Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: function and phylogenetic analysis

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO(2) uptake systems and two HCO(3)(-) transporters. We transformed a mutant impaired in CO(2) uptake and in cmpA-D encoding a HCO(3)(-)transporter with a transposon inactivation library, and we recovered mutants unable to take up HCO(3)(-) and grow in low CO(2) at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO(2), requires Na(+), and plays the major role in HCO(3)(-) uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na(+)/K(+)-translocating protein) abolished the ability of cells to grow at [Na(+)] higher than 100 mm and severely depressed the activity of the SbtA-mediated HCO(3)(-) transport. We propose that the SbtA-mediated HCO(3)(-) transport is driven by DeltamuNa(+) across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na(+)-dependent HCO(3)(-) transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO(2) supply is limited, or in Prochlorococcus strains that do not possess CO(2) uptake systems or Cmp-dependent HCO(3)(-) transport.     

Quenching of Chlorophyll a Fluorescence in Response to Na+-Dependent HCO3- Transport-Mediated Accumulation of Inorganic Carbon in the Cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the yield of chlorophyll a fluorescence decreased in response to the transport-mediated accumulation of intracellular inorganic carbon (CO2 + HCO3- + CO32- = dissolved inorganic carbon [DIC]) and subsequently increased to a near-maximum level following photosynthetic depletion of the DIC pool. When DIC accumulation was mediated by the active Na+-dependent HCO3- transport system, the initial rate of fluorescence quenching was found to be highly correlated with the initial rate of H14CO3- transport (r = 0.96), and the extent of fluorescence quenching was correlated with the size of the internal DIC pool (r = 0.99). Na+-dependent HCO3- transport-mediated accumulation of DIC caused fluorescence quenching in either the presence or absence of the CO2 fixation inhibitor glycolaldehyde, indicating that quenching was not due simply to NADP+ reduction. The concentration of Na+ required to attain one-half the maximum rate of H14CO3- transport, at 20 [mu]M external HCO3-, declined from 9 to 1 mM as the external pH increased from 8 to 9.6. A similar pH dependency was observed when fluorescence quenching was used to determine the kinetic constants for HCO3- transport. In cells capable of Na+-dependent HCO3- transport, both the initial rate and extent of fluorescence quenching increased with increasing external HCO3-, saturating at about 150 [mu]M. In contrast Na+-independent HCO3- transport-mediated fluorescence quenching saturated at an HCO3- concentration of about 10 [mu]M. It was concluded that measurement of chlorophyll a fluorescence emission provided a convenient, but indirect, means of following Na+-dependent HCO3- transport and accumulation in Synechococcus.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Plasmalemmal V-H(+)-ATPases regulate intracellular pH in human lung microvascular endothelial cells

The lung endothelium layer is exposed to continuous CO(2) transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na(+)/H(+) exchanger and HCO(3)(-)-dependent H(+)-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H(+)-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H(+)-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na(+) and HCO(3)(-) that were similar to those observed in the presence of either Na(+), or Na(+) and HCO(3)(-). The Na(+)- and HCO(3)(-)-independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H(+)-ATPase inhibitor. These studies show a Na(+)- and HCO(3)(-)-independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases.     

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.     

Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na(+)/H(+) exchange to regulate pH. HCO(3)(-)-dependent buffering and HCO(3)(-)-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pH(i)) was measured using the H(+)-sensitive fluoroprobe BCECF. In solutions lacking HCO(3)(-)/CO(2), pH(i) was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na(+)-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H(+)-ATPase, was also apparent. In solutions containing HCO(3)(-)/CO(2), pH(i) was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO(3)(-)/CO(2)-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO(3)(-)/CO(2), although under these conditions it was again largely dependent on Na(+) ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO(3)(-)-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na(+)/H(+) exchanger together with a H(+)-ATPase. The absence of extrinsic (HCO(3)(-)/CO(2)) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.     

Active transport of CO(2) and bicarbonate is induced in response to external CO(2) concentration in the green alga Chlorella kessleri

The time-course of induction of CO(2) and HCO(3)- transport has been investigated during the acclimation of high CO(2)-grown Chlorella kessleri cells to dissolved inorganic carbon (DIC)-limited conditions. The rate of photosynthesis of the cells in excess of the uncatalysed supply rate of CO(2) from HCO(3)- was taken as an indicator of HCO(3)- transport, while a stimulation of photosynthesis on the addition of bovine carbonic anhydrase was used as an indicator of CO(2) transport. The maximum rate of photosynthesis (Pmax) was similar for high CO(2)-grown and low CO(2)-grown cells, but the apparent whole cell affinity for DIC and CO(2) of high CO(2)-grown cells was found to be about 30-fold greater than in air-grown cells, which indicates a lower affinity for DIC and CO(2). It was found that HCO(3)- and CO(2) transport were induced in 5.5 h in cells acclimating to air in the light and in the presence and absence of 21% O(2), which indicates that a change in the CO(2)/O(2) ratio in the acclimating medium does not trigger induction of DIC transport. No active DIC transport was detected in high CO(2)-grown cells maintained on high CO(2) for 5.5 h in the presence of 5 mM aminooxyacetate, an aminotransferase inhibitor. These results indicate no involvement of photorespiration in triggering induction. Active DIC transport induction was inhibited in cells treated with 5 microgram ml(-1) cycloheximide, but was unaffected by chloramphenicol treatment, indicating that the induction process requires de novo cytoplasmic protein synthesis. The total DIC concentration eliciting the induction and repression of CO(2) and HCO(3)- transport was higher at pH 7.5 than at pH 6.6. The concentrations of external CO(2) required for the induction and repression of DIC transport were 0 and 120 microM, respectively, and was independent of the pH of the acclimation medium. Prolonged exposure to a critical external CO(2) concentration elicits the induction of DIC transport in C. kessleri.     

Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths

Intracellular pH (pH(i)), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H(+) ions (e.g. produced by respiration) are extruded by transporters such as Na(+)/H(+) exchange, or neutralized by HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (～10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) transporters and as a mobile buffer for facilitating extracellular diffusion of H(+) ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor growths and may be important for cancer disease progression.     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (II. Evidence for Induction in Response to External CO2 Concentration)

The critical species and concentrations of dissolved inorganic carbon (DIC) required for the induction of DIC transport during adaptation to low CO2 were determined for the green alga Chlorella ellipsoidea. The concentration of dissolved CO2 needed for the induction of both CO2 and HCO3- transport was independent of pH during adaptation, whereas the total DIC concentration required increased at alkaline pH. At pH 7.5, the minimum equilibrium DIC concentration at which high CO2 characteristics were maintained, i.e. transport was repressed, was 2100 [mu]M, whereas the maximum equilibrium DIC concentration below which DIC transport was fully induced (DICIND) was 500 [mu]M. Intracellular DIC concentration during adaptation to DICIND decreased temporarily after 2 h to 60% of the maximum level but recovered after 3 h of adaptation. After 3 h of adaptation to DICIND, cells exhibited maximum O2 evolution rate at DICIND. When cells partially adapted to DICIND were returned to high CO2, there was an immediate halt to the induction of transport and a gradual decrease in transport capacity over 23 h. The capacity for the induction of transport was unaffected by the absence of light. These results indicate that changes in the internal DIC pool during adaptation to low CO2 do not trigger the induction of DIC transport and that the induction is not light dependent. Induction of DIC transport in C. ellipsoidea appears to occur in response to the continuous exposure of cells to a critical CO2 concentration in the external medium.     

Cloning and characterization of an electrogenic Na/HCO3- cotransporter from the squid giant fiber lobe

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na(+)-driven Cl-HCO(3)(-) exchanger, sqNDCBE--related to the SLC4 superfamily and cloned from giant fiber lobe cDNA--is the only HCO(3)(-)-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5' and 3' directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na(+)-coupled SLC4 transporters. However, when we measure pH(i) and membrane potential--or use two-electrode voltage clamping to measure currents--on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO(3)(-) cotransporter, NBCe. That is, exposure to extracellular CO(2)/HCO(3)(-) not only causes a fall in pH(i), followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pH(i) recovery and current require HCO(3)(-) and Na(+), and are blocked by DIDS. Furthermore, neither K(+) nor Li(+) can fully replace Na(+) in supporting the pH(i) recovery. Extracellular Cl(-) is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.     

Acid and base secretion in the Calu-3 model of human serous cells

Submucosal glands are the primary source of airway mucus, a critical component of lung innate defenses. Airway glands are defective in cystic fibrosis (CF), showing a complete absence of secretion to vasoactive intestinal peptide or forskolin, which increase intracellular cAMP concentration. This defect is attributed to gland serous cells, which express the cystic fibrosis transmembrane conductance regulator. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO(3)(-), with HCO(3)(-) secretion predominating for forskolin stimulation and Cl(-) secretion predominating for stimuli that open basolateral K(+) channels to hyperpolarize the cells. We used pH stat and ion substitution experiments to clarify the mechanisms and consequences of these two modes of secretion. We confirm that Calu-3 cells secrete primarily HCO(3)(-) in response to forskolin. Unexpectedly, HCO(3)(-) secretion continued in response to K(+) channel openers, with Cl(-) secretion being added to it. Secretion of HCO(3)(-) from hyperpolarized cells occurs via the conversion of CO(2) to HCO(3)(-) and is reduced by approximately 50% with acetazolamide. A gap between the base equivalent current and short-circuit current was observed in all experiments and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (possibly H(+)-K(+)-ATPase), which partially neutralized the secreted HCO(3)(-). The conjoint secretion of HCO(3)(-) and H(+) may help explain the puzzling finding that mucus secreted from normal and CF glands has the same acidic pH as does mucus from glands stimulated with forskolin or ACh. It may also help explain how human airway glands produce mucus that is hypotonic.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.     

pH sensitivity of the Na+:HCO3- cotransporter in basolateral membrane vesicles isolated from rabbit kidney cortex.

HCO3- exit across the basolateral membrane of the kidney proximal tubule cell is mediated via an electrogenic Na+:HCO3- cotransporter. We have studied the effect of pH on the activity of this cotransport system in basolateral membrane vesicles isolated from rabbit renal cortex. At constant internal pH 6.0, increasing the external pH and [HCO3-] increased the rate of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 22Na+ influx into the vesicles. To determine the role of internal pH on the activity of the Na+:HCO3- cotransport system, the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange was measured in the absence of an initial pH and [HCO3-] gradient (pH(i) = pH(o), 5% CO2). Increasing the pH from 6.8 to 7.2 increased whereas, increasing the pH from 7.4 to 8.0 decreased the rate of 22Na+ influx via this exchange. Increasing pH at constant [HCO3-] (pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 10% CO2) reduced the influx of 22Na+ via HCO3-dependent Na(+)-Na+ exchange. Increasing pH at constant [CO3(2-)](pH(i) = pH(o) = 8.0, 1.5% CO2 versus pH(i) = pH(o) = 7.2, 60% CO2) was associated with reduced 22Na+ uptake. Decreasing the pH (pH(i) = pH(o) = 6.3, 60% CO2 versus pH(i) = pH(o) = 7.2, 5% CO2) was associated with a reduced rate of HCO3(-)-dependent Na(+)-Na+ exchange. We conclude that the Na+:HCO3- cotransporter displays a significant pH sensitivity profile with the cotransporter being more functional at pH 7.0-7.4 and less active at more acid or alkaline pH. In addition, the results suggest that the pH sensitivity arises at the inner surface of the basolateral membrane.     

Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD)

Epithelial Na(+) channel (ENaC)-mediated Na(+) absorption and BK channel-mediated K(+) secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO(3)(-) reabsorption in the proximal tubule due to changes in both luminal Na(+)/H(+) exchanger 3 and H(+)-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289-F296, 2006), we sought to test the hypothesis that flow also regulates H(+)-ATPase activity in the CCD. H(+)-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH(4)Cl prepulse technique). H(+)-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pH(i)) recovery in the absence of luminal K(+), bilateral Na(+), and CO(2)/HCO(3)(-), from a nadir pH of ～6.2. We found that 1) an increase in luminal flow rate from ～1 to 5 nl·min(-1)·mm(-1) stimulated H(+)-ATPase activity, 2) flow-stimulated H(+) pumping was Ca(2+) dependent and required microtubule integrity, and 3) basal and flow-stimulated pH(i) recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H(+)-ATPase activity in the rabbit CCD and that H(+)-ATPases therein are present in both principal and intercalated cells.     

Mechanisms of Mg(2+) transport in cultured ruminal epithelial cells

Net Mg(2+) absorption from the rumen is mainly mediated by a transcellular pathway, with the greater part (62%) being electrically silent. To investigate this component of Mg(2+) transport, experiments were performed with isolated ruminal epithelial cells (REC). Using the fluorescent indicators mag-fura 2, sodium-binding benzofuran isophthalate, and 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, we measured the intracellular free Mg(2+) concentration ([Mg(2+)](i)), the intracellular Na(+) concentration ([Na(+)](i)), and the intracellular pH (pH(i)) of REC under basal conditions, after stimulation with butyrate and HCO(-)(3), and after changing the transmembrane chemical gradients for Mg(2+), H(+), and Na(+). REC had a mean resting pH(i) of 6.83 +/- 0.1, [Mg(2+)](i) was 0.56 +/- 0. 14 mM, and [Na(+)](i) was 18.95 +/- 3.9 mM. Exposure to both HCO(-)(3) and HCO(-)(3)/butyrate led to a stimulation of Mg(2+) influx that amounted to 27.7 +/- 5 and 29 +/- 10.6 microM/min, respectively, compared with 15 +/- 1 microM/min in control solution. The increase of [Mg(2+)](i) was dependent on extracellular Mg(2+) concentration ([Mg(2+)](e)). Regulation of pH(i) has been demonstrated to be Na(+) dependent and is performed, for the most part, by a Na(+)/H(+) exchanger. The recovery of pH(i) was fully blocked in nominally Na(+)-free media, even if [Mg(2+)](e) was stepwise increased from 0 to 7.5 mM. However, an increase of [Mg(2+)](i) was observed after reversing the transmembrane Na(+) gradient. This rise in [Mg(2+)](i) was pH independent, K(+) insensitive, dependent on [Mg(2+)](e), imipramine and quinidine sensitive, and accompanied by a decrease of [Na(+)](i). The results are consistent with the existence of a Na(+)/Mg(2+) exchanger in the cell membrane of REC. The coupling between butyrate, CO(2)/HCO(-)(3), and Mg(2+) transport may be mediated by another mechanism, perhaps by cotransport of Mg(2+) and HCO(-)(3).     

Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (I. Time Course of Induction of the Two Systems)

Changes in the physiological properties of the green alga Chlorella ellipsoidea (UTEX 20) were determined during adaptation from high CO2 to air. Cells of C. ellipsoidea, grown in high CO2, had an extremely low affinity for dissolved inorganic carbon (DIC). However, high-affinity DIC transport was induced rapidly after switching to air, which caused a massive decrease in the DIC concentration in the medium. Rates of O2 evolution without added carbonic anhydrase (CA) were compared with calculated rates of uncatalyzed CO2 formation in the medium as a measure of active HCO3-uptake. Cells were found to be able to use HCO3- after 5 h of adaptation and this capacity increased during the next 17 h. The stimulation of O2 evolution upon CA addition was used as a measurement of active CO2 transport: such stimulation occurred 2 h after transfer and increased during the next 5 h. Increases in O2 evolution rates were correlated closely with an increasing capacity to accumulate intracellular pools of acid-labile DIC and with decreases in K1/2(CO2) and CO2-compensation point of the cells. Treatment of cells with cycloheximide (5 [mu]g mL-1) during adaptation completely inhibited DIC transport induction, whereas treatment with chloramphenicol (400 [mu]g mL-1) had no effect, indicating the requirement for cytoplasmic protein synthesis in the induction. These results suggest that both CO2 and HCO3- transport are induced upon transfer of cells from high CO2 to air and that there is a temporal separation between the induction of the two systems.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Diversity of inorganic carbon acquisition mechanisms by intact microbial mats of Microcoleus chthonoplastes (Cyanobacteriae, Oscillatoriaceae)

The dissolved inorganic carbon (DIC) acquisition mechanisms were researched in intact microbial mats dominated by the cyanobacteria Microcoleus chthonoplastes Thuret, by determining the effect on photosynthesis of different inhibitors. The microbial mats exhibited high affinity for DIC at alkaline pH, with K(m(DIC)) values similar to the ones described for pure cultures of cyanobacteria and algae in which carbon concentrating mechanisms have been researched. Besides, the photosynthesis was non-sensitive to pH changes within the range of 5.6-9.6, indicating that HCO(3)(-) was the main DIC source used for photosynthesis. The M. chthonoplastes mats featured external and internal carbonic anhydrase (CA) activity as measured in intact cells and cell extracts, respectively. Acetazolamide (AZ, which slowly enters the cell and then inhibits mainly the external CA) and ethoxyzolamide (EZ, which inhibits both external and internal CA) reduced significantly the oxygen evolution rates, demonstrating that the CA was implied in the DIC acquisition. Vanadate inhibited photosynthesis by 60% although its application, when CA being inhibited (i.e. after applying AZ + EZ), did not produce any additional effect. It could indicate that ATPase-dependent HCO(3)(-) use occurred and also that this putative mechanism was coupled with CA-like activity at the plasma membrane. The involvement of Na(+)-dependent HCO(3)(-) transporters in DIC acquisition was also inferred as monensin and 4-4'-diisothiocyanatostibilene-2,2'-disulfonate (DIDS) reduced photosynthesis by 70%. DIDS produced a strong inhibitory effect even after application of AZ + EZ + vanadate, indicating that this mechanism was not related to CA activity. The microbial mats become subject to very unfavourable conditions for Rubisco carboxylation at their natural habitats (e.g. external pH of 10.5 and O(2) concentration doubled with respect to saturation concentration); therefore, this putative diversity of DIC acquisition mechanisms could ensure their growth under these extreme conditions.     

Effects of extracellular HCO3(-) on fatigue, pHi, and K+ efflux in rat skeletal muscles.

Elevated plasma HCO(3)(-) can improve exercise endurance in humans. This effect has been related to attenuation of the work-induced reduction in muscle pH, which is suggested to improve performance via at least two mechanisms: 1) less inhibition of muscle enzymes and 2) reduced opening of muscle K(ATP) channels with less ensuing reduction in excitability. Aiming at determining whether the ergogenic effect of HCO(3)(-) is related to effects on muscles, we examined the effect of elevating extracellular HCO(3)(-) from 25 to 40 mM (pH from 7.4 to 7.6) on fatigue, intracellular pH (pH(i)), and K(+) efflux in isolated rat skeletal muscles contracting isometrically. Fatigue induced by 30-Hz stimulation at 30 and 37 degrees C was similar between soleus muscles incubated in high and normal HCO(3)(-) concentrations. In extensor digitorum longus muscles stimulated at 60 Hz, elevated HCO(3)(-) did not affect fatigue at 30 degrees C. In soleus muscles, 30-Hz stimulation induced a approximately 0.2 unit reduction in pH(i), as determined by using the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. This reduction in pH(i) was not affected by elevated HCO(3)(-). Estimation of K(+) efflux using (86)Rb(+) showed that elevated HCO(3)(-) did not affect K(+) efflux at rest or during contractions. Similarly, other modifications of the intra- and extracellular pH had little effect on K(+) efflux during contraction. In conclusion, elevated extracellular HCO(3)(-) had no significant effect on muscle fatigue, pH(i), and K(+) efflux. These findings indicate that alternative mechanisms must be considered for the ergogenic effect of HCO(3)(-) observed in integral exercise studies.