RESUMEN
Introduction: Hip spica casting is an important component of treatment for developmental dysplasia of the hip (DDH) and popular treatment method for femur fractures in children. Breakage at the hip region is a relatively common problem of this cast. We have developed a three-slab technique of hip spica application using fibreglass as the cast material. The purpose of this review was to evaluate the physical durability of the spica cast and skin complications with its use. Methodology: A retrospective review of children with various conditions requiring hip spica immobilisation which was applied using our method. Study duration was from 1st of January 2014 until 31st December 2015. Our main outcomes were cast breakage and skin complications. For children with hip instability, the first cast would be changed after one month, and the second cast about two months later. Results: Twenty-one children were included, with an average age of 2.2 years. The most common indication for spica immobilisation was developmental dysplasia of the hip. One child had skin irritation after spica application. No spica breakage was noted. Conclusion: This study showed that the three-slab method of hip spica cast application using fibreglass material was durable and safe with low risk of skin complications.
RESUMEN
BACKGROUND AND PURPOSE: Functional tissue engineering of the gastrointestinal (GI) tract is a complex process aiming to aid the regeneration of structural layers of smooth muscle, intrinsic enteric neuronal plexuses, specialized mucosa, and epithelial cells as well as interstitial cells. The final tissue-engineered construct is intended to mimic the native GI tract anatomically and physiologically. Physiological functionality of tissue-engineered constructs is of utmost importance while considering clinical translation. The construct comprises of cellular components as well as biomaterial scaffolding components. Together, these determine the immune response a tissue-engineered construct would elicit from a host upon implantation. Over the last decade, significant advances have been made to mitigate adverse host reactions. These include a quest for identifying autologous cell sources like embryonic and adult stem cells, bone marrow-derived cells, neural crest-derived cells, and muscle derived-stem cells. Scaffolding biomaterials have been fabricated with increasing biocompatibility and biodegradability. Manufacturing processes have advanced to allow for precise spatial architecture of scaffolds to mimic in vivo milieu closely and achieve neovascularization. This review will focus on the current concepts and the future vision of functional tissue engineering of the diverse neuromuscular structures of the GI tract from the esophagus to the internal anal sphincter.
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Intestinos/anatomía & histología , Intestinos/fisiología , Regeneración/fisiología , Medicina Regenerativa , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles , Humanos , Células Madre/fisiologíaAsunto(s)
Envejecimiento/fisiología , Tracto Gastrointestinal/anatomía & histología , Tracto Gastrointestinal/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Animales , Epigenómica , Enfermedades Gastrointestinales/patología , Enfermedades Gastrointestinales/fisiopatología , Tracto Gastrointestinal/fisiopatología , Humanos , Longevidad , Contracción Muscular/fisiología , Músculo Liso/fisiología , Neuroglía/fisiología , Neuronas/citología , Neuronas/fisiología , Comunicación Paracrina , Sensación/fisiología , Transducción de Señal/fisiologíaRESUMEN
The recruitment of signal transduction molecules to the membrane is crucial for the efficient coupling of extracellular signals and contractile response. The trafficking is dynamic. We have investigated a possible cross talk between agonist-induced association of translocated RhoA and translocated protein kinase C-alpha (PKC-alpha) and a role for heat shock protein 27 (HSP27) in mediating this interaction. Immunoprecipitation with HSP27 monoclonal antibody followed by immunoblotting with either RhoA antibody or PKC-alpha antibody indicated that acetylcholine induced associations of HSP27-RhoA and HSP27-PKC-alpha in the membrane fraction but not in the cytosolic fraction. Immunoprecipitation with anti-RhoA monoclonal antibody followed by immunoblotting with PKC-alpha antibody indicated that acetylcholine induced a significant complexing of RhoA-PKC-alpha in the membrane fraction but not in the cytosolic fraction. In summary, the data indicate that agonist-induced contraction is associated with 1) association of translocated RhoA with HSP27 on the membrane, 2) association of translocated PKC-alpha with HSP27 on the membrane, and 3) association of PKC-alpha with RhoA on the membrane. The data suggest an important role for HSP27 in modulating a multiprotein complex that includes translocated RhoA and PKC-alpha.
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Colon/metabolismo , Isoenzimas/fisiología , Músculo Liso/metabolismo , Proteínas de Neoplasias/fisiología , Proteína Quinasa C/fisiología , Proteína de Unión al GTP rhoA/fisiología , Acetilcolina/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Western Blotting , Ceramidas/farmacología , Colon/citología , Electroforesis en Gel de Poliacrilamida , Técnicas In Vitro , Isoenzimas/genética , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/enzimología , Proteínas de Neoplasias/genética , Pruebas de Precipitina , Proteína Quinasa C/genética , Proteína Quinasa C-alfa , Conejos , Transducción de Señal/fisiología , Translocación Genética/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
We studied the kinetics of [3H]lipopolysaccharide ([3H]LPS) (endotoxin) binding to Kupffer cells and hepatocytes at the level of the microtubular system after treatment with gadolinium chloride (GdCl(3)) and colchicine. Liver perfusion in Sprague-Dawley rats involves both portal vein and thoracic inferior vena cava cannulations as inlet and outlet, respectively. The subhepatic inferior vena cava is ligated to prevent perfusate leakage. Buffer containing 2% serum and [3H]LPS is administered at 1 ml/min and collected for 50 min. Rate constants for hepatocellular clearance of [3H]LPS in controls, colchicine-treated rats, GdCl(3)-treated rats, and colchicine plus GdCl(3)-treated rats are assessed using a simplified mathematical model. Forward-binding, reversal-binding, residency time, and influx rate constants are estimated. Results show that in GdCl(3)-treated rats, the hepatocytes effectively clear endotoxin from the circulation, and its ultimate binding affinity at the hepatocyte site is somewhat reduced compared to the Kupffer cells. In colchicine-treated rats, the disruption of the microtubule network altered [3H]LPS binding with Kupffer cells, suggesting that the microfilament-microtubular network also affects Kupffer cell function. Simultaneous treatments with colchicine and GdCl(3) increased the influx rate constant, suggesting that the compiled morphological alterations up-regulated endotoxin clearance by the liver, as indicated by a drastic increase in cellular vacuolation. In conclusion, the kinetics of the trafficking process of [3H]LPS clearance are regulated by apical-sinusoidal endocytotic and canalicular routes.
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Endotoxinas/farmacocinética , Macrófagos del Hígado/fisiología , Lipopolisacáridos/farmacocinética , Hígado/fisiología , Animales , Sitios de Unión , Colchicina/farmacología , Gadolinio/farmacología , Hepatocitos , Cinética , Microtúbulos/fisiología , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Insulin-resistance is associated with a number of disease states such as diabetes, syndrome X, and hypertension. These situations may be coupled to insulin-resistance through the insulin signaling system as a common pathway. The purpose of this study was to investigate the receptor binding alterations in streptozotocin-induced diabetic rats, spontaneously hypertensive rats and aortocaval shunted rats (eccentric cardiac hypertrophy). A physical model describing a 1:1 stoichiometry of ligand binding with its receptor is proposed describing reversible binding of [(125)I]insulin or [(125)I]IGF-1 at the microvascular endothelial as well as with the cardiac myocytes after CHAPS-treatment. Analysis of the collected effluents are curve-fitted with a conservation equation and a first-order Bessel function which allowed the calculation of the forward binding constants (k(n)), the reversible constants (k(-n)), the dissociation constants (k(d)) and the residency time constants (tau). The results showed that streptozotocin-induced diabetic rats showed insulin-resistance through alterations in the kinetics of insulin receptor binding. The normotensive controls of the spontaneously hypertension rats (SHR) carry themselves insulin-resistant receptors whose binding to insulin worsens in the hypertensive SHR. Negative cooperativity between insulin-like growth factor IGF-1 and insulin receptors could be a causative factor predisposing for insulin-resistance in the aortocaval shunted rats to insulin resistance. The defects may be occurring at the receptor level in insulin-dependent diabetes mellitus, Wistar-Kyoto rats and spontaneously hypertensive rats. In conclusion, alterations in the kinetics of insulin binding to its receptor seem to play a central role for the initiation of insulin-resistance during the various pathophysiological states.
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Cardiomegalia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Resistencia a la Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Receptor IGF Tipo 1/metabolismo , Animales , Antibacterianos , Femenino , Hipertensión/metabolismo , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Especificidad de la Especie , EstreptozocinaRESUMEN
Clinical evidence points to a role for angiotensin II (Ang II) in the post-infarction remodelling of cardiac hypertrophy. The present study was designed to investigate the remodelling process in an animal model of myocardial infarction (MI) using the following criteria: 1) histological studies to examine the re-vascularisation process and collagen deposition in different regions of the myocardium; 2) histological evidence to investigate the cell type distribution using cell-specific markers; 3) histological and Western blot analysis to localise Ang II receptor subtypes (AT(1)-receptor and AT(2)-receptor) and to study their regulation; 4) kinetics of the binding of Ang II to its receptors in a heart perfusion model; and 5) to assess the effect of the Ang II antagonist (losartan) on these parameters. MI was induced by ligation of the left anterior descending coronary artery of Sprague-Dawley rats. Four different animal groups were established: 1) sham-operated, non-treated; 2) sham-operated, treated with losartan; 3) myocardial infarct, non-treated; and 4) myocardial infarct, treated with losartan. In infarcted rat hearts, fibroblasts and collagen types I and III increased in the remnant viable region of the left ventricle compared with sham-operated rats. One month of losartan treatment in myocardial infarcted rats revealed insignificant changes in fibroblasts and collagen types I and III compared with sham controls. Also, myocardial infarction increased AT(1)-receptor protein levels compared with sham-operated controls, as judged by Western blotting. In losartan-treated myocardial infarct animals, no changes were detected at the level of AT(1)-receptor expression compared with non-treated myocardial infarct rats. Binding studies of Ang II on endothelial cell lining and directly on myocytes in sham-operated and infarcted perfused rat hearts revealed that, in myocardial infarcted-animals, Ang II binding affinity increased both in the endothelium and in myofibres. This may be considered a major putative effect of the peptide in potentiating the pharmacodynamics of hypertrophy. In losartan-treated myocardial infarcted-animals, a marked increase in the binding affinities of Ang II for the AT(2)-receptor subtype was observed. Hence, potential cardioprotective effects of the AT(1)-receptor antagonist are proposed.
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Angiotensina II/metabolismo , Matriz Extracelular/fisiología , Infarto del Miocardio/metabolismo , Animales , Biomarcadores , Cardiomegalia/patología , Cicatriz/patología , Circulación Coronaria/efectos de los fármacos , Femenino , Cinética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Perfusión , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
Behcet's disease is a chronic multisystem vasculitis that is frequent in Lebanon. The great arteries involvement is rare. We report here an unusual case of subclavian artery occlusion (pseudo-Takayasu) with a literature review.
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Síndrome de Behçet/complicaciones , Síndrome del Robo de la Subclavia/etiología , Adulto , Antiinflamatorios/uso terapéutico , Anticoagulantes/uso terapéutico , Síndrome de Behçet/diagnóstico , Síndrome de Behçet/tratamiento farmacológico , Quimioterapia Combinada , Humanos , Inmunosupresores/uso terapéutico , Masculino , Pronóstico , Radiografía , Esteroides , Síndrome del Robo de la Subclavia/diagnóstico por imagen , Arteritis de Takayasu/diagnóstico por imagen , Arteritis de Takayasu/etiologíaRESUMEN
Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that 1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide; 2) PKC-alpha and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-alpha translocation is inhibited by VIP; 3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.
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Colon/metabolismo , Proteínas Contráctiles/metabolismo , Proteínas de Choque Térmico/fisiología , Músculo Liso/metabolismo , Transducción de Señal/fisiología , Actinas/metabolismo , Animales , Azepinas/farmacología , Transporte Biológico , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Ceramidas/farmacología , Colon/citología , Colon/efectos de los fármacos , Citoesqueleto/metabolismo , Proteínas de Choque Térmico/metabolismo , Isoenzimas/metabolismo , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Miosinas/metabolismo , Naftalenos/farmacología , Pruebas de Precipitina , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa , Proteínas Proto-Oncogénicas c-raf/metabolismo , Conejos , Distribución Tisular , Tropomiosina/metabolismoRESUMEN
We have investigated the hypothesis that different contractile agonists activate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin (10(-7) M) induced a sustained increase in PI 3-kinase activity at both 30 s and 4 min of stimulation (151.5 +/- 8.5% at 30 s and 175.8 +/- 8.7% at 4 min, P < 0.005). Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein (3 microM) resulted in a significant inhibition of both C2 ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 microM), inhibited only C2 ceramide-induced PI 3-kinase activation and contraction. Western blotting using Src kinase antibody showed that C2 ceramide, not endothelin, stimulated the phosphorylation of Src kinase. Western blotting and immunoprecipitation with PI 3-kinase antibodies to the regulatory subunit p85 and the catalytic subunits p110alpha and p110gamma indicated that both endothelin and C2 ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110alpha and p110gamma, whereas C2 ceramide interacted only with the catalytic subunit p110alpha. In summary, C2 ceramide activated PI 3-kinase p110alpha subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C2 ceramide, was not mediated by the activation of Src kinase but was mediated by G protein activation of both p110alpha and p110gamma subunits (type IA and IB) of PI 3-kinase.
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Colon/fisiología , Endotelina-1/farmacología , Músculo Liso/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Esfingosina/análogos & derivados , Animales , Calcio/metabolismo , Cloruro de Calcio/farmacología , Células Cultivadas , Colon/enzimología , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Genisteína/farmacología , Cinética , Modelos Biológicos , Músculo Liso/enzimología , Conejos , Transducción de Señal/efectos de los fármacos , Esfingosina/farmacología , Factores de TiempoRESUMEN
We evaluated intracellular pathways responsible for the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini. Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting. When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected. CCK (10 pM-10 nM) and carbachol (0.1-100 microM) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min. High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1-1,000 nM) did not increase the intensities of the RhoA band, suggesting that stimulation of RhoA is mediated by the low-affinity CCK-A receptor. Although an increase in RhoA did not require the presence of extracellular Ca2+, the intracellular Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK and carbachol. The Gq protein inhibitor G protein antagonist-2A (10 microM) and the phospholipase C (PLC) inhibitor U-73122 (10 microM) markedly reduced RhoA bands in response to CCK. The protein kinase C (PKC) activator phorbol ester (10-1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 microM). The pp60(c-src) inhibitor herbimycin A (6 microM) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 microM) and the phosphoinositide 3-kinase inhibitor wortmannin (6 microM) had no effect. RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK stimulation. In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhibited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 exoenzyme at 1 ng/ml. We concluded that the small GTP-binding protein RhoA p21 exists in pancreatic acini and appears to be involved in the mediation of pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways are involved in the activation of PKC and Src cascades via Gq protein and PLC.
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Amilasas/metabolismo , Toxinas Botulínicas , Colecistoquinina/farmacología , Proteínas de Unión al GTP/metabolismo , Páncreas/fisiología , Proteína Quinasa C/metabolismo , Receptores de Colecistoquinina/fisiología , ADP Ribosa Transferasas/farmacología , Animales , Carbacol/farmacología , Permeabilidad de la Membrana Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , GTP Fosfohidrolasas/metabolismo , Genes src/efectos de los fármacos , Cinética , Masculino , Proteínas de la Membrana/metabolismo , Páncreas/citología , Páncreas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Colecistoquinina A , Receptores de Colecistoquinina/agonistas , Transducción de Señal , Sincalida/análogos & derivados , Sincalida/farmacología , Sulfonamidas/farmacología , Proteína de Unión al GTP rhoA , Proteína de Unión al GTP rhoBRESUMEN
Typhoid fever is still an endemic disease in Lebanon with a risk of mortality especially in the immunosuppressed patients. We report 3 atypical observations and discuss cardiopulmonary, neurological, hematologic and urinary manifestations of this disease.
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Enfermedades Endémicas , Infecciones Oportunistas/diagnóstico , Fiebre Tifoidea/diagnóstico , Adulto , Anciano , Diagnóstico Diferencial , Femenino , Humanos , MasculinoRESUMEN
The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2 ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60(src), respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10(-7) M) or ceramide (10(-7) M) ( approximately 30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60(src) inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60(src) and PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.
Asunto(s)
Toxinas Botulínicas , Citoesqueleto/metabolismo , Proteínas de Unión al GTP/fisiología , Proteínas de Choque Térmico/metabolismo , Contracción Muscular/fisiología , Músculo Liso/fisiología , ADP Ribosa Transferasas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Colon/citología , Colon/efectos de los fármacos , Colon/metabolismo , Endotelina-1/farmacología , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Conejos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Distribución Tisular , Proteína de Unión al GTP rhoARESUMEN
This work uses a new heart-perfusion technique to measure 125I-insulin binding on capillary endothelium and myofiber cell membranes in Wistar-Kyoto and spontaneously hypertensive rats. Ringer-Lock buffer was infused at a rate of 1 ml min-1 in the presence of 20 meq l-1 K+ and 125I-insulin through an aortic cannula. The effluent was collected through a catheter introduced into the right atrium. The capillary endothelial lining was removed by detergent treatment to expose the cardiac myocyte surfaces. A physical model describing a 1:1 binding stoichiometry of 125I-insulin with its receptors is proposed and the derived mathematical equations allow for the calculation of binding constants (kn), unbinding constants (k-n), dissociation constants (kd), and residency time constants (tau). The results showed that in the spontaneously hypertensive rats' hearts significant alterations were not noticed in the kinetics of insulin binding with its receptor at the capillary endothelial site compared to hearts of the normotensive control Wistar-Kyoto rats. However, at the myocyte site and in the spontaneously hypertensive rats, steric, configurational, and/or structural modifications for insulin binding with the receptor were observed as indicated by changes in insulin affinity for its receptor. Hence, alterations in insulin binding rather than reduction in insulin receptor number due to hyperinsulinemia, can be considered among the peculiarities of insulin resistance in the spontaneously hypertensive rats. Hyperinsulinemia, therefore, may be considered an upregulatory process as a consequence of insulin-resistance. The results support the hypothesis that insulin-resistance on the myocytes could be a pathophysiologic defect in insulin-receptor structure, function and affinity, and therefore myocardial function.
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Hipertensión/metabolismo , Insulina/metabolismo , Miocardio/ultraestructura , Receptor de Insulina/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Ácidos Cólicos/farmacología , Detergentes/farmacología , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Femenino , Hiperinsulinismo/metabolismo , Insulina/farmacocinética , Resistencia a la Insulina/fisiología , Radioisótopos de Yodo , Cómputos Matemáticos , Modelos Biológicos , Miocardio/metabolismo , Perfusión , Unión Proteica , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
Peptide delivery toward its targets in an intact organ is equally as important as its routing from the systemic circulation to cell surface receptor sites. A physical model pertinent to a heart perfusion technique in Sprague-Dawley rats is presented describing reversible binding of angiotensin II and/or antagonist (DUP 753, losartan) with the microvascular endothelial receptor subtypes as well as with the cardiac myocyte receptor subtypes that are exposed to the perfusate by CHAPS-treatment. Analysis of the collected effluents are curve-fitted with a conservation equation and a first-order Bessel function. The results suggest that angiotensin II delivery and binding to the pool of receptor subtypes both at the level of the microvascular endothelium and cardiac myocyte sites differ marginally in binding affinities. The findings postulate that angiotensin II can have access to the myocyte site in an intact heart by an endothelial angiotensin II-receptor-internalization process. In addition, considering that the AT1- and AT2-receptor subtypes are present in equal proportions and have equal binding affinities with angiotensin II, the results of the 3H-DUP 753 binding indicated approximately 3-3.5 times higher affinity to the AT1-receptors subtype than angiotensin II at both the endothelial and myocyte sites. In the presence of losartan, angiotensin II binding showed higher affinity with the exposed unopposed AT2-receptor subtype than with the receptor pool, which could be due to alterations in the AT2-receptor structure and configuration. This increase in the binding affinity of angiotensin II with the AT2-receptor subtype may be categorized under the direct effect of the AT1-antagonist modality in producing cardioprotective effects.
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Angiotensina II/metabolismo , Endotelio Vascular/metabolismo , Miocardio/metabolismo , Vasoconstrictores/metabolismo , Animales , Técnicas In Vitro , Losartán/metabolismo , Masculino , Modelos Biológicos , Ratas , Ratas Sprague-DawleyRESUMEN
Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60(src) tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60(src) and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.
Asunto(s)
Colon/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Secuencia de Aminoácidos , Androstadienos/farmacología , Animales , Benzoquinonas , Calcio/farmacología , Cromonas/farmacología , Colon/efectos de los fármacos , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Cinética , Lactamas Macrocíclicas , Modelos Biológicos , Datos de Secuencia Molecular , Morfolinas/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Fosforilación , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Quinonas/farmacología , Conejos , Rifabutina/análogos & derivados , Esfingosina/farmacología , Especificidad por Sustrato , Wortmanina , Dominios Homologos srcRESUMEN
The source of early production of sn-1,2-diacylglycerol (DAG) has for a long time been exclusively linked to hydrolysis of phosphatidylinositol 4,5-diphosphate, which on receptor activation is hydrolyzed into DAG and inositol 1,4,5-trisphosphate. We have investigated the origin of lipid sources of DAG production in smooth muscle cells, in response to contraction induced by peptide agonists. We have performed a quantitative analysis of the molecular species of DAG formed in relation to the known molecular composition of parent phospholipids. The molecular species of phospholipids are sufficiently unique that the phospholipid origin of DAGs and its quantitative contribution to their formation can be measured by HPLC. Cell suspensions (10-15 x 10(6) cells/ml) from the circular muscle of rabbit rectosigmoid were incubated in the presence of the contractile peptide agonist bombesin (BB) at 10(-6) M. Reactions were stopped at different time intervals from 30 s to 4 min. DAGs were extracted, purified by TLC, and benzoylated with benzoic anhydride. The benzoylated DAGs were first purified by TLC and then by normal phase HPLC before they were injected onto a reverse-phase column and eluted isocratically. Furthermore, phospholipids in the lipid extract [phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)] were purified by TLC and similarly analyzed after hydrolysis to DAGs with phospholipase C (PLC). The DAG molecular species profiles for PI, PC, PS, and PE were all unique. Contraction of cells with BB gave noticeable increases (17-55%) in newly formed DAGs. The major phospholipid source of the newly formed DAGs at 30 s was only approximately 30% from PI, and the remainder was from PC. In contrast, after 4 min of BB stimulation, a decrease was seen in newly formed DAGs in the peak specific for PI hydrolysis. The data suggest that BB-induced contraction by activation of PLCs results in hydrolysis of different phospholipids. The DAGs formed as a result are qualitatively and quantitatively distinct. This could be the basis for the kinetically different pattern of sustained contraction observed with BB.
Asunto(s)
Bombesina/farmacología , Colon Sigmoide/metabolismo , Diglicéridos/biosíntesis , Músculo Liso/metabolismo , Fosfolípidos/biosíntesis , Recto/metabolismo , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Colon Sigmoide/efectos de los fármacos , Diglicéridos/química , Diglicéridos/aislamiento & purificación , Cinética , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Fosfatidilinositoles/biosíntesis , Fosfatidilserinas/biosíntesis , Fosfolípidos/química , Conejos , Recto/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo CRESUMEN
Circular smooth muscle cells from the feline newborn antrum, unlike the adult, are unable to respond to myogenic agonists in the absence of extracellular calcium or to exogenous inositol 1,4,5-trisphosphate (IP3). This study examined the reasons behind the relative inaccessibility of intracellular calcium stores in the newborn period. IP3 binding was determined in antral smooth muscle homogenates from adult cats and newborns by evaluating the competitive binding of D-myo-[3H]IP3 and unlabeled IP3. Receptor density (Bmax) (fmol/mg of protein) and binding affinity (Kd) were determined. The Kd was similar in adults (31 +/- 4 nM) and newborns (28 +/- 7 nM); however, the Bmax was markedly decreased in the newborn (647 +/- 181.0 fmol/mg) compared with the adult (1755 +/- 275 fmol/mg). In adult and newborn antral cells, thapsigargin, which causes a net release of Ca2+ from intracellular stores by inhibiting Ca(2+)-ATPase-dependent reuptake activity, caused an early contraction at 30 s that was maintained for at least 20 min. We conclude that, in the newborn, dynamic intracellular calcium stores are present in the smooth muscle of the feline antrum and that differences in accessibility of intracellular calcium stores may be related to changes in the release of calcium from IP3-sensitive stores.
Asunto(s)
Calcio/metabolismo , Antro Pilórico/crecimiento & desarrollo , Antro Pilórico/metabolismo , Animales , Animales Recién Nacidos , Canales de Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Gatos , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/biosíntesis , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Desarrollo de Músculos , Músculo Liso/efectos de los fármacos , Músculo Liso/crecimiento & desarrollo , Músculo Liso/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Tapsigargina/farmacologíaRESUMEN
Insulin-like growth factor (IGF) binding protein 5 (IGFBP-5) mRNA was studied in intestines of rats with peptidoglycan-polysaccharide enterocolitis by Northern analysis and in situ hybridization. IGFBP-5 mRNA was increased 2.4 +/- 0.5-fold in inflamed rat colon compared with controls and was highly expressed in smooth muscle. Cultured rat intestinal smooth muscle cells were used to study the regulation of IGFBP-5 and type I collagen synthesis. IGF-I (100 ng/ml) increased IGFBP-5 mRNA (1.9 +/- 0.1-fold) and collagen type alpha1(I) mRNA (1.6 +/- 0.2-fold) in cultured smooth muscle cells. IGF-I induced a dose- and time-dependent increase in IGFBP-5 in conditioned medium by Western ligand blot and by immunoblot. IGF-I did not affect the IGFBP-5 mRNA decay rate after transcriptional blockade. Cycloheximide abolished IGFBP-5 mRNA. In conclusion, IGFBP-5 mRNA is expressed by intestinal smooth muscle and is increased during chronic inflammation. IGF-I increases IGFBP-5 and collagen mRNAs in intestinal smooth muscle cells.
Asunto(s)
Colágeno/biosíntesis , Colon/metabolismo , Enterocolitis/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/farmacología , Músculo Liso/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Células Cultivadas , Colon/citología , Colon/efectos de los fármacos , Colon/patología , Cicloheximida/farmacología , Enterocolitis/inducido químicamente , Enterocolitis/patología , Femenino , Hibridación in Situ , Inflamación , Músculo Liso/citología , Músculo Liso/patología , Peptidoglicano , Polisacáridos Bacterianos , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas Lew , Valores de Referencia , Factores de TiempoRESUMEN
A heart-perfusion technique was employed to measure 125I-insulin binding on capillary endothelial and myocyte cell membranes in Sprague-Dawley rats. Animals were anesthetized, and the anterior chest wall excised to expose the mediastinal contents. The right and left superior and inferior venae cavae were dissected and tied, and another tie was passed around the aorta. A polyethylene catheter was introduced into the aortic lumen from cephalad to caudad to sit with its tip above the aortic valve. Another catheter was introduced into the cavity of the right atrium and both were anchored by sutures. Oxygenated Ringer-Lock buffer containing 20 mM/L K+ and 125I-insulin was perfused at a rate of 1 mL/min via the aortic catheter. Concomitantly, the distal ascending aorta and venae cavae were ligated. The effluent was collected from the right atrial catheter at the same infusion rate. Animals were divided into two groups, the normal group and streptozotocin-induced diabetic group. Heart perfusion was done on both groups either without or after treatment with detergent (CHAPS) to remove the capillary endothelial lining. A physical model for 125I-insulin sequestration as a ligand to its receptors on endothelial and/or myocyte plasma membranes was proposed. The model described a reversible binding of ligand on cellular surface receptor concentration to fit a conservation equation and a first order Bessel function. The binding constants (kn), reversal constants (k-n), dissociation constants kd = k-n/kn, and residency time constants tau = 1/k-n of 125I-insulin in normal untreated, normal CHAPS-treated, diabetic untreated, and diabetic CHAPS-treated hearts were estimated using a theoretically generated curve-fit to the data. Since insulin receptor binding on the capillary endothelial cell surfaces may serve to transport insulin from the intravascular to the subendothelial space, and since streptozotocin-induced diabetes was shown to diminish receptor autophosphorylation and kinase activity and hence internalization of insulin, then one can conclude the following from the data. In the normal heart, removal of the capillary endothelial lining with CHAPS did not alter kn, k-n, kd, and tau of insulin binding as compared to the normal untreated, whereas in the diabetic untreated heart these constants were altered, compared to the diabetic treated. Furthermore, the kn and k-n values in the diabetic CHAPS-treated hearts were the same as for the normals untreated and CHAPS-treated, respectively. In conclusion, the dissociation constants and residency time constants of all groups indicated the possible existence of two types of insulin receptors: the capillary endothelial cell surface insulin receptors with lower residency time (low affinity receptor or combination of insulin and IGF-1 receptors) and the myocyte plasma membrane insulin receptors with higher residency times (high affinity).