RESUMO
During pregnancy, the uterus undergoes major functional and structural remodelling. It is well known that during the major part of pregnancy, the myometrium normally remains relatively quiescent but is able to generate powerful contractions at the time of parturition. However, the intracellular molecular events regulating myometrial contractility during pregnancy still remain poorly understood. We applied differential gene expression screening using cDNA array technology to probe myometrium samples from non-pregnant and mid-pregnant (15 days) rabbits. Among the differentially expressed genes, the farnesylated small G-protein of the Rho family, Rnd3, was found to be upregulated (3.6-fold) at mid-pregnancy. Upregulation of Rnd3 was confirmed at the protein level by a 3.4-fold increase in Rnd3 expression in mid-pregnant myometrium. Measurements of contractile properties of beta-escin permeabilized smooth muscle strips revealed that the upregulation of Rnd3 correlated with an inhibition of RhoA-Rho kinase-mediated Ca2+ sensitization at mid-pregnancy. Treatment of muscle strips from mid-pregnant myometrium with the farnesyl-transferase inhibitor manumycin A (10 muM) led to the recovery of RhoA-Rho kinase-dependent Ca2+ sensitization. At late pregnancy (31 days), upregulation of RhoA and Rho kinase expression was associated with an increase in Ca2+ sensitivity of contractile proteins that was inhibited by the Rho kinase inhibitor Y-27632 (10 muM). These data thus demonstrate the time-dependent regulation of the RhoA-Rho kinase-mediated Ca2+ sensitization during the course of pregnancy. The depression of this mechanism at mid-pregnancy followed by its constitutive activation near term is associated with a co-ordinated modulation of Rnd3, RhoA and Rho kinase expression. The RhoA-Rho kinase signalling pathway and its regulators might thus represent potential targets for the development of new treatments for pre-term labour.
Assuntos
Cálcio/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Miométrio/fisiologia , Prenhez/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Western Blotting , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Proteínas Ativadoras de GTPase/genética , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Contração Isométrica/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/biossíntese , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genética , Regulação para Cima/fisiologia , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Rolling of leukocytes at the surface of the vascular endothelium is a prerequisite for a subsequent firm adhesion, particularly the slow rolling appearing on ELAM CD62E. Therefore, it may be considered that increasing the rolling velocities should be a precise therapeutic target in clinical situations where leukocytes accumulate, mainly venous and arterial ischaemia. METHODS: Human neutrophils were allowed to flow on endothelial HUVECs, with and without 4 hours interleukin-1alpha activation, the cells having or not been incubated with INO5042 anti-inflammatory drug. Under a mean shear-stress of 2 dyn/cm(2), rollers and stickers were identified and quantified, using a video-camera and picture analysing software. RESULTS: When the drug had been added to endothelial cells a shift of velocities was observed towards fast speeds (from 3-5 to 7-11 microm/sec). The same results was significantly found when neutrophils, alone or along with endothelium, had been submitted to the drug, the number of stickers and rollers beeing reduced as well. Finally, such a precise pharmacological method proved efficient to detect the exact mechanism of INO5042 on white cell adhesion.
