Gastric HCO3-stimulated ATPase: Evidence against its microsomal localization in rat fundus mucosa

Rat gastric mucosa was shown to contain a Mg²⁺-dependent ATPase which is stimulated by HCO₃⁻ at pH 8–9.Triton X-100 solubilizes this HCO₃⁻-stimulated, Mg²⁺-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3).The gastric mucosa was resolved into five subcellular fractions by differential centrifugation. A large granule fraction (Fraction M), 28 000 g · min, was characterized by cytochrome c oxidase (marker enzyme for mitochondria). A microsomal fraction (Fraction P), 2 760 000 g · min, was characterized by 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) (plasma membrane).The Mg²⁺-dependent ATPase was demonstrated to have a bimodal mitochondrial membranous localization: 24% of its activity is associated with cytochrome c oxidase, and 75% with 5′-nucleotidase(5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) at pH 8.The HCO₃⁻ addition resulted in two opposite effects: (1) a strong stimulation (84%) in Fraction M; (2) a slight inhibition (12%) in Fraction P.Fraction M was subfractionated by equilibration on a sucrose gradient. It gave rise to a homogeneous mitochondrial (d, 1.17–1.21) Mg²⁺-dependent ATPase, closely associated with cytochrome c oxidase. This ATPase is strongly stimulated (×2) by HCO₃⁻. The subfractionation of Fraction P gave rise to two distinct ATPases: (1) the major one is associated with membranous (d, 1.10–1.15) material marked by 5′-nucleotidase and is slightly inhibited by HCO₃⁻; (2) the other is associated with denser (d, 1.17–1.21) material and is stimulated by HCO₃⁻.The bicarbonate-stimulated fraction of the Mg²⁺-dependent ATPase activity found in the gastric microsomal fraction is assumed to arise from mitochondrial cross-contamination. Further support comes from the optimal HCO₃⁻ concentration. In addition, SCN⁻ is shown to specifically inhibit the ATPase of Fraction M.From these results it appears that the implication of HCO₃⁻-stimulated ATPase in the gastric secretion of H⁺ is not as clear as had been suggested. However, in the view of an ATPase-supported model for H⁺ secretion, attention can be directed towards the Mg²⁺-dependent ATPase found to be associated with microsomes.     

Effect of pH on transepithelial electrical parameters in seawater-adapted eel intestine

The sensitivity to external pH of Cl^- absorption was studied in isolated stripped intestinal mucosa of the eel, Anguilla anguilla, mounted in Ussing chambers. Short-circuit current, transepithelial potential difference and conductance were measured in bathing solutions containing various combinations of HCO₃ ^--concentration (0–25 mmol·l^-1), partial pressure of CO₂ (0–76 mm Hg) and pH (6.9–7.9). A linear relationship was found between pH and short-circuit current in the range of pH studied both in HCO₃ ^-/CO₂ Ringer and in Hepes Ringer. The pH effect was almost completely reversible. It was not affected by the presence of mucosal Ba²⁺ (10^-3 mol·l^-1) but it was inhibited by the presence of luminal (10^-5 mol·l^-1) or serosal (10^-4 mol·l^-1) bumetanide. The results obtained suggest that the Cl^- absorption in the European eel intestine is pH sensitive. The data do not indicate whether the pH affects directly the Na⁺–K⁺–Cl^- cotransport and/or the basolateral Cl^- conductance or other mechanisms indirectly linked to Cl^- absorption.Abbreviations g _t transepithelial conductance - Hepes N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid - I _sc short circuit current - R _t transepithelial resistence - V _t transepithelial potential difference     

Acid-base status in plasma of trout and eel in hypocapnic and normocapnic conditions

Summary The effects of a three week period in hypocapnia (Pw_{CO 2}<0.05 Torr; pH=9.2) at 15 °C on acid-base status (Pa_{CO 2}, pHa, [HCO₃ ^–] and [CO₃ ^{– –}]) and ventilation were investigated in eel and trout and compared to the results obtained after the same period in normocapnia (Pw_{CO 2}=0.25 Torr; pH=8.1). These blood characteristics were continuously measured during the 12-hour period immediately after the transition from hypocapnia to normocapnia, and from normocapnia to hypocapnia.The results show that when transferred from hypocapnic to normocapnic water or conversely, the trout maintains a constant value of pHa by adjusting plasma carbonate and bicarbonate levels andPa_{CO 2}.In eel subjected to the same conditions it was observed that pHa and plasma carbonates level vary whilstPa_{CO 2} remains unchanged.These results are discussed on the basis of either an exchange of HCO₃ ^– ions between intra- and extracellular compartments or a modification of the ionic exchange in branchial cells.Equipe de Recherche associée du Centre National de la Recherche Scientifique N^o 070622     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

The kinetics of anion equilibrium exchange across the red blood cell membrane as measured by means of35S thiocyanate

Summary Up to a SCN^– concentration of about 110mm, the concentration dependence of SCN^– equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits arans acceleration by Cl^– and can be inhibited by H₂DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced intrans acceleration experiments with Cl^– and insensitive to H₂DIDS but susceptible to inhibition by phloretin. At SCN^– concentrations exceeding 110mm, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered.The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0mm at pH 7.4 to 13.3mm at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19×10^–12 to 22×10^–12 moles×cm^–2×sec^–1. The nonsaturable flux component also increases slightly.In accordance with previous observations of Wieth (J. Physiol. (London) 207:563–580, 1970), we find that SCN^– increases K⁺ and Na⁺ permeability. The induced cation-permeability is considerably smaller than the SCN^– exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former.     

Blood-respiratory and acid–base changes during extended diving in the bimodally respiring freshwater turtle<Emphasis Type="Italic"> Rheodytes leukops</Emphasis>

Changes in blood-gas, acid–base, and plasma-ion status were investigated in the bimodally respiring turtle, Rheodytes leukops, during prolonged dives of up to 12 h. Given that R. leukops routinely submerges for several hours, the objective of this study was to determine whether voluntarily diving turtles remain aerobic and simultaneously avoid hypercapnic conditions over increasing dive lengths. Blood PO₂, PCO₂, and pH, as well as plasma concentrations of lactate, glucose, Na⁺, K⁺, Cl^–, total Ca, and total Mg were determined in venous blood collected from the occipital sinus. Blood PO₂ declined significantly with dive length; however, oxy-haemoglobin saturation remained greater than 30% for all R. leukops sampled. No changes were observed in blood PCO₂, pH, [HCO₃^–], or plasma glucose, with increasing dive length. Despite repeated dives lasting more than 2 h, plasma lactate remained less than 3 mmol l^–1 for all R. leukops sampled, indicating the absence of anaerobiosis. Compensatory acid–base adjustments associated with anaerobiosis (e.g. declining [Cl^–], increasing total [Ca] and [Mg]) were likewise absent, with plasma-ion concentrations remaining stable with increasing dive length. Results indicate that R. leukops utilises aquatic respiration to remain aerobic during prolonged dives, thus effectively avoiding the development of a metabolic and respiratory acidosis.     

Specific ion effect as a probable cause of growth inhibition under saline conditions

Summary The replacement of carboxy-proton of IAA, plays an important role in the process of growth under saline conditions. Appreciable changes in pH have ben observed in the presence of CO₃ ^–2 and HCO₃ ^–, and increase in pH appears to be caused owing to the formation of unstable complex of IAA, which soon hydrolyzes and gives alkalinity. The pH change thus achieved favours IAA oxidase activity; therefore, suppression of growth is resulted in CO₃ ^–2 and HCO₃ ^– medium. In the presence of SO₄ ^–2 and Cl^– salts slight lowering of pH takes place which does not favour IAA oxidase activity. It is therefore, presumed that anion (CO₃ ^–2, HCO₃ ^–, SO₄ ^–2 and Cl^–) controls the process of growth and development through the alteration of pH of the medium.     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish

The purpose of this study was to investigate the characteristics of carbonic anhydrase (CA) and the Cl⁻/HCO₃⁻ exchanger (Band 3; AE1) in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish, in order to further understand the strategies of blood CO₂ transport in lower vertebrates and gain insights into the evolution of the vertebrate erythrocyte proteins, CA and Band 3. A significant amount of CA activity was measured in the erythrocytes of bowfin (70 mmol CO₂ min⁻¹ ml⁻¹), although it appeared to be lower than that in the erythrocytes of teleost fish. The turnover number (K_cat) of bowfin erythrocyte CA was intermediate between that of the slow type I CA isozyme in agnathans and elasmobranchs and the fast type II CA in the erythrocytes of the more recent teleost fishes, but the inhibition properties of bowfin erythrocyte CA were similar to the fast mammalian CA isozyme, CA II. In contrast to previous findings, a plasma CA inhibitor was found to be present in the blood of bowfin. Bowfin erythrocytes were also found to possess a high rate of Cl⁻/HCO₃⁻ exchange (6 nmol HCO₃⁻ s⁻¹ cm⁻²) that was sensitive to DIDS. Visualization of erythrocyte membrane proteins by SDS-PAGE revealed a major band in the 100 kDa range for the trout, which would be consistent with the anion exchanger. In contrast, the closest major band for the membranes of bowfin erythrocytes was around the 140 kDa range. Taken together, these results suggest that the strategy for blood CO₂ transport in bowfin is probably similar to that in most other vertebrates despite several unique characteristics of erythrocyte CA and Band 3 in these primitive fish.     

Cellular mechanism of HCO 3 − and Cl− transport in insect salt gland

Summary Active HCO ₃ ^t- secretion in the anterior rectal salt gland of the mosquito larva,Aedes dorsalis, is mediated by a 11 Cl^–/HCO ₃ ^– exchanger. The cellular mechanisms of HCO ₃ ^– and Cl^– transport are examined using ion- and voltage-sensitive microelectrodes in conjunction with a microperfused preparation which allowed rapid saline changes. Addition of DIDS or acetazolamide to, or removal of CO₂ and HCO ₃ ^– from, the serosal bath caused large (20 to 50 mV) hyperpolarizations of apical membrane potential (V _a) and had little effect on basolateral potential (V _bl). Changes in luminal Cl^– concentration alteredV _a in a repid, linear manner with a slope of 42.2 mV/decaloga _Cl ^l –. Intracellular Cl^– activity was 23.5mm and was approximately 10mm lower than that predicted for a passive distribution across the apical membrane. Changes in serosal Cl^– concentration had no effect onV _bl, indicating an electrically silent basolateral Cl^– exit step. Intracellular pH in anterior rectal cells was 7.67 and the calculated was 14.4mm. These results show that under control conditions HCO₃ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. A tentative cellular model is proposed in which Cl enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl^–-selective channel, and HCO ₃ ^– exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of basolateral Cl^– exit and HCO₃ entry, and the effects of serosal addition of the Cl^–/HCO₃ exchange inhibitor, DIDS, on and transepithelial potential (V _ic) suggest strongly that the basolateral membrane is the site of a direct coupling between Cl^– and HCO ₃ ^– movements.     

Luminal alkalinization in the intestine of the goby

Summary The rate of luminal alkalinization in vitro byGillichthys mirabilis posterior intestine as measured by a manual pH stat technique was 0.70±0.05 Equiv/cm² h; acidification of the mucosal medium was never observed. The rate of HCO ₃ ^– secretion (J ^HCO ₃) was reduced by ouabain, serosally-applied DIDS, removal of serosal HCO ₃ ^– and replacement of media Cl^– with gluconate. HCO ₃ ^– secretion was enhanced replacement of Cl^– with isethionate and unaffected by mucosal DIDS, furosemide or acetazolamide.J ^HCO ₃ was reduced at mucosal pH above or below 7.5. These results support active HCO ₃ ^– secretion via a Cl^–/HCO ₃ ^– exchange mechanism on the basolateral membrane and a conductive exit pathway for HCO ₃ ^– , H⁺ or OH^– on the apical membrane.Abbreviations DIDS diisothiocyanostilbene-2,2-disulfonic acid - TEP transepithelial potential - GBR Gillichthyts bicarbonate Ringer - GUR Gillichthys unbuffered, bicarbonate-free Ringer - GER Gillichthys EPPS-buffered, bicarbonate-free Ringer - EPPS N-(2-hydroxyethyl)piperazine-N-3-propanesulfonic acid     

Solubilization and functional reconstitution of a chloride channel fromTorpedo californica electroplax

Summary Chloride channels were detergent-extracted fromTorpedo electroplax plasma membrane vesicles and reconstituted into liposomes by rapid detergent removal and a freeze-thawsonication procedure. Concentrative uptake of³⁶Cl^–, driven by a Cl^– gradient was used to determine conductance properties of reconstituted channels. Chloride flux assayed by this method is strongly selective for Cl^– over cations, is blocked by SCN^–, inactivated by treatment with DIDS, and exhibits an anion selectivity sequence Cl^–>Br^–>F^–>SO ₄ ^2– , as does the voltagegated Cl^– channel fromTorpedo observed in planar lipid bilayers. The channels are localized to the noninnervated face of the electrocyte, and a novel trapped-volume method is used to estimate a channel density on the order of 500 pmol/mg protein. An initial fractionation of the membrane extract by anion exchange chromatography yields fivefold enrichment of the channel activity.     

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO₃, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO₃⁻, but inhibited by Cl⁻, indicating that Cl⁻ stimulation is a distinguishing property of the pea mitochondrial ATPase complex.     

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.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Bicarbonate-stimulated ATPase in the renal proximal tubule luminal (brush border) memebrane.

Brush border membranes of the rabbit renal tubule have an ATPase which was stimulated 60% by 50 mm HCO₃^?. The K_a for HCO₃^? was 36 mm. Kinetic studies of the “HCO₃^?-ATPase” indicate that HCO₃^? had no effect on the K_m for ATP and ATP did not alter the K_a for HCO₃^?. Several anions, notably SO₃^2?, also accelerated the rate of dephosphorylation of ATP. The V for “SO₃^2?-ATPase” was fivefold greater than that for “HCO₃^?-ATPase.” The K_a for SO₃^2? was 0.78 mm. Other anions including Cl^? and phosphates, did not enhance ATPase activity. Thus, of the anions present in the glomerular filtrate in appreciable concentrations only HCO₃^? stimulated the luminal membrane enzyme. The anion-stimulated ATPase activity increased sharply from pH 6.1 to 7.1 and moderately with higher pH. The renal ATPase was not inhibited by SCN^? nor methyl sulfonyl chloride and was relatively insensitive to oligomycin and quercetin. Carbonyl cyanide p-trifluoromethoxy phenylhydrazone increased the basal rate of the membranal ATPase, suggesting that the ATPase activity is limited by transmembrane H⁺ flux. Carbonic anhydrase significantly increased the HCO₃^?-stimulated ATPase activity. This increment was blocked by Diamox. These findings provide evidence consistent with the hypothesis that the brush border membrane ATPase is involved in the extrusion of H⁺ from tubular cell to lumen and support suggested interrelationships between HCO₃^?-stimulated ATPase, H⁺ secretion, and bicarbonate transport in the kidney.     

Comparative properties of sensitive to GABA<Subscript>A</Subscript>-ergic ligands Cl<Superscript>−</Superscript>, HCO<Subscript>3</Subscript><Superscript>−</Superscript>-activated Mg<Superscript>2+</Superscript>-ATPase from brain plasma membranes of fish and rats

Action of Cl^? + HCO₃ ^?1 ions on Mg²⁺-ATPase from brain plasma membranes of fish and rats has been studied. Maximal effect of the anions on the “basal” Mg²⁺-ATPase activity is revealed in the presence of 10 mM Cl^? and 3 mM HCO₃ ^?1 at physiological values of pH of incubation medium. The studied Cl^?, HCO₃ ^?-activated Mg²⁺-ATPases of both animal species, by their sensitivity to SH-reagents (5,5-dithio-bis-nitrobenzoic acid, N-ethylmaleimide), oligomycin, and orthovanadate, are similar to transport ATPase of the P-type, but differ from them by molecular properties and by sensitivity to ligands of GABA_A-receptors. It has been established that the sensitive to GABA_A-ergic ligands, Cl^?, HCO₃ ^?-activated Mg²⁺-ATPase from brain of the both animal species is protein of molecular mass around 300 kDa and of Stock’s radius 5.4 nm. In fish the enzyme is composed of one major unit of molecular mass approximately 56 kDa, while in rats-of three subunits of molecular masses about 57, 53, and 45 kDa. A functional and structural coupling of the ATP-hydrolyzing areas of the studied enzyme to sites of binding of GABA_A-receptor ligands is suggested.     

Exchange of HCO 3 − for monovalent anions across the human erythrocyte membrane

Summary A stopped-flow rapid reaction apparatus was used for measuring changes in extracellular pH (pH _o ) of red cell suspensions under conditions wheredpH _o /dt was determined by the rate of HCO ₃ ^– /X ^– exchange across the membrane (X ^–=Cl^–, Br^–, F^–, I^–, NO ₃ ^– or SCN^–). The rate of the exchange at 37°C decreased forX ^– in the order: Cl^–>Br^–>F^–>I^–>NO ₃ ^– >SCN^–, with rate constants in the ratios 10.860.770.550.520.31. When HCO ₃ ^– is exchanged for Cl^–, Br^–, F^–, NO ₃ ^– or SCN^–, a change in the rate-limiting step of the process takes place at a transition temperature (T _T ) between 16 and 26°C. In I^– medium, however, no transition temperature is detected between 3 and 42°C. AlthoughT _T varies withX ^–, the activation energies both above and belowT _T are similar for Cl^–, Br^–, NO ₃ ^– and F^–. The values of activation energy are considerably higher whenX ^–=I^– or SCN^–. The apparent turnover numbers calculated for HCO ₃ ^– /X ^– exchange (except forX ^–=I^–) at the correspondingT _T ranged from 140 to 460 ions/site ·sec for our experimental conditions. These findings suggest that: (i) HCO ₃ ^– /X ^– exchange for allX ^– studied takes place via the rapid anion exchange pathway; (ii) the rate of HCO ₃ ^– /X ^– exchange is influenced by the specific anions involved in the 11 obligatory exchange; and (iii) the different transition temperatures in the Arrhenius diagrams of the HCO ₃ ^– /X ^– exchange do not seem to be directly related to a critical turnover number, but may be dependent upon the influence ofX ^– on protein-lipid interactions in the red blood cell membrane.     

HCO3 ?/Cl? Exchange Inactivation and Reactivation during Mouse Oocyte Meiosis Correlates with MEK/MAPK-Regulated Ae2 Plasma Membrane Localization

Background

Germinal Vesicle (GV) stage mouse oocytes in first meiotic prophase exhibit highly active HCO₃ ⁻/Cl⁻ exchange—a class of transport nearly ubiquitously involved in regulation of intracellular pH and cell volume. During meiosis, however, oocyte HCO₃ ⁻/Cl⁻ exchange becomes inactivated during first metaphase (MI), remains inactive in second metaphase (MII), and is reactivated only after egg activation. Previous work using pharmacological manipulations had indicated that activity of the MEK/MAPK signaling pathway was negatively correlated with HCO₃ ⁻/Cl⁻ exchange activity during meiosis. However, the mechanism by which the exchanger is inactivated during meiotic progression had not been determined, nor had the role of MEK/MAPK been directly established.

Methodology/Principal Findings

Expression of a constitutively active form of MEK (MAP kinase kinase), which prevented the normal downregulation of MAPK after egg activation, also prevented reactivation of HCO₃ ⁻/Cl⁻ exchange. Conversely, suppression of endogenous MAPK activity with dominant negative MEK activated the normally quiescent HCO₃ ⁻/Cl⁻ exchange in mature MII eggs. A GFP-tagged form of the HCO₃ ⁻/Cl⁻ exchanger isoform Ae2 (Slc4a2) was strongly expressed at the GV oocyte plasma membrane, but membrane localization decreased markedly during meiotic progression. A similar pattern for endogenous Ae2 was confirmed by immunocytochemistry. The loss of membrane-localized Ae2 appeared selective, since membrane localization of a GFP-tagged human dopamine D1 receptor did not change during meiotic maturation.

Conclusions

Direct manipulation of MAPK activity indicated that GFP-tagged Ae2 localization depended upon MAPK activity. Inactivation of HCO₃ ⁻/Cl⁻ exchange during the meiotic cell cycle may therefore reflect the loss of Ae2 from the oocyte plasma membrane, downstream of MEK/MAPK signaling. This identifies a novel role for MEK/MAPK-mediated cytostatic factor (CSF) activity during meiosis in membrane protein trafficking in mouse oocytes, and shows for the first time that selective retrieval of membrane proteins is a feature of meiosis in mammalian oocytes.     

Effects of rapid transfer from sea water to fresh water on respiratory variables,blood acid-base status and O2 affinity of haemoglobin in Atlantic salmon (Salmo salar L.)

Summary Oxygen consumption, gill ventilation, blood acid-base/ionic status and haemoglobin oxygen affinity were studied in seawater-adapted adult salmon (Salmo salar) during five weeks after transfer into fresh water. Freshwater exposure induced the following changes: Standard oxygen consumption ( ) and ventilatory flow ( ) decreased markedly during the first days after transfer, then decreased more gradually until a new steady-state was achieved at which and were about 80% and 56% of the control values, respectively. The marked increase in oxygen extraction coefficient (Ew _{O 2}) and the marked decrease in the oxygen convection requirement ( ) were associated with a reduction in the partial pressure of carbon dioxide in arterial blood (Pa _{CO 2}), in spite of a decrease of both ventilatory flow and water CO₂ capacitance. These results suggested that transfer into fresh water induced an increase in branchial diffusive conductance. A biphasic pattern was observed in the time-course of the changes in both plasma ion concentration and acid-base status. During the first 10 days, plasma Na⁺, K⁺, and Cl^– concentrations fell abruptly, then more gradually. [Cl^–] decreased more than [Na⁺] resulting in a progressive increase in the [Na⁺]/[Cl^–] ratio. During the second phase of acclimation to fresh water plasma Na⁺, K⁺, and Cl^– concentrations progressively increased. [Cl^–] increased more than [Na⁺], so that [Na⁺]/[Cl^–] ratio decreased. Transfer into fresh water did not significantly change plasma lactate concentration. Upon exposure to fresh water, blood pH increased from 7.94±0.04 to 8.43±0.06 at day 10 and then decreased to 8.08±0.03 at day 34. The increase in blood pH induced by transfer to fresh water initially represented a mixed metabolic/respiratory alkalosis. However, after 15 days Pa _{CO 2} had returned to pretransfer values and the alkalosis was purely metabolic. The metabolic component of the alkalosis was associated with appropriate changes in the plasma strong ion difference (S.I.D.). Blood alkalosis moved the oxygen dissociation curve to the left, so that P₅₀ was decreased by 30% below the value in seawater for the maximal increase in blood pH. This rise in haemoglobin affinity for O₂, associated with a marked increase in blood buffer capacity, are regarded as adaptative processes allowing the salmon to cope with the markedly increased energy expenditure required for upstream migration.