CaMKIIδ Met281/282 oxidation is not required for recovery of calcium transients during acidosis.

CaMKII is needed for the recovery of Ca2+ transients during acidosis but also mediates postacidic arrhythmias. CaMKIIδ can sustain its activity following Met281/282 oxidation. Increasing cytosolic Na+ during acidosis as well as postacidic pH normalization should result in prooxidant conditions within the cell favoring oxidative CaMKIIδ activation. We tested whether CaMKIIδ activation through Met281/282 oxidation is involved in recovery of Ca2+ transients during acidosis and promotes cellular arrhythmias post-acidosis. Single cardiac myocytes were isolated from a well-established mouse model in which CaMKIIδ was made resistant to oxidative activation by knock-in replacement of two oxidant-sensitive methionines (Met281/282) with valines (MM-VV). MM-VV myocytes were exposed to extracellular acidosis (pHo 6.5) and compared to wild type (WT) control cells. Full recovery of Ca2+ transients was observed in both WT and MM-VV cardiac myocytes during late-phase acidosis. This was associated with comparably enhanced sarcoplasmic reticulum Ca2+ load and preserved CaMKII specific phosphorylation of phospholamban at Thr17 in MM-VV myocytes. CaMKII was phosphorylated at Thr287, but not Met281/282 oxidized. In line with this, postacidic cellular arrhythmias occurred to a similar extent in WT and MM-VV cells, whereas inhibition of CaMKII using AIP completely prevented recovery of Ca2+ transients during acidosis and attenuated postacidic arrhythmias in MM-VV cells. Using genetically altered cardiomyocytes with cytosolic expression of redox-sensitive green fluorescent protein-2 coupled to glutaredoxin 1, we found that acidosis has a reductive effect within the cytosol of cardiac myocytes despite a significant acidosis-related increase in cytosolic Na+. Our study shows that activation of CaMKIIδ through Met281/282 oxidation is neither required for recovery of Ca2+ transients during acidosis nor relevant for postacidic arrhythmogenesis in isolated cardiac myocytes. Acidosis reduces the cytosolic glutathione redox state of isolated cardiac myocytes despite a significant increase in cytosolic Na+. Pharmacological inhibition of global CaMKII activity completely prevents recovery of Ca2+ transients and protects from postacidic arrhythmias in MM-VV myocytes, which confirms the relevance of CaMKII in the context of acidosis.NEW & NOTEWORTHY The current study shows that activation of CaMKIIδ through Met281/282 oxidation is neither required for CaMKII-dependent recovery of Ca2+ transients during acidosis nor relevant for the occurrence of postacidic cellular arrhythmias. Despite a usually prooxidant increase in cytosolic Na+, acidosis reduces the cytosolic glutathione redox state within cardiac myocytes. This novel finding suggests that oxidation of cytosolic proteins is less likely to occur during acidosis.

Developmental loss, but not pharmacological suppression, of renal carbonic anhydrase 2 results in pyelonephritis susceptibility.

Carbonic anhydrase II knockout (Car2-/-) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2-/- and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2-/- versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2-/- kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.

The role of carbonic anhydrase IX in cancer development: links to hypoxia, acidosis, and beyond.

Cancer development is a complex process that follows an intricate scenario with a dynamic interplay of selective and adaptive steps and an extensive cast of molecules and signaling pathways. Solid tumor initially grows as an avascular bulk of cells carrying oncogenic mutations until diffusion distances from the nearest functional blood vessels limit delivery of nutrients and oxygen on the one hand and removal of metabolic waste on the other one. These restrictions result in regional hypoxia and acidosis that select for adaptable tumor cells able to promote aberrant angiogenesis, remodel metabolism, acquire invasiveness and metastatic propensity, and gain therapeutic resistance. Tumor cells are thereby endowed with capability to survive and proliferate in hostile microenvironment, communicate with stroma, enter circulation, colonize secondary sites, and generate metastases. While the role of oncogenic mutations initializing and driving these processes is well established, a key contribution of non-genomic, landscaping molecular players is still less appreciated despite they can equally serve as viable targets of anticancer therapies. Carbonic anhydrase IX (CA IX) is one of these players: it is induced by hypoxia, functionally linked to acidosis, implicated in invasiveness, and correlated with therapeutic resistance. Here, we summarize the available experimental evidence supported by accumulating preclinical and clinical data that CA IX can contribute virtually to each step of cancer progression path via its enzyme activity and/or non-catalytic mechanisms. We also propose that targeting tumor cells that express CA IX may provide therapeutic benefits in various settings and combinations with both conventional and newly developed treatments.

Transgenic expression of carbonic anhydrase III in cardiac muscle demonstrates a mechanism to tolerate acidosis.

Carbonic anhydrase III (CAIII) is abundant in liver, adipocytes, and skeletal muscles, but not heart. A cytosolic enzyme that catalyzes conversions between CO2 and HCO3- in the regulation of intracellular pH, its physiological role in myocytes is not fully understood. Mouse skeletal muscles lacking CAIII showed lower intracellular pH during fatigue, suggesting its function in stress tolerance. We created transgenic mice expressing CAIII in cardiomyocytes that lack endogenous CAIII. The transgenic mice showed normal cardiac development and life span under nonstress conditions. Studies of ex vivo working hearts under normal and acidotic conditions demonstrated that the transgenic and wild-type mouse hearts had similar pumping functions under normal pH. At acidotic pH, however, CAIII transgenic mouse hearts showed significantly less decrease in cardiac function than that of wild-type control as shown by higher ventricular pressure development, systolic and diastolic velocities, and stroke volume via elongating the time of diastolic ejection. In addition to the effect of introducing CAIII into cardiomyocytes on maintaining homeostasis to counter acidotic stress, the results demonstrate the role of carbonic anhydrases in maintaining intracellular pH in muscle cells as a potential mechanism to treat heart failure.

ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl- cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

Hypoxia-related carbonic anhydrase IX expression is associated with unfavourable response to first-line therapy in classical Hodgkin's lymphoma.

AIMS: The present study evaluates the impact of hypoxia-related carbonic anhydrase IX and XII isoenzyme expression as a basic adaptive mechanism to neutralise intracellular acidosis in classical Hodgkin's lymphoma (cHL). METHODS AND RESULTS: Eighty-one primary biopsies and 15 relapsed tissue samples diagnosed with cHL were analysed for necrosis, CAIX and CAXII expression and cell proliferation to compare hypoxia-related histological and functional data with survival characteristics. Variable, but highly selective cell membrane CAIX expression could be demonstrated in Hodgkin-Reed-Sternberg (HRS) cells in 39 of 81 samples (48.1%), while virtually no staining presented in their microenvironment. In contrast, CAXII expression in HRS cells could be demonstrated in only 18 of 77 samples (23.4%), with significant stromal positivity (50 of 77, 64.9%). The CAIX+ positive phenotype was strongly associated with lymphocyte depletion (four of four, 100%) and nodular sclerosis (29 of 51, 56.9%) subtypes. CAIX/Ki-67 dual immunohistochemistry demonstrated suppressed cell proliferation in CAIX+ positive compared to CAIX- negative HRS cells (P < 0.001). Seventy-two months' progression-free survival (PFS) was significantly lower for the CAIX positive group (0.192) compared with the CAIX negative group (0.771) (P < 0.001), while the overall survival (OS) did not differ (P = 0.097). CONCLUSION: Hypoxic stress-related adaptation - highlighted by CAIX expression - results in cellular quiescence in HRS cells, potentially contributing to the short-term failure of the standard chemotherapy in cHL.

Carbonic anhydrase IX is a critical determinant of pulmonary microvascular endothelial cell pH regulation and angiogenesis during acidosis.

Carbonic anhydrase IX (CA IX) is highly expressed in rapidly proliferating and highly glycolytic cells, where it serves to enhance acid-regulatory capacity. Pulmonary microvascular endothelial cells (PMVECs) actively utilize aerobic glycolysis and acidify media, whereas pulmonary arterial endothelial cells (PAECs) primarily rely on oxidative phosphorylation and minimally change media pH. Therefore, we hypothesized that CA IX is critical to PMVEC angiogenesis because of its important role in regulating pH. To test this hypothesis, PMVECs and PAECs were isolated from Sprague-Dawley rats. CA IX knockout PMVECs were generated using the CRISPR-Cas9 technique. During serum-stimulated growth, mild acidosis (pH 6.8) did not affect cell counts of PMVECs, but it decreased PAEC cell number. Severe acidosis (pH 6.2) decreased cell counts of PMVECs and elicited an even more pronounced reduction of PAECs. PMVECs had a higher CA IX expression compared with PAECs. CA activity was higher in PMVECs compared with PAECs, and enzyme activity was dependent on the type IX isoform. Pharmacological inhibition and genetic ablation of CA IX caused profound dysregulation of extra- and intracellular pH in PMVECs. Matrigel assays revealed impaired angiogenesis of CA IX knockout PMVECs in acidosis. Lastly, pharmacological CA IX inhibition caused profound cell death in PMVECs, whereas genetic CA IX ablation had little effect on PMVEC cell death in acidosis. Thus CA IX controls PMVEC pH necessary for angiogenesis during acidosis. CA IX may contribute to lung vascular repair during acute lung injury that is accompanied by acidosis within the microenvironment.

Acidic environment activates inflammatory programs in fibroblasts via a cAMP-MAPK pathway.

The tissue micromilieu in disorders (inflammation, ischemia, tumor) often shows pronounced metabolic acidosis that may alter signaling and transcriptional activity in resident cells which can be of special importance for omnipresent fibroblasts. In the present study we investigated the impact of metabolic acidosis on rat fibroblasts with special emphasis on their role in inflammation by regulation of TNF-α, MCP-1, COX-2 and iNOS expression and the signaling pathways involved. Extracellular acidosis led to an enhanced expression of TNF-α, COX-2 and iNOS in parallel to an activation of p38 and ERK1/2 kinases that was not observed by sole intracellular acidosis. Accordingly, the protein amounts of TNF-α and COX-2 as well as the production of nitrate and nitrite were elevated. Acidosis-induced expression of COX-2 and iNOS depended on p38 kinase, but not on ERK1/2. In contrast acidosis-induced TNF-α expression was independent of both kinases. Although GPR4, GPR68 and GPR132 are expressed in fibroblasts, the involvement of these potential candidate pH sensors could be ruled out since no acidosis-induced elevation in intracellular cAMP or free calcium content was observed. Furthermore our data show that MAPK activation by an acidic micromilieu depends on Ser/Thr phosphatase activity, but not on the production of reactive oxygen species and is sensitive to cAMP antagonism by Rp-cAMPS. In conclusion, our results show that an acidic microenvironment induces a differential transcriptional program of pathological relevant genes in fibroblasts via the cAMP-phosphatase-MAPK pathway and thereby generates a parainflammatory situation that can result in tissue remodeling.

Role of CaMKII in post acidosis arrhythmias: a simulation study using a human myocyte model.

Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. However, the molecular mechanisms underlying these arrhythmias are still unclear. To better understand this process, acidosis produced by CO2 increase from 5% to 30%, resulting in intracellular pH (pHi) change from 7.15 to 6.7, was incorporated into a myocyte model of excitation-contraction coupling and contractility, including acidotic inhibition of L-type Ca(2+) channel (I(CaL)), Na(+)-Ca(2+) exchanger, Ca(2+) release through the SR ryanodine receptor (RyR2) (I(rel)), Ca(2+) reuptake by the SR Ca(2+) ATPase2a (I(up)), Na(+)-K(+) pump, K(+) efflux through the inward rectifier K(+) channel and the transient outward K(+) flow (I(to)) together with increased activity of the Na(+)-H(+) exchanger (I(NHE)). Simulated CaMKII regulation affecting I(rel), I(up), I(CaL), I(NHE) and I(to) was introduced in the model to partially compensate the acidosis outcome. Late Na(+) current increase by CaMKII was also incorporated. Using this scheme and assuming that diastolic Ca(2+) leak through the RyR2 was modulated by the resting state of this channel and the difference between SR and dyadic cleft [Ca(2+)], postacidotic delayed after depolarizations (DADs) were triggered upon returning to normal pHi after 6 min acidosis. The model showed that DADs depend on SR Ca(2+) load and on increased Ca(2+) leak through RyR2. This postacidotic arrhythmogenic pattern relies mainly on CaMKII effect on I(CaL) and I(up), since its individual elimination produced the highest DAD reduction. The model further revealed that during the return to normal pHi, DADs are fully determined by SR Ca(2+) load at the end of acidosis. Thereafter, DADs are maintained by SR Ca(2+) reloading by Ca(2+) influx through the reverse NCX mode during the time period in which [Na(+)]i is elevated.

The mechanism underlying the effects of the cell surface ATP synthase on the regulation of intracellular acidification during acidosis.

The F1F0 ATP synthase has recently become the focus of anti-cancer research. It was once thought that ATP synthases were located strictly on the inner mitochondrial membrane; however, in 1994, it was found that some ATP synthases localized to the cell surface. The cell surface ATP synthases are involved in angiogenesis, lipoprotein metabolism, innate immunity, hypertension, the regulation of food intake, and other processes. Inhibitors of this synthase have been reported to be cytotoxic and to induce intracellular acidification. However, the mechanisms by which these effects are mediated and the molecular pathways that are involved remain unclear. In this study, we aimed to determine whether the inhibition of cell proliferation and the induction of cell apoptosis that are induced by inhibitors of the cell surface ATP synthase are associated with intracellular acidification and to investigate the mechanism that underlines the effects of this inhibition, particularly in an acidic tumor environment. We demonstrated that intracellular acidification contributes to the cell proliferation inhibition that is mediated by cell surface ATP synthase inhibitors, but not to the induction of apoptosis. Intracellular acidification is only one of the mechanisms of ecto-ATP synthase-targeted antitumor drugs. We propose that intracellular acidification in combination with the inhibition of cell surface ATP generation induce cell apoptosis after cell surface ATP synthase blocked by its inhibitors. A better understanding of the mechanisms activated by ecto-ATP synthase-targeted cancer therapies may facilitate the development of potent anti-tumor therapies, which target this enzyme and do not exhibit clinical limitations.

Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10.

It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier-disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier-protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that, within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4-sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of acute respiratory distress syndrome patients.

Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase.

The Sod2 gene for Mn-superoxide dismutase (MnSOD), an intramitochondrial free radical scavenging enzyme that is the first line of defense against superoxide produced as a byproduct of oxidative phosphorylation, was inactivated by homologous recombination. Homozygous mutant mice die within the first 10 days of life with a dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, and metabolic acidosis. Cytochemical analysis revealed a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required for normal biological function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide.

Creatine kinase overexpression improves ATP kinetics and contractile function in postischemic myocardium.

Reduced myofibrillar ATP availability during prolonged myocardial ischemia may limit post-ischemic mechanical function. Because creatine kinase (CK) is the prime energy reserve reaction of the heart and because it has been difficult to augment ATP synthesis during and after ischemia, we used mice that overexpress the myofibrillar isoform of creatine kinase (CKM) in cardiac-specific, conditional fashion to test the hypothesis that CKM overexpression increases ATP delivery in ischemic-reperfused hearts and improves functional recovery. Isolated, retrograde-perfused hearts from control and CKM mice were subjected to 25 min of global, no-flow ischemia and 40 min of reperfusion while cardiac function [rate pressure product (RPP)] was monitored. A combination of (31)P-nuclear magnetic resonance experiments at 11.7T and biochemical assays was used to measure the myocardial rate of ATP synthesis via CK (CK flux) and intracellular pH (pH(i)). Baseline CK flux was severalfold higher in CKM hearts (8.1 ± 1.0 vs. 32.9 ± 3.8, mM/s, control vs. CKM; P < 0.001) with no differences in phosphocreatine concentration [PCr] and RPP. End-ischemic pH(i) was higher in CKM hearts than in control hearts (6.04 ± 0.12 vs. 6.37 ± 0.04, control vs. CKM; P < 0.05) with no differences in [PCr] and [ATP] between the two groups. Post-ischemic PCr (66.2 ± 1.3 vs. 99.1 ± 8.0, %preischemic levels; P < 0.01), CK flux (3.2 ± 0.4 vs. 14.0 ± 1.2 mM/s; P < 0.001) and functional recovery (13.7 ± 3.4 vs. 64.9 ± 13.2%preischemic RPP; P < 0.01) were significantly higher and lactate dehydrogenase release was lower in CKM than in control hearts. Thus augmenting cardiac CKM expression attenuates ischemic acidosis, reduces injury, and improves not only high-energy phosphate content and the rate of CK ATP synthesis in postischemic myocardium but also recovery of contractile function.

Pathogenesis of pseudohypoaldosteronism type 2 by WNK1 mutations.

PURPOSE OF REVIEW: Pseudohypoaldosteronism type 2 (PHA2) is a rare autosomal dominant form of human arterial hypertension, associated with hyperkalemia and hyperchloremic metabolic acidosis. WNK1 and WNK4 are two of the genes mutated in PHA2 patients. This review focuses on the mechanisms by which deletions of the first intron of WNK1 found in PHA2 patients trigger the disease. RECENT FINDINGS: The WNK1 gene gives rise to a ubiquitous kinase (L-WNK1) and to a shorter kinase-defective isoform, KS-WNK1 (for kidney-specific WNK1), expressed only in the distal convoluted tubule (DCT) and connecting tubule. WNK1 first intron deletion leads to overexpression of L-WNK1 in the DCT and ubiquitous ectopic expression of KS-WNK1. The increased expression of L-WNK1 in the DCT results in increased activity of the Na-Cl cotransporter (NCC) and thus hypervolemia and hypertension. Contrarily, the mechanisms underlying the hyperkalemia and metabolic acidosis remain unclear. SUMMARY: As particularly small doses of thiazide diuretics, inhibitors of NCC activity, correct both the blood pressure and metabolic disorders in PHA2 patients, it was believed that increased NCC was directly responsible for all PHA2 features. Studies performed in mouse models of KS-WNK1 inactivation or WNK4-related PHA2, however, have revealed that the situation is much more complex.

Rab11b and its effector Rip11 regulate the acidosis-induced traffic of V-ATPase in salivary ducts.

Redistribution of acid-base transporters is a crucial regulatory mechanism for many types of cells to cope with extracellular pH changes. In epithelial cells, however, translocation of acid-base transporters ultimately leads to changes in vectorial transport of H+ and HCO3-. We have previously shown that the bicarbonate-secreting epithelium of salivary ducts responds to changes of systemic acid-base balance by adaptive redistribution of H+ and HCO3- transporters, thereby influencing the ionic composition and buffering capacity of saliva. However, the specific proteins involved in regulated vesicular traffic of acid-base transporters are largely unknown. In the present study we have investigated the impact of Rab11 family members on the acidosis-induced trafficking of the vacuolar-type H+-ATPase (V-ATPase) in salivary duct cells in vitro using the human submandibular cell line of ductal origin HSG as an experimental model. The results show that Rab11b is expressed in salivary ducts and exhibits a significantly higher co-localization with V-ATPase than Rab11a and Rab25. We also show that Rab11 but not Rab25 interacts with the ε subunit of V-ATPase. Extracellular acidosis up-regulates Rab11b expression and protein abundance in HSG cells and causes translocation of the V-ATPase from intracellular pools toward the plasma membrane. Loss-of-function experiments using specific siRNA either against Rab11b or against its effector Rip11 prevent acidosis-induced V-ATPase translocation. These data introduce Rab11b as a crucial regulator and Rip11 as mediator of acidosis-induced V-ATPase traffic in duct cells of submandibular gland.

Heme oxygenase-1 does not mediate the effects of extracellular acidosis on vascular smooth muscle cell proliferation, migration, and susceptibility to apoptosis.

BACKGROUND: Unbalanced vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis contribute to vascular disorders such as atherosclerosis, restenosis, and pulmonary hypertension. The effect of extracellular acidosis (EA) on VSMC homeostasis is incompletely understood but we previously reported that EA increases heme oxygenase-1 (HO-1) expression in VSMCs. Since HO-1 regulates VSMC proliferation and apoptosis we sought to define the role of HO-1 in VSMC responses to EA. METHODS: Mouse aortic smooth muscle cells (MASMCs) were isolated from wild-type and HO-1-null mice. Cell proliferation and migration assays were done in a physiologic pH (7.4) or EA (pH 6.8). VSMC apoptosis in response to hydrogen peroxide was assessed by JC-1 staining, caspase-3 cleavage, annexin V, and Hoechst staining. RESULTS: Wild-type MASMCs showed decreased proliferation and migration at pH 6.8 compared to pH 7.4. This observation was also true in HO-1-null MASMCs. Although wild-type and HO-1-null cells showed differences in the mode and kinetics of cell death, both genotypes exhibited increased susceptibility to hydrogen peroxide-induced apoptosis at pH 6.8 compared to 7.4. CONCLUSIONS: EA inhibits VSMC proliferation and migration and increases susceptibility to oxidant-induced apoptosis. These effects of acidosis on VSMC homeostasis are independent of HO-1.

Acute and chronic acidosis influence on antioxidant equipment and transport proteins of rat jejunal enterocyte.

Acidosis elicits the formation of oxidants and, in turn, ROS (reactive oxygen species)-induced intestinal diseases cause acidosis. This research investigated whether both acute and chronic acidosis influence the antioxidant enzymatic equipment of rat jejunocyte, including γ-GT activity, involved in GSH (glutathione) homoeostasis. Lipid peroxidation level and the expressions of (Na+, K+)-ATPase and GLUT2 were also investigated. The possible influence of acidosis on ROS action was tested. Isolated apical membranes, everted sac preparations and homogenates from acidotic rats were used. γ-GT activity is inhibited after incubation of isolated membranes at acidic pH, but using the whole intestinal tract this inhibition disappears, while SOD (superoxide dismutase) and GR (glutathione reductase) activities are enhanced. Also, in conditions of chronic acidosis, γ-GT activity is unaffected, but no variations of antioxidant activities are apparent. (Na+, K+)-ATPase expression increases, while GLUT2 decreases in acidotic animals. Lipid peroxidation level is unaffected by acidosis. H2O2 inhibits γ-GT activity only in isolated membranes; in the whole tissue, it enhances CAT (catalase) and SOD activities and reduces GLUT2 expression. The pattern of responses to oxidant agents is unaffected by acidosis. Although jejunum seems quite resistant to acidosis, results, suggesting specific responses to this condition, may direct further research on antioxidant supplementation.

Succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) deficiency: A rare and potentially fatal metabolic disease.

Succinyl-CoA:3-oxoacid coenzyme A transferase deficiency (SCOTD) is a rare autosomal recessive disorder of ketone body utilization caused by mutations in OXCT1. We performed a systematic literature search and evaluated clinical, biochemical and genetic data on 34 previously published and 10 novel patients with SCOTD. Structural mapping and in silico analysis of protein variants is also presented. All patients presented with severe ketoacidotic episodes. Age at first symptoms ranged from 36 h to 3 years (median 7 months). About 70% of patients manifested in the first year of life, approximately one quarter already within the neonatal period. Two patients died, while the remainder (95%) were alive at the time of the report. Almost all the surviving patients (92%) showed normal psychomotor development and no neurologic abnormalities. A total of 29 missense mutations are reported. Analysis of the published crystal structure of the human SCOT enzyme, paired with both sequence-based and structure-based methods to predict variant pathogenicity, provides insight into the biochemical consequences of the reported variants. Pathogenic variants cluster in SCOT protein regions that affect certain structures of the protein. The described pathogenic variants can be viewed in an interactive map of the SCOT protein at https://michelanglo.sgc.ox.ac.uk/r/oxct. This comprehensive data analysis provides a systematic overview of all cases of SCOTD published to date. Although SCOTD is a rather benign disorder with often favourable outcome, metabolic crises can be life-threatening or even fatal. As the diagnosis can only be made by enzyme studies or mutation analyses, SCOTD may be underdiagnosed.

The renal H,K-ATPases.

PURPOSE OF REVIEW: We integrate recent evidence that demonstrates the importance of the gastric (HKalpha1) and nongastric (HKalpha2)-containing hydrogen potassium adenosine triphosphatases (H,K-ATPases) on physiological function and their role in potassium (K), sodium (Na), and acid-base balance. RECENT FINDINGS: Previous studies focused on the primary role of H,K-ATPases as a mechanism of K conservation during states of K deprivation. Both isoforms function in H secretion and K absorption in vivo during K deprivation, but recent findings show that these pumps also function in acid secretion in animals fed normal K-replete diets. The complicated pharmacological inhibition of both pumps is reviewed. Interestingly, HKalpha2-null mice have a reduced expression and activity of the renal epithelial Na channel alpha subunit in the colon. When the human nongastric isoform was studied in a heterologous expression system with its cognate beta subunit (NaKbeta1), the pump exhibited substantial Na affinity at the 'K'-binding site. Evidence cited herein raises the possibility that either directly or indirectly the renal HKalpha2-containing H,K-ATPase may affect Na balance. SUMMARY: Both H,K-ATPase isoforms are active in normal animals and not just under conditions of K depletion. The possibility that either one or both isoforms contribute to Na absorption, particularly in humans, raises important clinical implications for these pumps in the kidney.

Protein kinase B/Akt phosphorylates and inhibits the cardiac Na+/H+ exchanger NHE1.

Sarcolemmal Na(+)/H(+) exchanger (NHE) activity is mediated by NHE isoform 1 (NHE1), which is subject to regulation by protein kinases. Our objectives were to determine whether NHE1 is phosphorylated by protein kinase B (PKB), identify any pertinent phosphorylation site(s), and delineate the functional consequences of such phosphorylation. Active PKBalpha phosphorylated in vitro a glutathione S-transferase (GST)-NHE1 fusion protein comprising amino acids 516 to 815 of the NHE1 carboxyl-terminal regulatory domain. PKBalpha-mediated phosphorylation of GST-NHE1 fusion proteins containing overlapping segments of this region localized the targeted residues to the carboxyl-terminal 190 amino acids (625 to 815) of NHE1. Mass spectrometry and phosphorylation analysis of mutated (Ser-->Ala) GST-NHE1 fusion proteins revealed that PKBalpha-mediated phosphorylation of NHE1 occurred principally at Ser648. Far-Western assays demonstrated that PKBalpha-mediated Ser648 phosphorylation abrogated calcium-activated calmodulin (CaM) binding to the regulatory domain of NHE1. In adult rat ventricular myocytes, adenovirus-mediated expression of myristoylated PKBalpha (myr-PKBalpha) increased cellular PKB activity, as confirmed by increased glycogen synthase kinase 3beta phosphorylation. Heterologously expressed myr-PKBalpha was present in the sarcolemma, colocalized with NHE1 at the intercalated disc regions, increased NHE1 phosphorylation, and reduced NHE1 activity following intracellular acidosis. Conversely, pharmacological inhibition of endogenous PKB increased NHE1 activity following intracellular acidosis. Our data suggest that NHE1 is a novel PKB substrate and that its PKB-mediated phosphorylation at Ser648 inhibits sarcolemmal NHE activity during intracellular acidosis, most likely by interfering with CaM binding and reducing affinity for intracellular H(+).