Transepithelial Na+ transport and the intracellular fluids: A computer study

Summary Computer simulations of tight epithelia under three experimental conditions have been carried out, using the rheogenic nonlinear model of Lew, Ferreira and Moura (Proc. Roy. Soc. London. B 206:53–83, 1979) based largely on the formulation of Koefoed-Johnsen and Ussing (Acta Physiol. Scand.42:298–308, 1958). First, analysis of the transition between the short-circuited and open-circuited states has indicated that (i) apical Cl^– permeability is a critical parameter requiring experimental definition in order to analyze cell volume regulation, and (ii) contrary to certain experimental reports, intracellular Na⁺ concentration (c _Na ^c ) is expected to be a strong function of transepithelial clamping voltage. Second, analysis of the effects of lowering serosal K⁺ concentration (c _K ^s ) indicates that the basic model cannot simulate several well-documented observations; these defects can be overcome, at least qualitatively, by modifying the model to take account of the negative feedback interaction likely to exist between the apical Na⁺ permeability andc _Na ^c . Third, analysis of the effects induced by lowering mucosal Na⁺ concentration (c _Na ^m ) strongly supports the concept that osmotically induced permeability changes in the apical intercellular junctions play a physiological role in conserving the body's stores of NaCl. The analyses also demonstrate that the importance of Na⁺ entry across the basolateral membrane is strongly dependent upon transepithelial potential,c _Na ^m andc _K ^s ; under certain conditions, net Na⁺ entry could be appreciably greater across the basolateral than across the apical membrane.     

Intracellular ionic activities in frog skin

Summary Asymmetrical displacement currents are measured in the absence and in the presence of the lipophilic anion dipicrylamine (DPA) in the extracellular solution of nerve fibres of the frogRana esculenta. DPA (30nM-3 M) enhances the current by a component that has the properties expected for a translocation current of DPA ion across the lipid membrane. Analysis in terms of a single-barrier model yields the translocation rate constant (k), the total surface density of DPA absorbed to the membrane (N _t ), and the equidistribution voltage (). The value ofk of about 10⁴ s^–1 is similar to that for a solvent-free artificial bilayer formed by the Montal-Mueller method. The surface densityN _t varies with the DPA concentration as it does in the artificial bilayer, but is about tenfold smaller at all concentrations. The DPA ions sense an intrinsic electric field that is offset by a transmembrane voltage between 0 and 30 mV (inside positive). The part of the axolemma probed by the DPA ion appears as a thin (<2.5 nm), fluid bilayer of lipids. DPA ions seem, however, to be excluded from the major part of the axolemma as if this area is occupied by integral proteins or negative charges.     

Contributions of secondary active transport processes to membrane potentials

Summary Equations are developed to examine the effects of secondary active transport processes on the steady-state membrane potential of symmetrical cells. It is shown that, with suitable modifications, equations of the type developed by Goldman, Hodgkin and Katz may be derived to accommodate the contributions to the membrane potential of both electroneutral and electrogenic transporters. Where the membrane potential is function of the dominant medium ions (Na, K, and Cl), other contributions can come only from an electrogenic Na pump and from neutral co- and counter-transporters if, and only if, these involve the dominant ions. Experimental approaches to measure the parameters necessary to solve the equations developed here are discussed.     

Effects of bicarbonate on fluid and electrolyte transport by guinea pig and rabbit gallbladder: Stimulation of absorption

Summary The effect of bicarbonate (HCO₃) on fluid absorption by guinea pig gallbladder was investigatedin vitro. Stimulation of fluid absorption was concentration dependent resulting in a fourfold increase in transport over the range 1 to 50mm. Phosphate, Tris, glycodiazine and glutamine buffers failed to substitutte for HCO₃ in stimulating absorption. Unidirectional²²Na fluxes were measured across short-circuited sheets of guinea pig and rabbit gallbladders mounted in Ussing-type chambers. In both species the net Na flux was unaffected by serosal HCO₃ alone but was stimulated by addition of HCO₃ to the mucosal bathing solution. Transepithelial electrical potential difference in rabbit gallbladder was about 1.4 mV (lumen positive) when HCO₃ was present in the mucosal or in both compartments. This fell to 0.2 mV under HCO₃-free conditions or when HCO₃ was present only in the serosal solution. The respective values for guinea pig gallbladder were –1.6 and –0.6 mV (lumen negative). HCO₃ stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 7.8, an effect resulting mainly from a reduction inJ _mis ^Na . Tris buffer (25mm) inhibited HCO₃-dependent fluid absorption in this species completely at pH 8.5 and partially at 7.5. These results indicate that HCO₃ stimulates gallbladder transport in both species by an action from the mucosal side. This effect cannot be attributed to simple buffering of H⁺ but may be explained by the participation of HCO₃ in the maintenance of intracellular H⁺ for a Na/H-exchange.     

Fluid transport and dimensions of epithelial cells and intercellular spaces in frog gallbladder

Summary Morphologic findings of widely dilated intercellular spaces in fluid transporting epithelia have been claimed as evidence for the existence of an epithelial compartment in which the coupling between solute and water fluxes takes place. The validity of using epithelial geometry in sectioned material as an argument can be questioned. The present report describes the morphological appearance of frog gallbladder epithelium — normal and ouabain-treated — in the living state in vitro and after fixation, dehydration and embedding. Gallbladder segments were photographed in the living state and at the end of each step of the preparative procedure. Direct observations of whole-mounted gallbladder segments were carried out, taking advantage of the possibility of optical sectioning and high resolution by Nomarski-microscopy. The same specimens were then sectioned and examined by conventional light and electron microscopy. The observations were quantitated and showed that the epithelial cells of normal and ouabain-treated gallbladders experienced an average linear shrinkage down to 70% of their length in Ringer's solution, which corresponds to a volume shrinkage down to 35%. Moreover, dilated lateral intercellular spaces appeared during the dehydration and embedding procedure in normal but only very moderately or not at all in ouabain-treated gallbladder specimens.     

Effect of acidic pH on the ability of Clostridium sporogenes MD1 to take up and retain intracellular potassium

At pH values >5.5, Clostridium sporogenes MD1 accumulated potassium even though it had little protonmotive force, but an ATPase inhibitor (N, N'- dicyclohexylcarbodiimide) prevented this uptake. The results suggested that potassium transport was ATP-driven, and a protonophore (3, 3', 4', 5 - tetrachlorosalicylanilide) did not eliminate uptake. However, potassium uptake could also be driven by an artificial pH gradient, and in this case the protonophore acted as an inhibitor. These latter results indicated that the cells also had a protonmotive force-driven transporter. When the pH <5.1, the cells could not retain potassium, rapid efflux was observed, and intracellular volume collapsed.     

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

Summary We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (V _bl) in rabbit proximal convoluted tubule (PCT. Sl segment). The present study is aimed at understanding the origin of the membrane repolarization following the intial effect of addition of luminal cotransported solutes. Luminal addition of 10–15mMl-alanine produced a rapid and highly significant depolarization ofV _bl (20.3±1.1 mV,n=15) which was transient and associated with an increase in the fractional K⁺ conductance of the basolateral membrane (t _K) from 8 to 29% (P<0.01,n=6). Despite the significant increase int _K, the repolarization was only slightly reduced by the presence of basolateral Ba²⁺ (2mM,n=6) or quinine (0.5 mM,n=5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4isothiocyamostilbene-2,2-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pH _i ) determined with the fluorescent dye 2, 7-bis-2-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF), averaged 7.39±0.02 in control solution (n=9) and increased to 7.50±0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16±0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K⁺ conductance but also through a cellular acidification reducing the basolateral HCO ₃ ^– exit through the electrogenic Na-3(HCO₃) cotransport mechanism.     

Measurement of intracellular potassium activity in protoplasts of Acer pseudoplatanus: origin of their electropositivity

Membrane resistances, electrical potentials and intracellular K⁺ activity have been studied in protoplasts of Acer pseudoplatanus L. using microelectrodes. The plasmalemma appears to be damaged during protoplast processing, so that it is almost completely depolarized. The positive polarization of the protoplasts is a property of their tonoplast. The osmotic shock of general dilution of the medium brings about a slight positive polarization of the protoplasts, which probably is the consequence of a dilution of the ionic contents of the cell. A rapid dilution of extracellular potassium produces the same effect by a considerable efflux of K⁺, which changes the concentration gradient so that electropositivity is accentuated. In general, certain states of shock can cause such polarization. These results, particularly the development of internal potassium activity, help in understanding the initial cause of the positive polarization that is sometimes observed in protoplasts.     

Sheep twin chimaeras with admixtures of red cell amino acid and potassium transport phenotypes

A pair of sheep twins each had two populations of red cells. Population 1 was positive for antigens Aa, Ma and Mb, was low-potassium type, possessed an amino acid transport system and was lysine-negative phenotype. Population 2 was negative for antigens Aa, and Mb, was high-potassium type, lacked the amino acid transport system and was lysine-positive phenotype. Population 2 disappeared from both sheep over a period of 8 years.     

Differential effects of turgor on sucrose and potassium transport at the tonoplast and plasma membrane of sugar beet storage root tissue

When turgor was increased, by decreasing the concentration of mannitol bathing discs of sugar beet storage root tissue, the rates of sucrose and potassium uptake into the vacuole were decreased. At all external mannitol concentrations the rate of sucrose and potassium uptake across the plasma membrane was an order of magnitude greater than the rate of quasi-steady uptake into the vacuole, implying a very large efflux. Efflux of both sucrose and potassium was increased at high turgor. However, while increasing turgor decreased the rate of K⁺ uptake, the rate of sucrose uptake at the plasma membrane increased with time. Compartmental analysis of tracer exchange kinetics was used to determine unidirectional K⁺ fluxes. From these results, it was estimated that the increase in K⁺ efflux accompanying a 1.5 MPa increase in turgor could lead to a net increase of 140mol^?3h^?1 in the external potassium concentration. It is suggested that the turgor-imposed increase in solute efflux is a means of regulating intracellular osmotic pressure and/or turgor in sugar beet storage roots, but that sucrose is preferentially retrieved from the apoplast, even under conditions of excessively high turgor. However, much of this sucrose is probably lost from the cell, implying a ‘futile’ sucrose transport cycle at the plasma membrane. The turgor-stimulated leak of potassium could play a major role in the regulation of turgor pressure in sugar beet storage root tissue.     

电压激活的钾通道阻断剂抑制山莨菪碱松弛去甲肾上腺素预收缩的兔主动脉平滑肌

研究显示,山莨菪碱预处理不改变高钾引起的兔主动脉环收缩,但可明显减弱去甲肾上腺素(noradrenaline,NA)、组织胺或5-羟色胺引起的收缩,且其减弱作用不受去除血管内皮影响。本实验观察了几种钾通道阻断剂对山良菪碱松弛：NA预收缩的兔主动脉环的影响。结果表明,1、3、10μmol／L山莨菪碱作用8min,可使0．01μmol／L NA预收缩的兔主动脉环松弛(P＜O．01)。10mmol／L,CsCl、1mmol／L 4-氨基吡啶、10μmol／L BaCl2、10μmol/L格列本脲、3μmol／L charybdotoxin和3μmol／L蜂毒明从分别与0．0lμmol／L NA同时加入,可增强后者收缩兔主动脉环的作用(P＜0．01)。10、30mmol／L CsCl或10、30mmol／L 4-氨基吡啶存在时,10μmol／L山茛菪碱对NA预收缩的兔主动脉环的松弛作用减弱,松弛率与对照组比较分别有极显著差异(P＜0．01);10、30μmol／L BaCl2,10、30μmol/L格列本脲,3μmol/L charybdotoxin或3μmol／L蜂毒明肽存在时,山莨菪碱对NA预收缩的兔主动脉环的松弛作用不受影响(P＞O．05)。本研究表明,电压激活的钾通道阻断剂抑制山莨菪碱松弛NA预收缩的兔主动脉平滑肌,初步提示血管平滑肌细胞膜上电压激活的钾通道参与山莨菪碱扩血管作用。     

Sulfate transport in rabbit ileum: Characterization of the serosal border anion exchange process

Summary The preceding paper [30] shows that transepithelial ileal SO₄ transport involves Na-dependent uptake across the ileal brush border, and Cl-dependent efflux across the serosal border. The present study examines more closely the serosal efflux process. Transepithelial mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) across rabbit ileal mucosa were determined under short-circuit conditions. SO₄ was present at 0.22mm. In standard Cl, HCO₃ Ringer's,J _ms ^SO4 was 81.3±5.3 (1se) andJ _ms ^SO4 was 2.5±0.2 nmol cm^–2 hr^–1 (n=20). Serosal addition of 4-acetamido-4-isothiocyanostilbene-22-disulfonate (SITS), 44-diisothiocyanostilbene-22-disulfonate (DIDS) or 1-anilino-8-naphthalene-sulfonate (ANS) inhibited SO₄ transport, SITS being the most potent. Several other inhibitors of anion exchange in erythrocytes and other cells had no effect on ileal SO₄ fluxes. In contrast to its effect on SO₄ transport, SITS (500 m) did not detectably alter Cl transport.Replacement of all Cl, HCO₃ and PO₄ with gluconate reducedJ _ms ^SO4 by 70% and increasedJ _ms ^SO4 by 400%. A small but significantJ _net ^SO4 remained.J _ms ^SO4 could be increased by addition to the serosal side of Cl, Br, I, NO₃ or SO₄. The stimulatory effect of all these anions was saturable and SITS-inhibitable. The maximalJ _ms ^SO4 in the presence of Cl was considerably higher than in the presence of SO₄ (73.1 and 42.2 nmol. cm^–2 hr^–1, respectively;p<0.001). TheK _1/2 value for Cl was 7.4mm, 10-fold higher than that for SO₄ (0.7mm). Omitting HCO₃ and PO₄ had no measurable effects on SO₄ fluxes.This study shows that (i) SO₄ crosses the serosal border of rabbit ileal mucosa by anion exchange; (ii) the exchange process is inhibited by SITS, DIDS and ANS, but not by several other inhibitors of anion exchange in other systems; (iii) SO₄ may exchange for Cl, Br, I, NO₃ and SO₄ itself, but probably not for HCO₃ or PO₄; (iv) kinetics of the exchange system suggest there is a greater affinity for SO₄ than for Cl, although the maximal rate of exchange is higher in the presence of Cl; and, finally (v) SITS has little or no effect on net Cl transport.     

Effect of zinc on L-threonine transport across the jejunum of rabbit

Zinc is an essential trace element for life. Many metalloenzymes involved in the metabolism of carbohydrates, lipids, protein, and nucleic acids require zinc for their functions. The aim of this study was to characterize how zinc acts on the intestinal amino acid absorption in rabbit. Results obtained show that zinc inhibits both L-threonine accumulation in the jejunum tissue, and mucosal-to-serosal transepithelial flux of this amino acid in a dose-dependent way. The inhibition does not increase by a 10-min previous intestinal exposure of the mucosa to the heavy metal, and is not reversed by washing the intestinal tissue with saline solution or 10mM EDTA, but is appreciably reversed with 10mM dithioerythritol. Zinc seems not to modify amino acid diffusion across the intestinal epithelium. The inhibition of intestinal amino acid transport by zinc seems to be of a competitive type, and appears to be a result of impairment of the active transport that is altered by its binding to proteins (prevailing to thiol groups) of the brush-border membrane of enterocytes.     

Uphill transport of water by electroosmosis

Summary Lepismatidae are able to gain water from subsaturated atmosphere above a relative humidity of 45%, surmounting a water potential difference of at least 1.1×10⁸ Pa (1,100 bar). This extraordinary task is performed by the monolayered epithelium of the posterior rectum. The particle coat of the folded apical membrane of this epithelium suggests the presence of the electrogenic, lumen-directed cation transport, which is commonly found in insects. Assuming this kind of transport and considering the anatomy of the organ, a working hypothesis for this hyposmotic water transport has been developed: The electrogenic cation transport maintains the circulation of the transported ion species across the apical membrane; the voltagedriven inward current transfers water by electroosmosis against its chemical potential from the extracellular space into the cytoplasm. Voltage and current measurements and synchronous measurements of water flow across the epithelium of the posterior rectum ofLepisma saccharina strongly corroborate this hypothesis. The transepithelial voltage is up to 200 mV (lumen positive); the short-circuit current averages 200 A per cm² of the epithelium. Both depend acutely on oxidative metabolism as does spontaneous water uptake. Exogenous transepithelial current (I) induces, independently of anoxia, a proportional change in volume flow (J _v). The induced flow has the direction of the cation flow. Its mean coupling ratio (J _v/I) is 1.5×10^–9m³/A·sec corresponding to 7 to 8 H₂O per positive unit charge. Critical evaluation of experimental data reveals that water uptake by electroosmosis may quantitatively account forin vivo performance without requiring any unusual assumption.     

The cytosol receptors for progesterone in the different parts of rabbit fallopian tube and uterus during ovum transport

Progesterone receptors were determined in the cytosol from the ampulla, ampullaryisthmic junction and isthmus of rabbit fallopian tube and uterus of estrus and pregnant rabbits. The receptor levels when compared among its various anatomical segments, were the same in ampulla, isthums and uterus but maximum in ampullary-isthmic junction. Significant differences were observed in mated animals at 14, 24, 34, 48, 70 and 144 h after coitus. The receptor concentrations in portions of the fallopian tube showed no significant change between 14 and 24 h after coitus, except for a decrease in ampullary-isthmic junction at 24 h. At 34 h the concentration of receptor further decreased in all parts of the tube. At 48 and 70 h after coitus, receptor concentrations decreased gradually in ampulla and ampullary-isthmic junction, while isthmus showed a gradual increase. At 144 h, the receptor concentration showed no further change in ampulla and ampullary-isthmic junction; however, isthmus showed a decline. The uterine receptor concentration declined steadily from estrus till 70 h after coitus, however, it was increased at 144 h. The dissociation constant (K_d) of cytosol receptor in all the tissues at estrus and during early pregnancy was found similar. The implications of these changes in relation to the normal ovum transport have been correlated in this paper.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Summary The intracellular pH (pH _i ) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H⁺ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH _i of cells placed in an acidic medium (pH _o below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH _o is acutely reduced to 5.5, pH _i falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H⁺. The unidirectional influx of H⁺ exhibits saturation kinetics with respect to extracellular [H⁺]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK _m is 0.74 ± 0.09 × 10^–6 m.Steady-state cells with pH _i above 6.8 continuously extrude H⁺ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH _i 6.3) when returned to pH _o 7.3 medium respond by transporting H⁺, resulting in a rapid rise in pH _i . The halftime for this process is 1.09 ± 0.22 min. The H⁺ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH _i to its steady-state value.     

Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Activity assay of membrane transport proteins

Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters, transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiologi-cal assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.     

ATP-dependent carbon transport in perfused Chara cells

Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO₂ and HCO₃^- are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO₂ and HCO₃^- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2-deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H⁺_-ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H⁺_-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied ¹⁴C intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or vanadate. The results indicate that Chara cells possess CO₂ and HCO₃^- transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m ^-3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m^-3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m^-3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no effect on the dark-carbon fixation.