RESUMEN
Our laboratory has identified at least two types of vascular smooth muscle cells (VSMCs) that exist in canine arteries and veins: type 1 cells, located in the media express muscle specific proteins but do not proliferate in culture; and type 2 cells, located in both media and adventitia, do not express muscle specific protein but proliferate in culture. Plasma membrane Ca(2+)-ATPases (PMCAs) have been implicated in proliferation control. The present study examines the expression of PMCA isoforms and calmodulin-binding domain splice variants in these two types of canine VSMCs. PMCA protein was found in both type 1 and type 2 cells. Reverse transcriptase-polymerase chain reaction assays were developed for canine PMCA calmodulin-binding domain splice variants. We cloned and sequenced isolates corresponding to PMCA1b, 4a and 4b from canine VSMCs. PMCA 2 and 3 were not detected. Freshly isolated type 1 cells expressed PMCA 1b, 4a and 4b, while freshly isolated type 2 cells expressed PMCA1b and 4b. Upon placement in culture, type 2 cells originating from either carotid artery or saphenous vein demonstrated a time-dependent upregulation of PMCA4a mRNA. Treatment with the phosphoinositide 3-kinase inhibitor wortmannin produced concentration-dependent inhibition of both PMCA4a upregulation and [(3)H]thymidine incorporation. These findings suggest a role for phosphoinositide 3-kinase in regulating PMCA expression, which may be important in the control of Ca(2+)-sensitive VSMC functions.
Asunto(s)
ATPasas Transportadoras de Calcio/biosíntesis , Arterias Carótidas/enzimología , Músculo Liso Vascular/enzimología , Vena Safena/enzimología , Empalme Alternativo , Secuencia de Aminoácidos , Androstadienos/farmacología , Animales , Secuencia de Bases , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Arterias Carótidas/citología , Proteínas de Transporte de Catión , Membrana Celular/enzimología , Células Cultivadas , Cartilla de ADN , ADN Complementario , Perros , Inhibidores Enzimáticos/farmacología , Femenino , Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Fenotipo , ATPasas Transportadoras de Calcio de la Membrana Plasmática , ARN Mensajero , Vena Safena/citología , Homología de Secuencia de Aminoácido , WortmaninaRESUMEN
Intercellular junctions have long been considered the main sites through which adherent neutrophils (PMNs) penetrate the endothelium. Tight junctions (TJs; zonula occludens) are the most apical component of the intercellular cleft and they form circumferential belt-like regions of intimate contact between adjacent endothelial cells. Whether PMN transmigration involves disruption of the TJ complex is unknown. We report here that endothelial TJs appear to remain intact during PMN adhesion and transmigration. Human umbilical vein endothelial cell (HUVEC) monolayers, a commonly used model for studying leukocyte trafficking, were cultured in astrocyte-conditioned medium to enhance TJ expression. Immunofluorescence microscopy and immunoblot analysis showed that activated PMN adhesion to resting monolayers or PMN migration across interleukin-1-treated monolayers does not result in widespread proteolytic loss of TJ proteins (ZO-1, ZO-2, and occludin) from endothelial borders. Ultrastructurally, TJs appear intact during and immediately following PMN transendothelial migration. Similarly, transendothelial electrical resistance is unaffected by PMN adhesion and migration. Previously, we showed that TJs are inherently discontinuous at tricellular corners where the borders of three endothelial cells meet and PMNs migrate preferentially at tricellular corners. Collectively, these results suggest that PMN migration at tricellular corners preserves the barrier properties of the endothelium and does not involve widespread disruption of endothelial TJs.
Asunto(s)
Movimiento Celular , Endotelio Vascular/citología , Neutrófilos/citología , Uniones Estrechas/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Conductividad Eléctrica , Endopeptidasas/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Técnica de Fractura por Congelación , Calor , Humanos , Interleucinas/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Microscopía Fluorescente , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/ultraestructura , Ocludina , Fosfoproteínas/metabolismo , Factor de Activación Plaquetaria/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/ultraestructura , Proteína de la Zonula Occludens-1 , Proteína de la Zonula Occludens-2RESUMEN
Several hypertensive states are associated with resistance to insulin-induced glucose disposal and insulin-induced vasodilation. Insulin can inhibit vascular smooth muscle (VSM) contraction at the level of the VSM cell, and resistance to insulin's inhibition of VSM cell contraction may be of pathophysiological importance. To understand the VSM cellular mechanisms by which insulin resistance leads to increased VSM contraction, we sought to determine how insulin inhibits contraction of normal VSM. It has been shown that insulin lowers the contractile agonist-stimulated intracellular Ca2+ (Ca2+i) transient in VSM cells. In this study, our goal was to see whether insulin inhibits VSM cell contraction at steps distal to Ca2+i and, if so, to determine whether the mechanism is dependent on nitric oxide synthase (NOS) and cGMP. Primary cultured VSM cells from canine femoral artery were bathed in a physiological concentration of extracellular Ca2+ and permeabilized to Ca2+ with a Ca2+ ionophore, either ionomycin or A-23187. The resultant increase in Ca2+i contracted individual cells, as measured by photomicroscopy. Preincubating cells with 1 nM insulin for 30 min did not affect basal Ca2+i or the ionomycin-induced increase in Ca2+i, as determined by fura 2 fluorescence measurements, but it did inhibit ionomycin- and A-23187-induced contractions by 47 and 51%, respectively (both P < 0.05). In the presence of 1.0 microM ionized Ca2+, ionomycin-induced contractions were inhibited by insulin in a dose-dependent manner. In the presence of ionomycin, insulin increased cGMP production by 43% (P < 0.05). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), a selective inhibitor of guanylate cyclase that blocked cGMP production in these cells, completely blocked the inhibition by insulin of ionomycin-induced contraction. It was found that the cells expressed the inducible isoform of NOS. NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester (0.1 mM), inhibitors of NOS, did not affect ionomycin-induced contraction but prevented insulin from inhibiting contraction. We conclude that insulin stimulates cGMP production and inhibits VSM contraction in the presence of elevated Ca2+i. This inhibition by insulin of VSM contraction at sites where Ca2+i could not be rate limiting is dependent on NOS and cGMP.
Asunto(s)
Calcio/metabolismo , Insulina/farmacología , Membranas Intracelulares/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Animales , Células Cultivadas , GMP Cíclico/biosíntesis , Perros , Femenino , Masculino , Contracción Muscular/fisiología , Músculo Liso Vascular/química , Óxido Nítrico Sintasa/fisiología , Óxido Nítrico Sintasa de Tipo II , Concentración OsmolarAsunto(s)
ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Empalme Alternativo , Animales , Línea Celular , Células HeLa , Humanos , Intrones , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/metabolismo , Sustancias Macromoleculares , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/química , Spodoptera , TransfecciónRESUMEN
The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Ratas , Alineación de Secuencia , Análisis de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/inmunologíaRESUMEN
We have assessed the Na pump alpha-subunit isoform content utilizing site directed antibodies in two vascular smooth muscle (VSM) preparations known to contain functional Na pump sites, VSM microsomal fractions (Na+, K(+)-ATPase) and intact primary confluent cells (ouabain inhibited 86Rb uptake). A comparison of isoform content was made with kidney microsomes. Both VSM and kidney microsomes contained a full length alpha 1 subunit (approximately 100 kDa) as well as a truncated subunit, alpha 1T (approximately 66 kDa). SDS treatment of VSM microsomes effected an increase in Na+, K(+)-ATPase and a retention of alpha 1T. SDS treated kidney microsomes retained the alpha 1 isoform and Na+, K(+)-ATPase. Confluent VSM cells showed no detectable alpha 1, only alpha 1T. In the absence of detectable full length alpha 1, the alpha 1T protein may represent a functional Na pump component in canine VSM.