Cl−HCO3 exchange in rat renal basolateral membrane vesicles

Pathways for HCO₃⁻ transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl⁻---HCO₃⁻ exchange was assessed directly by ³⁶Cl⁻ tracer flux measurements and indirectly by determinants of acridine orange absorbance changes. Under 10% CO₂/90% N₂ the imposition of an outwardly directed HCO₃⁻ concentration gradient (pH_o 6/pH_i 7.5) stimulated Cl⁻ uptake compared to Cl⁻ uptake under 100% N₂ in the presence of a pH gradient alone. Mediated exchange of Cl⁻ for HCO₃ was suggested by the HCO₃⁻ gradient-induced concentrative accumulation of intravesicular Cl⁻. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO₃⁻ gradient-driven Cl⁻ uptake further suggesting chemical as opposed to electrical Cl−HCO₃⁻ exchange coupling. Although basolateral membrane vesicle Cl⁻ uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl conductive pathway served to distinguish this mode of Cl⁻ translocation from HCO₃ gradient-driven Cl⁻ uptake. No evidence for cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pH_o 7.5/pH_i 6), a HCO₃⁻ dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl⁻ concentration gradient. The basolateral membrane vesicle origin of the observed Cl⁻−HCO₃⁻ exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl⁻ on HCO₃⁻ gradient-driven Na⁺ uptake suggesting a basolateral membrane Na⁺−HCO₃⁻ for Cl⁻ exchange mechanism, no effect of Na⁺ on Cl−HCO₃⁻ exchange was observed in the present study.     

ATP-dependent chloride transport in plasma membrane vesicles from Aplysia intestine

A Cl⁻-stimulated ATPase activity, which is sensitive to both thiocyanate and vanadate, has been localized to the plasma membrane of Aplysia enterocytes. Utilizing plasma membrane vesicles from Aplysia enterocytes, ATP stimulated Cl⁻ uptake to approximately 2.5-times that of control in a Na⁺, K⁺ and HCO₃⁻-free medium. This ATP-dependent Cl⁻ uptake was sensitive to both thiocyanate and vanadate. These results are consistent with the hypothesis that the active Cl⁻ absorptive process in Aplysia intestine could be a Cl⁻-stimulated ATPase found in the enterocyte plasma membrane.     

Molecular Characterization and In Situ Localization of a Mouse Retinal Taurine Transporter

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na⁺- and Cl^?-dependent [³H]taurine transport activity in Xenopus laevis oocytes that saturated with an average K_m of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl^? was varied, a single saturation process with an average apparent equilibrium constant (K_Cl?) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na⁺ was varied (apparent equilibrium constant, K_Na+～54.8 mM). Analyses of the Na⁺- and Cl^?-concentration dependence data suggest that at least two Na⁺ and one Cl^? are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β-alanine > l -diaminopropionic acid > guanidinoethane sulfonate > β-guanidinopropionic acid > chloroquine > γ-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.     

Potassium-Induced Stimulation of Glutamate Uptake in Mouse Cerebral Astrocytes: The Role of Intracellular pH

Abstract: The Na⁺-glutamate cotransporters are believed to countertransport OH^? and K⁺. Previous evidence that the velocity of glutamate uptake can exceed the acid extrusion capacity of astrocytes raised the question of whether intracellular pH can become rate limiting for glutamate uptake. Cytoplasmic buffering capacity and acid extrusion in astrocytes are partially HCO₃^? dependent. Also, it was reported recently that raising extracellular [K⁺] alkalinizes astrocyte cytoplasm by an HCO₃^?-dependent mechanism. Here, we have compared glutamate uptake in HCO₃^?-buffered and HCO₃^?-depleted solutions at varying [K⁺]. We observed a pronounced stimulation of glutamate uptake by extracellular K⁺ (3–24 mM) that was substantially HCO₃^? dependent and affected preferentially the uptake of high concentrations (>25 µM) of glutamate. Stimulation of uptake by low extracellular [K⁺] (1.5–3 mM) was less dependent on HCO₃^?. Potassium-induced stimulation of uptake was weaker in rat astrocyte cultures than in mouse. The effects of Ba²⁺ and amiloride on glutamate uptake, as well as the HCO₃^?-dependent stimulatory effects of K⁺ and the species difference, all related consistently to effects on intracellular pH. The effects on uptake, however, were much larger than predicted by the associated changes in electrochemical gradient of OH^?. A “bimodal” scheme for glutamate transport can account qualitatively for the observed correlation between intracellular pH and velocity of glutamate uptake.     

Poliovirus-induced intracellular alkalinization involves a proton ATPase and protein phosphorylation

We reported previously that poliovirus infection induces alkalinization in HeLa cells and that an alkaline intracellular pH (pHi) promoted viral replication. Additional experiments were carried out to understand the underlying mechanism. Virus-infected or control monolayer cultures were incubated with nominally bicarbonate-free Eagle's minimal essential medium (MEM) buffered with N-2-hydroxyethylpiperazine-N-3-ethanesulfonic acid (HEPES), and immediately following preincubations, changes in pHi were monitored via benzoic acid uptake around 2 h postinfection. The absence of pH increase in cells infected with ultraviolet light-inactivated virus (UV-virus) indicated that viral gene expression was required for this effect. On the other hand, lack of effect of 3 mM guanidine, an inhibitor of poliovirus-specific RNA but not protein synthesis, suggested that translation of input viral genome RNA is sufficient for the pH increase. Activation of Na⁺/H⁺ exchange, Cl^?HCO^?₃ exchange, or H⁺-ATPase was considered as possible mechanisms by which alkalinization occurs in virus-infected cells. Na⁺/H⁺ exchange was excluded because the pH effect occurred in a Na⁺/H⁺ exchange deficient HeLa cell mutant. Similarly, Cl^?/HCO^?₃ exchange was excluded because virus-specific alkalinization was evident in the presence of Cl^? or bicarbonate deficient medium and was not associated with an increase in HCO^?₃ uptake or a decrease in Cl^? uptake. Lack of dependence on Na⁺, abrogation by 10 μM 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), and resistance to 1 mM vandate suggested that this effect was due to the activation of a vacuolar-type (V) proton ATPase. Studies using protein kinase inhibitors indicated that activation of the ATPase in virus-infected cells probably involved protein kinase C-mediated phosphorylation. © 1993 Wiley-Liss, Inc.     

Functional expression of the murine D2, D3 and D4 dopamine receptors in Xenopus laevis oocytes

The different murine D2-type dopamine receptors (D_2L, D_2S, D_3L, D_3S, and D₄) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [¹²⁵I]sulpride. They were all shown to activate Cl⁻ influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na⁺/K⁺/2Cl⁻ cotransporter component of the Cl⁻ influx from the total unidirectional Cl⁻ influx. The D_3L subtype was found to operate exclusively through the bumetanide-insensitive Cl⁻ influx whereas the other D2-type receptors acted on the Na⁺/K⁺/2Cl⁻ cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na⁺/K⁺/2Cl⁻ cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.     

Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes

To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit CI⁻ secretion, their effects on CI⁻ transport and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl⁻ sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca²⁺-sensitive fluorescent probe, fura-2AM were used to assess Cl⁻ transport and [Ca²⁺]_i, respectively. Basal Cl⁻ transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl⁻ channel blocker diphenylamine-2-carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na⁺/K⁺/2Cl⁻ cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I⁻ > F⁻ ≥ Cl⁻ > Br⁻. DPC inhibited I⁻ influx by ∼50%, F⁻ influx by 80%, Cl⁻ influx by 85%, and Br⁻ influx by 90%. Furosemide significantly inhibited influx of Br⁻ (84%) and Cl⁻ (81%) but not of F⁻ and I⁻. The effects of agents known to alter biological response by increasing [Ca²⁺]_i in other epithelial systems were used to stimulate Cl⁻ transport. Cl⁻ influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca²⁺ ionophore A23187 (2.05 ± 0.40). The Cl⁻ permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl⁻ influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl⁻ permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl⁻ transport by activating the Na⁺/K⁺/2Cl⁻ cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl⁻ channels and the cotransporter, and their actions are cyclooxygenase-independent. © 1996 Wiley-Liss, Inc.     

Measurement of intracellular chloride activity in mouse liver slices with microelectrodes

Steady-state membrane potential (V_m) and intracellular Cl⁻ activity (a_Clⁱ) were measured with double-barreled Cl⁻-selective microelectrodes in mouse liver slices. In bathing solutions (33.8° C) containing pyruvate, glutamate, fumarate, and glucose, V_m and a_Clⁱ were −27.6 ± 1.0 mV and 32.6 ± 1.5 mM, respectively. This apparent value of a_Clⁱ exceeded the level required for passive distribution of this ion (a_Cl^eq = 26.4 ± 1.3 mM) by 6.2 ± 1.0 mM. This difference was essentially unchanged in experiments where (i) Na⁺ was replaced by choline, (ii) HCO₃⁻ was removed, and (iii) Cl⁻ was replaced by gluconate. These data argue against the presence of Na⁺- or HCO₃⁻-coupled Cl⁻ transport mechanisms in the plasma membrane of mouse liver cells. This implies that a_Clⁱ is in fact at equilibrium and interference with the response of Cl⁻-selective microelectrodes by intracellular anions is responsible for the apparent difference between a_Clⁱ and a_Cl^eq. We found that Cl⁻-selective microelectrodes containing Corning 477315 ligand are sensitive to taurocholate, a representative bile salt. Their selectivity to taurocholate is about 60-times their selectivity towards Cl⁻. This suggests that interference of bile acids at concentrations normally present in hepatocytes with determinations of a_Clⁱ can account for the apparent difference a_Clⁱ − a_Cl^eq.     

Understanding the gastrointestinal physiology and responses to feeding in air-breathing Anabantiform fishes

The Mekong Delta is host to a large number of freshwater species, including a unique group of facultative air-breathing Anabantiforms. Of these, the striped snakehead (Channa striata), the climbing perch (Anabas testudineus), the giant gourami (Osphronemus goramy) and the snakeskin gourami (Trichogaster pectoralis) are major contributors to aquaculture production in Vietnam. The gastrointestinal responses to feeding in these four species are detailed here. Relative intestinal length was lowest in the snakehead, indicating carnivory, and 5.5-fold greater in the snakeskin, indicating herbivory; climbing perch and giant gourami were intermediate, indicating omnivory. N-waste excretion (ammonia-N + urea-N) was greatest in the carnivorous snakehead and least in the herbivorous snakeskin, whereas the opposite trend was observed for net K⁺ excretion. Similarly, the more carnivorous species had a greater stomach acidity than the more herbivorous species. Measurements of acid–base flux to water indicated that the greatest postprandial alkaline tide occurred in the snakehead and a potential acidic tide in the snakeskin. Additional findings of interest were high levels of both PCO₂ (up to 40 mmHg) and HCO₃⁻ (up to 33 mM) in the intestinal chyme of all four of these air-breathing species. Using in vitro gut sac preparations of the climbing perch, it was shown that the intestinal net absorption of fluid, Na⁺ and HCO₃⁻ was upregulated by feeding but not net Cl⁻ uptake, glucose uptake or K⁺ secretion. Upregulated net absorption of HCO₃⁻ suggests that the high chyme (HCO₃⁻) does not result from secretion by the intestinal epithelium. The possibility of ventilatory control of PCO₂ to regulate postprandial acid–base balance in these air-breathing fish is discussed.     

Activation of Cl− channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts

Ionic channels regulated by extracellular Ca²⁺ concentration ([Ca²⁺]₀) were examined in freshly isolated rabbit osteoclasts. K⁺ current was suppressed by intracellular and extracellular Cs⁺ ions. In this condition, high [Ca²⁺]₀ evoked an outwardly rectifying current with a reversal potential of about −25 mV. When the concentration of extracellular Cl⁻ ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4′,4-diisothiocyanostilbene-2′,2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl⁻ channels. Cd²⁺ or Ni²⁺ caused a transient activation of the Cl⁻ current in contrast to the sustained activation elicited by Ca²⁺. Intracellular 20 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl⁻ current. Either when the osmolarity of extracellular medium was increased, or when 100 μM cAMP was dissolved in the patch pipette solution, high [Ca²⁺]₀ still elicited the Cl⁻ current, indicating that the divalent cation-induced Cl⁻ current was carried through Ca²⁺-activated Cl⁻ channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl⁻ current, indicating that the activation of Cl⁻ current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl⁻ channels under whole cell clamp. Intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) activated the Cl⁻ current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl⁻ current was mediated through a G protein, and that an increase in [Ca²⁺]_i was critical for the activation of Cl⁻ channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl⁻ current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca²⁺-activated Cl⁻ channels. © 1996 Wiley-Liss, Inc.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Carbonic anhydrase,a respiratory enzyme in the gills of the shore crabCarcinus maenas

This paper summarizes investigations on the enzyme carbonic anhydrase (CA) in the gills of the osmoregulating shore crabCarcinus maenas. Carbonic anhydrase, an enzyme catalyzing the reversible hydration of CO₂ to HCO₃ ⁻ and H⁺, is localized with highest activities in the posterior salt-transporting gills of the shore crab- and here CA activity is strongly dependent on salinity. Contrary to the earlier hypothesis established for the blue crabCallinectes sapidus that cytoplasmic branchial CA provides the counter ions HCO₃ ⁻ and H⁺ for apical exchange against Na⁺ and Cl⁻, the involvement of CA in NaCl uptake mechanisms can be excluded inCarcinus. Differential and density gradient centrifugations indicate that branchial CA is a predominantly membrane-associated protein. Branchial CA was greatly inhibited by the sulfonamide acetazolamide (AZ) K_i=2.4·10⁻⁸ mol/l). Using the preparation of the isolated perfused gill, application of 10⁻⁴ mol/l AZ resulted in an 80% decrease of CO₂/HCO₃ ⁻ excretion. Thus we conclude that CA is localized in plasma membranes, maintaining the CO₂ gradient by accelerating adjustment of the pH-dependent CO₂/HCO₃ ⁻ equilibrium.     

Anion uptake and acid-base and ionic effects during isolated and combined exposure to hypercapnia and nitrite in the freshwater crayfish, Astacus astacus

Nitrite influx into crayfish showed saturation kinetics, supporting a carrier-mediated uptake. Addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS: at 10⁻⁵, 10⁻⁴ and 10⁻³M) and bumetanide (at 10⁻⁵ M and 10⁻⁴ M) to the ambient water did not significantly affect nitrite influx. Rather than suggesting that neither Cl⁻/HCO₃ ⁻ exchange nor K⁺/Na⁺/2Cl⁻ cotransport were involved in the transport, this may reflect that the gill cuticle has a low permeability to the pharmacological agents, or that the sensitivity of the transport mechanism to the inhibitors is low. Nitrite accumulation in the haemolymph was significantly decreased during hypercapnic conditions compared to normocapnic conditions. This supports the idea that an acid–base regulatory decrease in Cl⁻(influx)/HCO₃ ⁻ (efflux) induced by hypercapnia should decrease NO₂ ⁻ uptake if NO₂ ⁻ and Cl⁻ share this uptake route. The respiratory acidosis caused by exposure to hypercapnia alone was partially compensated by HCO₃ ⁻ accumulation in the haemolymph. Combined exposure to hypercapnia and nitrite improved pH recovery, mainly by augmenting the [HCO₃ ⁻] increase, but also by decreasing haemolymph PCO₂. Exposure to nitrite in normocapnic water induced an initial increase in haemolymph [HCO₃ ⁻] and later also a decrease in PCO₂. Thus, the improved acid-base compensation during combined hypercapnia and nitrite exposure was an amplification of this nitriteinduced response. Haemolymph base excess rose much more than haemolymph [Ca], suggesting that transfer of acid-base equivalents between animal and water was more important than H⁺ buffering by exoskeletal CaCO₃ in mediating the increase in haemolymph [HCO₃ ⁻]. Accepted: 27 June 2000     

Cl− influx across posterior gills of the Chinese crab (Eriocheir sinensis): potential energization by a V-type H+-ATPase

Cl⁻ absorption across isolated, perfused gills of freshwater adapted Chinese crabs (Eriocheir sinensis) was analysed by measuring transepithelial potential differences (PD_te) and radioactive tracer fluxes across isolated, perfused posterior gills. Applying hemolymph-like NaCl salines on both sides of the epithelium PD_te amounted to −30±1 mV (n=14). Undirectional Cl⁻ influxes of 470±38 and effluxes of 245±27 μmol·hr⁻¹·g⁻¹ wet weight (ww) (n=14) resulted in a Cl⁻ net influx of 226±31 μmol·hr⁻¹·g⁻¹ ww. Symmetrical substitution of Na⁺ by choline resulted in a substantial hyperpolarisation of the gill. Cl⁻ influx was unchanged under these conditions. However, net influx of Cl⁻ decreased by 40%, due to an increase of the Cl⁻ efflux.Nevertheless, a significant Cl⁻ net influx remained which was independent of the presence of Na⁺. When 2 mmol/l ouabain were added to the internal perfusion medium, PD_te increased, although the fluxes remained unchanged. Following external application of 1μmol/l of the V-type H⁺-ATPase inhibitor bafilomycin, A_l PD_te and Cl⁻ effluxes were not significantly affected. However, Cl⁻ influxes decreased. These findings suggest that Cl⁻ can be taken up independently of Na⁺ and that active Na⁺ independent Cl⁻ uptake across the posterior gill of Eriocheir sinensis is probably driven by a V-type H⁺-ATPase localized in the apical membrane.     

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl−/HCO3− exchanger,AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO₂ to HCO₃^?+H⁺. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl^?/HCO₃^? exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl^?/NO₃^? exchange assays, which were independent of CA activity, and in Cl^?/HCO₃^? exchange assays. Transport was measured by following changes of intracellular [Cl^?] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl^?/NO₃^? exchange activity with EC₅₀ values in the range 0.22–2.8 μM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 μM of each compound was only 22–53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl^?/HCO₃^? exchange assays, which depend on functional CA to produce transport substrate, 40 μM celecoxib inhibited AE1 by 62±4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K⁺] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na⁺] was slightly lower than the extracellular [Na⁺] and was maintained at this level through a vanadate-sensitive transport system. Na⁺ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na⁺ uptake when compared with furosemide-sensitive K⁺ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na⁺, K⁺, and Cl^? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na⁺, K⁺, Cl^?, H⁺ and HCO₃ ^? fluxes are regulated in these cells.