Cytosol alkalization induces cytosolic and mitochondrial calcium elevation in human cells.

Rapid and parallel secretion of lysosomal beta-N-acetylglucosaminidase and preloaded fluorescein-labelled dextran was initiated in macrophages by agents affecting intracellular pH (methylamine, chlorpromazine, and the ionophores monensin and nigericin). In order to evaluate the relative role of changes in lysosomal and cytosolic pH, these parameters were monitored by using pH-sensitive fluorescent probes [fluorescein-labelled dextran or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein]. All agents except chlorpromazine caused large increases in lysosomal pH under conditions where they induced secretion. By varying extracellular pH and ion composition, the changes in lysosomal and cytosolic pH could be dissociated. Secretion was then found to be significantly modulated by changes in cytosolic pH, being enhanced by alkalinization and severely inhibited by cytosolic acidification. However, changes in cytosolic pH in the absence of stimulus were unable to initiate secretion. Dissociation of the effects on lysosomal and cytosolic pH was also achieved by combining stimuli with either nigericin or acetate. Further support for a role of intracellular pH in the control of lysosomal enzyme secretion was provided by experiments where bicarbonate was included in the medium. The present study demonstrates that an increase in lysosomal pH is sufficient to initiate lysosomal enzyme secretion in macrophages and provides evidence for a significant regulatory role of cytosolic pH.

Role of mitochondrial membrane permeability transition in p-hydroxybenzoate ester-induced cytotoxicity in rat hepatocytes

Is the Failing Heart Starved of Mitochondrial Calcium?

An elevation in cytosolic free calcium (Cai) produced by cellular ATP depletion may contribute to the initiation of cytotoxic events in renal ischemia. To evaluate whether ATP depletion results in a rise in Cai we examined the effect of cyanide and 2-deoxy-D-glucose on the Cai of Madin-Darby canine kidney cells. Exposure to the metabolic inhibitors resulted in a rise in Cai from 112 +/- 11 to 649 +/- 99 nM in 15 min. This combination of metabolic inhibitors also resulted in a decrement of cell ATP to 11 +/- 2% of control by 15 min. Experiments that were performed with other metabolic inhibitors confirm that the increment in Cai is due to inhibition of ATP synthesis. With the removal of cyanide and 2-deoxy-D-glucose, Cai recovered to 101 +/- 16 nM. In the absence of extracellular calcium activity (Ca0), Cai declined from 127 +/- 7 to 38 +/- 6 nM, whereas with cyanide plus 2-deoxy-D-glucose in the absence of Ca0 the Cai rose from 108 +/- 21 to 151 +/- 28 nM. Because the rise in Cai produced by ATP depletion in the absence of Ca0 is significantly less than that which occurs in the presence of Ca0, influx of Ca0 is necessary for the maximal rise of Cai. The rise in Cai that occurred in the absence of Ca0 suggests that the release of calcium from intracellular stores contributes to the increment in Cai seen with ATP depletion. TMB-8, an inhibitor of calcium release from intracellular stores, blunted the rise in Cai by nearly 50%. Neither verapamil nor nifedipine inhibited the rise in Cai. This study demonstrates that ATP depletion induced by the metabolic inhibitors cyanide and 2-deoxy-D-glucose is associated with a rapid and reversible increase in Cai. Both Ca0 influx and Cai redistribution contribute to this rise.

A Microfluorometric Method for Simultaneous Measurement of Changes in Cytosolic Free Calcium Concentration and pH in Single Cardiac Myocytes.

ATPase in the plasmalemma. Thus recovery from acid load in the cytosol can be performed by proton translocaShort-term light-induced pH changes were measured tion across one of the adjacent membranes, tonoplast or by means of pH-selective microelectrodes in the uni- plasmalemma, or, alternatively, by a biochemical pH-stat cellular green alga Eremosphaera viridis. Cytosolic pH mechanism. It is still unknown which mechanisms contribchanges were always transient and reversible within ute to the pH-stat mechanism under varying conditions. 1‐3 min. Taking into account the low-pass filtering by One way to approach this question is to investigate the the experimental set-up, the light-dependent cytosolic light-induced changes of cytosolic pH. They provide a pH changes could be described by a sum of two expo- physiological approach to disturbing cytosolic pH and to nential functions. By mathematical analysis it is dem- investigate short-term pH regulation. These changes have onstrated that the transient nature of light-dependent been observed in a number of plant species and share pH changes was due to a consecutive pH-stat common characteristics. Darkening of green plant cells regulation and not to a parallel light-triggered reaction. induces a transient acidification and illumination causes The short-term pH regulation depended linearly on the a transient alkalization (Felle and Bertl, 1986; Steigner pH-deviation with no indication of a feedback control et al., 1988; Okazaki et al., 1994). These pH deviations loop. Vacuolar light-dependent pH changes were were shown to be caused by the light-dependent proton measured to investigate further which pH-stat mech- translocation across the thylakoid membrane. Proton anisms are responsible for the back regulation of the fluxes across the chloroplast envelope propagate the pH light-dependent cytosolic pH changes. Vacuolar pH deviations from the chloroplast stroma to the cytosol changes were too small to explain the recovery of the ( Thaler et al., 1992; Hansen et al., 1993). cytosolic pH after darkening or illumination by H+ The transient character of these light-dependent pH fluxes across the tonoplast. changes is not understood. It is generally assumed that eYcient short-term pH-control mechanisms are respons

In vitro synergism between LFA-1 targeting leukotoxin (Leukothera™) and standard chemotherapeutic agents in leukemia cells

Intact hair cells of young rice (Oryza sativa L.) and maize roots (Zea mays L.), grown without external nitrogen, were specifically loaded with 2[prime],7[prime]-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein acetoxymethyl ester to monitor fluorescence ratio cytosolic pH changes in response to external ammonia (NH4+/NH3) application. In neutral media, cytosolic pH of root hairs was 7.15 [plus or minus] 0.13 (O. sativa) and 7.08 [plus or minus] 0.11 (Z. mays). Application of 2 mM ammonia at external pH 7.0 caused a transient cytosolic alkalization (7.5 [plus or minus] 0.15 in rice; 7.23 [plus or minus] 0.13 in maize). Alkalization increased with an increase of external pH; no pH changes occurred at external pH 5.0. The influx of 13N-labeled ammonia in both plant species did not differ between external pH 5.0 and 7.0 but increased significantly with higher pH. Pretreatment with 1 mM 1-methionine sulfoximine significantly reduced the ammonia-elicited pH increase in rice but not in maize. Application of 2 mM methylammonia only caused a cytosolic pH increase at high external pH; the increase in both species compared with the ammonia-elicited alkalization in 1-methionine sulfoximine-treated roots. The differential effects indicate that cytosolic alkalization derived from (a) NH3 protonation after passive permeation of the plasma membrane and, particularly in rice, (b) additional proton consumption via the glutamine synthetase/glutamate synthase cycle.

A large number of hormones and other agonists bring about their intracellular effects through an elevation of cytosolic free Ca2+ concentration. The mechanism by which this elevation of cytosolic Ca2+ is achieved recently became much clearer as a result of the elucidation of the role of inositol lipids in transmitting hormonal signals to the interior of the cell (see Berridge, 1984; Berridge and Irvine, 1984; Williamsonet al., 1985 for reviews). It is now widely accepted that the primary event following receptor activation is the stimulation of an inositol lipid-specific phospholipase C that cleaves phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] to yield inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol. Evi dence obtained using subcellular systems has indicated that a GTP-binding protein (G protein) is probably involved in coupling the occupied receptor to the phospholipase C (Cockcroft, 1987). Once formed in this primary reaction, both Ins(1,4,5)P3and diacylglycerol have distinct second messenger functions. Diacylglycerol is a potent activator of protein kinase C (Nishizuka, 1984) and Ins(1,4,5)P3is able to trigger Ca2+ release from an intracellular ATP-dependent Ca2+ storage pool (Strebet al., 1983; Josephet al., 1984).

The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.

The mechanisms underlying rewarming shock (cardiac failure during rewarming from profound hypothermia) are not known. Increased myocardial Ca2+ levels and altered mitochondrial ultrastructure have been reported. We hypothesized that mitochondrial buffering of cytosolic Ca2+ contributes to cardiac dysfunction. Enzymatically-dissociated rat cardiac myocytes loaded with Ca2+ indicators fluo-3 (cytosolic) and rhod-2 (mitochondrial) were electrically stimulated (0.5 Hz) and visualized using real time confocal microscopy. Myocytes were cooled from 37 to 15 °C over 1h, maintained at 15 °C for 30min, and rewarmed over 1h back to 37 °C. Hypothermia increased baseline mitochondrial Ca2+ (134% of normothermic control), which returned to control levels after rewarming. In contrast, rewarming elevated cytosolic Ca2+ significantly compared to controls (126% vs. 94%). During hypothermia both cytosolic and mitochondrial peak Ca2+, time to peak, and time to 50% decay increased by 135%, 150% and 200%, respectively, but returned to control levels during rewarming. These data indicate that during hypothermia, mitochondria buffer cytosolic Ca2+, and during rewarming, mitochondrial Ca2+ is redistributed to the cytosol, leading to continued elevation in cytosolic Ca2+. The temporary Ca2+ overload in mitochondria may further affect subsequent energy production and myocyte function. Supported by Mayo Foundation Clinical Research grants, and NIH grants HL74309 and GM56686.

The objective of this study was to determine the relationship between cytosolic pH and vesicular pH during ATP depletion. Using digitized video microscopy and single, cultured rat hepatocytes, cytosolic pH and vesicular pH were quantitated by ratio imaging of BCECF (2′, 7′ biscarboxyethyl-5, 6-carboxyfluorescein) fluorescence and fluoresceindextran fluorescence, respectively. Basal value for cytosolic pH was 7.26 and basal value for vesicular pH was 4.86. During ATP depletion by metabolic inhibition with KCN plus iodoacetic acid or antimycin A, cytosolic pH decreased 0.71 units to 6.55. In separate experiments under identical conditions, vesicular pH increased 1.59 units to 6.45, suggesting that protons were leaking from acidic vesicles during ATP depletion. Fluorescein-dextran fluorescence remained punctate, indicating that the rise in vesicular pH was due to an efflux of protons from vesicles and not loss of vesicle integrity. To determine whether efflux of protons from acidic vesicles can acidify cytosolic pH, we used two maneuvers that result in leakage of protons from acidic vesicles without significantly decreasing cellular ATP: (a) hypotonic stress in K+ free media and (b) exposure of the cells to the H+ -ATPase inhibitor NBD-Cl. Both hypotonic stress and NBD-Cl decreased cytosolic pH 0.4 units to 6.86 and increased vesicular pH 2.0 units to 6.76, resulting in near-equilibration of cytosolic pH and vesicular pH. Thus an efflux of protons from intracellular compartments will acidify cytosolic pH of hepatocytes (pH 6.86), but not to the same degree as ATP depletion (pH 6.55). Calculations based on buffering capacities and relative volumes of the cytosol and acidic compartments suggest that efflux of protons from acidic compartments into the cytosol may account for up to 20% of the decrease of cytosolic pH during the ATP depletion of anoxia observed in hepatocytes. (Hepatology 1991;14:626-633.)

Based on molecular and physiological resemblance, the mechanism that controls drug bioavailability and toxicity also shares strong similarities to the one that controls drug resistance. In both cases, this mechanism relies on the expression of drug transporters and the physico-chemical properties of drugs, which together alter the intracellular accumulation of chemicals in cells or tissues. However, a parameter that is central and has received great attention in the field of bioavailability, but almost none in the field of drug resistance, is the molecular weight of drugs. In the former area, it is well known that to achieve a reasonable bioavailability, drugs must have-among other properties-a molecular weight less than 500, known as Lipinski's 2nd rule. Accordingly, it is worth questioning whether a similar rule exists in the field of drug resistance and what subsequent mechanism would control the membrane permeability to drugs as a function of their molecular weight. I demonstrate here that cytosolic pH fixes the molecular weight of drugs entering cells, by altering the cell membrane mechanical properties and that, both cytosolic pH and membrane mechanical properties are needed and sufficient to explain doxorubicin resistance levels in different cancerous cell lines. Finally, I discuss the efficiency of a drug handling activity by transporters in MDR and suggest ways to control drug delivery mechanically. In addition, and for the first time, the literal expression of a Law similar to Lipinski's 2nd rule will be described as a function of cytosolic pH and lipid number asymmetry.

Import of Firefly Luciferase into Mammalian Peroxisomes in Vivo Requires Nucleoside Triphosphates

Calcium has been largely hypothesized to be involved in the arbuscular mycorrhizal (AM) signal trans- duction. A transient elevation in cytosolic Ca 2+ concentration is considered one of the earliest responses detected in plant cells challenged with different stimuli. In this paper the detection of Ca 2+ transients was selected as a tool to analyse whether plant cells perceive diffusible molecules released by the AM fungus Glomus intraradices .A naequo- rin-transformed line of soybean cells was used as experimental system. When cells were treated with the medium in which Gl. intraradices spores germinated, a rapid and significant increase in cytosolic Ca 2+ level was induced and dis- sipated within 30 min. The medium did not induce either intracellular accumulation of reactive oxygen species or cell death. A detectable Ca 2+ elevation was triggered also by the medium of fungal extraradical mycelium, associated to mycorrhizal hairy roots of carrot. These results indicate that Gl. intraradices releases diffusible molecules that are sensed by host plant cells through a Ca 2+ -mediated signalling during different steps of its life cycle.