RESUMEN
Regulation of myometrial functions during gestation, labor and birth are in the forefront of research in reproductive sciences. The complexity of the problem is reflected by our scant understanding of the intimate cellular and molecular events underlying these phenomena, despite extensive efforts spanning several decades. Unlike other smooth muscles, the myometrium is, to a large extent, under hormonal control. Of these, the steroid hormones, progesterone and estrogen, play dominant roles in terms of uterine growth, the maintenance of quiescence during gestation and the preparation of the uterus for labor and delivery. In addition to steroid hormones, there are a number of factors that modulate myometrial contractility (oxytocin, prostaglandins, endothelin, platelet activating factor) and relaxation (corticotropin releasing hormone, prostacyclin, nitric oxide). Although notable advances have been made towards understanding some of the key steps in receptor signaling that define the actions of these factors, a good deal of new information is needed to fully understand this fundamental life process. Pharmaceutical agents have been used extensively to induce labor or to prolong pregnancy in the case of preterm labor that represents the major cause of perinatal morbidity and mortality. Because preterm labor is a syndrome of multiple etiologies, pharmacologic agents will have to be targeted accordingly. This review attempts to present a critical overview of these topics.
Asunto(s)
Miometrio/citología , Miometrio/fisiología , Animales , Estrógenos/farmacología , Estrógenos/fisiología , Femenino , Humanos , Miometrio/efectos de los fármacos , Parto/efectos de los fármacos , Parto/fisiología , Embarazo , Mantenimiento del Embarazo/efectos de los fármacos , Mantenimiento del Embarazo/fisiología , Progesterona/farmacología , Progesterona/fisiologíaRESUMEN
Prostaglandins are important regulators of many aspects of reproductive processes from ovulation, fertilization and pregnancy recognition to labor and parturition. These biologically potent compounds are members of the large family of eicosanoids, derived from polyunsaturated fatty acids, principally arachidonic acid, found in the membrane phospholipids of virtually every cell of the human body, accounting for the ubiquity of prostaglandins, which act in a paracrine or autocrine fashion via discrete receptors. The availability of specific prostaglandins in various cells and tissues depends on the presence and activity of specific enzymes that convert a common precursor to the end product, as well as on the rate of enzymatic or spontaneous inactivation of the bioactive compounds. Here we offer a brief review of the regulation of prostaglandin generation in human uterine tissues, focusing on their role in labor and parturition at term and preterm.
Asunto(s)
Homeostasis , Prostaglandinas/biosíntesis , Útero/metabolismo , Ácido Araquidónico/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Trabajo de Parto/fisiología , Trabajo de Parto Prematuro , Fosfolipasas/metabolismo , Embarazo , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/fisiologíaRESUMEN
PROBLEM: The objective of this study was to evaluate the possible role of pertussis toxin (PTX)-sensitive G-protein(s) in interleukin-1beta (IL-1) signaling in human myometrial cells (HMC). METHOD: Primary cultures of HMC were stimulated with human recombinant IL-1 alone or in combination with PTX. Prostaglandin (PG) E2 in the medium was measured by radioimmunoassay, cyclooxygenase type 2 (Cox-2) and IkappaB by western analysis, and the activities of two members of the mitogen-activated protein kinase (MAPK) family of enzymes, ERK-2 and JNK, by the phosphorylation of appropriate substrates. RESULTS: IL-1 increased PGE2 output during an 18-hr long incubation by 21.7-fold (n = 5 experiments). This increase was inhibited by 57% after pretreatment overnight with PTX. IL-1-induced expression of Cox-2 protein was also suppressed to a similar degree in PTX-treated HMC cultures. Degradation of the nuclear factor kappa B (NF-kappaB)-inhibiting protein (IkappaB), a critical step in IL-1 signaling to the nucleus, was significantly inhibited by PTX, as was IL-1-induced activation of ERK-2 and JNK. CONCLUSIONS: It is suggested that the occupied IL-1 receptor-generated signal in HMC is transmitted by multiple pathways. One is coupled to a PTX-sensitive G-protein upstream from the MAPK phosphorylation cascade. This, in turn, may interact with another signaling pathway, the activation of NF-kappaB, via the phosphorylation of the IkappaB kinase complex.
Asunto(s)
Dinoprostona/biosíntesis , Interleucina-1/farmacología , Miometrio/efectos de los fármacos , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacología , Células Cultivadas , Femenino , Humanos , Modelos Biológicos , Miometrio/citología , Premenopausia , Transducción de SeñalRESUMEN
OBJECTIVE: Up-regulation of prostaglandin production by gestational tissues in the setting of intrauterine infection has been implicated as an important contributor to preterm labor and parturition. In this study we investigated the possible role of the nuclear transcription factor NF-kappaB in interleukin-1 signaling, leading to the expression of cyclooxygenase 2 and prostaglandin production in human myometrial cell cultures. STUDY DESIGN: Human myometrial smooth muscle cells from an immortalized line were used as a model system between passages 20 and 35. Growth-arrested cell cultures were stimulated with human recombinant interleukin 1, and the activation of NF-kappaB was assessed by the degradation of the inhibitory protein IkappaB-alpha (Western analysis), as well as by nuclear binding of NF-kappaB by using an electrophoretic mobility shift assay. The abundance of cyclooxygenase-2 messenger ribonucleic acid and protein was measured by Northern and Western analyses, whereas prostaglandin (prostaglandin I(2 ) and prostaglandin E(2 )) production was determined by specific radioimmunoassays. RESULTS: Within 15 minutes of stimulation with interleukin 1, 90% of IkappaB-alpha was degraded. This was temporally associated with nuclear translocation and binding of NF-kappaB. Within 30 minutes, cyclooxygenase 2 messenger ribonucleic acid appeared, with steady-state levels increasing up to 4 hours. This was followed by an up to 80-fold increase in cyclooxygenase 2 protein and a corresponding time-dependent increase in prostaglandin production. When IkappaB-alpha degradation was blocked with calpain I inhibitor, NF-kappaB translocation, cyclooxygenase 2 messenger ribonucleic acid and protein expression, and prostaglandin synthesis were also inhibited. CONCLUSION: Stimulation of human myometrial cells with interleukin 1 leads to rapid activation of the transcription factor NF-kappaB, which is functionally linked to the expression of cyclooxygenase 2 messenger ribonucleic acid, protein, and prostaglandin synthesis.
Asunto(s)
Expresión Génica , Interleucina-1/farmacología , Isoenzimas/genética , Miometrio/enzimología , FN-kappa B/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Northern Blotting , Western Blotting , Línea Celular Transformada , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Epoprostenol/biosíntesis , Femenino , Humanos , Cinética , Proteínas de la Membrana , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
OBJECTIVE: The objective of our study was to test the hypothesis that oxytocin promotes prostaglandin production by up-regulating cyclooxygenase-2 via activation of mitogen-activated protein kinase cascade in human myometrial cells. STUDY DESIGN: Confluent cultures of human myometrial cells obtained from uterine specimens of premenopausal women undergoing hysterectomy were serum starved for 48 hours before oxytocin stimulation. Prostacyclin levels, as 6-keto-prostaglandin F(1) (alpha), were measured by radioimmunoassay, and the cellular cyclooxygenase-2 protein content was determined by Western blot. Mitogen-activated protein kinase activity was assessed by measuring the phosphorylation of myelin basic protein. RESULTS: In a time- and dose-dependent manner oxytocin promoted prostacyclin production in human myometrial cells. Maximal responses were observed after 8 hours of stimulation at a dose of 100 nmol/L. This effect was mainly due to the expression of cyclooxygenase-2 protein. Within 5 minutes oxytocin significantly stimulated mitogen-activated protein kinase, as compared with the expression in untreated controls. The maximal increase in enzyme activity (2.5-fold) was obtained at 45 minutes. A selective inhibitor of mitogen-activated protein kinase activation (PD98059), as well as herbimycin, a tyrosine kinase inhibitor, and the transcriptional blocker actinomycin D, suppressed oxytocin-induced cyclooxygenase-2 expression and prostacyclin production. The stimulatory action of oxytocin was also sensitive to inhibition by pertussis toxin but appeared to be independent of protein kinase C activation. CONCLUSION: Our data indicate a largely unrecognized signal transduction mechanism for oxytocin, involving G-protein-coupled activation of mitogen-activated protein kinase and cyclooxygenase-2 gene expression, leading to increased prostaglandin production in human myometrial cells. This signaling pathway complements the rapid activation of the phosphoinositide cycle and may be responsible for sustained release of prostaglandins in uterine tissues, promoting labor and parturition.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Isoenzimas/metabolismo , Miometrio/efectos de los fármacos , Oxitocina/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/metabolismo , Transducción de Señal , Western Blotting , Línea Celular , Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Proteínas de la Membrana , Miometrio/enzimología , Premenopausia , Radioinmunoensayo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Bacterial products are thought to induce labor by stimulating the production of pro-inflammatory cytokines and prostaglandins in gestational tissues, leading to the onset of preterm parturition. Progesterone withdrawal is a prerequisite of parturition in many species. Yet a role for progesterone in the mechanisms responsible for preterm parturition, in the setting of infection, is unclear. The current studies were conducted to determine if a fall in serum progesterone concentrations occurs before the onset of bacterial product-induced preterm parturition in animals. Accordingly, pregnant mice at day 15 (70% gestation) were injected i.p. with Escherichia coli lipopolysaccharide (LPS; 50 microg/mouse) and timed-pregnant rabbits were inoculated transcervically with a suspension of E. coli to cause an ascending intrauterine infection. Control animals in both groups received equal volumes of sterile phosphate-buffered saline (PBS) solution. Blood specimens were collected at regular intervals and serum progesterone levels were determined by RIA. Within 14 h of LPS administration, mice delivered their pups. The median concentrations of serum progesterone were significantly lower at 1 h, 4 h, 10 h, and at the onset of preterm parturition (11-12 h) after LPS injection, compared to that in animals given PBS. Similarly, E. coli-inoculated rabbits delivered 1-2 days posttranscervical inoculation and demonstrated 60% decrease in serum progesterone within 12-24 h of inoculation compared to those given PBS. Parturition in both control groups occurred at term, following typical progesterone withdrawal. It is concluded that LPS administration to pregnant mice and ascending intrauterine infection in pregnant rabbits is associated with a dramatic fall in serum progesterone concentrations prior to the onset of parturition.
Asunto(s)
Infecciones por Escherichia coli , Lipopolisacáridos , Trabajo de Parto Prematuro/sangre , Trabajo de Parto Prematuro/microbiología , Progesterona/sangre , Animales , Escherichia coli , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Embarazo , Conejos , Enfermedades Uterinas/sangre , Enfermedades Uterinas/microbiologíaRESUMEN
There is growing evidence that proteinuric hypertension of pregnancy (preeclampsia) is associated with endothelial dysfunction. The aim of this study was to evaluate the effects of serum from preeclamptic patients on basal and agonist-stimulated prostacyclin production by human umbilical vein endothelial cells (HUVEC) in culture and to compare these to the effects of serum from normal pregnant and nonpregnant women. During a 24 h incubation of HUVEC with 20% of preeclampsia serum, baseline prostacyclin output was significantly (P < 0.01) increased over the control groups. However, this response was attenuated by extending the exposure to 72 h. Histamine, thrombin and the calcium ionophore, A23187, all acutely increased prostacyclin production, but the increase relative to baseline levels was greatest in HUVEC preincubated for 24 h in normal serum transiently promotes prostacyclin production in HUVEC derived from normal pregnancies, preeclampsia serum transiently promotes prostacyclin production in HUVEC derived from normal pregnancies, and 2) the relative increase in response to agonists is reduced by preeclampsia serum, compared to normal pregnancy sera.
Asunto(s)
Endotelio Vascular/metabolismo , Epoprostenol/biosíntesis , Preeclampsia/sangre , Calcimicina/farmacología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Femenino , Histamina/farmacología , Humanos , Embarazo , Trombina/farmacología , Venas UmbilicalesRESUMEN
The objective of this study was to characterize interleukin-1, -6, and -8 (IL-1-, IL-6-, and IL-8)-induced prostacyclin (PGI2 as 6-keto PGF1 alpha) and prostaglandin E2 (PGE2) production in primary cultures of human myometrial cells. Prostaglandins (PGs) released into the culture media were quantitated by specific radioimmunoassays. IL-1, but not IL-6 or IL-8, caused a dose- and time-dependent increase in the production of both PGI2 and PGE2. Half-maximally stimulating doses (EC50) of IL-1 were about 0.1 ng/ml, and maximal responses were observed at 1-10 ng/ml, amounting to 15- to 23-fold increases over unstimulated controls. The action of IL-1 was greatly potentiated by the protein kinase C-activating phorbol ester, TPA, and inhibited by actinomycin D and cycloheximide. IL-1-induced PG production was also suppressed by dexamethasone, by the natural IL-1 receptor antagonist (IL-1ra), and by transforming growth factor1 beta (TGF1 beta). It is concluded that IL-1 is a potent agonist of PG synthesis in human myometrial cells, acting by a mechanism dependent on the synthesis of new proteins, presumably key enzymes (phospholipase A2 and/or cyclo-oxygenase-2). This study has added further support to the notion that the myometrium serves as a target for the inflammatory cytokine, IL-1, and thereby may be affected directly, thus promoting preterm labor associated with intrauterine infection.
Asunto(s)
Dinoprostona/biosíntesis , Epoprostenol/biosíntesis , Interleucinas/farmacología , Miometrio/efectos de los fármacos , 6-Cetoprostaglandina F1 alfa/biosíntesis , Análisis de Varianza , Células Cultivadas , Cicloheximida/farmacología , Dactinomicina/farmacología , Sinergismo Farmacológico , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/farmacología , Interleucina-6/farmacología , Interleucina-8/farmacología , Miometrio/citología , Miometrio/metabolismo , Radioinmunoensayo , Proteínas Recombinantes/farmacología , Sialoglicoproteínas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Homogenized first trimester human placenta exhibits both Ca(2+)-dependent (90-95 per cent) and Ca(2+)-independent (5-10 per cent) nitric oxide (NO)-synthesizing activities. Addition of tetrahydrobiopterin (BH4) to homogenates containing Ca2+ in maximally activating concentrations (> 0.5 microM) results in a further 2-2.5-fold activation of NO synthesis, with half-maximal stimulation observed at 26 +/- 8.2 microM BH4 (mean +/- SEM, n = 4). Chelation of Ca2+ in the medium abolishes the stimulatory effect, indicating that only a Ca2(+)-dependent NO-synthase (NOS) isoform is activated by BH4. Based on our previous findings, we suggest that this isoform is the endothelial or Type III NOS. Importantly, BH4 has no significant effect on the Ca2(+)-dependency of NOS activity, the apparent Km values for Ca2+ are comparable in the absence (1.8 +/- 0.4 microM, mean +/- SEM, n = 6) or presence (2.5 +/- 0.6 microM, mean +/- SEM, n = 6) of 50 microM BH4. The BH4 content of these placentae is 207.4 +/- 86.7 pmol/g wet tissue (mean +/- s.d., n = 9), therefore, BH4 added to the homogenate does not simply restore the concentrations that occur endogenously. The results provide the first evidence that in the early human placenta, a constitutively expressed CA 2(+)-dependent NOS isoform is stimulated by exogenous BH4, raising the possibility that BH4 is an important regulator of NOS activity in this tissue. This novel aspect of the NO-generating pathway may have implications in the aetiology and treatment of pregnancy-induced hypertension and pre-eclampsia.
Asunto(s)
Biopterinas/análogos & derivados , Calcio/farmacología , Óxido Nítrico/biosíntesis , Placenta/efectos de los fármacos , Placenta/metabolismo , Arginina/metabolismo , Biopterinas/farmacología , Citrulina/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , NADP/farmacología , Óxido Nítrico Sintasa/metabolismo , EmbarazoRESUMEN
Calcium plays a pivotal role in the contraction-relaxation cycle of uterine smooth muscle. We investigated the effects of three uterine agonists; prostaglandin F2 alpha (PGF2 alpha), oxytocin and platelet activating factor (PAF) on intracellular levels of Ca ([Ca2+]i) in intact human myometrial cells and 45Ca2+ efflux from permeabilized myocytes. We observed that, whereas oxytocin and PAF activated the phosphoinositide cycle, generating inositol triphosphate to mobilize intracellular Ca2+, PGF2 alpha acted mainly to enhance Ca2+ influx. Oxytocin-, but not PGF2 alpha-elicited responses were suppressed by pertussis toxin. It is concluded that all three agonists act by regulating [Ca2+]i in myometrial cells, but the signal transduction mechanism of PGF2 alpha differs from that of oxytocin and PAF.
Asunto(s)
Calcio/metabolismo , Miometrio/fisiología , Transducción de Señal , Permeabilidad de la Membrana Celular , Células Cultivadas , Dinoprost/farmacología , Femenino , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Miometrio/efectos de los fármacos , Oxitocina/farmacología , Fosfatidilinositoles/metabolismo , Factor de Activación Plaquetaria/farmacologíaRESUMEN
The objectives of this study were to evaluate and compare the actions of endothelin-1 (ET-1), oxytocin, prostaglandin F2 alpha (PGF2 alpha) and inositol 1,4,5-trisphosphate (IP3) on 45Ca2+ mobilization in permeabilized rat myometrial cells and to examine the activation of the inositol lipid cycle in intact myocytes. Cells were isolated from late pregnant rat myometrium and used as confluent monolayers after a single passage. All four agonists caused a biphasic release of 45Ca2+ from non-mitochondrial pool(s), with the rank order of potency: oxytocin > PGF2 alpha > ET-1 > IP3. Inhibitors of phospholipase C blocked ET-1- and oxytocin-promoted but not PGF2 alpha-promoted 45Ca2+ efflux. Similarly, heparin, an IP3 receptor blocker, failed to inhibit PGF2 alpha-induced 45Ca2+ release while inhibiting the action of the other agonists. Endothelin-1 and oxytocin stimulated inositol phosphate accumulation at concentrations similar to those that promoted 45Ca2+ efflux, whereas about 100 times higher concentrations of PGF2 alpha were needed to activate this signaling pathway in intact cells. It is concluded that the primary action of PGF2 alpha in myometrial cells is to enhance Ca2+ influx, whereas oxytocin and ET-1 receptors are coupled to phospholipase C, generating IP3 and raising the intracellular concentration of free Ca2+ from intracellular as well as extracellular sources.
Asunto(s)
Dinoprost/farmacología , Endotelinas/farmacología , Miometrio/metabolismo , Oxitocina/farmacología , Transducción de Señal/efectos de los fármacos , Adenosina Trifosfato/farmacología , Animales , Calcimicina/farmacología , Calcio/metabolismo , Radioisótopos de Calcio , Activación Enzimática/efectos de los fármacos , Femenino , Inositol 1,4,5-Trifosfato/farmacología , Fosfatos de Inositol/metabolismo , Miometrio/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Rojo de Rutenio/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
The objective of this study was to test the hypothesis that, in human myometrial cells (HMC), PGF2 alpha and oxytocin promote the release of arachidonic acid (AA) which, in turn, acts to mobilize intracellular Ca2+. Primary monolayer cultures of HMC were labeled with [3H]arachidonic acid ([3H]AA) to isotopic equilibrium before exposure to PGF2 alpha or oxytocin. Radiolabeled phospholipids were separated on thin layer chromatography and quantitated by scintillation counting. Prostanoids were analyzed by high performance liquid chromatography. Calcium release was quantitated in digitonin-permeabilized myocytes preloaded with 45Ca, in the presence of ATP and ruthenium red. PGF2 alpha (10(-7) M) caused a rapid (peaking at 2 min), and significant (P < 0.01) increase in [3H]AA release that was derived selectively from phosphatidylethanolamine (PE), indicative of phospholipase A2 activation. Oxytocin caused a rapid (30 s) and significant increase in diacylglycerol, concomitant with a drop in phosphoinositides, as well as an increase in [3H]AA and a fall in PE and phosphatidylcholine. Exogenous AA caused a rapid and dose-related efflux of 45Ca2+, which was not inhibited by blockers of AA metabolism, or by heparin that abolished inositol 1,4,5-trisphosphate-induced 45Ca2+ release. It is concluded that PGF2 alpha and oxytocin promote, by different mechanisms, the release of AA, which in turn may amplify their action by enhancing Ca2+ mobilization from the sarcoplasmic reticulum, thereby fulfilling the role of intracellular signaling molecule in human myometrium.
Asunto(s)
Ácido Araquidónico/fisiología , Miometrio/metabolismo , Transducción de Señal , Ácido Araquidónico/farmacología , Calcio/metabolismo , Radioisótopos de Calcio , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dinoprost/farmacología , Femenino , Humanos , Oxitocina/farmacología , Fosfatidiletanolaminas/metabolismo , TritioRESUMEN
OBJECTIVE: Our purpose was to test the hypothesis that chronic inhibition of nitric oxide synthesis in pregnant rats can produce a preeclampsia-like syndrome. STUDY DESIGN: Pregnant rats were instrumented on day 14 of gestation (parturition day 21 to 22) and infused continuously through a venous catheter with L-nitro-arginine, a potent inhibitor of nitric oxide synthase, or with sterile saline solution from day 18 until 24 hours post partum. A group of virgin rats was treated identically. Blood pressure was recorded in unrestrained animals with an aortic catheter for 30 minutes before infusion and repeated each day throughout the experiment. Urinary albumin, platelet count, weight of newborn pups, blood chemistry, and several other parameters were determined. Data were analyzed by one-way, repeated-measures analysis of variance, with Dunnett's t test or by Student t test. RESULTS: Mean arterial pressure increased from 102.6 +/- 2.8 to a mean maximum of 152.5 +/- 7.3 on the second day of infusion and remained in this range until delivery, after which it fell significantly, in spite of continuing infusion of L-nitro-arginine. This treatment increased urinary albumin (milligrams per 24 hours) from 8.3 +/- 1.5 to 56.3 +/- 14.3 in gravid and from 8.2 +/- 0.8 to 18.2 +/- 2.4 in virgin rats. Weight of newborn pups was reduced by L-nitro-arginine from 5.62 +/- 0.10 to 3.37 +/- 0.32 gm (p < 0.005) without affecting time of delivery or litter size. Platelet count was reduced 58% in gravid and 50% in virgin rats. CONCLUSION: Chronic inhibition of nitric oxide synthesis in gravid rats leads to sustained hypertension, proteinuria, thrombocytopenia, and intrauterine growth retardation, providing a simple animal model for preeclampsia.
Asunto(s)
Retardo del Crecimiento Fetal/etiología , Hipertensión/etiología , Óxido Nítrico/antagonistas & inhibidores , Complicaciones del Embarazo/etiología , Proteinuria/etiología , Trombocitopenia/etiología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Creatinina/orina , Femenino , Hematócrito , NG-Nitroarginina Metil Éster , Recuento de Plaquetas/efectos de los fármacos , Potasio/orina , Embarazo , Ratas , Ratas Wistar , Sodio/orina , gamma-Glutamiltransferasa/orinaRESUMEN
We evaluated the effects of two protein kinase C (PKC) inhibitors, staurosporine (ST) and H-7, on LH-activated phospholipase C and adenylate cyclase activity by measuring the production of inositol phosphates (IP) and cAMP in freshly dispersed granulosa cells from mature preovulatory follicles of laying hens. ST and H-7 dose-dependently potentiated LH-stimulated IP generation, whereas a protein kinase A (PKA) inhibitor (H-8) had no effect. The PKC activator, phorbol ester TPA (50 nM), significantly inhibited LH-stimulated IP production, which was completely prevented by ST. Both ST and H-7, while having no effect on basal cAMP levels, significantly and dose-dependently potentiated LH-stimulated, but not forskolin-stimulated cAMP production. However, progesterone production in response to LH, forskolin, and 8-Br-cAMP was inhibited in granulosa cells preincubated for 30 min with H-7 or ST. H-7 and ST had no effect on 25-hydroxycholesterol- and pregnenolone-supported progesterone production. These results support a negative feedback role for PKC in LH-initiated signal transduction in avian granulosa cells. PKC blockade removes the inhibitory effect on LH-stimulated phospholipase C and adenylate cyclase activity. The inhibitory effect of H-7 and ST on progesterone synthesis could be attributed to inhibition of PKA and/or steps proximal to cholesterol side-chain cleavage.
Asunto(s)
Pollos/metabolismo , Células de la Granulosa/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Alcaloides/farmacología , Animales , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Sinergismo Farmacológico , Femenino , Fosfatos de Inositol/metabolismo , Isoquinolinas/farmacología , Piperazinas/farmacología , Progesterona/biosíntesis , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The aim of this study was to produce viable chicks by in vitro fertilization and transfer of fertilized ova to the oviduct of recipient hens. Out of a total of 76 transferred ova, 53 were laid with fully calcified shells, 31 of which were fertile (58%). Despite the high rate of embryonic loss, six live chicks were hatched from 12 fertile ova exposed to 0.05 ml of semen (1:200 dilution). Nine healthy chicks were hatched from ten control ova which were recovered from the oviduct following artificial insemination and subsequent transfer to recipient hens. This experimental approach provides a useful model for production of transgenic chicks.
Asunto(s)
Pollos/fisiología , Transferencia de Embrión , Fertilización In Vitro/métodos , Animales , Animales Modificados Genéticamente , FemeninoRESUMEN
OBJECTIVE: Our aim was to evaluate the effects of the cytokines interleukin-1 and tumor necrosis factor on arachidonic acid release in human myometrial cells. STUDY DESIGN: Primary monolayer cultures of human myometrial cells prelabeled with tritiated arachidonic acid were exposed to interleukin-1 or tumor necrosis factor for varying periods and the release of tritiated arachidonic acid and its loss from phospholipids were measured by radiochromatography. To gain some information on the biologic action of interleukin-1 the contractile response to oxytocin was measured in myometrial strips preincubated with this cytokine. Data were statistically evaluated with analysis of variance or Student's test. RESULTS: Both cytokines caused a dose-dependent increase in tritiated arachidonate release that was suppressed by the protein synthesis inhibitor cycloheximide. Tritiated arachidonic acid release was maximal after 24 hours of stimulation with interleukin-1. Both interleukin-1 and tumor necrosis factor stimulated the release of the isotopically labeled fatty acid from phosphatidylcholine. In addition, interleukin-1 also increased the loss of arachidonic acid from phosphatidic acid and significantly potentiated the oxytocin-evoked myometrial contractility. CONCLUSIONS: Both interleukin-1 and tumor necrosis factor enhance arachidonic acid release, probably by inducing the synthesis of phospholipase A2 and possibly other enzymes involved in the metabolism of phospholipids. In turn, arachidonic acid itself may act as a second messenger, synergizing with other uterotonic agents, as well as serving as the precursor for prostaglandins and various other bioactive eicosanoids.
Asunto(s)
Ácido Araquidónico/biosíntesis , Interleucina-1/fisiología , Miometrio/metabolismo , Fosfolípidos/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Análisis de Varianza , Animales , Células Cultivadas , Cicloheximida/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Miometrio/citología , Oxitocina/farmacología , Embarazo , Premenopausia , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Contracción Uterina/efectos de los fármacosRESUMEN
PROBLEM: The objectives of this study were to evaluate interleukin-1 (IL-1) binding and some postreceptor actions of this cytokine and tumor necrosis factor (TNF) in human myometrial cells (HMC). METHOD: Monolayer cultures of HMC were used to characterize binding and to measure cyclic (c)AMP, prostaglandin (PG)E2, and PGI2 production. Membrane preparations were used to assess ADP-ribosylation and for immunoblotting. RESULTS: HMC were found to specifically bind [125I]IL-1 with an apparent Kd of 2 x 10(-10) M. Incubation of HMC with IL-1 or TNF caused a time-dependent and dose-dependent accumulation of cAMP, as well as a significant potentiation of forskolin-promoted cAMP production. These cytokines also increase PGE2 and PGI2 output, independently of the activation of adenylyl cyclase. IL-1 treatment had no measurable effect either on cholera toxin-mediated and pertussis toxin-mediated ADP-ribosylation, or on the amount of Gi proteins, as assessed by immunoblotting using a polyclonal antibody. CONCLUSIONS: It is suggested that IL-1 and TNF may activate one or more isoforms of the catalytic component of adenylyl cyclase, raising intracellular cAMP.
Asunto(s)
Interleucina-1/metabolismo , Miometrio/fisiología , Transducción de Señal/fisiología , Adenosina Difosfato Ribosa/metabolismo , Adenilil Ciclasas/metabolismo , Células Cultivadas , AMP Cíclico/biosíntesis , Dinoprostona/biosíntesis , Epoprostenol/biosíntesis , Femenino , Humanos , Immunoblotting , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
OBJECTIVE: With N omega-nitro-L-arginine, a potent inhibitor of nitric oxide synthesis, we tested the hypothesis that nitric oxide plays a functional role in the blunted pressor responsiveness seen during pregnancy. STUDY DESIGN: A group of six pregnant rats were instrumented on the fourteenth day of gestation and studied on days 19 and 20, as well as 7 days post partum. Another group of six virgin rats were similarly prepared and used 5 days after surgery. Blood pressure and heart rate were monitored in conscious freely moving animals before and during the administration of drugs or placebo. Results were analyzed, by one-way repeated-measures analysis of variance, with Dunnett's t test, or by paired t test where applicable. RESULTS: Basal mean arterial pressure and heart rate were 90.8 +/- 3.0 mm Hg and 330 +/- 6 beats/min in pregnant animals and 107.1 +/- 3.2 mm Hg and 315 +/- 7 beats/min in nonpregnant animals. Pressor responses to angiotensin II, vasopressin, and norepinephrine were attenuated in gravid animals. Infusion of N omega-nitro-L-arginine significantly and in a dose-dependent manner increased mean arterial pressure and reduced heart rate. These effects could be completely reversed by L-arginine administration. Changes in mean arterial pressure were higher during pregnancy as compared with postpartum values. N omega-nitro-L-arginine infusion potentiated pressor responses to all three vasopressors, resulting in dose-response curves that were significantly shifted to the left, making them virtually identical in pregnant and postpartum rats. CONCLUSION: Our data support the emerging view that nitric oxide plays a key role in the regulation of blood pressure during pregnancy.
Asunto(s)
Arginina/análogos & derivados , Presión Sanguínea/efectos de los fármacos , Óxido Nítrico/antagonistas & inhibidores , Preñez/fisiología , Vasoconstrictores/farmacología , Angiotensina II/farmacología , Animales , Arginina/farmacología , Sinergismo Farmacológico , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Nitroarginina , Norepinefrina/farmacología , Embarazo , Ratas , Ratas Endogámicas , Vasopresinas/farmacologíaRESUMEN
Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.
Asunto(s)
Calcio/farmacología , AMP Cíclico/metabolismo , Células de la Granulosa/metabolismo , Hormona Luteinizante/farmacología , Aves de Corral/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Calcimicina/farmacología , Calmodulina/farmacología , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Ionomicina/farmacología , Trifluoperazina/farmacologíaRESUMEN
When freshly dispersed granulosa cells from the largest preovulatory follicle were incubated in the presence of very low density (VLDL), low density (LDL), or high density (HDL) lipoproteins isolated from sera of laying hens, production of both basal and LH-stimulated progesterone was significantly increased in a dose-related manner. VLDL, the principal transporter of cholesterol to the ovum, appeared to be the most efficacious. A highly significant potentiation of the steroidogenic action of 8-bromo-cyclic adenosine monophosphate and forskolin was also observed. Human LDL, and especially HDL, caused significant stimulation of progesterone production by these cells. It is suggested that the release of cholesterol from lipoproteins taken up by granulosa cells raises the precursor pool for steroidogenesis. This mechanism is further enhanced by a cyclic AMP-mediated mechanism.