The role of soluble adenylyl cyclase in sensing and regulating intracellular pH.

Soluble adenylyl cyclase (sAC) differs from transmembrane adenylyl cyclases (tmAC) in many aspects. In particular, the activity of sAC is not regulated by G-proteins but by the prevailing bicarbonate concentrations inside cells. Therefore, sAC serves as an exquisite intracellular pH sensor, with the capacity to translate pH changes into the regulation of localization and/or activity of cellular proteins involved in pH homeostasis. In this review, we provide an overview of literature describing the regulation of sAC activity by bicarbonate, pinpointing the importance of compartmentalization of intracellular cAMP signaling cascades. In addition, examples of processes involving proton and bicarbonate transport in different cell types, in which sAC plays an important regulatory role, were described in detail.

The role of Na+-coupled bicarbonate transporters (NCBT) in health and disease.

Cellular and organism survival depends upon the regulation of pH, which is regulated by highly specialized cell membrane transporters, the solute carriers (SLC) (For a comprehensive list of the solute carrier family members, see: https://www.bioparadigms.org/slc/ ). The SLC4 family of bicarbonate (HCO3-) transporters consists of ten members, sorted by their coupling to either sodium (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE), chloride (AE1, AE2, AE3), or borate (BTR1). The ionic coupling of SLC4A9 (AE4) remains controversial. These SLC4 bicarbonate transporters may be controlled by cellular ionic gradients, cellular membrane voltage, and signaling molecules to maintain critical cellular and systemic pH (acid-base) balance. There are profound consequences when blood pH deviates even a small amount outside the normal range (7.35-7.45). Chiefly, Na+-coupled bicarbonate transporters (NCBT) control intracellular pH in nearly every living cell, maintaining the biological pH required for life. Additionally, NCBTs have important roles to regulate cell volume and maintain salt balance as well as absorption and secretion of acid-base equivalents. Due to their varied tissue expression, NCBTs have roles in pathophysiology, which become apparent in physiologic responses when their expression is reduced or genetically deleted. Variations in physiological pH are seen in a wide variety of conditions, from canonically acid-base related conditions to pathologies not necessarily associated with acid-base dysfunction such as cancer, glaucoma, or various neurological diseases. The membranous location of the SLC4 transporters as well as recent advances in discovering their structural biology makes them accessible and attractive as a druggable target in a disease context. The role of sodium-coupled bicarbonate transporters in such a large array of conditions illustrates the potential of treating a wide range of disease states by modifying function of these transporters, whether that be through inhibition or enhancement.

CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants.

BACKGROUND: Many affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. METHODS: Next-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). RESULTS: Of 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. CONCLUSIONS: CFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators.

Binding Sites of Bicarbonate in Phosphoenolpyruvate Carboxylase.

Phosphoenolpyruvate carboxylase (PEPC) is used in plant metabolism for fruit maturation or seed development as well as in the C4 and crassulacean acid metabolism (CAM) mechanisms in photosynthesis, where it is used for the capture of hydrated CO2 (bicarbonate). To find the yet unknown binding site of bicarbonate in this enzyme, we have first identified putative binding sites with nonequilibrium molecular dynamics simulations and then ranked these sites with alchemical free energy calculations with corrections of computational artifacts. Fourteen pockets where bicarbonate could bind were identified, with three having realistic binding free energies with differences with the experimental value below 1 kcal/mol. One of these pockets is found far from the active site at 14 Å and predicted to be an allosteric binding site. In the two other binding sites, bicarbonate is in direct interaction with the magnesium ion; neither sequence alignment nor the study of mutant K606N allowed to discriminate between these two pockets, and both are good candidates as the binding site of bicarbonate in phosphoenolpyruvate carboxylase.

The Role of the Endocrine System in the Regulation of Acid-Base Balance by the Kidney and the Progression of Chronic Kidney Disease.

Systemic acid-base status is primarily determined by the interplay of net acid production (NEAP) arising from metabolism of ingested food stuffs, buffering of NEAP in tissues, generation of bicarbonate by the kidney, and capture of any bicarbonate filtered by the kidney. In chronic kidney disease (CKD), acid retention may occur when dietary acid production is not balanced by bicarbonate generation by the diseased kidney. Hormones including aldosterone, angiotensin II, endothelin, PTH, glucocorticoids, insulin, thyroid hormone, and growth hormone can affect acid-base balance in different ways. The levels of some hormones such as aldosterone, angiotensin II and endothelin are increased with acid accumulation and contribute to an adaptive increase in renal acid excretion and bicarbonate generation. However, the persistent elevated levels of these hormones can damage the kidney and accelerate progression of CKD. Measures to slow the progression of CKD have included administration of medications which inhibit the production or action of deleterious hormones. However, since metabolic acidosis accompanying CKD stimulates the secretion of several of these hormones, treatment of CKD should also include administration of base to correct the metabolic acidosis.

Role of CFTR in diabetes-induced pancreatic ductal fluid and HCO3 - secretion.

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO3 - secretion, CFTR activity, and the expression of CFTR, Na+ /H+ exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue.

Bicarbonate secretion and acid/base sensing by the intestine.

The transport of bicarbonate across the enterocyte cell membrane regulates the intracellular as well as the luminal pH and is an essential part of directional fluid movement in the gut. Since the first description of "active" transport of HCO3- ions against a concentration gradient in the 1970s, the fundamental role of HCO3- transport for multiple intestinal functions has been recognized. The ion transport proteins have been identified and molecularly characterized, and knockout mouse models have given insight into their individual role in a variety of functions. This review describes the progress made in the last decade regarding novel techniques and new findings in the molecular regulation of intestinal HCO3- transport in the different segments of the gut. We discuss human diseases with defects in intestinal HCO3- secretion and potential treatment strategies to increase luminal alkalinity. In the last part of the review, the cellular and organismal mechanisms for acid/base sensing in the intestinal tract are highlighted.

Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast.

CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.

Simultaneous fMRI and metabolic MRS of hyperpolarized [1-13C]pyruvate during nicotine stimulus in rat.

Functional MRI (fMRI) and MRS (fMRS) can be used to noninvasively map cerebral activation and metabolism. Recently, hyperpolarized 13C spectroscopy and metabolic imaging have provided an alternative approach to assess metabolism. In this study, we combined 1H fMRI and hyperpolarized [1-13C]pyruvate MRS to compare cerebral blood oxygenation level-dependent (BOLD) response and real-time cerebral metabolism, as assessed with lactate and bicarbonate labelling, during nicotine stimulation. Simultaneous 1H fMRI (multislice gradient echo echo-planar imaging) and 13C spectroscopic (single slice pulse-acquire) data were collected in urethane-anaesthetized female Sprague-Dawley rats (n = 12) at 9.4 T. Animals received an intravenous (i.v.) injection of either nicotine (stimulus; 88 µg/kg, n = 7, or 300 µg/kg, n = 5) or 0.9% saline (matching volume), followed by hyperpolarized [1-13C]pyruvate injection 60 s later. Three hours later, a second injection was administered: the animals that had previously received saline were injected with nicotine and vice versa, both followed by another hyperpolarized [1-13C]pyruvate i.v. injection 60 s later. The low-dose (88 µg/kg) nicotine injection led to a 12% ± 4% (n = 7, t-test, p ~ 0.0006 (t-value -5.8, degrees of freedom 6), Wilcoxon p ~ 0.0078 (test statistic 0)) increase in BOLD signal. At the same time, an increase in 13C-bicarbonate signal was seen in four out of six animals. Bicarbonate-to-total carbon ratios were 0.010 ± 0.004 and 0.018 ± 0.010 (n = 6, t-test, p ~ 0.03 (t-value -2.3, degrees of freedom 5), Wilcoxon p ~ 0.08 (test statistic 3)) for saline and nicotine experiments, respectively. No increase in the lactate signal was seen; lactate-to-total carbon was 0.16 ± 0.02 after both injections. The high (300 µg/kg) nicotine dose (n = 5) caused highly variable BOLD and metabolic responses, possibly due to the apparent respiratory distress. Simultaneous detection of 1H fMRI and hyperpolarized 13C-MRS is feasible. A comparison of metabolic response between control and stimulated states showed differences in bicarbonate signal, implying that the hyperpolarization technique could offer complimentary information on brain activation.

Secretin: a hormone for HCO3- homeostasis.

Secretin is a key hormone of the intestinal phase of digestion which activates pancreatic, bile duct and Brunner gland HCO3- secretion. Recently, the secretin receptor (SCTR) was also found in the basolateral membrane of the beta-intercalated cell (B-IC) of the collecting duct. Experimental addition of secretin triggers a pronounced activation of urinary HCO3- excretion, which is fully dependent on key functional proteins of the B-IC, namely apical pendrin and CFTR and the basolateral SCTR. Recent studies demonstrated that the SCTR knock-out mouse is unable to respond to an acute base load. Here, SCTR KO mice could not rapidly increase urine base excretion, developed prolonged metabolic alkalosis and exhibited marked compensatory hypoventilation. Here, we review the physiological effects of secretin with distinct focus on how secretin activates renal HCO3- excretion. We describe its new function as a hormone for HCO3- homeostasis.

Functional activation of pyruvate dehydrogenase in human brain using hyperpolarized [1-13 C]pyruvate.

PURPOSE: Pyruvate, produced from either glucose, glycogen, or lactate, is the dominant precursor of cerebral oxidative metabolism. Pyruvate dehydrogenase (PDH) flux is a direct measure of cerebral mitochondrial function and metabolism. Detection of [13 C]bicarbonate in the brain from hyperpolarized [1-13 C]pyruvate using carbon-13 (13 C) MRI provides a unique opportunity for assessing PDH flux in vivo. This study is to assess changes in cerebral PDH flux in response to visual stimuli using in vivo 13 C MRS with hyperpolarized [1-13 C]pyruvate. METHODS: From seven sedentary adults in good general health, time-resolved [13 C]bicarbonate production was measured in the brain using 90° flip angles with minimal perturbation of its precursors, [1-13 C]pyruvate and [1-13 C]lactate, to test the hypothesis that the appearance of [13 C]bicarbonate signals in the brain reflects the metabolic changes associated with neuronal activation. With a separate group of healthy participants (n = 3), the likelihood of the bolus-injected [1-13 C]pyruvate being converted to [1-13 C]lactate prior to decarboxylation was investigated by measuring [13 C]bicarbonate production with and without [1-13 C]lactate saturation. RESULTS: In the course of visual stimulation, the measured [13 C]bicarbonate signal normalized to the total 13 C signal in the visual cortex increased by 17.1% ± 15.9% (p = 0.017), whereas no significant change was detected in [1-13 C]lactate. Proton BOLD fMRI confirmed the regional activation in the visual cortex with the stimuli. Lactate saturation decreased bicarbonate-to-pyruvate ratio by 44.4% ± 9.3% (p < 0.01). CONCLUSION: We demonstrated the utility of 13 C MRS with hyperpolarized [1-13 C]pyruvate for assessing the activation of cerebral PDH flux via the detection of [13 C]bicarbonate production.

Dynamic T 2 * relaxometry of hyperpolarized [1-13 C]pyruvate MRI in the human brain and kidneys.

PURPOSE: This study aimed to quantify T 2 * $$ {T}_2^{\ast } $$ for hyperpolarized [1-13 C]pyruvate and metabolites in the healthy human brain and renal cell carcinoma (RCC) patients at 3 T. METHODS: Dynamic T 2 * $$ {T}_2^{\ast } $$ values were measured with a metabolite-specific multi-echo spiral sequence. The dynamic T 2 * $$ {T}_2^{\ast } $$ of [1-13 C]pyruvate, [1-13 C]lactate, and 13 C-bicarbonate was estimated in regions of interest in the whole brain, sinus vein, gray matter, and white matter in healthy volunteers, as well as in kidney tumors and the contralateral healthy kidneys in a separate group of RCC patients. T 2 * $$ {T}_2^{\ast } $$ was fit using a mono-exponential function; and metabolism was quantified using pyruvate-to-lactate conversion rate maps and lactate-to-pyruvate ratio maps, which were compared with and without an estimated T 2 * $$ {T}_2^{\ast } $$ correction. RESULTS: The T 2 * $$ {T}_2^{\ast } $$ of pyruvate was shown to vary during the acquisition, whereas the T 2 * $$ {T}_2^{\ast } $$ of lactate and bicarbonate were relatively constant through time and across the organs studied. The T 2 * $$ {T}_2^{\ast } $$ of lactate was similar in gray matter (29.75 ± 1.04 ms), white matter (32.89 ± 0.9 ms), healthy kidney (34.61 ± 4.07 ms), and kidney tumor (33.01 ± 2.31 ms); and the T 2 * $$ {T}_2^{\ast } $$ of bicarbonate was different between whole-brain (108.17 ± 14.05 ms) and healthy kidney (58.45 ± 6.63 ms). The T 2 * $$ {T}_2^{\ast } $$ of pyruvate had similar trends in both brain and RCC studies, reducing from 75.56 ± 2.23 ms to 22.24 ± 1.24 ms in the brain and reducing from 122.72 ± 9.86 ms to 57.38 ± 7.65 ms in the kidneys. CONCLUSION: Multi-echo dynamic imaging can quantify T 2 * $$ {T}_2^{\ast } $$ and metabolism in a single integrated acquisition. Clear differences were observed in the T 2 * $$ {T}_2^{\ast } $$ of metabolites and in their behavior throughout the timecourse.

Structural mechanism of Escherichia coli cyanase.

Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate-dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate-recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5-1.9 Šresolution using synchrotron X-rays and an X-ray free-electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate-binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate-binding and reaction process of cyanase.

Clinically Translatable Hyperpolarized 13C Bicarbonate pH Imaging Method for Use in Prostate Cancer.

Solid tumors such as prostate cancer (PCa) commonly develop an acidic microenvironment with pH 6.5-7.2, owing to heterogeneous perfusion, high metabolic activity, and rapid cell proliferation. In preclinical prostate cancer models, disease progression is associated with a decrease in tumor extracellular pH, suggesting that pH imaging may reflect an imaging biomarker to detect aggressive and high-risk disease. Therefore, we developed a hyperpolarized carbon-13 MRI method to image the tumor extracellular pH (pHe) and prepared it for clinical translation for detection and risk stratification of PCa. This method relies on the rapid breakdown of hyperpolarized (HP) 1,2-glycerol carbonate (carbonyl-13C) via base-catalyzed hydrolysis to produce HP 13CO32-, which is neutralized and converted to HP H13CO3-. After injection, HP H13CO3- equilibrates with HP 13CO2 in vivo and enables the imaging of pHe. Using insights gleaned from mechanistic studies performed in the hyperpolarized state, we solved issues of polarization loss during preparation in a clinical polarizer system. We successfully customized a reaction apparatus suitable for clinical application, developed clinical standard operating procedures, and validated the radiofrequency pulse sequence and imaging data acquisition with a wide range of animal models. The results demonstrated that we can routinely produce a highly polarized and safe HP H13CO3- contrast agent suitable for human injection. Preclinical imaging studies validated the reliability and accuracy of measuring acidification in healthy kidney and prostate tumor tissue. These methods were used to support an Investigational New Drug application to the U.S. Food and Drug Administration. This methodology is now ready to be implemented in human trials, with the ultimate goal of improving the management of PCa.

Does MPK4/12-HT1 function as a CO2/bicarbonate sensor to regulate the stomatal conductance under high CO2 levels?

KEY MESSAGE: Recently, a HT1 protein has been identified which causes continuous opening of stomata because of its kinase activity. However, reversible interaction between MAP4/12 and HT1 protein acts as a CO2/bicarbonate sensor and causes the closing of stomata by inhibiting HT1 kinase activity.

Substrate binding and inhibition of the anion exchanger 1 transporter.

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.

The K-Cl cotransporter-3 in the mammalian kidney.

PURPOSE OF REVIEW: We recently localized a new K-Cl cotransporters-3 (KCC3) transporter to the apical membrane of type-B intercalated cells. This gives us an opportunity to revisit the roles of the KCC3 in kidney and integrate the new findings to our current knowledge of the biology of the bicarbonate secreting cells. RECENT FINDINGS: Here, we review the basic properties of the K-Cl cotransporter with a particular attention to the responsiveness of the transporter to cell swelling. We summarize what is already known about KCC3b and discuss new information gained from our localizing of KCC3a in type-B intercalated cells. We integrate the physiology of KCC3a with the main function of the type-B cell, that is, bicarbonate secretion through the well characterized apical Cl-/HCO3- exchanger and the basolateral Na-HCO3 cotransporter. SUMMARY: Both KCC3b and KCC3a seem to be needed for maintaining cell volume during enhanced inward cotransport of Na-glucose in proximal tubule and Na-HCO3 in intercalated cells. In addition, apical KCC3a might couple to pendrin function to recycle Cl-, particularly in conditions of low salt diet and therefore low Cl- delivery to the distal tubule. This function is critical in alkalemia, and KCC3a function in the pendrin-expressing cells may contribute to the K+ loss which is observed in alkalemia.

Combined genomics to discover genes associated with tolerance to soil carbonate.

Carbonate-rich soils limit plant performance and crop production. Previously, local adaptation to carbonated soils was detected in wild Arabidopsis thaliana accessions, allowing the selection of two demes with contrasting phenotypes: A1 (carbonate tolerant, c+) and T6 (carbonate sensitive, c-). Here, A1(c+) and T6(c - ) seedlings were grown hydroponically under control (pH 5.9) and bicarbonate conditions (10 mM NaHCO3 , pH 8.3) to obtain ionomic profiles and conduct transcriptomic analysis. In parallel, A1(c+) and T6(c - ) parental lines and their progeny were cultivated on carbonated soil to evaluate fitness and segregation patterns. To understand the genetic architecture beyond the contrasted phenotypes, a bulk segregant analysis sequencing (BSA-Seq) was performed. Transcriptomics revealed 208 root and 2503 leaf differentially expressed genes in A1(c+) versus T6(c - ) comparison under bicarbonate stress, mainly involved in iron, nitrogen and carbon metabolism, hormones and glycosylates biosynthesis. Based on A1(c+) and T6(c - ) genome contrasts and BSA-Seq analysis, 69 genes were associated with carbonate tolerance. Comparative analysis of genomics and transcriptomics discovered a final set of 18 genes involved in bicarbonate stress responses that may have relevant roles in soil carbonate tolerance.

Redox basis of photosynthesis inhibition at supra-optimal bicarbonate in mesophyll protoplasts of Arabidopsis thaliana.

We examined the patterns of photosynthetic O2 evolution at 1 mM (optimal) and 10 mM (supra-optimal) bicarbonate in mesophyll protoplasts of Arabidopsis thaliana. The photosynthetic rate of protoplasts reached the maximum at an optimal concentration of 1 mM bicarbonate and got suppressed at supra-optimal levels of bicarbonate. We examined the basis of such photosynthesis inhibition by mesophyll protoplasts at supra-optimal bicarbonate. The wild-type protoplasts exposed to supra-optimal bicarbonate showed up signs of oxidative stress. Besides the wild-type, two mutants were used: nadp-mdh (deficient in chloroplastic NADP-MDH) and vtc1 (deficient in mitochondrial ascorbate biosynthesis). The protoplasts of the nadp-mdh mutant exhibited a higher photosynthetic rate and greater sensitivity to supra-optimal bicarbonate than the wild-type. The ascorbate-deficient vtc1 mutant had a low photosynthetic rate and no significant inhibition at high bicarbonate. The nadp-mdh mutants had elevated activities, protein, and transcript levels of key antioxidant enzymes. On the other hand, the antioxidant enzyme systems in vtc1 mutants were not much affected at supra-optimal bicarbonate. We propose that the inhibition of photosynthesis at supra-optimal bicarbonate depends on the redox state of mesophyll protoplasts. The robust antioxidant enzyme systems in protoplasts of nadp-mdh mutant might be priming the plants to sustain high photosynthesis at supra-optimal bicarbonate.