Local Acidosis Research Articles

Structures of Human Proton-Activated Chloride Channel (PAC) Reveal Mechanism of pH Sensing and Gating

Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. A family of chloride channels activated by extracellular acidic pH has been implicated in acidosis-induced cell death by mediating Clˉ influx and subsequent cell swelling. PAC currents are widespread and display characteristic biophysical properties, including strong outwardly rectifying current-voltage (I-V) relationship, time-dependent activation at positive membrane potentials, and anion permeability sequence of Iˉ > Brˉ > Clˉ. The molecular identity of PAC was unknown until we recently identified a novel gene PACC1 (TMEM206) encoding the PAC channel. PAC shares no sequence homology to other membrane proteins, representing a completely new ion channel family. By performing cryo-EM and electrophysiology, we present two structures of human PAC, a high-pH (pH 8) resting closed state and a low-pH (pH 4) proton-bound non-conducting state, and reveal critical functional residues involved in pH-dependent activation and ion selectivity. We show a unique desensitization of PAC upon prolonged treatment at pH 4. The trimeric PAC contains a transmembrane domain (TMD) and an extracellular domain (ECD). We observed conformational change in the ECD-TMD interface and the TMD between the pH 8 and pH 4 structures. A titratable residue H98 from the ECD-TMD interface is involved in PAC pH sensing. The selectivity filter of PAC is located in the intracellular side of TM2, in which a charge reversal mutant (K319E) converts PAC to a cation-selective channel. Our data provide the structural basis to understand the proton-dependent gating mechanisms of PAC channel.

Clinical Value of 3D-Printed Navigation Technology Combined with Neuroendoscopy for Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is the most common form of hemorrhagic stroke with high morbidity and mortality. Rapid and massive bleeding may compress the brain tissue, causing space-occupying and pathological effects, such as reduced local cerebral blood flow, acidosis, and inflammatory and immune responses. Although the development of minimally invasive technique provides a new option for the treatment of ICH, their application is limited due to the difficulty in achieving accurate puncture localization under the guidance of the marks on CT. We selected 30 patients treated with neuroendoscopic surgery guided by 3D-printed navigation technology (experimental group) and 30 patients treated with neuroendoscopic surgery guided by hand-painted on the patient's body surface according to the marks on CT (control group). Our results showed that patients in the experimental group had a lower number of intraoperative punctures, shorter operation time, less intraoperative blood loss, higher hematoma clearance rate, and smaller volume of perihematomal edema than the patients in the control group. Moreover, patients in the experimental group had higher Glasgow Coma Scale score at discharge, shorter postoperative hospitalization time and ICU stay, and a lower rate of postoperative complications, despite the lack of statistically significant differences. In addition, no statistically significant differences were observed in mortality and Glasgow Outcome Scale score between the two groups. In conclusion, 3D-printed navigation technology used for the neuroendoscopic hematoma removal is a more reliable and less invasive approach in the treatment of ICH. This technique has great application prospects and deserves promotion in the future clinical practice.

Acidic microenvironment induction of interleukin-8 expression and matrix metalloproteinase-2/-9 activation via acid-sensing ion channel 1 promotes breast cancer cell progression.

The cancer microenvironment exhibits local acidosis compared with the surrounding normal tissue. Many reports have shown that acidosis accelerates the invasiveness and metastasis of cancer, yet the underlying molecular mechanisms remain unclear. In the present study, we focused on acid-induced functional changes through acid receptors in breast cancer cells. Acidic treatment induced interleukin (IL)-8 expression in MDA-MB-231 cells and promoted cell migration and invasion. The acidic microenvironment elevated matrix metalloproteinase (MMP)-2 and MMP-9 activity, and addition of IL-8 had similar effects. However, inhibition of IL-8 suppressed the acid-induced migration and invasion of MDA-MB-231 cells. MDA-MB-231 cells express various acid receptors including ion channels and G protein-coupled receptors. Interestingly, acidic stimulation increased the expression of acid-sensing ion channel 1 (ASIC1), and acid-induced IL-8 was significantly decreased by ASIC1 knockdown. Moreover, phosphorylation of nuclear factor (NF)-κB was induced by acidic treatment, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that IL-8 induction by an acidic microenvironment promotes breast cancer development and that ASIC1 might be a novel therapeutic target for breast cancer metastasis.

Invasive Assessment of Hemodynamic, Metabolic and Ionic Consequences During Blood Flow Restriction Training.

Purpose: Medically recommended training often faces the dilemma that necessary mechanical intensities for muscle adaptations exceed patients' physical capacity. In this regard, blood flow restriction (BFR) training is becoming increasingly popular because it enables gains in muscle mass and strength despite using low-mechanical loads combined with external venous occlusion. Since the underlying mechanisms are still unknown, we applied invasive measurements during exercise with and without BFR to promote physiological understanding and safety of this popular training technique.Methods: In a randomized cross-over design, ten healthy men (28.1 ± 6.5 years) underwent two trials of unilateral biceps curls either with (BFR) and without BFR (CON). For analysis of changes in intravascular pressures, blood gases, oximetry and electrolytes, an arterial and a venous catheter were placed at the exercising arm before exercise. Arterial and venous blood gases and intravascular pressures were analyzed before, during and 5 min after exercise.Results: Intravascular pressures in the arterial and venous system were more increased during exercise with BFR compared to CON (p < 0.001). Furthermore, arterial and venous blood gas analyses revealed a BFR-induced metabolic acidosis (p < 0.05) with increased lactate production (p < 0.05) and associated elevations in [K+], [Ca2+] and [Na+] (p < 0.001).Conclusion: The present study describes for the first time the local physiological changes during BFR training. While BFR causes greater hypertension in the arterial and venous system of the exercising extremity, observed electrolyte shifts corroborate a local metabolic acidosis with concurrent rises in [K+] and [Na+]. Although BFR could be a promising new training concept for medical application, its execution is associated with comprehensive physiological challenges.

Transplanted Kidney Increases Nitric Oxide Formation With Metabolic Acidosis Reduction.

As a vasodilator, nitric oxide is considered to play a significant role in the homeostatic regulation of renal hemodynamics. To test the hypothesis that a kidney graft is capable of producing nitric oxide immediately after renal transplant surgery, we examined the possibility that it positively affects local metabolic acidosis. In kidney transplant recipients, we analyzed renal vein and central vein blood samples, which reflect local and systemic metabolic alterations, respectively. Samples were taken immediately after kidney recirculation (that is, the first blood passing through after clamps are released) and at 5, 15, and 30 minutes thereafter. Levels of nitric oxide metabolites (nitrites, nitrates, and their sum), malondialdehyde (an indicator of oxidative damages), and parameters of acid-base balance (pH level, actual excess base, hemoglobin, actual bicarbonate, partial pressure of carbon dioxide, partial pressure of oxygen) were analyzed. Living kidney donors (the recipients' parents) were controls. In renal vein samples, nitrates and the sum of nitrites and nitrates were significantly higher than that shown in control (P < .001) and central vein (P < .05) samples, suggesting an immediate increase in nitric oxide production in the transplanted organ. Metabolic acidosis occurred in both the renal and central vein, indicated by decreased pH and actual bicarbonate level as well as by negative actual base excess level. Only in the renal vein was an increased nitrite and nitrate associated with a reduction of negative actual excess base, thereby suggesting a decrease in anion formation. Transplanted kidneys increase nitric oxide production immediately after organ transplant surgery, which positively affects local metabolic acidosis. The mechanism for this effect is likely local circulation improvement.

Effects Of Different Intensity And Duration Of Warm-up On Hemodynamics, Jump Power, And Flexibility.

PURPOSE: Tabata protocol (TP), usually consisting of eight to nine bouts of 20-sec of maximal exercise with 10-sec rest, is time-efficient intervention with both aerobic and anaerobic benefits. This study investigated the effectiveness of different variations of TP as a warm-up procedure. METHODS: Twenty-five healthy subjects (13 females and 12 males) participated in this study. Participants performed 6 randomized exercise sessions separated by at least 48 hours. The exercise sessions involved 3-min (TP3-20:10; TP3-30:10), 5-min (TP5-20:10; TP5-30:10) or 8-min (TP8-20:10; TP8-30:10) consecutive bodyweight squats of either 20-sec workout with 10-sec rest (20:10) or 30-sec workout with 10-sec rest (30:10). Heart rate (HR), blood pressure (BP), thigh skin surface temperature (TT), vertical jump performance (VJ), and flexibility (F) were measured before and after execution of the protocols. Countermovement jump was used to measure VJ and sit-and-reach test was used for measuring F. RESULTS: Two-way ANOVA demonstrated significant condition∗time interaction (p<0.01) and time main effect (p<0.01) for F. Significant condition∗time interaction (p<0.01) and condition (p<0.01) and time main effects (p<0.01) were observed for HR. There were significant main effects for time with the post-test demonstrating higher values than the pre-test values for both SBP and DBP (p<0.01). Significant time main effect (p<0.01) was also noted for TT indicating reduction in TT following exercise bouts. CONCLUSIONS: The findings are suggestive of a decrease in F following a higher duration of exercise (TP8-20:10 and TP8-30:10). This may be ascribed to greater accumulation of metabolites (lactic acid, ammonia, and hydrogen ion) in the working muscles, which may alter Type III and IV afferent neural activity to increase pain perception. Local tissue acidosis also stimulates bradykinin release, which may contribute to the transmission of nociceptive signals from skeletal muscle. Additionally, higher duration of exercise may increase cortisol level that decreases the pain threshold level. Therefore, the decreases in flexibility may be explained by one or a combination of metabolic, hormonal, and neurobiological changes stimulating the brain to inhibit the muscular response.

Injectable and Self-Healing Nanocomposite Hydrogels with Ultrasensitive pH-Responsiveness and Tunable Mechanical Properties: Implications for Controlled Drug Delivery.

Injectable, self-healing, and pH-responsive hydrogels are great intelligent drug delivery systems for controlled and localized therapeutic release. Hydrogels that show pH-sensitive behaviors in the mildly acidic range are ideal to be used for the treatment of regions showing local acidosis like tumors, wounds and infections. In this work, we present a facile preparation of an injectable, self-healing, and supersensitive pH-responsive nanocomposite hydrogel based on Schiff base reactions between aldehyde-functionalized polymers and amine-modified silica nanoparticles. The hydrogel shows fast gelation within 10 s, injectability, and rapid self-healing capability. Moreover, the hydrogel demonstrates excellent stability under neutral physiological conditions, while a sharp gel-sol transition is observed, induced by a faintly acidic environment, which is desirable for controlled drug delivery. The pH-responsiveness of the hydrogel is ultrasensitive, where the mechanical properties, hydrolytic degradation, and drug release behaviors can alter significantly when subjected to a slight pH change of 0.2. Additionally, the hydrogel's mechanical and pH-responsive properties can be readily tuned by its composition. Its excellent biocompatibility is confirmed by cytotoxicity tests toward human dermal fibroblast cells (HDFa). The novel injectable, self-healing, and sensitive pH-responsive hydrogel serves as a promising candidate as a localized drug carrier with controlled delivery capability, triggered by acidosis, holding great promise for cancer therapy, wound healing, and infection treatment.

Mechanisms underlying the stimulatory effect of inhaled sulfur dioxide on vagal bronchopulmonary C-fibres.

Brief inhalation of SO2 of concentration >500p.p.m. triggered a pronounced stimulatory effect on vagal bronchopulmonary C-fibres in anaesthetized rats. This stimulatory effect was drastically diminished by a pretreatment with NaHCO3 that raised the baseline arterial pH, suggesting a possible involvement of acidification of airway fluid and/or tissue generated by inhaled SO2 . The stimulation was completely abolished by pretreatment with antagonists of both acid-sensing ion channels and transient receptor potential vanilloid type-1 receptors, indicating that this effect was caused by acid activation of these cation channels expressed in airway sensory nerves. This conclusion was further supported by the results obtained from studies in isolated rat vagal bronchopulmonary sensory neurones and also in the cough response to SO2 inhalation challenge in awake mice. These results provide new insight into the underlying mechanism of harmful irritant effects in the respiratory tract caused by accidental exposure to a high concentration of SO2 . Inhalation of sulfur dioxide (SO2 ) triggers coughs and reflex bronchoconstriction, and stimulation of vagal bronchopulmonary C-fibres is primarily responsible. However, the mechanism underlying this stimulatory effect is not yet fully understood. In this study, we tested the hypothesis that the C-fibre stimulation was caused by SO2 -induced local tissue acidosis in the lung and airways. Single-unit activities of bronchopulmonary C-fibres in response to inhalation challenges of SO2 (500-1500p.p.m., 10 breaths) were measured in anaesthetized rats. Inhalation of SO2 reproducibly induced a pronounced and sustained stimulation (lasting for 15-60s) of pulmonary C-fibres in a concentration-dependent manner. This stimulatory effect was significantly attenuated by an increase in arterial pH generated by infusion of sodium bicarbonate (NaHCO3 ), and completely abrogated by a combined pretreatment with amiloride (an antagonist of acid-sensing ion channels, ASICs) and AMG8910 (a selective antagonist of the transient receptor potential vanilloid type-1 receptor, TRPV1). Furthermore, in isolated rat vagal pulmonary sensory neurones, perfusion of an aqueous solution of SO2 evoked a transient increase in the intracellular Ca2+ concentration; this response was also markedly diminished by a pretreatment with amiloride and AMG8910. In addition, inhalation of SO2 consistently evoked coughs in awake mice; responses were significantly smaller in TRPV1-/- mice than in wild-type mice, and almost completely abolished after a pretreatment with amiloride in TRPV1-/- mice. These results suggested that the stimulatory effect of inhaled SO2 on bronchopulmonary C-fibres was generated by acidification of fluid and/or tissue in the lung and airways, which activated both ASICs and TRPV1 expressed in these sensory nerves.

The N-Terminal Region of a Ph-Responsive Peptide Controls Its Interaction with Phosphatidylserine-Containing Bilayers

Local acidosis is common in solid tumors, and provides a promising target for peptide-based anticancer treatments. To exploit this characteristic, the acidity-triggered rational membrane (ATRAM) peptide was designed to insert selectively into lipid bilayers when the surrounding media is acidic. Insertion of ATRAM into the bilayer as a transmembrane α-helix involves translocation of the C-terminus across the membrane, such that the N-terminus is free to interact with lipids on the outer leaflet. Since many cancers overexpose on the outer membrane leaflet the negatively charged lipid phosphatidylserine (PS), we propose that mutations at the non-inserting N-terminus would modify ATRAM's interactions with model cancer cell membranes. Here, we used two ATRAM variants, termed K2-ATRAM (L2K/L5K) and Y-ATRAM (L2Y), to explore the effects of positively charged or mildly hydrophobic amino acids near the N-terminus. The presence of PS in the membrane enhanced membrane partitioning for K2-ATRAM but decreased it for Y-ATRAM. However, screening with high-salt conditions showed that the effects of PS on the pK of insertion are multifactorial and cannot explained by electrostatics alone. These results suggest that studies using only neutral bilayers may fail to indicate the true interactions of anticancer peptides with tumor cells, which contain negatively charged phospholipids. Additionally, the different behaviors of the ATRAM variants demonstrate how the non-inserting N-terminal region can be tailored to adjust the specificity of ATRAM for target membranes. This information will aid the design of new ATRAMs and other peptides that are tailored to the pH and lipid compositions of a variety of diseases.

Acid-Sensing Ion Channels: Novel Mediators of Cerebral Vascular Responses.

Precise regulation of cerebral blood flow is critical for normal brain function. Insufficient cerebral blood flow contributes to brain dysfunction and neurodegeneration. Carbon dioxide (CO2), via effects on local acidosis, is one of the most potent regulators of cerebral blood flow. Although a role for nitric oxide in intermediate signaling has been implicated, mechanisms that initiate CO2-induced vasodilation remain unclear. Acid-sensing ion channel-1A (ASIC1A) is a proton-gated cation channel that is activated by extracellular acidosis. Based on work that implicated ASIC1A in the amygdala and bed nucleus of the stria terminalis in CO2-evoked and acid-evoked behaviors, we hypothesized that ASIC1A might also mediate microvascular responses to CO2. To test this hypothesis, we genetically and pharmacologically manipulated ASIC1A and assessed effects on CO2-induced dilation of cerebral arterioles in vivo. Effects of inhalation of 5% or 10% CO2 on arteriolar diameter were greatly attenuated in mice with global deficiency in ASIC1A (Asic1a-/-) or by local treatment with the ASIC inhibitor, psalmotoxin. Vasodilator effects of acetylcholine, which acts via endothelial nitric oxide synthase were unaffected, suggesting a nonvascular source of nitric oxide may be key for CO2 responses. Thus, we tested whether neurons may be the cell type through which ASIC1A influences microvessels. Using mice in which Asic1a was specifically disrupted in neurons, we found effects of CO2 on arteriolar diameter were also attenuated. Together, these data are consistent with a model wherein activation of ASIC1A, particularly in neurons, is critical for CO2-induced nitric oxide production and vasodilation. With these findings, ASIC1A emerges as major regulator of microvascular tone.

PAC, an evolutionarily conserved membrane protein, is a proton-activated chloride channel.

Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. However, the molecular mechanisms of the cellular response to acid are not fully understood. We performed an unbiased RNA interference screen and identified PAC (TMEM206) as being essential for the widely observed proton-activated Cl- (PAC) currents (I Cl,H). Overexpression of human PAC in PAC knockout cells generated I Cl,H with the same characteristics as the endogenous ones. Zebrafish PAC encodes a PAC channel with distinct properties. Knockout of mouse Pac abolished I Cl,H in neurons and attenuated brain damage after ischemic stroke. The wide expression of PAC suggests a broad role for this conserved Cl- channel family in physiological and pathological processes associated with acidic pH.

The modulation of acid-sensing ion channel 1 by PcTx1 is pH-, subtype- and species-dependent: Importance of interactions at the channel subunit interface and potential for engineering selective analogues

Acid-sensing ion channels (ASICs) are primary acid sensors in the mammalian nervous system that are activated by protons under conditions of local acidosis. They have been implicated in a range of pathologies including ischemic stroke (ASIC1a subtype) and peripheral pain (ASIC1b and ASIC3). Although the spider venom peptide PcTx1 is the best-studied ASIC modulator and is neuroprotective in rodent models of ischemic stroke, little experimental work has been done to examine its molecular interaction with human ASIC1a or the off-target ASIC1b. The complementary face of the acidic pocket binding site of PcTx1 is where these channels differ in sequence. We show here that although PcTx1 is 10-fold less potent at human ASIC1a than the rat channel, the apparent affinity for the two channels is comparable. We examined the pharmacophore of PcTx1 for human ASIC1a and rat ASIC1b, and show that inhibitory and stimulatory effects at each ASIC1 variant is driven mostly by a shared set of core peptide pharmacophore residues that bind to the thumb domain, while peptide residues that interact with the complementary face of the biding site underlie species and subtype-dependent differences in activity that may allow manipulation of ASIC1 variant selectivity. Finally, the stimulatory effect of PcTx1 on rat ASIC1a when applied under mildly alkaline pH correlates with low receptor occupancy. These new insights into the interactions between PcTx1 with ASIC1 subtypes demonstrates the complexity of its mechanism of action, and highlights important implications to consider when using PcTx1 as a pharmacological tool to study ASIC function.

The Effects of Systemic and Local Acidosis on Insulin Resistance and Signaling.

Most pathological conditions that cause local or systemic acidosis by overcoming the buffering activities of body fluids overlap with those diseases that are characterized by glucose metabolic disorders, including diabetes mellitus, inflammation, and cancer. This simple observation suggests the existence of a strong relationship between acidosis and insulin metabolism or insulin receptor signaling. In this review, we summarized the current knowledge on the activity of insulin on the induction of acidosis and, vice versa, on the effects of changes of extracellular and intracellular pH on insulin resistance. Insulin influences acidosis by promoting glycolysis. Although with an unclear mechanism, the lowering of pH, in turn, inhibits insulin sensitivity or activity. In addition to ketoacidosis that is frequently associated with diabetes, other important and more complex factors are involved in this delicate feedback mechanism. Among these, in this review we discussed the acid-mediated inhibiting effects on insulin binding affinity to its receptor, on glycolysis, on the recycling of glucose transporters, and on insulin secretion via transforming growth factor β (TGF-β) activity by pancreatic β-cells. Finally, we revised current data available on the mutual interaction between insulin signaling and the activity of ion/proton transporters and pH sensors, and on how acidosis may enhance insulin resistance through the Nuclear Factor kappa B (NF-κB) inflammatory pathway.

Proton Sensor GPR68 Is Essential to Maintain Myeloid Malignancies

Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML.Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function.In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway.In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. DisclosuresNo relevant conflicts of interest to declare.

Roles of Mitogen-Activated Protein Kinases in Osteoclast Biology.

Bone undergoes continuous remodeling, which is homeostatically regulated by concerted communication between bone-forming osteoblasts and bone-degrading osteoclasts. Multinucleated giant osteoclasts are the only specialized cells that degrade or resorb the organic and inorganic bone components. They secrete proteases (e.g., cathepsin K) that degrade the organic collagenous matrix and establish localized acidosis at the bone-resorbing site through proton-pumping to facilitate the dissolution of inorganic mineral. Osteoporosis, the most common bone disease, is caused by excessive bone resorption, highlighting the crucial role of osteoclasts in intact bone remodeling. Signaling mediated by mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, has been recognized to be critical for normal osteoclast differentiation and activation. Various exogenous (e.g., toll-like receptor agonists) and endogenous (e.g., growth factors and inflammatory cytokines) stimuli contribute to determining whether MAPKs positively or negatively regulate osteoclast adhesion, migration, fusion and survival, and osteoclastic bone resorption. In this review, we delineate the unique roles of MAPKs in osteoclast metabolism and provide an overview of the upstream regulators that activate or inhibit MAPKs and their downstream targets. Furthermore, we discuss the current knowledge about the differential kinetics of ERK, JNK, and p38, and the crosstalk between MAPKs in osteoclast metabolism.

Building on the Shoulders of Giants: Is the use of Early Spontaneous Ventilation in the Setting of Severe Diffuse Acute Respiratory Distress Syndrome Actually Heretical?

Acute respiratory distress syndrome (ARDS) is not a failure of the neurological command of the ventilatory muscles or of the ventilatory muscles; it is an oxygenation defect. As positive pressure ventilation impedes the cardiac function, paralysis under general anaesthesia and controlled mandatory ventilation should be restricted to the interval needed to control the acute cardio-ventilatory distress observed upon admission into the critical care unit (CCU; "salvage therapy" during "shock state"). Current management of early severe diffuse ARDS rests on a prolonged interval of controlled mechanical ventilation with low driving pressure, paralysis (48 h, too often overextended), early proning and positive end-expiratory pressure (PEEP). Therefore, the time interval between arrival to the CCU and switching to spontaneous ventilation (SV) is not focused on normalizing the different factors involved in the pathophysiology of ARDS: fever, low cardiac output, systemic acidosis, peripheral shutdown (local acidosis), supine position, hypocapnia (generated by hyperpnea and tachypnea), sympathetic activation, inflammation and agitation. Then, the extended period of controlled mechanical ventilation with paralysis under general anaesthesia leads to CCU-acquired pathology, including low cardiac output, myoneuropathy, emergence delirium and nosocomial infection. The stabilization of the acute cardio-ventilatory distress should primarily itemize the pathophysiological conditions: fever control, improved micro-circulation and normalized local acidosis, 'upright' position, minimized hypercapnia, sympathetic de-activation (normalized sympathetic activity toward baseline levels resulting in improved micro-circulation with alpha-2 agonists administered immediately following optimized circulation and endotracheal intubation), lowered inflammation and 'cooperative' sedation without respiratory depression evoked by alpha-2 agonists. Normalised metabolic, circulatory and ventilatory demands will allow one to single out the oxygenation defect managed with high PEEP (diffuse recruitable ARDS) under early spontaneous ventilation (airway pressure release ventilation+SV or low-pressure support). Assuming an improved overall status, PaO2/FiO2≥150-200 allows for extubation and continuous non-invasive ventilation. Such fast-tracking may avoid most of the CCU-acquired pathologies. Evidence-based demonstration is required.

Time-course evaluation of intestinal structural disorders in a porcine model of intra-abdominal hypertension by mechanical intestinal obstruction

BackgroundA mechanical intestinal obstruction (MIO) can generate intraabdominal hypertension (IAH) that is life threatening. The intestines are very sensitive to IAH since the low splanchnic perfusion causes intestinal hypoxia, local acidosis and bacterial translocations. This may lead to acute intestinal distress syndrome (AIDS). The identification of intestinal injuries during IAH and its correlation with clinical parameters as the abdominal perfusion pressure (APP), the gastric intramucosal pH (pHi) and lactic acid (Lc) are still unknown. This study aimed to evaluate the sequence of intestinal histopathological findings in an MIO model and to analyze potential relationships with parameters currently used in clinical practice (APP, pHi and Lc).Material and methodsTwenty pigs were divided into three groups: a control group (n = 5) and two experimental groups with 20 mmHg (G1, n = 10) and 30 mmHg (G2, n = 5) of IAH by MIO. The pressures were maintained for 3 hours, except in 5 animals in G1 where it was maintained for 5 hours. The APP, pHi and LA were recorded and biopsies of the terminal ileum were taken every 30 minutes in all groups. The intestinal damage was graded according to the Park Score.ResultsIntestinal injuries were found in 42.9% of pigs in the experimental groups. The lesions were independent of the level and duration of IAH. Although APP and pHi were slightly lower in injured animals (I +) of G1 and G2, there were no significant differences among those uninjured (I-). Lc was significantly increased in all I+ pigs from the onset of IAH.ConclusionThe IAH by MIO causes intestinal lesions from the first 30 minutes with concurrent decreases in APP and pHi and increases in Lc. Lc could be the best clinical parameter related to intestinal damages with a clear difference between I + and I- animals.

Mild acidosis delays neutrophil apoptosis via multiple signaling pathways and acts in concert with inflammatory mediators.

Accumulating evidence indicates development of local extracellular acidosis in inflamed tissues in response to infection and tissue injury. Activation of infiltrating neutrophils contributes to a transient decrease in pH, which, in turn, triggers innate immunity. In this study, we investigated the impact of extracellular acidosis on neutrophil apoptosis, a critical determinant of the outcome of the inflammatory response and analyzed the underlying signaling pathways. Culture of human isolated neutrophils in mildly acidotic conditions (pH 6.5-7.0) resulted in activation of NF-κB; intracellular accumulation of cAMP; and phosphorylation of Akt, ERK, and p38 MAPK; and preservation of Mcl-1 expression. Consequently, extracellular acidosis prevented disruption of mitochondrial transmembrane potential and translocation of cytochrome c and apoptosis-inducing factor from the mitochondria to cytoplasm and nuclei, respectively and inhibited caspase-3 activity. Pharmacological inhibition of ERK, PI3K, NF-κB, or PKA partially reversed survival cues by extracellular acidosis and redirected neutrophils to apoptosis. Conversely, dibutyryl cAMP (100-500 μM) delayed apoptosis of neutrophils cultured at pH 7.4. Extracellular acidosis-generated survival cues were additive to the potent prosurvival signals from bacterial DNA, LPS, modified C-reactive protein, and serum amyloid A. Acidosis increased CpG DNA uptake by neutrophils and augmented phosphorylation of ERK and Akt, leading to preservation of Mcl-1 expression. Our results identified extracellular acidosis as a survival signal for neutrophils by suppressing the constitutive apoptotic machinery and suggest that transient decreases in local pH can enhance neutrophil responses to inflammatory stimuli, thereby contributing to amplification or prolongation of the inflammatory response.

Intratumoral acidosis fosters cancer-induced bone pain through the activation of the mesenchymal tumor-associated stroma in bone metastasis from breast carcinoma.

Cancer-induced bone pain (CIBP) is common in patients with bone metastases (BM), significantly impairing quality of life. The current treatments for CIBP are limited since they are often ineffective. Local acidosis derived from glycolytic carcinoma and tumor-induced osteolysis is only barely explored cause of pain. We found that breast carcinoma cells that prefer bone as a metastatic site have very high extracellular proton efflux and expression of pumps/ion transporters associated with acid-base balance (MCT4, CA9, and V-ATPase). Further, the impairment of intratumoral acidification via V-ATPase targeting in xenografts with BM significantly reduced CIBP, as measured by incapacitance test. We hypothesize that in addition to the direct acid-induced stimulation of nociceptors in the bone, a novel mechanism mediated by the acid-induced and tumor-associated mesenchymal stroma might ultimately lead to nociceptor sensitization and hyperalgesia. Consistent with this, short-term exposure of cancer-associated fibroblasts, mesenchymal stem cells, and osteoblasts to pH 6.8 promotes the expression of inflammatory and nociceptive mediators (NGF, BDNF, IL6, IL8, IL1b and CCL5). This is also consistent with a significant correlation between breakthrough pain, measured by pain questionnaire, and combined high serum levels of BDNF and IL6 in patients with BM, and also by immunofluorescence staining showing IL8 expression that was more in mesenchymal stromal cells rather than in tumors cells, and close to LAMP-2 positive acidifying carcinoma cells in BM tissue sections.In summary, intratumoral acidification in BM might promote CIBP also by activating the tumor-associated stroma, offering a new target for palliative treatments in advanced cancer.

Regional acidosis locally inhibits but remotely stimulates Ca2+ waves in ventricular myocytes

AimsSpontaneous Ca2+ waves in cardiomyocytes are potentially arrhythmogenic. A powerful controller of Ca2+ waves is the cytoplasmic H+ concentration ([H+]i), which fluctuates spatially and temporally in conditions such as myocardial ischaemia/reperfusion. H+-control of Ca2+ waves is poorly understood. We have therefore investigated how [H+]i co-ordinates their initiation and frequency.Methods and resultsSpontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricular myocytes, subjected to modest Ca2+-overload. Whole-cell intracellular acidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologically blocking sarcolemmal Na+/H+ exchange (NHE1) prevented this stimulation, unveiling inhibition by H+. Acidosis also increased Ca2+ wave velocity. Restricting acidosis to one end of a myocyte, using a microfluidic device, inhibited Ca2+ waves in the acidic zone (consistent with ryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell. This remote stimulation was absent when NHE1 was selectively inhibited in the acidic zone. Remote stimulation depended on a locally evoked, NHE1-driven rise of [Na+]i that spread rapidly downstream.ConclusionAcidosis influences Ca2+ waves via inhibitory and stimulatory signals (the latter facilitating intracellular Ca2+-loading through modulation of sarcolemmal Na+/Ca2+ exchange activity). During spatial [H+]i-heterogeneity, -inhibition dominates in acidic regions, while rapid diffusion stimulates waves in downstream, non-acidic regions. Local acidosis thus simultaneously inhibits and stimulates arrhythmogenic Ca2+-signalling in the same myocyte. If the principle of remote H+-stimulation of Ca2+ waves also applies in multicellular myocardium, it raises the possibility of electrical disturbances being driven remotely by adjacent ischaemic areas, which are known to be intensely acidic.