Intracellular Carbonic Anhydrase Research Articles

Periplasmic carbonic anhydrase CAH1 contributes to high inorganic carbon affinity in Chlamydomonas reinhardtii.

Carbonic anhydrase (CA), an enzyme conserved across species, is pivotal in the interconversion of inorganic carbon (Ci; CO2 and HCO3-). Compared to the well-studied intracellular CA, the specific role of extracellular CA in photosynthetic organisms is still not well understood. In the green alga Chlamydomonas (Chlamydomonas reinhardtii), carbonic anhydrase 1 (CAH1), located at the periplasmic space, is strongly induced under CO2-limiting conditions by the Myb transcription factor LCR1. While the lcr1 mutant shows decreased Ci-affinity, the detailed mechanisms behind this phenomenon are yet to be elucidated. In this study, we aimed to unravel the LCR1-dependent genes essential for maintaining high Ci-affinity. To achieve this, we identified a total of 12 LCR1-dependent inducible genes under CO2-limiting conditions, focusing specifically on the most prominent ones - CAH1, LCI1, LCI6, and Cre10.g426800. We then created mutants of these genes using the CRISPR-Cas9 system, all from the same parental strain, and compared their Ci-affinity. Contrary to earlier findings that reported no reduction in Ci-affinity in the cah1 mutant, our cah1-1 mutant exhibited a decrease in Ci-affinity under high HCO3-/CO2-ratio conditions. Additionally, when we treated wild-type cells with a CA inhibitor with low membrane permeability, a similar reduction in Ci-affinity was observed. Moreover, the addition of exogenous CA to the cah1 mutant rescued the decreased Ci-affinity. These results, highlighting the crucial function of the periplasmic CAH1 in maintaining high Ci-affinity in Chlamydomonas cells, provide insights into the functions of periplasmic CA in algal carbon assimilation.

Revealing the environmental hazard posed by biodegradable microplastics in aquatic ecosystems: An investigation of polylactic acid's effects on Microcystis aeruginosa

The influence of petroleum-based microplastics (MPs) on phytoplankton has been extensively studied, while research on the impact of biodegradable MPs, derived from alternative plastics to contest the environmental crisis, remains limited. This study performed a 63 days co-incubation experiment to assess the effect of polylactic acid MPs (PLA-MPs) on the growth, physiology, and carbon utilization of M. aeruginosa and the change in PLA-MPs surface properties. The results showed that despite PLA-MPs induced oxidative stress and caused membrane damage in M. aeruginosa, the presence of PLA-MPs (10, 50, and 200 mg/L) triggered significant increases (p < 0.05) in the density of M. aeruginosa after 63 days. Specifically, the algal densities upon 50 and 200 mg/L PLA-MPs exposure were increased by 20.91% and 36.31% relative to the control, respectively. Meanhwhile, the reduced C/O ratio on PLA-MPs surface and change in PLA-MPs morphological characterization, which is responsible for substantially increase in the aquatic dissolved inorganic carbon concentration during the co-incubation, implying the degradation of PLA-MPs; thus, provided sufficient carbon resources that M. aeruginosa could assimilate. This was in line with the declined intracellular carbonic anhydrase content in M. aeruginosa. This study is the first attempt to uncover the interaction between PLA-MPs and M. aeruginosa, and the finding that their interaction promotes the degrading of PLA-MPs meanwhile favoring M. aeruginosa growth will help elucidate the potential risk of biodegradable MPs in aquatic environment.

Enhalus acoroides efficiently alleviate ocean acidification by shifting modes of inorganic carbon uptake and increasing photosynthesis when pH drops

Ocean acidification (OA) is causing increasing ecological damage, so it is worthwhile to find efficient and environmentally friendly ways to mitigate OA. The mechanism of inorganic carbon (Ci) absorption and the ability to mitigate OA of the tropical seagrass Enhalus acoroides were investigated in this study. At 2.2 mM Ci concentration, its CO2 fixation efficiency increased to 81.89 t CO2/year/Ha under pH 6.5 from 27.59 t CO2/year/Ha at pH 8.2, and even reached 88.11 t CO2/year/Ha at pH 6.5 with unlimited Ci availability, made possible by three pathways for Ci utilization, which included absorbing CO2 directly, transforming HCO3− into CO2 through extracellular carbonic anhydrase, and absorbing HCO3− directly by anion-exchange protein then transforming it to CO2 through intracellular carbonic anhydrase, as verified by inhibitor addition experiments. The carbon fixation rate increased with decreasing pH, suggesting a greater CO2 absorbing capacity for E. acoroides under acidic conditions, which further demonstrates the possibility of mitigating OA and increasing carbon fixation through conserving and restoring E. acoroides meadows. Due to the strong carbon absorption capacity of E. acoroides, it is very important to strengthen the artificial restoration of E. acoroides seagrass meadows in the environmental management of the coastline.

Evidencing the role of carbonic anhydrase in the formation of carbonate minerals by bacterial strains isolated from extreme environments in Qatar

Calcium carbonate, one of the most abundant minerals in the geological records is considered as primary source of the carbon reservoir. The role of microorganisms in the biotic precipitation of calcium carbonate has been extensively investigated, especially at extreme life conditions. In Qatar, Sabkhas which are microbial ecosystems housing biomineralizing bacteria, have been carefully studied as unique sites of microbial dolomite formation. Dolomite (CaMg(CO3)2 is an important carbonate mineral forming oil reservoir rocks; however, dolomite is rarely formed in modern environments. The enzyme carbonic anhydrase is present in many living organisms, performs interconversion between CO2 and the bicarbonate ion. Thus, carbonic anhydrase is expected to accelerate both carbonate rock dissolution and CO2 uptake at the same time, serving as carbonite source to carbonites-forming bacteria. This study gathered cross-linked data on the potential role of the carbonic anhydrase excreted by mineral-forming bacteria, isolated from two different extreme environments in Qatar. Dohat Faishakh Sabkha, is a hypersaline coastal Sabkha, from where various strains of the bacterium Virgibacillus were isolated. Virgibacillus can -not only-mediate carbonate mineral formation, but also contributes to magnesium incorporation into the carbonate minerals, leading to the formation of high magnesium calcite. The latter is considered as precursor for dolomite formation. In addition, bacterial strains isolated from marine sediments, surrounding coral reef in Qatar sea, would provide additional knowledge on the role of carbonic anhydrase in mineral formation. Here, the quantification of the two mostly described activities of carbonic anhydrase; esterase and hydration reactions were performed. Mineral-forming strains were shown to exhibit high activities as opposed to the non-forming minerals, which confirms the relation between the presence of active carbonic anhydrase combined with elevated metabolic activity and the biomineralizing potential of the bacterial strains. The highest specific intracellular carbonic anhydrase activity; as both esterase and hydration (i.e., 66 ± 3 and 583000 ± 39000 WAU/108 cells respectively), was evidenced in mineral-forming strains as opposed to non-mineral forming strains (i.e., 6 ±. 0.5 and 1223 ± 61 WAU/108cells) respectively. These findings would contribute to the understanding of the mechanism of microbially mediated carbonate precipitation. This role may be both in capturing CO2 as source of carbonate, and partial solubilization of the formed minerals allowing incorporation of Mg instead of calcium, before catalyzing again the formation of more deposition of carbonates.

The roles of plasma accessible and cytosolic carbonic anhydrases in bicarbonate (HCO3-) excretion in Pacific hagfish (Eptatretus stoutii).

Pacific hagfish (Eptatretus stoutii) are marine scavengers and feed on decaying animal carrion by burrowing their bodies inside rotten carcasses where they are exposed to several threatening environmental stressors, including hypercapnia (high partial pressures of CO2). Hagfish possess a remarkable capacity to tolerate hypercapnia, and their ability to recover from acid-base disturbances is well known. To deal with the metabolic acidosis resulting from exposure to high CO2, hagfish can mount a rapid elevation of plasma HCO3- concentration (hypercarbia). Once PCO2 is restored, hagfish quickly excrete their HCO3- load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO3- dehydration into CO2 at the hagfish gills. We aimed to characterize the role of branchial CA in CO2/HCO3- clearance from the plasma at the gills of E. stoutii, under control and high PCO2 (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO3- excretion by measuring in situ [14C]-HCO3- fluxes. To accomplish this, we employed a novel surgical technique of individual gill pouch arterial perfusion combined with perifusion of the gill afferent to efferent water ducts. [14C]-HCO3- efflux was measured at the gills of fish exposed to control, hypercapnic (48h) and recovery from hypercapnia conditions (6h), in the presence of two well-known pharmacological inhibitors of CA, the membrane impermeant C18 (targets membrane bound, plasma accessible CA) and membrane-permeant acetazolamide, which targets all forms of CA, including extracellular and intracellular cytosolic CAs. C18 did not affect HCO3- flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO3- fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO3- flux by 38%. The same pattern was observed for fish in recovery, where in all three experimental conditions, there was no significant inhibition of plasma-accessible CA. We also observed no change in CA enzyme activity (measured in vitro) in any of the experimental PCO2 conditions. In summary, our data suggests that there are additional pathways for HCO3- excretion at the gills of hagfish that are independent of plasma-accessible CA.

Initiation of efficient C4 pathway in response to low ambient CO2 during the bloom period of a marine dinoflagellate.

Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the global ocean. Despite the great ecological significance, the photosynthetic carbon acquisition by dinoflagellates is still poorly understood. The pathways of photosynthetic carbon assimilation in a marine dinoflagellate Prorocentrum donghaiense under both in situ and laboratory-simulated bloom conditions were investigated using a combination of metaproteomics, qPCR, stable carbon isotope and targeted metabolomics approaches. A rapid consumption of dissolved CO2 to generate high biomass was observed as the bloom proceeded. The carbon assimilation genes and proteins including intracellular carbonic anhydrase 2, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase and RubisCO as well as their enzyme activities were all highly expressed at the low CO2 level, indicating that C4 photosynthetic pathway functioned in the blooming P. donghaiense cells. Furthermore, δ13 C values and content of C4 compound (malate) significantly increased with the decreasing CO2 concentration. The transition from C3 to C4 pathway minimizes the internal CO2 leakage and guarantees efficient carbon fixation at the low CO2 level. This study demonstrates the existence of C4 photosynthetic pathway in a marine dinoflagellate and reveals its important complementary role to assist carbon assimilation for cell proliferation during the bloom period.

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2, intracellular carbonic anhydrase (CAi) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2-(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.

Induction of vitamin B12 to purify biogas slurry and upgrade biogas using co-culture of microalgae and fungi.

Different gradient concentrations of vitamin B12 (0, 10, 100, 1,000ngL-1 ) were used in the symbiosis system (Chlorella vulgaris-Ganoderma lucidum or Chlorella vulgaris-Pleurotus ostreatus) to assess their effect on simultaneous purification of biogas and removal of nutrients in biogas slurry using co-culture of microalgae and fungi. When B12 was added to the symbiosis system, biomass growth, intracellular carbonic anhydrase activity (CA), chlorophyll a content (CHL-a), photosynthetic characteristics of the two cultivation system, and removal efficiency of nutrients in biogas slurry and CO2 in biogas were significantly higher than those in the control group. The optimal concentration of B12 was determined to be 100ngL-1 considering the removal efficiency of nutrients and CO2 . Maximum mean chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP), and CO2 removal efficiencies were 75.98±6.26%, 78.46±6.21%, 80.21±6.83% and 61.08±5.21% in Chlorella vulgaris-Ganoderma lucidum, respectively. This study showed the potential of microalgae and fungi symbiosis system with B12 addition for nutrient removal and biogas upgrading. PRACTITIONER POINTS: Vitamin B12 had positive effects on algal-fungal pellets growth. The optimal vitamin B12 concentration was 100ngL-1 . The highest CO2 remove rate was 61.08% by G.lucidum/C.vulgaris pellets. Vitamin B12 significantly improved photosynthetic performance of pellets.

Intracellular Binding/Unbinding Kinetics of Approved Drugs to Carbonic Anhydrase II Observed by in-Cell NMR.

Candidate drugs rationally designed in vitro often fail due to low efficacy in vivo caused by low tissue availability or because of unwanted side effects. To overcome the limitations of in vitro rational drug design, the binding of candidate drugs to their target needs to be evaluated in the cellular context. Here, we applied in-cell NMR to investigate the binding of a set of approved drugs to the isoform II of carbonic anhydrase (CA) in living human cells. Some compounds were originally developed toward other targets and were later found to inhibit CAs. We observed strikingly different dose- and time-dependent binding, wherein some drugs exhibited a more complex behavior than others. Specifically, some compounds were shown to gradually unbind from intracellular CA II, even in the presence of free compound in the external medium, therefore preventing the quantitative formation of a stable protein–ligand complex. Such observations could be correlated to the known off-target binding activity of these compounds, suggesting that this approach could provide information on the pharmacokinetic profiles of lead candidates at the early stages of multitarget drug design.

Effect of GR24 concentrations on biogas upgrade and nutrient removal by microalgae-based technology

Freshwater microalgae Chlorella vulgaris was cultured and induced with strigolactone (GR24) to simultaneously eliminate nutrients in biogas slurry and purify biogas. Treatment with 10−7 M GR24 yielded maximum growth rate and mean daily productivity for algae at 0.187 ± 0.06 d−1 and 0.097 ± 0.008 g L−1 d−1, respectively. Results from chlorophyll fluorescence transients method demonstrated that moderate concentration of GR24 could enhance the photosynthetic performance of microalgae. In addition, GR24 affected intracellular carbonic anhydrase activity and chlorophyll-a content. Maximum chemical oxygen demand, total nitrogen, and CO2 removal efficiencies were 78.62 ± 2.36%, 76.47 ± 1.53% and 64.05 ± 1.15% with 10−7 M GR24 induction, respectively. Further, highest total phosphorus removal efficiency (80.27 ± 1.93%) was observed at 10−9 M. The optimal GR24 concentration range was determined to be between 10−9 and 10−7 M in consideration with nutrient and CO2 removal efficiencies.

Overexpression of Chlamydomonas reinhardtii LCIA (CrLCIA) gene increases growth of Nannochloropsis salina CCMP1776

Most aquatic photosynthetic organisms have developed inorganic carbon-concentrating mechanisms (CCMs) to compensate for the kinetic constraints of CO2 concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which functions in the first steps of carbon fixation. CCMs, which are widespread in various types of algae, evolved independently among algal lineages and accordingly play a fundamental role in algal photosynthesis, metabolism, growth, and biomass production. Nannochloropsis, a marine eustigmatophycean microalga, is a candidate organism for biofuel production due to its high lipid content; however, inorganic carbon (Ci) availability in Nannochloropsis cells is not sufficient for complete carbon fixation due to inherently weak CCM machinery, including a CO2-leaking HCO3− pump. CrLCIA, a member of the formate-nitrite transporter family, functions as a HCO3− transporter in Chlamydomonas reinhardti. Thus, in this study, we overexpressed the CrLCIA gene heterologously in N. salina CCMP1776 in an attempt to reinforce its bicarbonate transport activity. CrLCIA expression in N. salina increased intracellular Ci and carbonic anhydrase (CA) activity, resulting in increased growth (30%) and biomass (2-fold). These results indicate that constitutively expressed CrLCIA leads to increased Ci uptake and CA activity, contributing to high availability of CO2 for photosynthesis under low CO2 conditions. The total fatty acid (FA) per cell mass in the transgenic lines was similar to that of wild-type Nannochloropsis cells, indicating that total FA productivity in the transgenic lines is increased approximately 2-fold. These findings will be useful to improve the CCM function for high biomass and lipid production using genetic modification tools in Nannochloropsis species, contributing to improved biodiesel production.

Photosynthetic acclimation of terrestrial and submerged leaves in the amphibious plant Hygrophila difformis.

Hygrophila difformis, a heterophyllous amphibious plant, develops serrated or dissected leaves when grown in terrestrial or submerged conditions, respectively. In this study, we tested whether submerged leaves and ethylene-induced leaves of the heterophyllous, amphibious plant H. difformis have improved photosynthetic ability under submerged conditions. Also, we investigated how this amphibious plant photosynthesizes underwater and whether a HCO3− transport system is present. We have analysed leaf morphology, measured underwater photosynthetic rates and HCO3− affinity in H. difformis to determine if there are differences in acclimation ability dependent on growth conditions: terrestrial, submerged, terrestrial treated with ethylene and submerged treated with an ethylene inhibitor. Moreover, we measured time courses for changes in leaf anatomical characteristics and underwater photosynthesis in terrestrial leaves after submersion. Compared with the leaves of terrestrially grown plants, leaf thickness of submerged plants was significantly thinner. The stomatal density on the abaxial surface of submerged leaves was also reduced, and submerged plants had a significantly higher O2 evolution rate. When the leaves of terrestrially grown plants were treated with ethylene, their leaf morphology and underwater photosynthesis increased to levels comparable to those of submerged leaves. Underwater photosynthesis of terrestrial leaves was significantly higher by 5 days after submersion. In contrast, leaf morphology did not change after submergence. Submerged leaves and submerged terrestrial leaves were able to use bicarbonate but submerged terrestrial leaves had an intermediate ability to use HCO3− that was between terrestrial leaves and submerged leaves. Ethoxyzolamide, an inhibitor of intracellular carbonic anhydrase, significantly inhibited underwater photosynthesis in submerged leaves. This amphibious plant acclimates to the submerged condition by changing leaf morphology and inducing a HCO3− utilizing system, two processes that are regulated by ethylene.

Modeling of pH regulation in tumor cells: Direct interaction between proton-coupled lactate transporters and cancer-associated carbonic anhydrase.

The most aggressive tumor cells, which often reside in a hypoxic environment, can release vast amounts of lactate and protons via monocarboxylate transporters (MCTs). This additional proton efflux exacerbates extracellular acidification and supports the formation of a hostile environment. In the present study we propose a novel, data-based model for this proton-coupled lactate transport in cancer cells. The mathematical settings involve systems coupling nonlinear ordinary and stochastic differential equations describing the dynamics of intra- and extracellular proton and lactate concentrations. The data involve time series of intracellular proton concentrations of normoxic and hypoxic MCF-7 breast cancer cells. The good agreement of our final model with the data suggests the existence of proton pools near the cell membrane, which can be controlled by intracellular and extracellular carbonic anhydrases to drive proton-coupled lactate transport across the plasma membrane of hypoxic cancer cells.

Intracellular carbonic anhydrase from Citrobacter freundii and its role in bio-sequestration

The aim of this work was to study the CO2 bio-sequestration application of indigenous Citrobacter species and its carbonic anhydrase (CA). Intracellular CA was purified from Citrobacter freundii (CF; accession no: MH283871) isolated from limestone rock site in Kumaun region of Indian Himalaya studied for the sequestration of carbon dioxide and the formation of calcite. CF showed maximum CA enzyme activity at 11.3 EU/ml at pH 7.0 and 37 °C. Hydration of CO2 into carbonate was characterized by calcite phase of calcium carbonate using absorption spectroscopy and imaging technique. Purified CA showed a significantly high CO2 sequestration capacity of 230 mg CaCO3/mg of purified as compared to crude enzyme (50 mg CaCO3/ml of enzyme).

Correlation of carbonic anhydrase and Rubisco in the growth and photosynthesis in the diatom Phaeodactylum tricornutum

Carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are critical enzymes involved in the CO2-concentrating mechanism (CCM) and carbon fixation in phytoplankton. These enzymes play key roles in photosynthetic carbon acquisition and fixation in diatoms. However, the potential synergistic interactions of the said enzymes remain little understood. In the present study, effects of CA inhibitors on the growth, photosynthetic rate, and Rubisco activities in the diatom Phaeodactylum tricornutum were investigated. Our results showed that the growth and photosynthetic O2 evolution rate of P. tricornutum were significantly inhibited by the intracellular CA (iCA) inhibitor ethoxzolamide (EZ), but not by extracellular CA (eCA) inhibitor acetazolamide (AZ) in the long term. This suggests that the eCA was not necessarily essential for growth and photosynthesis, but rather it was the iCA activity that was more crucial in P. tricornutum. We also found that there is a significant positive correlation between Rubisco and CA activities, suggesting that the two carbon acquisition and fixation components of photosynthesis may have undergone co-evolution to optimize fundamental trade-offs. These results further indicate that the roles of eCA and iCA may also differ among diatom species or strains, and that their functions are tightly correlated with other carbon fixation components.

A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells.

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme's intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.

Elevated Inorganic Carbon Concentrating Mechanism Confers Tolerance to High Light in an Arctic Chlorella sp. ArM0029B.

Microalgae and higher plants employ an inorganic carbon (Ci) concentrating mechanism (CCM) to increase CO2 availability to Rubisco. Operation of the CCM should enhance the activity of the Calvin cycle, which could act as an electron sink for electrons generated by photosynthesis, and lower the redox status of photosynthetic electron transport chains. In this study, a hypothesis that microalgal cells with fully operating CCM are less likely to be photodamaged was tested by comparing a Chlorella mutant with its wild type (WT). The mutant acquired by screening gamma-ray-induced mutant libraries of Chlorella sp. ArM0029B exhibited constitutively active CCM (CAC) even in the presence of additional Ci sources under mixotrophic growth conditions. In comparison to the WT alga, the mutant named to constitutively active CCM1 (CAC1) showed more transcript levels for genes coding proteins related to CCM such as Ci transporters and carbonic anhydrases (CA), and greater levels of intracellular Ci content and CA activity regardless of whether growth is limited by light or not. Under photoinhibitory conditions, CAC1 mutant showed faster growth than WT cells with more PSII reaction center core component D1 protein (encoded by psbA), higher photochemical efficiency as estimated by the chlorophyll fluorescence parameter (Fv/Fm), and fewer reactive oxygen species (ROS). Interestingly, high light (HL)-induced increase in ROS contents in WT cells was significantly inhibited by bicarbonate supplementation. It is concluded that constitutive operation of CCM endows Chlorella cells with resistance to HL partly by reducing the endogenous generation of ROS. These results will provide useful information on the interaction between CCM expression, ROS production, and photodamage in Chlorella and related microalgae.

Does Aerobic Respiration Produce Carbon Dioxide or Hydrogen Ion and Bicarbonate?

Maintenance of intracellular pH is critical for clinical homeostasis. The metabolism of glucose, fatty acids, and amino acids yielding the generation of adenosine triphosphate in the mitochondria is accompanied by the production of acid in the Krebs cycle. Both the nature of this acidosis and the mechanism of its disposal have been argued by two investigators with a long-abiding interest in acid-base physiology. They offer different interpretations and views of the molecular mechanism of this intracellular pH regulation during normal metabolism. Dr. John Severinghaus has posited that hydrogen ion and bicarbonate are the direct end products in the Krebs cycle. In the late 1960s, he showed in brain and brain homogenate experiments that acetazolamide, a carbonic anhydrase inhibitor, reduces intracellular pH. This led him to conclude that hydrogen ion and bicarbonate are the end products, and the role of intracellular carbonic anhydrase is to rapidly generate diffusible carbon dioxide to minimize acidosis. Dr. Erik Swenson posits that carbon dioxide is a direct end product in the Krebs cycle, a more widely accepted view, and that acetazolamide prevents rapid intracellular bicarbonate formation, which can then codiffuse with carbon dioxide to the cell surface and there be reconverted for exit from the cell. Loss of this "facilitated diffusion of carbon dioxide" leads to intracellular acidosis as the still appreciable uncatalyzed rate of carbon dioxide hydration generates more protons. This review summarizes the available evidence and determines that resolution of this question will require more sophisticated measurements of intracellular pH with faster temporal resolution.

CO2 permeability and carbonic anhydrase activity of rat cardiomyocytes.

To determine the CO2 permeability (PCO2 ) of plasma membranes of cardiomyocytes. These cells were chosen because heart possesses the highest rate of O2 consumption/CO2 production in the body. Cardiomyocytes were isolated from rat hearts using the Langendorff technique. Cardiomyocyte suspensions exhibited a vitality of 2-14% and were studied by the previously described mass spectrometric 18 O-exchange technique deriving PCO2 . We showed by mass spectrometry and by carbonic anhydrase (CA) staining that non-vital cardiomyocytes are free of CA and thus do not contribute to the mass spectrometric signal, which is determined exclusively by the fully functional vital cardiomyocytes. Lysed cardiomyocytes yielded an intracellular CA activity for vital cells of 5070; that is, the rate of CO2 hydration inside the cell is accelerated 5071-fold. Using this number, analyses of the mass spectrometric recordings from cardiomyocyte suspensions yield a PCO2 of 0.10 cm s-1 (SD ± 0.06, n = 15) at 37 °C. In comparison with the PCO2 of other cells, this value is quite high and about identical to that of the human red cell membrane. As no major protein CO2 channels such as aquaporins 1 and 4 are present in rat cardiac sarcolemma, the high PCO2 of this membrane is likely due to its low cholesterol content of about 0.2 (mol cholesterol)·(mol total membrane lipids)-1 . Previous work predicted a PCO2 of ≥0.1 cm s-1 from this level of cholesterol. We conclude that the low cholesterol establishes a PCO2 high enough to render the membrane resistance to CO2 diffusion almost negligible, even under conditions of maximal O2 consumption of the heart.

Effects of ocean acidification on the physiological performance and carbon production of the Antarctic sea ice diatom Nitzschia sp. ICE-H

Ocean acidification (OA) resulting from increasing atmospheric CO2 strongly influences marine ecosystems, particularly in the polar ocean due to greater CO2 solubility. Here, we grew the Antarctic sea ice diatom Nitzschia sp. ICE-H in a semicontinuous culture under low (~400ppm) and high (1000ppm) CO2 levels. Elevated CO2 resulted in a stimulated physiological response including increased growth rates, chlorophyll a contents, and nitrogen and phosphorus uptake rates. Furthermore, high CO2 enhanced cellular particulate organic carbon production rates, indicating a greater shift from inorganic to organic carbon. However, the cultures grown in high CO2 conditions exhibited a decrease in both extracellular and intracellular carbonic anhydrase activity, suggesting that the carbon concentrating mechanisms of Nitzschia sp. ICE-H may be suppressed by elevated CO2. Our results revealed that OA would be beneficial to the survival of this sea ice diatom strain, with broad implications for global carbon cycles in the future ocean.