El peróxido de hidrógeno como mediador en el proceso de contracción-relajaciónestudios "in vitro" e "in vivo"de contracción-relajación

Laburpena

The hydrogen peroxide (H2O2) is one of the most important reactive oxygen species (ROS). It is involved in multitude of intracellular process such as angiogenesis, apoptosis, cell survival, hyperplasia, atherosclerosis, cellular contraction and cellular relaxation. Some of these process, the roll of the H2O2 is well known however in other the effect of the H2O2 remains unknown. Throughout this thesis, analyzed the effect of the H2O2 in the contraction and relaxation process, in specify the myo-inositol 1,4,5- trisphosphate (IP3) pathway and the cyclic guanilate monophosphate (cGMP) pathway respectively. As well, we studied the hydrogen peroxide in the hypertension mediated by L-NAME. Stimulation of cell surface receptors results in the formation of the second messenger IP3 via the activation of phospholipase C. IP3 mobilizes intracellular calcium by binding to a family of receptors (IP3Rs) that act as ligand-gated calcium channels. IP3Rs are tetramers, and full-length sequences of at least three different isoforms have been identified by molecular cloning.Both homo- and heterotetramers are found in cells expressing more than one isoform. The acute regulation of IP3Rs occurs primarily through feedback effects of cytosolic Ca2+ and/or by phosphorylation of the receptor. However, prolonged exposure of cells to agonists has also been shown to alter the expression of IP3R protein levels. In the present study, we analyzed the effect of H2O2 in IP3R level. Chronic stimulation of VSMC by H2O2 resulted in the down-regulation of both type I and type III myo-inositol 1,4,5-trisphosphate receptors (IP3Rs). H2O2- induced down-regulation of IP3Rs could be detected within 4 h and resulted in an inhibition of IP3-induced Ca2+ release from permeabilized cells. The proteasomal inhibitor MG132 completely prevented H2O2-mediated down-regulation of IP3Rs. However, the stimulation with H2O2 did not increase the amount of IP3R immunoprecipitated by anti-ubiquitin antibodies. On the other hand, we analyzed the roll of the H2O2 in the ANGII- induced down regulation of IP3Rs. The NAD(P)H inhibitors did not block the ANGII- induced down regulation in the long-term stimulation, however the catalase prevented the down-regualtion in the acute stimulation with ANGII. We conclude that H2O2 stimulated IP3R degradation involves enhanced degradation by the proteasome pathway, but independent of the ubiquitination process; and the H2O2 generation is necessary in the ANGII- induced down regulation. Cardiovascular disorders are characterized by impaired vasodilatory responses after acetylcholine administration. This abnormal response has been usually termed endothelial dysfunction, and is considered to be one of the main pathogenic mechanisms responsible for the abnormal hemodynamic status of patients with hypertension, diabetes or atherosclerosis. Acetylcholine induces the synthesis of nitric oxide (NO) by endothelial cells, and a decreased synthesis of NO or an increased inactivation of this molecule has been proposed as the main cause of endothelial dysfunction. However, alternative mechanisms may also be proposed. NO induces cell relaxation by interacting with its intracellular receptor, soluble guanylate cyclase (sGC), which leads to an increased intracellular cyclic guanosine monophosphate (cGMP) concentration. Hence a reduction in sGC content or an abnormal response of the enzyme after its interaction with NO could also be involved in the previously mentioned vascular dysfunction. Some studies have shown a decreased sGC content in the vascular walls of animals with experimental hypertension or atherosclerosis. The sGC deficiency seems to be a more generalized phenomenon, since an attenuated glomerular cGMP production and renal vasodilation in streptozotocin-induced diabetic rats has also been demonstrated. In the present study, we propose that the H2O2 might modulate the sGC content in contractile cells. For that purpose, we tested the effect of this compound on the sGC content of cultured vascular smooth muscle cells (VSMC). We observed an increased in the β1 subunit levels of sGC, while the α1 subunit remained without changes. This change in the β1 subunit was accompanied with an increased in its activity, measuring like cGMP production and phosphorilation of VASP. By other hand, the H2O2 stimulation did not modify the β1 subunit expression, analyzed by luciferasa activity. This suggests the possibility H2O2-mediated up regulation of the β1 subunit is involved an increased of the mRNA stability, or a decreased of the degradation rate by the 26S proteasome. Finally, we have examined the effect of endogenously produced H2O2 on BP using a transgenic mouse model that overexpresses catalase. We observed that the systolic blood pressure (SBP) of the transgenic mice (CAT) is similar to that of their wild-type littermates (WT). However, CAT mice showed a significantly reduced pressor response to L-NAME when compared to wild-type control mice. In addition, we observed that L-NAME significantly increased oxidative damage obtained from wildtype mice but did not alter from CAT mice. These results suggest that induction of H2O2 in the arterial wall is a mechanism by which L-NAME increase blood pressure.