The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis.

Bicarbonate (HCO3-) ions maintain pH homeostasis in eukaryotic cells and serve as a carbonyl donor to support cellular metabolism. However, whether the abundance of HCO3- is regulated or harnessed to promote cell growth is unknown. The mechanistic target of rapamycin complex 1 (mTORC1) adjusts cellular metabolism to support biomass production and cell growth. We find that mTORC1 stimulates the intracellular transport of HCO3- to promote nucleotide synthesis through the selective translational regulation of the sodium bicarbonate cotransporter SLC4A7. Downstream of mTORC1, SLC4A7 mRNA translation required the S6K-dependent phosphorylation of the translation factor eIF4B. In mTORC1-driven cells, loss of SLC4A7 resulted in reduced cell and tumor growth and decreased flux through de novo purine and pyrimidine synthesis in human cells and tumors without altering the intracellular pH. Thus, mTORC1 signaling, through the control of SLC4A7 expression, harnesses environmental bicarbonate to promote anabolic metabolism, cell biomass, and growth.

Carbon signaling protein SbtB possesses atypical redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA.

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

The electroneutral Na+-HCO3- cotransporter NBCn1 (SLC4A7) modulates colonic enterocyte pHi, proliferation, and migration.

NBCn1 (SLC4A7) is one of the two major Na+-HCO3- cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pHi), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells, NBCn1 mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pHi) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic Nbcn1-/- enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers.NEW & NOTEWORTHY The transporter NBCn1 plays a central role in maintaining homeostasis within Caco2BBe colonic epithelial cells through its regulation of intracellular pH, matrix adhesion, migration, and proliferation. These observations yield valuable insights into the molecular mechanism of the aberrant upregulation of this transporter in human colorectal cancers.

Metabolic acidosis in chronic kidney disease: mere consequence or also culprit?

Metabolic acidosis is a frequent complication in non-transplant chronic kidney disease (CKD) and after kidney transplantation. It occurs when net endogenous acid production exceeds net acid excretion. While nephron loss with reduced ammoniagenesis is the main cause of acid retention in non-transplant CKD patients, additional pathophysiological mechanisms are likely inflicted in kidney transplant recipients. Functional tubular damage by calcineurin inhibitors seems to play a key role causing renal tubular acidosis. Notably, experimental and clinical studies over the past decades have provided evidence that metabolic acidosis may not only be a consequence of CKD but also a driver of disease. In metabolic acidosis, activation of hormonal systems and the complement system resulting in fibrosis have been described. Further studies of changes in renal metabolism will likely contribute to a deeper understanding of the pathophysiology of metabolic acidosis in CKD. While alkali supplementation in case of reduced serum bicarbonate < 22 mmol/l has been endorsed by CKD guidelines for many years to slow renal functional decline, among other considerations, beneficial effects and thresholds for treatment have lately been under intense debate. This review article discusses this topic in light of the most recent results of trials assessing the efficacy of dietary and pharmacological interventions in CKD and kidney transplant patients.

Strengthening the basics: acids and bases influence vascular structure and function, tissue perfusion, blood pressure, and human cardiovascular disease.

Acids and their conjugate bases accumulate in or dissipate from the interstitial space when tissue perfusion does not match the metabolic demand. Extracellular acidosis dilates most arterial beds, but associated acid-base disturbances-e.g., intracellular acidification and decreases in HCO3- concentration-can also elicit pro-contractile influences that diminish vasodilation and even dominate in some vascular beds to cause vasoconstriction. The ensemble activities of the acid-base-sensitive reactions in vascular smooth muscle and endothelial cells optimize vascular resistance for blood pressure control and direct the perfusion towards active tissue. In this review, we describe the mechanisms of intracellular pH regulation in the vascular wall and discuss how vascular smooth muscle and endothelial cells sense acid-base disturbances. We further deliberate on the functional effects of local acid-base disturbances and their integrated cardiovascular consequences under physiological and pathophysiological conditions. Finally, we address how mutations and polymorphisms in the molecular machinery that regulates pH locally and senses acid-base disturbances in the vascular wall can result in cardiovascular disease. Based on the emerging molecular insight, we propose that targeting local pH-dependent effectors-rather than systemic acid-base disturbances-has therapeutic potential to interfere with the progression and reduce the severity of cardiovascular disease.

Cerebrospinal fluid pH regulation.

The cerebrospinal fluid (CSF) fills the brain ventricles and the subarachnoid space surrounding the brain and spinal cord. The fluid compartment of the brain ventricles communicates with the interstitial fluid of the brain across the ependyma. In comparison to blood, the CSF contains very little protein to buffer acid-base challenges. Nevertheless, the CSF responds efficiently to changes in systemic pH by mechanisms that are dependent on the CO2/HCO3- buffer system. This is evident from early studies showing that the CSF secretion is sensitive to inhibitors of acid/base transporters and carbonic anhydrase. The CSF is primarily generated by the choroid plexus, which is a well-vascularized structure arising from the pial lining of the brain ventricles. The epithelial cells of the choroid plexus host a range of acid/base transporters, many of which participate in CSF secretion and most likely contribute to the transport of acid/base equivalents into the ventricles. This review describes the current understanding of the molecular mechanisms in choroid plexus acid/base regulation and the possible role in CSF pH regulation.

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.

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.

Generalized Propensity Score Methods to Assess CKD-Associated Physiologic Factors and Risk of Kidney Failure in the Chronic Renal Insufficiency Cohort (CRIC) Study.

Epidemiologic studies have identified many biochemical risk factors for chronic kidney disease (CKD) progression that are correlates of kidney function, termed here 'CKD-associated physiologic factors'. Uncertainty remains if these factors are risk factors or risk markers accounting for aspects of kidney function not otherwise captured. We aimed to use flexible machine learning, a dynamic covariate history including kidney function informative markers, and generalized propensity score (GPS) weighting, to better control confounding for such exposures. We studied 3,052 adults with CKD in the Chronic Renal Insufficiency Cohort Study. We established a 2-year run-in period and assembled 90 variables that characterize variability and trends of selected CKD-associated physiologic factors and confounders. Using SuperLearner, we created a GPS for each CKD-associated physiologic factor and performed GPS-weighted Cox regressions. For context, we also evaluated results from traditional multivariable Cox proportional hazards models as in prior studies. Similar to traditional approaches, bicarbonate, calcium, potassium, hemoglobin, and PTH were each associated with risk of kidney failure using GPS weighting. The GPS approach detected non-linear associations in many factors, some of which were not detected with traditional models. We conclude that many associations between CKD-associated physiologic factors and kidney outcomes remain strong after GPS weighting.

Genomic investigation and clinical correlates of the in vitro ß-lactam: NaHCO3 responsiveness phenotype among methicillin-resistant Staphylococcus aureus isolates from a randomized clinical trial.

NaHCO3 responsiveness is a novel phenotype where some methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibit significantly lower minimal inhibitory concentrations (MIC) to oxacillin and/or cefazolin in the presence of NaHCO3. NaHCO3 responsiveness correlated with treatment response to ß-lactams in an endocarditis animal model. We investigated whether treatment of NaHCO3-responsive strains with ß-lactams was associated with faster clearance of bacteremia. The CAMERA2 trial (Combination Antibiotics for Methicillin-Resistant Staphylococcus aureus) randomly assigned participants with MRSA bloodstream infections to standard therapy, or to standard therapy plus an anti-staphylococcal ß-lactam (combination therapy). For 117 CAMERA2 MRSA isolates, we determined by broth microdilution the MIC of cefazolin and oxacillin, with and without 44 mM of NaHCO3. Isolates exhibiting ≥4-fold decrease in the MIC to cefazolin or oxacillin in the presence of NaHCO3 were considered "NaHCO3-responsive" to that agent. We compared the rate of persistent bacteremia among participants who had infections caused by NaHCO3-responsive and non-responsive strains, and that were assigned to combination treatment with a ß-lactam. Thirty-one percent (36/117) and 25% (21/85) of MRSA isolates were NaHCO3-responsive to cefazolin and oxacillin, respectively. The NaHCO3-responsive phenotype was significantly associated with sequence type 93, SCCmec type IVa, and mecA alleles with substitutions in positions -7 and -38 in the regulatory region. Among participants treated with a ß-lactam, there was no association between the NaHCO3-responsive phenotype and persistent bacteremia (cefazolin, P = 0.82; oxacillin, P = 0.81). In patients from a randomized clinical trial with MRSA bloodstream infection, isolates with an in vitro ß-lactam-NaHCO3-responsive phenotype were associated with distinctive genetic signatures, but not with a shorter duration of bacteremia among those treated with a ß-lactam.

Role of pH Regulatory Proteins and Dysregulation of pH in Prostate Cancer.

Prostate cancer is the fourth most commonly diagnosed cancer, and although it is often a slow-growing malignancy, it is the second leading cause of cancer-associated deaths in men and the first in Europe and North America. In many forms of cancer, when the disease is a solid tumor confined to one organ, it is often readily treated. However, when the cancer becomes an invasive metastatic carcinoma, it is more often fatal. It is therefore of great interest to identify mechanisms that contribute to the invasion of cells to identify possible targets for therapy. During prostate cancer progression, the epithelial cells undergo epithelial-mesenchymal transition that is characterized by morphological changes, a loss of cell-cell adhesion, and invasiveness. Dysregulation of pH has emerged as a hallmark of cancer with a reversed pH gradient and with a constitutively increased intracellular pH that is elevated above the extracellular pH. This phenomenon has been referred to as "a perfect storm" for cancer progression. Acid-extruding ion transporters include the Na+/H+ exchanger NHE1 (SLC9A1), the Na+HCO3- cotransporter NBCn1 (SLC4A7), anion exchangers, vacuolar-type adenosine triphosphatases, and the lactate-H+ cotransporters of the monocarboxylate family (MCT1 and MCT4 (SLC16A1 and 3)). Additionally, carbonic anhydrases contribute to acid transport. Of these, several have been shown to be upregulated in different human cancers including the NBCn1, MCTs, and NHE1. Here the role and contribution of acid-extruding transporters in prostate cancer growth and metastasis were examined. These proteins make significant contributions to prostate cancer progression.

Ion Channels, Transporters, and Sensors Interact with the Acidic Tumor Microenvironment to Modify Cancer Progression.

Solid tumors, including breast carcinomas, are heterogeneous but typically characterized by elevated cellular turnover and metabolism, diffusion limitations based on the complex tumor architecture, and abnormal intra- and extracellular ion compositions particularly as regards acid-base equivalents. Carcinogenesis-related alterations in expression and function of ion channels and transporters, cellular energy levels, and organellar H+ sequestration further modify the acid-base composition within tumors and influence cancer cell functions, including cell proliferation, migration, and survival. Cancer cells defend their cytosolic pH and HCO3- concentrations better than normal cells when challenged with the marked deviations in extracellular H+, HCO3-, and lactate concentrations typical of the tumor microenvironment. Ionic gradients determine the driving forces for ion transporters and channels and influence the membrane potential. Cancer and stromal cells also sense abnormal ion concentrations via intra- and extracellular receptors that modify cancer progression and prognosis. With emphasis on breast cancer, the current review first addresses the altered ion composition and the changes in expression and functional activity of ion channels and transporters in solid cancer tissue. It then discusses how ion channels, transporters, and cellular sensors under influence of the acidic tumor microenvironment shape cancer development and progression and affect the potential of cancer therapies.

Optimizing wheat productivity through integrated management of irrigation, nutrition, and organic amendments.

Enhancing wheat productivity by implementing a comprehensive approach that combines irrigation, nutrition, and organic amendments shows potential for collectively enhancing crop performance. This study examined the individual and combined effects of using irrigation systems (IS), foliar potassium bicarbonate (PBR) application, and compost application methods (CM) on nine traits related to the growth, physiology, and yield of the Giza-171 wheat cultivar. Analysis of variance revealed significant (P ≤ 0.05) main effects of IS, PBR, and CM on wheat growth, physiology, and yield traits over the two growing seasons of the study. Drip irrigation resulted in a 16% increase in plant height, leaf area index, crop growth rate, yield components, and grain yield compared to spray irrigation. Additionally, the application of foliar PBR at a concentration of 0.08 g/L boosted these parameters by up to 22% compared to the control. Furthermore, the application of compost using the role method resulted in enhanced wheat performance compared to the treatment including mix application. Importantly, the combined analysis revealed that the three-way interaction between the three factors had a significant effect (P ≤ 0.05) on all the studied traits, with drip irrigation at 0.08 g PBR rate and role compost application method (referred as Drip_0.08g_Role) resulting in the best performance across all traits, while sprinkle irrigation without PBR and conventional mixed compost method (referred as sprinkle_CK_Mix) produced the poorest results. This highlights the potential to synergistically improve wheat performance through optimized agronomic inputs.

The immunomodulatory effect of oral NaHCO3 is mediated by the splenic nerve: multivariate impact revealed by artificial neural networks.

Stimulation of the inflammatory reflex (IR) is a promising strategy for treating systemic inflammatory disorders. Recent studies suggest oral sodium bicarbonate (NaHCO3) as a potential activator of the IR, offering a safe and cost-effective treatment approach. However, the mechanisms underlying NaHCO3-induced anti-inflammatory effects remain unclear. We investigated whether oral NaHCO3's immunomodulatory effects are mediated by the splenic nerve. Female rats received NaHCO3 or water (H2O) for four days, and splenic immune markers were assessed using flow cytometry. NaHCO3 led to a significant increase (p < 0.05, and/or partial eta squared > 0.06) in anti-inflammatory markers, including CD11bc + CD206 + (M2-like) macrophages, CD3 + CD4 + FoxP3 + cells (Tregs), and Tregs/M1-like ratio. Conversely, proinflammatory markers, such as CD11bc + CD38 + TNFα + (M1-like) macrophages, M1-like/M2-like ratio, and SSChigh/SSClow ratio of FSChighCD11bc + cells, decreased in the spleen following NaHCO3 administration. These effects were abolished in spleen-denervated rats, suggesting the necessity of the splenic nerve in mediating NaHCO3-induced immunomodulation. Artificial neural networks accurately classified NaHCO3 and H2O treatment in sham rats but failed in spleen-denervated rats, highlighting the splenic nerve's critical role. Additionally, spleen denervation independently influenced Tregs, M2-like macrophages, Tregs/M1-like ratio, and CD11bc + CD38 + cells, indicating distinct effects from both surgery and treatment. Principal component analysis (PCA) further supported the separate effects. Our findings suggest that the splenic nerve transmits oral NaHCO3-induced immunomodulatory changes to the spleen, emphasizing NaHCO3's potential as an IR activator with therapeutic implications for a wide spectrum of systemic inflammatory conditions.

Measuring beta-lactam minimum inhibitory concentrations in Staphylococcus aureus in the clinical microbiology laboratory: pinning the tail on the donkey.

Significant shortcomings have been identified in standard methods of susceptibility testing in bacteriological media, not only because the media fails to recapitulate the in vivo environment, but susceptibility testing itself fails to capture sub-MIC effects that significantly attenuate bacterial virulence properties. Until susceptibility testing conditions better recapitulate the in vivo environment, attempts to establish the quantitative relevance of beta-lactam MIC using current clinical microbiology standards in Staphylococcus aureus infections will likely prove unsuccessful.

Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

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.

Biallelic variants in SLC4A10 encoding a sodium-dependent bicarbonate transporter lead to a neurodevelopmental disorder.

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.

Bicarbonate is a key regulator but not a substrate for O2 evolution in Photosystem II.

Photosystem II (PSII) uses light energy to oxidize water and to reduce plastoquinone in the photosynthetic electron transport chain. O2 is produced as a byproduct. While most members of the PSII research community agree that O2 originates from water molecules, alternative hypotheses involving bicarbonate persist in the literature. In this perspective, we provide an overview of the important roles of bicarbonate in regulating PSII activity and assembly. Further, we emphasize that biochemistry, spectroscopy, and structural biology experiments have all failed to detect bicarbonate near the active site of O2 evolution. While thermodynamic arguments for oxygen-centered bicarbonate oxidation are valid, the claim that bicarbonate is a substrate for photosynthetic O2 evolution is challenged.