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.     

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells

Uptake studies with²²Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na⁺ transport. A large part of Na⁺ uptake was sensitive to amiloride, quinidine and harmaline. Na⁺ uptake was stimulated by intracellular acidification (using the NH ₄ ⁺ prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentK _M for Na⁺ from 38 to 86 mM without considerable changes inV _max. In the presence of 5 mM Na⁺ half maximal inhibition of amiloride sensitive Na⁺ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na⁺ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H⁺ seemed to be competitive with aK _i of 20–40 nM. 10 M amiloride increased the apparentK _M for Na⁺ from 33 mM to 107 mM, whileV _max remained nearly unchanged. IC₅₀ for amiloride was 6 M at 5 mM Na⁺ and 36 M in the presence of 150 mM Na⁺. Thus, amiloride behaves as a competitive inhibitor with aK _i of about 5 M. The affinities of Na⁺ to the transport site (K _M16 mM), to the inhibitory site for protons (K _M21 mM), and to the inhibitory site for amiloride (K _M26 mM) were in the same order of magnitude.In summary, we have presented evidence for the presence of a Na⁺/H⁺ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na⁺, H⁺ and amiloride.     

Preparation of a bioartificial kidney using tubular epithelial cells, and an evaluation of Na+ active transport and morphological changes

We intend to develop a bioartificial kidney using tubular epithelial cells and artificial membranes, and to evaluate the reabsorptive function of the confluent layers. Madin-Darby canine kidney (MDCK) cells were cultured on a nucleopore polycarbonate membrane for up to 4 weeks after confluence to examine the influence of culture period on their properties, such as the localization of Na⁺/K⁺-ATPase and active Na⁺ transport. The results were as follows. Ouabain-sensitive Na⁺ active transport declined at 3 to 4 weeks after confluence in each matrix. The localization of Na⁺/K⁺-ATPase indicated depolarization in the cell membrane 3 to 4 weeks after confluence. Prolongation of the culture period increased the formation of an upheaving cell mass after the formation of the confluent monolayer. Scanning electron microscopy revealed fewer microvilli and more flat cells after 3 to 4 weeks of confluency. We conclude that the decline of Na⁺ active transport in the MDCK cells was due to both the formation of multilayers and a decline of cell function throughout the long period of culture following the formation of the confluent monolayers. Further study for selection of membrane material, the extracellular matrix, and species of cells should be continued. Laboratories for Structure and Function Research Department of Physiology     

Spontaneous membrane potential oscillations in Madin-Darby canine kidney cells transformed by alkaline stress

High pH is known to be associated with normal cell growth and neoplastic transformation. We observed that Madin-Darby canine kidney (MDCK) cells grown under sustained alkaline stress (pH 7.7) develop foci composed of spindle-shaped cells lacking contact inhibition and exhibiting only poor adhesion to the culture support. Foci-developing (F) cells were cloned and grown in control medium (pH 7.4), where they maintained their neoplastic features indicating a stable pH-induced genetic transformation. After F cells had been fused to giant cells with polyethylene glycol, the cell membrane potential (V _m) was measured by means of microelectrodes. In contrast to non-transformed MDCK cells, V _M of F cells showed spontaneous biorhythmicity caused by periodic opening of Ca²⁺-activated K⁺ channels. Spiking activity was blunted by the Ca²⁺ channel blocker nifedipine, by the K⁺ channel blocker Ba²⁺, by the Na⁺/H⁺ exchange blocker amiloride and its analogue ethylisopropylamiloride, and by an extracellular pH of 7.6 and 6.8. We conclude that MDCK cells transformed by sustained alkaline stress have lost their stable plasma membrane potential but, instead, exhibit endogenous Ca²⁺- and pH-sensitive oscillations.     

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     

Signaling pathways involved with the stimulatory effect of Angiotensin II on vacuolar H+-ATPase in proximal tubule cells

It has been documented that angiotensin II (ANG II) (10⁻⁹ M) stimulates proton extrusion via H⁺-adenosine triphosphatase (ATPase) in proximal tubule cells. In the present study, we investigated the signaling pathways involved in the effects of ANG II on H⁺-ATPase activity and on the cytosolic free calcium concentration in immortalized rat proximal tubule cells, a permanent cell line derived from rat proximal tubules. The effects of ANG on pH_i and [Ca⁺²]_i were assessed by the fluorescent probes, 2′,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxy-methyl ester and fluo-4-acetoxy-methyl ester, in the absence of Na⁺ to block the Na⁺/H⁺ exchanger. In the control situation, the pH recovery rate following intracellular acidification with NH₄Cl was 0.073±0.011 pH units/min (n=12). This recovery was significantly increased with ANG II (10⁻⁹ M), to 0.12±0.015 pH units/min, n=10. This last effect was also followed by a significant increase of Ca⁺² _i, from 99.72±1.704 nM (n=21) to 401.23±33.91 nM (n=39). The stimulatory effect of ANG II was blocked in the presence of losartan, an angiotensin II subtype 1 (AT₁) receptor antagonist. H89 [protein kinase A (PKA) inhibitor] plus ANG II had no effect on the pH recovery. Staurosporine [protein kinase C (PKC) inhibitor] impaired the effect of ANG II. Phorbol myristate acetate (PKC activator) mimicked in part the stimulatory effect of ANG II, but reduced Ca⁺² _i. 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (intracellular calcium chelator) alone reduced the pH_i recovery rate below control levels and impaired the effect of ANG II, in a way similar to that of trimethoxy benzoate (a blocker of Ca⁺² _i mobilization). We conclude that ANG II regulates rat proximal tubule vacuolar H⁺-ATPase by a PKA-independent mechanism and that PKC and intracellular calcium play a critical role in this regulation.     

Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues

We have investigated the possible existence of a Na⁺/H⁺ ion exchanger in the frog skin epithelium by using isotopic methods and two amiloride analogues: 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and phenamil. We found phenamil to be a specific blocker of sodium entry to its cellular transport compartment since it inhibited both the transepithelial Na⁺ influxes (J ₁₃) with aK _I of 4·10^–7 mol/l and the Na⁺ pool (control: 77±4 neq·h^–1·cm^–2; phenamil: 21±1 neq·h^–1·cm^–2). On the contrary EIPA (10^–5 mol/l) had no effect onJ ₁₃ nor on the apical Na⁺ conductance. Acidification of the epithelium by passing from a normal Ringer (25 mmol/l HCO ₃ ^– , 5% CO₂, pH 7.34) to a HCO ₃ ^– -free Ringer (5% CO₂, pH 6.20) while blocking the Na⁺ conductance with phenamil, produced a large stimulation of Na⁺ influxes exclusively across the basolateral membranes (J ₃₂), after return to a normal Ringer (J ₃₂=706±76 and 1635±199 neq·h^–1·cm^–2 in control and acid-loaded epithelia respectively). The stimulation ofJ ₃₂ was initiated when the epithelia were acid-loaded with Ringer of pH lower than 6.90 and was blocked by amiloride (K _I=7·10^–6 mol/l) and EIPA (K _I=5·10^–7 mol/l) whereas phenamil had no effect. In na⁺-loaded epithelia (ouabain treated) the Na⁺ efflux across the basolateral membranes was stimulated by an inwardly directed proton gradient and was blocked by EIPA (10^–5 mol/l) or amiloride (10^–4 mol/l), a result suggesting reversibility of the mechanism. We conclude that a Na⁺ permeability mediated by a Na⁺/H⁺ ion exchanger exists in the basolateral membranes, which is stimulated by intracellular acidification and is sensitive to amiloride or EIPA. This exchanger is proposed to be involved in intracellular pH regulation.     

Hypertonicity in fused Madin-Darby canine kidney cells: transient rise in NaHCO3 followed by sustained KCl accumulation

We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium⁺ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84±2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na⁺ and HCO ₃ ^– concentrations was observed. It was followed by a sustained increase in intracellular K⁺ and Cl^– concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K⁺ accumulation significantly, whereas the H⁺ /K⁺-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19±2 mV, owing to the decrease of the ratio of Cl^– conductance to K⁺ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na⁺/H⁺ exchange in MDCK cells; (b) transient alteration of intracellular Na⁺ and pH stimulates Na⁺/K⁺-ATPase and Cl^–/HCO ₃ ^– exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl^–/K⁺ conductance ratio of the plasma membrane.     

Diversity of the mammalian sodium/proton exchanger SLC9 gene family

Sodium/proton antiporters or exchangers (NHE) are integral membrane proteins present in most, if not all, living organisms. In mammals, these transporters chiefly catalyze the electroneutral exchange of Na(+) and H(+) down their respective concentration gradients and are crucial for numerous physiological processes, ranging from the fine control of intracellular pH and cell volume to systemic electrolyte, acid-base and fluid volume homeostasis. NHE activity also facilitates the progression of other cellular events such as adhesion, migration, and proliferation. Thus far, eight distinct NHE genes (NHE1/SLC9A1-NHE8/SLC9A8) and several pseudogenes have been identified in the human genome. The functional genes encode proteins of varying primary sequence identity (25-70%), but share a common predicted secondary structure comprising 12 conserved membrane-spanning segments at the amino-terminus and a more divergent, cytoplasmically-oriented, carboxy-terminus. They show considerable heterogeneity in their patterns of tissue/cell expression and membrane localization. Functional studies have revealed further differences in their kinetic properties, sensitivity to pharmacological antagonists, and regulation by diverse hormonal and mechanical stimuli. Altered NHE activity has been linked to the pathogenesis of several diseases, including essential hypertension, congenital secretory diarrhea, diabetes, and tissue damage caused by ischemia/reperfusion. Further characterization of their functional properties should lead to a better understanding of their unique contributions to human health and disease.     

Ba2+ and amiloride uncover or induce a pH-sensitive and a Na+ or non-selective cation conductance in transitional cells of the inner ear

The membrane potential V _m the cytosolic pH (pH_i), the transference numbers (t) for K⁺, Cl^– and Na⁺/ non-selective cation (NSC) and the pH-sensitivity of V _m were investigated in transitional cells from the vestibular labyrinth of the gerbil. V _m, pH_i, , and the pH_i sensitivity of V _m were under control conditions were –92±1 mV (n=89 cells), pH_i 7.13±0.07 (n=11 epithelia), 0.87±0.02 (n=22), 0.02±0.01 (n=19), 0.01±0.01 (n=24) and –5 mV/pH unit (n=13 cells/n=11 epithelia), respectively. In the presence of 100 mol/l Ba²⁺ the corresponding values were: –70±1 mV (n=32), pH_i 7.16±0.08 (n=6), 0.31±0.05 (n=4), 0.06±0.01 (n=6), 0.20±0.03 (n=10) and -16 mV/pH-unit (n=15/n=6). In the presence of 500 mol/l amiloride the corresponding values were: –72±2mV (n=34), pH_i 7.00±0.07 (n=5), 0.50±0.04 (n=6), 0.04±0.01 (n=11), 0.28±0.04 (n=9) and –26 mV/pH-unit (n=20/n=5). In the presence of 20 mmol/l propionate plus amiloride the corresponding values were: –61±2 mV (n=27), pH_i 6.72±0.06 (n=5), 0.30±0.02 (n=6), 0.06±0.01 (n=5) and 0.40±0.02 (n=8), respectively. V _m was depolarized and and pH_i decreased due to (a) addition of 1 mmol/l amiloride in 150 mmol/l Na⁺ by 38±1 mV (n=8), from 0.82±0.02 to 0.17±0.02 (n=8) and by 0.13±0.01 pH unit (n=6), respectively; (b) reduction of [Na⁺] from 150 to 1.5 mmol/l by 3.3±0.5 mV (n=30), from 0.83±0.02 to 0.75±0.04 (n=9) and by 0.33±0.07 pH unit (n=4), respectively and (c) addition of 1 mmol/l amiloride in 1.5 mmol/l Na⁺ by 20±1 mV (n=11) and from 0.83±0.03 to 0.53±0.02 (n=5), respectively. These data suggest that the K⁺ conductance is directly inhibited by amiloride and Ba²⁺ and that Ba²⁺ and amiloride uncover or induce a pH-sensitive and a Na⁺/NSC conductance which may or may not be the same entity.Some of the data have been presented at various meetings and appear in abstract form in [31, 35, 37]     

Endothelin-1 blunts transepithelial transport and differentiation of Madin-Darby canine kidney cells

We investigated the effects of endothelin-1 (ET-1) on Madin-Darby canine kidney (MDCK) cells, a cell line originating from the renal collecting duct. The activity of transepithelial transport was assessed as the rate of dome formation in monolayers grown on solid support. The pH value of the dome fluid (dome pH) was measured by means of pH-selective microelectrodes. Differentiation of monolayer cells was estimated as the peanut-lectin(PNA)-binding capacity of the apical membrane. Confluent monolayers were incubated for 12–72 h in serum-free medium at various concentrations of ET-1. Exposure to 1 nmol/l ET-1 reduced dome formation by a maximum of 41±8% (n=4; P<0.02) after 24 h. ET-1 (10 nmol/l; 24 h) decreased dome pH from 7.52±0.02 (n=53) to 7.36±0.03 (n=51; P<0.02). Apical application of amiloride (1 mmol/l) reduced dome pH in both ET-1-treated and non-treated domes to essentially the same level, 7.25±0.03 (n=19) and 7.23±0.03 (n=17) respectively. ET-1 (10 nmol/l; 24 h) reduced PNA-binding capacity by 19±3% (n=5; P < 0.02). Moreover, ET-1 prevented the increase in PNA binding (+53±7%; n=5) induced by 0.1 mol/l aldosterone. We conclude that ET-1 inhibits transepithelial transport and PNA binding via inhibition of apical Na⁺/H⁺ exchange, thus antagonizing aldosterone action in MDCK cells.     

KCNQ-encoded channels regulate Na+ transport across H441 lung epithelial cells

H441 cells are a model of absorptive airway epithelia that are characterised by a pronounced apical Na⁺ flux through amiloride-sensitive Na⁺ channels. The flux of Na⁺ is intimately linked to Na⁺ handling by the cell as well as the membrane potential across the apical membrane. As KCNQ-encoded K⁺ channels influence chloride secretion in gastrointestinal epithelia, the goal of the present study was to ascertain the expression of KCNQ genes in H441 cells and determine the functional role of the expression products. Message for KCNQ3 and KCNQ5 was detected by RT-polymerase chain reaction and the translated proteins were observed by immunocytochemistry. Ussing experiments showed that the pan-KCNQ channel blocker XE991, but not KCNQ1 selective blockers, reduced the short circuit current and the amiloride-sensitive component. These data show for the first time that potassium channels encoded by KCNQ3 or KCNQ5 are crucial determinants of epithelial Na⁺ flux.     

Mechanisms of intracellular pH regulation in the hamster inner medullary collecting duct perfused in vitro

To examine the mechanisms of H⁺ transport in the mid-inner medullary collecting duct of hamsters, we measured the intracellular pH (pH_i) in the in vitro perfused tubules by microscopic fluorometry using 2,7-bis(carboxyethyl)-carboxyfluorescein (BCECF) as a fluorescent probe. In the basal condition, pH_i was 6.74±0.04 (n=45) in HCO ₃ ^– -free modified Ringer solution. Either elimination of Na⁺ from the bath or addition of amiloride (1 mM) to the bath produced a reversible fall in pH_i After acid loading with 25 mM NH₄Cl, pH_i spontaneously recovered with an initial recovery rate of 0.096±0.012 (n=23) pH unit/min. In the absence of ambient Na⁺, after removal of NH ₄ ⁺ , the pH_i remained low (5.95±0.10, n=8) and showed no signs of recovery. Subsequent restoration of Na⁺ only in the lumen had no effect on pH_i. However, when Na⁺ in the bath was returned to the control level, pH_i recovered completely. Amiloride (1 mM) in the bath completely inhibited the Na⁺-dependent pH_i recovery. Furthermore, elimination of Na⁺ from the bath, but not from the lumen, decreased pH_i from 6.97±0.07 to 6.44±0.05 (n=12) in the HCO ₃ ^– /Ringer solution or 6.70±0.03 to 6.02±0.05 (n=8) in the HCO ₃ ^– free solution. pH_i spontaneously returned to 6.76±0.08 with a recovery rate of 0.017±0.5 pH unit/min in the presence of CO₂/HCO ₃ ^– , whereas it did not recover in the absence of CO₂/HCO ₃ ^– . Although elimination of ambient Na⁺ depolarized the basolateral membrane voltage (V _B) from –78±1.2 to –72 ±0.6 mV (n=5, P<0.01), the level of V _B was not sufficient to explain the pH_i recovery solely by HCO ₃ ^– entry driven by the voltage. These results indicate that (a) pH_i of the inner medullary collecting duct is regulated mainly by a Na⁺/H⁺ exchanger in the basolateral membranes, (b) no apparent Na⁺-dependent H⁺ transport system exists in the luminal membranes and (c) Na⁺-independent H⁺ transport may also operate in the presence of CO₂/HCO ₃ ^– Preliminary data were reported at the Conference on Bicarbonate, Chloride, and Proton Transport Systems, New York, USA, in January 1989     

Characterization of acetate uptake by the colonic epithelial cells of the rat

The characteristics of acetate uptake by colonic epithelial cells of the rat were studied. Clear saturation kinetics of acetate uptake were not observed in these experiments at either 0° C or 30° C. A decrease in the pH of the medium markedly increased the acetate uptake. The activation energy for acetete uptake derived from an Arrhenius plot was about 6.1 kcal/mole. Among the inhibitors tested, no effective inhibition of acetate uptake at 0° C was observer. Metabolic inhibitors severely inhibited transport at 30° C. Inhibition of acetate uptake by other short chain fatty acids, which was non-competitive, was observed. The finding that efflux from the cells was stimulated in the presence of compounds such as pyruvate and bicarbonate supported the notion of a close interrelationship between weak electrolyte transports in vivo. Although the H⁺ gradient across the cell membrane is suggested to be one of the factors determining the uptake rate, it seems difficult to explain all the results in this way.     

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.     

Effects of forskolin on crypt cells of rat distal colon

Na⁺-K⁺-2Cl^– cotransport activity has previously been shown to depend on both intracellular ATP and Mg²⁺, but the mechanisms remain unknown. Cotransport in avian erythrocytes can be stimulated by a variety of agents including cAMP and permeant serine/ threonine phosphatase inhibitors and is inhibited by prior depletion of either ATP with antimycin A, or Mg²⁺ by incubation in A23187 plus EDTA. However, when cells were first stimulated with cAMP rather than calyculin A then subjected to either depletion strategy, a differential effect was found. The phosphatase-inhibitor-treated cells were resistant to subsequent ATP or Mg²⁺ depletion while cAMP-treated cells were sensitive to both treatments. Parallel examination of protein phosphorylation confirmed that ATP or Mg²⁺ depletion leads to dephosphorylation of membrane proteins in cAMP-treated but not in calyculin-A-treated cells. These results suggest that, for cotransport, ATP and Mg²⁺ are required primarily to maintain the system in a phosphorylated state rather than as direct modulators. The relative effectiveness of okadaic acid (EC₅₀ 630 nM) and calyculin A (EC₅₀ 8 nM) in stimulating the cotransporter indicate that a type-1 protein phosphatase is probably responsible for dephosphorylating the system. Cells stimulated by hypertonicity were also resistant to ATP or Mg²⁺ depletion suggesting that the mechanism of shrinkage-induced cotransport stimulation might also involve protein phosphatase modulation.     

Molecular origin of Na/Li exchanger: Evidence against the involvement of major cloned erythrocyte transporters

Numerous studies have demonstrated heightened Na⁺/Li⁺ countertransport (NLCT) activity in erythrocytes of patients with essential hypertension or diabetic nephropathy. The same carrier also contributes to the therapeutic action of lithium salt, widely used in the treatment of psychiatric disorders. However, the molecular origin of NLCT remains unknown. This study examined the role of major ion transporters in NLCT by comparative analysis of its activity and that of ion transporters providing inwardly directed ⁸⁶Rb, ²²Na and ³²P fluxes. NLCT was below the detection limit in rat erythrocytes and ∼50-fold higher in rabbits compared to humans. Unlike NLCT, the activities of Na⁺,K⁺-ATPase, Na⁺,K⁺,2Cl⁻ cotransporter and anion exchanger were somewhat similar in the erythrocytes of these species, whereas Na⁺,P_i cotransport was in 1:2:6 proportion in rats, humans and rabbits, respectively. Loading of erythrocytes with Li⁺ for NLCT measurement did not affect the activity of Na⁺,P_i cotransporter. Keeping in mind that NLCT is much higher in rabbits vs humans and rats, we compared the set of membrane proteins in these species using 2-dimensional gel electrophoresis. This approach revealed 174 common spots, whereas 132 proteins were detected only in human and rabbit erythrocyte membranes. Among these proteins, we found 17 spots whose expression was higher by more than 5-fold in rabbit compared to human erythrocytes. Thus, our results argue against the involvement of major ion transporters in NLCT. They also show that comparative proteomics is a potent tool to identify the molecular origin of this carrier.