RESUMO
Inhibitors of Na(+)/H(+) exchanger (NHE) 1 have been shown to exert protective effects on various myocardial injuries. In this study, we characterized the pharmacodynamic properties of new guanidine NHE1 inhibitors (cariporide, sabiporide, KR-32511, KR-32570, and KR-33028) to analyze their myocardial protective effects. Although NHE1 is the major NHE isoform in cardiomyocytes, IC(50)values of these chemicals tested in rat cardiomyocytes were significantly different from those in PS120/hNHE1 cells where human NHE1 is heterologously expressed. In rat cardiomyocytes, KR-32570 and KR-33028 exhibited the highest potencies and their IC(50)values were 7+/-2 nM and 9+/-3 nM, respectively. The IC(50)values of all the chemicals tested on rat submandibular gland NHE2 were in the micromolar range, and they showed no inhibitory effects on hNHE3 and epithelial Na(+) channels up to 30 microM, suggesting a high selectivity toward NHE1. Sabiporide and KR-32570 exhibited slow dissociation kinetics with NHE1 inhibition persisting even after rinsing-out. When the cytoprotective effects of chemicals against hypoxic damage of rat cardiomyocytes were examined, the order of potency was KR-32570>or=KR-33028>sabiporide>cariporide>KR-32511. This order was exactly the same as that for the NHE1 inhibition in rat cardiomyocytes and did not correlate with any other properties, including the slow dissociation kinetics. Taken together, these results suggest that KR-32570 and KR-33028 are potent candidates for cardioprotective agents, and that the IC(50) in the target organ is the most critical factor governing the cytoprotective effects of NHE1 inhibitors.
Assuntos
Cardiotônicos/farmacologia , Guanidinas/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Furanos/farmacologia , Humanos , Cinética , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Trocador 1 de Sódio-Hidrogênio , Relação Estrutura-Atividade , Glândula Submandibular/citologia , Sulfonas/farmacologia , Tiofenos/farmacologiaRESUMO
BACKGROUND: The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Naâº,Kâº-ATPase. The catalytic subunit of the Naâº,Kâº-ATPase includes several functional domains that determine its enzymatic and trafficking properties. METHODOLOGY/PRINCIPAL FINDINGS: Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Naâº,Kâº-ATPase interacting protein. PP-2A C-subunit interacted with the Naâº,Kâº-ATPase, but not with the homologous sequences of the Hâº,Kâº-ATPase. We confirmed that the Naâº,Kâº-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Naâº,Kâº-ATPase trafficking through direct association. PP2A inhibits association between the Naâº,Kâº-ATPase and arrestin, and diminishes the effect of arrestin on Naâº,Kâº-ATPase trafficking. GRK phosphorylates the Naâº,Kâº-ATPase and PP2A can at least partially reverse this phosphorylation. CONCLUSIONS/SIGNIFICANCE: Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase.
Assuntos
Arrestina/metabolismo , Proteína Fosfatase 2/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Ligação Competitiva , Células COS , Chlorocebus aethiops , Quinases de Receptores Acoplados a Proteína G/metabolismo , Deleção de Genes , Humanos , Imunoprecipitação , Rim/enzimologia , Camundongos , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Ratos , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo , Cricetinae , Deleção de Genes , Genes Reporter , Humanos , Cinética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
BACKGROUND & AIMS: Protease-activated receptor 2 can be stimulated by interstitially released trypsin during acute inflammation of the pancreas. In this study, we investigated the roles of pancreatic and circulatory protease-activated receptor 2 in the pathogenesis of acute pancreatitis by using in vitro and in vivo model systems. METHODS: Physiological and pathologic effects of protease-activated receptor 2 activation were measured in isolated pancreatic cells and in rats with experimental pancreatitis. Consequences of protease-activated receptor 2 activation on the systemic and inflammatory responses were measured after treatments with trypsin or protease-activated receptor 2-activating peptide. RESULTS: Stimulation of protease-activated receptor 2 in rat pancreatic acinar cells activated short-lasting (Ca(2+) signaling) and long-lasting (extracellular signal-related kinase) signaling pathways and protected the cells against bile-induced cell damage. More importantly, protease-activated receptor 2 activation ameliorated the pathologic effects observed in the in vivo model of cerulein-induced pancreatitis. Trypsin in the circulation of rats with taurocholate-induced severe acute pancreatitis reached levels sufficient to activate endothelial and immune cells to stimulate nitric oxide and interleukin-8 production, respectively. Most notably, activation of systemic protease-activated receptor 2 by circulating protease-activated receptor 2 agonists induced a hemodynamic response pattern similar to that observed in rats with severe acute pancreatitis. The effects of protease-activated receptor 2 agonists and acute pancreatitis were not additive. CONCLUSIONS: These findings suggest that protease-activated receptor 2 may have a dual role in acute pancreatitis: protecting acinar and duct cells against pancreatitis-induced cell damage while mediating or aggravating the systemic complications of acute pancreatitis, which are the major cause of mortality in the early phase of necrotizing pancreatitis.
Assuntos
Pancreatite/metabolismo , Pancreatite/patologia , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Doença Aguda , Animais , Pressão Sanguínea , Sobrevivência Celular/fisiologia , Células Cultivadas , Ceruletídeo , Endopeptidases/farmacologia , Expressão Gênica , Técnicas In Vitro , Masculino , Monócitos/patologia , Pâncreas/citologia , Ductos Pancreáticos/citologia , Pancreatite/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Tripsina/sangueRESUMO
Pancreatic duct cells secrete bicarbonate-rich fluids, which are important for maintaining the patency of pancreatic ductal trees as well as intestinal digestive function. The bulk of bicarbonate secretion in the luminal membrane of duct cells is mediated by a Cl(-)-dependent mechanism (Cl(-)/HCO(3)(-) exchange), and we previously reported that the mechanism is CFTR-dependent and cAMP-activated (Lee, M. G., Choi, J. Y., Luo, X., Strickland, E., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 14670-14677). In the present study, we provide comprehensive evidence that calcium signaling also activates the same CFTR- and Cl(-)-dependent HCO(3)(-) transport. ATP and trypsin evoked intracellular calcium signaling in pancreatic duct-derived cells through the activation of purinergic and protease-activated receptors, respectively. Cl(-)/HCO(3)(-) exchange activity was measured by recording pH(i) in response to [Cl(-)](o) changes of the perfusate. In perfusate containing high concentrations of K(+), which blocks Cl(-) movement through electrogenic or K(+)-coupled pathways, ATP and trypsin highly stimulated luminal Cl(-)/HCO(3)(-) exchange activity in CAPAN-1 cells expressing wild-type CFTR, but not in CFPAC-1 cells that have defective (DeltaF508) CFTR. Notably, adenoviral transfection of wild-type CFTR in CFPAC-1 cells completely restored the stimulatory effect of ATP on luminal Cl(-)/HCO(3)(-) exchange. In addition, the chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid (BAPTA) treatment abolished the effect of calcium agonists on luminal Cl(-)/HCO(3)(-) exchange. These results provide a molecular basis for calcium-induced bicarbonate secretion in pancreatic duct cells and highlight the importance of CFTR in epithelial bicarbonate secretion induced by various stimuli.
Assuntos
Cálcio/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Ductos Pancreáticos/metabolismo , Trifosfato de Adenosina/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Bicarbonatos/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/citologia , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Ductos Pancreáticos/citologia , Potássio/metabolismo , Receptor PAR-1 , Receptores Purinérgicos/metabolismo , Receptores de Trombina/metabolismo , Tripsina/metabolismoRESUMO
Accumulating evidence suggests that protein-protein interactions play an important role in transepithelial ion transport. In the present study, we report on the biochemical and functional association between cystic fibrosis transmembrane conductance regulator (CFTR) and a PDZ domain-containing protein Shank2. Exploratory reverse transcription-PCR screening revealed mRNAs for several members of PDZ domain-containing proteins in epithelial cells. Shank2, one of these scaffolding proteins, showed a strong interaction with CFTR by yeast two-hybrid assays. Shank2-CFTR interaction was verified by co-immunoprecipitation experiments in mammalian cells. Notably, this interaction was abolished by mutations in the PDZ domain of Shank2. Protein phosphorylation, HCO(3)(-) transport and Cl(-) current by CFTR were measured in NIH 3T3 cells with heterologous expression of Shank2. Of interest, expression of Shank2 suppressed cAMP-induced phosphorylation and activation of CFTR. Importantly, loss of Shank2 by stable transfection of antisense-hShank2 plasmid strongly increased CFTR currents in colonic T84 cells, in which CFTR and Shank2 were natively expressed. Our results indicate that Shank2 negatively regulates CFTR and that this may play a significant role in maintaining epithelial homeostasis under normal and diseased conditions such as those presented by secretory diarrhea.