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1.
J Mol Cell Cardiol ; 87: 180-91, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26283571

RESUMO

Interendothelial junctions play an important role in the maintenance of endothelial integrity and the regulation of vascular functions. We report here that cationic amino acid transporter-1 (CAT-1) is a novel interendothelial cell adhesion molecule (CAM). We identified that CAT-1 protein localized at cell-cell adhesive junctions, similar to the classic CAM of VE-cadherin, and knockdown of CAT-1 with siRNA led to an increase in endothelial permeability. In addition, CAT-1 formed a cis-homo-dimer and showed Ca(2+)-dependent trans-homo-interaction to cause homophilic cell-cell adhesion. Co-immunoprecipitation assays showed that CAT-1 can associate with ß-catenin. Furthermore, we found that the sub-cellular localization and function of CAT-1 are associated with cell confluency, in sub-confluent ECs CAT-1 proteins distribute on the entire surface and function as L-Arg transporters, but most of the CAT-1 in the confluent ECs are localized at interendothelial junctions and serve as CAMs. Further functional characterization has disclosed that extracellular L-Arg exposure stabilizes endothelial integrity via abating the cell junction disassembly of CAT-1 and blocking the cellular membrane CAT-1 internalization, which provides the new mechanisms for L-Arg paradox and trans-stimulation of cationic amino acid transport system (CAAT). These results suggest that CAT-1 is a novel CAM that directly regulates endothelial integrity and mediates the protective actions of L-Arg to endothelium via a NO-independent mechanism.


Assuntos
Permeabilidade Capilar/genética , Transportador 1 de Aminoácidos Catiônicos/biossíntese , Transportador 1 de Aminoácidos Catiônicos/metabolismo , Adesão Celular/genética , Animais , Arginina/metabolismo , Transportador 1 de Aminoácidos Catiônicos/genética , Junções Comunicantes/genética , Junções Comunicantes/patologia , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Óxido Nítrico/metabolismo , Suínos , beta Catenina/metabolismo
2.
Mol Cell Biochem ; 360(1-2): 309-20, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21948261

RESUMO

We previously reported that the vasoactive peptide 1 (P1, "SSWRRKRKESS") modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-α is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were incubated in culture medium containing Rhodamine red-labeled P1 (100 µM) for 0-120 min. Confocal examinations indicate that P1 internalization is time-dependent and reaches a plateau at 60 min. Caveolae disruption by methyl-ß-cyclodextrin (CD) and filipin (FIL) inhibited the internalization of P1 in ECs suggesting that P1 internalizes via caveolae. P1-stimulation also enhances phosphorylation of caveolar PKC-α and increases intracellular calcium (Ca(2+)) release in intact cells suggesting that P1 internalization is regulated by PKC-α in ECs. To confirm the roles of increased phosphorylation of PKC-α and Ca(2+) release in internalization of P1, PKC-α modulation by phorbol ester (PMA), PKC-α knockdown, and Ca(2+) scavenger BAPTA-AM model systems were used. PMA-stimulated phosphorylation of caveolar PKC-α is associated with significant reduction in P1 internalization. In contrast, PKC-α deficiency and reduced phosphorylation of PKC-α enhanced P1 internalization. P1-mediated increased phosphorylation of PKC-α appears to be associated with increased intracellular calcium (Ca(2+)) release since the Ca(2+) scavenger BAPTA-AM enhanced P1 internalization. These data indicate that caveolar integrity and P1-mediated increased phosphorylation of caveolar PKC-α play crucial roles in the regulation of P1 internalization in lung ECs.


Assuntos
Cavéolas/enzimologia , Peptídeos Penetradores de Células/metabolismo , Células Endoteliais/metabolismo , Pulmão/citologia , Peptídeos/metabolismo , Proteína Quinase C-alfa/metabolismo , Animais , Sinalização do Cálcio , Cavéolas/efeitos dos fármacos , Caveolina 1/metabolismo , Células Cultivadas , Endocitose/efeitos dos fármacos , Células Endoteliais/enzimologia , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Filipina/farmacologia , Fosforilação , Proteína Quinase C-alfa/genética , Suínos , Acetato de Tetradecanoilforbol/farmacologia , beta-Ciclodextrinas/farmacologia
3.
Biochim Biophys Acta ; 1800(9): 906-11, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20594946

RESUMO

BACKGROUND: The H,K-ATPase, consisting of α and ß subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα1 and HKα2 encoded by different genes. The ouabain-resistant gastric HKα1-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα2-H,K-ATPase from different species shows poor primary structure conservation of the HKα2 subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα1, HKα2, or HKbeta genes. METHODS: Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα1, HKα2, or HKß knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined. RESULTS: The colonic H,K-ATPase demonstrated slightly greater affinity for K(+) than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα2 KO but was observed in HKß KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα1-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα2-H,K-ATPase was not inhibited by these compounds. CONCLUSIONS: These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride. GENERAL SIGNIFICANCE: Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.


Assuntos
Colo/enzimologia , Resistência a Medicamentos/fisiologia , Inibidores Enzimáticos/farmacologia , Mucosa Gástrica/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Inibidores da Bomba de Prótons , Amilorida/farmacologia , Animais , Domínio Catalítico/genética , Resistência a Medicamentos/efeitos dos fármacos , ATPase Trocadora de Hidrogênio-Potássio/genética , Imidazóis/farmacologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/fisiologia , Ouabaína/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Vanadatos/farmacologia
4.
Am J Physiol Renal Physiol ; 298(2): F408-15, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19923411

RESUMO

In the collecting duct (CD), H-K-ATPases function in cation reabsorption and H secretion. This study evaluated H-K-ATPase-mediated H secretion along the mouse CD, measured as EIPA- and luminal bafilomycin A(1)-insensitive intracellular pH (pH(i)) recovery from acute H loading (NH(4)) using BCECF. pH(i) recovery was measured in 1) microperfused cortical, outer medullary, and inner medullary CDs (CCD, OMCD, and IMCD) from C57BL/6J mice fed a normal diet and 2) common murine CD cell lines. H-K-ATPase activity along the native, microperfused CD was greatest in the CCD, less in the OMCD, and least in the IMCD (0.10 +/- 0.02, 0.04 +/- 0.01, and 0.01 +/- 0.002 U/min, respectively). H-K-ATPase activity was 0.30 +/- 0.03 and 0.26 +/- 0.03 in A- and B-type ICs, respectively, and was sensitive to Sch-28080 or ouabain. pH(i) recovery was greatest in the OMCD(1) cell line (0.25 +/- 0.01) and less in mpkCCD(c14) (0.17 +/- 0.01), mIMCD-K2 (0.12 +/- 0.01), and mIMCD-3 (0.05 +/- 0.01) cells. EIPA inhibited the majority of pH(i) recovery in these cells (100%, 64%, 75%, and 80% in mpkCCD(c14), OMCD(1), mIMCD-K2, and mIMCD-3, respectively). In OMCD(1) cells, where EIPA-insensitive pH(i) recovery was greatest, H-K-ATPase activity was 0.10 +/- 0.01 and was significantly inhibited (80%) by Sch-28080. We conclude that 1) H-K-ATPase-mediated H secretion in the native mouse CD is greatest in the ICs of the CCD, 2) A- and B-type ICs possess HKalpha(1) and HKalpha(2) H-K-ATPase activity, and 3) the OMCD(1) cell line best exhibits H-K-ATPase.


Assuntos
Ácidos/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Túbulos Renais Coletores/metabolismo , Animais , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Feminino , ATPase Trocadora de Hidrogênio-Potássio/genética , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Isoenzimas/genética , Córtex Renal/citologia , Medula Renal/citologia , Túbulos Renais Coletores/citologia , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Ouabaína/farmacologia , Perfusão/métodos , Inibidores da Bomba de Prótons , RNA Mensageiro/metabolismo , Distribuição Tecidual
5.
Biophys J ; 97(6): 1578-85, 2009 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-19751662

RESUMO

Tension generation in endothelial cells of the aorta, spleen, and eye occurs in actin stress fibers, and is necessary for normal cell function. Sarcomeres are the tension-generating units of actin stress fibers in endothelial cells. How sarcomeres generate and maintain tension in stress fibers is not well understood. Using femtosecond laser ablation, we severed living stress fibers and measured sarcomere contraction under zero tension. The length of the sarcomere decreased in two phases: an instantaneous initial response, followed by a slower change in length attributed to myosin activity. The latter phase ceased abruptly after a minimum sarcomere length was reached, suggesting a rigid resistance that prevents further contraction. Furthermore, severed, contracted stress fibers did not relax when treated with myosin inhibitors, indicating that contracted stress fibers do not store elastic potential energy. These novel measurements combined with modeling suggest that myosin-generated forces in adjacent sarcomeres are directly in balance, and argue against sarcomere models with springlike elements in parallel with myosin contractile elements. We propose a new model for tension generation in the sarcomere, which provides a mechanistic interpretation for our observations and previous observations of inhomogeneous sarcomere contraction and apparent stress fiber viscoelastic behavior.


Assuntos
Células Endoteliais/citologia , Sarcômeros/metabolismo , Animais , Fenômenos Biomecânicos , Bovinos , Elasticidade , Células Endoteliais/metabolismo , Terapia a Laser , Modelos Lineares , Modelos Biológicos , Miosinas/metabolismo , Fibras de Estresse/metabolismo , Fatores de Tempo
6.
Mediators Inflamm ; 2009: 535348, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19325914

RESUMO

Macrophage migration inhibitory factor (MIF), an inflammatory cytokine, and its receptor CD74 are upregulated by bladder inflammation. MIF-mediated signal transduction involves binding to cell-surface CD74, this study documents, in vivo, MIF-CD74 interactions at the urothelial cell surface. N-hydroxysulfosuccinimide biotin ester-labeled surface urothelial proteins in rats treated either with saline or substance P (SP, 40 microg/kg). The bladder was examined by histology and confocal microscopy. Biotinylated proteins were purified by avidin agarose, immunoprecipitated with anti-MIF or anti-CD74 antibodies, and detected with strepavidin-HRP. Only superficial urothelial cells were biotinylated. These cells contained a biotinylated MIF/CD74 cell-surface complex that was increased in SP-treated animals. SP treatment increased MIF and CD74 mRNA in urothelial cells. Our data indicate that intraluminal MIF, released from urothelial cells as a consequence of SP treatment, interacts with urothelial cell-surface CD74. These results document that our previously described MIF-CD74 interaction occurs at the urothelial cell surface.


Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas de Membrana/metabolismo , Substância P/metabolismo , Urotélio/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Biotinilação , Antígenos de Histocompatibilidade Classe II/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Ratos , Ratos Sprague-Dawley , Urotélio/citologia
7.
Am J Pathol ; 172(3): 839-48, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18276778

RESUMO

Intimal hyperplasia of autologous vein grafts is a critical problem affecting the long-term patency of many types of vascular reconstruction. Within intimal hyperplasia lesions, smooth muscle cells are a major component, playing an essential role in the pathological process. Given that bone marrow-derived cells may differentiate into smooth muscle cells in the neointima of injured arteries, we hypothesized that the bone marrow may serve as a source for some of the smooth muscle cells within intimal hyperplasia lesions of vein grafts. To test this hypothesis, we used an established mouse model for intimal hyperplasia in wild-type mice that had been transplanted with bone marrow from a green fluorescent protein (GFP+/+) transgenic mouse. High-resolution confocal microscopy analysis performed 2 and 8 weeks after grafting demonstrated expression of GFP in 5.4 +/- 0.8% and 11.9 +/- 2.3%, respectively, of smooth muscle cells within intimal hyperplasia lesions. By 16 weeks, GFP expression in smooth muscle cells was not detected by immunohistochemistry; however, real-time PCR revealed that 20.2 +/- 1.7% of the smooth muscle cells captured from the neointima lesion by laser capture microdissection at 16 weeks contained GFP DNA. Our results suggest that bone marrow-derived cells differentiated into smooth muscle cells within the intimal lesion and may provide a novel clinical approach for decreasing intimal hyperplasia in vein grafts.


Assuntos
Transplante de Medula Óssea/fisiologia , Túnica Íntima/patologia , Veias/transplante , Animais , Proliferação de Células , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hiperplasia/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microdissecção , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Fatores de Tempo , Transplante Autólogo , Resultado do Tratamento
8.
Am J Physiol Renal Physiol ; 294(3): F621-7, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18057185

RESUMO

Two classes of H pumps, H-K-ATPase and H-ATPase, contribute to luminal acidification and HCO(3) transport in the collecting duct (CD). At least two H-K-ATPase alpha-subunits are expressed in the CD: HKalpha(1) and HKalpha(2). Both exhibit K dependence but have different inhibitor sensitivities. The HKalpha(1) H-K-ATPase is Sch-28080 sensitive, whereas the pharmacological profile of the HKalpha(2) H-K-ATPase is not completely understood. The present study used a nonpharmacological, genetic approach to determine the contribution of HKalpha(1) and HKalpha(2) to cortical CD (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. Intracellular pH (pH(i)) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type mice and mice of the same strain lacking expression of HKalpha(1), HKalpha(2), or both H-K-ATPases (HKalpha(1,2)). A-type and B-type ICs were differentiated by luminal loading with BCECF-AM and peritubular chloride removal from CO(2)/HCO(3)-buffered solutions to identify the membrane locations of Cl/HCO(3) exchange activity. H-ATPase- and Na/H exchange-mediated H transport were inhibited with bafilomycin A(1) (100 nM) and EIPA (10 microM), respectively. Here, we report 1) initial pH(i) and buffering capacity were not significantly altered in the ICs of HKalpha-deficient mice, 2) either HKalpha(1) or HKalpha(2) deficiency resulted in slower acid extrusion, and 3) A-type ICs from HKalpha(1,2)-deficient mice had significantly slower acid extrusion compared with A-type ICs from HKalpha(1)-deficient mice alone. These studies are the first nonpharmacological demonstration that both HKalpha(1) and HKalpha(2) contribute to H secretion in both A-type and B-type ICs in animals fed a normal diet.


Assuntos
Ácidos/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/deficiência , Túbulos Renais Coletores/metabolismo , Prótons , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Antiarrítmicos/farmacologia , Dieta , Feminino , Genótipo , ATPase Trocadora de Hidrogênio-Potássio/genética , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Túbulos Renais Coletores/citologia , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores
9.
Am J Physiol Renal Physiol ; 293(5): F1751-7, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17686958

RESUMO

This study determined whether nucleotides that bind to purinergic receptors (P2R) regulate the expression or function of serum- and glucocorticoid-inducible kinase-1 (SGK1) in mouse renal inner medullar collecting duct cells (mIMCD-3). The SGK1 protein was detected by Western blotting. A significant reduction of cytosolic SGK1 expression was observed in the cells pretreated with P2R agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and the reduction could be reversed by P2R antagonists. This reduction was also observed in cells that were pretreated with agonists for P2R subtypes. Using ELISA, we observed a reduced SGK1 kinase activity in ATPgammaS-pretreated cells. This effect was reversed by P2R antagonists. Furthermore, an increase of SGK1 kinase activity in aldosterone-pretreated cells was suppressed by ATPgammaS. These studies demonstrate for the first time that SGK1 can be downregulated by nucleotides in renal collecting duct epithelial cells, likely via the activation of P2R, and suggest that activation of renal purinergic signaling regulates a SGK1-dependent pathway that is known to modulate ion transport in the renal collecting duct.


Assuntos
Regulação para Baixo/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Túbulos Renais Coletores/metabolismo , Nucleotídeos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Aldosterona/farmacologia , Animais , Linhagem Celular , Citosol/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Medula Renal , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Camundongos , Nucleotídeos/farmacologia , Antagonistas Purinérgicos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ácidos Sulfônicos/farmacologia , Suramina/farmacologia
10.
Biochem Biophys Res Commun ; 359(3): 438-44, 2007 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-17560948

RESUMO

We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.


Assuntos
Sinalização do Cálcio , Túbulos Renais Coletores/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , RNA Mensageiro/genética , Receptores Purinérgicos P2/classificação , Receptores Purinérgicos P2/genética
11.
Br J Pharmacol ; 148(5): 732-40, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16715118

RESUMO

1. Myristoylated pseudosubstrate of PKCzeta (mPS) - a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCzeta pseudosubstrate region -- is considered a selective cell-permeable inhibitor of PKCzeta. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2. mPS at micromolar concentrations (1-10 microM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell-permeable version of PKCzeta pseudosubstrate fused to the HIV-TAT membrane-translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3. In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4. Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5. Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKC should be done with caution, especially in endothelial cells.


Assuntos
Células Endoteliais/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3-L1 , Animais , Cálcio/fisiologia , Células Cultivadas , GMP Cíclico/biossíntese , Humanos , Isoenzimas/química , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação/efeitos dos fármacos , Placebos/farmacologia , Proteína Quinase C/química , Proteína Quinase C/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Suínos , Vasodilatação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Vascul Pharmacol ; 43(5): 336-45, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16257270

RESUMO

In addition to its role as a vasoconstrictor, angiotensin II also acts as a potent growth factor by activating several tyrosine kinases, including Jak2. Interestingly, Jak2 has been linked to similar cardiovascular pathologies as have been previously linked to the renin-angiotensin system. Identifying the downstream targets of Jak2 via the AT(1) receptor may therefore elucidate its role in the progression of various pathologies. Previously, microarray analysis from our laboratory identified the Type 1 inositol 1,4,5 trisphosphate (IP(3)) receptor as a potential target of Jak2 following chronic stimulation by angiotensin II. Therefore, we hypothesized that Jak2 regulates IP(3) receptor expression in response to angiotensin II. To test this hypothesis, rat aortic smooth muscle (RASM) cells over-expressing a dominant negative (DN) Jak2 protein were used. The Jak2-dependent signaling in these cells is reduced approximately 90% when compared to RASM control cells. Analysis of protein expression showed that the IP(3) receptor was degraded approximately 2-fold (P<0.05) in cells lacking functional Jak2 within 1 h of treatment by angiotensin II. Notably, degradation of the IP(3) receptor was reversible since protein levels were restored to normal following 2 h of recovery from angiotensin II. To eliminate the possibility of clonal artifact in the DN cells, wild type RASM cells were treated with the Jak2 pharmacological inhibitor, AG490. We found that angiotensin II treatment degraded IP(3) receptor in AG490-treated cells, but not in the vehicle controls. Treatment with lactacystin, a proteasome inhibitor, completely blocked angiotensin II-mediated degradation of IP(3) receptor, thereby suggesting that the degradation occurs through a proteasome-dependent mechanism. Moreover, the degradation of IP(3) receptor in DN cells correlated with a significant loss of intracellular calcium mobilization when treated with angiotensin II (DN 27.4+/-1.1% vs. WT 42.2+/-4.7%; n=5, P=0.002). We next examined through what mechanism Jak2 regulates the IP(3) receptor. When wild type RASM cells were treated with PP2, an Src-family inhibitor, IP(3) receptor expression was markedly reduced. Since previous data show that Fyn, a downstream target of Jak2, is able to phosphorylate the IP(3) receptor at Tyr 353, we believe our data suggest that Jak2 prevents the angiotensin II-mediated IP(3) receptor degradation through the activation of Fyn. In conclusion, these data suggest that Jak2 has a protective role in maintaining IP(3) receptor expression, potentially through activation of Fyn and subsequent phosphorylation of the IP(3) receptor.


Assuntos
Angiotensina II/antagonistas & inibidores , Canais de Cálcio/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Western Blotting , Cálcio/fisiologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Janus Quinase 2 , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirfostinas/farmacologia
13.
Genomics ; 85(3): 352-9, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15718102

RESUMO

We have cloned and characterized the gene encoding the porcine cationic amino acid transporter, member 1 (CAT-1) (HGMW-approved gene symbol SLC7A1) from porcine pulmonary artery endothelial cells. The porcine SLC7A1 encodes 629 deduced amino acid residues showing a higher degree of sequence similarity with the human counterpart (91.1%) than with the rat (87.3%) and mouse (87.6%) counterparts. Confocal microscopic examination of porcine CAT-1-GFP-expressing HEK293 cells revealed that porcine CAT-1 localizes on the plasma membrane. Amino acid uptake studies in Xenopus oocytes injected with cRNA encoding this protein demonstrated transport properties consistent with system y(+). Radiation hybrid mapping data indicate that the porcine SLC7A1 maps to the distal end of the short arm of pig chromosome 11 (SSC11). This map location is consistent with the known conservation of genome organization between human and pig and provides further confirmation that we have characterized the porcine orthologue of the human SLC7A1.


Assuntos
Transportador 1 de Aminoácidos Catiônicos/genética , Mapeamento Cromossômico , Suínos/genética , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Primers do DNA , Humanos , Dados de Sequência Molecular , Óxido Nítrico/biossíntese , Xenopus laevis
14.
Am J Physiol Renal Physiol ; 287(2): F204-14, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15068972

RESUMO

Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular Ca(2+) signaling and downstream cellular function. The purpose of this study was to determine whether P2 receptors were present in mIMCD-3 cells, a mouse inner medullary collecting duct cell line, and if so, to examine their link with intracellular Ca(2+) homeostasis. To monitor intracellular Ca(2+) concentration ([Ca(2+)](i)), experiments were conducted using the fluorescent dye fura 2. ATP (0.1-100 microM) produced a dose-dependent increase in [Ca(2+)](i) in a physiological Ca(2+)-containing solution, with an EC(50) of 2.5 microM. The P2-receptor antagonist PPADS reduced the effect of ATP on [Ca(2+)](i), and the P1-receptor agonist adenosine caused only a small increase in [Ca(2+)](i). Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [Ca(2+)](i), indicating P2Y receptors were involved in this process. In a Ca(2+)-free bath solution, thapsigargin and ATP induced intracellular Ca(2+) release from an identical pool. Nucleotides caused an increase in [Ca(2+)](i) in the potency order of UTP = ATP > ATP gamma S > ADP > UDP that is best fitted with the P2Y(2) subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca(2+)](i) as did ATP, a small but sustained increase in [Ca(2+)](i) occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca(2+) influx. The sustained increase in [Ca(2+)](i) could be blocked by either nonselective cation channel blockers Gd(3+) or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd(3+) or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca(2+)](i) was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X(1) and P2Y(2) receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular Ca(2+) signaling.


Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Espaço Extracelular/metabolismo , Túbulos Renais Coletores/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sequência de Bases , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Membranas Intracelulares/metabolismo , Medula Renal , Túbulos Renais Coletores/citologia , Camundongos , Dados de Sequência Molecular , Concentração Osmolar , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Biochemistry ; 41(41): 12457-66, 2002 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-12369836

RESUMO

We have characterized single and double mutations in the M1-M2 segment of an inwardly rectifying K(+) channel, Kir2.1, using the cell-attached configuration of the patch-clamp technique. These mutations generated novel N-glycosylation sites at positions 128, 140, 143, and 147. Previously, we showed that these mutants were glycosylated, functional, and at the cell surface, which indicated that the putative pore-forming segment, including the invariant G(Y/F)G sequence of K(+) channels, was extracellular [Schwalbe, R. A., Rudin, A., Xia, S.-L., and Wingo, C. S. (2002) J. Biol. Chem. 277, 24382-24389]. In this study, three conductance states, corresponding to the main open state and two subconductance states, were identified in WT Kir2.1 channels expressed in infected Sf9 cells. Kir2.1 channels with mutations in the M1-M2 linker had at least one distinguishable conductance state of WT channels. In addition, these mutations altered the transitions, duration, and frequency of the defined populations of permeating and nonpermeating states. Of note, S128N had permeation rates similar to those of WT Kir2.1, but the total duration of the lower subconductance state was 3-5 times longer. Mutations in the signature sequence, I143N/Y145T, produced channels with permeation rates similar to those of the main open state and lower subconductance state of WT Kir2.1; however, the frequencies of these states were substantially different. These results demonstrate a novel functional role of the M1-M2 segment in regulating the transitions of the Kir2.1 channel and therefore suggest that this segment is a critical structural determinant in adjustments of pore conformations. Additionally, our results indicate that these mutants are correctly folded and thus further substantiate that the M1-M2 segment, including the G(Y/F)G sequence, is topologically extracellular.


Assuntos
Ativação do Canal Iônico/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Mutação Puntual , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Asparagina/genética , Cisteína/genética , Glicosilação , Isoleucina/genética , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/biossíntese , Fenilalanina/genética , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Conformação Proteica , Estrutura Terciária de Proteína/genética , Serina/genética , Spodoptera/genética , Treonina/genética , Fatores de Tempo , Tirosina/genética
17.
Am J Physiol Cell Physiol ; 283(4): C1080-9, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12225972

RESUMO

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.


Assuntos
Cálcio/metabolismo , Endotélio Vascular/metabolismo , Canais Iônicos/metabolismo , Óxido Nítrico/metabolismo , Regulação para Cima/fisiologia , Animais , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde , Immunoblotting , Líquido Intracelular/metabolismo , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Doadores de Óxido Nítrico/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Artéria Pulmonar , RNA Mensageiro/antagonistas & inibidores , Suínos , Transfecção , Regulação para Cima/efeitos dos fármacos
18.
J Biol Chem ; 277(27): 24382-9, 2002 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-11991952

RESUMO

Inwardly rectifying K+ channels or Kirs are a large gene family and have been predicted to have two transmembrane segments, M1 and M2, intracellular N and C termini, and two extracellular loops, E1 and E2, separated by an intramembranous pore-forming segment, H5. H5 contains a stretch of eight residues that are similar in voltage-dependent K+ channels, Kvs, and this stretch is called the signature sequence of K+ channels. Because mutations in this sequence altered selectivity in Kvs, it has been designated as the selectivity filter. Previously, we used N-glycosylation substitution mutants to map the extracellular topology of a weak inwardly rectifying K+ channel, Kir1.1 or ROMK1, and found that the entire H5 segment was extracellular. We now report utilization of introduced N-glycosylation sites, NX(S/T), at positions Ser(128) in E1, and Gln(140), Ileu(143), and Phe(147) in the H5 sequence of a strong inwardly rectifying K+ channel, Kir2.1. Furthermore, we show that biotinylated channel proteins with N-linked oligosaccharides attached at positions 140 and 143 in the signature sequence are located at the cell surface. Mutant channels were functional as detected by whole-cell and single-channel recordings. Unlike Kir1.1, position Lys(117) was not occupied. We conclude that, for yet another K+ channel, the invariant G(Y/F)G sequence is extracellular rather than intramembranous.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Glutamina , Glicosilação , Isoleucina , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Fenilalanina , Reação em Cadeia da Polimerase , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Recombinantes/metabolismo , Serina , Spodoptera , Transfecção
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