Cell Acidification Research Articles

UV-B overexposure induces programmed cell death in a BY-2 tobacco cell line

There is not a great deal of information available on the effects of overexposure of ultraviolet B (UV-B) on programmed plant cell death in cell cultures. Applying fluorescent microscopy, typical apoptotic morphological features such as cell shrinkage, condensation of chromatin in perinuclear areas and formation of micronuclei were all observed in UV-B treated BY-2 tobacco cells. Moreover, cell acidification as an apoptosis-related process was also detected in irradiated cells. Because of the wide variety of morphological changes that have been associated with UV-B-caused cell apoptosis, it is difficult to attribute any of these changes to a particular molecular mechanism. DNA laddering and TUNEL assays also indicated a dose-dependent specific nucleosomal DNA fragmentation under ultraviolet irradiation. These observations clearly indicate that UV-B irradiation can initiate apoptotic processes in plant cells.

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH(i) in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na(+)/H(+) antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH(i) decreased sharply, and recovery required both Na(+)/H(+) antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn(2+) resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn(2+). Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis.

Change in intracellular pH causes the toxic Ca2+ entry via NCX1 in neuron- and glia-derived cells.

Brain hypoxia or ischemia causes acidosis and the intracellular accumulation of Ca(2+) in neuron. The aims of the present study were to elucidate the interaction between intracellular pH and Ca(2+) during transient acidosis and its effects on the viability of neuronal and glial cells. Intracellular Ca(2+) and pH were measured using the fluorescence of fura-2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester in neuroblastoma (IMR-32), glioblastoma (T98G), and astrocytoma (CCF-STTG1) cell lines. The administration of 5 mM propionate caused intracellular acidification in IMR-32 and T98G cells but not in CCF-STTG1 cells. After the removal of propionate, the intracellular pH recovered to the resting level. The intracellular Ca(2+) transiently increased upon the removal of propionate in IMR-32 and T98G cells but not in CCF-STTG1 cells. The transient Ca(2+) increase caused by the withdrawal of intracellular acidification was abolished by the removal of external Ca(2+), diminished by a reduction of external Na(+), and inhibited by benzamil. Transient acidosis caused cell death, whereas the cells were more viable in the absence of external Ca(2+). Benzamil alleviated cell death caused by transient acidosis in IMR-32 and T98G cells but not in CCF-STTG1 cells. These results suggest that recovery from intracellular acidosis causes a transient increase in cytosolic Ca(2+) due to reversal of Ca(2+) transport via Na(+)/Ca(2+) exchanger coactivated with Na(+)/H(+) exchanger, which can cause cell death.

Hydrogen sulfide regulates intracellular pH in rat primary cultured glia cells

Intracellular pH (pH(i)) plays an important role in the regulation of central nervous system function. In the present study, we examined whether hydrogen sulfide (H(2)S), a recently recognized neuromodulator, regulates pH(i) in rat primary cultured glia cells. pH(i) was measured with a fluorescent sensitive dye, BCECF-AM. Activities of Cl(-)/HCO(3)(-) exchanger and Na(+)/H(+) exchanger were examined by assessing their capacities to load or extrude H(+) upon NH(4)Cl pulse load. We found that NaHS, a H(2)S donor, decreased pH(i) in a concentration-dependent manner ranging from 10 to 200muM in the primary cultured microglia. Blockade of the Cl(-)/HCO(3)(-) exchanger with, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or Na(+)/H(+) exchanger with 5-N-methyl-N-isobutylamiloride (MIA) significantly attenuated the pH(i)-lowering effect of NaHS. Moreover, NaHS significantly increased the activity of Cl(-)/HCO(3)(-) exchanger but inhibited that of Na(+)/H(+) exchanger. The pH regulatory effect of H(2)S was also observed in primary cultured astrocytes, but not in SH-SY5Y neuronal cells. In conclusion, we found for the first time that H(2)S induced intracellular acidification in glia cells via regulation of the activities of Cl(-)/HCO(3)(-) exchanger and Na(+)/H(+) exchanger. The present study may provide new evidence for H(2)S to serve as a neuromodulator and offer a potential approach for the treatment of neurological diseases.

Inhibition of spoilage mould conidia by acetic acid and sorbic acid involves different modes of action, requiring modification of the classical weak-acid theory

Fungal spoilage of many foods is prevented by weak-acid preservatives such as sorbic acid or acetic acid. We show that sorbic and acetic acids do not both inhibit cells by lowering of internal pH alone and that the “classical weak-acid theory” must be revised. The “classical weak-acid theory” suggests that all lipophilic acids with identical pK a values are equally effective as preservatives, causing inhibition by diffusion of molecular acids into the cell, dissociation, and subsequent acidification of the cytoplasm. Using a number of spoilage fungi from different genera, we have shown that sorbic acid was far more toxic than acetic acid, and no correlation existed between resistance to acetic acid and resistance to sorbic acid. The molar ratio of minimum inhibitory concentrations (MICs) (acetic: sorbic) was 58 for Paecilomyces variotii and 14 for Aspergillus phoenicis. Using flow cytometry on germinating conidia of A spergillus niger, acetic acid at pH 4.0 caused an immediate decline in the mean cytoplasmic pH (pH i) falling from neutrality to ~ pH4.7 at the MIC (80 mM). Sorbic acid also caused a rapid but far smaller drop in pH i, at the MIC (4.5 mM); the pH remained above pH 6.3. Over 0–5 mM, a number of other weak acids caused a similar fall in cytoplasmic pH. It was concluded that while acetic acid inhibition of A. niger conidia was due to cytoplasmic acidification, inhibition by sorbic acid was not. A possible membrane-mediated mode of action of sorbic acid is discussed.

Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase

Disassembly of the yeast V-ATPase into cytosolic V(1) and membrane V(0) sectors inactivates MgATPase activity of the V(1)-ATPase. This inactivation requires the V(1) H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V(1) and V(0) subunits were identified by two-hybrid assay. The B subunit of the V(1) catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V(1) subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V(0) subunit Vph1p. V(1)-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V(1) when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V(1) lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V(1) was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V(1) complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V(1)-ATPase activity and precludes V(0) interactions.

Acidification of damaged cells is a consequence of damage to the gastric epithelium, and may contribute to surface pH increases after damage

Focal damage of the gastric surface epithelium causes a transient rise in extracellular pH (pHo) over the damage site, prior to epithelial repair. To identify sources of the pHo rise we measured intracellular pH (pHi) of cells in and around the damaged area. The exposed gastric mucosa of anesthetized mice was loaded with 5 μM SNARF‐5F‐AM, and tissue imaged on a two‐photon microscope stage, during tissue superfusion with buffered pH 3 saline. A 2‐3 cell region of the gastric epithelium was photodamaged (710 nm, 350 mW for 5‐10 sec). Damage was monitored as loss of NAD(P)H autofluorescence and disrupted cell structure (confocal reflectance). pHi was measured near the damage (5‐10 μm), far from the damage (100‐150 μm), or in the actual damaged area. Before damage, initial pHi values were similar in all cell groups (~pH 7.1). After damage, pHi values were similar in near and far cells (7.17 ± 0.06 and 7.15 ± 0.04), but decreased to 6.45 ± 0.08 in damaged cells (n=4). Adding 20 μM iv EIPA or 100 μM luminal SITS did not significantly change initial pHi values in any cell type. After damage, EIPA selectively acidified damaged cells vs control (6.00 ± 0.09 vs. 6.45 ± 0.08, n=4), but SITS did not affect damaged cell acidification (6.59 ± 0.3, n=4). We conclude that damaged gastric epithelial cells may activate basolateral Na/H exchange as part of transepithelial alkali secretion that causes the luminal pHo increase seen after epithelial damage.

Defective organellar acidification as a cause of cystic fibrosis lung disease: reexamination of a recurring hypothesis.

The cellular mechanisms by which loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel produce cystic fibrosis (CF) lung disease remain uncertain. Defective organellar function has been proposed as an important determinant in the pathogenesis of CF lung disease. According to one hypothesis, reduced CFTR chloride conductance in organelles in CF impairs their acidification by preventing chloride entry into the organelle lumen, which is needed to balance the positive charge produced by proton entry. According to a different hypothesis, CFTR mutation hyperacidifies organelles by an indirect mechanism involving unregulated sodium efflux through epithelial sodium channels. There are reports of defective Golgi, endosomal and lysosomal acidification in CF epithelial cells, defective phagolysosomal acidification in CF alveolar macrophages, and organellar hyperacidification in CF respiratory epithelial cells. The common theme relating too high or low organellar pH to cellular dysfunction and CF pathogenesis is impaired functioning of organellar enzymes, such as those involved in ceramide metabolism and protein processing in epithelial cells and antimicrobial activity in alveolar macrophages. We review here the evidence for defective organellar acidification in CF. Significant technical and conceptual concerns are discussed regarding the validity of data showing too high/low organellar pH in CF cells, and rigorous measurements of organellar pH in CF cells are reviewed that fail to support defective organellar acidification in CF. Indeed, there is an expanding body of evidence supporting the involvement of non-CFTR chloride channels in organellar acidification. We conclude that biologically significant involvement of CFTR in organellar acidification is unlikely.

KCNQ1 loss-of-function mutation impairs gastric acid secretion in mice

The KCNQ1 channel is abundantly expressed in the gastric parietal cells. Although the functional coupling of KCNQ1 with the H(+)/K(+)-ATPase has already been confirmed on the basis of pharmacological kinetics, the effect of a KCNQ1 loss-of-function mutation on gastric acidification remains unclear. In this study, parietal cells and gastric glands from both C57BL/6 J mice (normal control) and J343 mice (mice with a KCNQ1 loss-of-function mutation) were isolated to study the effects of KCNQ1 on gastric acidification. We found that the mutation limited intracellular acidification of parietal cells and H(+) secretion of the stomach in response to histamine. Thus, a KCNQ1 loss-of-function mutation may impair gastric acid secretion.

Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

Neurotensin signaling induces intracellular alkalinization and interleukin-8 expression in human pancreatic cancer cells

Pancreatic adenocarcinomas express neurotensin receptors in up to 90% of cases, however, their role in tumor biology and as a drug target is not clear. In the present study, a stable neurotensin (NT) analog induced intracellular calcium release and intracellular alkalinization in BxPC-3 and PANC-1 pancreatic cancer cells that was abolished by inhibitors of NT receptor (NTR) and sodium–proton exchanger 1 (NHE1), amiloride and SR 142948, respectively. Activation of NHE1 involved increased phosphorylation of dimethylfumarate-sensitive mitogen- and stress-activated kinase 1/2 (MSK1/2). NTR signaling appears to promote a metastatic phenotype in pancreatic cancer cells by induction of localized extracellular acidification in normoxic cells, preceeding acidosis induced by hypoxia and switch to glycolysis in addition to increased expression of interleukin-8 (IL-8).

Multiple mechanisms of germ cell loss in the perinatal mouse ovary

In the perinatal ovary of most mammals, external and internal factors establish a primordial follicle reserve that specifies the duration of the reproductive lifespan of a given species. We analyzed the mechanism of follicle loss and survival in C57BI/6 mice using static and dynamic assays of apoptosis, autophagy, and ovarian morphogenesis. We confirm an initial loss soon after birth, when about 44% of the germ cells detectable at the end of the fetal period abruptly disappear. The observations that (1) few germ or somatic cells were apoptotic in newborn ovaries, (2) vitally stained organ cultures exhibit active extrusion of non-apoptotic germ cells and (3) germ-cell lysosome amplification occurs at birth suggested that additional mechanisms are involved in perinatal germ cell loss. Newborn mouse ovaries cultured in the pH sensitive dye lysotracker red exhibit an increased incidence of acidified non-apoptotic germ cells when maintained in the absence but not in the presence of serum, implying a role for autophagy in germ cell attrition. Inhibitors of autophagy, but not apoptosis, reduce germ cell acidification induced by serum starvation in ovary organ cultures and protein mediators of both autophagy and apoptosis are expressed at birth. From these findings we suggest that multiple perinatal mechanisms establish the primordial follicle reserve in mice.

Calmodulin Association with Connexin32-derived Peptides Suggests trans-Domain Interaction in Chemical Gating of Gap Junction Channels

Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca2+ binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1–22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca2+ dissociation rate constants k3,4, 1.7 ± 0.5 s–1) than to the N-lobe (k1,2, 10.8 ± 1.3 s–1). In contrast, peptides representing the C-terminal tail domain (residues 208–227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k3,4, 2.6 ± 0.1 s–1 or k1, 13.8 ± 0.5 s–1 and k2, 1000 s–1). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1–21 and 216–227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.

A novel design of multifunctional integrated cell-based biosensors for simultaneously detecting cell acidification and extracellular potential

The paper discussed a novel design of multifunctional cell-based biosensors for simultaneously detecting cell acidification and extracellular potential. Employing living cells such as cardiac myocytes as a source for the light addressable potentiometric sensor (LAPS) array, this cell-based biosensor was able to monitor both the acidification and extracellular potential in parallel. For LAPS array fabrication, part of the silicon base was heavily doped with boron to form separate testing areas. Detecting system was built involving lock-in amplifier and digital demodulation with FFT methods. This LAPS array showed a good sensitivity of 53.9 mV/pH to H + with good linearity. Each testing area for extracellular potential detection was decreased to 200 μm × 200 μm in size to obtain a better sensitivity. Experiment results showed that this LAPS array could monitor the acidification of cells as well as the extracellular potential with good sensitivity. This novel integrated biosensor will be useful for multi-parameter extracellular monitoring and can possibly be a platform for drug screening.

Protein kinase C modulates synaptic vesicle acidification in a ribbon type nerve terminal in the retina

The driving force for neurotransmitter accumulation into synaptic vesicles is provided by the generation of a transmembrane electrochemical gradient (ΔμH+) that has two components: a chemical gradient (ΔpH, inside acidic) and an electrical potential across the vesicular membrane (ΔΨ, inside positive). This gradient is generated in situ by the electrogenic vacuolar H +-ATPase, which is responsible for the acidification and positive membrane potential of the vesicle lumen. Here, we investigate the modulation of vesicle acidification by using the acidic-organelle probe LysoTracker and the pH-sensitive probe LysoSensor at goldfish Mb-type bipolar cell terminals. Since phosphorylation can modulate secretory granule acidification in neuroendocrine cells, we investigated if drugs that affect protein kinases modulate LysoTracker staining of bipolar cell terminals. We find that protein kinase C (PKC) activation induces an increase in LysoTracker-fluorescence. By contrast, protein kinase A (PKA) or calcium/calmodulin kinase II (CaMKII) activation or inhibition did not change LysoTracker-fluorescence. Using a pH-dependent fluorescent dye (LysoSensor) we show that the PKC activation with PMA induces an increase in LysoSensor-fluorescence, whereas the inactive analog 4alpha-PMA was unable to cause the same effect. This increase induced by PMA was blocked by PKC inhibitors, calphostin C and staurosporine. These results suggest that phosphorylation by PKC may increase synaptic vesicle acidification in retinal bipolar cells and therefore has the potential to modulate glutamate concentrations inside synaptic vesicles.

Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.

Suppression of the proliferation of cancer cell lines, KB-3-1 and K562 cells preceded by a decrease in intracellular pH caused by phenoxazine derivatives

The intracellular pH (pHi) of cancer cells such as the KB-3-1 (human epidermoid carcinoma cell line) and K562 cells (human chronic myeloid leukemia cell line) cultured in a medium (pH 7.4) was found to be much higher (pH 7.65 and 7.8, respectively) than that of normal cells (pHi is usually < or =7.2). When a phenoxazine derivative, 2-aminophenoxazine-3-one (Phx-3) or 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1) was added to these cells, pHi rapidly decreased within 20 min, dose-dependently, though the extent of the decrease of pHi was significantly larger for Phx-3 (a decrease of 0.9 units) than for Phx-1 (a decrease of 0.4 units). Phx-3 and Phx-1 caused the proliferative suppression of these cells 24 h after the addition, dose-dependently. The anti-proliferative effects of Phx-3 on KB-3-1 and K562 cells were far greater than those of Phx-1. It was proposed that the proliferative suppression of KB-3-1 and K562 cells caused by Phx-3 and Phx-1, may be preceded by a rapid and extensive decrease in pHi, which possibly influenced the intracellular homeostasis, finally causing the suppressed proliferation and apoptosis of these cancer cells. The present results suggest that the anti-cancer effects of Phx-3 and Phx-1 may be strengthened by the intracellular acidification of cancer cells by these compounds.

Inhibition of Endosome-Lysosome System Acidification Enhances Porcine Circovirus 2 Infection of Porcine Epithelial Cells

Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH(4)Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH(4)Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% +/- 110%, 1,212% +/- 34%, and 1,100% +/- 179%, respectively, in porcine kidney (PK-15) cells; by 128% +/- 7%, 158% +/- 3%, and 142% +/- 11% in swine kidney cells; by 160% +/- 28%, 446% +/- 50%, and 162% +/- 56% in swine testicle (ST) cells; and by 313% +/- 25%, 611% +/- 86%, and 352% +/- 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibition of endosome-lysosome system acidification.

Peritoneal acidosis mediates immunoprotection in laparoscopic surgery

We have shown previously that abdominal insufflation with CO(2) increases serum levels of IL-10 and TNFalpha and increases survival among animals with lipopolysaccharide (LPS)-induced sepsis, even after a laparotomy. We demonstrated previously that the effect of CO(2) is not from changes in systemic pH, although the peritoneum is locally acidotic during abdominal insufflation with CO(2) even when systemic pH is corrected. We hypothesized that acidification of the peritoneum via means other than CO(2) insufflation would produce alterations in the inflammatory response similar to those associated with CO(2) pneumoperitoneum. In total, 42 rats were randomized into 7 groups (n = 6): 1) LPS only, 2) anesthesia control, 3) helium pneumoperitoneum, 4) CO(2) pneumoperitoneum, 5) buffered mild acid lavage, 6) buffered strong acid lavage, and 7) buffered strong acid lavage + helium pneumoperitoneum. Animals received anesthesia with vaporized isoflurane (except the LPS-only group) and their respective abdominal treatment (pneumoperitoneum and/or lavage) for 30 min followed immediately by stimulation with systemic LPS (1 mg/kg, IV). Blood was harvested via cardiac puncture 60 min after LPS injection, and serum levels of IL-10 and TNFalpha levels were determined by enzyme-linked immunosorbent assay. Mean peritoneal pH decreased (P < .05) after CO(2) pneumoperitoneum, buffered strong acid lavage, and buffered strong acid lavage + helium pneumoperitoneum, and it decreased (P = .1) after helium pneumoperitoneum alone and buffered mild acid lavage. IL-10 levels were increased (P < .01), and TNFalpha levels decreased (P < .001) among animals with acidic peritoneal cavities compared with animals with pH-normal peritoneal cavities. Decreasing peritoneal pH correlated with both increasing IL-10 levels (r = -.465, P < .01) and decreasing TNFalpha levels (r = 0.448, P < .01). Among animals with peritoneal acidosis, there were no differences in levels of IL-10 or TNFalpha regardless of insufflation status (P > .05 for both cytokines). Acidification of the peritoneal cavity whether by abdominal insufflation or by peritoneal acid lavage increases serum IL-10 and decreases serum TNFalpha levels in response to systemic LPS challenge. The degree of peritoneal acidification correlates with the degree of inflammatory response reduction. These results support the hypothesis that pneumoperitoneum-mediated attenuation of the inflammatory response after laparoscopic surgery occurs via a mechanism of peritoneal cell acidification.

Role of kidney chloride channels in health and disease

Chloride channels are expressed along the entire mammalian nephron. They participate in transepithelial chloride transport, cell volume regulation and acidification of intracellular vesicles. Some chloride channels are constitutively active and others are regulated by either second messengers such as cAMP or Ca(++) or secondary to changes in membrane potential. The molecular identities of a number of chloride channels within the kidney are still unknown. Abnormalities in chloride channel expression and function in the kidney can cause a range of disorders such as autosomal recessive Dent's disease, Bartter's syndrome, renal tubular acidosis and diabetes insipidus. The purpose of this review is to give an overview of the chloride channels in the kidney and to focus on the function of renal chloride channels as revealed by diseases associated with channel dysfunction.