| Urotensin â…¡ (Uâ…¡) is a cyclic neuropeptide that was first isolated from telst fish some35years ago. It is a potent vasoactive peptides identified, and its vasoactive effects are reported to be dependent both on species and on anatomical location of the vessels, suggesting a complex role for this peptide in cardiovascular homeostasis. Recent evidence suggests that brain Uâ…¡ as a neuromodulator plays an important role in cardiovascular activity via sympathoexcitatory pathway. However, the mechanisms underlying the central cardiovascular effects of Uâ…¡ remain largely unknown. Since a growing body of evidence now indicates that reactive oxygen species (ROS) is an important mediator of sympathoexcitation, in the present study, we tested the hypothesis that ROS mediates the central cardiovascular effects of U â…¡ by activating NADPH oxidase and thus an increase in the production of ROS. By using combined morphological, functional, and molecular biological techniques, we aimed to explore the mechanism of cantrl cardiovascular effects of Uâ…¡.Urethane-chloralose-anesthetized SHR and WKY rats were used in all experiments. To examine the differences in distributions of Uâ…¡ receptor (UT) in the brain between these two species, immunocytochemistry was employed. The results showed that expression of UT immunoreactive cells was observed in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS) of both SHR and WKY rats, however, the mean optic density (MOD) was significantly higher in SHR than that in WKY rats (p<0.05). Western blot analysis was used to confirm these observations. It was demonstrated that UT protein was detected from ventrolateral medulla and hypothalamus, the MOD was also higher in the ventrolateral medulla of SHR than that in WKY rats (p<0.05). We also identified Uâ…¡ content in brain by using radioimmunoassay. U â…¡ was detected in the ventrolateral medulla, midbrain and hypothalamus, the content of U â…¡ in hypothalamus was higher in SHR than that in WKY rats (p<0.05). These results indicate that the expression of UT and the content of Uâ…¡ were higher in SHR than in WKY rats, suggesting the possibility that U â…¡ might be involved in the genesis of hypertension in the spontaneously hypertensive rat.Immunofluorescence double-labeling combined with confocal microscopy was used to identify the cellular distribution of UT. Colocalization of UT protein and Neu, a neuron marker, was identified in the RVLM. However, colocalization of UT protein and GFAP, a glia marker, was not identified in RVLM.. The results suggest that UT is mainly present in the neurons of RVLM.To clarify whether ROS mediates the central cardiovascular action of Uâ…¡, intracerbroventricular (icv) infusion and microinjection were employed in SHR and WKY rats. The results showed that:1. icv Uâ…¡ significantly increased mean arterial pressure (MAP) and heart rate (HR)(P<0.05), these effects of Uâ…¡ were significantly more pronounced in SHR than in WKY (P<0.05). This finding is consistent with the prevous morphological observations.2. In SHR rat, icv urantide alone can significantly decreased MAP. Pretreatment with urantide (a UT antagonist, icv) or Tempol (an SOD mimetic, icv) eliminated the effects of U â…¡.3. Microinjection of Uâ…¡ (500pmol) into the RVLM significantly increased mean arterial pressure (MAP, P<0.05), pretreatment with urantide, or Tempol (50nmol), Apocynin (5nmol)(an NADPH oxidase inhibitor) also eliminated the effects of U â…¡ (P<0.05). These results indicate that ROS may mediate the effects of U â…¡ and that the RVLM is possibly the brain areas of the central cardiovascular effects of U â…¡.We also observed the brain ROS level by using dihydroehidium (DHE) staining in brain slices. The results revealed that the level of superoxide production in the RVLM was significantly higher in SHR than in WKY rats (P<0.05), icv U â…¡ significantly increased superoxide production in the RVLM of SHR (P<0.05). Furthemore, we detected NADPH oxidase activity to clarify whether superoxide production was NADPH oxidase-derived by using lucigenin-enhanced chemilumnescence assay. NADPH oxidase activity was significantly greater in the RVLM of SHR, compared with that in WKY rats (P<0.05); icv Uâ…¡ significantly increased the activity of NADPH oxidase in the RVLM of SHR (P<0.05), the augmented NADPH oxidase activity was markedly inhibited by Apocynin (P<0.05). These results indicate that NADPH-derived ROS possibly plays an important role in the cardiovascular effects of central Uâ…¡.To elucidate the signaling pathway of NADPH-deri ved ROS induced by central Uâ…¡, i mmunofluorescence double-labeling combined with confocal microscopy was used to observe the colocalization of UT and NADPH subunit. The results showed that most of NADPH memberane-bound subunit gp91phox or cytosolic subunit p47phox in RVLM were double-labeled with UT respectively. This finding provides a structural basis for the interaction bewteen NADPH oxidase and Uâ…¡. Futhermore, we employed real-time PCR and Western blotting to detect the mRNA level of NADPH subunit gp91phox, p47phox and the phosphorylation of serine residues of p47phox induced by Uâ…¡. The results demonstrated that icv Uâ…¡ significantly increased gp91phox or p47phox mRNA level in the RVLM. More importantly, a significant elevation in phosporylated P47phox was detected5min after microinjection of Uâ…¡ into the RVLM. Taking together, these results suggest that Uâ…¡ may induce serine phoaphrylation of p47phox subunit and then NADPH oxidase-derived superoxide.In conclusion, the present study identified that a novel signaling pathway for the cardiovascular effects of central Uâ…¡. It follows that by acting Uâ…¡ receptor in the RVLM, Uâ…¡ increases blood pressure, which is possibly mediated by NADPH oxidase-derived superoxide following the induction of of serine phoaphrylation of the p47phox subunit. Increased sensitivity of UT in SHR is patially responsible for the development of hypertension in this genetic hypertensive rat. |
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