Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pH_c) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pH_c by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pH_c causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pH_c in regulating the metabolic program of yeast cells.     

Effects of exogenous phytohormones on intracellular pH of Petunia hybrida pollen grains

The effects of exogenous phytohormones (IAA, ABA, and GA₃) on the intracellular (cytoplasmic) pH (pH_c) in ungerminating and germinating petunia (Petunia hybrida L.) pollen grains were studied. The pH_c values were measured with fluorescein diacetate. In ungerminating pollen grains, all phytohormones reduced pH_c relatively rapidly; after 10–15 min, initial value was restored. In germinating pollen grains, IAA and ABA induced a relatively rapid cytosol alkalization, which was not reversed during experiment. GA₃ acidified the cytosol, i.e., exerted the effect similar to that in ungerminating pollen grains. Sodium orthovanadate suppressed completely the hormone-induced pH_c shift toward alkaline values in germinating pollen grains, whereas this inhibitor did not affect pH_c in the absence of phytohormones. Sodium orthovanadate also slowed the recovery of pH_c after hormone-induced cytoplasm acidification in ungerminating pollen grains, reduced pH_c in control ungerminating grains, and weakened substantially the effects of all phytohormones on these pollen grains. On the basis of these results, we suggested that physiological activities of phytohormones in this system were mediated by pH_c modulation, namely, a transient disturbance in the cytosolic pH homeostasis, which could trigger further phytohormone-induced cell responses. We concluded that a hormone-induced cytoplasm alkalization in pollen grains was mediated by the activity of their plasma membrane H⁺-ATPase and that this proton pump was involved in the control of pH_c in both germinating and ungerminating pollen grains.     

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.     

Altered proton extrusion in cells adapted to growth at low extracellular pH

Intracellular pH (pH_i) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pH_i in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pH_i relative to the extracellular pH (pH_e). To test the ability of low pH_e adapted cells to respond to a pH_i lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pH_i during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pH_e = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pH_i was consistently elevated in adapted cells as compared with cells grown at normal pH_e = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pH_i of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na⁺/H⁺ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl⁻/HCO⁻₃ exchange inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification. J. Cell. Physiol. 173:397–405, 1997. © 1997 Wiley-Liss, Inc.     

Upregulation of Na/H exchanger by the AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O₂ deficiency glycolytic degradation of glucose leads to formation of lactate and H⁺, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H⁺ export. The present study explored whether AMPK influences Na⁺/H⁺ exchanger (NHE) activity and/or Na⁺-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH_i) was estimated utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺-dependent re-alkalinization (ΔpH_i) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH_i and ΔpH_i in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly decreased by AMPK inhibitor compound C (10 μM). The effect of AICAR on pH_i and ΔpH_i was blunted in the presence of the Na⁺/H⁺ exchanger inhibitor cariporide (10 μM), but not by the H⁺ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.     

Reduction of Proteinuria through Podocyte Alkalinization

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pH_i). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pH_i.     

Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V‐ATPase

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP‐dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V‐ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V‐ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V‐ATPase is also regulated by glucose in the Min6 β‐cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose‐sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.     

Independent and Additive Effects of Glutamic Acid and Methionine on Yeast Longevity

It is established that glucose restriction extends yeast chronological and replicative lifespan, but little is known about the influence of amino acids on yeast lifespan, although some amino acids were reported to delay aging in rodents. Here we show that amino acid composition greatly alters yeast chronological lifespan. We found that non-essential amino acids (to yeast) methionine and glutamic acid had the most significant impact on yeast chronological lifespan extension, restriction of methionine and/or increase of glutamic acid led to longevity that was not the result of low acetic acid production and acidification in aging media. Remarkably, low methionine, high glutamic acid and glucose restriction additively and independently extended yeast lifespan, which could not be further extended by buffering the medium (pH 6.0). Our preliminary findings using yeasts with gene deletion demonstrate that glutamic acid addition, methionine and glucose restriction prompt yeast longevity through distinct mechanisms. This study may help to fill a gap in yeast model for the fast developing view that nutrient balance is a critical factor to extend lifespan.     

Sensitivity of sea urchin fertilization to pH varies across a natural pH mosaic

In the coastal ocean, temporal fluctuations in pH vary dramatically across biogeographic ranges. How such spatial differences in pH variability regimes might shape ocean acidification resistance in marine species remains unknown. We assessed the pH sensitivity of the sea urchin Strongylocentrotus purpuratus in the context of ocean pH variability. Using unique male–female pairs, originating from three sites with similar mean pH but different variability and frequency of low pH (pH_T ≤ 7.8) exposures, fertilization was tested across a range of pH (pH_T 7.61–8.03) and sperm concentrations. High fertilization success was maintained at low pH via a slight right shift in the fertilization function across sperm concentration. This pH effect differed by site. Urchins from the site with the narrowest pH variability regime exhibited the greatest pH sensitivity. At this site, mechanistic fertilization dynamics models support a decrease in sperm–egg interaction rate with decreasing pH. The site differences in pH sensitivity build upon recent evidence of local pH adaptation in S. purpuratus and highlight the need to incorporate environmental variability in the study of global change biology.     

Auxin causes oscillations of cytosolic free calcium and pH inZea mays coleoptiles

In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pH_c), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pH_c/, _m, Ca²⁺/ _m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3–7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations in _m, pH_c and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. The _m oscillations are attributed to changes in activity of the H⁺-extrusion pump at the plasmalemma, triggered off by pH and controlled by pH regulation (pH oscillation). The origin of the pH_c and Ca²⁺ changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca²⁺ changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.Abbreviations and symbols FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - pH_c cytosolic pH - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - _m membrane potential difference (mV)     

Extension of Yeast Chronological Lifespan by Methylamine

Background

Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth.

Methodology/Principal Findings

The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase.

Conclusion/Significance

We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.     

Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell

The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl⁻) in regulation of lysosomal acidification [intra-lysosomal pH (pH_lys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl⁻ condition elevated pH_lys and reduced the intra-lysosomal Cl⁻ concentration ([Cl⁻]_lys) via reduction of cytosolic Cl⁻ concentration ([Cl⁻]_c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G₀/G₁ arrest without induction of apoptosis. We also studied effects of direct modification of H⁺ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H⁺-ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na⁺/H⁺ exchanger (NHE)] elevated pH_lys and decreased [Cl⁻]_lys associated with inhibition of cell proliferation via induction of G₀/G₁ arrest similar to the culture under a low Cl⁻ condition. However, unlike low Cl⁻ condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl⁻]_c compared with low Cl⁻ condition. These observations suggest that the lowered [Cl⁻]_c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl⁻ is a key factor of lysosome acidification and autophagy.     

Light-Induced Changes in Cytosolic pH in Leaf Cells of Egeria densa: Measurements with pH-Sensitive Microelectrodes

Light-induced changes of cytosolic pH (pH_c) and the plasmalemmapotential (E_m) in dark-adapted leaf cells of the aquatic plant,Egeria densa were measured simultaneously with double-barreledpH-sensitive microelectrodes. Upon illumination, pH_c increasedtransiently and then decreased to a level that was lower thanthe original value, while the plasmalemma was greatly hyperpolarizedafter an initial small depolarization. DCMU inhibited the light-inducedchanges in both pH_c and E_m. DCMU acted without directly inhibitingthe electrogenic proton pump in the plasmalemma since a decreasein pH_c caused by treatment with butyrate (H⁺-loading) hyperpolarizedthe plasmalemma in DCMU-pretreated cells. N.N-Dicyclohexylcarbodiimide(DCCD) also inhibited the light-induced changes in both pH_cand E_m. This result may be explained by direct inhibition ofthe proton pump in the plasmalemma by DCCD since the decreasein pH_c caused by butyrate did not induce membrane hyperpolarizationin DCCD-treated leaf cells. Fusicoccin induced membrane hyperpolarizationand slight acidification of the cytosol. DCCD inhibited thefusicoccin-induced changes in both pH_c and E_m. The mechanismof the light-induced changes in pH_c is discussed in relationto activities of the proton pump in the plasmalemma and photosynthesis. (Received January 10, 1994; Accepted June 9, 1994)     

Light-induced cytoplasmic pH changes and their interrelation to the activity of the electrogenic proton pump in Riccia fluitans

In green thallus cells of the aquatic liverwort Riccia fluitans light-induced pH changes have been measured, using a turgor-resistant pH-sensitive microelectrode. (1) Light-off/-on causes oscillations of the cytoplasmic pH (pH_c), as well as of the membrane potential difference across the plasmalemma (ψ). Beside the well-known ψ_m changes, the first detectable pH_c change following light-off is a transient acidification of about 0.3 pH units, whereas light-on causes a transient alkalinization of roughly 0.4 pH units. (2) 1 μM DCMU eliminates these transients. (3) In the presence of 0.2 mM procaine, which alkalizes the cytoplasm to over pH 8, the light-induced ψ_m transients are enhanced, but are almost absent, if pH_c is acidified to 6.9 by 1 mM acetate. It is suggested that the transient light-induced changes in pH_c are caused by light-dependent proton translocation across the thylakoid membranes, and it is concluded that the subsequent changes in ψ_m are essentially the result of altered activities of the electrogenic proton pump in the plasmalemma, due to the observed fluctuations of its substrate, the proton.     

Ocean acidification and kelp development: Reduced pH has no negative effects on meiospore germination and gametophyte development of Macrocystis pyrifera and Undaria pinnatifida

The absorption of anthropogenic CO₂ by the oceans is causing a reduction in the pH of the surface waters termed ocean acidification (OA). This could have substantial effects on marine coastal environments where fleshy (non‐calcareous) macroalgae are dominant primary producers and ecosystem engineers. Few OA studies have focused on the early life stages of large macroalgae such as kelps. This study evaluated the effects of seawater pH on the ontogenic development of meiospores of the native kelp Macrocystis pyrifera and the invasive kelp Undaria pinnatifida, in south‐eastern New Zealand. Meiospores of both kelps were released into four seawater pH treatments (pH_T 7.20, extreme OA predicted for 2300; pH_T 7.65, OA predicted for 2100; pH_T 8.01, ambient pH; and pH_T 8.40, pre‐industrial pH) and cultured for 15 d. Meiospore germination, germling growth rate, and gametophyte size and sex ratio were monitored and measured. Exposure to reduced pH_T (7.20 and 7.65) had positive effects on germling growth rate and gametophyte size in both M. pyrifera and U. pinnatifida, whereas, higher pH_T (8.01 and 8.40) reduced the gametophyte size in both kelps. Sex ratio of gametophytes of both kelps was biased toward females under all pH_T treatments, except for U. pinnatifida at pH_T 7.65. Germling growth rate under OA was significantly higher in M. pyrifera compared to U. pinnatifida but gametophyte development was equal for both kelps under all seawater pH_T treatments, indicating that the microscopic stages of the native M. pyrifera and the invasive U. pinnatifida will respond similarly to OA.     

Persistence of Streptococcus mutans in Stationary-Phase Batch Cultures and Biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (±8) days, and an average of 2.7 × 10⁵ CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.     

The Role of the Plasma Membrane Proton Pump in Short-term pH Regulation in the Aquatic Liverwort Riccia fluitans L.

The question is raised, to what extent is the plasma membraneproton pump involved in short-term pH regulation of plant cells?For this purpose the cytosolic pH (pH_c) of Riccia fluitans rhizoidand thallus cells has been measured continuously using pH-sensitivemicroelectrodes (Felle and Bertl, 1986a). It is demonstratedthat pH perturbations (light, weak acids, external pH) in bothdirections are completely or at least partly eliminated withinminutes. The pH_c recovery occurs regardless of the activationstate of the proton pump. The proton pump reacts to changesin cytosolic pH as expected, namely with increased proton extrusionto decreased pH_c; however, changes in pump activity (fusicoccin,CCCP, cyanide) do not necessarily result in cytosolic pH shifts.These results suggest that several proton transport mechanisms(including the proton pump) co-operate in the restoration ofa perturbed cytosolic pH. It is concluded, however, that theproton pump, although most important for the energization ofthe plasma membrane, does not regulate cytosolic pH.