Increases in Intracellular pH and Ca2+ are Essential for K+ Channel Activation After Modest `Physiological' Swelling in Villus Epithelial Cells

We studied the relationship between changes in intracellular pH (pH _i ), intracellular Ca²⁺([Ca²⁺] _i ) and charybdotoxin sensitive (CTX) maxi-K⁺ channels occurring after modest `physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH <sub>i</sub> and [Ca<sup>2+</sup>] <sub>i</sub> by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 × isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 × isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 × isotonic dilution, the pH <sub>i</sub> alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this ΔpH <sub>i</sub> and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH<sub>4</sub>Cl permitted complete RVD which could be inhibited by CTX. The rate of <sup>86</sup>Rb efflux which also increased after this 0.93 × isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na<sup>+</sup>-free medium. Modest swelling transiently increased [Ca<sup>2+</sup>] <sub>i</sub> and Ca<sup>2+</sup>-free medium or blocking alkalinization by MIA or Na<sup>+</sup>-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca<sup>2+</sup>-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca<sup>2+</sup>] <sub>i</sub> and volume changes became sensitive to CTX. We conclude that both alkalinization of pH <sub>i</sub> and increased [Ca<sup>2+</sup>] <sub>i</sub> observed with `physiological' volume increase are essential for the activation of CTX-sensitive maxi-K⁺ channels required for RVD. Received: 30 March 1999/Revised: 6 July 1999     

P2Y(2) receptor-mediated inhibition of amiloride-sensitive short circuit current in M-1 mouse cortical collecting duct cells

Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y₂ receptors stimulate luminal Ca²⁺-activated Cl⁻ channels and inhibit Na⁺ transport. Here we address the mechanism of ATP-mediated inhibition of Na⁺ transport. M-1 cells had a transepithelial voltage (V _te ) of −31.4 ± 1.3 mV and a transepithelial resistance (R _te ) of 1151 ± 28 Ωcm². The amiloride-sensitive short circuit current (I _sc ) was −28.0 ± 1.1 μA/cm². The ATP-mediated activation of Cl⁻ channels was inhibited when cytosolic Ca²⁺ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca²⁺]_i increase was paralleled by a rapid and transient R _te decrease (297 ± 51 Ωcm²). In the presence of CPA, basolateral ATP led to an R _te increase by 144 ± 17 Ωcm² and decreased V _te from −31 ± 2.6 to −26.6 ± 2.5 mV. I _sc dropped from −28.6 ± 2.4 to −21.6 ± 1.9 μA/cm². Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na⁺ absorption. Lowering [Ca²⁺]_i by removal of extracellular Ca²⁺ did not alter the ATP effect. PKC inhibition or activation were without effect. Na⁺ absorption was activated by pH_i alkalinization and inhibited by pH_i acidification. ATP slightly acidified M-1 cells by 0.05 ± 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y₂ receptors inhibits Na⁺ absorption. This effect is not mediated via [Ca²⁺]_i, does not involve PKC and is to a small part mediated via intracellular acidification. Received: 9 February 2001/Revised: 17 May 2001     

Variations of Intracellular pH in Human Erythrocytes via K+(Na+)/H+ Exchange Under Low Ionic Strength Conditions

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K⁺ efflux and Cl⁻ loss. When human erythrocytes were suspended in a physiological NaCl solution (pH _o = 7.4), the measured pH _i was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH _i to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl⁻ content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H⁺ influx with the measured unidirectional K⁺ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H⁺ influx and a K⁺ efflux in LIS solution of 108.2 ± 20.4 mmol (l _cells hr)⁻¹ and 98.7 ± 19.3 mmol (l _cells hr)⁻¹, respectively. For bovine and porcine erythrocytes, in LIS media, H⁺ influx and K⁺ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K⁺(Na⁺)/H⁺ exchanger, inhibited the K⁺ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K⁺(Na⁺)/H⁺ exchanger in the human erythrocyte membrane. Received: 22 December 1999/Revised: 10 April 2000     

Ca2+/Calmodulin Kinase II and Decreases in Intracellular pH are Required to Activate K+ Channels After Substantial Swelling in Villus Epithelial Cells

To assess the activation of the charybdotoxin-insensitive K⁺ channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH (pH _i ), intracellular calcium ([Ca²⁺] _i ) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume changes. Fluorescence spectroscopy was used to measure pH _i and [Ca²⁺] _i of cells in suspension, loaded with 2,7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca²⁺] _i to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification of ≃0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 μm), an inhibitor of Ca²⁺/calmodulin kinase II, blocked RVD. Gramicidin (0.5 μm) bypassed this inhibition suggesting that the K⁺ channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase; inhibition of RVD generated by increasing the K⁺ gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 μm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K⁺ channels utilized for RVD after substantial (>7%) `nonphysiological' volume increases requires phosphorylation mediated by Ca²⁺/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca²⁺] _i are a critical determinant of this activation. Received: 30 March 1999/Revised: 6 July 1999     

Effect of Trace Levels of Nigericin on Intracellular pH and Acid-Base Transport in Rat Renal Mesangial Cells

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pH _i ) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pH _i in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH _i decrease elicited by removing extracellular Na⁺, (ii) an increase in acid extrusion during the pH _i increase caused by elevating extracellular [K⁺], and (iii) an acid shift in the pH _i dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pH _i dependence of Na-H exchange, computed by adding the pH _i dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment. Received: 14 October 1998/Revised: 2 March 1999     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

Activators of Epithelial Na+ Channels Inhibit Cytosolic Feedback Control. Evidence for the Existence of a G Protein-Coupled Receptor for Cytosolic Na+

We have previously shown that epithelial Na⁺ channels in mouse mandibular gland duct cells are controlled by cytosolic Na⁺ and Cl⁻, acting, respectively, via G _o and G _i proteins. Since we found no evidence for control of epithelial Na⁺ channels by extracellular Na⁺ ([Na⁺] _o ), our findings conflicted with the long-held belief that Na⁺ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na⁺] _o . Instead, we now show that PCMPS acts by rendering epithelial Na⁺ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na⁺ and Cl⁻ on Na⁺ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na⁺ channels by cytosolic Na⁺, while leaving inhibition by cytosolic Cl⁻ unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of G _o in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na⁺ receptor. Received: 1 October 1997/Revised: 24 December 1997     

Effects of Hyperthermia on Intracellular Chloride

Hyperthermia induces transient changes in [Na⁺] _i and [K⁺] _i in mammalian cells. Since Cl⁻ flux is coupled with Na⁺ and K⁺ in several processes, including cell volume control, we have measured the effects of heat on [Cl⁻] _i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl⁻] _i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl⁻] _i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na⁺] _i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl⁻] _i depended upon time and temperature. The [Cl⁻] _i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl⁻] _i increase was completely blocked. Since furosemide inhibits the Na⁺/K⁺/2Cl⁻ cotransporter, Cl⁻ channels, and even Cl⁻HCO₃ exchange, these ion transporters may be involved in the heat-induced increase in [Cl⁻] _i . Received: 15 June 1995/Revised: 9 April 1996     

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions

Previous squid-axon studies identified a novel K/HCO₃ cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K⁺ and HCO⁻ ₃ out of the cell, tending to lower intracellular pH (pH_i). With an inwardly directed K/HCO₃ gradient, the cotransporter mediates a net uptake of alkali (i.e., K⁺ and HCO⁻ ₃ influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA⁺) inhibit the inwardly directed cotransporter by interacting at the intracellular K⁺ site. We computed the equivalent HCO⁻ ₃ influx (J _HCO3) mediated by the cotransporter from the rate of pH_i increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH_i 8.0, using a dialysis fluid (DF) free of K⁺, Na⁺ and Cl⁻. Our standard artificial seawater (ASW) also lacked Na⁺, K⁺ and Cl⁻. After halting dialysis, we introduced an ASW containing 437 mm K⁺ and 0.5% CO₂/12 mm HCO⁻ ₃, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pH_i decrease, due to CO₂ influx, followed by a slower plateau-phase pH_i increase, due to inward cotransport of K⁺ and HCO⁻ ₃. With no QA⁺ in the DF, J _HCO3 was ∼58 pmole cm⁻² sec⁻¹. With 400 mm tetraethylammonium (TEA⁺) in the DF, J _HCO3 was virtually zero. The apparent K _i for intracellular TEA⁺ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K⁺ channels. Introducing 100 mm inhibitor into the DF reduced J _HCO3 to ∼20 pmole cm⁻² sec⁻¹ for tetramethylammonium (TMA⁺), ∼24 for TEA⁺, ∼10 for tetrapropylammonium (TPA⁺), and virtually zero for tetrabutylammonium (TBA⁺). The apparent K _i value for TBA⁺ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA⁺), with an apparent K _i of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO₃ influx in the potency sequence PPTEA⁺ > TBA⁺ > TPA⁺ > TEA⁺≅ TMA⁺. The identification of inhibitors of the K/HCO₃ cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter. Received: 21 November 2000/Revised: 14 May 2001     

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na⁺]_i in these cells loaded with the Na⁺ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na⁺]_i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA₂ activity (10 μm AACOCF₃) obviated EGF-induced increases in [Na⁺]_i. In contrast, PGE₂ (10 μg/ml) and cAMP (2 mm) increased [Na⁺]_i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na⁺]_i. Similarly, EGF failed to increase [Na⁺]_i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na⁺]_i followed by a decline towards its baseline value. EGF-induced increases in [Na⁺]_i were unaltered by inhibition of K⁺ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA₂ activity. Their stimulation increases PGE₂ and cAMP levels resulting in PKA and NKCC activation. Received: 18 December 2000/Revised: 24 May 2001     

Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver

Although both amiloride- and phloretin-sensitive Na⁺/Li⁺ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li⁺ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li⁺ by hepatocytes, preloaded with Na⁺ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K _m 1.2 mm Li⁺, V _max 40 μmol · (kg dry wt · min)⁻¹, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H⁺ was curvilinear suggesting a modifier role of [H⁺] _i . This system persisted in Na⁺-depleted cells, although with apparent K _m 3.8 mm. (b) A phloretin-sensitive component, with K _m 1.2 mm, V _max 21 μmol · (kg · min)⁻¹, was unaffected by pH but was inactive in Na⁺-depleted cells. Phloretin inhibited Li⁺ uptake and Na⁺ efflux in parallel. (c) A residual uptake increased linearly with the external Li⁺ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li⁺ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H⁺]. Received: 8 April 1999/Revised: 19 July 1999     

Surface pH at the Basolateral Membrane of the Caecal Mucosa of Guinea Pig

Since the major mechanisms responsible for regulation of intracellular pH of enterocytes are located in the basolateral membrane, respective effects may be expected on pH in the compartment near the basolateral membrane. A method was established to estimate the pH at the basolateral membrane (pH _b ) of isolated caecal epithelia of guinea pig using pH-sensitive fluorescein attached to lectin (lens culinaris). In the presence of bicarbonate and a perfusion solution-pH of 7.4, pH _b was 7.70 ± 0.15. In the absence of bicarbonate or chloride as well as by inhibition of the basolateral Cl⁻-HCO⁻ ₃ exchange with H₂-DIDS, pH _b was reduced near to solution-pH. Inhibition of the basolateral Na⁺-H⁺ exchanger by adding a sodium- and bicarbonate-free, low-buffered solution increased pH _b . Decrease of pH of serosal perfusion solution to 6.4 provoked a similar decrease of pH _b to solution pH. Short-chain fatty acids (SCFA) added to the mucosal solution caused a slight decrease of pH _b . SCFA added to the serosal side alkalized pH _b . However, in the presence of bicarbonate pH _b returned quickly to the initial pH _b , and after removal of SCFA a transient acidification of pH _b was seen. These responses could not be inhibited by MIA or H₂-DIDS. We conclude that no constant pH-microclimate exists at the basolateral side. The regulation of the intracellular pH of enterocytes reflects pH _b . The slightly alkaline pH _b is due to the bicarbonate efflux. Data support the presence of an SCFA⁻-HCO⁻ ₃ exchange. Received: 17 December 1998/Revised: 24 February 1999     

Effects of Internal K+ and ABA on the Voltage- and Time-dependence of the Outward K+-rectifier in Vicia Guard Cells

One of the main effects of abscisic acid (ABA) is to induce net loss of potassium salts from guard cells enabling the stomata to close. K⁺ is released from the vacuole into the cytosol and then to the extracellular space. The effects of increasing cytosolic K⁺ on the voltage- and time-dependence of the outwardly rectifying K⁺-current (I _K,out) in guard cell protoplasts (GCP) was examined in the whole-cell configuration of the patch-clamp technique. The same quantitative analysis was performed in the presence of ABA at different internal K⁺ concentrations ([K⁺] _i ). Varying [K⁺] _i in the patch pipette from 100 to 270 mm increased the magnitude of I _K,out in a nonlinear manner and caused a negative shift in the midpoint (V _0.5) of its steady-state activation curve. External addition of ABA (10–20 μm) also increased the magnitude of I _K,out at all [K⁺] _i , but caused a shift in V _0.5 of the steady-state activation curve only in those GCP loaded with 150 mm internal K⁺ or less. Indeed, V _0.5 did not shift upon addition of ABA when the [K⁺] _i was above 150 mm and up to 270 mm, i.e., the shift in V _0.5 caused by ABA depended on the [K⁺] _i . Both increase in [K⁺] _i and external addition of ABA, decreased (by ≈ 20%) the activation time constant (τ _n ) of I _K,out. The small decrease in τ _n , in both cases, was found to be independent of the membrane voltage. The results indicate that ABA mimics the effect of increasing cytoplasmic K⁺, and suggest that ABA may increase I _K,out and alter V _0.5 of its steady-state activation curve via an enhancement in cytosolic K⁺. This report describes for the first time the effects of [K⁺] _i on the voltage- and time-dependence of I _K,out in guard cells. It also provides an explanation for the quantitative (total membrane current) and qualitative (current kinetics) differences found between intact guard cells and their protoplasts. Received: 1 December 1995/Revised: 8 May 1996     

H+ ATPase and Cl− Interaction in Regulation of MDCK Cell pH

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na⁺-H⁺ exchange, H⁺–K⁺ ATPase and Cl⁻/HCO⁻ ₃ exchange. In this work we studied the functional activity of a vacuolar H⁺-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H⁺ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH⁺ ₄ pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na⁺ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10⁻⁸ m (a specific inhibitor of the vacuolar H⁺-ATPase) was added to the zero Na⁺ solution. When using a solution with zero Na⁺, low K⁺ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H⁺–K⁺-ATPase. This result was confirmed by the use of 5 × 10⁻⁵ m Schering 28080. The Na⁺ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10⁻⁵ m (a specific blocker of Cl⁻ channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl⁻/normal Na⁺ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na⁺ and Cl⁻ free solution. From these results we conclude that: (i) MDCK cells have two Na⁺-independent mechanisms of pHi recovery, a concanamycin sensitive H⁺-ATPase and a K⁺ dependent, Schering 28080 sensitive H⁺–K⁺ ATPase; and, (ii) pHi recovery in Na⁺-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl⁻–free medium. This finding supports a role for chloride in the function of the H⁺ ATPase, which might be electrical shunting or a biochemical interaction. Received: 24 October 1997/Revised: 19 February 1998     

A Membrane-delimited Effect of Internal pH on the K+ Outward Rectifier of Vicia Faba Guard Cells

ABA stimulation of outward K⁺ current (I _K,out) in Vicia faba guard cells has been correlated with a rise in cytosolic pH (pH _i ). However, the underlying mechanism by which I _K,out is affected by pH _i has remained unknown. Here, we demonstrate that pH _i regulates outward K⁺ current in isolated membrane patches from Vicia faba guard cells. The stimulatory effect of alkalinizing pH _i was voltage insensitive and independent of the two free calcium levels tested, 50 nm and 1 μm. The single-channel conductance was only slightly affected by pH _i . Based on single-channel measurements, the kinetics of time-activated whole-cell current, and the analysis of current noise in whole-cell recordings, we conclude that alkaline pH _i enhances the magnitude of I _K,out by increasing the number of channels available for activation. The fact that the pH _i effect is seen in excised patches indicates that signal transduction pathways involved in the regulation of I _K,out by pH _i , and by implication, components of hormonal signal transduction pathways that are downstream of pH _i , are membrane-delimited. Received: 5 June 1996/Revised: 1 August 1996     

Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells

Stimulation with leukotriene D₄ (LTD₄) (3–100 nm) induces a transient increase in the free intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in Ehrlich ascites tumor cells. The LTD₄-induced increase in [Ca²⁺] _i is, however, significantly reduced in Ca²⁺-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD₄ concentration (3 nm) does not result in any detectable increase in [Ca²⁺] _i . Addition of LTD₄ (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD₄-induced (100 nm) acceleration of the RVD response is also seen in Ca²⁺-free medium and also at 3 nm LTD₄, indicating that LTD₄ can open K⁺- and Cl⁻-channels without any detectable increase in [Ca²⁺] _i . Buffering cellular Ca²⁺ with BAPTA almost completely blocks the LTD₄-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca²⁺] _i level after BAPTA-loading or buffering of [Ca²⁺] _i seems to inhibit the LTD₄-induced stimulation of the RVD response even though the LTD₄-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca²⁺] _i . The LTD₄ receptor antagonist L649,923 (1 μm) completely blocks the LTD₄-induced increase in [Ca²⁺] _i and inhibits the RVD response as well as the LTD₄-induced acceleration of the RVD response. When the LTD₄ receptor is desensitized by preincubation with 100 nm LTD₄, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD₄ plays a role in the activation of the RVD response. LTD₄ seems to activate K⁺ and Cl⁻ channels via stimulation of a LTD₄ receptor with no need for a detectable increase in [Ca²⁺] _i . Received: 25 September 1995/Revised: 25 January 1996     

Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary Epithelium

Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pH _i ) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pH _i was measured using the fluorescent dye BCECF and the pH _i responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pH _i changes elicited by bicarbonate/CO₂ removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH _i . We considered whether CA-IV might facilitate H⁺ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mm) reduced pH _i 0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH _i reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pH _i reduction. DBI also reduced by ∼40% the rate of pH _i recovery in cells acidified by an ammonium chloride (20 mm) prepulse; a reduction in pH _i recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH _i alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H⁺ and HCO⁻ ₃ with CO₂ in the unstirred layer outside the plasma membrane, preventing local accumulation of H⁺ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH _i changes that might alter the function of other ion transporters and channels in the NPE. Received: 24 April 1997/Revised: 4 November 1997     

Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions

In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for studying H⁺- and Na⁺-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (P_i) was accumulated by two kinetically discrete H⁺/P_i-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P_i concentrations. The high-affinity system, subjected to regulation by both extracellular P_i availability and intracellular polyphosphate stores, is mobilized during P_i-starvation. In cells grown at pH 9.5–10, P_i uptake is mediated by several kinetically discrete Na⁺-dependent systems that are specifically activated by Na⁺ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P_i concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P_i-starvation and appear to be controlled by extracellular P_i. They represent the first examples of high-capacity, Na⁺-driven P_i transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H⁺- and Na⁺-coupled P_i transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H⁺-coupled P_i transport systems are predominant. The contribution of the Na⁺/P_i cotransport systems to the total cellular P_i uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher. Received: 15 December 2000/Revised: 14 May 2001     

II. Voltage-activated Proton Currents in Human THP-1 Monocytes

Depolarization-activated H⁺-selective currents were studied using whole-cell and excised-patch voltage clamp methods in human monocytic leukemia THP-1 cells, before and after being induced by phorbol ester to differentiate into macrophage-like cells. The H⁺ conductance, g _H, activated slowly during depolarizing pulses, with a sigmoidal time course. Fitted by a single exponential following a delay, the activation time constant, τ_act was roughly 10 sec at threshold potentials, decreasing at more positive potentials. Tail currents upon repolarization decayed mono-exponentially at all potentials. The tail current time constant, τ_tail, was voltage dependent, decreasing with hyperpolarization from 2–3 sec at 0 mV to ∼200 msec at −100 mV. Surprisingly, although τ_act depended strongly on pH _o , τ_tail was completely independent of pH _o . H⁺ currents were inhibited by Zn²⁺. Increasing pH _o or decreasing pH _i shifted the voltage-activation relationship to more negative potentials, tending to activate the g _H at any given voltage. Studied in excised, inside-out membrane patches, H⁺ currents were larger and activated much more rapidly at lower bath pH (i.e., pH _i ). In THP-1 cells differentiated into macrophages, the H⁺ current density was reduced by one-half, and τ_act was slower by about twofold. The properties of H⁺ channels in THP-1 cells and in other macrophage-related cells are compared. Received: 19 September 1995/Revised: 14 March 1996     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999