Decrease In pHi Research Articles

Changes in Doppler parameters of portal pressure after interventional management of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has many options for management; some of them are complicated by development of portal hypertension (PHT). Doppler ultrasound is an effective method to diagnose and monitor PHT changes after HCC ablation procedures. The aim of this study is to investigate changes in portal pressure hemodynamics of HCC patients following treatment with different interventional strategies: radiofrequency ablation (RFA), microwave ablation (MWA), and transarterial chemoembolization (TACE). A total of 60 patients with HCC were divided into three main groups, and each group received a different type of therapy (RFA, MWA, and TACE). Full medical record and basic investigations were performed including Doppler ultrasound and upper GIT endoscopy for evaluation of PHT parameters, and then repeated after three months of ablation. RFA is associated with the increased splenic artery resistive index, while MWA has no significant impact on PHT indices. TACE has led to a marked increase in liver vascular index with significant decrease in hepatic artery resistive index and PHI after treatment. No significant changes in esophageal varices were observed by upper GIT endoscopy following all ablation methods. RFA is quite safe but associated with degree of PHT. On the contrary, TACE is associated with improved PHT parameters. MWA has no significant association to development of PHT following the technique. Doppler ultrasound could be used as a reliable and effective method of evaluation of PHT post ablation for HCC.

Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3- release from HCO3--secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na+-H+ exchanger (NHE) to pHi regulation for HCO3- secretion by cAMP and Ca2+ signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca2+-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3- secretion.

Influence of tetrapeptide tuftsin on intracellular pH of mouse peritoneal macrophages

It has been shown by the method of probe microfluorimetry of single cells that tuftsin at concentrations of 0.1 and 1.0 μg/mL, increasing the phagocytic activity of murine peritoneal macrophages, causes biphasic changes in the intracellular pH (pHi) over time. First there was a decrease in pHi, reaching the limit in 5 min of incubation. Then, the pHi value increased until reaching the maximum 30 min after the interaction between the cells and the agent. Afterwards, the observed parameter (pHi) did not vary up to minute 55. It was shown that there was no increase in intracellular pH on the addition of the Na+/H+-exchange blocker ethylisopropylamiloride to the incubation medium in the presence of tuftsin. This fact suggests that the observed increase in pHi during tuftsin treatment in the second phase of cell response is associated with the Na+/H+ exchange system.

Extracellular acidification leads to mitochondrial depolarization with following free radical formation in rat brain synaptosomes

Brain ischemia is accompanied by lowering of pHo and pHi. We investigated an influence of of acidosis on free radical formation in synaptosomes. Three models were used. 1) Strong extracellular acidification down to pHo 6.0. 2) Moderate extracellular acidification down to pHo 7.0 3) Intracellular acidification induced by addition of 1 mM amiloride corresponding to pHi decrease down to 6.65. We have shown that both types of extracellular acidification, but not intracellular acidification, increase DCFDA fluorescence by calcium-independent way that reflects free radical formation. These three treatments induce the rise of the dihydroethidium fluorescence that reports synthesis of superoxide anion. However, the impact of low pHi on superoxide anion synthesis was less than induced by moderate extracellular acidification. Mitochondrial uncoupler CCCP did not inhibit an increase of fluorescence of both dyes at pHo 6.0. In contrast, superoxide anion synthesis at pHo 7.0 was almost completely eliminated by CCCP. Furthermore, using fluorescent dyes JC-1 and rhodamine-123, we confirmed that decrease of pHo leads to mitochondria depolarization. Low pHi was not effective. Iron chelator deferoxamine and antioxidant ionol are inhibits pH-induced increase of DCFDA fluorescence, but does not influenced mitochondria depolarization. We are failed to found sodium influx monitored by fluorescent dye Sodium Green in case of low pHo. Involving of plasma membrane receptor which is distinct from acid-sensitive ion channels (ASIC) and electron transport chain of mitochondria for moderate acidification can be suggested. Action of strong acidification seems to be mediated by release of iron from proteins. We have shown that low pHo led to oxidative stress in neuronal presynaptic endings that might underlie the long term irreversible changing in synaptic transmission.

ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH.

BackgroundChemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. Carbonic anhydrase 9 (CA9) has been shown to be upregulated in the drug-resistant tongue cancer cell line Tca8113/PYM and to be associated with drug resistance. However, the mechanisms regulating CA9 expression and its role in drug resistance remain unclear.MethodsBioinformatic and experimental analysis involving ChIP and luciferase reporter assays were used to validate Zinc finger E-box-binding homeobox 1 (ZEB1) as a transcriptional regulator of CA9. Gene expression and protein levels were evaluated by quantitative RT-PCR and western blotting, respectively. Sensitivity to chemotherapy was examined using the MTS assay and Hoechst staining and analysis caspase-3 activity to evaluate changes in apoptosis. Intracellular pH (pHi) was measured using fluorescent pH-indicator BCECF-AM. Protein expression in patient tissue samples was examined by immunohistochemistry and survival of tongue cancer patients from which these samples were derived was also analyzed.ResultsZEB1 bound to the promoter of CA9 to positively regulate CA9 expression in tongue cancer cells. Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells. CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation. Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation. Importantly, a positive correlation was observed between ZEB1 and CA9 protein expression in tongue cancer tissues, and expression of these proteins associated with a poor prognosis for patients.ConclusionOur finding that tumor cells regulate pHi in response to chemotherapy provides new insights into mechanisms of drug resistance during cancer treatment. Identification of the ZEB1–CA9 signaling axis as a biomarker of poor prognosis in tongue cancer will be valuable in future development of therapeutic strategies aimed at improving treatment efficacy, especially in terms of drug resistance associated with this disease.

Impact of Calcineurin Homologous Protein and NHE1 on Lung Cancer Cell Adaptation to Hypoxia and Serum Deprivation

The Sodium Hydrogen Exchanger Isoform 1 (NHE1) is vital in maintaining pH homeostasis and is regulated by several proteins including both isoforms of Calcineurin Homologous Protein 1 and 2 (CHP1 and CHP2). The metabolic rate of cancer cells is elevated and in hypoxic and serum deprived developing tumors intracellular pH (pHi) is significantly decreased. Cells must adopt survival mechanisms to escape these conditions. Earlier work suggests that CHP1 is required for basic NHE1 proton transport while CHP2 increases the proton transport kinetics. CHP2 expression may activate NHE1 allowing for pHi homeostasis. Therefore, we investigated CHP binding to NHE1 as a means for adapting to hypoxic and serum deprivation conditions. Using Quartz Crystal Microbalance (QCMd) we measured protein‐protein interactions between NHE1 and CHP1 or CHP2. We also cultured H1299 cells with chemically induced hypoxia using dimethyloxaloylglycine (DMOG) and with serum deprivation in H1299 lung cancer cells. Cell exposed to hypoxia and serum deprivation showed a decreased pHi 0.2‐0.4 pH units. When NHE1 is inhibited by 5‐(N‐Ethyl‐N‐isopropyl) amiloride (EIPA) the decrease in pHi was significantly lower than non EIPA treated cells, indicating NHE1 supports cell viability. The impact of hypoxia and serum deprivation on intracellular pH was also investigated in H1299 cells with reduced NHE1 or CHP2 expression (shRNA knockdown). Together this work suggests that CHP2 plays an important role in cell survival in lung tumor development.

Na+/H+ Exchange Blockers Reveal the Existence of a Skeletal Muscle Ca2+/H+ Exchanger, which is Altered in Malignant Hyperthermia Muscle Cells

In resting skeletal muscle fibers intracellular pH (pHi) is kept constant at a relatively alkaline level. The transporters involved in maintaining muscle pHi at rest are the Na+/H+ exchange system (NHE), and to a lesser extent the Na+- and Cl--dependent bicarbonate dependent transport systems. Many studies conducted in nerve and smooth cells have suggested a link between changes in intracellular free Ca2+ ([Ca2+]i) and changes in intracellular pH (pHi) suggesting a Ca2+/H+ exchange (Daugirdas et al, 1995). Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered by inhalational anesthetics and depolarizing neuromuscular blocking agents. We have measured [Ca2+]i and pHi simultaneously in Wt and heterozygous R163C myotubes using double barreled Ca2+-selective and single barreled pH+-selective microelectrodes. MH cells have a higher [Ca2+]i and a lower pHi than Wt cells. Treatment of both MH and Wt cells with the NHE inhibitors dimethylamiloride (DMA) or cariporide, caused an increase in [Ca2+]i and a decrease in pHi in a dose dependent manner. These effects were more prominent in MH than Wt myotubes. YM-244769, a high affinity reverse mode NCX3 blocker, did not modify [Ca2+]i overload or the drop in pHi elicited by DMA or cariporide in either cell type, suggesting that the change in [Ca2+]i was not the consequence to an activation of the reverse form of the exchanger. Gd3+ and dantrolene respectively were able to partially inhibit or fully reverse the DMA or cariporide-mediated elevation of [Ca2+]i and acidification in both types of cells. These results suggest the existence of a Ca2+/H+ exchange in skeletal myotubes, which appears to be altered in MH muscle cells.

Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid-base disturbances in the armoured catfish, Pterygoplichthys pardalis.

Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid-base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2-3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15-7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid-base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.

Extracellular H+ induces Ca2+ signals in respiratory chemoreceptors of zebrafish.

Neuroepithelial cells (NECs) of the fish gill are respiratory chemoreceptors that detect changes in O2 and CO2/H(+) and are homologous to type I cells of the mammalian carotid body. In zebrafish (Danio rerio), stimulation of NECs by hypoxia or hypercapnia initiates inhibition of K(+) channels and subsequent membrane depolarisation. The goal of the present study was to further elucidate, in zebrafish NECs, the signalling pathways that underlie CO2/H(+) sensing and generate intracellular Ca(2+) ([Ca(2+)]i) signals. Breathing frequency was elevated maximally in fish exposed to 5 % CO2 (~37.5 mmHg). Measurement of [Ca(2+)]i in isolated NECs using Fura-2 imaging indicated that [Ca(2+)]i increased in response to acidic hypercapnia (5 % CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5 % CO2, pH 7.6). Measurement of intracellular pH (pHi) using BCECF demonstrated a rapid decrease in pHi in response to acidic and isohydric hypercapnia, while isocapnic acidosis produced a smaller change in pHi. Intracellular acidification was reduced by the carbonic anhydrase inhibitor, acetazolamide, without affecting [Ca(2+)]i responses. Moreover, intracellular acidification using acetate (at constant extracellular pH) was without effect on [Ca(2+)]i. The acid-induced increase in [Ca(2+)]i persisted in the absence of extracellular Ca(2+) and was unaffected by Ca(2+) channel blockers (Cd(2+), Ni(2+) or nifedipine). The results of this study demonstrate that, unlike type I cells, extracellular H(+) is critical to the hypercapnia-induced increase in [Ca(2+)]i in NECs. The increase in [Ca(2+)]i occurs independently of pHi and appears to originate primarily from Ca(2+) derived from intracellular stores.

Upregulation of NHE1 protein expression enables glioblastoma cells to escape TMZ-mediated toxicity via increased H+extrusion, cell migration and survival

Sodium-hydrogen exchanger isoform 1 (NHE1) plays a role in survival and migration/invasion of several cancers and is an emerging new therapeutic target. However, the role of NHE1 in glioblastoma and the interaction of NHE1 expression and function in glioblastoma cells with cytotoxic temozolomide (TMZ) therapy remain unknown. In this study, we detected high levels of NHE1 protein only in primary human glioma cells (GC), glioma xenografts and glioblastoma, but not in human neural stem cells or astrocytes. GC exhibited an alkaline resting pHi (7.46±0.04) maintained by robust NHE1-mediated H(+) extrusion. GC treatment with TMZ for 2-24h triggered a transient decrease in pHi, which recovered by 48h and correlated with concurrent upregulation of NHE1 protein expression. NHE1 protein was colocalized with ezrin at lamellipodia and probably involved in GC migration. The TMZ-treated GC exhibited increased migration and invasion, which was attenuated by addition of NHE1 inhibitor HOE-642. Most importantly, NHE1 inhibition prevented prosurvival extracellular signal-regulated kinase activation and accelerated TMZ-induced apoptosis. Taken together, our study provides the first evidence that GC upregulate NHE1 protein to maintain alkaline pHi. Combining TMZ therapy with NHE1 inhibition suppresses GC migration and invasion, and also augments TMZ-induced apoptosis. These findings strongly suggest that NHE1 is an important cytoprotective mechanism in GC and presents a new therapeutic strategy of combining NHE1 inhibition and TMZ chemotherapy.

Benzimidazole derivative, BMT-1, induces apoptosis in multiple myeloma cells via a mitochondrial-mediated pathway involving H+/K+-ATPase inhibition

2-(1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol (BMT-1), a bicyclic compound, belongs to the benzimidazole group and consists of the fusion of benzene and imidazole. The objective of the present study was to assess the effect of BMT-1 on the proliferation of multiple myeloma (MM) cells and identify whether BMT-1 induces apoptosis in MM cells. Our results showed a dose- and time-dependent decrease in the proliferation of MM cells treated with BMT-1. Further studies revealed that the antiproliferative effects of BMT-1 were caused by induction of apoptosis with activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in MM cells. In addition, BMT-1 induced the loss of mitochondrial membrane potential resulting in the activation of caspase-8 and-9. Furthermore, the MM cells treated with BMT-1 showed a more acidic intracellular pH (pHi) as indicated by a lower FL1/FL2 ratio caused by inhibition of H+/K+-ATPase. Collectively, these findings demonstrated that a decrease in pHi, caused by H+/K+-ATPase inhibition induced by BMT-1, triggered the dysfunction of the mitochondria resulting in the apoptosis of MM cells. Therefore, BMT-1 may be used as a lead compound for the design and development of new agents with which to treat MM and other forms of cancer.

Depolarization of plasma membrane of rat brain synaptosomes at extra- and intracellular acidification

To elucidate whether a decrease in pHo and pHi has the same effect on presynaptic plasma membrane depolarization, we compared the effects of 1 mM of amiloride which decreased pHi down to 6.65 and incubation medium acidification down to pHo 7.0 and 6.0 on DiSC3(5) dye fluorescence. Measurements were performed with and without the mitochondrial toxins: rotenone (10 μM) + oligomycin (5 μg/mL). In the presence of toxins the change in DiSC3(5) fluorescence was specific for plasma membrane potential. In the second case, DiSC3(5) signal represented the total potential of mitochondria and plasma membrane. It was shown that decreasing of pHo works both in the presence of mitochondrial toxins and also in their absence. Amiloride is able to increase DiSC3(5) fluorescence only in the presence of the mixture of rotenone + oligomycin. Therefore, unlike the mitochondrial potential, the plasma membrane potential is sensitive to a decrease in pHi and pHo.

The distinct roles of anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1) in fluid and electrolyte absorption in the murine small intestine

The mixing of gastric and pancreatic juice subjects the jejunum to unique ionic conditions with high luminal CO2 tension and HCO3 − concentration. We investigated the role of the small intestinal apical anion exchangers PAT-1 (Slc26a6) and DRA (Slc26a3) in basal and CO2/HCO3 −-stimulated jejunal fluid absorption. Single pass perfusion of jejunal segments was performed in anaesthetised wild type (WT) as well as in mice deficient in DRA, PAT-1, Na+/H+ exchanger 3 (NHE3) or NHE2, and in carbonic anhydrase II (CAII). Unbuffered saline (pH 7.4) perfusion of WT jejunum resulted in fluid absorption and acidification of the effluent. DRA-deficient jejunum absorbed less fluid than WT, and acidified the effluent more strongly, consistent with its action as a Cl−/HCO3 − exchanger. PAT-1-deficient jejunum also absorbed less fluid but resulted in less effluent acidification. Switching the luminal solution to a 5 % CO2/HCO3 − buffered solution (pH 7.4), resulted in a decrease in jejunal enterocyte pHi in all genotypes, an increase in luminal surface pH and a strong increase in fluid absorption in a PAT-1- and NHE3- but not DRA-, CAII, or NHE2-dependent fashion. Even in the absence of luminal Cl−, luminal CO2/HCO3 − augmented fluid absorption in WT, CAII, NHE2- or DRA-deficient, but not in PAT-1- or NHE3-deficient mice, indicating the likelihood that PAT-1 serves to import HCO3 − and NHE3 serves to import Na+ under these circumstances. The results suggest that PAT-1 plays an important role in jejunal Na+HCO3 – reabsorption, while DRA absorbs Cl− and exports HCO3 − in a partly CAII-dependent fashion. Both PAT-1 and DRA significantly contribute to intestinal fluid absorption and enterocyte acid/base balance but are activated by different ion gradients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1381-2) contains supplementary material, which is available to authorized users.

Intracelluar pH (pHi) Measurements in the In Vitro Tadpole Brainstem: Direct Correlations between Changes in pHi and Ventilation

Central respiratory chemoreceptors measure pH in the brain stem and are an integral part of the neural circuitry that modulates respiratory rhythmogenesis, specifically in response to hypercapnic acidosis. Central respiratory chemore- ceptor membrane potential and/or action potential firing rate are altered in response to changes in intracellular pH (pHi), which changes with the hydration of CO2 in both the extracellular and intracellular space, however the role of cellular changes in chemoreceptor properties on respiratory motor output has been difficult to identify. We studied whole nerve respiratory activity while simultaneously visualizing pHi dynamics using the pH-sensitive dye, BCECF, in the spontane- ously active in vitro tadpole brainstem. The isolated, superfused tadpole brainstem is well oxygenated and retains synaptic connectivity among respiratory central pattern generators, central respiratory chemoreceptors, and respiratory motor neu- ronsunder physiological conditions, where mammalian preparations do not. An ammonium prepulse was used to selec- tively induce a decrease in pHi. Our results show intracellular pH is regulated differently in cells located in chemosensi- tive and non-chemosensitive regions of the tadpole brainstem during hypercapnia. We were also able to show an inverse correlation between pHi in cells located in chemosensitive regions of the tadpole brainstem and whole nerve respiratory- related activity. Using this approach, the microenvironment of individual cells may be manipulated while monitoring real time changes in pHi, neuronal activity and ventilatory-related activity to elucidate the role of a variety of signals in elicit- ing changes in ventilation.

Transient outwardly rectifying A currents are involved in the firing rate response to altered CO2 in chemosensitive locus coeruleus neurons from neonatal rats

The effect of hypercapnia on outwardly rectifying currents was examined in locus coeruleus (LC) neurons in slices from neonatal rats [postnatal day 3 (P3)-P15]. Two outwardly rectifying currents [4-aminopyridine (4-AP)-sensitive transient current and tetraethyl ammonium (TEA)-sensitive sustained current] were found in LC neurons. 4-AP induced a membrane depolarization of 3.6 ± 0.6 mV (n = 4), while TEA induced a smaller membrane depolarization of 1.2 ± 0.3 mV (n = 4). Hypercapnic acidosis (HA) inhibited both currents. The maximal amplitude of the TEA-sensitive current was reduced by 52.1 ± 4.5% (n = 5) in 15% CO2 [extracellular pH (pHo) 7.00, intracellular pH (pHi) 6.96]. The maximal amplitude of the 4-AP-sensitive current was reduced by 34.5 ± 3.0% (n = 6) in 15% CO2 (pHo 7.00, pHi 6.96), by 29.4 ± 6.8% (n = 6) in 10% CO2 (pHo 7.15, pHi 7.14), and increased by 29.0 ± 6.4% (n = 6) in 2.5% CO2 (pHo 7.75, pHi 7.35). 4-AP completely blocked hypercapnia-induced increased firing rate, but TEA did not affect it. When LC neurons were exposed to HA with either pHo or pHi constant, the 4-AP-sensitive current was inhibited. The data show that the 4-AP-sensitive current (likely an A current) is inhibited by decreases in either pHo or pHi. The change of the A current by various levels of CO2 is correlated with the change in firing rate induced by CO2, implicating the 4-AP-sensitive current in chemosensitive signaling in LC neurons.

Effects of non-steroidal estrogen diethylstilbestrol on pH and ion transport in the mantle epithelium of a bivalve Anodonta cygnea

Freshwater bivalves are used as sentinel organisms to detect pollutants effects in the aquatic environment due to their sedentary nature, filter-feeding behaviour. We aimed to determine the in vivo, ex vivo and in vitro influence of Diethylstilbestrol (DES), a widely used synthetic non-steroidal estrogen and endocrine disruptor, in Anodonta cygnea shell growth mechanisms. For that, in vivo exposure to DES (0.75μM) during 15 days, in vitro and ex vivo exposure of outer mantle epithelium (OME) cells to DES (0.75μM), were performed followed by study of short-circuit current (Isc), transepithelial potential (Vt) and transepithelial conductance (Gt) as well as identification of membrane transport systems and intracellular pH (pHi). Our results show that in vivo exposure to DES decreases in 30% the OME Isc and ex vivo addition of DES to the basolateral side of OME also induced Isc decrease. Several membrane transporters such as V-type ATPases, Na+/H+ exchangers, Na+–K+ pump, Na+-driven and Na+-independent HCO3−/Cl− transporters and Na+/HCO3− co-transporter were identified as responsible for pHi maintenance in OME and noteworthy, DES caused a pHi decrease in OME cells similar to the effect observed when OME cells were exposed to 4,4′-diisothiocyanostilbene disulfonic acid (DIDS), an inhibitor of several bicarbonate membrane transporters. The addition of DIDS after OME cells exposure to DES did not cause any alteration. We concluded that DES is able to modulate membrane ion transport and pHi in the OME of A. cygnea and that this effect seems to be due to inhibition of HCO3−/Cl− co-transporters present on the basolateral membrane.

Yeast adaptation to weak acids prevents futile energy expenditure

Weak organic acids (WOAs) are widely used preservatives to prevent fungal spoilage of foods and beverages. Exposure of baker's yeast Saccharomyces cerevisiae to WOA leads to cellular acidification and anion accumulation. Pre-adaptation of cultures reduced the rate of acidification caused by weak acid exposure, most likely as a result of changes in plasma membrane or cell wall composition. In order to adapt to sublethal concentrations of the acids and grow, yeast cells activate ATP consuming membrane transporters to remove protons and anions. We explored to what extent ATP depletion contributes to growth inhibition in sorbic or acetic acid treated cells. Therefore, we analyzed the effect of the reduction of proton and anion pumping activity on intracellular pH (pHi), growth, and energy status upon exposure to the hydrophilic acetic acid (HA) and the lipophilic sorbic acid (HS). ATP concentrations were dependent on the severity of the stress. Unexpectedly, we observed a stronger reduction of ATP with growth reducing than with growth inhibitory concentrations of both acids. We deduce that the not the ATP reduction caused by proton pumping, but rather the cost of sorbate anion pumping contributes to growth inhibition. A reduction of proton pumping activity may reduce ATP consumption, but the resulting decrease of pHi affects growth more. ATP utilization was differentially regulated during moderate and severe stress conditions. We propose that the energy depletion alone is not the cause of growth inhibition during HA or HS stress. Rather, the cells appear to reduce ATP consumption in high stress conditions, likely to prevent futile cycling and maintain energy reserves for growth resumption in more favorable conditions. The mechanism for such decision making remains to be established.

Activation of ciliary beating by a mucolytic agent, ambroxol, in bronchioles of mice

Ambroxol has been used as a mucolytic agent in pulmonary diseases. In this study, we studied the effects of ambroxol on ciliary beat frequency (CBF) and ciliary bend amplitude (CBA) of the bronchioles. Mice were anestetized by pentobarbital sodium (70mg/kg, ip) and bronchiolar ciliary cells were isolated from lungs by an elastase treatment. In a HCO3−‐containing solution, ambroxol increased CBA and CBF in a concentration dependent manner: ambroxol (10μM) increased CBA and CBF by 30 % within 15 min. However, in a HCO3−‐free solution, it did not induce any increase in CBA and CBF, although the switch from the HCO3−‐containing solution to the HCO3−‐free solution increased them. Measurement of intracellular pH (pHi) demonstrated that ambroxol (10 μM) increased pHi gradually in bronchiolar ciliary cells. These suggest that an increase in pHi induced by ambroxol increases CBA and CBF. To confirm the effects of pHi on CBA and CBF, an NH4+ pulse was applied. An increase in pHi increased CBA and CBF, whereas a decrease in pHi decreased them. Diisothiocyanostilbene‐disulfonic acid (DIDS) abolished the increases in CBA and CBF induced by ambroxol. In bronchiolar ciliary cells, ambroxol increases pHi via activation of HCO3−/Cl− exchanges, which increases CBA and CBF.

Basigin interacts with both MCT1 and MCT2 in murine spermatozoa

Lactate is provided to spermatogenic cells by Sertoli cells as an energy substrate and its transport is regulated by H(+)-monocarboxylate co-transporters (MCTs). In the case of several cell types it is known that MCT1 is associated with basigin and MCT2 with embigin. Here we demonstrate co-localization and co-immunoprecipitation of basigin with both MCT1 and MCT2 in sperm, whereas no interaction with embigin was detectable. An investigation of the functional activity of MCT proteins revealed that it was mainly the application of L-lactate which resulted in a decrease in pH(i) . The pH(i) changes were blocked with α-cyano-4-OH cinnamate and the preference for L-lactate-as opposed to D-Lactate-was demonstrated by the determination of ATP after exposure to both lactate isomers. We propose that basigin interacts with MCT1 and MCT2 to locate them properly in the membrane of spermatogenic cells and that this may enable sperm to utilize lactate as an energy substrate contributing to cell survival.

A New view of Insulin Action

Insulin not only stimulates the Na-K-pump,but by stimulating the Na:H exchange pump increases intracellular pH, pHi. Fidelman et al (1982) found, as predicted by thermodynamic theory, the effect of insulin on glycolysis varied linearly with log[Na]o with the insulin effect being converted from stimulation of glycolysis to a 51% inhibition at 0.12 mM Nao.Zierler & Rabinowitz (1964) demonstrated in the forearm of human males that levels of insulin too low to affect glucose uptake, as little as 38 micro units per ml, was sufficient to stimulate the Na-K-pump, as reflected by increased potassium uptake, while producing no effect upon glucose uptake. This one experiment forces us to realize that the main action of insulin is not to regulate blood glucose levels, but to regulate the Na-K-pump and Na:H exchange. Since the Na-K-pump uses about 25% of the ATP production in a resting muscle, stimulation of the Na-K-pump increases the consumption of ATP so much that at high levels of stimulation, it is necessary to get more glucose into the cell to manufacture more ATP.Since insulin decreases Nai, elevates pHi, and increases production of ATP, one would expedt that the decreased insulin, such as seen in diabetes or in fasting would result in the reverse of these changes. Lowering plasma levels of insulin in rats by small doses of streptozotocin or by fasting produced an increase in Nai of about 30%, a decrease in pHi of 0.15 units, and a 24% decrease in ATPi.The action of thiazide diuretics to cause type 2 diuretics has been shown to be due to their ability to cause potassium loss. Not surprisingly, diets with large amounts of potassium and small amounts of sodium have been shown to reverse diabetes.