Intracellular pH topography of Penicillium cyclopium protoplasts. Maintenance of Δ pH by both passive and active mechanisms

The intracellular pH distribution in protoplasts of Penicillium cyclopium has been studied using the recently developed fluorescent probe microscopic technique. The technique gives detailed pH maps of the interior of the protoplasts with the exception of vacuoles (no fluorescence signal from vacuoles was observed). In the cytoplasm two separate layers were distinguished: a thinner outer layer with acidic pH (around 5) and the larger core region with near neutral pH. The pH of the core region is decreased by the addition of uncouplers, inhibitors of respiration and during the uptake of l-phenylalanine. These compounds do not change the pH of the surface layer, which is, however, acidified by addition of vanadate, an inhibitor of the proton pump of the plasmalemma. We suggest that the pH of the surface layer is maintained by the combined effects of a Donnan distribution of protons (bound to postulated anion binding proteins) and the proton extrusion via the plasmalemma proton pump. This mechanism explains the protection of the cytoplasmic core of acidophilic eukaryotes from the influence of the usually acidic environment.     

Confocal pH Topography in Plant Cells — Acidic Layers in the Peripheral Cytoplasm and the Apoplast

The distribution of intracellular pH was studied in cultured cells of Gossypium hirsutum by con-focal pH topography using the fluorescent probe car-boxy SNARF1 and a ratio imaging procedure. The resulting pH maps can visualize pH differences with an accuracy of 0.1 unit in the investigated range between 7.5 and 5.6. They reveal the following characteristic features of the Gossypium cells: – the pH of the cytoplasmic core regions ranges from near 7.4 in younger to near 6.0 in older cells; – vacuoles show the expected acidity with pH < 5.6; – the cell wall/apoplastic region is acidic with a pH near 5.6 or below, especially in young, growing cells; – interestingly, acidic areas appear also at the periphery of the cytoplasm, i.e. beneath the plasma membrane. They remain stable in the presence of 5/μmol/I of the protonophore CCCP. Acidic layers of peripheral cytoplasm were also detected in protoplasts of Penicillium cyclopium, i.e. eukaryotic cells of simpler structure, which served as a reference object. This ronfirms earlier findings obtained with classical fluorescence microscopy and another fluoroprobe (fluorescein diacetate). Though additional experimental support is needed, low pH regions at the cytoplasm/plasma membrane interface should be considered a real contribution to the pH control of plant and fungal cells, facilitating e.g. the maintenance of cytosolic pH in acidic environments.     

Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles

Putrescine and spermidine uptake in carrot (Daucus carota L., cv “Tip top”) protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K_m = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca²⁺ affected only surface binding but not transport into the cells. Nonpermeant polycations such as La³⁺ and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.     

Isolation and Characterization of Vacuoles from Melilotus alba Mesophyll

Methods for the preparation of protoplasts and vacuoles from mesophyll tissues of sweet clover (Melilotus alba Desr.) are described. Vacuoles are obtained using a new procedure which involves lysis of the plasmalemma during a brief centrifugation of protoplasts through a diethylaminoethyl dextran layer. This method combines the release of vacuoles and their purification in one step. The contamination of vacuole preparations was found to be low, as judged by enzymic markers and microscopic inspection. The method described is rapid and gives a good yield of vacuoles without causing changes in osmotic pressure. Several hydrolases were found to be located in vacuoles from sweet clover, which were also examined for their amino acid content.     

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)     

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.     

An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

The fusicoccin-stimulated phosphorylation of a 33 KDa polypeptide in cells of Acer pseudoplatanus as influenced by extracellular and intracellular pH

Abstract In a previous study, it was shown that the fungal toxin fusicoccin (FC) is able to stimulate the in vivo phosphorylation of a 33 KDalton polypeptide (33 KP) independently of protein synthesis. Here we show that the stimulation by FC of both proton efflux and 33 KP phosphorylation are strongly enhanced when the external medium contains K⁺ or Na⁺, suggesting that the two phenomena are related. The stimulatory effect of FC is higher in unbuffered than in buffered media; moreover, in the absence of FC, 33 KP is more phosphorylated at an acidic than at a basic pH of the medium, suggesting that the effect of FC may depend, to a certain extent, on the acidification of the free space caused by FC-promoted proton efflux. Treatments reported to alter the intracellular pH influence 33 KP phosphorylation even more strongly than the external pH does. The acidifying agents isobutyric acid and trimethylacetic acid decrease 33 KP phosphorylation, while the alkalinizing agents, ammonia and procaine, increase it. FC partially counteracts the inhibition by the weak acids, whereas the stimulatory effect of FC is not additive with that of the weak bases. The results indicate that 33 KP phosphorylation senses both the external and internal pH. The stimulatory effect of cytoplasm-alkalinizing treatments, which mimics that of FC, agrees with the reported capacity of FC to cause cytoplasmic alkalinization, following activation of the plasmalemma proton pump.     

Regulation of plant elongation growth by surface and xylem proton pumps

The roles of plasmalemma electrogenic proton pumps in elongation growth of plant stems are discussed on the basis of growth-electrophysiological studies on hypocotyl segments ofVigna unguiculata. Plant stems usually have two spatially separated electrogenic proton pumps: the surface proton pump which is located on the surface membrane of the symplast and the xylem proton pump, on the cell membrane of the symplast/xylem apoplast boundary. The surface proton pump excretes protons into the surface cell wall layer and causes the loosening of the cell wall. The xylem proton pump excretes protons into the xylem apoplast and drives the uptake of solute and water into the symplastvia secondary and/or tertiary active mechanisms: the proton cotransport system and the apoplast canal system. Both the surface and the xylem proton pumps are active during elongation growth because both the yielding of cell wall loosening and the uptake of water are necessary for continued elongation growth.     

Evidence that an ATPase functions in the maintenance of the acidic pH of the hamster sperm acrosome

The hamster sperm acrosome exhibits a transmembrane proton concentration gradient (inside acidic). The gradient was dissipated by valinomycin and the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) together, but not by either alone. Several anion transport inhibitors, when utilized in the presence of FCCP, also eliminated the proton gradient. These experiments demonstrate that a modified Donan-type equilibrium dependent upon selective permeability of membranes to protons has no role in maintenance of the acidic pH of the acrosome. N,N'-Dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofuran, inhibitors of the mitochondrial proton-translocating ATPase, dissipated the proton concentration gradient when FCCP was present. Oligomycin and ouabain had no effect, either in the presence or absence of FCCP. Our experimental evidence suggests that an ATP-dependent proton pump is functioning in the maintenance of the acidic pH of the hamster sperm acrosome.     

Primaquine interferes with membrane recycling from endosomes to the plasma membrane through a direct interaction with endosomes which does not involve neutralisation of endosomal pH nor osmotic swelling of endosomes

The anti-malaria drug primaquine is a weak base which accumulates in endosomes in a protonated form and consequently neutralises the endosomal pH. Bafilomycin A1 prevents endosome acidification by inhibiting the vacuolar proton pump. Although both agents neutralise the endosomal pH, only primaquine has a strong inhibitory effect on recycling of endocytosed proteins to the plasma membrane (Van Weert et al. (1995), J. Cell Biol. 130, 821-834). This suggests that primaquine interferes with a parameter, other than endosomal pH, that is essential for membrane recycling. In the presence of 0.3 mM primaquine, endocytosed transferrin-receptors accumulated intracellularly, but not in the additional presence of bafilomycin A1. Thus, at relative low concentrations proton pump-driven accumulation of primaquine in endosomes was required to inhibit membrane recycling, suggesting that the target of primaquine is associated with endosomes. The inhibitory effect of 1 mM primaquine on transferrin receptor recycling was not reversed by the additional presence of bafilomycin A1, indicating that osmotic swelling of endosomes due to accumulation of protonated primaquine could also not explain its effect. To study endosome swelling morphologically, we introduce a novel technique for fluorescent labelling of endosomes involving HRP-catalysed biotinylation. In the presence of 0.2 mM primaquine endosomal vacuoles with diameters up to 2 microm were observed. Endosome swelling was not observed when in addition to primaquine also bafilomycin A1 was present, supporting the notion that vacuolar proton pump activity lowers the dose response for primaquine. Factors that are crucial for membrane recycling and may be affected by primaquine are discussed.     

Influence of unsaturated fatty acids in chloroplasts. Shift of the pH optimum of electron flow and relations to deltapH, thylakoid internal pH and proton uptake.

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to deltapH, the proton pump and the pH of the inner thylakoid space (pHi). The deltapH and pHi were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6-dichlorophenolindophenol) coupled or non-coupled; (b) photosystem II (from H2O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the deltapH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the deltapH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosytems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the deltapH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo. As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5-5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), deltapH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtile equilibrium between the factors is disturbed.     

pH dependence of the flash-induced S-state transitions in the oxygen-evolving center of photosystem II from Thermosynechoccocus elongatus as revealed by Fourier transform infrared spectroscopy

pH dependence of the efficiencies of the flash-induced S-state transitions in the oxygen-evolving center (OEC) was studied by means of Fourier transform infrared (FTIR) difference spectroscopy using photosystem II (PSII) core complexes from the thermophilic cyanobacterium Thermosynechoccocus elongatus. The PSII core complexes dark-adapted at different pHs in the presence of ferricyanide as an electron acceptor were excited by four consecutive saturating laser flashes, and FTIR difference spectra induced by each flash were recorded in the region of 1800-1200 cm(-1). Each difference spectrum was fitted with a linear combination of standard spectra measured at pH 6.0, which represent the spectra upon individual S-state transitions, and the transition efficiencies were estimated from the fitting parameters. It was found that the S1 --> S2 transition probability is independent of pH throughout the pH region of 3.5-9.5, while the S2 --> S3, S3 --> S0, and S0 --> S1 transition probabilities decrease at acidic pH with pK values of 3.6 +/- 0.2, 4.2 +/- 0.3, and 4.7 +/- 0.5, respectively. These findings, i.e., the pH-independent S1 --> S2 transition probability and the pK values for the inhibition in the acidic range of the other three transitions, were in good agreement with recent results obtained by electron paramagnetic resonance measurements for PSII-enriched membranes of spinach [Bernát, G., Morvaridi, F., Feyziyev, Y., and Styring, S. (2002) Biochemistry 41, 5830-5843]. On the basis of this correspondence for quite different types of PSII preparations exhibiting marked difference in the pH dependence of the apparent proton release pattern, it is concluded that the inhibition of the S2 --> S3, S3 --> S0, and S0 --> S1 transitions in the acidic region is an inherent property of the OEC. This feature probably reflects proton release from substrate water in these three transitions. On the other hand, all of the S-state transitions remained generally efficient up to pH 9.5 in the alkaline region, except for a slight decrease of the S3 --> S0 transition probability above pH 8 (pK approximately 10). This observation partly differs from the tendency reported for spinach preparations, suggesting that a mechanism different from that in the acidic region is responsible for the transition efficiencies in the alkaline region.     

The apoplastic pH of the Zea mays root cortex as measured with pH-sensitive microelectrodes: aspects of regulation

In the root cortex of Zea mays the apoplastic pH and aspects of its regulation were investigated using pH-sensitive microelectrodes. To measure the pH directly in different cell layers of the apoplast sharp double-barrelled electrodes were applied, whereas blunt pH-electrodes were used simultaneously to measure the pH at the root surface. Recordings carried out 8-10 mm behind the root tip show that the apoplastic pH is maintained between 5.1 and 5.6, depending on the given experimental conditions, i.e. varying external [K⁺], [Ca²⁺], pH, weak buffering, as well as perfusion of the test medium. When the medium pH (bulk) differs considerably from the apoplastic pH, a small pH gradient is built up between the root surface (unstirred layer) and the outer cortex layers. In a standing medium these gradients equilibrate. The apoplastic pH responds to increases in external [K⁺] and [CA²⁺] with an acidification, which is attributed to ion-exchange properties of the cell wall constituents. Stimulation of proton pump activity with fusicoccin acidifies the apoplast from pH 5.6 to pH 4.8, while deactivation of the pump with cyanide/salicylhydroxamic acid increases the pH of the apoplast from 5.6 to 6.2, and further to pH 6.6 with CCCP. The Ca²⁺ channel antagonists nifedipine and La³⁺ also increase the apoplastic pH. It is suggested that not only the proton pump, but also the cation channels may contribute to the regulation of the apoplastic pH.Keywords: Apoplast, ion-selective microelectrodes, pH, unstirred layer, Zea mays, root.      

Plasmalemma fine structure in isolated tobacco mesophyll protoplasts

Summary Tobacco mesophyll protoplasts have been examined by electron microscopy during isolation procedures and after 24 hours culture in a medium known to support cell wall regeneration. During isolation the plasmalemma shows little structural differentiation apart from the formation of small vacuoles in the cytoplasm. After 24 hours of culture, several types of activity are seen at the plasmalemma surface. Microtubules, profiles of endoplasmic reticulum, electron dense granules and coated vesicles are associated with the inner surface of the membrane. External to the plasmalemma fibrillar structures occur, both as extensive networks and as individual fibrils apparently associated with the membrane itself. Techniques and criteria for electron microscopy are presented, and the results discussed in terms of plasmalemma function and the regeneration of the cell wall.     

Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts,vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H⁺-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.Abbveviations CAM Crassulacean acid metabolism - H⁺-ATPase proton-translocating ATPase     

Cytoplasmic pH Regulation in Acer pseudoplatanus Cells: II. Possible Mechanisms Involved in pH Regulation during Acid-Load

Qualitative and quantitative aspects of the mechanisms involved in the regulation of cytoplasmic pH during an acid-load have been studied in Acer pseudoplatanus cells. Two main processes, with about the same relative importance, account for the removal of H⁺ from the cytoplasm, namely a `metabolic consumption' of protons and the excretion of protons or proton-equivalents out of the cells. The metabolic component corresponds to a change in the equilibrium between malate synthesis and degradation leading to a 30% decrease of the malate content of the cells during the period of cytoplasmic pH regulation. Various conditions which severely inhibit the activity of the plasmalemma proton pump ATPase reduce, at most by 50%, the excretion of H⁺. This suggests that, besides the plasmalemma proton-pump, other systems are involved in the excretion of proton-equivalents. Indirect information on qualitative and quantitative features of these systems is described, which suggests the involvement of Na⁺ and HCO₃⁻ exchanges in the regulation of cytoplasmic pH of acid-loaded cells.     

Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa L.)

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.     

Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas

The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.