Independent HCO3 Research Articles

Mechanisms of bicarbonate secretion in the equine colon ex vivo.

To examine bicarbonate (HCO3-) secretion ex vivo in the equine large colon to determine any differences between the right dorsal colon (RDC) and right ventral colon (RVC). The effect of phenylbutazone (PBZ) on HCO3- secretion was examined in the RDC. 14 healthy horses. In anesthetized horses (n = 10), segments of mucosa from RDC and RVC were harvested to measure HCO3- secretion ex vivo with the pH Stat method. The effect of PBZ on HCO3- secretion in the RDC was studied in 4 additional horses. Three distinct mechanisms of HCO3- secretion previously described in a murine model were confirmed in the equine colon. The RDC had a greater capacity for electrogenic, Cl--independent HCO3- secretion than the RVC (P = 0.04). In the RDC, all HCO3- secretion was decreased by PBZ (P < 0.02) but was not studied in the RVC because of low baseline secretion. Secretion of HCO3- by the RDC could play a pivotal role in equine colon physiology, because intense microbial fermentation in this site could require HCO3- secretion to buffer short-chain fatty acids. Inhibition of this secretion by PBZ could interfere with mucosal buffering and predispose to changes associated with right dorsal colitis.

Combining multiple nutrient stresses and bicarbonate addition to promote lipid accumulation in the diatom RGd-1

Algal biofuels represent a renewable, potentially viable, solution to mitigate transportation fuel demands. A novel diatom strain, RGd-1, isolated from Yellowstone National Park, produces high concentrations of lipids that can be converted to biodiesel. To increase the cell concentration and determine optimal conditions for growth, RGd-1 was grown without added Si, in the presence of four Si concentrations within the soluble range (0.5–2mM), and one above the soluble range (2.5mM). Medium Si concentrations and intracellular triacylglycerol (TAG) content were monitored daily by inductively coupled plasma mass spectrometry and Nile Red fluorescence, respectively (end-point TAG values were measured using gas chromatography). Si depletion with or without combined nitrate (NO3−) limitation was shown to induce TAG accumulation. Additionally, the effects of sodium bicarbonate (NaHCO3) supplementation were examined on cultures grown using two NO3− concentrations (2.94 and 1mM NO3−), which also resulted in increased TAG accumulation. It was determined that utilizing a combination of two independent physiological stresses and HCO3− supplementation resulted in the highest total and per cell TAG accumulation.

Effects of non-steroidal estrogen diethylstilbestrol on pH and ion transport in the mantle epithelium of a bivalve Anodonta cygnea

Freshwater bivalves are used as sentinel organisms to detect pollutants effects in the aquatic environment due to their sedentary nature, filter-feeding behaviour. We aimed to determine the in vivo, ex vivo and in vitro influence of Diethylstilbestrol (DES), a widely used synthetic non-steroidal estrogen and endocrine disruptor, in Anodonta cygnea shell growth mechanisms. For that, in vivo exposure to DES (0.75μM) during 15 days, in vitro and ex vivo exposure of outer mantle epithelium (OME) cells to DES (0.75μM), were performed followed by study of short-circuit current (Isc), transepithelial potential (Vt) and transepithelial conductance (Gt) as well as identification of membrane transport systems and intracellular pH (pHi). Our results show that in vivo exposure to DES decreases in 30% the OME Isc and ex vivo addition of DES to the basolateral side of OME also induced Isc decrease. Several membrane transporters such as V-type ATPases, Na+/H+ exchangers, Na+–K+ pump, Na+-driven and Na+-independent HCO3−/Cl− transporters and Na+/HCO3− co-transporter were identified as responsible for pHi maintenance in OME and noteworthy, DES caused a pHi decrease in OME cells similar to the effect observed when OME cells were exposed to 4,4′-diisothiocyanostilbene disulfonic acid (DIDS), an inhibitor of several bicarbonate membrane transporters. The addition of DIDS after OME cells exposure to DES did not cause any alteration. We concluded that DES is able to modulate membrane ion transport and pHi in the OME of A. cygnea and that this effect seems to be due to inhibition of HCO3−/Cl− co-transporters present on the basolateral membrane.

Rescue of epithelial HCO3− secretion in murine intestine by apical membrane expression of the cystic fibrosis transmembrane conductance regulator mutant F508del

This study investigated whether expression of the common cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508del in the apical membrane of enterocytes confers increased bicarbonate secretory capacity on the intestinal epithelium of F508del mutant mice compared to that of CFTR knockout (KO) mice. CFTR KO mice, F508del mutant mice (F508del) and wild-type (WT) littermates were bred on the FVB/N background. F508del isolated brush border membrane (BBM) contained approximately 5-10% fully glycosylated band C protein compared to WT BBM. Similarly, the forskolin (FSK)-induced, CFTR-dependent short-circuit current (I(sc)) of F508del mucosa was approximately 5-10% of WT, whereas the HCO(3)(-) secretory response ( ) was almost half that of WT in both duodenum and mid-colon studied in vitro and in vivo. While WT intestine retained full FSK-induced in the absence of luminal Cl(-), the markedly higher than I(sc) in F508del intestine was dependent on the presence of luminal Cl(-), and was blocked by CFTR inhibitors. The Ste20-related proline-alanine-rich kinases (SPAK/OSR1), which are downstream of the with-no-lysine (K) protein kinases (WNK), were rapidly phosphorylated by FSK in WT and F508del, but significantly more slowly in CFTR KO intestine. In conclusion, the data demonstrate that low levels of F508del membrane expression in the intestine of F508del mice significantly increased FSK-induced HCO(3)(-) secretion mediated by Cl(-)/HCO(3)(-) exchange. However, in WT mucosa FSK elicited strong SPAK/OSR1 phosphorylation and Cl(-)-independent HCO(3)(-) efflux. This suggests that therapeutic strategies which deliver F508del to the apical membrane have the potential to significantly enhance epithelial HCO(3)(-) secretion.

Neuronal and non‐neuronal steady‐state pHi and recovery from NH4+‐induced acid loads

Regulation of neuronal function requires a tightly controlled intracellular pH (pHi) in both neurons and non‐neuronal cells by the major pHi‐regulating transport proteins, the sodium hydrogen exchangers (NHE's) and sodium‐coupled bicarbonate transporters (NCBT's). The regulation of steady‐state pHi was examined in neurons and non‐neuronal cells (presumably astrocytes) in the same primary culture of rat hippocampus. Fluorescent recordings using BCECF, a pH‐sensitive dye were taken while perfusing cultured cells on gridded coverslips, for later identified using immunocytochemistry. Neurons have a relatively wide range of steady‐state pHi values in a non‐CO2/HCO3− buffer, with a mean of 7.40, whereas non‐neuronal cells have a more narrow steady‐state range, with a mean of 7.25. When examining the recovery rate (dpHi/dt) from an NH4+‐induced acid load in the absence of CO2/HCO3−, we did not observe much difference between cell populations within the pHi range 6.9–7.1. At more acidic values, non‐neuronal cells have a greater dpHi/dt. In the presence of CO2/HCO3−, the dpHi/dt in both cell populations increased, although the increase in non‐neuronal cells was more pronounced. In summary, the recovery of pHi from NH4+‐induced acid loads in both neurons and non‐neuronal cells requires both HCO3− independent transporters (presumably NHEs) and HCO3−‐dependent transporters (presumably NCBTs).

Effect of Cl− on photosynthetic bicarbonate uptake in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803

Photosynthetic inorganic carbon utilization was investigated in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803 grown in standing culture. Photosynthetic rates for the two algae reached about 10 times the theoretical CO2 supply rate at low dissolved inorganic carbon (DIC) of 100 μmol/L, and the rates were unaffected by the addition of 20 mmol/L Na+, indicating that the two algae possessed Na+-independent HCO 3 − utilization for photosynthesis under low DIC. Their photosynthetic rates at low DIC were inhibited by higher Cl− and the degrees of inhibition were increased with the rise of Cl− concentration, and in the presence of Diphenylamine-2-carboxylate (DPC), a reported Cl− channel inhibitor, the rates decreased by 74%–82%, implying that putative DPC-sensitive Cl− channels participate in Na+-independent HCO 3 − uptake for photosynthesis. The experiment of intracellular 14C-DIC accumulation for photosynthesis showed that internal DIC pools decreased by about 80% with 200 μmol/L DPC and by 64%–70% with 100 mmol/L Cl−. The experiment of chlorophyll a fluorescence quenching showed that initial rates and extents of fluorescence quenching obviously decreased with 200 μmol/L DPC or 100 mmol/L Cl−. The two experiments gave further evidence that putative DPC-sensitive Cl− channels participate in Na+-independent HCO 3 − uptake for photosynthesis in the two algae grown in standing culture.

Intracellular pH regulation in human Sertoli cells: role of membrane transporters

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Intracellular pH (pHi) is an important parameter in cell physiology regulating namely cell metabolism and differentiation. However, pHi regulation mechanisms in Sertoli cells have not yet been systematically elucidated. In this work, pHi was determined in primary cultures of human Sertoli cells. Sertoli cells were exposed to weak acids, which caused a rapid acidification of the intracellular milieu. pHi then recovered by a mechanism that was shown to be particularly sensitive to the presence of the inhibitor DIDS (4,4'-diisothiocyanostilbene disulfonic acid). In the presence of amiloride and PSA (picrylsulfonic acid), pHi recovery was also significantly affected. These results indicate that, in the experimental conditions used, pHi is regulated by the action of an Na(+)-driven HCO(3)(-)/Cl(-) exchanger and an Na(+)/HCO(3)(-) co-transporter and also by the action of the Na(+)/H(+) exchanger. On the other hand, pHi recovery was only slightly affected by concanamycin A, suggesting that V-Type ATPases do not have a relevant action on pHi regulation in human Sertoli cells, and was independent of the presence of bumetanide, suggesting that the inhibition of the Na(+)/K(+)/Cl(-) co-transporter does not affect pHi recovery, not even indirectly via the shift of ionic gradients. Finally, pHi was shown to be sensitive to the removal of external Cl(-), but not of Na(+) or K(+), evidencing the presence of a membrane Cl(-)-dependent base extruder, namely the Na(+)-independent HCO(3)(-)/Cl(-) exchanger, and its role on pHi maintenance on these cells.

Oxidative stress and α1‐adrenoceptor‐mediated stimulation of the Cl−/HCO3− exchanger in immortalized SHR proximal tubular epithelial cells

This study evaluated the signalling coupled to the alpha1-adrenoceptor-induced stimulation of the Cl-/HCO3- exchanger in hypertension. The Na+ -independent HCO3- transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). Noradrenaline increased Cl-/HCO3- exchanger activity with EC50 values of 0.6 and 5.3 microM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl-/HCO3- exchanger activity in SHR and WKY cells (EC50 of 2.6 and 4.9 microM, respectively). Phenylephrine-mediated increase in Cl-/HCO3- exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of alpha1A- and alpha1B-adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H2O2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H2O2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl-/HCO3- exchanger activity. H2O2-dependent stimulation of Cl-/HCO3- exchange activity was significantly higher in SHR than in WKY cells. Differences between WKY and SHR cells on their sensitivity to alpha1-adrenoceptor stimulation did not correlate with the abundance of alpha1A- and alpha1B-adrenoceptors and may be related to the increased generation of H2O2, which may amplify the response downstream of alpha1-adrenoceptor activation.

The CO2‐concentrating mechanism in the bloom‐forming cyanobacterium Microcystis aeruginosa (Cyanophyceae) and effects of UVB radiation on its operation1

The bloom‐forming cyanobacterium Microcystis aeruginosa (Kütz.) Kütz. 854 was cultured with 1.05 W · m−2 ultraviolet‐B radiation (UVBR) for 3 h every day, and the CO2‐concentrating mechanism (CCM) within this species as well as effects of UVBR on its operation were investigated. Microcystis aeruginosa 854 possessed at least three inorganic carbon transport systems and could utilize external HCO3− and CO2 for its photosynthesis. The maximum photosynthetic rate was approximately the same, but the apparent affinity for dissolved inorganic carbon was significantly decreased from 74.7 μmol · L−1 in the control to 34.7 μmol · L−1 in UVBR‐treated cells. At 150 μmol · L−1 KHCO3 and pH 8.0, Na+‐dependent HCO3− transport contributed 43.4%–40.2% to the photosynthesis in the control and 34.5%–31.9% in UVBR‐treated cells. However, the contribution of Na+‐independent HCO3− transport increased from 8.7% in the control to 18.3% in UVBR‐treated cells. The contribution of CO2‐uptake systems showed little difference: 47.9%–51.0% in the control and 49.8%–47.2% in UVBR‐treated cells. Thus, the rate of total inorganic carbon uptake was only marginally affected, although UVBR had a differential effect on various inorganic carbon transporters. However, the number of carboxysomes in UVBR‐treated cells was significantly decreased compared to that in the control.

Acute inflammation alters bicarbonate transport in mouse ileum

T-cell mediated acute inflammation of the ileum may occur during Crohn's disease exacerbations. During ileal inflammation, absorption of nutrients and electrolytes by villus cells is decreased with a concomitant increase in crypt and/or villus fluid secretion. These alterations lead to fluid accumulation and the subsequent diarrhoea. Net intestinal fluid secretion consists of HCO3--rich plasma-like fluid. However, the regulation and mechanisms of HCO3- secretion in normal and acutely inflamed ileum are not clearly understood. To study this phenomenon, anti-CD3 monoclonal antibody (mAb)- induced in vivo ileal inflammatory mouse models was used for in vitro functional studies with Ussing chamber and pH stat techniques. Three hours after anti-CD3 mAb injection, ileal mucosa stripped of muscular and serosal layers showed a significant increase in short circuit current (Isc) (0.58+/-0.07 microEq h(-1) cm2 versus 1.63+/-0.14 microEq h(-1) cm2). The cAMP-stimulated Isc component was sensitive to glibenclamide but not to DIDS, suggesting that a cystic fibrosis transmembrane conductance regulator (Cftr)-mediated anion conductance was responsible. Basal Cl--dependent HCO3- secretion, measured using a pH stat technique, was decreased significantly in anti-CD3-injected mice, with a simultaneous increase in Cl--independent HCO3- secretion that was also inhibited by glibenclamide. Experiments using Cftr-/- mice showed neither an increase in Isc nor an increase in HCO3- secretion, confirming the role for Cftr protein in stimulating anion secretion following anti-CD3 treatment. Western blot analysis indicated that Cftr protein levels were unaltered by anti-CD3 treatment, at least acutely. Finally, an immunoassay for cAMP showed significant increases in intracellular cAMP in villus cells, but not in crypt cells. These studies therefore suggest a shift from a predominantly electroneutral Cl-HCO3- exchange in normal mice, to a predominantly electrogenic anion secretion including HCO3- that occurs via functional Cftr during anti-CD3-mediated acute inflammation.

Evidence for K+-dependent HCO3- utilization in the marine diatom Phaeodactylum tricornutum.

Photosynthetic utilization of inorganic carbon in the marine diatom Phaeodactylum tricornutum was investigated by the pH drift experiment, measurement of K(1/2) values of dissolved inorganic carbon (DIC) with pH change, and comparison of the rate of photosynthesis with the rate of the theoretical CO(2) formation from uncatalyzed HCO(3)(-) conversion in the medium. The higher pH compensation point (10.3) and insensitivity of the photosynthetic rate to acetazolamide indicate that the alga has good capacity for direct HCO(3)(-) utilization. The photosynthetic rate reached 150 times the theoretical CO(2) supply rate at 100 micromol L(-1) DIC (pH 9.0) in the presence of 10 mmol L(-1) K(+) and 46 times that in the absence of K(+), indicating that for pH 9.4-grown P. tricornutum, HCO(3)(-) in the medium is taken up through K(+)-dependent and -independent HCO(3)(-) transporters. The K(1/2) (CO(2)) values at pH 8.2 were about 4 times higher than those at pH 9.0, whereas the K(1/2) (HCO(3)(-)) values at pH 8.2 were slightly lower than those at pH 9.0 whether without or with K(+), providing further evidence for the presence of the two HCO(3)(-) transport patterns in this alga. Photosynthetic rate and affinity for HCO(3)(-) in the presence of K(+), respectively, were about 2- and 7-fold higher than those in the absence of K(+), indicating that K(+)-dependent HCO(3)(-) transport is a predominant pattern of HCO(3)(-) cellular uptake in low DIC concentration. However, as P. tricornutum was cultured at pH 7.2 or 8.0, photosynthetic affinities to HCO(3)(-) were not affected by K(+), implying that K(+)-dependent HCO(3)(-) transport is induced when P. tricornutum is cultured at high alkaline pH.

SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl−/HCO3− exchange, and is inhibited by NH4+

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.

Utilization of inorganic carbon in the edible cyanobacterium Ge‐Xian‐Mi (Nostoc) and its role in alleviating photo‐inhibition

ABSTRACTThe present work investigated the inorganic carbon (Ci) uptake, fluorescence quenching and photo‐inhibition of the edible cyanobacterium Ge‐Xian‐Mi (Nostoc) to obtain an insight into the role of CO2 concentrating mechanism (CCM) operation in alleviating photo‐inhibition. Ge‐Xian‐Mi used HCO3– in addition to CO2 for its photosynthesis and oxygen evolution was greater than the theoretical rates of CO2 production derived from uncatalysed dehydration of HCO3–. Multiple transporters for CO2 and HCO3– operated in air‐grown Ge‐Xian‐Mi. Na+‐dependent HCO3– transport was the primary mode of active Ci uptake and contributed 53–62% of net photosynthetic activity at 250 µmol L−1 KHCO3 and pH 8.0. However, the CO2‐uptake systems and Na+‐independent HCO3– transport played minor roles in Ge‐Xian‐Mi and supported, respectively, 39 and 8% of net photosynthetic activity. The steady‐state fluorescence decreased and the photochemical quenching increased in response to the transport‐mediated accumulation of intracellular Ci. Inorganic carbon transport was a major factor in facilitating quenching during the initial stage and the initial rate of fluorescence quenching in the presence of iodoacetamide, an inhibitor of CO2 fixation, was 88% of control. Both the initial rate and extent of fluorescence quenching increased with increasing external dissolved inorganic carbon (DIC) and saturated at higher than 200 µmol L−1 HCO3–. The operation of the CCM in Ge‐Xian‐Mi served as a means of diminishing photodynamic damage by dissipating excess light energy and higher external DIC in the range of 100–10000 µmol L−1 KHCO3 was associated with more severe photo‐inhibition under strong irradiance.

Three distinct mechanisms of HCO3- secretion in rat distal colon.

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

Effects of carbon nutrition on the physiological expression of HCO3- transport and the CO2-concentrating mechanism in the Cyanobacterium chlorogloeopsis sp. ATCC 27193.

We have examined the effect of inorganic and organic carbon nutrition on the physiological expression of HCO3- transport and the CO2-concentrating mechanism (CCM) in the nutritionally versatile cyanobacterium Chlorogloeopsis sp. ATCC 27193. Cells grown under photoautotrophic conditions in the presence of limiting or replete levels of inorganic carbon (Ci), or grown under mixotrophic (light) or chemoheterotrophic (dark) conditions in the presence of sucrose retained both active CO2 and Na(+)-independent HCO3- transport activity. However, two distinct effects on the kinetic properties of HCO3- transport were observed, which segregated on the basis of phototrophic and chemoheterotrophic growth in the dark. In the former, the apparent substrate affinity of the HCO3- transport system (K0.5) varied (12-fold) in response to the growth Ci or mixotrophy while the maximum rate of HCO3- transport was approximately constant. In the latter case, the K0.5 value was unchanged from the starting value (35 microM) of Ci-limited photoautotrophic cells used to initiate the dark-grown cultures, but transport capacity declined 3-fold. Modulation of the K0.5 (HCO3- transport) value required light. Cellular carboxysome content was unaffected by growth under any of the regimes employed and these structures were the predominant location of ribulose-1,5-bisphosphate carboxylase/oxygenase, as indicated by immunogold electron microscopy. Mixotrophic and chemoheterotrophic growth resulted in a diminished ability to concentrate Ci internally and a reduction in Ci accumulation ratios at low external Ci concentrations. The relationship between photosynthetic carbon fixation and the internal Ci pool varied by 2-fold, with high-Ci-grown cells being the most efficient and mixotrophically grown cells the least, indicating that there was limited capacity to modulate this relationship in response to changes in carbon nutrition. Within broad limits this relationship appeared to be a fixed trait of the strain and an important factor in determining growth rate.

Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na+/H+exchanger

The present study was aimed at evaluating the role of D(1)- and D(2)-like receptors and investigating whether inhibition of Na(+) transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na(+)/H(+) exchanger, inhibition of the basolateral Na(+)-K(+)-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D(1)- and D(2)-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC(50) = 220 +/- 2 nM), marked intracellular acidification (IC(50) = 58 +/- 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 +/- 4.5% reduction) without affecting intracellular pH recovery after CO(2) removal. These results agree with the view that dopamine, through the activation of D(1)- but not D(2)-like receptors, inhibits both the Na(+)/H(+) exchanger (0.001933 +/- 0.000121 vs. 0.000887 +/- 0.000073 pH unit/s) and Na(+)-K(+)-ATPase without interfering with the Na(+)-independent HCO transporter. It is concluded that dopamine, through the action of D(1)-like receptors, inhibits both the Na(+)/H(+) exchanger and Na(+)-K(+)-ATPase, but its marked acidifying effects result from inhibition of the Na(+)/H(+) exchanger only, without interfering with the Na(+)-independent HCO transporter and Na(+)-K(+)-ATPase.

Proton secretion in the male reproductive tract: involvement of Cl--independent HCO-3 transport.

The lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile. An acidic luminal fluid is required for the maintenance of sperm quiescence and for the prevention of premature activation of acrosomal enzymes during their storage in the cauda epididymis and vas deferens. We have previously demonstrated that a vacuolar H+-ATPase [proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in the corpus and cauda epididymis and that this PP is responsible for the majority of proton secretion in the proximal vas deferens. We now show that PP-rich cells in the vas deferens express a high level of carbonic anhydrase type II (CAII) and that acetazolamide markedly inhibits the rate of proton secretion by 46.2 +/- 6.1%. The rate of acidification was independent of Cl- and was strongly inhibited by SITS under both normal and Cl--free conditions (50.6 +/- 5.0 and 57. 5 +/- 6.0%, respectively). In the presence of Cl-, diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited proton secretion by 63.7 +/- 11.3% when applied together with DPC. In Cl--free solution, DPC markedly inhibited proton efflux by 45.1 +/- 7.6%, SITS produced an additional inhibition of 18.2 +/- 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII plays a major role in proton secretion by the proximal vas deferens. Acidification does not require the presence of Cl-, but DPC-sensitive Cl- channels might contribute to basolateral extrusion of HCO-3 under Cl--free conditions. The inhibition by SITS observed under both normal and Cl--free conditions indicates that a Cl-/HCO-3 exchanger is not involved and that an alternative HCO-3 transporter participates in proton secretion in the proximal vas deferens.

Concurrent and independent HCO3- and Cl- secretion in a human pancreatic duct cell line (CAPAN-1).

The present study investigated both HCO-3 and Cl- secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (Isc) technique. In Cl-/HCO-3-containing solution, secretin (1 microM) or forskolin (10 microM) stimulated a biphasic rise in the Isc which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external Cl- abolished the initial transient phase in the forskolin-induced Isc while the plateau remained. In HCO-3/CO2-free solution, on the contrary, only the initial transient increase in Isc was prominent. Summation of the current magnitudes observed in Cl--free and HCO-3-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the current observed in Cl-/HCO-3-containing solution. Removal of external Na+ greatly reduced the initial transient rise in the forskolin-induced Isc response, and the plateau level observed under this condition was similar to that obtained in Cl--free solution, suggesting that Cl--dependent Isc was also Na+-dependent. Bumetanide (50 microM), an inhibitor of the Na+-K+-2Cl- cotransporter, and Ba2+ (1 mm), a K+ channel blocker, could reduce the forskolin-induced Isc obtained in Cl-/HCO-3-containing or HCO-3-free solution. However, they were found to be ineffective when external Cl- was removed, indicating the involvement of these mechanisms in Cl- secretion. On the contrary, the HCO-3-dependent (in the absence of external Cl-) forskolin-induced Isc could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 microM). Basolateral application of amiloride (100 microM) inhibited the Isc; however, a specific Na+-H+ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5-10 microM) was found to be ineffective, excluding the involvement of the Na+-H+ exchanger. However, an inhibitor of H+-ATPase, N-ethylmaleimide did suppress the Isc (IC50 = 22 microM). Immunohistochemical studies also confirmed the presence of a vacuolar type of H+-ATPase in these cells. H2DIDS (100 microM), an inhibitor of Na+-HCO-3 cotransporter, was without effect. Apical addition of Cl- channel blocker, diphenylamine-2,2'-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 microM) or SITS (100 microM), exerted an inhibitory effect on both Cl- and HCO-3-dependent forskolin-induced Isc responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting that HCO-3 secretion may be specialized to a certain population of cells. The present results suggest that both HCO-3 and Cl- secretion by the human pancreatic duct cells may occur concurrently and independently.

Photosynthesis and inorganic carbon acquisition in the cyanobacterium Chlorogloeopsis sp. ATCC 27193

The ability of the morphologically complex cyanobacterium Chlorogloeopsis sp. ATCC 27193 to actively transport and accumulate inorganic carbon (C1= CO2+ HCO3−+ CO32−) for photosynthetic CO2 fixation was investigated. Mass‐spectrometric assays revealed that Chlorogloeopsis cells grown under C1 limitation rapidly took up CO2 from the medium in a light‐dependent reaction which was independent of CO2 fixation. Ethoxyzolamide, a carbonic anhydrase (CA) inhibitor, inhibited CO2 transport. Since electrometric and mass‐spectrometric assays did not detect the presence of a periplasmic CA, it is suggested that CO2 transport was mediated by a CA‐like activity which converted CO2 to HCO3− during passage across the membrane. Radiochemical assays, using H14CO3 as substrate, showed that C3‐limited cells also had a high affinity (K0.5 HCO3−= 37 μM), Na+‐independent HCO3− uptake mechanism. HCO3−uptake was light dependent and occurred against its electrochemical potential indicating a carrier‐mediated, active transport process. The rate of Na+‐independent HCO3− transport was sufficient to account for the steady state rate of CO2 fixation. Although not absolutely required. Na+ did specifically enhance the rate of HCO3− transport by up to 2‐fold, but had no effect on the apparent affinity of the transport system for HCO3− Combined CO2 and HCO3− transport resulted in C1 accumulation as high as 25 mM and in excess of 300 times the external concentration. The C1 pool was the source of CO2 for photo‐synthetic fixation and was generated, presumably, by the dehydration of HCO3− catalyzed by an intracellular CA. The collective evidence indicates that Chlorogloeopsis has a physiologically functional CO2‐concentrating mechanism which is essential for photosynthesis.

Evidence for Cl(-)-independent HCO3- transport in basolateral membranes of Necturus oxyntopeptic cells

Luminal H+ secretion by gastric mucosa is accompanied by basolateral HCO3- release. A basolateral Cl-/HCO3- exchanger is known to mediate HCO3- extrusion from oxyntopeptic cells during resting and secretagogue-induced apical HCl secretion. From recent work, we hypothesized that there might be a Cl(-)-independent pathway for basolateral HCO3- exit in Necturus oxyntopeptic cells. In this study, we used a fluorescent pH indicator [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein] to evaluate Cl(-)-independent HCO3- transport across the basolateral membranes of intact oxyntopeptic cells. Removal of serosal Cl- increased intracellular pH (pHi) (7.05 to 7.25), consistent with Cl(-)-dependent HCO3- extrusion. Removal of serosal Na+ in the absence of Cl- resulted in significant acidification of pHi (7.10 to 6.89), but studies involving amiloride, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 0 HCO3(-)-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions suggest that Na(+)-dependent changes in pHi are due to Na+/H+ exchange. Our studies demonstrate a marked concentration-dependent alkalinization when tissues are exposed to increases in serosal K+. A substantial part of this alkalinization in response to increases in serosal K+ (pHi 7.00 to 7.46) appears to be a HCO3- exit pathway that is independent of both Na+ and Cl-, unaffected by bumetanide or amiloride, but sensitive to DIDS. We propose the presence of a Cl(-)- and Na(+)-independent K(+)-dependent HCO3- cotransporter in Necturus oxyntopeptic cell basolateral membranes.