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.

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.

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.

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.

p38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil.

Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal-regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.

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.

Stressor-driven extracellular acidosis as tumor inducer via aberrant enzyme activation: A review on the mechanisms and possible prophylaxis.

When the extracellular pH of human body vacillates in either direction, tissue homeostasis is compromised. Fluctuations in acidity have been linked to a wide variety of pathological conditions, including bone loss, cancer, allergies, and auto-immune diseases. Stress conditions affect oxygen tension, and the resultant hypoxia modulates the expression and/or activity of membrane-tethered transporters/pumps, transcription factors, enzymes and intercellular junctions. These modifications provoke erratic gene expression, aberrant tissue remodeling and oncogenesis. While the physiological optimization of pH in tissues is practically challenging, it is at least theoretically achievable and can be considered as a possible therapy to resolve a broad array of diseases.

Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis.

Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl- N'-nitro- N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl- N'-nitro- N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl- N'-nitro- N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

Neonatal multiorgan failure due to ACAD9 mutation and complex I deficiency with mitochondrial hyperplasia in liver, cardiac myocytes, skeletal muscle, and renal tubules.

Complex I deficiency causes Leigh syndrome, fatal infant lactic acidosis, and neonatal cardiomyopathy. Mutations in more than 100 nuclear DNA and mitochondrial DNA genes miscode for complex I subunits or assembly factors. ACAD9 is an acyl-CoA dehydrogenase with a novel function in assembly of complex I; biallelic mutations cause progressive encephalomyopathy, recurrent Reye syndrome, and fatal cardiomyopathy. We describe the first autopsy in fatal neonatal lethal lactic acidosis due to mutations in ACAD9 that reduced complex I activity. We identified mitochondrial hyperplasia in cardiac myocytes, diaphragm muscle, and liver and renal tubules in formalin-fixed, paraffin-embedded tissue using immunohistochemistry for mitochondrial antigens. Whole-exome sequencing revealed compound heterozygous variants in the ACAD9 gene: c.187G>T (p.E63*) and c.941T>C (p.L314P). The nonsense mutation causes late infantile lethality; the missense variant is novel. Autopsy-derived fibroblasts had reduced complex I activity (53% of control) with normal activity in complexes II to IV, similar to reported cases of ACAD9 deficiency.

Induction of Phosphoenolpyruvate Carboxykinase (PEPCK) during Acute Acidosis and Its Role in Acid Secretion by V-ATPase-Expressing Ionocytes.

Vacuolar-Type H(+)-ATPase (V-ATPase) takes the central role in pumping H(+) through cell membranes of diverse organisms, which is essential for surviving acid-base fluctuating lifestyles or environments. In mammals, although glucose is believed to be an important energy source to drive V-ATPase, and phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for gluconeogenesis, is known to be activated in response to acidosis, the link between acid secretion and PEPCK activation remains unclear. In the present study, we used zebrafish larva as an in vivo model to show the role of acid-inducible PEPCK activity in glucose production to support higher rate of H(+) secretion via V-ATPase, by utilizing gene knockdown, glucose supplementation, and non-invasive scanning ion-selective electrode technique (SIET). Zebrafish larvae increased V-ATPase-mediated acid secretion and transiently expression of Pck1, a zebrafish homolog of PEPCK, in response to acid stress. When pck1 gene was knocked down by specific morpholino, the H(+) secretion via V-ATPase decreased, but this effect was rescued by supplementation of glucose into the yolk. By assessing changes in amino acid content and gene expression of respective enzymes, glutamine and glutamate appeared to be the major source for replenishment of Krebs cycle intermediates, which are subtracted by Pck1 activity. Unexpectedly, pck1 knockdown did not affect glutamine/glutamate catalysis, which implies that Pck1 does not necessarily drive this process. The present study provides the first in vivo evidence that acid-induced PEPCK provides glucose for acid-base homeostasis at an individual level, which is supported by rapid pumping of H(+) via V-ATPase at the cellular level.

NADPH oxidase-2: linking glucose, acidosis, and excitotoxicity in stroke.

SIGNIFICANCE: Neuronal superoxide production contributes to cell death in both glutamate excitotoxicity and brain ischemia (stroke). NADPH oxidase-2 (NOX2) is the major source of neuronal superoxide production in these settings, and regulation of NOX2 activity can thereby influence outcome in stroke. RECENT ADVANCES: Reduced NOX2 activity can rescue cells from oxidative stress and cell death that otherwise occur in excitotoxicity and ischemia. NOX2 activity is regulated by several factors previously shown to affect outcome in stroke, including glucose availability, intracellular pH, protein kinase ζ/δ, casein kinase 2, phosphoinositide-3-kinase, Rac1/2, and phospholipase A2. The newly identified functions of these factors as regulators of NOX2 activity suggest alternative mechanisms for their effects on ischemic brain injury. CRITICAL ISSUES: Key aspects of these regulatory influences remain unresolved, including the mechanisms by which rac1 and phospholipase activities are coupled to N-methyl-D-aspartate (NMDA) receptors, and whether superoxide production by NOX2 triggers subsequent superoxide production by mitochondria. FUTURE DIRECTIONS: It will be important to establish whether interventions targeting the signaling pathways linking NMDA receptors to NOX2 in brain ischemia can provide a greater neuroprotective efficacy or a longer time window to treatment than provided by NMDA receptor blockade alone. It will likewise be important to determine whether dissociating superoxide production from the other signaling events initiated by NMDA receptors can mitigate the deleterious effects of NMDA receptor blockade.

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.

Transient acidosis during early reperfusion attenuates myocardium ischemia reperfusion injury via PI3k-Akt-eNOS signaling pathway.

In this paper, we concluded that transient acidosis reperfusion conferred cardioprotection against myocardial ischemia reperfusion injury in isolated rat hearts through activating PI3K-Akt-eNOS pathway.

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.

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.

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.