RESUMEN
The mechanisms related to hyperglycemia-induced pancreatic beta-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Delta psi (m)), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Delta psi (m), lead to an important pancreatic beta-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia.
Asunto(s)
Apoptosis/fisiología , Hiperglucemia/metabolismo , Membranas Mitocondriales/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Hiperglucemia/patología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Potenciales de la Membrana/fisiología , Microscopía Confocal , Membranas Mitocondriales/patología , Transporte de Proteínas/fisiología , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Glucose auto-oxidation may be a significant source of reactive oxygen species (ROS), and also be important in the lipid peroxidation process, accompanied by the release of toxic reactive products. We wanted to demonstrate that acrolein can be formed directly and actively from free fatty acids in a hyperglycemic environment. A suspension of linoleic and arachidonic acids (2.5 mM) was exposed to different glucose concentrations (5, 10 and 15 mmol/L) in vitro. The samples were extracted with organic solvents, partitioned, followed at 255-267 nm, and analysed using capillary electrophoresis and mass spectroscopy. The total release of aldehydes significantly (P < 0.01) increased from 1.0 to 5.1, 8.3 and 13.1 micromol/L after 6 hours of incubation, proportional to glucose concentrations. It was possible to verify a correlate hydroperoxide formation as well. Among the lipid peroxidation products, acrolein (5% of total) and its condensing product, 4-hydroxy-hexenal, were identified. From the results presented here, it was possible to demonstrate the production of acrolein, probably as a fatty acid product, due to free radicals generated from the glucose auto-oxidation process. The results led us to propose that acrolein, which is one of the most toxic aldehydes, is produced during hyperglycemic states, and may lead to tissue injury, as one of the initial problems to be linked to high levels of glucose in vivo.
Asunto(s)
Acroleína/metabolismo , Ácidos Grasos Insaturados/metabolismo , Glucosa/farmacología , Peroxidación de LípidoRESUMEN
The frequency of macroprolactinemia related to the presence of anti-PRL autoantibodies in the serum of 209 healthy women at different stages of pregnancy was studied. Measurements were taken of serum PRL concentrations before and after chromatographic separation (gel filtration and affinity with proteins A and G) and extraction of free PRL with polyethylene glycol (PEG). Sera from 8 of the 209 women (3.8%) were found to have a significantly high proportion of precipitated PRL by PEG (macroprolactinemia); in these patients, gel filtration showed that a substantial amount of big big PRL (molecular mass >100 kDa) was present (19.0--78.2% vs. 3.8-4.9%, P = 0.009 in normal pregnant women with a normal proportion of precipitated PRL by PEG). The presence of macroprolactinemia was attributable to anti-PRL autoantibodies in 5 of the 8 women. Comparison of serum levels of direct and free PRL between women with macroprolactinemia related to anti-PRL autoantibodies and women without macroprolactinemia showed significant differences (direct PRL: 270.2 +/- 86.9 vs. 203.4 +/- 69.0 microg/L, P = 0.04; and free PRL: 107.0 +/- 75.9 vs. 173.3 +/- 67.6 microg/L, P = 0.002). On the other hand, there was no difference between women with macroprolactinemia not related to anti-PRL autoantibodies and women with macroprolactinemia caused by anti-PRL autoantibodies, nor was there a difference between women with macroprolactinemia not related to anti-PRL autoantibodies and women without macroprolactinemia. There was a positive correlation between titers of the anti-PRL autoantibody and serum PRL levels (r = 0.82, P = 0.09). The presence of the anti-PRL autoantibody had no relation to the patient's age, stage of gestation, or number of previous pregnancies. We concluded that the frequency of macroprolactinemia was 3.8% among healthy, pregnant women, which was caused by a anti-PRL autoantibodies in 62.5% of the cases. The autoantibodies were found in the bloodstream, forming a PRL-IgG complex, in accordance with the following observations: 1) immunoreactive PRL on gel filtration was eluted in the fractions corresponding to the molecular mass of IgG (150 kDa); 2) a significantly high proportion of immunoreactive PRL was retained on an affinity gel for IgG (proteins A and G); and 3) a significantly high proportion of serum PRL bound to IgG was precipitated by protein A. There was a positive correlation between titers of anti-PRL autoantibodies and serum PRL levels. Serum levels of total PRL were higher, and serum levels of free PRL were lower, in pregnant women with anti-PRL autoantibodies than in pregnant women without macroprolactinemia.
Asunto(s)
Autoanticuerpos/sangre , Embarazo/inmunología , Prolactina/sangre , Prolactina/inmunología , Adulto , Femenino , Humanos , Embarazo/sangre , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Tercer Trimestre del Embarazo , Valores de ReferenciaRESUMEN
In an aqueous system, the oxidation of the erythrocyte membrane by the singlet oxygen formed during the photoactivation of the rose bengal coloring was examined. The effects of the singlet oxygen on lipids and proteins were studied through the simultaneous quantification of peroxidation products, lipoperoxides and carbonyl groups, the oxidation of protein SH groups and the activity of the glyceraldehyde 3-phosphate dehydrogenase (G3PD) associated with the erythrocyte membrane. The antioxidant activity of melatonin was tested and compared to that of two antioxidants in extreme cases of hydrosolubility, ascorbate and beta-carotene, with the purpose of comparing the protective ability of melatonin against singlet oxygen. The results show the expected effect even at low (0.125-0.75 mM; 0.015-0.90 mM, respectively) for ascorbate and beta-carotene, antioxidants known to possess important antioxidant qualities against singlet oxygen. It is shown that melatonin, under the conditions described, and at the concentrations at which the other two compounds are efficacious, not only confers little antioxidant protection, but that a pro-oxidant tendency was proven both on lipids and proteins, as well as on G3PD enzymatic activity. The results show that the antioxidant protective effect that melatonin can exert on biological systems is probably not by a direct interaction with oxidant species, but probably, as has been previously proposed, through the regulation of antioxidant defense systems. The formation of secondary oxidation products, such as melatonin-derived endoperoxides, may explain the evidence found on pro-oxidant qualities of this molecule.
Asunto(s)
Membrana Eritrocítica/química , Melatonina/farmacología , Oxidantes/farmacología , Oxígeno/sangre , Adulto , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Glucosafosfato Deshidrogenasa/sangre , Humanos , Peroxidación de Lípido/efectos de los fármacos , Lípidos de la Membrana/sangre , NAD/sangre , Fotoquímica , Rosa Bengala/química , Oxígeno Singlete , Solubilidad , Compuestos de Sulfhidrilo/sangre , beta Caroteno/farmacologíaRESUMEN
The effect of Nw-nitro-L-arginine on embryonic implantation and cGMP carbonyl group concentration was assessed at the rat implantation site (IS) and non-implantation site (NIS). The intraluminal administration of 25 microg (2.3 mM) of Nw-nitro-L-arginine inhibited implantation in 34.7% and embryo survival (100%), while in addition, decreasing cGMP concentration both at the site (1664.2 +/- 333.8 pmoles/mg of protein for the control and 1321 +/- 384.3 for those treated), as well as at the NIS (1203.7 +/- 200 to 780.2 +/- 168.5). Carbonyl group concentration was considerably less at the implantation site treated with Nw-nitro-L-arginine than in the control (0.062 +/- 0.012 nmoles/mg of protein and 0.45 +/- 0.1, respectively). Nonetheless, the NIS was not significantly different (0.12 +/- 0.04 and 0.15 +/- 0.05). Our results show that a nitric oxide (NO) dependent system parallel to the formation of cGMP and protein peroxidation products is important at the blastocyst implantation site in order for the endometrium to acquire the necessary properties for an adequate receptivity.
Asunto(s)
GMP Cíclico/metabolismo , Implantación del Embrión/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Preñez/metabolismo , Animales , AMP Cíclico/biosíntesis , Desarrollo Embrionario y Fetal/efectos de los fármacos , Endometrio/efectos de los fármacos , Endometrio/enzimología , Femenino , Indicadores y Reactivos , Nitroarginina/farmacología , Oxidación-Reducción , Peróxidos/metabolismo , Fenilhidrazinas , Embarazo , Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The peroxidation products, the free radicals, and the antioxidants compounds notable increase during ovulation, implantation, and pregnancy evolution. Superoxide anion (O2-) rise six time more on the proestrous, than on other stages, while its regulation enzyme, the superoxide dismutase (SOD), decreases. The presence of superoxide anion is related with the edema and the cellular proliferation on the estrous. Superoxide anion is also connected with the increase of fluidity and polarity of the membranes during the implantation. As the pregnancy elapse the lipoperoxides products and the antioxidant compounds augment suggesting that lipoperoxides evoke defense mechanism in a way that, at the end of the pregnancy, the antioxidants exceed peroxidative phenomena. By the other hand, the nitric oxide radical has gained great importance during the pregnancy because it is considered one of the most powerful relaxants of smooth muscle. The inhibition of its synthesis provokes similar signs to the preeclampsia and tis administration can revert many vascular alterations. The role of free radicals is not limited to dangerous effects, but it also, in adequate concentrations, includes the as stimulators of grown factors, and participants of the membranes fluidity.
Asunto(s)
Peroxidación de Lípido , Especies Reactivas de Oxígeno/metabolismo , Reproducción/fisiología , Animales , Implantación del Embrión/fisiología , Estro/fisiología , Femenino , Radicales Libres/metabolismo , Humanos , Embarazo , Ratas , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismoRESUMEN
Follicular fluid (FF) is of great importance in Assisted Procreation Programmes, because it constitutes the micro-environment of the follicle that regulates the development of the oocyte and participates in the capacitation of the spermatozoa. The biochemical composition of FF is influenced by the state of follicle maturation and reciprocally the content of the fluid predicts the success of the subsequent follicular maturation and pregnancy. In most of the species the high concentrations of estradiol are common in the follicles of intermediate maturity, the progesterone in the mature ones and the androgens in the atresics ones. Estradiol concentration is associated with the fertilizing capacity of the oocytes and its metabolites stimulate the progesterone production. Moreover, the FF regulates the action of the gonadotrophins, because it contains factors that help the union of these with its receptor. The FF contributes providing factors that inhibit and stimulate the meiosis. It contains factors that stimulates oocyte maturation, or that block the ability of the cortical granules to modify the components of the pellucid zone. The exposition of spermatozoons with this fluid helps the acrosomal reaction, the spermatic motility and the ovum penetration.
Asunto(s)
Líquido Folicular/fisiología , Fase Folicular , Oocitos/fisiología , Gonadotropina Coriónica/fisiología , Femenino , Humanos , Inseminación Artificial , Masculino , Embarazo , Espermatozoides/fisiologíaRESUMEN
The purpose of the study was to demonstrate that the effect of colchicine on the implantation and embryo development in rat is caused in part by its action on lysosome translocation to the perinuclear region. The subcellular enzymatic distribution of two lysosomal enzymes, acid phosphatase (E.C.3.1.3.2.) and beta-glucuronidase (E.C.3.2.1.3.1.), were measured. The right uterine horn was treated during preimplantation with colchicine (2 micrograms/kg of body weight on day 4) and the left with lumicolchicine (control). In the control horn, the nuclear activity of lysosomal enzymes was significantly higher in the implantation site tissue than the treated horn (colchicine) (p < 0.01). There were no modifications on the undecidualized endometrium under colchicine treatment. Simultaneously to this result, implantation and embryonic development were abolished. From the results presented herein, it is proposed that the inhibitory effect of colchicine is due to an inadequate biochemical differentiation at the implantation site, related to both arrest of cell division (mitosis is arrested in methaphase) and inhibition of the lysosomal movement toward the nucleus.
Asunto(s)
Fosfatasa Ácida/análisis , Colchicina/farmacología , Implantación del Embrión/fisiología , Desarrollo Embrionario y Fetal/fisiología , Glucuronidasa/análisis , Lisosomas/enzimología , Útero/enzimología , Fosfatasa Ácida/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Animales , Núcleo Celular/enzimología , Estudios de Cohortes , Colchicina/administración & dosificación , Implantación del Embrión/efectos de los fármacos , Desarrollo Embrionario y Fetal/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/enzimología , Epitelio/metabolismo , Femenino , Glucuronidasa/efectos de los fármacos , Glucuronidasa/metabolismo , Lisosomas/fisiología , Ratas , Células del Estroma/efectos de los fármacos , Células del Estroma/enzimología , Células del Estroma/metabolismo , Útero/efectos de los fármacos , Útero/ultraestructuraRESUMEN
The cytoskeleton in the endometrium, takes part not only in all the mechanic functions of the cell, but because of movement and location of healthy organelles and proteins, it also takes part in the metabolism. The endometrial epithelium, because of its morphology and its supposed cellular homogeneity, has been studied more than the stroma. It is known that intermedium filaments show a characteristic pattern of typical distribution and expression of the cellular type. During pregnancy and pseudopregnancy, in the apical region of the epithelial cells, both, luminal and glandular, there is an abundance of keratin in the basolateral region; while the vimentin is abundant only in the luminal epithelial cells and it increases in the implantation day. In humans and rats, the desmin only expresses during the decidual response. It is considered that intermedium filaments have a role in the polarity changes of the membrane. The microfilaments (MF) are related with the regulation of the cellular morphology and movement. In the luminal epithelium the MF play a role in the transformations of the uterine surface like the microvilli. The microtubules in the endometrium and other organs play an important role in the organelles position like lysosomes, mitochondria and Golgi complex. Also it is proved that take part in the DNA synthesis, because colchicine drug inhibits it.
Asunto(s)
Citoesqueleto/fisiología , Endometrio/citología , Endometrio/fisiología , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Animales , Citoesqueleto/ultraestructura , ADN/biosíntesis , Decidua/citología , Decidua/metabolismo , Decidua/fisiología , Desmina/metabolismo , Desmina/fisiología , Implantación del Embrión , Endometrio/metabolismo , Células Epiteliales , Epitelio/metabolismo , Epitelio/fisiología , Femenino , Humanos , Filamentos Intermedios/fisiología , Filamentos Intermedios/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/fisiología , Microvellosidades/fisiología , Orgánulos/fisiología , Orgánulos/ultraestructura , Embarazo , Ratas , Vimentina/metabolismo , Vimentina/fisiologíaRESUMEN
The role of lysosomes in the intracellular mechanism of action of several steroid an proteic hormones has been demonstrated. In presence of the specific hormone the target cell induce membranal changes and the lysosomes are moved toward the nucleus; after this the lysosomal enzymes are released in the perinuclear space. For the moment it is not possible to know the biochemical role of this enzymatic activities upon the nucleic acids function and des-repretion process of specific genes, but the inhibition of lysosomes movement utilizing hormone antagonist or dexamethasone inhibits some reproductive process like the implantation of the mammalian egg. We present herein a review related with the mode action of some hormones through the lysosomes in reproductive processes.
Asunto(s)
Hormonas/fisiología , Lisosomas/fisiología , Reproducción , Hormona Adrenocorticotrópica/fisiología , Animales , AMP Cíclico/fisiología , Implantación del Embrión , Estrógenos/fisiología , Femenino , Gonadotropinas/fisiología , Humanos , Hidrolasas/metabolismo , Técnicas In Vitro , Lisosomas/enzimología , Lisosomas/genética , Ovario/fisiología , Hormonas Hipofisarias/fisiología , Embarazo , Prolactina/fisiología , Ratas , Translocación Genética , Útero/fisiología , Vasopresinas/fisiologíaRESUMEN
In order to learn the mechanism of action of dexamethasone administration as an efficient inhibitor of estrogen activity in different tissues, the subcellular enzymatic distribution of two lysosomal enzymes: acid phosphatase (E.C.3.1.3.2.) and beta glucuronidase (E.C.3.2.1.3.1.) were measured. The rats were treated during preimplantation with dexamethasone (0.8 mg on days 3 and 4) or saline (controls). In the control group, the nuclear activity of lysosomal enzymes was significantly less in the implantation site tissue than in the treated group (p < 0.05). There were no modifications on the undecidualized endometrium under the steroid treatment. The lysosomal subfraction showed an opposite response. The steroid treatment produced an increase of activity in the decidualized tissue (1.9 +/- 0.4 to 4.9 +/- 0.4) while the nuclear enzymatic activity decreased under treatment; and simultaneously, the embryonic development was 100% abolished. From the results presented herein, it is proposed that the inhibitory effect of dexamethasone upon implantation is due to an inadequate biochemical differentiation at the implantation site, related to the inhibition of lysosomal movement toward the nucleus, and consequently to lysosomal enzymatic release and metabolic role.
Asunto(s)
Dexametasona/farmacología , Implantación del Embrión/efectos de los fármacos , Desarrollo Embrionario y Fetal/efectos de los fármacos , Lisosomas/fisiología , Fosfatasa Ácida/análisis , Animales , Implantación del Embrión/fisiología , Desarrollo Embrionario y Fetal/fisiología , Endometrio/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Femenino , Glucuronidasa/análisis , Lisosomas/enzimología , Embarazo , RatasRESUMEN
OBJECTIVE: To test the Szego hypothesis of increased beta-glucuronidase and acid phosphatase activities in hormone target tissues. METHODS: The presence of beta-glucuronidase and acid phosphatase activities in nuclear subcellular fractions obtained from decidual (implantation site) and stromal (nonimplantation zone) tissues was demonstrated by both biochemical measurements and ultramicrographic analysis utilizing a histochemical reaction. RESULTS: Acid phosphatase was almost twice as abundant in nuclei and lysosomes of epithelial cells (implantation sites), and beta-glucuronidase also was significantly more active in nuclei from epithelial and decidual tissues than in nonimplantation tissue. CONCLUSION: Our results, utilizing the implantation process as experimental model, support the Szego hypothesis of the lysosomal role in hormonal mechanisms of action.
Asunto(s)
Fosfatasa Ácida/análisis , Núcleo Celular/enzimología , Implantación del Embrión/fisiología , Glucuronidasa/análisis , Lisosomas/enzimología , Animales , Decidua/enzimología , Decidua/ultraestructura , Epitelio/enzimología , Epitelio/ultraestructura , Femenino , Histocitoquímica , Masculino , Microscopía Electrónica , Embarazo , Ratas , Células del Estroma/enzimología , Células del Estroma/ultraestructura , Fracciones Subcelulares/enzimología , Útero/enzimología , Útero/ultraestructuraRESUMEN
OBJECTIVE: The purpose of this study was to investigate the activity of gamma-glutamyl transpeptidase (gamma-GTP, E.C. 2.3.2.2) in rat endometrium (day 5 of pregnancy). Since gamma-GTP is an enzyme involved in the translocation of amino acids from fluids toward tissues, these substrates are necessary for anabolic processes. METHODS AND RESULTS: The presence of statistically higher activity of gamma-GTP in rat (Sprague-Dawley) implantation sites (1.06 nmol/mg protein/min) than in nondecidualized (0.87 nmol/mg protein/min) tissues was demonstrated. The intrauterine administration of L-serine-borate complex (5 mM) during day 5 of pregnancy arrested 91.6% of rat embryonic development (day 18). This inhibitory effect was not present when borate or L-serine was administered separately. The L-serine-borate complex also inhibited (by 88%) the gamma-GTP in vitro. CONCLUSION: The inhibition of gamma-GTP by L-serine-borate complex might be considered as a new approach to the arrest of biological processes in differentiation or development.