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

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

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO3 Cotransport

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na⁺-driven influx of HCO₃ ⁻ causes the increase in intracellular pH (pH_i) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO₂/17 mM HCO₃ ⁻. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na⁺-driven Cl-HCO₃ exchanger, an electrogenic Na/HCO₃ cotransporter, or an electroneutral Na/HCO₃ cotransporter. To determine if the transporter is a Na⁺-driven Cl-HCO₃ exchanger, we depleted the cells of intracellular Cl⁻ by incubating them in a Cl⁻-free solution for an average of ∼11 min. We verified the depletion with the Cl⁻-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl⁻-depleted cells, the pH_i still increases after one or more exposures to CO₂/HCO₃ ⁻. Furthermore, the pH_i decrease elicited by external Na⁺ removal does not require external Cl⁻. Therefore, the transporter cannot be a Na⁺-driven Cl-HCO₃ exchanger. To determine if the transporter is an electrogenic Na/ HCO₃ cotransporter, we measured pH_i and plasma membrane voltage (V_m) while removing external Na⁺, in the presence/absence of CO₂/HCO₃ ⁻ and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO₂/HCO₃ ⁻ solutions contained 20% CO₂ and 68 mM HCO₃ ⁻, pH 7.3, to maximize the HCO₃ ⁻ flux. In pH_i experiments, removing external Na⁺ in the presence of CO₂/HCO₃ ⁻ elicited an equivalent HCO₃ ⁻ efflux of 281 μM s⁻¹. The HCO₃ ⁻ influx elicited by returning external Na⁺ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V_m change was 3.3 mV. Indeed, we found that removing external Na⁺ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO₂/ HCO₃ ⁻. Thus, the Na/HCO₃ cotransporter is electrogenic. Because a cotransporter with a Na⁺:HCO₃ ⁻ stoichiometry of 1:3 or higher would predict a net HCO₃ ⁻ efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO₃ cotransporter with a stoichiometry of 1:2.     

HCO3?-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO₃⁻-independent intracellular pH (pH_i) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pH_i in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H⁺ pump by Cl⁻ removal or via the selective inhibitor bafilomycin had only a small effect upon resting pH_i and recovery following an acid load. In contrast, resting pH_i in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pH_i recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO₃⁻-independent pH_i regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.     

The Stoichiometry between CO(2) and H Fluxes Involved in the Transport of Inorganic Carbon in Cyanobacteria

The pH of the medium during CO₂ uptake into the intracellular inorganic carbon (C_i) pool of a high CO₂-requiring mutant (E₁) and wild type of Anacystis nidulans R2 was measured. Experiments were performed under conditions where photosynthetic CO₂ fixation is inhibited. There was an acidification of the medium during CO₂ uptake in the light and an alkalization during CO₂ efflux after darkening. A one to one stoichiometry existed between the amounts of H⁺ appearing in the medium and CO₂ taken up into the intracellular C_i pool, regardless of the carbon species transported. The results indicate that (a) CO₂ is taken up simultaneously with an efflux of equimolar H⁺, probably produced as a result of CO₂ hydration during transport and (b) HCO₃⁻ produced by hydration of CO₂ in the medium was transported into the cells without accompanying net flux of H⁺ or OH⁻. The influx and efflux of C_i during C_i transport produced nonequilibrium between CO₂ and HCO₃⁻ in the medium, with the concentration of HCO₃⁻ being higher than that expected under equilibrium conditions. The nonequilibrium was present even under the conditions where the influx of C_i is compensated by its efflux. The direction of this nonequilibrium suggested that efflux of HCO₃⁻ occurs during uptake of C_i.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm

HCO₃⁻ is a key factor in the regulation of sperm motility. High concentrations of HCO₃⁻ in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO₂ to HCO₃⁻, represent potential candidates in the regulation of the HCO₃⁻ homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO₃⁻ homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO₃⁻-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO₃⁻ or CO₂ that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO₃⁻. These results suggest that CAII and CAIV are required for optimal fertilization.     

Energy Sources for HCO3? and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 625

Light-dependent inorganic C (C_i) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO₂ or HCO₃⁻ uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular C_i pool of 20 mm, even though CO₂ fixation was completely inhibited, indicating that cyclic electron flow was involved in the C_i-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar C_i accumulation was observed, suggesting that linear electron flow could support the transport of C_i. When carbonic anhydrase was not present, initial CO₂ uptake was greatly reduced and the extracellular [CO₂] eventually increased to a level higher than equilibrium, strongly suggesting that CO₂ transport was inhibited and that C_i accumulation was the result of active HCO₃⁻ transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, C_i transport and accumulation were inhibited by inhibitors of CO₂ transport, such as COS and Na₂S, whereas Li⁺, an HCO₃⁻-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, C_i transport and accumulation were not inhibited by COS and Na₂S but were inhibited by Li⁺. These results suggest that CO₂ transport is supported by cyclic electron transport and that HCO₃⁻ transport is supported by linear electron transport.     

Ethoxyzolamide Inhibition of CO(2) Uptake in the Cyanobacterium Synechococcus PCC7942 without Apparent Inhibition of Internal Carbonic Anhydrase Activity

In high inorganic carbon grown (1% CO₂ [volume/volume]) cells of the cyanobacterium Synechococcus PCC7942, the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZ), was found to inhibit the rate of CO₂ uptake and to reduce the final internal inorganic carbon (C_i) pool size reached. The relationship between CO₂ fixation rate and internal C_i concentration in high C_i grown cells was little affected by EZ. This suggests that in intact cells internal CA activity was unaffected by EZ. High C_i grown cells readily took up CO₂ but had little or no capacity for HCO₃⁻ uptake. These cells appear to possess a CO₂ utilizing C_i pump that has a CA-like function associated with the transport step such that HCO₃⁻ is the species delivered to the cell interior. This CA-like step may be the site of inhibition by EZ. Low C_i grown cells possess both CO₂ uptake and HCO₃⁻ uptake activities and EZ inhibited both activities to a similar degree, suggesting that a common step in CO₂ and HCO₃⁻ uptake (such as the C_i pump) may have been affected. The inhibitor had no apparent effect on internal CO₂/HCO₃⁻ equilibria (internal CA function) in low C_i grown cells.     

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis

The nature of the inorganic carbon (C_i) species actively taken up by cyanobacteria CO₂ or HCO₃⁻ has been investigated. The kinetics of CO₂ uptake, as well as that of HCO₃⁻ uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO₂ was higher than that for HCO₃⁻. Though the calculated V_max was the same in both cases, the maximum rate of uptake actually observed was higher when HCO₃⁻ was supplied. C_i uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO₂ was the species supplied. Observations of photosynthetic rate as a function of intracellular C_i level (following supply of CO₂ or HCO₃⁻ for 5 seconds) led to the inference that HCO₃⁻ is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.

On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C_i transport apparatus binds CO₂ and releases HCO₃⁻ at or near a membrane porter. The latter transports HCO₃⁻ ions to the cell interior.

     

Characterization of the na-requirement in cyanobacterial photosynthesis

The Na⁺ requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li⁺ concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O₂ evolution) as a function of Na⁺ concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na⁺ (K_½[Na⁺]) were calculated. The value of both the low and the high K_½(Na⁺) was dependent upon extracellular DIC concentration. The low K_½(Na⁺) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K_½(Na⁺) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na⁺ and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na⁺ and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO₂ and HCO₃⁻ transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO₂ and HCO₃⁻ transport systems required Na⁺, directly or indirectly, for full activity. However, micromolar levels of Na⁺ were required for CO₂ transport while millimolar levels were required for HCO₃⁻ transport. These levels corresponded to those found for the low and high K_½(Na⁺) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO₂ transport increased with increased DIC concentration relative to HCO₃⁻ transport. This change was reflected in a decrease in the Na⁺ concentration required for maximum photosynthesis, in accord with the lower Na⁺-requirement for CO₂ transport. Lithium competitively inhibited Na⁺-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na⁺-dependent HCO₃⁻ transport. Lithium had little effect on CO₂ transport and only a small effect on the size of the pool it generated. Thus, CO₂ transport did not require a functional HCO₃⁻ transport system for full activity. Based on these observations and the differential requirement for Na⁺ in the CO₂ and HCO₃⁻ transport system, it was proposed that CO₂ and HCO₃⁻ were transported across the membrane by different transport systems.     

Mechanism of Photosynthetic Carbon Dioxide Uptake by the Red Macroalga, Chondrus crispus

The aim of this study was to determine how Chondrus crispus, a marine red macroalga, acquires the inorganic carbon (C_i) it utilizes for photosynthetic carbon fixation. Analyses of C_i uptake were done using silicone oil centrifugation (using multicellular fragments of thallus), infrared gas analysis, and gas chromatography. Inhibitors of carbonic anhydrase (CA), the band 3 anion exchange protein and Na⁺/K⁺ exchange were used in the study. It was found that: (a) C. crispus does not accumulate C_i internally above the concentration attainable by diffusion; (b) the initial C_i fixtion rate of C. crispus fragments saturates at approximately 3 to 4 millimolar C_i; (c) CA is involved in carbon uptake; its involvement is greatest at high HCO₃⁻ and low CO₂ concentration, suggesting its participation in the dehydration of HCO₃⁻ to CO₂; (d) C. crispus has an intermediate C_i compensation point; and (e) no evidence of any active or facilitated mechanism for the transport of HCO₃⁻ was detected. These data support the view that photosynthetic C_i uptake does not involve active transport. Rather, CO₂, derived from HCO₃⁻ catalyzed by external CA, passively diffuses across the plasma membrane of C. crispus. Intracellular CA also enhances the fixation of carbon in C. crispus.     

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO₃^?) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO₃^? by the surface‐bound enzyme carbonic anhydrase (CA_ext). Here, we examined other putative HCO₃^? uptake mechanisms in M. pyrifera under pH_T 9.00 (HCO₃^?: CO₂ = 940:1) and pH_T 7.65 (HCO₃^?: CO₂ = 51:1). Rates of photosynthesis, and internal CA (CA_int) and CA_ext activity were measured following the application of AZ which inhibits CA_ext, and DIDS which inhibits a different HCO₃^? uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO₃^? uptake by M. pyrifera is via an AE protein, regardless of the HCO₃^?: CO₂ ratio, with CA_ext making little contribution. Inhibiting the AE protein led to a 55%–65% decrease in photosynthetic rates. Inhibiting both the AE protein and CA_ext at pH_T 9.00 led to 80%–100% inhibition of photosynthesis, whereas at pH_T 7.65, passive CO₂ diffusion supported 33% of photosynthesis. CA_int was active at pH_T 7.65 and 9.00, and activity was always higher than CA_ext, because of its role in dehydrating HCO₃^? to supply CO₂ to RuBisCO. Interestingly, the main mechanism of HCO₃^? uptake in M. pyrifera was different than that in other Laminariales studied (CA_ext‐catalyzed reaction) and we suggest that species‐specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO₃^?:CO₂ due to ocean acidification.     

Evidence for Na-Independent HCO(3) Uptake by the Cyanobacterium Synechococcus leopoliensis

At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na⁺. The rate of photosynthesis greatly exceeded the CO₂ supply rate and indicated that HCO₃⁻ was taken up by a Na⁺-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na⁺ and exceeded the CO₂ supply rate by 8 to 45 times. The apparent photosynthetic affinity (K_½) for DIC was high (6-40 micromolar) and was not markedly affected by Na⁺ concentration, whereas with air-grown cells K_½ (DIC) decreased by more than an order of magnitude in the presence of Na⁺. Lithium, which inhibited Na⁺-dependent HCO₃⁻ uptake in air-grown cells, had little effect on Na⁺-independent HCO₃⁻ uptake by standing culture cells. A component of total HCO₃⁻ uptake in standing culture cells was also Na⁺-dependent with a K_½ (Na⁺) of 4.8 millimolar and was inhibited by lithium. Analysis of ¹⁴C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na⁺-independent CO₂ transport system. The conversion from Na⁺-independent to Na⁺-dependent HCO₃⁻ uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO₃⁻ for subsequent photosynthetic fixation.     

The Role of Carbonic Anhydrase 9 in Regulating Extracellular and Intracellular pH in Three-dimensional Tumor Cell Growths

We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of ∼300 μm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH_i) and pH in the restricted extracellular spaces (pH_e). With no CA9 expression, spheroids developed very low pH_i (∼6.3) and reduced pH_e (∼6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH_i = ∼6.6), whereas extracellular acidity was enhanced (pH_e = ∼6.6), so that radial pH_i gradients were smaller and radial pH_e gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH_e indicates the enzyme is facilitating CO₂ excretion from cells (by converting vented CO₂ to extracellular H⁺), rather than facilitating membrane H⁺ transport (such as H⁺ associated with metabolically generated lactic acid). This latter process requires titration of exported H⁺ ions with extracellular HCO₃⁻, which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH_e will depend on the cellular CO₂/lactic acid emission ratio (set by local oxygenation and membrane HCO₃⁻ uptake). Our results suggest that CO₂-producing tumors may express CA9 to facilitate CO₂ excretion, thus raising pH_i and reducing pH_e, which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.The carbonic anhydrases (CAs)³ are a family of enzymes that reversibly catalyze CO₂ hydration to H⁺ and HCO₃⁻ (1, 2). Recent studies have identified several CA isoforms, such as CA4, CA9, CA12, and CA14, with extracellular-facing catalytic sites (2). Many cells express extracellular CA (CA_e) isoforms, but their physiological role remains unclear. In particular, the strong link between cancer and CA9 expression (1–5) has provoked great interest in the role of CA_e in tumor biology.Based on their topology, CA_e isoforms are likely to regulate the concentration of extracellular H⁺, CO₂, and HCO₃⁻. Cell metabolism drives transmembrane fluxes of H⁺ ions, CO₂ and HCO₃⁻, and can provide substrate for the CA_e-assisted reaction. For example, CO₂ is released from aerobically respiring cells. By consuming or producing H⁺ ions, the CA_e-catalyzed reaction will affect extracellular pH (pH_e). Many membrane proteins are modulated by pH_e (6–8). Some of these are acid/base transporters that regulate intracellular pH (pH_i) (9). Such modulation allows pH_e to cross-talk with pH_i (10, 11), thus helping to shape the plethora of effects that pH_i has on cellular physiology (3, 9, 12, 13). Extracellular pH can also affect tissue structure through the release or modulation of proteolytic enzymes that act on the extracellular matrix (14, 15). In addition, the pH_e-pH_i difference is important in determining the distribution of membrane-permeant weak acids/bases, which include many drugs used clinically (e.g. doxorubicin).A complete understanding of pH regulation at tissue level requires characterization of events occurring within cells, at their surface membrane, and in the surrounding extracellular space. To date, many pH studies have treated the extracellular space as an infinite, well-stirred, and equilibrated compartment of constant pH. This condition is compatible with experimentally superfused, isolated cells, but it may not apply to all cells in situ. Blood plasma is a major component of extracellular fluid. In health, plasma pH is regulated to ∼7.4 by the lungs and kidneys, acting in concert to remove excess acid/base that has been added to blood from dietary or cellular sources. Tissue fluid occupies the gap between plasma and cells (with the exception of blood-borne cells). Under conditions of ideal diffusive coupling between cells and capillaries, pH_e in tissue fluid would be held close to plasma pH. However, pH_e close to the cell surface can diverge from 7.4, particularly when the cell-capillary distance is increased (e.g. as a result of poor blood perfusion), when the excreted acid/base load is elevated, or when the local buffering capacity is compromised.Regulation of pH_e is particularly important in tumors because these are characterized by a high metabolic rate (16, 17) and abnormal blood perfusion (18, 19). Studies have shown that tumors develop low pH_e (∼6.9) in response to the mismatch between metabolic demand and the capacity to remove metabolic waste products (14, 18, 20). Tumors can survive in considerably more acidic interstitium than their non-neoplastic counterparts, partly because of their ability to maintain a favorably alkaline pH_i for growth and development (21). It has been argued that tumors can survive selectively by maintaining a level of pH_e that is lethal to normal cells but not sufficiently acidic to kill the tumor itself (2, 14, 22).A major fraction of cell-derived acid is excreted in the form of CO₂, generated directly from the Krebs cycle or from titration of intracellular H⁺ with HCO₃⁻. To maintain a steep outward gradient for CO₂ excretion, extracellular CO₂ must not accumulate. This can be achieved by venting CO₂ to the nearest capillary or by reacting CO₂ locally to produce H⁺ and HCO₃⁻. The balance between these two fluxes is set by the diffusion distance and CO₂ hydration kinetics, respectively. Diffusion is anecdotally considered to be fast. However, over long distances, CO₂ diffusion may be slower than its local reactive flux. Assuming a CO₂ diffusion coefficient, D_CO2, of 2500 μm²/s and a spontaneous CO₂ hydration rate, k_f, of 0.14 s⁻¹ (23), local CO₂ consumption by reaction will be faster than CO₂ diffusion over distances >190 μm (√(2 × D_CO2/k_f)). The reactive flux can be augmented enzymatically by CA_e, to increase further the importance of reactive versus diffusive consumption of CO₂. If, for instance, hydration is catalyzed 10-fold, reactive CO₂ removal would exceed diffusive CO₂ removal over distances of >60 μm.The remainder of transmembrane acid efflux takes the form of lactic acid, generated from anaerobic respiration or aerobic glycolysis (Warburg effect) (16). Lactic acid efflux can be accelerated if its extracellular concentration is kept low by diffusive dissipation or by CA_e-catalyzed extracellular titration of H⁺ with HCO₃⁻. It is important to note that CA_e-catalyzed hydration of extracellular CO₂ will reduce pH_e, whereas titration of extracellular lactic acid by HCO₃⁻ (to form CO₂, a weaker acid) will raise pH_e. Therefore, the capacity of CA_e to regulate pH_e will depend on the chemistry of the excreted acid. In most healthy tissues at rest, the majority of cellular acid is emitted as CO₂. Recent work on tumors also suggests a dominance of CO₂ over lactic acid (22, 24).The role for CA_e in facilitating CO₂ removal has been demonstrated for CA4 in skeletal muscle (25) and proposed for CA9 in tumors (2, 26). Furthermore, CA9 expression is strongly up-regulated in hypoxia (5), providing a mechanism by which CA9 levels are linked to diffusion distance. A consequence of facilitated CO₂ removal is the attainment of a more uniformly alkaline pH_i across the tissue. We demonstrated this recently in three-dimensional in vitro tissue models imaged for pH_i (23). One prediction from that study is that CA9, although reducing pH_i nonuniformity, will give rise to local extracellular acidity, particularly at the core of multicellular growths.If pH_e is indeed acidified by CA9, the enzyme expression may be doubly beneficial for CO₂-excreting tumors: it will help to attain (i) a favorable alkaline pH_i for growth and (ii) an acidic pH_e to facilitate invasiveness. Clinically, CA9 may serve as a target for drugs. In the present work, we image pH_e using a novel, membrane-impermeant fluorescent pH dye in multicellular spheroid growths (∼35,000 cells) derived from the colon carcinoma cell line HCT116. We demonstrate a key role for CA9 in regulating both pH_i and pH_e. Furthermore, we show that, even in the hypoxic core of spheroids, the principal substrate for CA9 is cell-excreted CO₂ and that the precise effect of CA9 on pH_e depends on the relative efflux from cells of lactic acid versus CO₂.     

Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625

The active transport and intracellular accumulation of HCO₃⁻ by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na⁺.Na⁺-dependent HCO₃⁻ accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O₂ evolution and CO₂ fixation. However, when Synechococcus was grown in standing culture, high rates of HCO₃⁻ transport and photosynthesis were observed in the absence of added Na⁺. The internal HCO₃⁻ pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO₃⁻ transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO₃⁻ from the medium in the absence of Na⁺ was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO₂ transport involving a high-affinity transport system which was reversibly inhibited by H₂S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO₂ transport system, whereas Na⁺-dependent and Na⁺-independent HCO₃⁻ transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO₃⁻ was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO₂ fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO₃⁻ + CO₂. The pattern of fluorescence quenching observed in response to added HCO₃⁻ and Na⁺ in air-grown and standing culture cells was highly characteristic for Na⁺-dependent and Na⁺-independent HCO₃⁻ accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO₃⁻ transport and the dynamics of intracellular HCO₃⁻ accumulation and dissipation.     

Inorganic Carbon Uptake during Photosynthesis : II. Uptake by Isolated Asparagus Mesophyll Cells during Isotopic Disequilibrium

The species of inorganic carbon (CO₂ or HCO₃⁻) taken up a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO₂ or HCO₃⁻ transport, during steady state photosynthesis, is achieved by monitoring the changes (by ¹⁴C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO₂ or HCO₃⁻ transport) and experimental time-courses of ¹⁴C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO₂, rather than HCO₃⁻, is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO₂ is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO₃⁻ transport, as the incorporation of ¹⁴C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO₂ uptake alone. The contribution of HCO₃⁻ to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO₃⁻ concentration. The evidence for direct HCO₃⁻ transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of ¹⁴C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO₂, which is partially alleviated by a high extracellular concentration of HCO₃⁻.     

Intracellular pH regulation and buffer capacity in CO2/HCO− 3-buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO⁻ ₃-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH_i) was monitored by continuous spectrofluorometric recording from cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH=7.70 were transferred to normal CO₂/HCO⁻ ₃-buffered medium {P _CO2=3.71 mmHg, [HCO⁻ ₃]= 6.1 mmol l⁻¹, extracellular pH (pH_e)=7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (∼7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5–8.0 slykes) based on intracellular [HCO⁻ ₃], and was unaffected by most drugs and treatments. However, after transfer to high P _CO2=11.15 mmHg, [HCO⁻ ₃]= 18.2 mmol l⁻¹ at the same pH_e=7.70, the final regulated pHi was elevated (∼7.53). The rate of correction of alkalosis caused by washout of this high P _CO2, high-HCO⁻ ₃ medium was unaffected by removal of extracellular Cl⁻. Removal of extracellular Na⁺ lowered resting pH_i and greatly inhibited the rate of pH_i recovery from acidosis. Bafilomycin A₁ (3 μmol l⁻¹) had no effect on these responses. However amiloride (0.2 mmol l⁻¹) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH_i, the latter differing from the response in HEPES where amiloride also lowered resting pH_i. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l⁻¹) did not affect resting pH_i but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl⁻ also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO⁻ ₃)_N co-transport and/or Na⁺-dependent HCO⁻ ₃/Cl⁻ exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to “housekeeping” pH_i regulation in gill cells in CO₂/HCO⁻ ₃ media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl⁻/HCO⁻ ₃ exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media. Accepted: 30 November 1999     

Uptake of HCO3? and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.     

Role of Na+/ H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO₂ on intracellular pH (pH_i) and mechanisms that mediate pH_i recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pH_i was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH_i in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH₄ ⁺/NH₃-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pH_i recovery from an NH₄ ⁺/NH₃-induced acid load by 58% and 70%, respectively. pH_i recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO₂/HCO₃ ⁻-buffered (5%/20 mM) solution caused a rapid decrease of pH_i (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pH_i recovery by 88%. The results indicate that intracellular acidification by butyrate and CO₂ is effectively counter-regulated by an Na⁺/H⁺ exchanger and by DIDS-sensitive, HCO₃ ⁻-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH_i homeostasis of ruminal epithelial cells. Accepted: 8 March 2000     

Carbon Oxysulfide Is an Inhibitor of Both CO(2) and HCO(3) Uptake in the Cyanobacterium Synechococcus PCC7942

Carbon oxysulfide (COS) was reinvestigated as an inhibitor of active inorganic carbon transport in cells of Synechococcus PCC7942 adapted to growth at low inorganic carbon. COS inhibited both CO₂ and HCO₃⁻ transport processes in a reversible (in the short term) and mixed competitive manner. The inhibition of COS was established using both silicone oil centrifugation experiments and O₂-evolution studies. The K_i for COS inhibition was 29 micromolar for CO₂ transport and 110 micromolar for HCO₃⁻ transport. These results support a model of inorganic carbon transport with a central CO₂ pump and an inducible HCO₃⁻ utilizing accessory protein which supplies CO₂ to the primary pump.