The activity of vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.

The vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V-0 and V-1 subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V1 components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V-1 translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Master of Science / Department of Entomology / Kristopher S. Silver / Kun Yan Zhu / The yellow fever mosquito, Aedes aegypti, is a significant vector of several viral diseases, including Zika, dengue fever, yellow fever, and chikungunya. Since vaccines are not currently available for these viruses, control of the disease vectors by using insecticides is the most common practice for preventing disease. As a result, Ae. aegypti has developed resistance against many of the most commonly used insecticides, including organophosphates and pyrethroids. The rise in resistance in vector mosquitoes requires the search for new control strategies, such as RNA interference (RNAi), to manage mosquito populations. Vacuolar H[sup plus]+-ATPase (V-ATPase), a multi-subunit enzyme involved in many cellular processes, including membrane energization, acidification of organelles, and entry of dengue virus into the cytoplasm, is a potential target for RNAi, though little is known about its genetic structure or expression patterns in Ae. aegypti. In this study, I performed genome-wide surveys to identify the genes encoding different subunits of the V-ATPase protein complex, partially characterized the molecular properties and expression patterns of selected V-ATPase subunit genes, and tested the feasibility of using oral-based delivery of nanoparticles formed from double-stranded RNA (dsRNA) and chitosan to suppress the expression of selected V-ATPase subunit genes in Ae. aegypti. My genome-wide surveys revealed that Ae. aegypti V-ATPase consists of 13 different subunits (A, B, C, D, E, F, G, H, a, c, c”, d, e) encoded by 14 genes. Analysis of exon-intron arrangements for each gene demonstrated that each V-ATPase subunit gene has between one (subunit c) and 12 (subunit C) exons, with most genes (11) having 3 to 6 exons. Subsequent phylogenetic analysis of the deduced amino acid sequences of each subunit showed that V-ATPase subunits A, B, C, F, G, H, and a exhibited high levels of conservation among all the examined species, but subunits D, E, c, c”, d, and e showed high conservation only among dipteran species. Analysis of the expression profiles in different tissues and developmental stages of three specific V-ATPase subunits (A, D, and H) showed that whereas the expression of these genes varied between tissues and developmental stages, the patterns of expression of subunits A, D, and H were very similar. The highest mRNA expression level was observed in Malpighian tubules in fourth-instar larvae. Interestingly, expression of subunits A, D, or H in different tissues of adults was highest in male hindgut versus Malpighian tubules in females. Feeding mosquito larvae with chitosan nanoparticles made with dsRNA complementary to subunits A, D, or H resulted in significant suppression of mRNA transcript levels of each of these subunits. Peak suppression of V-ATPase A, D, or H transcripts occurred on the fifth day, where the gene transcript level was suppressed by 66.0, 27.3, or 70.4%, respectively, as compared with those of the control. Additionally, feeding of dsRNA/chitosan nanoparticles targeting subunit D caused mortality starting on day 3, with cumulative larval mortality reaching 14.8% on the sixth day. These results suggest that oral delivery of dsRNA/chitosan nanoparticles can substantially suppress target gene expression in Ae. aegypti larvae. However, increasing RNAi efficiency in targeting V-ATPase subunit genes in mosquito larvae appears to be necessary in order to obtain higher larval mortality using oral delivery of dsRNA/chitosan nanoparticles.

Vor zehn Jahren wurde der (Pro)Renin-Rezeptor [(P)RR] entdeckt und als neuer Bestandteil des Renin-Angiotensin-Systems beschrieben. Neuere Studien ergaben, dass der (P)RR mit der vakuolären H+-ATPase (V-ATPase) assoziiert sein kann, weshalb er auch V-ATPase associated protein 2 (ATP6ap2) genannt wird. In Osteoklasten befinden sich V-ATPase hauptsächlich an der zur Knochenoberfläche gerichteten Plasmamembran und transportieren Protonen in den extrazellulären Raum. Mäuse mit genetischer Deletion verschiedener V-ATPase-Untereinheiten charakterisiert durch einen Anstieg von Knochenmasse (Osteopetrose). In der vorliegenden Arbeit fanden wir heraus, dass (P)RR stark in reifen Osteoklasten in vitro und in vivo exprimiert wird. Mäuse mit genetischer Deletion des (P)RR in Osteoklasten wurden durch einen komplexen Knochen-Phänotyp mit reduzierter Knochendichte charakterisiert. (P)RR-defiziten Osteoklasten wiesen vermehrte Differenzierung und/oder Aktivität in vitro und in vivo auf. Wir postulieren deshalb, dass der (P)RR die in der Plasmamembran lokalisierten V-ATPase nicht direkt reguliert, sondern mit der physiologischen Aktivität der Osteoklasten durch andere Mechanismen interferiert. Macrophagen sind speziell auf die Immunabwehr ausgerichtete Fresszellen (Phagozyten). Phagozytose ist ein wesentlicher Zellprozess der die V-ATPase in Lysosomen braucht um die eingeschlossenen Pathogen zu zerstören. Wir generierten transgene Ratten mit konditionellen knockdown von (P)RR unter Nutzung eines Doxyzyclin-induzierten shRNA-Expressionssystems. Eine effiziente (P)RR-Depletion in Makrophagen wurde durch Behandlung mit Doxyzyclin in vivo im Trinkwasser und in vitro im Kulturmedium erreicht. Die vorliegende Arbeit zeigt, dass die Verschiebung des vesikulären pHs erst ziemlich spät nach (P)RR-Depletion auftritt. Wir fanden heraus, dass (P)RR-Depletion weder Phagozytose noch Endozytose beeinträchtigte, sondern für das Recycling des Transferrin-Rezeptors zur Plasmamembran wichtig ist. / A decade ago, the (pro)renin receptor [(P)RR] was discovered and depicted as a new component of the renin-angiotensin system. However, recent studies have put in evidence that the (P)RR associate with and regulate the vacuolar H+-ATPase (V-ATPase), hence its other name vacuolar H+-ATPase associated protein 2 (ATP6ap2). In osteoclasts, V-ATPases are mainly located at the plasma membrane facing the bone surface and extrude protons into the extracellular space. Mice with genetic deletion of various V-ATPase subunits are characterized by an increase of bone mass (osteopetrosis). In this work, we found that the (P)RR is highly expressed in mature osteoclasts in vitro and in vivo. Mice with genetic deletion of the (P)RR in osteoclasts developed a complex bone phenotype characterized by a reduced bone density. Osteoclasts lacking (P)RR displayed increased differentiation and/or activity in vitro and in vivo. We therefore suggest that the (P)RR does not directly regulate V-ATPases located at the plasma membrane but rather interferes with osteoclast physiology through other mechanisms. Macrophages are professionalized phagocytes crucial for immune response. Phagocytosis is an essential cellular process, which requires lysosomal V-ATPases for degradation of engulfed pathogens. We generated transgenic rats with a conditional depletion of the (P)RR with the use of a doxycycline-induced shRNA expression system. Efficient (P)RR depletion in macrophages was accomplished by doxycycline treatment in vivo in drinking water and in vitro in culture medium. In this work, we found that the impairment of vesicular pH occurs lately after (P)RR deletion. Also, we found that (P)RR deletion did not impair neither phagocytosis nor endocytosis but rather perturbed the recycling of the transferrin receptor to the plasma membrane.

Este estudo investigou o efeito da glicose sobre a atividade e expressão da isoforma 1 do trocador Na+/H+ (NHE1) e da H+-ATPase do tipo vacuolar, em células MDCK (Mardin Darby Canine Kidney), linhagem derivada de rim de cão, que apresenta características similares às células principais e intercalares das porções distais do néfron. Por microscopia de fluorescência, se avaliou a velocidade de recuperação do pHi (dpHi/dt) e a capacidade tamponante (<font face=\"symbol\">bi). A partir desses parâmetros, se calculou o efluxo de H+ (JH+). Por Western blot, se avaliou a expressão de NHE1 e da subunidade E da H+-ATPase do tipo vacuolar. Resultados: Na condição controle o efluxo de H+ foi de 6.27 ± 0.51 mM/min (n = 9). O tratamento agudo com glicose (25 mM) aumentou o efluxo de H+ via NHE1, o qual foi modulado pela PI3 cinase. Na mesma condição, não se observou alterações na atividade da H+-ATPase. O tratamento crônico com glicose (25 mM) induziu significante aumento do efluxo de H+, via NHE1 e H+-ATPase. O efeito estimulador da glicose sobre a atividade de NHE1 e H+-ATPase foi dependente da atividade da p38 MAP cinase. Além disso, o tratamento crônico com glicose (25 mM) induziu fosforilação do sistema ezrin/radixin/moesin (ERM) e Akt. Conclusões: Nossos resultados indicam que no tratamento agudo com glicose (25 mM), o NHE1 foi modulado pela PI3 cinase. Contudo, no tratamento crônico com glicose (25 mM), a atividade do NHE1 foi modulada pelo sistema ERM/Akt e a atividade da H+-ATPase foi modulada pela p38 MAP cinase. / This study investigated the effect of glucose on the activity and expression of Na+/H+ exchanger isoform 1 (NHE1) and vacuolar H+-ATPase, in Mardin Darby Canine Kidney (MDCK) cells from dog kidney, with similar characteristics to principal and intercalated cells of the distal nephron. The pHi recovery rate (dpHi/dt) and the buffering capacity (<font face=\"symbol\">bi) was evaluated through fluorescence microscopy. From these parameters the H+ efflux (JH+) was calculated. By Western blot, the NHE1 and H+-ATPase (E subunit) expression was evaluated. Results: In the control situation the H+ efflux was 6.27 ± 0.51 mM/pH units (n = 9). Acute treatment with glucose (25 mM) increased the H+ efflux via NHE1, which was modulated by PI3 kinase. In the same condition, the H+-ATPase activity did not change. Chronic treatment with glucose (25 mM) induced significant increase in H+ efflux via NHE1 and H+-ATPase. The stimulatory effect of glucose on the NHE1 and H+-ATPase activity was dependent on p38 MAP kinase activity. Furthermore, chronic treatment with glucose (25 mM) induced Ezrin/radixin/moesin (ERM) and Akt phosphorylation. Conclusions: Our results indicate that during the acute treatment with glucose (25 mM), the NHE1 is modulated by PI3 kinase. However, during chronic treatment with glucose (25 mM), NHE1 activity was modulated by the ERM/Akt system and of H+-ATPase activity was modulated by p38 MAP Kinase.

Este estudo investigou o efeito da glicose sobre a atividade e expressão da isoforma 1 do trocador Na+/H+ (NHE1) e da H+-ATPase do tipo vacuolar, em células MDCK (Mardin Darby Canine Kidney), linhagem derivada de rim de cão, que apresenta características similares às células principais e intercalares das porções distais do néfron. Por microscopia de fluorescência, se avaliou a velocidade de recuperação do pHi (dpHi/dt) e a capacidade tamponante (<font face=\"symbol\">bi). A partir desses parâmetros, se calculou o efluxo de H+ (JH+). Por Western blot, se avaliou a expressão de NHE1 e da subunidade E da H+-ATPase do tipo vacuolar. Resultados: Na condição controle o efluxo de H+ foi de 6.27 ± 0.51 mM/min (n = 9). O tratamento agudo com glicose (25 mM) aumentou o efluxo de H+ via NHE1, o qual foi modulado pela PI3 cinase. Na mesma condição, não se observou alterações na atividade da H+-ATPase. O tratamento crônico com glicose (25 mM) induziu significante aumento do efluxo de H+, via NHE1 e H+-ATPase. O efeito estimulador da glicose sobre a atividade de NHE1 e H+-ATPase foi dependente da atividade da p38 MAP cinase. Além disso, o tratamento crônico com glicose (25 mM) induziu fosforilação do sistema ezrin/radixin/moesin (ERM) e Akt. Conclusões: Nossos resultados indicam que no tratamento agudo com glicose (25 mM), o NHE1 foi modulado pela PI3 cinase. Contudo, no tratamento crônico com glicose (25 mM), a atividade do NHE1 foi modulada pelo sistema ERM/Akt e a atividade da H+-ATPase foi modulada pela p38 MAP cinase. / This study investigated the effect of glucose on the activity and expression of Na+/H+ exchanger isoform 1 (NHE1) and vacuolar H+-ATPase, in Mardin Darby Canine Kidney (MDCK) cells from dog kidney, with similar characteristics to principal and intercalated cells of the distal nephron. The pHi recovery rate (dpHi/dt) and the buffering capacity (<font face=\"symbol\">bi) was evaluated through fluorescence microscopy. From these parameters the H+ efflux (JH+) was calculated. By Western blot, the NHE1 and H+-ATPase (E subunit) expression was evaluated. Results: In the control situation the H+ efflux was 6.27 ± 0.51 mM/pH units (n = 9). Acute treatment with glucose (25 mM) increased the H+ efflux via NHE1, which was modulated by PI3 kinase. In the same condition, the H+-ATPase activity did not change. Chronic treatment with glucose (25 mM) induced significant increase in H+ efflux via NHE1 and H+-ATPase. The stimulatory effect of glucose on the NHE1 and H+-ATPase activity was dependent on p38 MAP kinase activity. Furthermore, chronic treatment with glucose (25 mM) induced Ezrin/radixin/moesin (ERM) and Akt phosphorylation. Conclusions: Our results indicate that during the acute treatment with glucose (25 mM), the NHE1 is modulated by PI3 kinase. However, during chronic treatment with glucose (25 mM), NHE1 activity was modulated by the ERM/Akt system and of H+-ATPase activity was modulated by p38 MAP Kinase.