RESUMO
This study examined the effects of N-ethylmaleimide-sensitive factor (NSF) small interfering RNA (siRNA) on cardiac function following myocardial infarction (MI) in rats. Thirty-six adult Sprague Dawley rats were randomly divided into three equivalent groups. An acute MI model was established by ligating the anterior descending branch of the left coronary artery and confirmed by electrocardiogram. Recombinant NSF-siRNA adenovirus (experimental), negative adenovirus (control), and normal saline were injected near the infarcted area of the left ventricle in each respective group. The left ventricular ejection fraction (LVEF) was measured with a noninvasive ultrasonic cardiogram. Left ventricular end-diastolic pressure (LVEDP) and the maximum rate of rise in left ventricular pressure (+dp/dt max) were measured using the BL-420 Biological Functional Experimental System. Hearts were sectioned and stained with 2,3,5,-triphenyl tetrazolium chloride (TTC) to observe the MI area. Two weeks after surgery, LVEF in the experimental group (46.0 ± 7.5%) was higher than control (34.0 ± 6.0%) and saline (37.5 ± 4.5%) group LVEFs (P < 0.05), whereas LVEDP was the lowest in the experimental group (18.51 ± 6.87 vs 29.47 ± 9.94 and 26.58 ± 8.97 mmHg, respectively) (P < 0.05). The +dp/dt max was also higher in the experimental group (9.74 ± 1.16 vs 4.33 ± 1.19 and 5.24 ± 1.53 mmHg/s x 10(3), respectively) (P < 0.05); however, the MI area did not differ significantly between groups. Local injection of an adenovirus-mediated NSF-siRNA expression vector near infarcted areas improved cardiac function two weeks after MI, but had no impact on the MI area.
Assuntos
Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Proteínas Sensíveis a N-Etilmaleimida/genética , RNA Interferente Pequeno/genética , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Eletrocardiografia , Vetores Genéticos/genética , Masculino , Infarto do Miocárdio/diagnóstico , Ratos , Volume Sistólico , Função Ventricular Esquerda , Remodelação VentricularRESUMO
Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the cortical region of metaphase II oocytes, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model.
Assuntos
Exocitose/fisiologia , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Oócitos/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Animais , Exocitose/genética , Feminino , Fertilização in vitro , Camundongos , Proteínas Sensíveis a N-Etilmaleimida/genética , Oócitos/citologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genéticaRESUMO
The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.