RESUMEN

In brief: The cervix plays a crucial role not only in the maintenance of pregnancy but also during delivery, when it undergoes extensive changes. This study highlights the involvement of the endocannabinoidome in cervical remodeling, emphasizing its relevance in the shift from a nonpregnant to pregnant state and its potential contribution to preterm delivery in inflammatory contexts. Abstract: During pregnancy, the main role of the cervix is to isolate the fetus from outside pathogens and maintain the relatively closed system of uterine gestation. Conversely, toward the end of pregnancy, the cervix must be remodeled to increase flexibility and allow the delivery. This process is called cervical remodeling and dysregulation of the process plays a role in premature delivery. The endocannabinoidome plays an important role in several reproductive events; however, its function on cervical tissue throughout pregnancy is poorly understood. The goal of this study was to evaluate the presence and participation of the endocannabinoidome in lipopolysaccharide (LPS)-induced cervical changes. Therefore, we evaluated key components of the endocannabinoidome in cervical tissue from nonpregnant mice and pregnant mice with and without LPS treatment. Using mass spectrometric analysis, we found an increase in anandamide and 2-arachidonoylglycerol in the cervix of pregnant mice when compared to nonpregnant mice. We have also found a reduction in FAAH protein expression in these tissues. Furthermore, when treated with LPS, we observed a reduction in the cervical immunostaining with anti-CB1 and anti-CB2 antibodies. Likewise, using cervix explants from pregnant mice, we found that LPS significantly increased cervical metalloprotease activity and cyclooxygenase 2, which were subsequently modulated by cannabinoid receptor antagonists. Collectively, our findings suggest that an LPS-induced imbalance of cervix endocannabinoidome likely contributes to premature cervical remodeling, which is part of the key components that contribute to premature delivery.

Asunto(s)

Trabajo de Parto Prematuro , Nacimiento Prematuro , Embarazo , Humanos , Femenino , Ratones , Animales , Cuello del Útero/fisiología , Endocannabinoides/farmacología , Lipopolisacáridos/farmacología , Útero/metabolismo , Trabajo de Parto Prematuro/metabolismo , Nacimiento Prematuro/metabolismo

RESUMEN

Implantation-related events are crucial for pregnancy success. In particular, defects in vascular remodeling at the maternal-fetal interface are associated with spontaneous miscarriage and recurrent pregnancy loss. Physical activity and therapies oriented to reduce stress improve pregnancy outcomes. In animal models, environmental stimulation and enrichment are associated with enhanced well-being, cognitive function and stress resilience. Here, we studied whether the exposure of BALB/c mice to an enriched environment (EE) regulates crucial events during early gestation at the maternal-fetal interface. Pregnant BALB/c mice were exposed to the EE that combines non-invasive stimuli from the sensory pathway with voluntary physical activity. The pregnancy rate was evaluated. Implantation sites were investigated microscopically and macroscopically. Vascular adaptation parameters at the maternal-fetal interface were analyzed. We found that exposure to the EE prevented pregnancy loss between gestational days 7 and 15. Also, it increased the diameter of the uterine artery and decreased the wall:lumen ratio of the mesometrial decidual vessels, suggesting that EE exposure promotes vascular remodeling. Moreover, it increased nitric oxide synthase activity and inducible nitric oxide synthase expression, as well as prostaglandin F2a production and endoglin expression in the implantation sites. Exposure of pregnant females to the EE regulates uterine physiology, promoting vascular remodeling during early gestation. These adaptations might contribute to preventing embryo loss. Our results highlight the importance of the maternal environment for pregnancy success. The design of an 'EE-like' protocol for humans could be considered as a new non-pharmacologic strategy to prevent implantation failure and recurrent miscarriage.

Asunto(s)

Pérdida del Embrión , Remodelación Vascular , Animales , Decidua/metabolismo , Femenino , Humanos , Exposición Materna , Ratones , Ratones Endogámicos BALB C , Embarazo , Útero/metabolismo

RESUMEN

Maternal overnutrition negatively impacts the offspring's health leading to an increased risk of developing chronic diseases or metabolic syndrome in adulthood. What we eat affects the endocannabinoid system (eCS) activity, which in turn modulates lipogenesis and fatty acids utilization in hepatic, muscle, and adipose tissues. This study aimed to evaluate the transgenerational effect of maternal obesity on cannabinoid receptor 1 knock-out (CB1 KO) animals in combination with a postnatal obesogenic diet on the development of metabolic disturbances on their offspring. CB1 KO mice were fed a control diet (CD) or a high-fat diet (HFD; 33% more energy from fat) for 3 months. Offspring born to control and obese mothers were also fed with CD or HFD. We observed that pups born to an HFD-fed mother presented higher postnatal weight, lower hepatic fatty acid amide hydrolase activity, and increased blood cholesterol levels when compared to the offspring born to CD-fed mothers. When female mice born to HFD-fed CB1 KO mothers were exposed to an HFD, they gained more weight, presented elevated blood cholesterol levels, and more abdominal adipose tissue accumulation than control-fed adult offspring. The eCS is involved in several reproductive physiological processes. Interestingly, we showed that CB1 KO mice in gestational day 15 presented resistance to LPS-induced deleterious effects on pregnancy outcome, which was overcome when these mice were obese. Our results suggest that an HFD in CB1 receptor-deficient mice contributes to a "nutritional programming" of the offspring resulting in increased susceptibility to metabolic challenges both perinatally and during adulthood.

Asunto(s)

Lipopolisacáridos/efectos adversos , Obesidad Materna/genética , Receptor Cannabinoide CB1/genética , Animales , Animales Recién Nacidos , Dieta Alta en Grasa/efectos adversos , Femenino , Fenómenos Fisiologicos Nutricionales Maternos , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Ratones , Ratones Noqueados , Obesidad , Obesidad Materna/metabolismo , Embarazo , Resultado del Embarazo , Efectos Tardíos de la Exposición Prenatal/etiología , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/metabolismo , Receptor Cannabinoide CB1/metabolismo

RESUMEN

Maternal obesity has been shown to impact the offspring health during childhood and adult life. This study aimed to evaluate whether maternal obesity combined with postnatal exposure to an obesogenic diet could induce metabolic alterations in offspring. Female CD1 mice were fed a control diet (CD, 11.1% of energy from fat) or with a high-fat diet (HFD, 44.3% of energy from fat) for 3 months. After weaning, pups born from control and obese mothers were fed with CD or HFD for 3 months. Both mothers and offspring were weighted weekly and several blood metabolic parameters levels were evaluated. Here, we present evidence that the offspring from mothers exposed to a HFD showed increased acetylation levels of histone 3 on lysine 9 (H3K9) in the liver at postnatal Day 1, whereas the levels of acetylation of H4K16, dimethylation of H3K27, and trimethylation of H3K9 showed no change. We also observed a higher perinatal weight and increased blood cholesterol levels when compared to the offspring on postnatal Day 1 born from CD-fed mothers. When mice born from obese mothers were fed with HFD, we observed that they gained more weight, presented higher blood cholesterol levels, and abdominal adipose tissue than mice born to the same mothers but fed with CD. Collectively, our results point toward maternal obesity and HFD consumption as a risk factor for epigenetic changes in the liver of the offspring, higher perinatal weight, increased weight gain, and altered blood cholesterol levels.

Asunto(s)

Colesterol/sangre , Dieta Alta en Grasa/efectos adversos , Obesidad/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Fenómenos Fisiologicos de la Nutrición Prenatal , Animales , Metilación de ADN , Femenino , Histonas/metabolismo , Hígado/metabolismo , Ratones , Embarazo

RESUMEN

Preterm delivery is the leading cause of neonatal mortality and contributes to delayed physical and cognitive development in children. At present, there is no efficient therapy to prevent preterm labor. A large body of evidence suggests that intra-amniotic infections may be a significant and potentially preventable cause of preterm birth. This work assessed the effect of melatonin in a murine model of inflammation-associated preterm delivery which mimics central features of preterm infection in humans. For this purpose, preterm labor was induced in BALB/c mice by intraperitoneal injections of bacterial lipopolysaccharide (LPS) at 10.00 hr (10 µg LPS) and 13.00 hr (20 µg LPS) on day 15 of pregnancy. On day 14 of pregnancy, a pellet of melatonin (25 mg) had been subcutaneously implanted into a group of animals. In the absence of melatonin, a 100% incidence of preterm birth was observed in LPS-treated animals, and the fetuses showed widespread damage. By comparison, treatment with melatonin prevented preterm birth in 50% of the cases, and all pups from melatonin-treated females were born alive and their body weight did not differ from control animals. Melatonin significantly prevented the LPS-induced rises in uterine prostaglandin (PG) E2 , PGF2α, and cyclooxygenase-2 protein levels. In addition, melatonin prevented the LPS-induced increase in uterine nitric oxide (NO) production, inducible NO synthase protein, and tumor necrosis factor-alpha (TNFα) levels. Collectively, our results suggest that melatonin could be a new therapeutic tool to prevent preterm labor and to increase offspring survival.

Asunto(s)

Melatonina/uso terapéutico , Trabajo de Parto Prematuro/tratamiento farmacológico , Trabajo de Parto Prematuro/metabolismo , Sustancias Protectoras/uso terapéutico , Animales , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Femenino , Lipopolisacáridos/toxicidad , Melatonina/farmacología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Trabajo de Parto Prematuro/inducido químicamente , Trabajo de Parto Prematuro/prevención & control , Embarazo , Prostaglandinas/metabolismo , Sustancias Protectoras/farmacología

RESUMEN

BACKGROUND AND PURPOSE: Infections with a strain of Escherichia coli producing Shiga toxins could be one of the causes of fetal morbidity and mortality in pregnant women. We have previously reported that Shiga toxin type 2 (Stx2) induces preterm delivery in pregnant rats. In this study, we evaluate the role of TNF-α, PGs and NO in the Stx2-induced preterm delivery. EXPERIMENTAL APPROACH: Pregnant rats were treated with Stx2 (0.7 ng g(-1)) and killed at different times after treatment. Placenta and decidua were used to analyse NOS activity by the conversion of L-[(14)C]arginine into L-[(14)C]citrulline, levels of PGE(2) and PGF(2α) assessed by radioimmunoassay, and cyclooxygenase (COX) proteins by Western blot. TNF-α level was analysed in serum by ELISA and by cytotoxicity in L929 cells. The inhibitor of inducible NOS, aminoguanidine, the COX-2 inhibitor, meloxicam, and the competitive inhibitor of TNF-α, etanercept, were used alone or combined to inhibit NO, PGs and TNF-α production respectively, to prevent Stx2-induced preterm delivery. KEY RESULTS: Stx2 increased placental PGE(2) and decidual PGF(2α) levels as well as COX-2 expression in both tissues. Aminoguanidine and meloxicam delayed the preterm delivery time but did not prevent it. Etanercept blocked the TNF-α increase after Stx2 treatment and reduced the preterm delivery by approximately 30%. The combined action of aminoguanidine and etanercept prevented Stx2-induced preterm delivery by roughly 70%. CONCLUSION AND IMPLICATIONS: Our results demonstrate that the increased TNF-α and NO induced by Stx2 were the predominant factors responsible for preterm delivery in rats.

Asunto(s)

Dinoprost/biosíntesis , Dinoprostona/biosíntesis , Nacimiento Prematuro/inducido químicamente , Toxina Shiga II/toxicidad , Factor de Necrosis Tumoral alfa/sangre , Animales , Ciclooxigenasa 2/biosíntesis , Decidua/efectos de los fármacos , Decidua/enzimología , Decidua/metabolismo , Quimioterapia Combinada , Etanercept , Femenino , Guanidinas/administración & dosificación , Guanidinas/uso terapéutico , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/uso terapéutico , Óxido Nítrico/biosíntesis , Placenta/efectos de los fármacos , Placenta/enzimología , Placenta/metabolismo , Embarazo , Nacimiento Prematuro/sangre , Nacimiento Prematuro/metabolismo , Nacimiento Prematuro/prevención & control , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/administración & dosificación , Receptores del Factor de Necrosis Tumoral/uso terapéutico

RESUMEN

Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids' receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins' synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids' levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation.

Asunto(s)

Implantación del Embrión , Endocannabinoides/metabolismo , Lisofosfolípidos/metabolismo , Prostaglandinas/metabolismo , Amidohidrolasas/metabolismo , Animales , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Femenino , Hidrolasas Diéster Fosfóricas/análisis , Hidrolasas Diéster Fosfóricas/metabolismo , Embarazo , Ratas , Ratas Wistar , Receptores del Ácido Lisofosfatídico/análisis , Receptores del Ácido Lisofosfatídico/metabolismo , Útero/irrigación sanguínea , Útero/metabolismo

RESUMEN

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE(2) and PGF(2α) biosynthesis, by inhibiting PGE(2) production and increasing PGF(2α) levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.

Asunto(s)

Ácidos Araquidónicos/farmacología , Dinoprost/biosíntesis , Dinoprostona/biosíntesis , Lipopolisacáridos/farmacología , Útero/efectos de los fármacos , Útero/metabolismo , Animales , Ácidos Araquidónicos/administración & dosificación , Endocannabinoides/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Embarazo , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo

RESUMEN

Anandamide, an endocannabinoid, prostaglandins derived from cyclooxygenase-2 and nitric oxide synthesized by nitric oxide synthase (NOS), are relevant mediators of embryo implantation. We adopted a pharmacological approach to investigate if anandamide modulated NOS activity in the receptive rat uterus and if prostaglandins mediated this effect. As we were interested in studying the changes that occur at the maternal side of the fetal-maternal interface, we worked with uteri obtained from pseudopregnant rats. Females were sacrificed on day 5 of pseudopregnancy, the day in which implantation would occur, and the uterus was obtained. Anandamide (2 ng/kg, i.p.) inhibited NOS activity (P<0.001) and increased the levels of prostaglandin E(2) (P<0.001) and prostaglandin F(2α) (P<0.01). These effects were mediated via cannabinoid receptor type 2, as the pre-treatment with SR144528 (10 mg/kg, i.p.), a selective cannabinoid receptor type 2 antagonist, completely reverted anandamide effect on NOS activity and prostaglandin levels. The pre-treatment with a non-selective cyclooxygenase inhibitor (indomethacin 2.5mg/kg, i.p.) or with selective cyclooxygenase-2 inhibitors (meloxicam 4 mg/kg, celecoxib 3mg/kg, i.p.) reverted anandamide inhibition on NOS, suggesting that prostaglandins are derived from cyclooxygenase-2 mediated anandamide effect. Thus, anandamide levels seemed to modulate NOS activity, fundamental for implantation, via cannabinoid receptor type 2 receptors, in the receptive uterus. This modulation depends on the production of cyclooxygenase-2 derivatives. These data establish cannabinoid receptors and cyclooxygenase enzymes as an interesting target for the treatment of implantation deficiencies.

Asunto(s)

Ácidos Araquidónicos/farmacología , Dinoprost/metabolismo , Dinoprostona/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Alcamidas Poliinsaturadas/farmacología , Animales , Canfanos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Endocannabinoides , Femenino , Óxido Nítrico Sintasa/metabolismo , Seudoembarazo , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptor Cannabinoide CB2/efectos de los fármacos , Receptor Cannabinoide CB2/metabolismo , Útero/efectos de los fármacos , Útero/metabolismo

RESUMEN

The submandibular gland-derived tumor cell line SCA-9 is considered a useful tool to study the signaling pathways involved in proliferation, and their regulation, triggered by different stimuli. It is proposed that the non neuronal cholinergic system: acethylcholine, the enzymes that synthesize and degrade it, and the nicotinic and muscarinic receptors, play a key role in tumorigenesis. Here, we investigate the role of muscarinic receptors in SCA-9 cell proliferation, and the modulation of cholinergic signaling pathways exerted by the nuclear transcription factor κB (NF-κB). The activation of cholinergic receptors by carbachol (10⁻9M) increased cell proliferation (P<0.001). This was prevented by preincubating cells with the muscarinic antagonist atropine but not by mecamylamine, a nicotinic receptor blocker. Phospholipase C (PLC)/nitric oxide synthase (NOS)/arginase pathway is involved in this effect, since carbachol stimulated nitric oxide production, increased NOS2 and NOS3 expressions, urea production, and arginase II expression (P<0.001). Also, phospholipase A2 (PLA2)/cyclooxygenase (COX) pathway is up-regulated in carbachol-induced SCA-9 cell proliferation, because prostaglandin E2 liberation (P<0.001) is increased and COX-1 expression is turned up (P<0.001). Interactions between PLC/NOS/arginases and PLA2/COX pathways via its metabolites were detected. SCA-9 cells exhibit a constitutive activation of NF-κB, which regulates carbachol-induced NOS2 and 3, arginase II and COX-1 expressions. In addition, protein kinase C is involved in the up-regulation of NOS2 and arginase II enzymes induced by carbachol via NF-κB. In conclusion, the activation of cholinergic receptors in SCA-9 tumor cells promotes proliferation via muscarinic effector enzymes, and reveals the participation of NF-κB at this step of tumorigenesis.

Asunto(s)

Arginasa/metabolismo , Proliferación Celular/efectos de los fármacos , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Antineoplásicos/farmacología , Arginasa/antagonistas & inhibidores , Línea Celular Tumoral , Agonistas Colinérgicos/farmacología , Ciclooxigenasa 1/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Antagonistas Muscarínicos/farmacología , FN-kappa B/antagonistas & inhibidores , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Inhibidores de Fosfolipasa A2 , Fosfolipasas A2/metabolismo , Prostaglandina-Endoperóxido Sintasas/química , Receptores Colinérgicos/química , Receptores Muscarínicos/química , Transducción de Señal/efectos de los fármacos , Neoplasias de la Glándula Submandibular/tratamiento farmacológico , Neoplasias de la Glándula Submandibular/enzimología , Neoplasias de la Glándula Submandibular/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.

Asunto(s)

Ácidos Araquidónicos/farmacología , Trompas Uterinas/citología , Trompas Uterinas/metabolismo , Óxido Nítrico/metabolismo , Alcamidas Poliinsaturadas/farmacología , Transducción de Señal/efectos de los fármacos , Espermatozoides/citología , Animales , Bovinos , Comunicación Celular/efectos de los fármacos , Endocannabinoides , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Trompas Uterinas/efectos de los fármacos , Femenino , Hemoglobinas/farmacología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptor Cannabinoide CB1/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Canales Catiónicos TRPV/metabolismo

RESUMEN

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1) h(-1)) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.

Asunto(s)

Ácidos Araquidónicos/farmacología , Blastocisto/citología , Blastocisto/fisiología , Óxido Nítrico Sintasa/metabolismo , Alcamidas Poliinsaturadas/farmacología , Útero/efectos de los fármacos , Útero/enzimología , Animales , Benzamidas/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Carbamatos/farmacología , Implantación del Embrión , Endocannabinoides , Femenino , Inmunohistoquímica/métodos , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) infections could be one of the causes of fetal morbimortality in pregnant women. The main virulence factors of STEC are Shiga toxin type 1 and/or 2 (Stx1, Stx2). We previously reported that intraperitoneal (i.p.) injection of rats in the late stage of pregnancy with culture supernatant from recombinant E. coli expressing Stx2 and containing lipopolysaccharide (LPS) induces premature delivery of dead fetuses. It has been reported that LPS may combine with Stx2 to facilitate vascular injury, which may in turn lead to an overproduction of nitric oxide (NO). The aim of this study was to evaluate whether NO is involved in the effects of Stx2 on pregnancy. Pregnant rats were i.p. injected with culture supernatant from recombinant E. coli containing Stx2 and LPS (sStx2) on day 15 of gestation. In addition, some rats were injected with aminoguanidine (AG), an inducible isoform inhibitor of NO synthase (iNOS), 24 h before and 4 h after sStx2 injection. NO production was measured by NOS activity and iNOS expression by Western blot analysis. A significant increase in NO production and a high iNOS expression was observed in placental tissues from rats injected with sStx2 containing 0.7 ng and 2 ng Stx2/g body weight and killed 12 h after injection. AG caused a significant reduction of sStx2 effects on the feto-maternal unit, but did not prevent premature delivery. Placental tissues from rats treated with AG and sStx2 presented normal histology that was indistinguishable from the controls. Our results reveal that Stx2-induced placental damage and fetus mortality is mediated by an increase in NO production and that AG is able to completely reverse the Stx2 damages in placental tissues, but not to prevent premature delivery, thus suggesting other mechanisms not yet determined could be involved.

Asunto(s)

Óxido Nítrico/química , Toxina Shiga II/metabolismo , Animales , Peso Corporal , Chlorocebus aethiops , Escherichia coli/metabolismo , Femenino , Lipopolisacáridos/metabolismo , Masculino , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Placenta/metabolismo , Embarazo , Isoformas de Proteínas , Ratas , Células Vero

RESUMEN

Pregnancy maintenance is a very complex phenomenon, and the mechanisms that allow the survival of the fetus within the maternal uterus are still poorly understood. Our objectives were to analyze heme oxygenase (HO) activity and expression in the pregnant rat and to study its association with steroid hormones and prostaglandins. Uterine tissues were obtained from non-pregnant and from time-mated rats at days 5, 13, 18-22 of pregnancy and postpartum. HO activity was significantly higher at days 5 and 20 while HO-1 protein levels measured by Western blot, were significantly elevated from days 19 to 22. In ovariectomized rats, estrogen and progesterone in estrogenized animals increased HO activity and expression. Cyclooxygenase inhibitors augmented HO activity and HO-1 expression. Pre-incubation with prostaglandin F2alpha (PGF2alpha) diminished the enzymatic activity in ovariectomized rat uterus. Tin protoporphyrin IX, an HO inhibitor, significantly decreased uterine cGMP accumulation. Bilirubin decreased uterine thiobarbituric acid substances levels (an index of lipid peroxidation). These results demonstrate a uterine gestational pattern of HO activity and expression in the rat. In addition, these results suggest that uterine HO activity could regulate uterine quiescence in pregnancy via cGMP and it may contribute to the defense against oxidative stress.

Asunto(s)

Monóxido de Carbono/metabolismo , Hormonas Esteroides Gonadales/fisiología , Hemo Oxigenasa (Desciclizante)/metabolismo , Prostaglandinas/fisiología , Útero/enzimología , Animales , GMP Cíclico/metabolismo , Femenino , Hemo-Oxigenasa 1/metabolismo , Estrés Oxidativo/fisiología , Embarazo , Ratas , Ratas Wistar

RESUMEN

The aim of our study was to investigate if the lipopolysaccharide (LPS) differentially modulates throughout time the nitric oxide synthase (NOS) and cyclooxygenase (COX) enzymes in the estrogenized rat uterus. To study the effect of LPS throughout time on nitric oxide and prostaglandins production and on NOS and COX expression in the estrogenized rat uterus, females received 5 mg/kg intraperitoneally (i.p.) of LPS and were sacrificed 0.5, 1, 2, 3, 4 and 5 h post-administration. NO production was measured by arginine-citrulline conversion assay and prostaglandin E2/prostaglandin F2alpha by radioconversion. Enzyme expression was evaluated by Western blot analysis. The present work shows that LPS augmented NOS activity 3 h post-treatment and iNOS expression earlier, 2 h post-administration. On the other hand, the administration of LPS stimulated the production of prostaglandin E2/prostaglandin F2alpha and augmented the expression of COX-I 1 h after the treatment and of COX-II 2 h post-treatment. Meloxicam, a COX-II inhibitor, stimulated NO production in a group of rats injected i.p. with both LPS and the inhibitor and sacrificed 2 h after the treatment. These results indicate that, in the estrogenized rat uterus challenged with LPS, the early stimulation in the production of prostaglandins inhibited NOS activity, until the expression of the NOS isoforms is sufficient to overpass the inhibitory effect of the prostaglandins. The above findings suggest that the interaction between NOS and COX might be important in the regulation of physiopathologic events during pregnancy.

Asunto(s)

Dinoprost/metabolismo , Dinoprostona/metabolismo , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Útero/enzimología , Animales , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Estrógenos , Femenino , Meloxicam , Modelos Animales , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Wistar , Tiazinas/farmacología , Tiazoles/farmacología , Factores de Tiempo , Útero/efectos de los fármacos

RESUMEN

BACKGROUND/OBJECTIVE: The aim of our study was first to investigate if there exists an interaction between nitric oxide (NO) and prostaglandin (PG) generation in the estrogenized rat uterus challenged by lipopolysaccharide (LPS), and, secondly, which isoforms of nitric oxide synthase (NOS) and cyclooxygenase (COX) participate in this process. METHODS: To study the effect of LPS and to characterize the isoenzymes involved in the process, specific inhibitors of iNOS (aminoguanidine) and COX-II (meloxicam, nimesulide) and non-specific of COX (indomethacin) were injected intraperitoneally to determine their effect on NO and PG production, and on NOS and COX expression induced by LPS in estrogenized rat uterus. NO production was measured by arginine-citrulline conversion assay and PGE(2)/PGF(2alpha,)by radioconversion. Enzyme expression was evaluated by Western blot analysis. RESULTS: The present work shows that iNOS inhibitor, aminoguanidine, reduced NO and PGE(2)/PGF(2alpha) production induced by LPS injection. Aminoguanidine exerts its effect over the PG metabolism by inhibiting COX-II activity and expression. On the other hand, both indomethacin, a non-selective PG inhibitor, and meloxicam, a COX-II inhibitor, stimulated NO production and reduced PGE(2)/PGF(2alpha) generation. Indomethacin also reduced COX-II and iNOS expression. CONCLUSION: These results indicate that in the estrogenized rat uterus challenged with LPS, PG and NO interact affecting each other's metabolic pathways. The above findings indicate that the interaction between NOS and COX might be important in the regulation of physiopathologic events during pregnancy.

Asunto(s)

Isoenzimas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/biosíntesis , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/biosíntesis , Útero/efectos de los fármacos , Útero/enzimología , Animales , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprost/biosíntesis , Dinoprostona/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Pérdida del Embrión/enzimología , Pérdida del Embrión/fisiopatología , Estrógenos/metabolismo , Estrógenos/farmacología , Femenino , Guanidinas/farmacología , Indometacina/farmacología , Inflamación/inducido químicamente , Inflamación/enzimología , Inflamación/fisiopatología , Isoenzimas/antagonistas & inhibidores , Lipopolisacáridos , Meloxicam , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Embarazo , Ratas , Ratas Wistar , Sulfonamidas/farmacología , Tiazinas/farmacología , Tiazoles/farmacología , Útero/fisiopatología

RESUMEN

OBJECTIVES: Recent reports point to a role for the nitric oxide/nitric oxide synthase (NO/NOS) system in implantation. It has been suggested that inducible NOS expressed at peri-implantation would lead to enhanced NO production, which could promote the attachment of the blastocyst. Short-term administration of NO donors during the pre-implantation period reduced the pregnancy rate in a dose-dependent manner. Thus, it is thought that optimal levels of NO are critical for embryo implantation, so regulation of NOS must be crucial. Taking this into consideration, interleukin-10 (IL-10), synthesized and secreted by the embryo, could be modulating NOS during implantation. In this study we have investigated the in vitro effect of IL-10 on NOS in the uterus. METHODS: To determine the effect of IL-10, slices of uterus from estrogenized mice were pre-incubated for 60 min with different concentrations of IL-10 and NOS activity was measured. RESULTS: IL-10 (50 and 100 ng/ml in vitro) diminished NOS activity. The in vivo administration of lipopolysaccharide (LPS; 8 mg/kg) significantly increased the conversion of arginine into citrulline. This effect was abolished after 60 min of preincubation with IL-10 (100 ng/ml). The stimulatory effect of LPS and estrogen on NOS activity is exerted on the Ca-independent isoform and IL-10 in vitro abolished this increase. We observed that the uterus of pregnant mice on day 5 of gestation synthesized NO. This production was significantly inhibited by preincubation with IL-10 (100 ng/ml). CONCLUSIONS: This report demonstrates that IL-10 is capable of inhibiting NO synthesis in estrogenized, LPS-treated and pregnant rat uterus.

Asunto(s)

Interleucina-10/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico/biosíntesis , Útero/enzimología , Animales , Arginina/metabolismo , Citrulina/biosíntesis , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Estrógenos/metabolismo , Estrógenos/farmacología , Femenino , Interleucina-10/inmunología , Interleucina-10/farmacología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa/efectos de los fármacos , Embarazo , Útero/efectos de los fármacos , Útero/inmunología

RESUMEN

Repeated restraint stress (RRS) in male rats activated the pituitary adrenal system, as indicated by increases in adrenal weight and plasma corticosterone concentration that were accompanied by a decrease in constitutive nitric oxide synthase (cNOS), but not inducible NOS (iNOS). iNOS activated cyclooxgenase, causing elevated prostaglandin E(2) (PGE(2)) and F(2 alpha) in the adrenals, but had no effect on lipoxygenase. Administration of ethanol (ETOH) was also associated with elevated adrenal weight and a slight increase in corticosterone coupled with a decrease in both cNOS and iNOS and PGs in the adrenal. When ETOH was administered together with RRS, a decrease in iNOS and PGE release was noted consequent to a reduction in iNOS. Thus, ETOH probably reduced RRS-induced adrenocorticotropic hormone release. Adrenals were incubated in vitro to further evaluate the role of NO in these processes. Results indicated that NO released by sodium nitroprusside increased corticosterone release presumably by activating guanylyl cyclase with production of cyclic guanosine monophosphate (cGMP), because although NO also increased PGE release, PGE(2) (10(-5)-10(-9) M) decreased corticosterone release, an effect that was highly significant at a concentration of 10(-7) M PGE(2). ETOH (100 mM) had no effect on corticosterone release and did not block the increase in corticosterone caused by NO; however, ETOH reduced PGE release into the medium and blocked PGE(2) release induced by NO. Consequently, NO activated corticosterone release not by PGs, but by activation of guanylyl cyclase and release of cGMP. PGs have a negative feedback to suppress corticosterone release.

Asunto(s)

Etanol/farmacología , Óxido Nítrico Sintasa/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Estrés Psicológico/fisiopatología , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Animales , Corticosterona/metabolismo , Eicosanoides/metabolismo , Masculino , Óxido Nítrico Sintasa/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Restricción Física