCO 3 − -containing media

We studied the regulation of intracellular pH (pH_i) and the mechanisms of pH_i regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2,7-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pH_i was 7.00±0.02 in HCO ₃ ^- containing solutions at an extracellular pH of 7.35. Addition of 4, 4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) or amiloride decreased pH_i, as did removal of extracellular Na⁺, while removal of extracellular Cl^- was followed by an increase in pH_i. Following exposure to an acid transient induced by increasing the CO₂ content from 5 to 15%, pH_i rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min^-1 (corresponding to a mean acid extrusion rate of 6.3±0.36 mmolo 1^-1 min^-1). Regulation of pH_i was impaired when either amiloride or DIDS was added or Cl^- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pH_i regulation was completely blocked in the absence of extracellular Na⁺. The rapid regulation of pH_i normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na⁺, but was partially inhibited by DIDS and by the absence of extracellular Cl^-. The results are compatible with the presence of at least three different pH_i-regulating mechanisms: a Na⁺/H⁺ antiporter, a Na⁺-dependent HCO ₃ ^- /Cl^- exchanger (both regulating pH_i during a transient acidification), and a passive Cl^-/HCO ₃ ^- exchanger (regulating pH_i during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na⁺/HCO ₃ ^- symporter.     

Effect of rabbit duodenal mRNA on phosphate transport in Xenopus laevis oocytes: dependence on 1,25-dihydroxy-vitamin-D3

Expression of Na⁺-dependent transport of phosphate (P_i) was analysed in Xenopus laevis oocytes after injection of poly(A)-rich RNA isolated from the duodenal mucosa of rabbits with increased levels of 1,25-(OH)₂-VitD₃ (injection of vitamin D₃ or low-P_i diet) or from control animals. In parallel, the effect of elevated levels of vitamin D₃ was studied in isolated duodenal brush-border membrane vesicles. In brush-border membrane vesicles, the rate of Na⁺-P_i cotransport was found to be doubled after 1,25-(OH)₂-VitD₃ injections while Na⁺ — d-glucose cotransport (measured as a control) was not altered. In X. laevis oocytes, Na⁺-dependent P_i uptake was increased after injection of poly(A)-rich RNA isolated from duodenal mucosa of animals with increased levels of 1,25-(OH)₂-VitD₃ but not after injection of poly(A)-rich RNA isolated from control animals; between the two groups of mRNA no difference in the expression of the Na⁺ — d-glucose transport system was observed. Sucrose density gradient fractionation suggests that mRNA species related to the increased Na⁺-dependent P_i uptake are of average chain lengths between 2×10³ and 3×10³ bases (2–3 kb). It is concluded that in duodenal enterocytes 1,25-(OH)₂-VitD₃ increases the content of mRNA species of 2–3 kb that might be involved either directly in Na-P_i cotransport or at least in controlling its activity.