Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : I. Role of HCO3?

We studied the regulation of intracellular pH (pH_i) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH_i. Intrinsic buffering power (β_I) was 10.5 mM (pH unit)⁻¹ at pH_i 7.0, and decreased linearly with pH_i; the best-fit line to the data had a slope of −10.0 mM (pH unit)⁻². In the absence of HCO₃ ⁻, pH_i recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH_i 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH_i. Acid extrusion was inhibited 68% (pH_i 6.23) by substituting Li⁺ for Na⁺ in the bath solution. Switching from a CO₂/HCO₃ ⁻-free to a CO₂/HCO₃ ⁻-containing bath solution caused mean steady state pH_i to increase from 6.82 to 6.90, due to a Na⁺-driven HCO₃ ⁻ transporter. The HCO₃ ⁻-induced pH_i increase was unaffected by amiloride, but was inhibited 75% (pH_i 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pH_i 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO₂/HCO₃ ⁻-induced pH_i increase was blocked when external Na⁺ was replaced with N-methyl-d-glucammonium (NMDG⁺). In the presence of HCO₃ ⁻, the Na⁺-driven HCO₃ ⁻ transporter contributed to the pH_i recovery from an acid load. For example, HCO₃ ⁻ shifted the plot of acid-extrusion rate vs. pH_i by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO₃ ⁻ increased acid extrusion 3.8-fold (pH_i 6.20). When astrocytes were acid loaded in amiloride, with Li⁺ as the major cation, HCO₃ ⁻ failed to elicit a substantial increase in pH_i. Thus, Li⁺ does not appear to substitute well for Na⁺ on the HCO₃ ⁻ transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na⁺-driven HCO₃ ⁻ transporter are the predominant acid extruders in astrocytes.     

pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

pH _i recovery in acid-loaded Ehrlich ascites tumor cells and pH _i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO ₃ ^– -free media and at 25 mm HCO ₃ ^– , the measured pH _i (7.26 and 7.82, respectively) was significantly more alkaline than the pH _i . value calculated assuming the transmembrane HCO ₃ ^– gradient to be equal to the Cl^– gradient. Thus, pH _i in these cells is not determined by the Cl^– gradient and by Cl^–/HCO ₃ ^– exchange.pH _i recovery following acid loading by propionate exposure, NH ₄ ⁺ withdrawal, or CO₂ exposure is mediated by amiloride-sensitive Na⁺/H⁺ exchange in HCO₃ ^– free media, and in the presence of HCO ₃ ^– (25 mm) by DIDS-sensitive, Na⁺-dependent Cl^–/HCO ₃ ^– exchange. A significant residual pH _i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H⁺ pump in pH _i regulation. pH _i maintenance at steady-state involves both Na⁺/H⁺ exchange and Na⁺-dependent Cl^–/HCO ₃ ^– exchange.Acute removal of external Cl^– induces a DIDS-sensitive, Na⁺-dependent alkalinization, taken to represent HCO ₃ ^– influx in exchange for cellular Cl^–. Measurements of ³⁶Cl^– efflux into Cl^–-free gluconate media with and without Na⁺ and/or HCO ₃ ^– (10 mm) directly demonstrate a DIDS-sensitive, Na⁺ dependent Cl^–/HCO ₃ ^– exchange operating at slightly acidic pH _i (pH_o 6.8), and a DIDS-sensitive, Na⁺-independent Cl^–/HCO ₃ ^– exchange operating at alkaline pH _i (pH _o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).     

Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane

Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H⁺ (pH_i) and Cl^– (aCl_i) activities with double-barreled ion-selective microelectrodes. In a HCO ₃ ^– -free solution of pH 7.4 (HEPES/Tris buffered), pH_i was 7.25 andaCl_i was 33mm. By an exposure to a HCO ₃ ^– (25mm HCO ₃ ^– /5% CO₂, pH 7.4) solution for 15 min,aCl_i was decreased to 25mm and pH_i was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO ₃ ^– concentration [HCO ₃ ^– ]_i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO ₃ ^– -free solution reversed the changes inaCl_i and pH_i. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCl_i, pH_i, and [HCO ₃ ^– ]_i caused by admission/withdrawal of the HCO ₃ ^– , solution and decreased the buffer value. Replacement of all Cl^– with gluconate in the HCO ₃ ^– solution increased pH_i, and readmission of Cl^– decreased pH_i. The rates of these changes in pH_i were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO ₃ ^– solution, a stimulation of intracellular HCO ₃ ^– production by exposing the tissue to 25mm NH ₄ ⁺ increasedaCl_i significantly. While in the HCO ₃ ^– -free solution or in the HCO ₃ ^– , solution containing DIDS, exposure to NH ₄ ⁺ had little effect onaCl_i. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl^–/HCO ₃ ^– exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl^–/HCO ₃ ^– exchange is discussed.     

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

Hill Coefficient Analysis of Transmembrane Helix Dimerization

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Cytoplasmic pH (pH _i ) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However, membrane transport mechanisms involved in pH _i regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pH _i was determined using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pH _i by a mechanism that was shown to be sensitive to external Na⁺. pH _i recovery was also greatly reduced in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and amiloride. These results point toward the action of an Na⁺-driven HCO₃⁻/Cl⁻ exchanger and/or an Na⁺/HCO₃⁻ cotransporter and the action of the Na⁺/H⁺ exchanger on pH _i regulation in the experimental conditions used. pH _i recovery was only slightly affected by ouabain, suggesting that the inhibition of Na⁺/K⁺-ATPase affects recovery indirectly, possibly via the shift on the Na⁺ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor of the V-type ATPases, suggesting that these pumps do not have a relevant action on pH _i regulation in bovine Sertoli cells.     

Acid–base regulation in isolated gill cells of the goldfish (Carassius auratus)

Mechanisms of acid release and intracellular pH (pH_i) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pH_i was determined using the fluorescent probe 2,7-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na⁺ channel and Na⁺/H⁺ exchanger (NHE) inhibitor, had no effect on pH_i, but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH₄Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na⁺. Under steady-state conditions, 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid (DIDS), a HCO₃^–-transport inhibitor, caused a slow acidification of pH_i, and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A₁, an inhibitor of V-ATPase, had no effect on steady-state pH_i and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na⁺-dependent HCO₃^– transport is the dominant mechanism besides an NHE and V-ATPase to control pH_i in goldfish gill cells.Communicated by G. Heldmaier     

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     

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na⁺/ HCO ₃ ^? cotransporter. We investigated whether the activity of Na⁺/HCO ₃ ^? cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased ²²Na uptake in the presence of HCO₃ but not in the presence of gluconate, indicating that trypsin does not alter diffusive ²²Na uptake but directly stimulates the Na⁺/HCO ₃ ^? cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na⁺/HCO ₃ ^? cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na⁺/HCO ₃ ^? cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na⁺/HCO ₃ ^? cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na⁺/HCO ₃ ^? cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S?). With the addition of fraction S?, the CSU of cAMP-PKA failed to inhibit the Na⁺/HCO ₃ ^? cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na⁺/HCO ₃ ^? cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na⁺/HCO ₃ ^? cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

pH Regulation in tissue-cultured bovine lens epithelial cells

Summary The intracellular pH (pH _i ) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO ₃ ^- and 5% CO₂, pH 7.25, pH _i was 7.19±0.02 (sem, n = 95). On removing HCO ₃ ^- and CO₂ there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO ₃ ^- and CO₂ led to a rapid and pronounced fall in pH. Exposure to Na⁺-free AAH again led to a marked fall in pH _i , but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl^– in the presence of HCO ₃ ^- led to a rise in pH _i , while substitution in the absence of HCO ₃ ^- led to a fall in pH _i . The above data indicate a significant role for a sodium-dependent Cl^–-HCO ₃ ^- exchange mechanism in the regulation of pH _i . Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO ₃ ^- led to a marked fall in pH _i , indicating that a Na⁺/H⁺ exchange mechanism also has a significant role in the regulation of pH _i . There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH _i . Larger changes in pH _i were observed in control compared to HCO ₃ ^- -free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH _i with either NH ₄ ⁺ or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO ₃ ^- was 5 mm/pH unit (procaine). However, in the presence of HCO ₃ ^- and CO₂ the buffer capacity increases approximately fourfold, indicating that HCO ₃ ^- is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Rapid increase in pH set-point of the Na+-in-dependent chloride/bicarbonate antiporter in vero cells exposed to heat shock

Internal pH (pH _i ) is in Vero cells regulated mainly by three antiports. Na⁺/H⁺ antiport and Na⁺-dependent Cl⁺/HCO ₃ ⁺ antiport increase pH _i in acidified cells, and Na⁺-independent Cl⁺/HCO ₃ ⁺ antiport lowers pH _i in cells after alkalinization. The activities of the antiporters were altered in cells after exposure to 41–45°C. Under such conditions the Na⁺/H⁺ antiport and the Na⁺-dependent Cl⁺/HCO ₃ ⁺ antiport were both stimulated, whereas the Na⁺-independent Cl⁺/HCO ₃ ⁺ antiport was inhibited in such a way that a higher pH value was required to activate it. This alteration was also induced by some other forms of cellular stress, but did most likely not involve stress proteins as protein synthesis was not required. The possibility of regulation by alteration in protein phosphorylation is discussed.We are grateful to Mrs. Jorunn Jacobsen for her skillful handling of the cell cultures. This work was supported by the Norwegian Research Council for Science and Humanities, the Norwegian Cancer Society and the Lærdal Foundation.     

Proton Pump Activity of Mitochondria-rich Cells : The Interpretation of External Proton-concentration Gradients

We have hypothesized that a major role of the apical H⁺-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl⁻ via an apical Cl⁻/HCO₃ ⁻-exchanger. The activity of the H⁺ pump was studied by monitoring mucosal [H⁺]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 ± 0.07 (mean ± SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 ± 18 μm, corresponding to an estimated “unstirred layer” thickness of 329 ± 29 μm. Mucosal acidification was dependent on serosal pCO₂, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H⁺. The [H⁺] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H⁺]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H⁺ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace''s equation. Model calculations predicted that with a physiological density of MR cells, the [H⁺] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H⁺]–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H⁺]-gradient depended on the mucosal anion such as to indicate that base (HCO₃ ⁻) is excreted in exchange not only for Cl ⁻, but also for Br⁻ and I⁻, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.     

Effect of Prediabetes on Membrane Bicarbonate Transporters in Testis and Epididymis

The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. This includes the control of bicarbonate (HCO₃ ^?) concentration, which plays an essential role in the maintenance of extracellular and intracellular pH (pH_i) values. Diabetes mellitus alters pH_i regulation in mammalian cells, mainly by altering the activity of ion transporters, particularly HCO₃ ^?-dependent mechanisms. Yet, little is known about the effects of this pathology and its prodromal stage, prediabetes, on the membrane transport mechanisms of male reproductive tract cells. Herein, we analyzed protein and mRNA levels of the most relevant HCO₃ ^? transporters of the SLC4 family [anion exchanger 2 (AE2), Na⁺-driven Cl^?/HCO₃ ^? exchanger (NDCBE), electrogenic Na⁺/HCO₃ ^? cotransporter 1 (NBCe1), electroneutral Na⁺/HCO₃ ^? cotransporter 1 (NBCn1)] in the testis and epididymis of a prediabetic animal model. Firstly, we identified the HCO₃ ^? transporters of the SLC4 family, in both testicular and epididymal tissue. Secondly, although no alterations were detected in protein expression, mRNA levels of NBCe1, NBCn1 and NDCBE were significantly increased in the testis of prediabetic rats. On the other hand, in the epididymis, prediabetes caused an increase of AE2 and a decrease of NDCBE protein levels. These alterations may be translated into changes of HCO₃ ^? transepithelial epididymal fluxes in vivo, which may represent a threat for sperm survival. Moreover, these results provide evidence of the molecular mechanism that may be responsible for the significant increase in abnormal sperm morphology already reported in prediabetic rats.     

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

Summary In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a _i Cl^–), sodium (a _i Na⁺) and potassium (a _i K⁺), pH_i, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a _i Cl^–=35 meq/liter, a _i Na⁺=11 meq/liter and a _i K⁺=95 meq/liter. During bilateral substitution the latter two did not change while a _i Cl^– dropped to virtually zero.Mucosal membrane potentials (_ms) were: control,-53 mV; serosal substitution,-51 mV; bilateral substitution,-66 mV; while during mucosal substitution a transient depolarization occurred and the final steady state _ms was-66 mV.During control and bilateral substitution the transepithelial potentials (_ms) did not differ from zero. During unilateral substitutions _ms was small, in the order of magnitude of the errors in the liquid junction potentials near the measuring salt bridges.During bilateral substitution pH _i increased 0.4 pH units. Cellular volume decreased during mucosal substitution to 88% in 40 min; after serosal substitution it transiently increased, but the new steady-state value was not significantly above its control value.Three minutes after mucosal substitution ana _i Cl^– of approx. 10 meq/liter was measured.Chemical concentrations of Na, K and Cl were determined under control conditions and bilateral substitution. Cl concentrations were also measured as a function of time after unilateral substitutions.The data indicate an electrically silent chloride influx mechanism in the brush border membrane and an electrodiffusional chloride efflux in the basolateral membrane. A substantial bicarbonate permeability is present in the basolateral membrane. The results are in agreement with the observed changes in membrane resistances, volume changes and pH changes.     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

Renal cortical basolateral Na+/HCO 3 − cotransporter: I. Partial purification and reconstitution

The renal basolateral Na⁺/HCO ₃ ^– cotransporter is the main system responsible for HCO ₃ ^– transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na⁺/HCO ₃ ^– cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l--phosphatidylcholine (proteoliposomes). Na⁺/HCO ₃ ^– cotransporter activity was assessed as the difference in ²²Na uptake in the presence of HCO ₃ ^– and gluconate. The activity of the Na⁺/HCO ₃ ^– cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na⁺/HCO ₃ ^– cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na⁺/HCO ₃ ^– cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na⁺/HCO ₃ ^– cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na⁺/HCO ₃ ^– cotransporter and suggest that the 56 kD doublet protein may represent the Na⁺/HCO ₃ ^– cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.