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Highlights: [•] Copper transport in gill cells of a mangrove crab Ucides cordatus is dependent of calcium. [•] Copper transport mechanism is ATP-dependent. [•] Transport was monitored second by second during 300s. [Copyright &y& Elsevier]

Highlights: [•] Immunocytochemical localization of glycine in CA1 pyramidal neurons. [•] Co-storage of glycine in glutamatergic presynaptic terminals. [•] Activity-dependent release of glycine. [•] Functional expression of synaptic and extrasynaptic GlyRs. [ABSTRACT FROM AUTHOR]

Abstract: Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca2+, although the mechanisms mediating Ca2+ uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca2+, are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca2+, dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca2+. We will also address the potential role of vesicular Ca2+ in two major processes in secretory cells, vesicle movement and exocytosis. [Copyright &y& Elsevier]

Abstract: Fibroblast growth factors (FGFs) act as trophic factors during development and regeneration of the nervous system. FGFs mediate their responses by activation of four types of FGF receptors (FGFR1–4). FGFR1 is expressed in adult sensory neurons of dorsal root ganglia (DRG), and overexpression of FGFR1 enhances FGF-2-induced elongative axon growth in vitro. Ligand-induced activation of FGFR1 is followed by endocytosis and rapid lysosomal degradation. We previously reported that the lysosomal inhibitor leupeptin prevents degradation of FGFR1 and promotes FGF-2-induced elongative axon growth of DRG neurons overexpressing FGFR1. Therefore, we analyzed the effects of leupeptin on intracellular sorting of FGFR1 in PC12 pheochromocytoma cells and DRG neurons. Leupeptin increased colocalization of FGFR1 with lysosomes. Furthermore, leupeptin enhanced the cell surface localization of FGFR1 by increased receptor recycling and this effect was abolished by the recycling inhibitor monensin. In addition, a lysine mutant of FGFR1, which is preferentially recycled back to the cell surface, promoted elongative axon growth of DRG neurons similar to leupeptin. In contrast, the lysosomal inhibitor bafilomycin had no effect on surface localization of FGFR1, inhibited axon growth of DRG neurons and abolished the effects of leupeptin on receptor recycling. Together, our results strongly imply that increased recycling of FGFR1 promotes axon elongation, but not axonal branching, of adult DRG neurons in vitro. [Copyright &y& Elsevier]

We investigated transport of the organic anion Chlorophenol Red (CPR) in the tardigrade Halobiotus crispae using a new method for quantifying non-fluorescent dyes. We compared the results acquired from the tardigrade with CPR transport data obtained from Malpighian tubules of the desert locust Schistocerca gregaria. CPR accumulated in the midgut lumen of H. crispae, Indicating that organic anion transport takes place here. Our results show that CPR transport is inhibited by the mitochondrial un-coupler DNP (1 mmol l-1 81% reduction), the Na+/K+-ATPase inhibitor ouabain (10 mmol l-1 21% reduction) and the vacuolar H+-ATPase inhibitor bafilomycin (5 μmol l-1; 21% reduction), and by the organic anions PAH (10 mmol l-1 44% reduction) and probenecid (10 mmol l-1 61% reduction, concentration-dependent inhibition). Transport by locust Malpighian tubules exhibits a similar pharmacological profile, albeit with markedly higher concentrations of CPR being reached in S. gregaria. Immunolocalizatlon of the Na+/K+-ATPase α-subunit in S. gregaria revealed that this transporter is abundantly expressed and localized to the basal cell membranes. Immunolocalizatlon data could not be obtained from H. crispae. Our results indicate that organic anion secretion by the tardigrade midgut is transporter mediated with likely candidates for the basolateral entry step being members of the Oat and/or Oatp transporter families. From our results, we cautiously suggest that apical H+ and possibly basal Na+/W pumps provide the driving force for the transport; the exact coupling between electrochemical gradients generated by the pumps and transport of ions, as well as the nature of the apical exit step, are unknown. This study is, to our knowledge, the first to show active epithelial transport in tardigrades. [ABSTRACT FROM AUTHOR]

The involvement of a Na+/H+ exchanger (NHE) in mediating Na+ uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na+ uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na+ uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na+ uptake in fish reared in acidic water (pH ~ 4.0). A role for NHE in Na+ uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na+/H+ exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na+ uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na+ uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na+ uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H+-ATPase) significantly reduced Na+ uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na+ uptake in larval zebrafish in acidic water: one in which Na+ uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H+-ATPase. [ABSTRACT FROM AUTHOR]

Abstract: The rate of acid-stimulated and phenamil-sensitive sodium (Na+) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA− MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na+ uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H+-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na+ transport in fish). In contrast, Na+ uptake in PNA− MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0mM external Na+. Acid-stimulated Na+ transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na+ transport in fish, were also responsible for inhibiting acid stimulated Na+ uptake in PNA− MR cells, but by themselves had no effect on basal Na+ transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na+ uptake in PNA− MR cells in a dose-dependent manner. We also demonstrate rapid (<1min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA− MR cells, but not in PVC. These data lend further support to the idea of a PNA− MR cell type as the primary site for Na+ uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na+ across the apical surface of the fish gill. [Copyright &y& Elsevier]

Recently, the development of an IgE receptor (FcεRI)-targeted, pH-sensitive, surface-enhanced Raman spectroscopy (SERS) nanosensor has been demonstrated by Nowak-Lovato and Rector (Appl Spectrosc 63:387-395, ). The targeted nanosensor enables spatial and temporal pH measurements as internalized receptors progress through endosomal compartments in live cells. Trafficking of receptor-bound nanosensors was compared at physiological temperature (37 °C) versus room temperature (25 °C). As expected, we observed markedly slower progression of receptors through low-pH endocytic compartments at the lower temperature. We also demonstrate the utility of the nanosensors to measure directly changes in the pH of intracellular compartments after treatment with bafilomycin or amiloride. We report an increase in endosome compartment pH after treatment with bafilomycin, an H ATPase pump inhibitor. Decreased endosomal luminal pH was measured in cells treated with amiloride, an inhibitor of Na/H exchange. The decrease in amiloride-treated cells was transient, followed by a recovery period of approximately 15-20 min to restore endosomal pH. These experiments demonstrate the novel application of Raman spectroscopy to monitor local pH environment in live cells with the use of targeted SERS nanosensors. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 1193–1204. We have shown previously that the plecomacrolide antibiotics bafilomycin A1 and B1 significantly attenuate cerebellar granule neuron death resulting from agents that disrupt lysosome function. To further characterize bafilomycin-mediated cytoprotection, we examined its ability to attenuate the death of naïve and differentiated neuronal SH-SY5Y human neuroblastoma cells from agents that induce lysosome dysfunction in vitro, and from in vivo dopaminergic neuron death in C. elegans. Low-dose bafilomycin significantly attenuated SH-SY5Y cell death resulting from treatment with chloroquine, hydroxychloroquine amodiaquine and staurosporine. Bafilomycin also attenuated the chloroquine-induced reduction in processing of cathepsin D, the principal lysosomal aspartic acid protease, to its mature ‘active’ form. Chloroquine induced autophagic vacuole accumulation and inhibited autophagic flux, effects that were attenuated upon treatment with bafilomycin and were associated with a significant decrease in chloroquine-induced accumulation of detergent-insoluble α-synuclein oligomers. In addition, bafilomycin significantly and dose-dependently attenuated dopaminergic neuron death in C. elegans resulting from in vivo over-expression of human wild-type α-synuclein. Together, our findings suggest that low-dose bafilomycin is cytoprotective in part through its maintenance of the autophagy-lysosome pathway, and underscores its therapeutic potential for treating Parkinson’s disease and other neurodegenerative diseases that exhibit disruption of protein degradation pathways and accumulation of toxic protein species. [ABSTRACT FROM AUTHOR]

The article presents information on a study which determined the role of (pro)renin receptor [(P)RR]/ATP6AP2 in murine cardiomyocytes. The role of [(P)RR] in the activation of the local renin-angiotensin system (RAS) is discussed. Findings of the study showed that cardiomyocyte-specific ablation of Atp6ap2 led to lethal heart failure and that RAB7- and lysosomal-associated membrane protein 2 (LAMP2)-positive multivesicular vacuoles are found in the cardiomyocytes. The mechanism of actions of the gene product of ATP6AP2 are described.

Abstract: Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca2+ and Na+ channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca2+ release channels that respond to activation by the Ca2+ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled. [Copyright &y& Elsevier]

A new 16-membered diene macrolide, bafilomycin M ( 1 ), was produced from a Streptomyces sp. GIC10-1, which was originally isolated from a marine sponge Theonella sp. The structure of 1 was established by spectroscopic methods and this compound was found to exhibit cytotoxicity toward K-562, HL-60, SUPT-1, and LNCaP tumor cells with IC 50 values 0.060, 0.011, 0.047, and 0.389 μg/mL, respectively. [ABSTRACT FROM AUTHOR]

Summary: Acidity is one of the main features of the tumors. The V-ATPase is the primary responsible for the control of tumor microenvironment by proton extrusion to the extracellular medium. The acid environment favors tissue damage, activation of destructive enzymes in the extracellular matrix, the acquisition of metastatic cell phenotypes as well as increasing the destructive capacity. The application of specific inhibitors of V-ATPases, can decrease the acidity of tumor and may allow the reduction of tumor metastasis, acting on the survival of tumor cells and prevent the phenomena of chemoresistance. Among the most important inhibitors can be distinguished benzolactone enamides (salicylihalamide), lobatamide A and B, apicularen, indolyls, oximidine, macrolactone archazolid, lobatamide C, and cruentaren. The latest generation of inhibitors includes NiK12192, FR202126, and PPI SB 242784. The purpose of this paper is to describe the latest advances in the field of V-ATPase inhibitors, describe further developments related to the classic inhibitors, and discuss new potential applications of these drugs in cancer treatment. [Copyright &y& Elsevier]

The (pro)renin receptor ([P]RR) is a transmembrane protein that binds both renin and prorenin with high affinity, increasing the catalytic cleavage of angiotensinogen and signaling intracellularly through mitogen-activated protein kinase activation. Although initially reported as having no homology with any known membrane protein, other studies have suggested that the (P)RR is an accessory protein, named ATP6ap2, that associates with the vacuolar H(+)-ATPase, a key mediator of final urinary acidification. Using in situ hybridization, immunohistochemistry, and electron microscopy, together with serial sections stained with nephron segment-specific markers, we found that (P)RR mRNA and protein were predominantly expressed in collecting ducts and in the distal nephron. Within collecting ducts, the (P)RR was most abundant in microvilli at the apical surface of A-type intercalated cells. Dual-staining immunofluorescence demonstrated colocalization of the (P)RR with the B1/2 subunit of the vacuolar H(+)-ATPase, the ion exchanger that secretes H(+) ions into the urinary space and that associates with an accessory subunit homologous to the (P)RR. In collecting duct/distal tubule lineage Madin-Darby canine kidney cells, extracellular signal-regulated kinase 1/2 phosphorylation, induced by either renin or prorenin, was attenuated by the selective vacuolar H(+)-ATPase inhibitor bafilomycin. The predominant expression of the (P)RR at the apex of acid-secreting cells in the collecting duct, along with its colocalization and homology with an accessory protein of the vacuolar H(+)-ATPase, suggests that the (P)RR may function primarily in distal nephron H(+) transport, recently noted to be, at least in part, an angiotensin II-dependent phenomenon. [ABSTRACT FROM AUTHOR]

Abstract: Differing and sometimes conflicting data have been reported regarding several aspects of vaccinia virus (VV) entry. To address this, we used a β-galactosidase reporter virus to monitor virus entry into multiple cell types under varying conditions. Entry into HeLa, B78H1 and L cells was strongly inhibited by heparin whereas entry into Vero and BSC-1 cells was unaffected. Bafilomycin also exhibited variable and cell-type-specific effects on VV entry. Entry into B78H1 and BSC-1 cells was strongly inhibited by bafilomycin whereas entry into Vero and HeLa cells was only partially inhibited suggesting the co-existence of both pH-dependent and pH-independent VV entry pathways in these cell types. Finally, entry into HeLa, B78H1, L and BSC-1 cells exhibited a lag of 6–9 min whereas this delay was undetectable in Vero cells. Our results suggest that VV exploits multiple cell attachment and entry pathways allowing it to infect a broad range of cells. [Copyright &y& Elsevier]

Synaptic transmission depends on the continued availability of neurotransmitter-filled synaptic vesicles (SVs) for triggered release from presynaptic boutons. Surprisingly, small boutons in the brain, that already contain comparatively few SVs, are thought to retain the majority of these SVs in a "reserve" pool that is not mobilized under physiological conditions. Why such a scarce synaptic resource is normally inaccessible has been a matter of debate. Here, we readdress this issue by developing an electrophysiological approach for counting SVs released from boutons formed by a single, isolated neuron on itself ("autapses"). We show that, after treatment with Bafilomycin Al to prevent reloading of discharged SVs with glutamate, each SV is counted only once on first-time release. Hence, by integrating all autaptic currents as they run down overtime, we can estimate the total number of SVs released by a single neuron. This total can be normalized to the number of boutons on the neuron, giving the mean number of SVs released per bouton. We estimate that up to ≈~130 vesicles can be released per bouton over ≈10 mm of stimulation at 0.2 Hz. This number of vesicles represents a substantial proportion of the total number of SVs (100-200) that have been counted in these boutons by using electron microscopy. Thus, mild electrical stimulation, when maintained for sufficient time, causes the eventual release of many of the SVs in a bouton, including those in the putative reserve pool. This result suggests that SVs are functionally homogeneous in that the majority can contribute to basal synaptic transmission. [ABSTRACT FROM AUTHOR]

Abstract: A wave of β cell apoptosis occurs around 2 weeks of age in rats and mice. We have previously reported that exposure in utero to bafilomycin, a plecomacrolide antibiotic that inhibits the vacuolar (v)ATPase enzyme and contaminates the human diet, delays this neonatal wave and accelerates diabetes in non-obese diabetic (NOD) mice. Here we exposed C57BL/6J mice in utero to bafilomycin and assessed the effects on islet morphology, apoptosis and activation of cell survival signaling in β cells. The neonatal wave of β cell apoptosis was associated with high expression and low phosphorylation of the pro-apoptotic Bcl-2 family protein Bad, whereas after weaning (3 weeks of age) Bad was down-regulated and β cell apoptosis was low. In contrast, in bafilomycin-exposed mice the frequency of apoptotic β cells and the expression of Bad remained high after weaning. Bafilomycin exposure also inactivated the insulin/IGF signaling pathway intermediate, FoxO1, and increased the insulin content in neonatal islets. Thus, exposure in utero to bafilomycin disrupts the regulation of Bad in neonatal β cells, increases cell survival signaling and delays the neonatal wave of apoptosis. Increased expression of Bad in adult β cells provides an explanation for accelerated diabetes in bafilomycin-exposed NOD mice, whereby disruption of neonatal islet-cell turnover may render the adult β cells more susceptible to induced cell death. [Copyright &y& Elsevier]

Abstract: We employed optical detection of the lipophylic dye FM1-43 and focal recordings of quantal release to investigate how synapsin affects vesicle cycling at the neuromuscular junction of synapsin knockout (Syn KO) Drosophila. Loading the dye employing high K+ stimulation, which presumably involves the recycling pool of vesicles in exo/endocytosis, stained the periphery of wild type (WT) boutons, while in Syn KO the dye was redistributed towards the center of the bouton. When endocytosis was promoted by cyclosporin A pretreatment, the dye uptake was significantly enhanced in WT boutons, and the entire boutons were stained, suggesting staining of the reserve vesicle pool. In Syn KO boutons, the same loading paradigm produced fainter staining and significantly faster destaining. When the axon was stimulated electrically, a distinct difference in dye loading patterns was observed in WT boutons at different stimulation frequencies: a low stimulation frequency (3 Hz) produced a ring-shaped staining pattern, while at a higher frequency (10 Hz) the dye was redistributed towards the center of the bouton and the fluorescence intensity was significantly increased. This difference in staining patterns was essentially disrupted in Syn KO boutons, although synapsin did not affect the rate of quantal release. Stimulation of the nerve in the presence of bafilomycin, the blocker of the transmitter uptake, produced significantly stronger depression in Syn KO boutons. These results, taken together, suggest that synapsin maintains the reserve pool of vesicles and segregation between the recycling and reserve pools, and that it mediates mobilization of the reserve pool during intense stimulation. [Copyright &y& Elsevier]

Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical Cl-/HCO3- exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, JCl in mouse CCD through a pendrin-dependent mechanism. JCl and transepithelial voltage, VT, were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCI-replete diet or a NaCI-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaCl-replete diet, VT and JCl were not different from zero either in the presence or absence of angiotensin II (10-8 M) in the bath. Thus further experiments employed mice given the high-NaCI diet and furdsemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative VT and absorbed Cl-. With angiotensin II in the bath, Cl- absorption doubled although VT did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl- secretion and a VT of ~0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished VT although JCl fell only ~50%. Thus substantial Cl- absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the H+-ATPase inhibitor bafilomycin abolished benzamil-insensitive Cl- absorption. In conclusion, angiotensin II increases transcellular Cl- absorption in the CCD through a pendrin- and H+-ATPase-dependent process. [ABSTRACT FROM AUTHOR]

Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3-based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and ceil migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascalar endothelial cells. [ABSTRACT FROM AUTHOR]

The article presents a study which identified iejimalides (IEJLs), 24-membered macrolides, as potent osteoclast inhibitors. The effect of IEJLs on V-ATPases was investigated. A bafilomycin-resistant yeast mutant conferred IEJL resistance. The results show that IEJLs are novel V-ATPase inhibitors and that antitumor and antiosteoporotic activities are exerted through V-ATPase inhibition.

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development. [ABSTRACT FROM AUTHOR]

Abstract: Acrosomal protease activation is regarded as an important event triggered by acrosomal reaction and leading to sperm passage through zona pellucida. Mammalian acrosome has an internal acid pH that probably helps to maintain inactive proenzymes that otherwise could be precociously activated and prevent normal fertilization. In this work, we have studied the effect of bafilomycin A1, a potent and specific inhibitor of vacuolar H(+)-pump (V-ATPase), on acrosome reaction and proacrosin activation. We used the pH-sensitive probe Lysotracker Green DND-26 to monitor qualitatively intra-acrosomal pH in cauda epididymal mouse spermatozoa. Our results showed that loss of Lysotracker label induced by bafilomycin A1 (acrosome alkalinization) did not induce acrosome reaction or proacrosin activation. We also developed a new technique for imaging the acrosome, and for evaluating the acrosome reaction, in live mouse spermatozoa using Lysotracker DND-26. These results showed that the V-ATPase is a key regulator of mammalian acrosome pH, and that acrosome alkalinization is not the only prerequisite to activate proacrosin under in vivo conditions. Our results suggest that acrosome alkalinization and acrosome reaction are two processes that could be independently regulated during mammalian sperm capacitation. [Copyright &y& Elsevier]

Under anoxia, Artemia franciscana embryos downregulate metabolic processes and approach an ametabolic state. Entrance into this quiescent state is accompanied by a profound acidification of the intracellular space, and more than two decades of research now clearly demonstrates that this acidification is critical to metabolic downregulation in anoxic embryos. However, the proximal mechanisms responsible for the pH shift remain largely unidentified. Here, we report evidence demonstrating expression of the V-ATPase in encysted embryos and present an argument for its involvement in the intracellular acidification induced by anoxia. We identified a single B-subunit cDNA sharing the greatest degree of sequence similarity with 'generalist-type' homologues from mammals (brain-type) and invertebrates. Quantitative analysis of B-subunit mRNA demonstrates differential expression throughout early development, and western blot analyses confirm the expression of at least six V-ATPase subunits in both heavy membranes and microsomal vesicles. The critical need for proton pumping during the anoxia-tolerant stage of development is demonstrated by incubation with the V-ATPase inhibitor bafilomycin A1, which halts embryonic development. Importantly, net proton flux from V-ATPase-acidified compartments to the surrounding cytoplasm is likely under anoxia and may significantly contribute to the enigmatic acidification critical to quiescence. [ABSTRACT FROM AUTHOR]

The effects of bafilomycin, nocodazole, and reduced temperature on recycling and the lysosomal pathway have been investigated in various cultured cell lines and have been shown to vary dependent on the cell type examined. However, the way in which these treatments affect recycling and transport to lysosomes within the same cell line has not been analyzed. In the current study, we used fluorophore-labeled transferrin and dextran as typical markers for the recycling and the lysosomal pathways, respectively, to explore the morphology and the intravesicular pH of endocytic compartments in HeLa cells. The V-ATPase inhibitor bafilomycin selectively inhibited the transport of marker destined for lysosomal degradation in early endosomes, whereas the transport of transferrin to the perinuclear recycling compartment (PNRC) still occurred. The kinetics of transferrin acidification was found to be biphasic, indicative of fast and slow recycling pathways via early endosomes (pH 6.0) and PNRC (pH 5.6), respectively. Furthermore, the disruption of microtubules by nocodazole blocked the transport of transferrin to the PNRC in early endosomes and of lysosome-directed marker into endosomal carrier vesicles. In contrast, incubation at 20°C affected the lysosomal pathway by causing retention of internalized dextran in late endosomes and a delay in transferrin recycling. Taken together, these data clearly demonstrate, for the first time, that the transferrin recycling pathway and transport of endocytosed material to lysosomes are differentially affected by bafilomycin, nocodazole, and low temperature in HeLa cells. Consequently, these treatments can be applied to investigate whether internalized macromolecules such as viruses follow a recycling or degradative pathway. [ABSTRACT FROM AUTHOR]

Abstract: In the fly''s visual system, two classes of lamina interneuron, L1 and L2, cyclically change both their size and shape in a rhythm that is circadian. Several neurotransmitters and the lamina''s glial cells are known to be involved in regulating these rhythms. Moreover, vacuolar-type H+—ATPase (V-ATPase) in the optic lobe is thought also to participate in such regulation. We have detected V-ATPase-like immunoreactivity in the heads of both Drosophilla melanogaster and Musca domestica using antibodies raised against either the B- or H-subunits of V-ATPase from D. melanogaster or against the B-subunit from two other insect species Culex quinquefasciatus and Manduca sexta. In the visual systems of both fly species V-ATPase was localized immunocytochemically to the compound eye photoreceptors. In D. melanogaster immunoreactivity oscillated during the day and night and under constant darkness the signal was stronger during the subjective night than the subjective day. In turn, blocking V-ATPase by injecting a V-ATPase blocker, bafilomycin, in M. domestica increased the axon sizes of L1 and L2, but only when bafilomycin was applied during the night. As a result bafilomycin abolished the day/night difference in axon size in L1 and L2, their sizes being similar during the day and night. [Copyright &y& Elsevier]

Proton pumps participate in several aspects of endocytic protein trafficking. However, their involvement specifically in the GLUT4 pathway has been a matter of great controversy. Here, we report that incubation of 3T3-L1 adipocytes with specific inhibitors of V-type ATPase, concanamycin A and bafilomycin A1, inhibits insulin-regulated glucose transport and results in accumulation of GLUT4 in heavy, rapidly sedimenting intracellular membranes. Correspondingly, the amount of small responsive GLUT4 vesicles in concanamycin A- and bafilomycin A1-treated cells is decreased. We conclude that these drugs block translocation of GLUT4 in adipose cells by inhibiting formation of small insulin-responsive vesicles on donor intracellular membranes. At the same time, proton pump inhibitors do not affect insulin-dependent translocation of preexisting vesicles or GLUT4 sorting in recycling endosomes. On the contrary, wortmannin acutely inhibits insulin-dependent translocation of the preexisting vesicles but has no effect on vesicle formation. [ABSTRACT FROM AUTHOR]

Bafilomycin, a plecomacrolide produced by plant-pathogenic Streptomyces, contaminates tuberous vegetables and has adverse effects on β cells in adult mice. We therefore determined whether dietary bafilomycin influenced the progression of diabetes in the non-obese diabetic (NOD) mouse model of autoimmune Type 1 diabetes. Parent NOD mice were fed sub-toxic doses of bafilomycin in drinking water from conception until weaning, or various times after birth and blood glucose was monitored in the offspring. Pancreatic islets in neonatal offspring were examined histologically by quantitative morphometry and islet cell apoptosis was estimated by TUNEL assay. Exposure in utero to bafilomycin but not after birth significantly accelerated onset and increased the frequency of diabetes. In exposed mice, pancreatic islet organogenesis was disrupted, characterized by a striking increase in β-cell mass and a shift in timing of the normal wave of neonatal islet cell apoptosis from 2 weeks to 4 weeks of age. We postulate that accelerated onset and increased incidence of diabetes later in life result from disruption of the normal turnover of β cells in the neonatal pancreas. Since bafilomycin and related plecomacrolides contaminate Streptomyces-infected vegetables, dietary exposure during pregnancy could be an important and previously unsuspected environmental component of human Type 1 diabetes. [Copyright &y& Elsevier]

The incorporation of concanamycin A, a potent inhibitor of vacuolar ATPases, into membranes of dimyristoyl phosphatidylcholine has been studied by using EPR of spin-labelled lipid chains. At an inhibitor/lipid ratio of 1:1 mol/mol, concanamycin A broadens the chain-melting transition of the phospholipid bilayer membrane, and effects the lipid chain motion in the fluid phase. The outer hyperfine splitting of a spin label at the C-5 position and the line widths of a spin label at the C-14 position of the lipid chain are increased by concanamycin A. Considerably larger membrane perturbations are caused by equimolar admixture of a designed synthetic 5-(5,6-dichloro-2-indolyl)-2,4-pentadienoyl V-ATPase inhibitor. These results indicate that concanamycin A intercalates readily between the lipid chains in biological membranes, with minimal perturbation of the bilayer structure. Essentially identical results are obtained with concanamycin A added to preformed membranes as a concentrated solution in DMSO, or mixed with lipid in organic solvent prior to membrane formation. Therefore, the common mode of addition in V-ATPase inhibition assays ensures incorporation of concanamycin into the lipid bilayer milieu, which provides an efficient channel of access to the transmembrane domains of the V-ATPase. [Copyright &y& Elsevier]

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases. [Copyright &y& Elsevier]

Mammalian cells regulate their cytosolic pH through a variety of proton extruding and bicarbonate loading mechanisms. The human embryonic kidney cell line HEK 293 is thought to show some characteristics of proximal tubule cells. The present study was performed to investigate the activity of proton extruding mechanisms (i.e. Na+/H+ exchange and/or V-H+-ATPases) and the influence of intracellular pH (pHi) on the activation of these transport processes. At resting pHi (7.4) and in the absence of bicarbonate, removal of extracellular Na+ did not alter pHi. Intracellular acidification (pHi ∼6.2) after a NH4Cl prepulse (20 mM) followed by exposure to a Na+ free bath solution led to a slow pHi recovery (with a delay of 5 min), which was inhibited by the specific vacuolar H+-ATPase inhibitor bafilomyocin. There was no Na+- dependent pHi recovery upon exposure to Na+ after a short intracellular acidification (less than 5 min). However, when the intracellular acidification phase was extended, the activation of a Na+-dependent pHi recovery was seen. This Na+-dependent pHi recovery was inhibited by 30 µM EIPA but not by 2 µM EIPA or less, suggesting the involvement of NHE3. Western blot analysis confirmed the presence of NHE3 protein in HEK 293 cells. Disruption of the microtubular network by colchicine (20 µM) did not significantly inhibit the rate of Na+-independent or -dependent pHi recovery indicating that activation of both H+-ATPase, and the NHE were not due to stimulated trafficking of the transport proteins or some activators to the membrane. We conclude that a) the plasma membrane of HEK293 cells contains both, NHE and H+-ATPase and b) both H+ extrusion systems are inactive at neutral pHi, c) a decrease of cytosolic pH to 6.5 activates both transport proteins in a slow, time-dependent manner. [ABSTRACT FROM AUTHOR]

Lysosomes are known centers for sequestration of calcium and a variety of heavy metals in many invertebrate tissues, and as a result of this compartmentalization these organelles perform important detoxification roles in the animals involved. The present investigation uses a centrifugation method to isolate and purify hepatopancreatic lysosomes from the American lobster, Homarus americanus. Purified lysosomal preparations were used to characterize membrane transport mechanisms in these organelles for transferring and sequestering cytoplasmic copper following its absorption across the plasma membrane from dietary constituents. The copper-specific fluorescent dye, Phen Green, was employed to quantify transmembrane fluxes of this metal as has been recently used to investigate copper movements across hepatopancreatic mitochondrial and plasma membranes. Results indicated the presence of a vanadate-sensitive, calcium-stimulated, copper ATPase in the membranes of these organelles that displayed high affinity carrier-mediated transport kinetics and may significantly contribute to organismic copper homeostasis. Together with a putative bafilomycin-sensitive V-ATPase in the membrane of the same organelles, importing hydrogen ions into the organellar interior, this copper ATPase may function as part of a physiological mechanism for precipitate formation between metallic cations and anions. These ionic precipitate complexes may then act as a sink for excess metals and thereby reduce the circulating concentrations of these elements. [Copyright &y& Elsevier]