HCO3 Uptake Research Articles

Sources and pathways of carbon and nitrogen of macrophytes and sediments using stable isotopes in Al-Kharrar Lagoon, eastern Red Sea coast, Saudi Arabia.

Elemental ratios (δ13C, δ15N and C/N) and carbon and nitrogen concentrations in macrophytes, sediments and sponges of the hypersaline Al-Kharrar Lagoon (KL), central eastern Red Sea coast, were measured to distinguish their sources, pathways and see how they have been influenced by biogeochemical processes and terrestrial inputs. The mangroves and halophytes showed the most depleted δ13C values of -27.07±0.2 ‰ and -28.34±0.4 ‰, respectively, indicating their preferential 12C uptake, similar to C3-photosynthetic plants, except for the halophytes Atriplex sp. and Suaeda vermiculata which showed δ13C of -14.31±0.6 ‰, similar to C4-plants. Macroalgae were divided into A and B groups based on their δ13C values. The δ13C of macroalgae A averaged -15.41±0.4 ‰, whereas macroalgae B and seagrasses showed values of -7.41±0.8 ‰ and -7.98 ‰, suggesting uptake of HCO3- as a source for CO2 during photosynthesis. The δ13C of sponges was -10.7±0.3 ‰, suggesting that macroalgae and seagrasses are their main favoured diets. Substrates of all these taxa showed δ13C of -15.52±0.8 ‰, suggesting the KL is at present a macroalgae-dominated lagoon. The δ15N in taxa/sediments averaged 1.68 ‰, suggesting that atmospheric N2-fixation is the main source of nitrogen in/around the lagoon. The heaviest δ15N (10.58 ‰) in halophytes growing in algal mats and sabkha is possibly due to denitrification and ammonia evaporation. The macrophytes in the KL showed high C %, N %, and C/N ratios, but this is not indicated in their substrates due possibly to a rapid turnover of dense, hypersaline waters carrying most of the detached organic materials out into the Red Sea. The δ13C allowed separation of subaerial from aquatic macrophytes, a proxy that could be used when interpreting paleo-sea level or paleoclimatic changes from the coastal marine sediments.

Genome-wide characterization of the Triplophysa dalaica slc4 gene family and expression profiles in response to salinity changes

BackgroundThe solute carrier 4 (SLC4) gene family is involved in a variety of physiological processes in organisms and is essential for maintaining acid-base balance in the body. The slc4 genes have been extensively studied in mammals, and they play important roles in intracellular and extracellular pH regulation and in the secretion and uptake of HCO3− and other ions (Na+ and Cl−) between transepithelial cells in different tissues. This study identified and characterized the entire slc4 gene family of Triplophysa dalaica.ResultsFifteen slc4 genes were identified in the whole genome of Triplophysa dalaica in this study, including five copies of Na+-independent Cl−/HCO3− transporters, eight members of Na+-dependent HCO3− transporters, and two genes coding Na+-coupled borate transporters. The chromosomal location information, isoelectric points, and molecular weights of the 15 slc4 genes were analyzed. The results for gene structure, domain analysis, and phylogenetic relationships of this gene family showed that the slc4 genes (except for slc4a9, which is missing in teleosts) are significantly expanded in teleosts compared to higher vertebrates. This phenomenon suggests that the slc4 gene family played an important role in the transition from aquatic to terrestrial animals. RT-PCR results showed that different slc4 genes showed diversified expression patterns in the tissues of T. dalaica. For osmotic pressure regulating organs, slc4a1b, slc4a4b, slc4a7, and slc4a11a were highly expressed in gills. In the kidney, slc4a1a, slc4a3, and slc4a10b were highly expressed, suggesting that the slc4 genes play a specific role in the salinity adaptation of T. dalaica. Our study has deciphered the biological roles of the slc4 genes in maintaining ionic and acid-base homeostasis in teleost fishes and provides a foundation for future exploration of the highly differentiated gene family in Triplophysa.ConclusionsThe results are relevant for the breeding of alkali-tolerant varieties in saline-alkali areas for aquaculture. Our findings have important implications for the adaptation process of freshwater species to saline-alkali water.

Molecular characterization, immunofluorescent localization, and expression levels of two bicarbonate anion transporters in the whitish mantle of the giant clam, Tridacna squamosa, and the implications for light-enhanced shell formation

Giant clams conduct light-enhanced shell formation, which requires the increased transport of Ca2+ and inorganic carbon (Ci) from the hemolymph through the shell-facing epithelium of the whitish inner mantle to the extrapallial fluid where CaCO3 deposition occurs. The major form of Ci in the hemolymph is HCO3−, but the mechanisms of HCO3− transport through the basolateral and apical membranes of the shell-facing epithelial cells remain unknown. This study aimed to clone from the inner mantle of Tridacna squamosa the complete coding cDNA sequences of electrogenic Na+-HCO3−cotransporter 1 homolog (NBCe1-like-b) and electrogenic Na+-HCO3−cotransporter 2 homolog (NBCe2-like). NBCe1-like-b comprised 3360 bp, encoding a 125.7 kDa protein with 1119 amino acids. NBCe1-like-b was slightly different from NBCe1-like-a of the ctenidium reported elsewhere, as it had a serine residue (Ser1025), which might undergo phosphorylation leading to the transport of Na+: HCO3− at a ratio of 1: 2 into the cell. NBCe1-like-b was localized at the basolateral membrane of the shell-facing epithelial cells, and its gene and protein expression levels increased significantly in the inner mantle during illumination, indicating a role in the light-enhanced uptake of HCO3− from the hemolymph. The sequence of NBCe2-like obtained from the inner mantle was identical to that reported previously for the outer mantle. In the inner mantle, NBCe2-like had an apical localization in the shell-facing epithelial cells, and its protein abundance was upregulated during illumination. Hence, NBCe2-like might take part in the light-enhanced transport of HCO3− through the apical membrane of these cells into the extrapallial fluid.

An analysis of the variability in <i>δ</i><sup>13</sup>C in macroalgae from the Gulf of California: indicative of carbon concentration mechanisms and isotope discrimination during carbon assimilation

Abstract. The isotopic composition of carbon in macroalgae (δ13C) is highly variable, and its prediction is complex concerning terrestrial plants. The determinants of δ13C macroalgal variations were analyzed in a large stock of specimens that vary in taxa and morphology and were collected in shallow marine habitats in the Gulf of California (GC) with distinctive environmental conditions. A large δ13C variability (−34.6 ‰ to −2.2 ‰) was observed. Life-forms (taxonomy 57 %, morphology and structural organization 34 %) explain the variability related to carbon use physiology. Environmental conditions influenced the δ13C macroalgal values but did not change the physiology, which is most likely inherently species-specific. Values of δ13C were used as indicators of the presence or absence of carbon concentrating mechanisms (CCMs) and as integrative values of the isotope discrimination during carbon assimilation in the life cycle macroalgae. Based on δ13C signals, macroalgae were classified in three strategies relative to the capacity of CCM: (1) HCO3- uptake (δ13C > −10 ‰), (2) using a mix of CO2 and HCO3- uptake (-10<δ13C > −30 ‰), and (3) CO2 diffusive entry (δ13C < −30 ‰). Most species showed a δ13C that indicates a CCM using a mix of CO2 and HCO3- uptake. HCO3- uptake is also widespread among GC macroalgae, with many Ochrophyta species. Few species belonging to Rhodophyta relied on CO2 diffusive entry exclusively, while calcifying macroalgae species using HCO3- included only Amphiroa and Jania. The isotopic signature evidenced the activity of CCM, but it was inconclusive about the preferential uptake of HCO3- and CO2 in photosynthesis and the CCM type expressed in macroalgae. In the study of carbon use strategies, diverse, species-specific, and complementary techniques to the isotopic tools are required.

Capacitation-associated alkalization in human sperm is differentially controlled at the subcellular level.

Capacitation in mammalian sperm involves the accurate balance of intracellular pH (pHi), but the mechanisms controlling this process are not fully understood, particularly regarding the spatiotemporal regulation of the proteins involved in pHi modulation. Here, we employed an image-based flow cytometry technique combined with pharmacological approaches to study pHi dynamics at the subcellular level during capacitation. We found that, upon capacitation induction, sperm cells undergo intracellular alkalization in the head and principal piece regions. The observed localized pHi increases require the initial uptake of HCO3-, which is mediated by several proteins acting consistently with their subcellular localization. Hv1 proton channel (also known as HVCN1) and cAMP-activated protein kinase (protein kinase A, PKA) antagonists impair alkalization mainly in the principal piece. Na+/HCO3- cotransporter (NBC) and cystic fibrosis transmembrane regulator (CFTR) antagonists impair alkalization only mildly, predominantly in the head. Motility measurements indicate that inhibition of alkalization in the principal piece prevents the development of hyperactivated motility. Altogether, our findings shed light on the complex control mechanisms of pHi and underscore their importance during human sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Na+,HCO3--cotransporter NBCn1 (Slc4a7) accelerates ErbB2-induced breast cancer development and tumor growth in mice.

Metabolic acid production challenges cellular pH homeostasis in solid cancer tissue, and mechanisms of net acid extrusion represent promising new targets for breast cancer therapy. Here, we used genetically engineered mice to investigate the contribution of the Na+,HCO3--cotransporter NBCn1 (Slc4a7) to intracellular acid-base regulation in ErbB2-induced breast cancer tissue and the consequences of NBCn1 knockout for breast tumor development and growth. We demonstrate an approximately 2-fold increase of NBCn1 protein abundance in ErbB2-induced breast cancer tissue compared to normal breast tissue despite a 4-fold decrease in the NBCn1 mRNA level. In congruence, we show that NBCn1 facilitates net acid extrusion and elevates steady-state intracellular pH in breast cancer tissue. Disruption of NBCn1 expression delayed ErbB2-induced breast carcinogenesis from a median tumor-free survival of 9.5 months in wild-type mice to 12 months in NBCn1-knockout mice and decelerated the tumor growth rate by approximately 1/3. Glycolytic metabolism-evaluated based on the interstitial concentrations of lactate and glucose measured in microdialysates-was increased in breast cancer tissue compared to normal breast tissue, but was unaffected by NBCn1 knockout. Disruption of NBCn1 expression inhibited cell proliferation-evaluated by staining for the proliferative marker Ki67-particularly in central tumor areas with predicted increase in acid loading from glycolytic metabolism. In conclusion, NBCn1 regulates intracellular pH in ErbB2-induced breast cancer tissue by providing a pathway for cellular uptake of HCO3-, which can neutralize metabolic acidic waste products. Disrupting NBCn1 expression delays ErbB2-induced breast cancer development, inhibits cancer cell proliferation, and decelerates tumor growth.

Carbon Sources in Fruit Carbonate of Buglossoides arvensis and Consequences for 14C Dating

AbstractFruit carbonate of Buglossoides arvensis (syn. Lithospermum arvense) is a valuable dating and paleoenvironmental proxy for late Quaternary deposits and cultural layers because CaCO3 in fruit is assumed to be accumulated from photosynthetic carbon (C). However, considering the uptake of HCO3– by roots from soil solution, the estimated age could be too old depending on the source of HCO3– allocated in fruit carbonate. Until now, no studies have assessed the contributions of photosynthetic and soil C to the fruit carbonate. To evaluate this, the allocation of photo-assimilated carbon and root uptake of HCO3– was examined by radiocarbon (14C) labeling and tracing. B. arvensis was grown in carbonate-free and carbonate-containing soils (sand and loess, respectively), where 14C was provided as (1) 14CO2 in the atmosphere (5 times shoot pulse labeling), or (2) Na214CO3 in soil solution (root-labeling; 5 times by injecting labeled solution into the soil) during one month of fruit development. Distinctly different patterns of 14C distribution in plant organs after root- and shoot labeling showed the ability of B. arvensis to take up HCO3– from soil solution. The highest 14C activity from root labeling was recovered in roots, followed by shoots, fruit organics, and fruit carbonate. In contrast, 14C activity after shoot labeling was the highest in shoots, followed by fruit organics, roots and fruit carbonate. Total photo-assimilated C incorporated via shoot labeling in loess-grown plants was 1.51 mg lower than in sand, reflecting the presence of dissolved carbonate (i.e. CaCO3) in loess. Loess carbonate dissolution and root-respired CO2 in soil solution are both sources of HCO3– for root uptake. Considering this dilution effect by carbonates, the total incorporated HCO3– comprised 0.15% of C in fruit carbonate after 10 hr of shoot labeling. However, if the incorporated HCO3– during 10 hr of shoot labeling is extrapolated for the whole month of fruit development (i.e. 420-hr photoperiod), fruit carbonate in loess-grown plants incorporated approximately 6.3% more HCO3– than in sand. Therefore, fruit carbonates from plants grown on calcareous soils may yield overestimated 14C ages around 500 yr because of a few percentage uptake of HCO3– by roots. However, the age overestimation because of HCO3– uptake becomes insignificant in fruits older than approximately 11,000 yr due to increasing uncertainties in age determination.

PH determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanism

Many carbon-fixing bacteria rely on a CO2 concentrating mechanism (CCM) to elevate the CO2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). The CCM is postulated to simultaneously enhance the rate of carboxylation and minimize oxygenation, a competitive reaction with O2 also catalyzed by RuBisCO. To achieve this effect, the CCM combines two features: active transport of inorganic carbon into the cell and colocalization of carbonic anhydrase and RuBisCO inside proteinaceous microcompartments called carboxysomes. Understanding the significance of the various CCM components requires reconciling biochemical intuition with a quantitative description of the system. To this end, we have developed a mathematical model of the CCM to analyze its energetic costs and the inherent intertwining of physiology and pH. We find that intracellular pH greatly affects the cost of inorganic carbon accumulation. At low pH the inorganic carbon pool contains more of the highly cell-permeable H2CO3, necessitating a substantial expenditure of energy on transport to maintain internal inorganic carbon levels. An intracellular pH ≈8 reduces leakage, making the CCM significantly more energetically efficient. This pH prediction coincides well with our measurement of intracellular pH in a model cyanobacterium. We also demonstrate that CO2 retention in the carboxysome is necessary, whereas selective uptake of HCO3 (-) into the carboxysome would not appreciably enhance energetic efficiency. Altogether, integration of pH produces a model that is quantitatively consistent with cyanobacterial physiology, emphasizing that pH cannot be neglected when describing biological systems interacting with inorganic carbon pools.

Saldanha Bay, South Africa III: new production and carrying capacity for bivalve aquaculture

Measurements of NH4, NO3, urea and HCO3 uptake using 15N and 13C stable isotope tracers were undertaken in Saldanha Bay, South Africa, between January 2012 and January 2013. These studies provide the first direct measurements of N utilisation by the plankton in the bay. Primary production in the bay is driven predominantly by the advection of nutrients from the neighbouring shelf environment during upwelling events, with terrestrial and other sources providing minor inputs. New production (NO3-based) was calculated from the f-ratio and total primary production and was used to provide estimates of potential carrying capacity for bivalve culture. Despite the apparent light limitation of NO3 uptake in the winter, the availability of NO3 appeared to exert the major influence on new production throughout the year. In addition, new production was modulated by NH4 availability as shown by the suppression of NO3 uptake by concentrations higher than 1−1.5 mmol m−3. The estimated areal new production of 0.60 g C m−2 d−1 yielded a bay-wide annual estimate of 9 811 t C ha−1 y−1, slightly higher than previous calculations based on physical models. It is estimated that the total annual production of mussels and oysters, respectively, for a 1 000-ha cultivation area is approximately 40 000–53 000 t y−1 (mainly Mytilus galloprovincialis) and 4 600–6 000 t y−1 (Crassotrea gigas). The combined total production figures constitute only 24–31% of the surplus new production. A combined harvestable carrying capacity of 74 000–82 000 t y−1 can be calculated from this surplus. However, from a management and ecological perspective, bivalve culture should be limited to well below this theoretical maximum. Even with this constraint, there appears to be considerable scope for expansion of bivalve farming over the modest, present levels with little jeopardy to ecological integrity.

Cholecystokinin receptors regulate sperm protein tyrosine phosphorylation via uptake of HCO3-.

Cholecystokinin (CCK), a peptide hormone and a neurotransmitter, was detected in mature sperm two decades ago. However, the exact role of CCK and the types of CCK receptors (now termed CCK1 and CCK2) in sperm have not been identified. Here, we find that CCK1 and CCK2 receptors are immunolocalized to the acrosomal region of mature sperm. The antagonist of CCK1 or CCK2 receptor strongly activated the soluble adenylyl cyclase/cAMP/protein kinase A signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation in dose- and time-dependent manners. But these actions of stimulation were abolished when sperm were incubated in the medium in the absence of HCO3-. Further investigation demonstrated that the inhibitor of CCK1 or CCK2 receptor could accelerate the uptake of HCO3- and significantly elevate the intracellular pH of sperm. Interestingly, the synthetic octapeptide of CCK (CCK8) showed the same action and mechanism as antagonists of CCK receptors. Moreover, CCK8 and the antagonist of CCK1 or CCK2 receptor were also able to accelerate human sperm capacitation-associated protein tyrosine phosphorylation by stimulating the influx of HCO3-. Thus, the present results suggest that CCK and its receptors may regulate sperm capacitation-associated protein tyrosine phosphorylation by modulating the uptake of HCO3-.

Nr5a2 restrains an inflammatory program in the pancreas through the regulation of AP-1 activity

s / Pancreatology 15 (2015) S1eS141 S47 Results: HAT-7 cells formed epithelial layers with measureable TER on Transwell permeable supports, and expressed key tight junction proteins claudin-1, -4 and -8. Transport proteins described in maturation ameloblasts were also present in HAT-7 cells. By microfluorometry we found that the cells were polarized with a high apical membrane CO2 permeability and vigorous basolateral HCO3 uptake which was sensitive to Naþ withdrawal, to the carbonic anhydrase inhibitor acetazolamide and to H2DIDS inhibition. Measurements of transepithelial HCO3 transport showed a marked increase in response to Ca2þand cAMP-mobilizing stimuli. Taken together, these studies provide evidence for a regulated, vectorial, basolateral to apical bicarbonate transport in polarized HAT-7 cells. Conclusion: We propose that the HAT-7 cell line is a useful functional model for studying electrolyte transport of ameloblasts. Supported by NIH-NIDCR (5R01DE013508subaward:7743sc), TAMOP4.2.1/B-09/1/KMR-2010-0001, TAMOP-4.2.2/B-10/1-2010-0013.

Schwann cell-associated adhesion molecules beta-1-Integrin, L1CAM, N-Cadherin and PMP22 enhance pancreatic cancer cell adhesion during neural invasion in pancreatic cancer

s / Pancreatology 15 (2015) S1eS141 S47 Results: HAT-7 cells formed epithelial layers with measureable TER on Transwell permeable supports, and expressed key tight junction proteins claudin-1, -4 and -8. Transport proteins described in maturation ameloblasts were also present in HAT-7 cells. By microfluorometry we found that the cells were polarized with a high apical membrane CO2 permeability and vigorous basolateral HCO3 uptake which was sensitive to Naþ withdrawal, to the carbonic anhydrase inhibitor acetazolamide and to H2DIDS inhibition. Measurements of transepithelial HCO3 transport showed a marked increase in response to Ca2þand cAMP-mobilizing stimuli. Taken together, these studies provide evidence for a regulated, vectorial, basolateral to apical bicarbonate transport in polarized HAT-7 cells. Conclusion: We propose that the HAT-7 cell line is a useful functional model for studying electrolyte transport of ameloblasts. Supported by NIH-NIDCR (5R01DE013508subaward:7743sc), TAMOP4.2.1/B-09/1/KMR-2010-0001, TAMOP-4.2.2/B-10/1-2010-0013.

Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii

The supply of inorganic carbon (Ci; CO2 and HCO3 (-)) is an environmental rate-limiting factor in aquatic photosynthetic organisms. To overcome the difficulty in acquiring Ci in limiting-CO2 conditions, an active Ci uptake system called the CO2-concentrating mechanism (CCM) is induced to increase CO2 concentrations in the chloroplast stroma. An ATP-binding cassette transporter, HLA3, and a formate/nitrite transporter homolog, LCIA, are reported to be associated with HCO3 (-) uptake [Wang and Spalding (2014) Plant Physiol 166(4):2040-2050]. However, direct evidence of the route of HCO3 (-) uptake from the outside of cells to the chloroplast stroma remains elusive owing to a lack of information on HLA3 localization and comparative analyses of the contribution of HLA3 and LCIA to the CCM. In this study, we revealed that HLA3 and LCIA are localized to the plasma membrane and chloroplast envelope, respectively. Insertion mutants of HLA3 and/or LCIA showed decreased Ci affinities/accumulation, especially in alkaline conditions where HCO3 (-) is the predominant form of Ci. HLA3 and LCIA formed protein complexes independently, and the absence of LCIA decreased HLA3 mRNA accumulation, suggesting the presence of unidentified retrograde signals from the chloroplast to the nucleus to maintain HLA3 mRNA expression. Furthermore, although single overexpression of HLA3 or LCIA in high CO2 conditions did not affect Ci affinity, simultaneous overexpression of HLA3 with LCIA significantly increased Ci affinity/accumulation. These results highlight the HLA3/LCIA-driven cooperative uptake of HCO3 (-) and a key role of LCIA in the maintenance of HLA3 stability as well as Ci affinity/accumulation in the CCM.

Proximal Tubule Function and Response to Acidosis

The human kidneys produce approximately 160-170 L of ultrafiltrate per day. The proximal tubule contributes to fluid, electrolyte, and nutrient homeostasis by reabsorbing approximately 60%-70% of the water and NaCl, a greater proportion of the NaHCO3, and nearly all of the nutrients in the ultrafiltrate. The proximal tubule is also the site of active solute secretion, hormone production, and many of the metabolic functions of the kidney. This review discusses the transport of NaCl, NaHCO3, glucose, amino acids, and two clinically important anions, citrate and phosphate. NaCl and the accompanying water are reabsorbed in an isotonic fashion. The energy that drives this process is generated largely by the basolateral Na(+)/K(+)-ATPase, which creates an inward negative membrane potential and Na(+)-gradient. Various Na(+)-dependent countertransporters and cotransporters use the energy of this gradient to promote the uptake of HCO3 (-) and various solutes, respectively. A Na(+)-dependent cotransporter mediates the movement of HCO3 (-) across the basolateral membrane, whereas various Na(+)-independent passive transporters accomplish the export of various other solutes. To illustrate its homeostatic feat, the proximal tubule alters its metabolism and transport properties in response to metabolic acidosis. The uptake and catabolism of glutamine and citrate are increased during acidosis, whereas the recovery of phosphate from the ultrafiltrate is decreased. The increased catabolism of glutamine results in increased ammoniagenesis and gluconeogenesis. Excretion of the resulting ammonium ions facilitates the excretion of acid, whereas the combined pathways accomplish the net production of HCO3 (-) ions that are added to the plasma to partially restore acid-base balance.

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.

Inorganic carbon uptake and intracellular assimilation by subarctic Pacific phytoplankton assemblages

We report the results of inorganic carbon (C) uptake experiments and activity measurements for carbonic anhydrase, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), and phosphoenolpyruvate carboxylase (PEPC) in offshore and coastal regions of the eastern subarctic Pacific Ocean. HCO3- was the dominant source of inorganic C taken up by phytoplankton at all sampling locations, accounting for ∼60−90% of total C uptake. The uptake of HCO3- occurred primarily through a direct transport system, while indirect HCO3− use, mediated by extracellular carbonic anhydrase (eCA), played a minor role in the C uptake system. Direct HCO3- transport and eCA activity were not related to ambient nutrient or CO2 concentrations or to phytoplankton biomass (chlorophyll a [Chl a]) or primary productivity. There was significant variability in the biomass-normalized activities of Rubisco, PEPC, and total (intracellular and extracellular) carbonic anhydrase. The activities of all of the enzymes measured exhibited significant correlations with both CO2 concentrations and Chl a. PEPC activity averaged 20% of Rubisco activity (range 0.5-110%), and the PEPC : Rubisco ratio was positively correlated with CO2 concentrations and negatively correlated with Chl a. Carbonic anhydrase activity was strongly anticorrelated with CO2 and positively correlated with Chl a. The results provide evidence for the importance of CO2-regulated carbon concentrating mechanisms in marine waters.

Carbon acquisition mechanisms by planktonic desmids and their link to ecological distribution

To test if different inorganic carbon (Ci) uptake mechanisms underlie the ecological distribution pattern of planktonic desmids, we performed pH-drift experiments with 12 strains, belonging to seven species, originating from lakes of different pH. Staurastrum brachiatum Ralfs and Staurodesmus cuspidatus (Ralfs) Teil. var. curvatus (W. West) Teil., species confined to acidic, soft water habitats, showed remarkably different behavior in the pH drift experiments: S. brachiatum appeared to use CO2 only, whereas Staurodesmus cuspidatus appeared to use HCO3– as well. Staurastrum chaetoceras (Schr.) Smith and Staurastrum planctonicum Teil, species well-known for their abundant occurrence in alkaline waters, were the most effective at using HCO3–. Other species, to be encountered in both slightly acidic and slightly alkaline waters, took an intermediate position. Experiments using specific inhibitors suggested that Cosmarium abbreviatum Rac. var. planctonicum W. & G.S. West and S. brachiatum use CO2 by an active CO2 uptake mechanism, whereas S. chaetoceras and Staurodesmus cuspidatus showed an active HCO3– uptake pattern. Most likely, these active uptake mechanisms make use of H+-ATPase, as none of the desmids expressed significant carbonic anhydrase activity. A series of strains of Staurastrum planctonicum isolated from different habitats, all clustered in between the species using HCO3–, but no further differentiation was observed. Therefore, desmids cannot be simply characterized as exclusive CO2 users, and the ecological distribution pattern of a desmid species does not unequivocally link to a certain Ci uptake mechanism. Nevertheless, there does appear to be a general ecological link between a species' Ci uptake mechanism and its ecological distribution.Key words: pH drift, desmids, isolate variation, inorganic carbon acquisition.

HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

BackgroundBicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.ResultsRT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 μM adenosine or 10 μM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).ConclusionHCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

UPTAKE OF INORGANIC CARBON BY CLADOPHORA GLOMERATA (CHLOROPHYTA) FROM THE BALTIC SEA1

Carbon uptake in the green macroalga Cladophora glomerata (L.) Kütz. from the brackish Baltic Sea was studied by recording changes in pH, alkalinity, and inorganic carbon concentration of the seawater medium during photosynthesis. The use of specific inhibitors identified three uptake mechanisms: 1) dehydration of HCO3− into CO2 by periplasmic carbonic anhydrase, followed by diffusion of CO2 into the cell; 2) direct uptake of HCO3− via a 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonate‐sensitive mechanism; and 3) uptake of inorganic carbon by the involvement of a vanadate‐sensitive P‐type H+‐ATPase (proton pump). A decrease in the alkalinity of the seawater medium during carbon uptake, except when treated with vanadate, indicated a net uptake of the ionic species contributing to alkalinity (i.e. HCO3−, CO32−, and OH−) from the medium, where OH− influx is equivalent to H+ efflux. This would suggest that the proton pump is involved in HCO3− transport. We also show that the proton pump can be induced by carbon limitation. The inducibility of carbon uptake in C. glomerata may partly explain why this species is so successful in the upper littoral zone of the Baltic Sea. Usually, carbon limitation is not a problem in the upper littoral of the sea. However, it may occur frequently within dense Cladophora belts with high photosynthetic rates that create high pH and low carbon concentrations in the alga's microenvironment.

Strategies for carbon acquisition in the red marine macroalga Coccotylus truncatus from the Baltic Sea

Marine macroalgae need carbon-concentrating mechanisms because they have only limited access to CO2 in their natural environment. Previous studies have shown that one important strategy common to many algae is the activity of periplasmic carbonic anhydrases that catalyse the dehydration of HCO3 – into CO2. The latter can then cross the plasma membrane by passive diffusion. We hypothesised that an active (energy-consuming) mechanism might also be involved in the membrane transport of CO2, as is the case in a number of microalgae. Coccotylus truncatus was chosen as a model organism for this study because it belongs to a group of algae that usually lack direct HCO3 – uptake: sublittoral red algae. The method used to study carbon uptake was pH drift of the seawater medium surrounding the algae in a closed vessel, with and without the addition of specific inhibitors or proton buffers. Measured parameters included pH, total inorganic carbon and alkalinity of the seawater medium. Our results suggest that, in C. truncatus, periplasmic carbonic anhydrase as well as H+ extrusion, probably driven by a vanadate-sensitive P-type H+-ATPase (proton pump), are involved in CO2 uptake. No direct uptake of HCO3 – was discovered. This paper also presents data on the buffer capacity of several proton buffers and the carbon-uptake inhibitors acetazolamide, 4,4′-diisothiocyano-stilbene-2,2′-disulfonate (DIDS) and orthovanadate in Baltic Sea water with a salinity of 6.5 psu.