Effect Of Extracellular Acidosis Research Articles

Effect of Extracellular Acidosis on Functional Contribution of KATP and TASK-1 Potassium Channels to Vascular Tone Regulation in Early Postnatal Ontogenesis

Effect of Extracellular Acidosis on Functional Contribution of KATP and TASK-1 Potassium Channels to Vascular Tone Regulation in Early Postnatal Ontogenesis

Motility and Mechanical Properties of Dendritic Cells Deteriorated by Extracellular Acidosis

Dendritic cells (DCs) are the most powerful antigen-presenting cells known to date and play an important role in initiating and amplifying both innate and adaptive immune responses. Extracellular acidosis is an important hallmark of a variety of inflammatory processes and solid tumors. However, few studies have focused on the effect of extracellular acidosis on DCs and their functions. Cellular mechanical properties reflect the relationship between cell structure and function, including cytoskeleton (especially F-actin organization), membrane negative charges, membrane fluidity, and osmotic fragility. The study investigated the effects of extracellular acidosis on the DCs functions from the perspective of cellular migration and mechanical properties. The results showed that migration ability, F-actin contents, and membrane negative charges of DCs were reduced by extracellular acidosis no matter whether LPS stimulated its maturation or not. And these functions could not return to normal after removing acidic microenvironment, which revealed that the function impairment induced by extracellular acidosis might be irreversible. In addition, the proliferation capacity of stimulated allogeneic T cells was impaired by extracellular acidosis. Our results suggest extracellular acidosis may play an immunosuppressive role in DCs-mediated immune process.

Abstract 2407: Extracellular acidosis inhibits glucose starvation-induced lung cancer cell death by suppressing overall metabolic rate

Abstract Bioenergetic tumor metabolism is essential to meet ATP requirements for energy homeostasis. Cancer cells utilize aerobic glycolysis and proliferate continuously beyond the capacity of blood supply. These phenotypic alterations lead to various microenvironmental stresses, such as hypoxia, nutrient (glucose) deprivation, and extracellular acidosis, to which cancer cells must metabolically adapt in order to survive and proliferate. However, compared to hypoxia, relatively little is known about the metabolic adaptations in response to glucose deprivation and acidosis. In the present study, we explored the effects of extracellular acidosis on cancer metabolism under glucose deprivation stress, as extracellular acidosis and insufficient glucose supply usually co-exist in the tumor microenvironment. To this aim, A549 lung cancer cells were treated with acidosis, glucose deprivation, or a combination. Then, cell survival, total cellular ATP turnover, relative glycolytic and mitochondrial dependency and capacity, and de novo rRNA and protein synthesis were determined. We found that acidosis (pH 6.8) inhibited ATP depletion and cell death induced by glucose deprivation. The cytoprotective effect of acidosis against glucose deprivation-induced cell death was mediated by two distinct mechanisms. First, acidosis shifted cellular ATP production from glycolysis to OXPHOS that became independent of glucose uptake but dependent on glutaminolysis and fatty acid β-oxidation. Second, acidosis reduced ATP consumption by inhibiting de novo rRNA and protein synthesis that utilizes ATP. In contrast, the protective effect of acidosis on glucose starvation-induced cell death was independent of AMPK, PI3K, ERK, and p38 MAPK. Based on this mechanistic knowledge of acidosis-dependent metabolic alterations, we tested the effect of the mitochondrial complex I inhibitor metformin on death of lung cancer cells. Metformin and a combination of rotenone and AICAR mimicking an action of metformin induced ATP depletion and cell death in glucose-free medium, but under acidosis the cytotoxic effect of metformin was significantly attenuated because overall ATP turnover was reduced under low pH. These results suggest that during acidosis, cells are forced to a reduced glycolytic energy production, but the decrease in protein synthesis and ATP requirement serves as an adaptive response to glucose deprivation stress. This acidosis-dependent, glucose starvation-resistant phenotype may provide an adaptive strategy in an energy-restricted tumor microenvironment. Our findings highlight the importance of considering the two factors, acidosis and nutrient starvation in combination to understand their contributions to tumor metabolism. Citation Format: Yuki Iwai, Ryota Kikuchi, Yasutaka Watanabe, Nobuyuki Koyama, Koichi Hagiwara, Hiroyuki Nakamura, Kazutetsu Aoshiba. Extracellular acidosis inhibits glucose starvation-induced lung cancer cell death by suppressing overall metabolic rate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2407.

The effect of extracellular acidosis on the behaviour of mesenchymal stem cells in vitro.

The stem cell fraction of a cell population is finely tuned by stimuli from the external microenvironment. Among these stimuli, a decrease of extracellular pH (pHe) may occur in a variety of physiological and pathological conditions, including hypoxia and inflammation. In this study, by using bone marrow stem cells and dental pulp stem cells, we provided evidence that extracellular acidosis endows the maintenance of stemness in mesenchymal cells. Indeed, continuous exposure for 21 d to low pHe (6.5-6.8) conditions impaired the osteogenic differentiation of both cell types. Moreover, the exposure to low pHe, for 1 and up to 7 d, induced the expression of stemness-related genes and proteins, drove cells to reside in the quiescent G0 alert state and enhanced their ability to form floating spheres. The pre-conditioning with extracellular acidosis for 7 d did not affect the differentiation potential of dental pulp stem cells since, when the cells were cultured again at physiological pHe, their multilineage potential was almost unmodified. Our data provided evidence of the role of extracellular acidosis as a modulator of the stemness of mesenchymal cells. This condition is commonly found both in systemic and local bone conditions, hence underlining the relevance of this phenomenon for a better comprehension of bone healing and regeneration.

Extracellular acidosis contracts coronary but neither renal nor mesenteric artery via modulation of H+,K+-ATPase, voltage-gated K+ channels and L-type Ca2+ channels.

Extracellular acidosis (EA) jeopardizes the heart, whereas mild extracellular alkalinization is cardioprotective, but it remains elusive how the coronary artery (CA) responses to EA. In the present study, EA was demonstrated to induce contraction in rat coronary artery (RCA) in a manner dependent on extracellular pH (pHo, 7.2-6.6), whereas it did not affect the resting tone of either rat renal interlobe artery (RIA) or mesenteric artery (MA). The amplitude of contraction provoked by pHo6.8 was approximately equal to that induced by 60mmoll(-1) KCl at pHo7.4. Blockade of L-type voltage-gated Ca(2+) channels and inhibition of H(+),K(+)-ATPase attenuated the contraction, whereas inhibition of nitric oxide synthesis and endothelial denudation augmented it. A molecular probe study showed that EA acidified the cytosol of arterial smooth muscle cells (ASMCs) in RIA and MA, but alkalinized it in RCA. Extracellular acidosis elevated the intracellular Ca(2+) concentration exclusively in RCA ASMCs. Patch-clamp studies showed that EA enhanced L-type voltage-gated Ca(2+) channel currents in RCA ASMCs, but depressed the currents in MA ASMCs and did not affect the currents in RIA ASMCs. Extracellular acidosis depressed voltage-gated K(+) channel (KV) currents only in RCA ASMCs. Lansoprazole blunted all these observed effects of EA on RCA. Taken together, the present results demonstrate that the responses of RCA to EA are different from those of RIA and MA and suggest that activation of L-type voltage-gated Ca(2+) channels and H(+),K(+)-ATPase as well as depression of KV may, at least partly, underlie the EA-induced contraction in RCA.

Acidosis Sensing Receptor GPR65 Correlates with Anti-Apoptotic Bcl-2 Family Member Expression in CLL Cells: Potential Implications for the CLL Microenvironment.

The tumor microenvironment is generally an acidic environment, yet the effect of extracellular acidosis on chronic lymphocytic leukemia (CLL) is not well established. Here we are the first to report that the extracellular acid sensing G-protein coupled receptor, GPR65, is expressed in primary CLL cells where its level correlate strongly with anti-apoptotic Bcl-2 family member levels. GPR65 expression is found normally within the lymphoid lineage and has not been previously reported in CLL. We demonstrate a wide range of GPR65 mRNA expression among CLL 87 patient samples. The correlation between GPR65 mRNA levels and Bcl-2 mRNA levels is particularly strong (r=0.8063, p= <0.001). The correlation extends to other anti-apoptotic Bcl-2 family members, Mcl-1 (r=0.4847, p=0.0010) and Bcl-xl (r=0.3411, p=0.0252), although at lower levels of significance. No correlation is detected between GPR65 and levels of the pro-apoptotic proteins BIM, PUMA or NOXA. GPR65 expression also correlates with the favorable prognostic marker of 13q deletion. The present findings suggest the acid sensing receptor GPR65 may be of significance to allow CLL tolerance of extracellular acidosis. The correlation of GPR65 with Bcl-2 suggests a novel cytoprotective mechanism that enables CLL cell adaptation to acidic extracellular conditions. These findings suggest the potential value of targeting GPR65 therapeutically.

Acidosis induces relaxation mediated by nitric oxide and potassium channels in rat thoracic aorta

We investigated the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. The relaxation response to HCl-induced extracellular acidification (7.4 to 6.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10−6 M) or KCl (45mM). The vascular reactivity experiments were performed in endothelium-intact and denuded rings, in the presence or absence of indomethacin (10−5 M), L-NAME (10−4 M), apamin (10−6 M), and glibenclamide (10−5 M). The effect of extracellular acidosis (pH 7.0 and 6.5) on nitric oxide (NO) production was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5μM). The extracellular acidosis failed to induce any changes in the vascular tone of aortic rings pre-contracted with KCl, however, it caused endothelium-dependent and independent relaxation in rings pre-contracted with Phe. This acidosis induced-relaxation was inhibited by L-NAME, apamin, and glibenclamide, but not by indomethacin. The acidosis (pH 7.0 and 6.5) also promoted a time-dependent increase in the NO production by the isolated endothelial cells. These results suggest that extracellular acidosis promotes vasodilation mediated by NO, KATP and SKCa, and maybe other K+ channels in isolated rat thoracic aorta.

Extracellular pH affects inflammatory cell production of superoxide and nitric oxide.

Previous research has described how high cellular metabolism creates an acidic environment in inflammatory cells during respiratory burst. The aim of our work was to describe the acid-base dependence of exudate in superoxide (O2.-) and nitric oxide (NO.) generation by inflammatory cells from a carrageenan-granuloma. Although the carrageenan solution was alkaline (pH 7.74 when equilibrated with air) the exudate showed an acidification that stabilised at around 7 units of pH. A notable hypercapnia, but not hypoxia, was found in the exudate at up to 24 h. The effect of extracellular acidosis on O2.- and NO. production by inflammatory cells was also studied. The maximum O2.- production and the lowest levels of NO. were found at pH 7, which was closer to the pH of the granuloma-pouch. These results suggest that experiments with inflammatory cells ex vivo should be carried out at an identical pH to that found in vivo in order to reproduce the physiological mechanisms of free radical generation during inflammatory processes.

Acidosis Inhibits Gallbladder Contraction Mediated by Protein Kinase C Activation

The effects of extracellular acidosis on gallbladder contraction were investigated using gallbladder strips isolated from guinea pigs. In an acidic medium (pH 6.9), gallbladder contraction induced by histamine and prostaglandin E2 was significantly lower than that in a normal medium (pH 7.4). Acidosis affected neither gallbladder contraction induced by histamine in the absence of extracellular Ca2+ nor that induced by KCl. Acidosis significantly inhibited Ca2+-induced contraction in the presence of sodium fluoride and phorbol 12,13-dibutyrate but not that in the presence of KCl. Staurosporine (30 nM) significantly inhibited gallbladder contraction induced by histamine and prostaglandin E2, but not that by KCl. Histamine-induced contraction in the presence of staurosporine was not affected by acidosis. Acidosis significantly inhibited Ca2+-induced contraction in the presence of histamine but not that in the presence of both histamine and staurosporine. These results suggest that extracellular acidosis selectively inhibits gallbladder contraction mediated by protein kinase C activation.

The actions of extracellular H+ on the electrophysiological properties of isolated human detrusor smooth muscle cells.

1. The influence of extracellular pH changes on intracellular pH and [Ca2+], as well as on L-type Ca2+ currents, has been investigated in isolated human detrusor smooth muscle cells. 2. Alteration of extracellular pH by changing superfusate PCO2 also changed intracellular pH. A change of superfusate pH made by altering the [NaHCO3] at constant PCO2 was not reflected in a change in intracellular pH. 3. Extracellular acidosis attenuated the magnitude and rate of change of intracellular [Ca2+] evoked by raising the extracellular [KCl]. 4. Extracellular acidosis attenuated the rate of rise and amplitude of the action potential, as well as the magnitude of the L-type Ca2+ current. In the pH range 6.78-7.62 no alteration to the voltage dependence of Ca2+ current activation or inactivation was recorded. 5. A close proportional relationship between tension generated by multicellular strips and the magnitude of peak inward Ca2+ current in isolated cells was noted over a wide range of the two variables using a number of interventions, including alteration to extracellular pH, [Ca2+] and [Mg2+]. 6. Extracellular acidosis attenuated the magnitude of caffeine-dependent intracellular Ca2+ transients and the resting [Ca2+]i between transients. Acidosis was without effect on the rise of [Ca2+]i induced by carbachol. 7. The results suggest that the negative inotropic effect of extracellular acidosis can be accounted for by attenuation of the L-type Ca2+ current. The results also imply that intracellular stores are influenced by transmembrane Ca2+ fluxes at rest and that such fluxes are also attenuated by extracellular H+.

The effects of extracellular acidosis on neurons and glia in vitro.

Cerebral lactic acid, a product of ischemic anaerobic glycolysis, may directly contribute to ischemic brain damage in vivo. In this study we evaluated the effects of extracellular acid exposure on 7-day-old cultures of embryonic rat forebrain. Mixed neuronal and glial cultures were exposed to either lactic or hydrochloric acid to compare the toxicities of relatively permeable and impermeable acids. Neurons were relatively resistant to extra-cellular HCl acidosis, often surviving 10-min exposures to pH 3.8. In the same cultures, immunochemically defined astrocytes survived 10-min HCl exposures to a maximum acidity of pH 4.2. Similarly, axonal bundles defasciculated in HCl-titrated media below pH 4.4, although their constituent fibers often survived pH 3.8. Cell death occurred at higher pH in cultures subjected to lactic acidosis than in those exposed to HCl. Over half of forebrain neurons and glia subjected for 10 min to lactic acidification failed to survive exposure to pH 4.9. Longer 1-h lactic acid incubations resulted in cell death below pH 5.2. The potent cytotoxicity of lactic acid may be a direct result of the relatively rapid transfer of its neutral protonated form across cell membranes. This process would rapidly deplete intracellular buffer stores, resulting in unchecked cytosolic acidification. Neuronal and glial death from extracellular acidosis may therefore be a function of both the degree and the rapidity of intracellular acidification.

Cardiac performance in the in situ perfused fish heart during extracellular acidosis: interactive effects of adrenaline.

The physiological integrity of the in situ perfused heart of the ocean pout was established by its ability to maintain cardiac output (Q) over a range of work loads, and by the dependence of Q upon the filling pressure of the heart. Similar observations have been reported previously for the in situ perfused heart of the sea raven. Physiological levels of extracellular acidosis (pH 7.6/1% CO2 and pH 7.4/2% CO2) significantly depressed cardiac performance in sea raven and ocean pout hearts in situ. Negative chronotropic and inotropic responses were observed. Adrenaline (AD; 10(-7) M) under control conditions (pH 7.9/0.5% CO2) produced a sustained tachycardia. The tachycardia reduced filling time of the ventricle and stroke volume was compromised because of the constant preload to the heart. Consequently, AD produced only an initial, transient increase in stroke volume and Q. Thereafter, stroke volume was reduced in proportion with the increase in heart rate, and Q remained unchanged. The combined challenge of extracellular acidosis and AD demonstrated interactive effects between AD and acidosis in situ. Q and power output were maintained in both species at both levels of extracellular acidosis during the combined challenge. Thus AD alone can maintain (but not improve upon) basal Q during extracellular acidosis. The effects of extracellular acidosis, circulating catecholamines and venous return pressure to the heart are discussed in relation to the regulation of Q following exhaustive exercise.