Decrease In pHi Research Articles

Effects of extracellular metabolic acidosis and out-of-equilibrium CO2/HCO3 - solutions on intracellular pH in cultured rat hippocampal neurons.

Metabolic acidosis (MAc)-an extracellular pH (pHo) decrease caused by a [HCO3 -]o decrease at constant [CO2]o-usually causes intracellular pH (pHi) to fall. Here we determine the extent to which the pHi decrease depends on the pHo decrease vs the concomitant [HCO3 -]o decrease. We use rapid-mixing to generate out-of-equilibrium CO2/HCO3 - solutions in which we stabilize [CO2]o and [HCO3 -]o while decreasing pHo (pure acidosis, pAc), or stabilize [CO2]o and pHo while decreasing [HCO3 -]o (pure metabolic/down, pMet↓). Using the fluorescent dye 2',7'-bis-2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) to monitor pHi in rat hippocampal neurons in primary culture, we find that-in naïve neurons-the pHi decrease caused by MAc is virtually the sum of those caused by pAc (∼70%) + pMet↓ (∼30%). However, if we impose a first challenge (MAc1, pAc1, or pMet↓1), allow the neurons to recover, and then impose a second challenge (MAc2, pAc2, or pMet↓2), we find that pAc/pMet↓ additivity breaks down. In a twin-challenge protocol in which challenge #2 is MAc, the pHo and [HCO3 -]o decreases during challenge #1 must be coincident in order to mimic the effects of MAc1 on MAc2. Conversely, if challenge #1 is MAc, then the pHo and [HCO3 -]o decreases during challenge #2 must be coincident in order for MAc1 to produce its physiological effects during the challenge #2 period. We conclude that the history of challenge #1 (MAc1, pAc1, or pMet↓1)-presumably as detected by one or more acid-base sensors-has a major impact on the pHi response during challenge #2 (MAc2, pAc2, or pMet↓2).

Abstract C041: Emerging role of P2X receptors as Golgi pH regulators in pancreatic cancer

Abstract Cancer cells reprogram their metabolism to meet their elevated energy demands, favoring energy production via glycolysis even with functional mitochondria. Upregulated glycolysis results in rapid production of lactate and protons in the cytoplasm. This would normally reduce the intracellular pH (pHi); however, cancer cells employ mechanisms such as ion transporters that remove excess protons from the cytoplasm to maintain an alkaline pHi, allowing for cell growth, evasion of cell death and cell proliferation. Our previous studies have shown that in pancreatic ductal adenocarcinoma (PDAC), the Golgi apparatus is among the intracellular compartments that can uptake excess protons, giving PDAC cells a cell fitness advantage. The underlying mechanisms that drive Golgi acidification in PDAC are incompletely understood. Here, we set out to comprehensively identify novel modulators that can affect this process. By using a fluorescent-protein-based ratiometric pH biosensor targeted to the Golgi membrane, we screened thousands of chemical compounds for an impairment in Golgi acidification, which would lead to pHi dysregulation and cell death. One of our hits from the screen was an inhibitor of the P2X family of receptors (P2XRs). P2XRs consist of seven subtypes (P2X1-7) and are ATP-gated ion channels that mediate the passage of non-selective cations through the plasma membrane and intracellular compartments. P2XRs have also been shown to regulate the pHi. We are elucidating the mechanism by which P2XRs may regulate pHi via the Golgi in PDAC. Our preliminary results show that concomitant with the alkalinization of the Golgi, P2XR inhibition leads to a pronounced decrease of pHi and results in cell death in PDAC while normal untransformed pancreas cells are unaffected. These data suggest that P2XRs play a role in pHi regulation and cell fitness maintenance through a function in the Golgi. Targeting this metabolic reprograming dependency may serve as a novel therapeutic tool for PDAC. Citation Format: Cheska Marie Galapate, Koen Galenkamp, Charles Fisher, Susanne Heynen-Genel, Cosimo Commisso. Emerging role of P2X receptors as Golgi pH regulators in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C041.

Delivery of Melittin as a Lytic Agent via Graphene Nanoparticles as Carriers to Breast Cancer Cells.

Melittin, as an agent to lyse biological membranes, may be a promising therapeutic agent in the treatment of cancer. However, because of its nonspecific actions, there is a need to use a delivery method. The conducted research determined whether carbon nanoparticles, such as graphene and graphene oxide, could be carriers for melittin to breast cancer cells. The studies included the analysis of intracellular pH, the potential of cell membranes, the type of cellular transport, and the expression of receptor proteins. By measuring the particle size, zeta potential, and FT-IT analysis, we found that the investigated nanoparticles are connected by electrostatic interactions. The level of melittin encapsulation with graphene was 86%, while with graphene oxide it was 78%. A decrease in pHi was observed for all cell lines after administration of melittin and its complex with graphene. The decrease in membrane polarization was demonstrated for all lines treated with melittin and its complex with graphene and after exposure to the complex of melittin with graphene oxide for the MDA-MB-231 and HFFF2 lines. The results showed that the investigated melittin complexes and the melittin itself act differently on different cell lines (MDA-MB-231 and MCF-7). It has been shown that in MDA-MD-231 cells, melittin in a complex with graphene is transported to cells via caveolin-dependent endocytosis. On the other hand, the melittin-graphene oxide complex can reach breast cancer cells through various types of transport. Other differences in protein expression changes were also observed for tumor lines after exposure to melittin and complexes.

Antitumor effects of lactate transport inhibition on esophageal adenocarcinoma cells

As a consequence of altered glucose metabolism, cancer cell intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs). MCT 1 and MCT4 are frequently overexpressed in tumors, and recently, MCT inhibition has been reported to exert antineoplastic effects. In the present study, MCT1 and MCT4 levels were assessed in esophageal adenocarcinoma (EAC) cells and the effects of the MCT-1 selective inhibitor AZD3965, hypoxia, and a glucose overload were evaluated in vitro. Two EAC cell lines (OE33 and OACM5.1C) were treated with AZD3965 (10–100 nM) under different conditions (normoxia/hypoxia) and also different glucose concentrations, and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi), and lactate levels were evaluated. MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small proportion of OACM5.1C cells. Glucose addition did not have any effect on apoptosis nor cell proliferation. AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia. MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress. AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4 and also increased the sodium/hydrogen exchanger 1 (NHE-1) expression on these cells. These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition.

Beat-to-beat dynamic regulation of intracellular pH in cardiomyocytes

SummaryThe mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pHi) for normal cardiac function. Even though spatial pHi non-uniformity in cardiomyocytes has been documented, it remains unknown how pHi is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pHi transients, in synchrony with cardiomyocyte contractions. The pHi transients are regulated by pacing rate, Cl−/HCO3- transporters, pHi buffering capacity, and β-adrenergic signaling. Mitochondrial electron-transport chain inhibition attenuates the pHi transients, implicating mitochondrial activity in sculpting the pHi regulation. The pHi transients provide dynamic alterations of H+ transport required for ATP synthesis, and a decrease in pHi may serve as a negative feedback to cardiac contractions. Current findings dovetail with the prevailing three known dynamic systems, namely electrical, Ca2+, and mechanical systems, and may reveal broader features of pHi handling in excitable cells.

Modulation of acid extrusion and acid loading in neurons and astrocytes by RPTPγ and RPTPζ

Receptor protein tyrosine phosphatases γ and ζ (RPTPγ & RPTPζ) are transmembrane signaling proteins that, as monomers, catalyze dephosphorylation of tyrosine residues on cytosolic targets. RPTPγ and RPTPζ are unusual in having an extracellular carbonic-anhydrase–like domain (CALD). Carbonic anhydrases (CAs) catalyze the reaction, CO₂ + H₂O ⇌ HCO3– + H⁺, but CALDs are enzymatically inactive. Previous studies on renal proximal tubules (PTs) strongly suggest that RPTPγ is a dual extracellular CO₂/HCO₃⁻ sensor. RPTPγ—expressed on the PT basal (i.e., blood-side) membrane—is essential for modulating acid-base transport in response to changes in basolateral [CO₂] and [HCO₃⁻]. RPTPγ and RPTPζ are also present in the central nervous system (CNS), including mouse hippocampus (HC). RPTPγ is expressed primarily in neurons and RPTPζ, in astrocytes. Here, we investigate whether RPTPγ and RPTPζ in brain, like RPTPγ in kidney, play roles in sensing extracellular [CO₂]ₒ and [HCO₃⁻]ₒ and altering acid-base transport. We hypothesize that activation/termination of the intracellular phosphatase activity of these RPTPs regulates initial steps in signaling cascades that defend cells against intracellular pH (pHi) decreases triggered by metabolic acidosis (MAc, caused by ↓ [HCO₃⁻]ₒ) or respiratory acidosis (RAc, caused by ↑ [CO₂]ₒ). We propose that the RPTPs respond to ↑[CO₂]ₒ or ↓[HCO₃⁻]ₒ by promoting a shift from dimers to monomers, thereby dis-inhibiting the activity of the intracellular phosphatase domains. The ultimate result of the RPTP dis-inhibition would be increased acid-extrusion rate (JE) or decreasedacid-loading rate (JL), both of which would minimize the fall of pHi during a first exposure to MAc or RAc, and may promote adaptation during a second exposure. Immunocytochemistry of mixed HC neuron-astrocyte cultures from wild-type (WT) mice using a primary antibody against the RPTPγ fibronectin III domain shows that RPTPγ colocalizes with MAP2 in HC neurons but not with GFAP in HC astrocytes . RT-PCR cloning from mixed HC cultures identifies specific RPTPγ and RPTPζ variants. Presumably it is the neurons that express two identified RPTPγ variants: the full-length protein, encoded by all 30 ptprg exons (NM_008981), and a second newly-detected variant that corresponds to the hypothetical assembly X1 (XM_006517956). The HC co-cultures also express six RPTPζ variants: Three RPTPζ variants (V) have previously been validated in mice: VR3 (NM_001081306), VR4 (NM_001311064), and VR5 (NM_001361349). Three others were previously only reported as hypothetical assemblies: X2 (XM_006505013), X3 (XM_006505014), and X4 (XM_006505015). To assess the influence of RPTPζ on pHi homeostasis, we loaded cells with the pH-sensitive dye BCECF, digitally monitored dye fluorescence, and computed pHi of cells subjected to acid-base disturbances. In MAc-MAc protocols, the effects of the knockout (KO) of RPTPζ are unremarkable in both neurons and astrocytes. In RAc-RAc protocols, the KO has unremarkable effects on neurons. However, in astrocytes the RPTPζ-KO markedly increases both the rates and the magnitudes of pHi decrease during both RAc pulses. Thus, RPTPζ may stabilize pHi in astrocytes subjected to RAc.

Brain tumor acidification using drugs simultaneously targeting multiple pH regulatory mechanisms.

Non-invasively distinguishing aggressive from non-aggressive brain tumors is an important clinical challenge. Intracellular pH (pHi) regulation is essential for normal cell function and is normally maintained within a narrow range. Cancer cells are characterized by a reversed intracellular to extracellular pH gradient, compared to healthy cells, that is maintained by several distinct mechanisms. Previous studies have demonstrated acute pH modulation in glioblastoma detectable by chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) after blocking individual pH regulatory mechanisms. The purpose of the current study was to simultaneously block five pH regulatory mechanisms while also providing glucose as an energy substrate. We hypothesized that this approach would increase the acute pH modulation effect allowing the identification of aggressive cancer. Using a 9.4T MRI scanner, CEST spectra were acquired sensitive to pHi using amine/amide concentration independent detection (AACID). Twelve mice were scanned approximately 11 ± 1days after implanting 105 U87 human glioblastoma multiforme cells in the brain, before and after intraperitoneal injection of a combination of five drugs (quercetin, cariporide, dichloroacetate, acetazolamide, and pantoprazole) with and without glucose. Two hours after combination drug injection there was a significant 0.1 ± 0.03 increase in tumor AACIDvalue corresponding to a 0.4 decrease in pHi. After injecting the drug combination with glucose the AACID value increased by 0.18 ± 0.03 corresponding to a 0.72 decrease in pHi. AACID values were alsoslightly increased in contralateral tissue. The combined drug treatment with glucose produced a large acute CEST MRI contrast indicating tumor acidification, which could be used to help localize brain cancer and monitor tumor response to chemotherapy.

Consequences of the Endogenous N-Glycosylation of Human Ribonuclease 1.

Ribonuclease 1 (RNase 1) is the most prevalent human homologue of the archetypal enzyme RNase A. RNase 1 contains sequons for N-linked glycosylation at Asn34, Asn76, and Asn88 and is N-glycosylated at all three sites in vivo. The effect of N-glycosylation on the structure and function of RNase 1 is unknown. By using an engineered strain of the yeast Pichia pastoris, we installed a heptasaccharide (Man5GlcNAc2) on the side chain of Asn34, Asn76, and Asn88 to produce the authentic triglycosylated form of human RNase 1. As a glutamine residue is not a substrate for cellular oligosaccharyltransferase, we used strategic asparagine-to-glutamine substitutions to produce the three diglycosylated and three monoglycosylated forms of RNase 1. We found that the N-glycosylation of RNase 1 at any position attenuates its catalytic activity but enhances both its thermostability and its resistance to proteolysis. N-Glycosylation at Asn34 generates the most active and stable glycoforms, in accord with its sequon being highly conserved among vertebrate species. These data provide new insight on the biological role of the N-glycosylation of a human secretory enzyme.

The Monocarboxylate transporter inhibitor Quercetin induces intracellular acidification in a mouse model of Glioblastoma Multiforme: in-vivo detection using magnetic resonance imaging.

The response of tumor intracellular pH to a pharmacological challenge could help identify aggressive cancer. Chemical exchange saturation transfer (CEST) is an MRI contrast mechanism that is dependent on intracellular pH (pHi). pHi is important in the maintenance of normal cell function and is normally maintained within a narrow range by the activity of transporters located at the plasma membrane. In cancer, changes in pHi have been correlated with both cell proliferation and cell death. Quercetin is a bioflavonoid and monocarboxylate transporter (MCT) inhibitor. Since MCTs plays a significant role in maintaining pH balance in the tumor microenvironment, we hypothesized that systemically administered quercetin could selectively acidify brain tumors. The goals of the current study were to determine whether CEST MRI measurements sensitive to tumor pH could detect acidification after quercetin injection and to measure the magnitude of the pH change (ΔpH). Using a 9.4T MRI, amine and amide concentration independent detection (AACID) CEST spectra were acquired in six mice approximately 15 ± 1days after implanting 105U87 human glioblastoma multiforme cells in the brain, before and after administration of quercetin (dose: 200mg/kg) by intraperitoneal injection. Three additional mice were studied as controls and received only vehicle dimethyl sulfoxide (DMSO) injection. Repeated measures t-test was used to compare AACID changes in tumor and contralateral tissue regions of interest. Two hours after quercetin injection there was a significant increase in tumor AACID by 0.07 ± 0.03 corresponding to a 0.27 decrease in pHi, and no change in AACID in contralateral tissue. There was also a small average increase in AACID in tumors within the three mice injected with DMSO only. The use of the natural compound quercetin in combination with pH weighted MRI represents a unique approach to cancer detection that does not require injection of an imaging contrast agent.

Intestinal histopathological changes in a porcine model of pneumoperitoneum-induced intra-abdominal hypertension.

Low splanchnic perfusion is an immediate effect of pneumoperitoneum-induced intra-abdominal hypertension (IAH). Anatomical structure results in the intestinal mucosa being the area most sensitive to hypoperfusion. The relationship between intestinal injury and clinical parameters of tissue perfusion [abdominal perfusion pressure (APP), gastric intramucosal pH (pHi) and lactic acid (Lc)] has not been previously studied. This study aimed to monitorize intestinal pathogenesis through sequential ileal biopsies and to measure APP, pHi, and Lc levels at different pneumoperitoneum-induced intra-abdominal pressures (20, 30, and 40mmHg) to evaluate the potential relationships between them. Fifty pigs were divided into four groups; a control group (C) and three experimental groups with different pneumoperitoneum-induced levels [20mmHg (G20), 30mmHg (G30), and 40mmHg (G40)], that were maintained for 3 and 5h. APP, pHi, and Lc were measured and ileal biopsies taken laparoscopically every 30min. The mucosal damage was graded using the standardized Park's Score and animals were classified as injured (I+) or uninjured (I-). Different histopathological lesions were observed in groups G20, G30, and G40 but no damage observed in group C. A 33.3% of animals in G20 and G30 were I+ after 3h, while 93.3% were injured in G40. After 5h, histopathological lesions were no longer seen in some animals in G20 and only 10% were I+. Conversely, in G30 I+ pigs increased to 80% while those in G40 remained at 93.3% I+. The I+ animals had significantly lower APP and pHi than those I-. Lc was the clinical parameter that showed the earliest differences, with significantly higher figures in I+ animals. The evolution of intestinal injuries from pneumoperitoneum-induced IAH depends on the degree of IAP. These damages may be associated with decreases in APP and pHi, and increases in Lc.

In vivo detection of acute intracellular acidification in glioblastoma multiforme following a single dose of cariporide.

Glioblastoma is an aggressive brain cancer that is very difficult to treat. Clinically, it is important to be able to distinguish aggressive from non-aggressive brain tumors. Previous studies have shown that some drugs can induce a rapid change in intracellular pH that could help to identify aggressive cancer. The sodium proton exchanger (NHE1) plays a significant role in maintaining pH balance in the tumor microenvironment. Cariporide is a sodium proton exchange inhibitor that is well tolerated by humans in cardiac applications. We hypothesized that cariporide could selectively acidify brain tumors. The purpose of this study was to determine whether amine/amide concentration-independent detection (AACID) chemical exchange saturation transfer (CEST) MRI measurement of tumor pHi could detect acidification after cariporide injection. Using a 9.4T MRI scanner, CEST spectra were acquired in six mice approximately 14days after implanting 105 U87 human glioblastoma multiforme cells in the brain, before and after administration of cariporide (dose: 6mg/kg) by intraperitoneal injection. Three additional mice were studied as controls and received only vehicle injection (DMSO + PBS). Repeated measures t test was used to examine changes in tumor and contralateral tissue regions of interest. Two hours after cariporide injection, there was a significant 0.12 ± 0.03 increase in tumor AACID value corresponding to a 0.48 decrease in pHi and no change in AACID value in contralateral tissue. A small but significant increase of 0.04 ± 0.017 in tumor AACID value was also observed following vehicle injection. This study demonstrates that acute CEST MRI contrast changes, indicative of intracellular acidification, after administration of cariporide could help localize glioblastoma.

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.

Dichloroacetate induced intracellular acidification in glioblastoma: in vivo detection using AACID-CEST MRI at 9.4Tesla.

Intracellular pH (pHi) plays an important role in the maintenance of normal cell function, and is maintained within a narrow range by the activity of transporters located at the plasma membrane. Modulation of tumor pHi may influence proliferation, apoptosis, chemotherapy resistance, and thermosensitivity. Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is dependent on cellular pH. Amine and amide concentration-independent detection (AACID) is a recently developed CEST contrast method that is intracellular pH (pHi) weighted. Dichloroacetate (DCA) can alter tumor pHi by inhibiting the enzyme pyruvate dehydrogenase kinase causing reduced lactate (increasing pHi), or by decreasing the expression of monocarboxylate transporters and vacuolar ATPase leading to reduced pHi. Since the net in vivo effect of DCA on pHi is difficult to predict, the purpose of this study was to quantify the magnitude of acute pHi change in glioblastoma after a single DCA injection using AACID CEST MRI. Using a 9.4T MRI scanner, CEST spectra were acquired in six mice approximately 14 days after implanting 105 U87 human glioblastoma multiforme (GBM) cells in the brain, before and after intravenous injection of DCA (dose: 200mg/kg). Three additional mice received only phosphate buffered saline (PBS) injection and were studied as controls. Repeated measures t test was used to compare AACID changes in tumor and contralateral tissue regions of interest. One hour after DCA injection there was a significant increase in tumor AACID level by 0.04 ± 0.01 corresponding to a 0.16 decrease in pHi, and no change in AACID in contralateral tissue. Inspection of AACID maps following PBS injection showed no differences. The use of DCA to induce a tumor specific pH change detectable by AACID CEST MRI is consistent with previous studies that have shown similar effects for lonidamine and topiramate. This study demonstrates that a single dose of DCA can be used as a pharmacological challenge to induced rapid tumor intracellular acidification.

Characterization of pHi Regulation by NBCe1/SLC4A4 Variants with Known Clinical Phenotypes in Human Immortalized Trabecular Meshwork Cells with a Red‐Shifted Genetically‐Encoded pH‐Indicator “pHire”

The electrogenic Na+/HCO3− cotransporter (NBCe1/SLC4A4) maintains systemic acid/base balance by reabsorbing HCO3− in the renal proximal tubule but also contributes to intracellular pH (pHi) regulation in the eye, nervous system, pancreas, and intestine. Human recessive mutations lead to diverse phenotypes (glaucoma, proximal renal tubular acidosis, poor dentition) and mouse models suggest some organ manifestations are partially penetrant. The objective of this study was to develop a full spectrum genetically‐encoded live‐imaging toolkit which could be adapted to assay NBCe1‐mediated pHi regulation across a variety of cell types to further characterize how NBCe1 recessive mutations and polymorphisms functionally interact with each other and wild‐type (WT) NBCe1 to produce alterations in cellular acid/base physiology. WT human NBCe1 (WT‐NBCe1) and a recently characterized human recessive mutation (Mut‐NBCe1) were tagged with GFP. Each construct was co‐expressed via transfection in human immortalized trabecular meshwork cells (TM5) with “pHire”, a red‐shifted (556/630nm ex/em) pH‐sensitive variant of mApple, to assay NBCe1 function through real‐time quantification of pHi via widefield epifluorescent live‐imaging. pHire successfully reported pHi of TM5 cells in response to H+ loading with NH4Cl pulses. Calibration of pHire revealed an in vitro pKa of 7.44, a 5‐fold linear fluorescence response from pH 6.5 to 8.0, and stability following acid loads (pH 5.0) and long imaging protocols (30 min). pHi of TM5 cells fell in response to CO2/HCO3−— buffered solution and displayed a modest Na+‐dependent recovery (dpHi/dt = 0.02 ± 0.01 pH units min−1 in CO2/HCO3−, −0.06 ± 0.02 in Na+‐free), consistent with intrinsic TM5‐cell NBCe1 activity. Transfection of WT‐NBCe1 did not consistently alter HCO3− recovery but accelerated pHi decrease in Na+‐free solution by 10‐fold (1017 ± 3%) relative to neighboring untransfected cells. Transfection with E91R‐NBCe1 failed to similarly potentiate acidification upon Na+ withdrawal (117 ± 81%), and transfection with Mut‐NBCe1 suppressed Na+‐dependent acidification to 72 ± 28% of untransfected cells. These data suggest that although both Mut‐NBCe1 and E91R‐NBCe1 are functionally “transporter‐null” they possess different capacities to impair endogenous WT‐NBCe1, possibly through dimerization and localization deficits. Such differences could manifest discretely in specific cell types and may underlie the partial penetrance of some NBCe1‐associated phenotypes.Support or Funding InformationFunding: T32‐DK007013 to AJR, DK092408, DK100227 and R25‐DK101405 to MFR.

The Effect of SLC4A11 Mutations on Intracellular pH

SLC4A11 is a ubiquitously expressed transporter showing Na+‐OH−, NH3‐H+ cotransport, and water permeability. SLC4A11 mutations have been associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED2) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). To address the role of known human SLC4A11 mutations on cellular physiology we measured intracellular pH regulation in PS120 fibroblasts that have been stably transfected with SLC4A11 mutant variants.Mutations were made in the Wild‐Type (WT) construct phCMV2‐hSLC4A11 by site‐directed mutagenesis. Mutants with a single amino acid change were obtained and referred to as R125H, C386R, V507I and N639A respectively. PS120 cells were stably transfected with those constructs and empty vector (EV) using SuperFect, and selected by Geneticin. For intracellular pH (pHi) measurement, PS120 cells grown on coverslips were loaded with a pH‐sensitive fluorescent dye, 2′7′‐Bis‐(2‐Carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF‐AM). The coverslip was mounted in a perfusion chamber (37°C) on the stage of a fluorescence microscope. Perfusion was performed with a Bicarbonate‐free solution (BF) to obtain stable baseline pH, followed by pulsing cells with 10 mM NH4Cl for 5 minutes, and changed back to BF perfusion until pHi was stable. Fluorescence ratio (495/440 nm) was converted to pHi following calibration with high K+/Nigericin. Results are means ± SE from 3 trials.The baseline pHi was 7.211 ± 0.136, 7.877 ± 0.056, 7.391 ± 0.069, 7.226 ± 0.232, 7.504 ± 0.104 and 7.359 ± 0.221 for WT, EV, R125H, C386R, V507I and N639A, respectively. The pHi increase from NH4Cl pulse was 0.486 ± 0.014, 0.161 ± 0.018, 0.298 ± 0.05, 0.307 ± 0.07, 0.304 ± 0.011 and 0.442 ± 0.084 respectively. The rate of pHi increase (ΔpH/min) was 2.184 ± 0.33, 0.469 ± 0.037, 1.004 ± 0.124, 1.024 ± 0.362, 1.17 ± 0.142 and 1.818 ± 0.405, respectively. The subsequent rate of pHi decrease in the presence of NH4Cl was (ΔpH/min) 0.153 ± 0.038, 0.006 ± 0.012, 0.012 ± 0.005, 0.011 ± 0.003, 0.033 ± 0.002 and 0.009 ± 0.007, respectively. Removal of NH4Cl‐BF resulted in maximum acidification of 1.205 ± 0.122, 0.343 ± 0.074, 0.671 ± 0.019, 0.409 ± 0.122, 0.747 ± 0.041 and 0.748 ± 0.048 pH units. NH4Cl induced pHi changes were WT > N639A > V507I > R125H > C386R > EV.Expression of SLC4A11 WT and mutants lower resting pHi relative to EV, consistent with a H+ permeability. NH4Cl induced pHi changes were consistent with reduction of NH3:H+ linked cotransport in SLC4A11 mutants.Support or Funding InformationNIH R01 EY08834‐23 & P30EY019008

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk

The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability, membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3days incubation in control supernatant, while it was prevented for up to 10days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

Role of Cl- -HCO3- exchanger AE3 in intracellular pH homeostasis in cultured murine hippocampal neurons, and in crosstalk to adjacent astrocytes.

A polymorphism of human AE3 is associated with idiopathic generalized epilepsy. Knockout of AE3 in mice lowers the threshold for triggering epileptic seizures. The explanations for these effects are elusive. Comparisons of cells from wild-type vs. AE3-/- mice show that AE3 (present in hippocampal neurons, not astrocytes; mediates HCO3- efflux) enhances intracellular pH (pHi ) recovery (decrease) from alkali loads in neurons and, surprisingly, adjacent astrocytes. During metabolic acidosis (MAc), AE3 speeds initial acidification, but limits the extent of pHi decrease in neurons and astrocytes. AE3 speeds re-alkalization after removal of MAc in neurons and astrocytes, and speeds neuronal pHi recovery from an ammonium prepulse-induced acid load. We propose that neuronal AE3 indirectly increases acid extrusion in (a) neurons via Cl- loading, and (b) astrocytes by somehow enhancing NBCe1 (major acid extruder). The latter would enhance depolarization-induced alkalinization of astrocytes, and extracellular acidification, and thereby reduce susceptibility to epileptic seizures. The anion exchanger AE3, expressed in hippocampal (HC) neurons but not astrocytes, contributes to intracellular pH (pHi ) regulation by facilitating the exchange of extracellular Cl- for intracellular HCO3- . The human AE3 polymorphism A867D is associated with idiopathic generalized epilepsy. Moreover, AE3 knockout (AE3-/- ) mice are more susceptible to epileptic seizure. The mechanism of these effects has been unclear because the starting pHi in AE3-/- and wild-type neurons is indistinguishable. The purpose of the present study was to use AE3-/- mice to investigate the role of AE3 in pHi homeostasis in HC neurons, co-cultured with astrocytes. We find that the presence of AE3 increases the acidification rate constant during pHi recovery from intracellular alkaline loads imposed by reducing [CO2 ]. The presence of AE3 also speeds intracellular acidification during the early phase of metabolic acidosis (MAc), not just in neurons but, surprisingly, in adjacent astrocytes. Additionally, AE3 contributes to braking the decrease in pHi later during MAc in both neurons and astrocytes. Paradoxically, AE3 enhances intracellular re-alkalization after MAc removal in neurons and astrocytes, and pHi recovery from an ammonium prepulse-induced acid load in neurons. The effects of AE3 knockout on astrocytic pHi homeostasis in MAc-related assays require the presence of neurons, and are consistent with the hypothesis that the AE3 knockout reduces functional expression of astrocytic NBCe1. These findings suggest a new type of neuron-astrocyte communication, based on the expression of AE3 in neurons, which could explain how AE3 reduces seizure susceptibility.

Perfusion of the gastric conduit during esophagectomy.

In esophageal cancer surgery, perfusion of the gastric conduit is a critical issue. Measurement of gastric intramucosal pH (pHi) is a method to identify anaerobic metabolism as a sign of impaired perfusion. In this study we aimed to monitor changes in the perfusion of the gastric conduit at key steps during and after esophagectomy. pHi was measured per- and postoperatively using intermittent gastric tonometry in 32 patients undergoing open, 65%, or video-assisted thoracoscopic esophagectomy for esophageal cancer. Measurements focused on the surgical steps when the vascular supply to the gastric conduit was altered. A tonometry catheter was successfully placed in all patients and a decrease in pHi (mean±SD) was observed from baseline to after the division of the short gastric vessels (7.33±0.07 to 7.29± 0.07, P =0.005). A further reduction after the ligation of the left gastric artery (7.26±0.08, P <0.001) and after final linear stapling the gastric conduit (7.15±0.13, P <0.001) was observed. Two hours after surgery, pHi increased (7.24±0.09, P =0.002). In contrast to open surgery, a trend towards less reduction in pHi was seen in thoracoscopic surgery. Patients with anastomotic leaks had lower pHi on the first postoperative day (7.12±0.05 vs. 7.27±0.08, P =0.040). It can be concluded that each surgical step altering the vascular supply to the gastric conduit resulted in detectable changes, however transient, in pHi. Patients with low pHi on the first postoperative day were more prone to have clinically relevant anastomotic leaks.

Photodynamic acidification therapy to reduce triple negative breast cancer growth in vivo.

e12574Background: Breast cancer, though much researched, continues to claim the lives of more than half a million women worldwide each year; triple negative breast cancer (TNBC) accounts for 15-25% of these mortalities. Treatment options for TNBC are limited to surgery, radiation, and a select few chemotherapies, to which the cancer often becomes resistant. Cancer cells maintain a more basic intracellular pH (pHi ) than healthy cells, making disruption of pH dynamics a potential target for treatment. The ability to induce a decrease of pHi, in a focal manner, would allow for treatment of cancers with a poor response to standard treatments or in cases where standard therapy is not feasible due to comorbidities or the location of the tumor. Methods: In our lab we were able to drop pHi in MDA-MB-231 TNBC, leading to apoptosis, using a photosensitizing agent in vitro. This technique was then tested in vivo using MDA-MB-231 TNBC cells in mice; cells were injected into the mammary fat-pads of athymic female mic...

Is the electrogenic Na/HCO3 cotransporter a CO2 channel?

It has now become clear that not all gases cross all biological membranes simply by dissolving into the lipid phase of the membrane, but that in some cases dissolved gases cross membranes via channels formed by integral membrane proteins.Currently, we know of three families of gas channels—the aquaporins (AQPs), rhesus (Rh) proteins, and urea transporters (UTs)—that can augment CO2 and/or NH3 permeability. In this study, we investigate the possibility that a fourth family exists, exemplified by the electrogenic Na/HCO3 cotransporter (NBCe1, SLC4A4), which may be a CO2 channel.We investigate the CO2 permeability of NBCe1 by combining electrophysiology experiments with mathematical modeling. In the electrophysiology work, we use microelectrodes to monitor intracellular pH (pHi) of oocytes injected with water (controls) or cRNA encoding human NBCe1‐A, which transports one Na+ and one CO3= (carbonate) ion into the cell. We determine the maximal rate of pHi change, (dpHi/dt)max, as we switch the extracellular solution from CO2/HCO3−‐free to (1) equilibrated (EQ) CO2/HCO3− at pH 7.50, or (2) an out‐of‐equilibrium (OOE) solution at the same pH, but containing only CO2 (“pure CO2”), or (3) an OOE solution containing only HCO3− (“pure HCO3−”).We find that exposing control oocytes to “pure HCO3−” elicits virtually no change in pHi, whereas exposing them to “pure CO2” or EQ CO2/HCO3− causes pHi to fall rapidly and at nearly the same rate. Exposing NBCe1‐A oocytes to “pure HCO3−” causes pHi to rise moderately fast (CO3= uptake), whereas exposing them to “pure CO2” causes pHi to fall rapidly (CO2 uptake). Intuition suggests that exposing an NBCe1‐A oocyte to EQ CO2/HCO3− will produce a pHi trajectory halfway between those of “pure HCO3−” and “pure CO2”. Surprisingly, we find that exposing NBCe1‐A oocytes to EQ causes pHi to fall about as rapidly as with “pure CO2”. Thus, NBCe1‐A, in the presence of HCO3−, appears to increase CO2 permeability.To assist us in interpreting our pHi data, we extended our 3D reaction‐diffusion mathematical model of CO2 fluxes across the oocyte membrane to include NBCe1‐mediated transmembrane fluxes of CO3= (informed by two‐electrode voltage clamp data). The model can reproduce the pHi data over the first 15 sec of a physiological experiment, for both “pure HCO3−” and “pure CO2”. However, reproducing the fast pHi decrease of EQ CO2/HCO3− requires that we increase CO2 permeability, thereby supporting the hypothesis that NBCe1 may conduct CO2 while transporting CO3=.Support or Funding InformationSupported by grants from the Office of Naval Research (N00014‐11‐1‐0889, N00014‐14‐1‐0716) and NIH (U01‐GM111251, K01‐DK107787)