1.Bicarbonate reabsorption in proximal renal tubule: molecular mechanisms and metabolic acidosis.
Yi-Min GUO ; Ying LIU ; Li-Ming CHEN
Acta Physiologica Sinica 2014;66(4):398-414
HCO3(-) reabsorption in the renal tubules plays a critically important role in maintaining the global acid-base balance. Loss of HCO3(-) causes metabolic acidosis. Proximal renal tubule is the major site for HCO3(-) reabsorption, accounting for more than 80% of total HCO3(-) reabsorption along the nephron. Over the past more than half centuries, tremendous progresses have been made on understanding the molecular mechanisms underlying the HCO3(-) reabsorption in proximal tubules. The transepithelial movement of HCO3(-) involves the coordinated operation of machineries on both the apical and the basolateral membranes of the epithelial cells. On the apical domain, Na(+)-H(+) exchanger NHE3 and the vacuolar H(+)-ATPase are two major pathways mediating the apical uptake of HCO3(-)-related species. Taken together, NHE3 and H(+)-ATPase are responsible for about 80% of HCO3(-) reabsorption in the proximal tubule. The remaining 20% is likely mediated by pathways yet to be characterized. On the basolateral membrane, NBCe1 represents the only major known pathway mediating the extrusion of HCO3(-) coupled with Na(+) into the interstitial space. In the present article, we provide a historical view about the studies on the mechanisms of HCO3(-) reabsorption since 1940s. Moreover, we summarize the latest progresses emerging over the past decade in the physiological as well as pathological roles of acid-base transporters underlying the HCO3(-) reabsorption in proximal tubules.
Acidosis
;
physiopathology
;
Animals
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Bicarbonates
;
metabolism
;
Humans
;
Kidney Tubules, Proximal
;
physiopathology
;
Sodium-Hydrogen Exchangers
;
physiology
;
Vacuolar Proton-Translocating ATPases
;
physiology
2.Gene cloning and expression characteristics of vacuolar-type ATPase subunit B in Bombyx mori.
Huifang CHEN ; Xin WANG ; Kang XIE ; Yi LI ; Ping ZHAO
Chinese Journal of Biotechnology 2016;32(4):487-496
Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H⁺-transporting proteins. It keeps the proton balance by transporting H⁺ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and purified using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.
Animals
;
Bombyx
;
enzymology
;
Cloning, Molecular
;
Escherichia coli
;
metabolism
;
Insect Proteins
;
genetics
;
metabolism
;
Larva
;
Recombinant Proteins
;
genetics
;
metabolism
;
Silk
;
Vacuolar Proton-Translocating ATPases
;
genetics
;
metabolism
3.Effect of synergistic polarization macrophage modulated by N-terminal domain of a2 vacuolar ATPase and macrophage colony stimulating factor on proliferation of gastric cancer cells.
Dandan LIAN ; Guiliang MA ; Chen SUN ; Weizheng MAO
Chinese Journal of Gastrointestinal Surgery 2016;19(2):209-215
OBJECTIVETo investigate the synergistic effect between the N-terminus domain of the a2 isoform of vacuolar ATPase (a2NTD) and macrophage colony-stimulating factor (M-CSF) on modulating macrophage polarization and the impact of polarized macrophages on proliferation of gastric cancer cells.
METHODSPeripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. Then macrophages were randomly divided into four groups: the control group (RPMI 1640), the experimental group I (M-CSF 100 μg/L), the experimental group II (a2NTD 500 μg/L) and the experimental group III (a2NTD 500 μg/L plus M-CSF 100 μg/L). After stimulation for 48 hours, double color immunofluorescence cytochemistry was adopted to detect the expression of cell membrane molecules on macrophages; ELISA was used to measure the secretion of cytokines IL-10 and IL-12; CCK-8 assay was used to evaluate the impact of macrophages on proliferation ability of gastric cancer cell strain SGC-7901.
RESULTSThe expression of CD68, also known as macrophage surface antigen, was detected on macrophage membrane in all four groups (+). The mean absorbance (A) was 0.092 ± 0.005 in control group, 0.095 ± 0.006 in group I, 0.094 ± 0.005 in group II, 0.094 ± 0.005 in group III, and no significant differences were observed among 4 groups (all P>0.05). Meanwhile, the expression of CD206, which mainly exists on M2 macrophage membrane, was hard to detect in control group (-) with A 0.025 ± 0.004; it was normal in groupI and group II (+) with A 0.191 ± 0.012 in group I and 0.197 ± 0.136 in group II (P=0.212), and it was up-regulated significantly in group III (+++) with A 0.285 ± 0.011. There were significant differences between either two groups except group I and group II (all P<0.01). Secretion of IL-10 in group I and group II [(85.65 ± 13.64) ng/L and (87.77 ± 14.25) ng/L] was significantly higher compared with control group [(71.67 ± 7.56) ng/L, P<0.01]. Secretion of IL-12 in group I and group II [(9.91 ± 1.50) ng/L and (10.15 ± 1.80) ng/L] was significantly lower compared with control group [(16.87 ± 1.10) ng/L, P<0.01]. Secretion of IL-10 in group III [(116.98 ± 14.27) ng/L] was the highest, and secretion of IL-12 [(5.31 ± 0.88) ng/L] was the lowest (all P<0.01). There was a synergistic effect between a2NTD and M-CSF on the secretion of both IL-10 and IL-12. Elevated proliferation of gastric cancer cell strain SGC-7901 was detected in all four groups, in which group III showed the greatest impact compared with other 3 groups (P<0.01).
CONCLUSIONSa2NTD and M-CSF show a synergistic effect in modulating macrophage phenotype and the secretion of IL-10 and IL-12. The polarized macrophage can significantly enhance proliferation of gastric cancer cell strain SGC-7901.
Cell Proliferation ; Humans ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Macrophage Colony-Stimulating Factor ; pharmacology ; Macrophages ; cytology ; Phenotype ; Stomach Neoplasms ; pathology ; Tumor Cells, Cultured ; Vacuolar Proton-Translocating ATPases ; pharmacology
4.Ultracytochemical observation of the intracellular localization of H+-adenosine triphosphatase.
Shen-qiu LUO ; Zhi-yong KE ; Yan-meng LU
Journal of Southern Medical University 2011;31(8):1431-1433
OBJECTIVETo observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles.
METHODSThe localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods.
RESULTSH(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats.
CONCLUSIONThis finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.
Animals ; Cell Membrane ; enzymology ; Hepatocytes ; cytology ; enzymology ; ultrastructure ; Histocytochemistry ; methods ; Kidney ; cytology ; enzymology ; ultrastructure ; Lysosomes ; enzymology ; Male ; Organelles ; enzymology ; Rats ; Rats, Wistar ; Vacuolar Proton-Translocating ATPases ; metabolism
5.LASS2 interacts with V-ATPase and inhibits cell growth of hepatocellular carcinoma.
Ning TANG ; Jie JIN ; Yun DENG ; Rong-Hu KE ; Qiu-Jin SHEN ; Shao-Hua FAN ; Wen-Xin QIN
Acta Physiologica Sinica 2010;62(3):196-202
Homo sapiens longevity assurance homologue 2 (LASS2) is a novel gene isolated from a human liver cDNA library by our laboratory, and it is a human homologue of the yeast longevity assurance gene LAG1 (Saccharomyces cerevisiae longevity assurance gene). According to our previous results, LASS2 could interact with subunit c of vacuolar type H(+)-ATPase (V-ATPase), and the overexpression of LASS2 could inhibit the cell growth of a human hepatocellular carcinoma (HCC) cell line, SMMC-7721. In order to understand the role of the interaction between LASS2 and V-ATPase in HCC cell growth, we transiently transfected plasmid pCMV-HA2-LASS2 into HCCLM3, a HCC cell line without the significant expression of endogenous LASS2. The pH-sensitive fluorescence probes, BCECF and BCECF-AM, were used to measure the intracellular and extracellular H(+) concentrations of HCCLM3 cells respectively. The effect of LASS2 gene on apoptosis was evaluated with Annexin-V/FITC and propidium iodide (PI) by flow cytometry. Western blot was used to detect cytochrome c (Cyt c) in the cytosol and mitochondria, as well as pro-caspase-3 in cytosol. The results showed that the cell growth of LASS2-transfected HCCLM3 cells was significantly inhibited compared with that of the mock control. LASS2 transfection increased intracellular H(+) concentration of HCCLM3 cells, while decreased extracellular H(+) concentration. Moreover, LASS2 transfection significantly enhanced the apoptosis of HCCLM3 cells. In LASS2-transfected cells, the amounts of Cyt c increased in the cytosol, while decreased in the mitochondria. Meanwhile, the expression of pro-caspase-3 in the cytosolic extracts was decreased. These results implicate that LASS2 gene might increase intracellular H(+) of HCC cells via the interaction with V-ATPase, thereby inducing cell apoptosis through mitochondrial pathway.
Apoptosis
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Carcinoma, Hepatocellular
;
pathology
;
Caspase 3
;
metabolism
;
Cell Line, Tumor
;
Cell Proliferation
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Humans
;
Liver Neoplasms
;
pathology
;
Membrane Proteins
;
metabolism
;
RNA, Small Interfering
;
Sphingosine N-Acyltransferase
;
metabolism
;
Transfection
;
Tumor Suppressor Proteins
;
metabolism
;
Vacuolar Proton-Translocating ATPases
;
metabolism
6.Cell type specificity of signaling: view from membrane receptors distribution and their downstream transduction networks.
Ying HE ; Zhonghao YU ; Dongya GE ; Rui WANG-SATTLER ; Hans-Jürgen THIESEN ; Lu XIE ; Yixue LI
Protein & Cell 2012;3(9):701-713
Studies on cell signaling pay more attention to spatial dynamics and how such diverse organization can relate to high order of cellular capabilities. To overview the specificity of cell signaling, we integrated human receptome data with proteome spatial expression profiles to systematically investigate the specificity of receptors and receptor-triggered transduction networks across 62 normal cell types and 14 cancer types. Six percent receptors showed cell-type-specific expression, and 4% signaling networks presented enriched cell-specific proteins induced by the receptors. We introduced a concept of "response context" to annotate the cell-type dependent signaling networks. We found that most cells respond similarly to the same stimulus, as the "response contexts" presented high functional similarity. Despite this, the subtle spatial diversity can be observed from the difference in network architectures. The architecture of the signaling networks in nerve cells displayed less completeness than that in glandular cells, which indicated cellular-context dependent signaling patterns are elaborately spatially organized. Likewise, in cancer cells most signaling networks were generally dysfunctional and less complete than that in normal cells. However, glioma emerged hyper-activated transduction mechanism in malignant state. Receptor ATP6AP2 and TNFRSF21 induced rennin-angiotensin and apoptosis signaling were found likely to explain the glioma-specific mechanism. This work represents an effort to decipher context-specific signaling network from spatial dimension. Our results indicated that although a majority of cells engage general signaling response with subtle differences, the spatial dynamics of cell signaling can not only deepen our insights into different signaling mechanisms, but also help understand cell signaling in disease.
Cell Line
;
Databases, Protein
;
Gene Expression Profiling
;
Humans
;
Metabolic Networks and Pathways
;
Neoplasms
;
metabolism
;
pathology
;
Proteome
;
analysis
;
Receptors, Cell Surface
;
metabolism
;
Receptors, Tumor Necrosis Factor
;
metabolism
;
Signal Transduction
;
Vacuolar Proton-Translocating ATPases
;
metabolism
7.Numb activates the mTORC1 signaling pathway in proximal tubular epithelial cells by upregulating V1G1 expression.
Ze LIU ; Da YOU ; Yong LI ; Yong Mei HE ; A Fang LI ; Pan LI ; Chun Yan LI
Journal of Southern Medical University 2022;42(10):1462-1469
OBJECTIVE:
To investigate the role of Numb in regulating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway.
METHODS:
Male BALB/C mouse models of acute kidney injury (AKI) were subjected to intravenous injections of Numb-siRNA or NC-siRNA with or without intraperitoneal cisplatin injections. After the treatments, the expressions and distribution of Numb and megalin in the renal tissues of the mice were detected with immunohistochemistry, and the renal expressions of Numb, S6, p-S6, S6K1, p-S6K1, 4EBP1 and p-4EBP1 were examined with Western blotting. The proximal renal tubular epithelial cells were isolated from the mice transfected with Numb-siRNA for in vitro culture. In NRK-52E cells, the effects of amino acid stimulation, Numb knockdown, and V1G1 overexpression, alone or in combination, on expressions of Numb, S6 and p-S6 were detected with Western blotting; the expressions of AMPK and p-AMPK were also detected in transfected NRK-52E cells, mouse kidneys and cultured mouse renal tubular epithelial cells.
RESULTS:
In BALB/C mice, injection of Numb-siRNA caused significant reductions of Numb and p-S6 expressions without affecting megalin expression in the renal proximal tubules (P < 0.05). Cisplatin treatment obviously upregulated p-S6K1 and p-4EBP1 expressions in the kidneys of the mice (P < 0.05), and this effect was significantly inhibited by treatment with Numb-siRNA (P < 0.05). In NRK-52E cells, amino acid stimulation significantly upregulated the expression of p-S6 (P < 0.05), which was strongly suppressed by transfection with Numb-siRNA (P < 0.05). Numb knockdown inhibited AMPK activation in NRK-52E cells, mouse kidneys and primary proximal tubular epithelial cells (P < 0.05). Numb knockdown significantly downregulated V1G1 expression in NRK-52E cells (P < 0.05), and V1G1 overexpression obviously reversed the inhibitory effect of Numb-siRNA on S6 phosphorylation (P < 0.05).
CONCLUSION
Numb promotes the activation of mTORC1 signaling in proximal tubular epithelial cells by upregulating V1G1 expression.
Animals
;
Male
;
Mice
;
Amino Acids/pharmacology*
;
AMP-Activated Protein Kinases/metabolism*
;
Cisplatin/pharmacology*
;
Epithelial Cells
;
Low Density Lipoprotein Receptor-Related Protein-2/metabolism*
;
Mammals/metabolism*
;
Mechanistic Target of Rapamycin Complex 1/metabolism*
;
Membrane Proteins/metabolism*
;
Mice, Inbred BALB C
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Nerve Tissue Proteins/metabolism*
;
RNA, Small Interfering/metabolism*
;
Signal Transduction
;
Vacuolar Proton-Translocating ATPases/metabolism*
8.TMSG-1 and its roles in tumor biology.
Xiao-Yan XU ; Fei PEI ; Jiang-Feng YOU
Chinese Journal of Cancer 2010;29(7):697-702
TMSG-1 is a newly discovered tumor metastasis suppressor gene, which plays important roles in promoting apoptosis and inhibiting invasion and metastasis of tumor cells. The inhibitory function of TMSG-1 in tumor cells may be related to vacuolar H+-ATPase and ceramide, but the underlying mechanism remains unknown. Studies on TMSG-1 are limited worldwide, and only a research group in Shanghai and our group have recently studied on it. As a new research field, the function of TMSG-1 remains to be explored. This review discusses the discovery of TMSG-1, structure of its encoded protein, its roles and possible mechanism in inhibiting tumor invasion and metastasis.
Animals
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Apoptosis
;
drug effects
;
Cell Cycle
;
drug effects
;
Ceramides
;
chemical synthesis
;
pharmacology
;
Enzyme Activation
;
Humans
;
Membrane Proteins
;
metabolism
;
physiology
;
Neoplasm Invasiveness
;
Neoplasm Metastasis
;
Neoplasms
;
metabolism
;
pathology
;
Phosphorylation
;
Sphingosine N-Acyltransferase
;
metabolism
;
physiology
;
Tumor Suppressor Proteins
;
metabolism
;
physiology
;
Vacuolar Proton-Translocating ATPases
;
metabolism
9.Refractory Hypertension and Isosexual Pseudoprecocious Puberty Associated with Renin-Secreting Ovarian Steroid Cell Tumor in a Girl.
Sun Hee LEE ; Mi Seon KANG ; Gyeong Sin LEE ; Woo Yeong CHUNG
Journal of Korean Medical Science 2011;26(6):836-838
Steroid cell tumor, not otherwise specified (NOS), are rare ovarian tumor, in addition, it is more rare in children. The majority of these tumors produce several steroid hormones, particularly testosterone. Estrogen also secreted by steroid cell tumor, NOS, but it is uncommon. Furthermore, hypertension is an infrequent sign in steroid cell tumor, NOS. An 8.5-yr-old girl with hypertension and frequent vaginal spotting visited at our clinic. On laboratory evaluation, secondary hypertension due to an elevated plasma renin level and isosexual pseudoprecocious puberty was diagnosed. Right solid ovarian mass was detected in radiologic tests. She underwent a right ooporectomy and it revealed renin and progesterone receptor positive steroid cell tumor, NOS. After operation, her blood pressure returned to normal level and vaginal bleeding disappeared. Even though this case is very rare, when hypertension coincides with virilization or feminization, a renin-secreting ovarian steroid cell tumor, NOS, should be considered.
Child
;
Female
;
Humans
;
Hypertension/*etiology
;
Ovarian Neoplasms/complications/*diagnosis/pathology
;
Puberty, Precocious/enzymology/*etiology
;
Receptors, Cell Surface/metabolism
;
Receptors, Progesterone/metabolism
;
Renin/blood
;
Sex Cord-Gonadal Stromal Tumors/complications/*diagnosis/pathology
;
Steroids/biosynthesis
;
Tomography, X-Ray Computed
;
Vacuolar Proton-Translocating ATPases/metabolism
10.Molecular and functional comparisons of the vacuolar Na+/H+ exchangers originated from glycophytic and halophytic species.
Jin-yao LI ; Xiao-wei HE ; Li XU ; Jie ZHOU ; Ping WU ; Hui-xia SHOU ; Fu-chun ZHANG
Journal of Zhejiang University. Science. B 2008;9(2):132-140
A novel vacuolar Na+/H+ exchanger, CgNHX1, was cloned from a halophytic species Chenopodium glaucum by using reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique. Sequence alignment and phylogenetic analysis of 22 NHX genes from GenBank as well as the new CgNHX1 gene indicate that NHX genes shared a great degree of similarity, regardless of their glycophytic or halophytic origin. Expression of the CgNHX1 gene was induced by NaCl and peaked at 400 mmol/L NaCl. Overexpression of NHX1 genes in rice enhanced their tolerance to salt stress. However, there is no significant difference in salt tolerance among the transgenic rice plants overexpressing the NHX1 genes from either glycophytic or halophytic species. The Na+ content of both the wild type (WT) and transgenic plants increased when exposed to 50 and 100 mmol/L NaCl, and the Na+ concentration in transgenic plants was marginally higher than that of WT. Our data demonstrate that the overexpression of the NHX1 gene from either glycophytic or halophytic species resulted in the enhanced tolerance to salt stress at a similar level, suggesting that NHX gene per se might not be the reason accounting for the difference in salt tolerance between glycophytes and halophytes.
Amino Acid Sequence
;
Cloning, Molecular
;
DNA, Complementary
;
metabolism
;
Gene Expression Regulation, Plant
;
Molecular Sequence Data
;
Oryza
;
genetics
;
Phylogeny
;
Plant Proteins
;
genetics
;
Plants
;
genetics
;
metabolism
;
Plants, Genetically Modified
;
Protein Structure, Tertiary
;
Reverse Transcriptase Polymerase Chain Reaction
;
Salts
;
pharmacology
;
Sequence Homology, Amino Acid
;
Sodium Chloride
;
pharmacology
;
Vacuolar Proton-Translocating ATPases
;
chemistry
;
physiology