RESUMEN
In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP3) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Células Endoteliales/enzimología , Uniones Comunicantes/química , Miocitos del Músculo Liso/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Transporte Biológico , Bradiquinina/farmacología , Señalización del Calcio , Comunicación Celular/efectos de los fármacos , Técnicas de Cocultivo , GMP Cíclico/metabolismo , Diglicéridos/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Regulación de la Expresión Génica , Humanos , Inositol 1,4,5-Trifosfato , Compuestos Macrocíclicos/farmacología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/clasificación , Óxido Nítrico Sintasa de Tipo III/genética , Oxazoles/farmacología , Fenilefrina/farmacología , Fosfatidilserinas/metabolismo , Fosforilación , Cultivo Primario de Células , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Tapsigargina/farmacologíaRESUMEN
Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.