RESUMEN
BACKGROUND: Mammalian tissues contain a presumed endogenous Na+, K(+)-ATPase inhibitor that binds reversibly to the Na+ pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself. The objective of this study was to test the hypothesis that a biosynthetic pathway exists in mammalian tissues to produce a steroid derivative closely related to plant cardiac glycosides. METHODS AND RESULTS: Using bioinformatics and genomic techniques, Milan hypertensive rat tissues were studied because this strain has a 10-fold increase in hypothalamic ouabain-like compound that is linked to the pathogenesis of the hypertension. A putative steroid biosynthetic pathway was constructed and candidate genes encoding enzymes in this pathway were identified from sequence databases. Differential expression of selected genes in the pathway was studied by microarray analysis and quantitative polymerase chain reaction, with functional validation by gene silencing using small interfering RNAs. Marked upregulation of genes coding for P450 side chain cleavage and Delta5-3beta-hydroxysteroid dehydrogenase/Delta5-Delta4- isomerase enzymes in hypertensive hypothalamus but not adrenal was found, compared with normotensive Milan rats. Knockdown of the latter gene decreased production of ouabain-like factor from neural tissue. CONCLUSIONS: Our findings support the possibility that a unique steroid biosynthetic circuit exists in Milan rat brain, functioning independently from adrenal, which could account for the overproduction of the hypothalamic ouabain-like compound in this species.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Hipertensión/metabolismo , Hipotálamo/metabolismo , Complejos Multienzimáticos/genética , Ouabaína/metabolismo , Progesterona Reductasa/genética , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Esteroide Isomerasas/genética , Animales , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Reacción en Cadena de la Polimerasa , Interferencia de ARN , ARN Mensajero/análisis , RatasRESUMEN
Human DNA microarrays are used to study the spatiotemporal patterns of gene expression during the course of fetal monkey brain development. The 444 most dynamically expressed genes in four major brain areas are reported at five different fetal ages. The spatiotemporal profiles of gene expression show both regional specificity as well as waves of gene expression across the developing brain. These patterns of expression are used to identify statistically significant clusters of co-regulated genes. This study demonstrates for the first time in the primate the relevance, timing, and spatial locations of expression for many developmental genes identified in other animals and provides clues to the functions of many unknowns. Two different microarray platforms are used to provide high-throughput cross validation of the most important gene expression changes. These results may lead to new understanding of brain development and new strategies for treating and repairing disorders of brain function.