Photosynthetic bicarbonate utilization in the aquatic angiospermsPotamogeton andElodea

Summary As the CO₂ supply often limits photosynthesis a number of aquatic species use HCO ₃ ^– as carbon source as well. The use of HCO ₃ ^– leads to the production of one OH^– for every molecule/CO₂ fixed. The OH^– is excreted into the medium. We studied the mechanism of HCO ₃ ^– utilization in the leaves ofElodea andPotamogeton. In the so-called polar leaves of these plants the HCO ₃ ^– uptake takes place at the lower and OH^–-release at the upper epidermis. This flux of negative charge is balanced by a kation flux in the same direction. The use of HCO ₃ ^– and the influx of kations is accompanied by a pH drop. The release of OH^– and kations at the upper epidermis causes a raise of the pH there. The pH changes and the kation concentrations (in the present experiments K⁺) are measured by means of miniature electrodes. From this the CO₂ (including H₂CO₃), HCO ₃ ^– and CO ₃ ⁼ concentrations were calculated. When the light is turned on, after a dark period, the pH increases simultaneously at both sides for 5–10 minutes. During this so-called a-polar phase there is no K⁺ transport through the leaf. Experiments at different ambient pH's and comparison with other aquatic species shows that this initial pH raise results from CO₂ fixation. After 5–10 minutes the polar phase and HCO ₃ ^– utilization start. At the lower side the pH and [K⁺] drop, at the upper side pH and [K⁺] increase. During the a-polar phase [CO₂] at the lower epidermis decreased, as expected. Whereas in the a-polar phase the CO₂ concentration at this side very markedly increased. This sharp increase of [CO₂] may be explained either by CO₂ diffusion from the leaf cells previously taken up as HCO ₃ ^– or by a proton (H⁺) extrusion at the lower epidermis causing conversion of HCO ₃ ^– into CO₂ in the cell wall. This latter mechanism is discussed in more detail.     

Mechanism of acquisition of exogenous bicarbonate by internodal cells of Chara corallina

Electrophysiological measurements on internodal cells of the alga, Chara corallina Klein ex Willd., em. R.D.W., showed that the potential across the plasmalemma was sensitive to the level of exogenous HCO ₃ ^- . In alkaline solutions (pH 8) the membrane potential depolarized by 50–75 mV when exogenous HCO ₃ ^- was removed from the bathing medium. In the presence of exogenous HCO ₃ ^- , the membrane potential rapidly hyperpolarized when the cell was given a brief dark treatment; in the light the potential was approx.-240 mV; after the cell had been in the dark for 3–6 min the potential was -330 to -350 mV. In the absence of exogenous HCO ₃ ^- the potential only hyperpolarized slowly and to a much smaller extent when cells were placed in the dark. Upon re-illuminating the cell, the potential further hyperpolarized, transiently, and then rapidly depolarized back towards the light-adapted value. (These responses were only obtained when cells were not perturbed by microelectrode insertion into the vacuole.) Analysis of membrane potential and experiments with the extracellular vibrating electrode indicated a high level of correlation between the light- and dark-induced changes in membrane potential and extracellular currents. However, when experiments were conducted in HCO ₃ ^- -free media that contained 1.0 mM phosphate buffer, pH 8, it was found that the dark-induced hyperpolarization of the membrane potential and the light-dependent extracellular currents could be maintained in the absence of exogenous HCO ₃ ^- . These results are interpreted in terms of two basic models by which internodal cells of C. corallina may acquire exogenous HCO ₃ ^- for photosynthesis. They are consistent with HCO ₃ ^- being transported across the plasmalemma via an electrically neutral HCO ₃ ^- –H⁺ cotransport system. The hyperpolarizing response is thought to be the consequence of the operation of an electrogenic H⁺-translocating ATPase that has a transport stoichiometry of 1 H⁺ per ATP hydrolyzed.Abbreviation CPW/B artificial Chara pond water containing exogenous bicarbonate     

Short-chain fatty acids and CO2 as regulators of Na+ and Cl− absorption in isolated sheep rumen mucosa

Summary Unidirectional ²²Na⁺ and ³⁶Cl^– fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO₂/HCO _– ³ -containing and CO₂/HCO _– ³ -free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39 mM) significantly enhanced mucosal-toserosal Na⁺ absorption without affecting the Cl^– transport in the same direction. Short-chain fatty acid stimulation of Na⁺ transport was at least partly independent of Cl^– and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na⁺ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO₂ in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na⁺ flux. Along with its effect on sodium transport, raising the PCO₂ also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO₂ permeate the cell membrane and produce a raise in intracellular H⁺ concentration. This stimulates an apical Na⁺/H⁺ exchange, leading to increased Na⁺ transport. The stimulatory effect of CO₂ on Cl^– transport is probably mediated by a Cl^–/HCO _– ³ exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.Abbreviations DIDS 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid - G _t transepithelial conductance (mS·cm^-2) - HSCFA undissociated short-chain fatty acids - J _ms mucosal-to-serosal flux (Eq · cm^-2 · h^-1) - J _net net flux (Eq · cm^-2 · h^-1) - J _sm serosal-to-mucosal flux (Eq · cm^-2 · h^-1) - PD transepithelial potential difference (mV) - SCFA dissociated short-chain fatty acids - SCFA short-chain fatty acids     

Dependence of cell membrane conductances on bathing solution HCO 3 − /CO2 inNecturus gallbladder

Summary The effects of bathing solution HCO ₃ ^– /CO₂ concentrations on baseline cell membrane voltages and resistances were measured inNecturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO ₃ ^– /CO₂ Ringer's solutions (2.4mm HCO ₃ ^– /air or 1mm HEPES/air) or a high HCO ₃ ^– /CO₂ Ringer's (10mm HCO ₃ ^– /1% CO₂). The principal finding of these studies was that the apical membrane fractional resistance (fR _a) was higher in tissues bathed in the 10mm HCO ₃ ^– /CO₂ Ringer's, averaging 0.87±0.06, whereasfR _a averaged 0.63±0.07 and 0.48±0.08 in 2.4mm HCO ₃ ^– and 1mm HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical (R _a) and basolateral membrane (R _b) resistances in tissues bathed in 10mm HCO ₃ ^– /1% CO₂ Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO ₃ ^– /CO₂ Ringer's, the higher value offR _a was found to be due to both an increase inR _a and a decrease inR _b. The higher values offR _a and lower values ofR _b confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K⁺ and Cl^– substitutions. The results of these studies suggest that an electrodiffusive Cl^– transport mechanism exists at the basolateral membrane of tissues bathed in a 10mm HCO ₃ ^– /1% CO₂ Ringer's, which can explain in part the fall inR _b. The above observations are discussed in terms of a stimulatory effect of solution [HCO ₃ ^– /PCO₂ on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes.     

Electrogenic proton secretion in the Hindgut of the desert locust,Schistocerca gregaria

Summary The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. Previous studies conducted in the nominal absence of exogenous CO₂ and HCO ₃ ^– suggested that the acidification was due to a proton-secretory rather than bicarbonate-reabsorptive mechanism (Thomson, R.B., Speight, J.D., Phillips, J.E. 1988. J. Insect Physiol. 34:829–837). This conclusion was confirmed in the present study by demonstrating that metabolic CO₂ could not contribute sufficient HCO ₃ ^– to the lumen to account for the rates of rectal acidification observed under the nominally CO₂/ HCO ₃ ^– -free conditions used in these investigations.Rates of luminal acidification (J _H ⁺) were completely unaffected by changes in contraluminal pH, but could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of J _H ⁺ was not dependent on Na⁺, K⁺, Cl^–,Mg²⁺, or Ca²⁺ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10–16%) of J _H ⁺ measured under these conditions could be attributed to an apical amiloride-inhibitable Na⁺/H⁺ exchange mechanism.This work was supported by operating grants to J.E.P. and postgraduate scholarships to R.B.T. from Natural Sciences & Engineering Research Council, Canada.     

Bicarbonate transport inChara corallina: evidence for cotransport of HCO 3 − with H+

Summary Experiments were undertaken on the fresh water algaChara corallina to determine the form of inorganic carbon (CO₂ or HCO ₃ ^– ) which enters the cell during photosynthesis at alkaline pH. Recent proposals have centered on the possibility that proton efflux in alkaline solution is able to generate, in the immediate vicinity of the cell, a sufficiently low pH to raise the partial pressure of CO₂, and hence facilitate its passive permeation into the cell. Predictions have been made by modelling this situation (N.A. Walker, F.A. Smith & I.R. Cathers, 1980,J. Membrane Biol. 5751–58, J.M. Ferrier, 1980,Plant Physiol. 661198–1199), and these were tested by placing recessed-tip pH microelectrodes in the unstirred layer surrounding cells in stagnant solution (bulk pH 8.2, buffered only with 1mm HCO ₃ ^– ). Even as close as 2 m from the cell wall, the pH was typically 7.2 to 7.6 in the acid band center — over 1 pH unitgreater than that suggested by the models for CO₂ entry at the necessary rate for C-fixation. Further evidence for the entry of HCO ₃ ^– , rather than CO₂, at high solution pH was obtained from experiments in which the radial pH gradient in the unstirred layer was reduced. Buffer solutions containing 5mm phosphate or 5mm HEPES, raised the pH at the cell surface in the acid regions from around 7.2 to 7.8 or higher. This pH increase (reduction in acid gradient) would have greatly reduced the CO₂ level at the cell surface and should, therefore, have greatly reduced the CO₂-related¹⁴C-influx. However,¹⁴C-fixation was reduced by only 31% (phosphate) or 15% (HEPES), compared with buffer-free controls. Reduction of the unstirred layer thickness by fast solution flow resulted in a stimulation, and not a reduction, of¹⁴C-fixation. The similarity of our radial pH profiles near the wall with that predicted by the model (Walker et al., 1980) assuming H⁺–HCO ₃ ^– cotransport, together with the effects of buffer, and the results of increased solution flow rate, lead to the conclusion that cotransport of HCO ₃ ^– with H⁺ is the likely method of entry of inorganic carbon. Longitudinal pH profiles of theChara cell were obtained at a distance of 25 m from the wall. These revealed much sharper delineation of the acid and alkaline bands than has previously been possible with miniature pH electrodes. Profiles of local electric field, obtained with a vibrating probe, were in excellent agreement with the high resolution pH profiles. This supports the hypothesis that membrane proton transport has a role (direct) in the generation of the extracellular currents.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

Inorganic-carbon transport in some marine eukaryotic microalgae

Inorganic-carbon transport was investigated in the eukaryotic marine microalgaeStichococcus minor, Nannochloropsis oculata and aMonallantus sp. Photosynthetic O₂ evolution at constant inorganic-carbon concentration but varying pH showed thatS. minor had a greater capacity for CO₂ rather than HCO ₃ ^– utilization but forN. oculata andMonallantus HCO ₃ ^– was the preferred source of inorganic carbon. All three microalgae had a low affinity for CO₂ as shown by the measurement of inorganic-carbon-dependent photosynthetic O₂ evolution at pH 5.0. At pH 8.3, where HCO ₃ ^– is the predominant form of inorganic carbon, the concentration of inorganic carbon required for half-maximal rate of photosynthetic O₂ evolution [K _0.5 (CO₂)] was 53 M forMonallantus sp. and 125 M forN. oculata, values compatible with HCO ₃ ^– transport. Neither extra- nor intracellular carbonic anhydrase was detected in these three microalgal species. It is concluded that these microalgae lack a specific transport system for CO₂ but that HCO ₃ ^– transport occurs inN. oculata andMonallantus, and in the absence of intracellular carbonic anhydrase the conversion of HCO ₃ ^– to CO₂ may be facilitated by the internal pH of the cell.     

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.).     

Pathways for bicarbonate transfer across the serosal membrane of turtle urinary bladder: Studies with a disulfonic stilbene

Summary Bicarbonate is transferred across the serosal (S) membrane of the epithelial cells of the turtle bladder in two directions. Cellular HCO ₃ ^– generated behind the H⁺ pump moves across this membrane into the serosal solution. This efflux of HCO ₃ ^– is inhibited by SITS (4-isothiocyano-4-acetamido-2,2-disulfonic stilbene). When HCO ₃ ^– is added to the serosal solution it is transported across the epithelium in exchange for absorbed Cl^–. This secretory HCO ₃ ^– flow traverses the serosal cell membrane in the opposite direction. In this study the effects of serosal addition of 5×10^–4 m SITS on HCO ₃ ^– secretion and Cl^– absorption were examined. The rate of H⁺ secretion was brought to zero by an opposing pH gradient, and 20mm HCO ₃ ^– was added toS. HCO ₃ ^– secretion, measured by pH stat titration, was equivalent to the increase inMS Cl^– flux after HCO ₃ ^– addition. Neither theSM flux of HCO ₃ ^– nor theMS flux of Cl^– were affected by SITS. In the absence of electrochemical gradients, net Cl^– absorption was observed only in the presence of HCO ₃ ^– in the media; under such conditions, unidirectional and net fluxes of Cl^– were not altered by serosal or mucosal SITS. H⁺ secretion, however, measured simultaneously as the short-circuit current in ouabain-treated bladders decreased markedly after serosal SITS. The inhibition of the efflux of HCO ₃ ^– in series with the H⁺ pump and the failure of SITS to affect HCO ₃ ^– secretion and Cl^– absorption suggest that the epithelium contains at least two types of transport systems for bicarbonate in the serosal membrane.     

Fusion of cultured dog kidney (MDCK) cells: II. Relationship between cell pH and K+ conductance in response to aldosterone

Summary We have chosen the MDCK cell line to investigate aldosterone action on H⁺ transport and its role in regulating cell membrane K⁺ conductance (G _m ^K ). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K⁺ channels was tested in giant cells fused from individual MDCK cells. Cytoplasmic pH (pH _i ) andG _m ^K were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pH _i andG _m ^K (Hill coefficient 4.4±0.4), indicating multiple H⁺ binding sites at a single K⁺ channel. Application of aldosterone increased pH _i within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO₂/HCO ₃ ^– buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO₂/ HCO ₃ ^– is limited by the increased activity of a pH _i -regulating HCO ₃ ^– extrusion system. SinceG _m ^K is stimulated half-maximally at the pH _i of 7.18±0.04, internal H⁺ ions could serve as an effective intracellular signal for the regulation of transepithelial K⁺ flux.     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

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.     

Charasomes are not essential for photosynthetic utilization of exogenous HCO3 − inChara corallina

Summary Cells ofChara corallina grown under high CO₂ culture conditions were able to utilize exogenous HCO₃ ^– to give appreciable rates of net photosynthesis. Since these rates of photosynthesis could be detected within 10 min of being transferred from high-CO₂ to normal HCO₃ ^– (pH 8.2) culture conditions, it would appear that the HCO₃ ^–-accumulating system ofChara is not fully repressed under these high CO₂ culture conditions. The membrane potential of these cells also responded to light/dark treatments in a manner consistent with the operation of a HCO₃ ^– acquisition system. With prolonged exposure (2–6 days) to CPW/B, net photosynthesis continued to increase towards the expected control rate and, in parallel, the electrical responses elicited by light/dark treatments converged towards those obtained on control (CPW/B-grown)Chara cells. Charasomes were absent in CPW/CO₂-grownChara, but redeveloped in mature cells once the culture was returned to CPW/B conditions; a minimum period of 7 days in CPW/B was required before charasomes were detected in tissue examined in the transmission electron microscope. As the above-detailed physiological and electrophysiological features were observed with both axial and whorl cells ofChara in which charasomes were completely absent, we conclude that this specialized organelle is not an essential component for photosynthetic utilization of exogenous HCO₃ ^– in this species.Abbreviations CPW/B Chara pond water containing 1.0 mM NaHCO₃, pH8.2 - CPW/CO₂ Chara pond water containing dissolved CO₂, pH 5.5 - DIC dissolved in organic carbon - D.H. dark-induced membrane hyperpolarization - L.H. light-induced membrane hyperpolarization - TEM transmission electron microscopy     

Fish gill water boundary layer: a site of linkage between carbon dioxide and ammonia excretion

Summary Carbon dioxide excreted across fish gills is hydrated catalytically to form HCO ₃ ^– and H⁺ ions in water near the gill surface. We tested the possibility that CO₂ excretion is functionally linked to ammonia excretion through chemical reactions in the gill-water boundary layer. A bloodperfused trout head preparation was utilized in which the convective and diffusive components of branchial gas transfer were controlled. Pre-incubation of blood perfusate with the carbonic anhydrase inhibitor, acetazolamide, reduced both carbon dioxide and ammonia excretion in the blood-perfused preparation. Increasing the buffering capacity of inspired ventilatory water significantly reduced ammonia excretion, but carbon dioxide excretion was unaffected. Each of these experimental treatments significantly reduced the acidification of ventilatory water flowing over the gills. It is proposed that the catalysed conversion of excreted CO₂ to form HCO ₃ ^– and H⁺ ions provides a continual supply of H⁺ ions need for the removal of NH₃ as NH ₄ ⁺ . We suggest, therefore, that acidification of boundary layer water by CO₂ enhances blood-to-water NH₃ diffusion gradients and facilitates ammonia excretion.     

HCO 3 − -coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes

Summary Recent studies in hepatocytes indicate that Na⁺-coupled HCO ₃ ^– transport contributes importantly, to regulation of intracellular pH and membrane HCO ₃ ^– transport. However, the direction of net coupled Na⁺ and HCO ₃ ^– movement and the effect of HCO ₃ ^– on Na⁺ turnover and Na⁺/K⁺ pump activity are not known. In these studies, the effect of HCO ₃ ^– on Na⁺ influx and turnover were measured in primary rat hepatocyte cultures with²²Na⁺, and [Na⁺] _i was measured in single hepatocytes using the Na⁺-sensitive fluorochrome SBFI. Na⁺/K⁺ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na⁺-dependent or ouabain-suppressible⁸⁶Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K⁺ on membrane potential difference (PD) and [Na⁺] _i . In hepatocyte monolayers, HCO ₃ ^– increased²²Na⁺ entry and turnover rates by 50–65%, without measurably altering²²Na⁺ pool size or cell volume, and HCO ₃ ^– also increased Na⁺/K⁺ pump activity by 70%. In single cells, exposure to HCO ₃ ^– produced an abrupt and sustained rise in [Na⁺] _i , from 8 to 12mm. Na⁺/K⁺ pump activity assessed in single cells by PD excursions during transient K⁺ removal increased 2.5-fold in the presence of HCO ₃ ^– , and the rise in [Na⁺] _i produced by inhibition of the Na⁺/K⁺ pump was similarly increased 2.5-fold in the presence of HCO ₃ ^– . In intact perfused rat liver, HCO ₃ ^– increased both Na⁺/K⁺ pump activity and O₂ consumption. These findings indicate that, in hepatocytes, net coupled Na⁺ and HCO ₃ ^– movement is inward and represents a major determinant of Na⁺ influx and Na⁺/K⁺ pump activity. About half of hepatic Na⁺/K⁺ pump activity appears dedicated to recycling Na⁺ entering in conjunction with HCO ₃ ^– to maintain [Na⁺] _i within the physiologic range.     

Volume regulation in the early proximal tubule of theNecturus kidney

Summary The ability of early proximal tubule cells of theNecturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes.Necturus proximal tubule cells regulate volume in both hyperand hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO ₃ ^– dependent and is associated with a decrease in the relative K⁺ conductance of the basolateral cell membrane and a decrease in the resistance ratio,R _a /R _bl . Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO ₃ ^– but is also inhibited by 2mm Ba²⁺ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K⁺ conductance of the basolateral cell membrane and an increase inR _a /R _bl . Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO ₃ ^– removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO ₃ ^– -dependent transport systems and the basolateral K⁺ conductance.     

Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells

Summary We investigated intracellular pH (pH _i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4,5-dimethylfluorescein (CDMF). The steady-state pH _i was 7.09±0.04 (n = 12) in CO₂/ HCO ₃ ^– -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO₂/ HCO ₃ ^– and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH _i recovery, which was almost completely amiloride-sensitive in the absence of CO₂/ HCO ₃ ^– but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH _i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH _i recovery was almost completely mediated by Na⁺/H⁺ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH _i recovery was observed in CO₂/HCO ₃ ^– -buffered solution which was mediated by chloride-independent and chloride-dependent Na⁺ HCO ₃ ^– cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH _i recovery after NH₄Cl prepulse revealed V _max = 0.57 pH units/min, K _m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V _max = 0.19 pH units/min, K _m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na⁺/H⁺ exchange and chloride-independent and chloride-dependent Na⁺HCO ₃ ^– cotransport are involved in the pH _i regulation of cultured human ciliary muscle cells.The expert technical assistance of Astrid Krolik is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft grant DFG Wi 328/11.     

Differential regulation of Na+/H+ exchange and H+ -ATPase by pH and HCO 3 − in kidney proximal tubules

This study examines the effects of acute in vitro acid-base disorders on Na⁺/H⁺ and H⁺-ATPase transporters in rabbit kidney proximal tubules (PT). PT suspensions were incubated in solutions with varying acid base conditions for 45 min and utilized for brush border membrane (BBM) vesicles preparation. BBM vesicles were studied for Na⁺/H⁺ exchange activity (assayed by ²²Na⁺ influx) or abundance (using NHE-3 specific antibody) and H⁺-ATPase transporter abundance (using antibody against the 31 kDa subunit). The Na⁺/ H⁺ exchanger activity increased by 55% in metabolic acidosis (pH 6.5, HCO ₃ ^– 3 mm) and decreased by 41% in metabolic alkalosis (pH 8.0, HCO ₃ ^– 90 mm). The abundance of NHE-3 remained constant in acidic, control, and alkalotic groups. H⁺-ATPase abundance, however, decreased in metabolic acidosis and increased in metabolic alkalosis by 57% and 42%, respectively. In PT suspensions incubated in isohydric conditions (pH 7.4), Na⁺/H⁺ exchanger activity increased by 29% in high HCO ₃ ^– group (HCO ₃ ^– 96 mm) and decreased by 16% in the low HCO ₃ ^– groups (HCO ₃ ^– 7mm. The NHE-3 abundance remained constant in high, normal, and low [HCO ₃ ^– ] tubules. The abundance of H⁺-ATPase, however, increased by 82% in high [HCO ₃ ^– ] and decreased by 77% in the low [HCO ₃ ^– ] tubules. In PT suspensions incubated in varying pCO₂ and constant [HCO ₃ ^– ], Na⁺/H⁺ exchanger activity increased by 35% in high pCO₂ (20% pCO₂, respiratory acidosis) and decreased by 32% in low pCO₂ (1.5% pCO₂, respiratory alkalosis) tubules. The NHE-3 abundance remained unchanged in high, normal, and low pCO₂ tubules. However, the H⁺-ATPase abundance increased by 74% in high pCO₂ and decreased by 69% in low pCO₂ tubules.The results of these studies suggest that the luminal Na⁺/H⁺ exchanger is predominantly regulated by pH whereas H⁺-ATPase is mainly regulated by [HCO ₃ ^– ] and/ or pCO₂. They further suggest that the adaptive changes in H⁺-ATPase transporter are likely mediated via endocytic/exocytic pathway whereas the adaptive changes in Na⁺/H⁺ exchanger are via the nonendocytic/exocytic pathway.The excellent technical assistance of Yollanda J. Hattabaugh, Gwen L. Bizal, and L. Yang is greatly appreciated. Portions of these studies were presented at the annual meeting of the American Society of Nephrology, Boston, MA, November 1993, and published in abstract form (J.Am.Soc.Neph. 4:840A, 1993)These studies were supported by a Merit Review Grant from the Department of Veterans Affairs and a grant-in-aid from the American Heart Association (to M.S.), a Baxter Health Care Grant (to B.B.), and the National Institute of Health Grants DK 38510 (to E.B.C. and M.C.R.) and DK 42086 (to E.B.C.).     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.