Interaction of Respiration, Ion Regulation, and Acid-Base Balance in the Everyday Life of Aquatic Crustaceans

Extracellular and intracellular acid-base balance is necessaryfor the maintenance of normal metabolic processes. The primarysource of acid is metabolically produced CO₂, and the CO₂/HCO₃^–system is the most significant buffer. The regulation of acid-basebalance is complex, involving the interaction between respiratorygas exchange and ion transport. In aquatic crustaceans respirationis governed by the need to extract oxygen from water, an O₂-poormedium; thus, acid-base balance is maintained primarily throughion transport mechanisms. These mechanisms include Na⁺/H⁺ andCl^–/HCO₃^– exchange processes that are sensitiveto the extracellular acid-base status of the animal. In marinecrabs, ion regulation and acid-base balance are accomplishedby the posterior gills, while in freshwater species all gillsand the antennal gland perform these functions. Intracellularacid-base balance appears to be maintained primarily by iontransport across the cell membrane. Hemolymph pH varies inverselywith acclimation temperature and salinity. In both cases Pco₂remains nearly constant, and the pH change is a result of changesin hemolymph HCO₃^– concentrations brought about by ionexchange mechanisms. Environmental hypercapnia or hyperoxiainduces a repiratory acidosis characterized by increased Pco₂,low pH, and elevated HCO₃^–; this is partially compensatedfor by ion exchange processes that bring about a further increasein hemolymph HCO₃^–. Exercise causes a mixed respiratoryand metabolic acidosis with compensation via H⁺ ion excretionand hyperventilation.     

Ion Regulation in Crayfish: Freshwater Adaptations and the Problem of Molting

SYNOPSIS. Crayfish have a long evolutionary history in temperatefresh water (FW). Ion regulation is challenged by low externalconcentrations of Na, Cl, and Ca (<1 mM). In intermolt theprimary concern is Na and Cl balance; around ecdysis the emphasisswitches to Ca regulation as the cuticle is decalcified/calcified.Compared with marine crustaceans, intermolt crayfish maintaina reduced extracellular (EC) osmolality and have lower permeabilityto both ions and water. Hyperregulation involves active branchialuptake of Na and Cl and the unique ability to produce a hypotonicurine. Ion uptake involves apical electroneutral ion exchange(Na^$ for H^$; Cl^– for HCO₃–; counterions providedfrom CO₂ via carbonic anhydrase) followed by active basolateraltransport of Na via the Na pump, with Cl following passively.Reabsorption of 95% of filtered electrolytes at the antennalgland (kidney) involves similar subcellular mechanisms in amorphologically differentiated region of the distal tubule.Intermolt crayfish exhibit negative Ca balance (passive effluxunopposed by uptake) tolerable in view of the large cuticularCaCO₃ reserve. In premolt, cuticular Ca is reabsorbed. A smallamount is stored as gastroliths, the remainder is lost via branchialexcretion and in the discarded exuviae. At ecdysis, FW uptakegenerates the physical force for shedding, leaving the crayfishwith dilute hemolymph and a Ca deficiency. Levels of EC Na andCl are restored by intensive postmolt branchial uptake. Mineralizationof the soft exoskeleton involves remobilization of stored Caand branchial uptake of Ca and HCO₃. Transepithelial Ca transportinvolves Ca^2$ ATPase and Ca^2$/Na^$ exchange. The importance ofexternal electrolytes and pH in postmolt ion regulation is explored,as are some allometric considerations.     

Nongenomic regulation by aldosterone of the epithelial NHE3 Na(+)/H(+) exchanger

The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO₃^– absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na⁺/H⁺ exchangers (NHE). In the MTAL, apical NHE3 mediates H⁺ secretion necessary for HCO₃^– absorption; basolateral NHE1 influences HCO₃^– absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO₃^– absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na⁺/H⁺ exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na⁺/H⁺ exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in V_max. Inhibition of HCO₃^– absorption by aldosterone was not affected by 0.1 mM lumen Zn²⁺ or 1 mM lumen DIDS, arguing against the involvement of an apical H⁺ conductance or apical K⁺-HCO₃^– cotransport. These results demonstrate that aldosterone inhibits HCO₃^– absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger. thick ascending limb; acid-base transport; epithelial Na⁺ transport; kidney     

Photosynthetic Fixation of 14Carbon by Internodal Cells of Chara corallina

Maximum fixation rates of 120 and 60 pmol cm^–2 s ^–1wereobtained when exogenous carbon was supplied as ¹CO₂ and H¹⁴CO₃^–respectively. These values are considerably higher than thosepreviously reported for this species. A kinetic analysis wasperformed on this data. Substrate saturation in the concentrationrange 1.0–1.5 mM was observed for both CO₂ and HCO₃^– In the presence of exogenous CO², a linear relationship wasobserved between light intensity and fixation while the HCO₃^–relationship was slightly sigmoidal. Fixation saturated at intensitiesof 15–20 W m^–2 and 13–15 W m^–2 for exogenous¹⁴CO² and H¹⁴CO₃^–respectively. The presence, in this species, of an extremely active HCO₃^–transport system, situated in the plasmalemma, demonstratesthat when alkaline solutions are employed the involvement ofthis ion cannot be ignored during electrical studies on thismembrane. The maximum H¹⁴CO₃^– influxes obtained duringthis study are the largest ionic fluxes measured for any Characeanspecies. It was demonstrated that CO² for fixation can be supplied simultaneouslyby gaseous diffusion and HCO₃^– transport (cf. Raven, 1968).Inhibition of H¹⁴CO₃^– influx was observed in the presenceof Tris, Tricine, and borate buffers, and CO₃^{2 –} alsoappeared to act as a strong inhibitor. The possible mechanism(s)by which this inhibition occurs is discussed.     

Photosynthetic Carbon Sources of Stream Macrophytes

Rates of photosynthesis of four submerged stream macrophyteswere examined under varying pH and composition of inorganiccarbon species. Callitriche stagnatis and Sparganium simplexused only CO₂ for photosynthesis. Potamogeton crispus and P.pectinatus used HCO₃ in addition to CO₂, but with much lowerefficiency. The photosynthetic rates at air equilibrium anda total inorganic carbon concentration of 5.0 mM were 2–3times lower than maximum rates at CO2 saturation for the HCO₃users and 10–14 times lower for the CO₂ users. The CO₂compensation point of entire plants of Callitriche (2.5 µM)and Sparganium (6.0µM) was well below the equilibriumconcentration (15 µM). and the low saturation points (250–500µM) also pointed to efficient use of CO2. Callitricheand Sparganium compete successfully with HCO₃^– users inhardwater streams, which have a higher exchange and generationcapacity of CO2 than stagnant and more soft waters. Rates ofphotosynthesis of Potamogeton crispus and P. pectinatus decreasedat high pH. Depending on the two alternative hypotheses forHCO₃use, this decline can be explained by CO₃^––inhibition of HCO₃^– uptake or by increasing capacity tobuffer H+efflux from the plant. Habitats subject to high pH,e. g. small ponds with dense vegetation, may have a strong selectionfor efficient mechanisms of HCO₃ use. Key words: Photosynthesis, Macrophytes, Carbon-source     

Analogue Inhibition of the Active HCO-3 Transport Site in the Characean Plasma Membrane

Competitive inhibition of the HCO^–₃ transport site, atthe plasmalemma of Chara coraUina, by the CO^2–₃ ion isdemonstrated. This CO^2–₃ inhibition was used to demonstratethat HCO^–₃ ions enter the cell by facilitated ‘diffusion’when the HCO^–₃ transport system has been inactivated bytreatment with 10 mM K⁺. Use of CO^2–₃ as a HCO^–₃analogue is limited, however, because of the necessity to employsolutions of high pH. Inhibition was not observed in the presenceof a range of organic and inorganic acid anions. These resultsdemonstrate the stereo-specific nature of the HCO^–₃ bindingsite. A variety of amino compounds were found to inhibit H¹⁴CO^–₃influx. Inhibition appeared to be competitive, being completelyrelieved at higher substrate (HCO^–₃) concentrations. Asimple correlation was not found between the degree of inhibitionand the concentration of neutral base. A combination of thepresence of neutral base and experimental pH values of at least8·0 was required to produce the reactive species thatinhibited HCO^–₃ transport. This species is consideredto be the amino carbamate. These results are discussed withrespect to further HCO^–₃ analogue experiments.     

Transport and Fixation of Inorganic Carbon during Photosynthesis of Anabaena Grown under Ordinary Air. I. Active Species of Inorganic Carbon Utilized for Photosynthesis

Inorganic carbon transport during photosynthesis of cyanobacteriumAnabaena variabilis grown under ordinary air was investigatedby supplying ¹⁴CO₂ or H¹⁴CO₃^– solution to three differentstrains. Both CO₂ and HCO₃^– were accumulated within thealgal cells. In the cell suspension from which dissolved inorganiccarbon had been depleted by pre-illumination, CO₂ was transportedand accumulated faster than HCO₃^–. When the concentrationof HCO₃^– injected into the cell suspension of A. variabilisM3 was 25 times as high as that of CO2 (the expected ratio atequilibrium at pH 7.8), the initial rates of fixation of bothinorganic carbon species were practically the same. On the otherhand, when ¹⁴CO₂ or H¹⁴CO₃^– was added under steady statephotosynthetic conditions, both carbon species were transportedat similar rates. The ratio of fixed to transported carbon measuredafter the initial 5 s was only 23–27% regardless of thecarbon species supplied. This percentage is much lower thanthat reported for Chlorella cells. ¹ To whom reprint requests should be addressed (Received June 30, 1986; Accepted December 16, 1986)     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat     

Characterization of Photosynthetic 14Carbon Assimilation by Potamogeton lucens L.

Photosynthetic assimilation of exogenous ¹⁴CO₂ and H¹⁴CO₃^–by the aquatic angiosperm Potamogeton lucens L. is reported.Equivalent maximum rates of assimilation (1.5 µmol s^–1m^–2) were obtained in the presence of saturating levelsof ¹⁴CO₂ (1.0 mol m^–3, pH 5.3) or H¹⁴CO₃^– (1.5 molm^–3, pH, 9.2). Under subsaturating ¹⁴CO₂ levels, bothgaseous diffusion and H¹⁴CO₃^– transport were shown tooperate simultaneously, such that maximal photosynthetic rateswere established. An induction lag of approximately 3 min was observed when exogenous¹⁴CO₂ was assimilated. A longer lag of approximately 12 minwas required, however, before linear assimilation rates wereestablished when H¹⁴CO₃ acted as the carbon source. The light-activatedH¹⁴CO₃^– transport system was found to be quite labile.A brief (5 min) dark treatment returned the system to the inactivestate. Bicarbonate transport was shown to be competitively inhibitedby CO₃^2–ions. The possibility is discussed that this formof inhibition may be common to many HCO₃^– assimilators. Preliminary polar cation transport studies (from lower to upperleaf surface) indicated an almost exact one to one relationshipbetween the rates of Na⁺ influx and efflux and H¹⁴CO₃^–assimilation. The possible relationship(s) between these transportprocesses and the requirement for electrical neutrality is brieflydiscussed.     

Glucose 6-phosphate dehydrogenase from sweet potato: Effect of various ions and their ionic strength on enzyme activity

Activity of glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate:NADP oxidoreductase, EC 1.1.1.49 [EC] ) preparation from sweet potatoroot tissue was markedly altered in the presence of variousions. Cations or anions were effective in the following order:Na^$, K^$>Tris^$>NH₄^$>Mg^2$>Ca^2$, or Cl^–>NO₃^–,HPO₄^2–>SO₄^2–>HCO₃^–. Activity was inhibitedat high concentrations of Ca^2$, and HCO₃^–,. In an investigationon the dependence of the activity on pH, two activity peakswere clearly observed at low ionic strength. Ionic strength altered both the Km and Vmax for glucose 6-phosphate(G6P). A Lineweaver-Burk plot for the enzyme, with respect toG6P, showed a bimodal nature at low ionic strength; suggestingnegative cooperativity. Deviation from linearity of the plotwas less with an increase in the ionic strength. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113. (Received September 18, 1971; )     

The Role of Carbonic Anhydrase in Blood Ion and Acid-Base Regulation

The role of carbonic anhydrase (CA) in ion transport processesof aquatic and terrestrial arthropod species is reviewed. Inboth insects and crustaceans CA is found in a variety of iontransporting tissues. The bulk of CA activity in crustaceansis concentrated in the posterior gills, which are morphologicallyand biochemically adapted for ion transport. The enzyme canbe specifically localized to gill lamellae which contain largepopulations of salt transporting chloride cells. Enzyme activityin the posterior gills of species having the ability to regulateblood ion concentrations increases when these organisms areacclimated to environmental salinities in which they ion regulate.In stenohaline, ion conforming species branchial CA activityis uniformly low, being only 5–10% that in regulatingspecies. Studies on the blue crab, Callinectes sapidus, usingthe specific CA inhibitor acetazolamide have shown that theenzyme is indeed important in blood ion regulation. Blood Na^$and Cl^– concentrations are both severely lowered in drug-treatedanimals acclimated to low salinity, while they remain virtuallyunaffected in animals acclimated to high salinity, in whichthe animal is an ion conformer. High salinity acclimated crabstreated with acetazolamide do not survive transfer to low salinity,and mortality is related to a breakdown in the ion regulatorymechanism. Branchial CA most likely functions in the hydrationof respiratory CO₂ to H^$ and HCO₃^–, which serve as counterionsfor the active uptake of Na^$ and Cl^–, respectively. Interrestrial species the role of CA is unclear and merits furtherinvestigation.     

Utilization Modes of Inorganic Carbon for Photosynthesis in Various Species of Chlorella

Rates of CO₂ and HCC₃^– fixation in cells of various Chlorellaspecies in suspension were compared from the amounts of ¹⁴Cfixed during the 5 s after the injection of a solution containingonly ¹⁴CO₂ or H¹⁴CO₃^–. Results indicated that irrespectiveof the CO₂ concentration during growth, Chlorella vulgaris 11h and C. miniata mainly utilized CO₂, whereas C. vulgaris C-3,C. sp. K. and C. ellipsoidea took up HCO₃^– in additionto CO₂. Cells of C. pyrenoidosa that had been grown with 1.5%CO₂ (high-CO₂ cells) mainly utilized CO₂, whereas those grownwith air (low-CO₂ cells) utilized HCO₃^– in addition toCO₂. Cells that utilized HCO₃^– had carbonic anhydrase(CA) on their surfaces. The effects of Diamox and CA on the rates of CO₂ and HCO₃^–fixation are in accord with the inference that HCO₃^– wasutilized after conversion to CO₂ via the CA located on the cellsurface. CA was found in both the soluble and insoluble fractions;the CA on the cell surface was insoluble. Independent of the modes of utilization, the apparent K_m (NaHCO₃)for photosynthesis was much lower in low-CO₂ cells than in high-CO₂ones. The fact that the CA in the soluble fraction in C. vulgarisC-3 was closely correlated with the K_m(NaHCO₃) indicates thatsoluble CA lowers the K_m. ¹ Dedicated to the late Professor Joji Ashida, one of the foundersand first president of the Japanese Society of Plant Physiologists. ⁴ On leave from Research and Production Laboratory of Algology,Bulgarian Academy of Sciences, Sofia. (Received September 14, 1982; Accepted March 1, 1983)     

Kinetic Studies on the Active Species of Inorganic Carbon Absorbed by the Cells of Dunaliella tertiolecta

Cells of Dunaliella tertiolecta which had been grown in ordinaryair (low-CO₂ cells) had high carbonic anhydrase (CA) activityon the cell surface and mainly utilized HCO₃^– for photosynthesis.When CA activity on the cell surface was inhibited by Diamoxor subtilisin, the cells utilized CO₂. When bovine CA was added,the subtilisin-treated low-CO₂ cells utilized mainly HCO₃^–.When grown in air containing 2% CO₂, the cells had low CA activityon the cell surface, and preferred CO₂ to HCO₃^–. Kineticanalysis of these results indicated that low-CO₂ cells of D.tertiolecta absorb CO₂ which was converted from HCO₃^–via the CA located on the cell surface. (Received June 29, 1985; Accepted October 9, 1985)     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA

Human NBC3 is an electroneutral Na⁺/HCO₃^– cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO₃^– metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO₂ + H₂O HCO₃^– + H⁺ in many tissues. We investigated whether NBC3, like some Cl^–/HCO₃^– exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K_d = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 µM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH_i) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH_i recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [-³²P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO₃^– transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct. pH regulation; bicarbonate transport; metabolon     

The Mechanism of Photosynthetic Use of Bicarbonate by Hydrodictyon africanum

Hydrodictyon africanum can photosynthesize at high pH underconditions in which HCO₃^– rather than CO₂ is the carbonspecies entering the cell. A passive entry of HCO₃^– seemsunlikely; a metabolic HCO₃^– pump is proposed. It is possiblethat such a pump is related to a light-dependent reaction specificto the use of HCO₃^–. This reaction is dependent on photosystem2, but appears to be independent of ATP. These characteristicsare similar to those of active lightdependent Cl influx in H.africanum, and suggest a similar energy source for the two pumps.The HCO₃^– pump may be electrogenic.     

Active Uptake of CO2 by the Diatom Navicula pelliculosa

Mass spectrometry has been used to investigate the transportof CO₂ in the freshwater diatom Navicula pelliculosa. The timecourseof CO₂ formation in the dark after addition of 100 mmol m^–3dissolved inorganic carbon (DIC) to cell suspensions showedthat no external carbonic anhydrase (CA) was present in thesecells. Upon illumination, cells pre-incubated at pH 75 with100 mmol m^–3 DIC, removed almost all free CO₂ from themedium at an initial rate of 285 µmol CO₂ mg^–1Chl h^–1. Equilibrium between HCO₃^– and CO₂ in themedium occurred rapidly upon addition of bovine CA, showingthat CO₂ depletion resulted from a selective uptake of CO₂ ratherthan an uptake of all inorganic carbon species. However, photosyntheticO₂ evolution rate remained constant after CO₂ had been depletedfrom the medium indicating that photosynthesis is sustainedprimarily by active HCO₃^– uptake. Treatment of cells with2-iodoacetamide (83 mol m^–3) completely inhibited CO₂fixation but had little effect on CO₂ transport since initialrates of CO₂ depletion were about 81% that of untreated cells.Transfer of iodoacetamide-treated cells to the dark caused arapid increase in the CO₂ concentration in the medium largelydue to the efflux of the unfixed intracellular DIC pool whichwas found to be about 194 times the concentration of that inthe external medium. These results indicate that Navicula pelliculosaactively takes up molecular CO₂ against a concentration gradientby a process distinct from HCO₃^– transport. Key words: Dissolved inorganic carbon, carbonic anhydrase, bicarbonate transport, CO₂ transport, mass spectrometry     

Effects of Ammonia and Methylamine on Cl- Transport and on the pH Changes and Circulating Electric Currents Associated with HCO3- Assimilation in Chara corallina

Low concentrations of ammonia and methylamine greatly increaseCl^– influx into Chara corallina. Both amines have theirmaximum effect at pH 6.5–7.5. The amine stimulation ofCl^– influx is small below about pH 5.5. Above pH 8.5 theremay be inhibition of influx by amines. Concentrations of 10–25µM ammonia are sufficient to cause the maximum stimulationof Cl^– influx; the corresponding methylamine concentrationsare 0.1–0.2 mM. It is concluded that entry of amine cations(NH₄^$ and CH₃NH₃^$), rather than unionized bases (NH₃ and CH₃NH₂),causes Cl^– transport to be increased. Increases in rates of Cl^– transport are not necessarilyaccompanied by effects on HCO₃^$ assimilation and OH^– efflux.Measurements of localized pH differences at the cell surfaceand of circulating electric currents in the bathing solutionshow that these phenomena are only significantly affected byammonia at or above 50 µM and by methylamine at or above1.0 mM. The significance of the effects of amines is assessedin relation to current ideas about transport of Cl^–, HCO₃^–,and OH^–.     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse