ABSTRACT
Hydrogen sulphide (H2S) has been recently hypothesized to be an endogenous adipocyte-derived relaxing factor, evoking vasorelaxation of conductance and resistance vessels. Although the activation of ATP-sensitive potassium channels is known to play a central role in H2S-induced vasorelaxation, activation of vascular Kv7 voltage-gated potassium channels has also been suggested. To investigate this possibility, the ability of selective activators and blockers of distinct classes of potassium channels to affect vasodilation induced by the H2S-donor NaHS, as well as NaHS-induced Rb(+) efflux in endothelium-denuded rat aortic rings, was investigated. NaHS-induced changes of membrane potential were fluorimetrically assessed on human vascular smooth muscle (VSM) cells. Modulation of Kv7.4 channels by NaHS was assessed by electrophysiological studies, upon their heterologous expression in CHO cells. In isolated aortic rings, NaHS evoked vasorelaxing responses associated with an increase of Rb(+)-efflux. NaHS promoted membrane hyperpolarization of human VSM cells. These effects were antagonized by selective blockers of Kv7 channels. The H2S-donor caused a left-shift of current activation threshold of Kv7.4 channels expressed in CHO cells. Altogether, these results suggest that the activation of Kv7.4 channels is a key mechanism in the vascular effects of H2S. Given the relevant roles played by Kv7.4 channels in VSM contractility and by H2S in circulatory homeostasis regulation, these findings provide interesting insights to improve our understanding of H2S pathophysiology and to focus on Kv7.4 channels as novel targets for therapeutic approaches via the "H2S-system".
Subject(s)
Aorta/drug effects , Hydrogen Sulfide/pharmacology , KCNQ Potassium Channels/metabolism , Muscle, Smooth, Vascular/drug effects , Sulfides/pharmacology , Vasodilation/drug effects , Animals , Aorta/metabolism , Cell Line , Data Interpretation, Statistical , Dose-Response Relationship, Drug , Endothelium, Vascular/physiology , Humans , In Vitro Techniques , KCNQ Potassium Channels/biosynthesis , Male , Membrane Potentials/drug effects , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Rats , Rats, Wistar , Vasoconstrictor Agents/pharmacologyABSTRACT
AIMS: To characterize the bacterial community of taberna, an alcoholic traditional beverage from the Southern part of Mexico produced by the fermentation of the coyol palm sap (Acrocomia aculeate). METHODS AND RESULTS: Bacterial 16S rDNA libraries were constructed from metagenomic DNA extracted during the fermentation process at 0, 60 and 108 h. A total of 154 clones were sequenced, and 13, 10 and nine unique sequences were found at each sampling time. At the onset of the fermentation, Zymomonas mobilis, Fructobacillus spp., Pantoea agglomerans and other Gammaproteobacteria were detected. After 60 h, lactic acid bacteria were found and 30% of clones in the library were related to Lactobacillus nagelii, L. sucicola and L. sp. By the end of the experiment, i.e. after 108 h, the bacterial community included Z. mobilis, Lact. nagelii and Acetobacter pasteurianus. CONCLUSIONS: Our results suggest that Z. mobilis population represented an important proportion of the bacterial community (60-80%), as well as the lactobacilli during the fermentation process. The bacterial diversity was low and decreased as the fermentation progressed. SIGNIFICANCE AND IMPACT OF THE STUDY: This culture-independent study suggests that Z. mobilis and lactobacilli play an important role in the alcoholic fermentation of the taberna beverage.
Subject(s)
Arecaceae/microbiology , Bacteria/isolation & purification , Beverages/microbiology , Biodiversity , Bacteria/classification , Bacteria/genetics , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Fermentation , Mexico , Molecular Sequence Data , PhylogenyABSTRACT
AIM: To compare in vivo the accuracy of two electronic apex locators (EALs) by means of digital radiographic imaging system. METHODOLOGY: Electronic working lengths of 831 canals were determined with the DentaPort ZX and Raypex 5 apex locators and confirmed radiographically. The radiographic images acquired with the aid of a digital radiographic imaging system (VisualiX eHD; Gendex Dental Systems, Des Plaines, IL, USA) were blindly analysed by two independent evaluators. The distance between the file tip and the radiographic apex was measured using dedicated software (VixWin Pro, Gendex Dental Systems, Des Plaines, IL, USA) and the mean distance achieved between different tooth type and EALs were compared statistically. Statistical analyses were performed using the t-test for independent samples and one-way anova with the null hypothesis set as 5%. Positive or negative values were recorded when the file tip was detected beyond or short of the radiographic apex, respectively. RESULTS: The mean distance between file tip and radiographic apex were -1.08 +/- 0.73 and -1.0 +/- 0.67 mm considering DentaPort ZX and Raypex 5 groups, respectively, with no significant differences (P > 0.05). No statistically significant differences were found amongst the same tooth type when comparing both groups (P > 0.05) or amongst different teeth type in the same group (P > 0.05). CONCLUSIONS: Within the limitations of this in vivo study, the DentaPort ZX and Raypex 5 were similar in terms of accuracy.
Subject(s)
Dental Pulp Cavity/anatomy & histology , Odontometry/instrumentation , Tooth Apex/anatomy & histology , Adult , Aged , Dental Pulp Cavity/diagnostic imaging , Electric Impedance , Electrical Equipment and Supplies , Humans , Middle Aged , Radiography, Dental, Digital , Root Canal Preparation/instrumentation , Tooth Apex/diagnostic imaging , Young AdultABSTRACT
Extracts of neem (Azadirachta indica A. Juss.) and Gliricidia sepium Jacquin, locally known as 'mata-raton', are used to control pests of maize. Their application, however, is known to affect soil microorganisms. We investigated if these extracts affected emissions of methane (CH4), carbon dioxide (CO2) and nitrous oxide (N2O), important greenhouse gases, and dynamics of soil inorganic N. Soil was treated with extracts of neem, mata-raton or lambda-cyhalothrin, used as chemical control. The soil was amended with or without urea and incubated at 40% and 100% water holding capacity (WHC). Concentrations of ammonium (NH4+), nitrite (NO2(-)) and nitrate (NO3(-)) and emissions of CH4, CO2 and N2O were monitored for 7d. Treating urea-amended soil with extracts of neem, mata-raton or lambda-cyhalothrin reduced the emission of CO2 significantly compared to the untreated soil with the largest decrease found in the latter. Oxidation of CH4 was inhibited by extracts of neem in the unamended soil, and by neem, mata-raton and lambda-cyhalothrin in the urea-amended soil compared to the untreated soil. Neem, mata-raton and lambda-cyhalothrin reduced the N2O emission from the unamended soil incubated at 40%WHC compared to the untreated soil. Extracts of neem, mata-raton and lambda-cyhalothrin had no significant effect on dynamics of NH4(+), NO2(-) and NO(3)(-). It was found that emission of CO2 and oxidation of CH4 was inhibited in the urea-amended soil treated with extracts of neem, mata-raton and lambda-cyhalothrin, but ammonification, N2O emission and nitrification were not affected.
Subject(s)
Azadirachta/chemistry , Fabaceae/chemistry , Gases , Nitrogen/metabolism , Plant Extracts/pharmacology , Soil , Urea/metabolism , Carbon Dioxide/chemistry , Carbon Dioxide/metabolism , Greenhouse Effect , Methane/chemistry , Methane/metabolism , Nitrates/chemistry , Nitrates/metabolism , Nitrites/chemistry , Nitrites/metabolism , Nitrogen/chemistry , Plant Extracts/chemistry , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/metabolism , Soil MicrobiologyABSTRACT
An orthogonal experimental design L9 (3(4)) with 10 repetitions was used to investigate the effect of Glomus claroideum (0, 1 or 2g(-1) plant), G. fasciculatum (0, 1 or 2g plant(-1)), native diazotrophic bacteria (0, 10(3) and 10(5) UFC ml(-1)) and sheep manure vermicompost (0%, 5% and 10% v/v) on maize plant growth, N and P in leaves and mycorrhization percent. Vermicompost explained most of the variation found for leaf number, wet weight, stem height, and diameter. Both mycorrhizas increased the plant wet weight but G. fasciculatum the most. Mycorrhization increased the P content, but not the N content. Mycorrhizal colonization increased when diazotrophic bacteria and vermicompost were added. It was found that weight of maize plants cultivated in peat moss amended with vermicompost increased when supplemented with G. fasciculatum and diazotrophic bacteria.
Subject(s)
Bacteria/metabolism , Manure/microbiology , Mycorrhizae/metabolism , Soil , Zea mays/growth & development , Animals , SheepSubject(s)
Nervous System Diseases/genetics , Receptors, Glycine/genetics , Reflex, Startle , Codon, Nonsense , Humans , Infant , Male , PedigreeABSTRACT
The survival rate, shoot and root dry mass, shout number, plant growth, stem height and diameter, number of leaves and root length were measured in micropropagated plantlets of Alpinia purpurata (Red ginger) inoculated with Azospirillum sp. 11B and Azotobacter sp. Pachaz 008 at 10(7), 10(8) and 10(9) cells cm(-3) using a complete randomized experimental design. Inoculation ofA. purpurata plantlets with the Azospirillum sp. 11B or Azotobacter sp. PACHAZ 008 strains induced larger stem diameter, root dry mass, number of shoots and increased their survival rate from 77 to 100% compared to plantlets without inoculation, while other plant characteristics were not affected.
Subject(s)
Alpinia/growth & development , Azospirillum/physiology , Azotobacter/physiology , Alpinia/microbiologyABSTRACT
OBJECTIVE: Calcitonin gene-related peptide (CGRP) circulates in maternal circulation throughout pregnancy, and specific receptors for CGRP (CGRPrs) are expressed by human myometrium. Because CGRP induces a dose-dependent relaxation of human myometrium, we examined a role for CGRP in modulation of myometrial smooth muscle contractility during pregnancy and labor. The aim of the present study was to evaluate the changes of maternal serum CGRP levels during parturition, according to the mode of delivery and in relation to cervical dilatation. METHODS: Circulating CGRP levels were measured in the following groups of healthy women: nonpregnant women, during the follicular phase of the menstrual cycle (n = 19); at term pregnancy (39-40 weeks; n = 24); after elective cesarean delivery (39-40 weeks; n = 20); and at spontaneous vaginal delivery (39-40 weeks; n = 16). In a subgroup of women, blood samples were collected longitudinally throughout labor at various cervical dilatations in the progress of labor (n = 8). RESULTS: Pregnant women at term not in labor had significantly higher CGRP levels than nonpregnant women (P =.021). No significant difference was found between women who delivered vaginally and those who had elective cesarean, and there were no correlations between CGRP plasma levels and cervical dilatation. CONCLUSIONS: Parturition is characterized by no significant changes in maternal serum CGRP levels, and no significant correlation exists between plasma CGRP levels and cervical dilatation during labor.
Subject(s)
Calcitonin Gene-Related Peptide/blood , Delivery, Obstetric/methods , Labor, Obstetric/physiology , Adult , Cesarean Section , Female , Follicular Phase , Gestational Age , Humans , Labor Stage, First , PregnancyABSTRACT
The gene expression and synthesis of both constitutive and inducible heme oxygenase (HO) isoforms have been recently described in human placental cells, but the functional role(s) of this biochemical pathway in placental physiology and pathology is still unclear. In the present study, we have investigated whether HO activity is involved in the control of CRH secretion from trophoblast cells. Fluctuations in HO activity were induced in primary cultures of human trophoblast cells using well-known activators and inhibitors of HO, and the subsequent changes in CRH secretion were monitored measuring CRH immunoreactivity released into the incubation medium. It was found that the increase in HO activity induced by hemin or cobalt chloride (CoCl(2)) was associated with parallel significant increases in CRH release. This effect was probably caused by the gaseous HO end-product, carbon monoxide (CO), because it was blocked by the HO inhibitor tin-mesoporphyrin-9, but it was not mimicked by stable HO end-products, biliverdin and bilirubin. We have also investigated whether stimulation of CRH release induced by HO was mediated by the cyclooxygenase (COX) pathway. Indeed, hemin also caused significant increases in PGE2 release in this experimental paradigm. However, CoCl(2), which also enhances CRH release, had no stimulatory effect and actually inhibited PG secretion; moreover, a nonselective COX inhibitor, indomethacin, failed to counteract hemininduced CRH release. Taken collectively, these findings suggested that modulation of CRH secretion by the HO-CO system occurs through a mechanism independent of COX activity.
Subject(s)
Carbon Monoxide/metabolism , Corticotropin-Releasing Hormone/metabolism , Heme Oxygenase (Decyclizing)/metabolism , Trophoblasts/metabolism , Cells, Cultured , Humans , Prostaglandin-Endoperoxide Synthases/physiology , Signal Transduction/physiologyABSTRACT
The coculture of endometrial epithelial cells (EEC) with stromal cells (ESC) allows achievement of an improved in vitro system for studying interactions between cells via soluble signals. The purpose of this study was to investigate whether 17beta-estradiol and insulin can induce proliferation of EEC through ESC-secreted factors. No evidence of estrogen-induced EEC proliferation has been reported so far in the conventional culture methods. To this end, we used an in vitro bicameral coculture model where human EEC were grown on extracellular matrix-coated inserts applied in dishes containing ESC. Proliferation was assessed by tritiated thymidine incorporation. Homogeneity of endometrial cell populations was ascertained immunocytochemically. 17beta-estradiol did not induce any proliferative effect on EEC cultured alone. Endometrial epithelial cell proliferation was significantly enhanced in EEC/ESC cocultures; moreover, it was further increased by 17beta-estradiol addition. Insulin increased proliferation in EEC cultured alone, but again the effect was more pronounced in EEC/ESC cocultures. Coincubation of 17beta-estradiol and an antibody against insulin-like growth factor I (IGF I) led to neutralization of ESC-mediated EEC proliferation. This work provides evidence that the effect of 17beta-estradiol on human EEC proliferation may be mediated at least in part through ESC-secreted IGF I. We also showed that insulin effect is also partially due to ESC activation.
Subject(s)
Cell Communication/physiology , Endometrium/physiology , Estradiol/pharmacology , Adult , Antibodies, Monoclonal , Biocompatible Materials/pharmacology , Cell Communication/drug effects , Cell Division/drug effects , Cell Division/physiology , Coculture Techniques , Collagen/pharmacology , Drug Combinations , Endometrium/cytology , Endometrium/drug effects , Endometrium/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/physiology , Female , Humans , Immunohistochemistry , Insulin/pharmacology , Insulin-Like Growth Factor I/metabolism , Laminin/pharmacology , Proteoglycans/pharmacology , Stromal Cells/cytology , Stromal Cells/drug effects , Stromal Cells/metabolism , Stromal Cells/physiology , Thymidine/metabolismABSTRACT
We have previously shown that endothelin-1 (ET-1) is normally found in human luteal cells, where it is able to significantly inhibit both basal and hCG-induced progesterone production. To further expand our comprehension of the possible roles of endothelins (ETs) in luteal physiology, in this study we used primary cultures of luteal cells exposed to graded doses of ET-1 and ET-3; PGF(2alpha) and PGE(2) were assayed in the culture medium to investigate whether ETs also influence cyclooxygenase activity in these cells. We found that both ETs are able to significantly stimulate PGF(2alpha) and PGE(2) release in a dose- and time-dependent manner. ET-1 was always more effective than ET-3. Experiments with two endothelin receptor antagonists (the BQ485 and BQ788 compounds, which block the ET-A and ET-B receptors, respectively) showed that the two endothelins induce PG production through different receptors and signaling pathways. In conclusion, here we demonstrate the ability of ETs to influence PG synthesis and release from human luteal cells. As PGs are deeply involved in corpus luteum activity, and ETs were also able to influence progesterone production, the present new data suggest an interesting interplay among progesterone, PGs, and ETs in the control of corpus luteum physiology.
Subject(s)
Corpus Luteum/physiology , Dinoprost/biosynthesis , Dinoprostone/biosynthesis , Endothelin-1/pharmacology , Endothelin-3/pharmacology , Antihypertensive Agents/pharmacology , Cells, Cultured , Chorionic Gonadotropin/pharmacology , Corpus Luteum/drug effects , Corpus Luteum/metabolism , Cyclic AMP/pharmacology , Dose-Response Relationship, Drug , Female , Humans , Kinetics , Oligopeptides/pharmacology , Piperidines/pharmacology , Progesterone/metabolism , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Preeclampsia is a mainly vascular disease of pregnancy, probably caused by an imbalance between vasodilator and vasoconstrictor agents that results in generalized vasospasm and poor perfusion in many organs. Among these factors, endothelin-1 (ET-1), a potent vasoconstrictor, is highly increased in preeclamptic women, while nitric oxide (NO), a vasodilator of human utero-placental arteries, is reduced in the same patients. The present study was designed to investigate the interactions between ET-1 and the NO system in the feto-placental unit; to this purpose we also examined the messenger ribonucleic acid (mRNA) expression of ET-1, inducible NO synthase (iNOS), and endothelial NOS (eNOS) in human cultured placental trophoblastic cells obtained from preeclamptic (PE) and normotensive (NT) pregnancies. We also studied whether exogenous ET-1 may affect the expression of iNOS and eNOS in human placental trophoblastic cells. Interestingly, by Northern blot analysis we observed an increased ET-1 mRNA expression level in PE trophoblastic cells compared to NT trophoblastic cells. Furthermore, exogenous ET-1 (10(-7) mol/L) was able to up-regulate its own mRNA expression in both NT and PE trophoblastic cells. iNOS and eNOS mRNA expression was then detected, by semiquantitative PCR, in both NT and PE trophoblastic cells. PE trophoblastic cells expressed lower iNOS mRNA levels compared with NT pregnancies. On the contrary, eNOS mRNA expression was higher in PE trophoblastic cells than in NT cells. Moreover, in the presence of ET-1 we observed a decrease in iNOS and an increase in eNOS mRNA expression levels in both NT and PE trophoblastic cells compared with the respective untreated cells. In conclusion, we demonstrate that ET-1 expression is increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, is decreased; conversely, eNOS expression is increased. Finally, ET-1 is able to influence its own as well as NOS isoform expression in normal and PE trophoblastic cultured cells. These findings suggest the existence of a functional relationships between ET(s) and NOS isoforms that could constitute the biological mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation, which are characteristic of the pathophysiology of preeclampsia.
Subject(s)
Endothelin-1/genetics , Nitric Oxide Synthase/genetics , Placenta/metabolism , Pre-Eclampsia/genetics , Pre-Eclampsia/metabolism , Transcription, Genetic , Adult , Birth Weight , Female , Gestational Age , Humans , Infant, Newborn , Isoenzymes/genetics , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Pregnancy , Proteinuria , RNA, Messenger/genetics , Reference Values , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Corticotropin-releasing factor (CRF) plays a key role in the modulation of fetal-placental unit function during human pregnancy. CRF has a potent vasoactive action on fetal-placental circulation. As products secreted from endothelial cells affect vascular wall reactivity, we investigated whether cultured human umbilical vein endothelial cells (HUVEC) may represent a source and a target for CRF. With RT-PCR we showed that HUVEC express CRF and CRF receptor type 2 messenger ribonucleic acids. Cultured HUVEC also released CRF peptide in a time-dependent way, and the CRF release was differently regulated by various molecules. Dexamethasone decreased CRF release, whereas progesterone and 17beta-estradiol markedly increased it. Forskolin and PGF2alpha were potent stimulators of CRF release from HUVEC. Among the peptides, CRF secretion was stimulated by interleukin-1beta and by endothelin-1. Our study shows for the first time that HUVEC express CRF messenger ribonucleic acid and peptide as well as the CRF R2 gene, and that CRF release is differentially regulated by several distinct molecules. We here propose that CRF has a role in the regulation of the fetal-placental circulation.
Subject(s)
Corticotropin-Releasing Hormone/biosynthesis , Endothelium, Vascular/metabolism , Umbilical Veins/metabolism , Cells, Cultured , Corticotropin-Releasing Hormone/genetics , Cyclic AMP/physiology , Endothelium, Vascular/cytology , Estradiol/pharmacology , Female , Humans , Pregnancy , RNA, Messenger/analysis , Receptors, Corticotropin-Releasing Hormone/genetics , Umbilical Veins/cytologyABSTRACT
The existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins has been demonstrated as well as the ability of IGF-I to positively affect steroidogenesis in human granulosa cells. Furthermore, we recently showed that IGF-I and IGF-II stimulate progesterone secretion by human luteal cells. As the PGs, PGE2 and PGF2alpha, are classically known to have luteotropic and luteolytic effects, we wanted to determine whether the IGFs could affect the human luteal phase by influencing the PG system. For this reason, human luteal cells were cultured for different times (12, 24, and 48 h) with IGF-I, IGF-II (10-100 ng/mL), and GH (100 ng/mL), and both PGs were assayed in the medium culture. We found that both IGF-I and IGF-II were able to stimulate PGE2 synthesis in a time- and dose-dependent way, whereas they both inhibited PGF2alpha production. GH, too, significantly reduced PGF2alpha synthesis; this effect was IGF-I mediated because it was reverted by increasing dilutions of an anti-IGF-I antibody. On the contrary, no GH effect was observed on PGE2 production. In conclusion, based on these data and on our previous results, we speculate that IGFs could influence luteal steroidogenesis through PG system.
Subject(s)
Corpus Luteum/metabolism , Dinoprost/biosynthesis , Dinoprostone/biosynthesis , Insulin-Like Growth Factor II/pharmacology , Insulin-Like Growth Factor I/pharmacology , Adult , Cells, Cultured , Dose-Response Relationship, Drug , Female , Human Growth Hormone/pharmacology , Humans , Insulin-Like Growth Factor I/administration & dosage , Insulin-Like Growth Factor II/administration & dosage , Kinetics , Luteal PhaseABSTRACT
PROBLEM: The effect of anticardiolipin antibodies (ACAs) on basal- and growth factor-stimulated prolactin and insulin-like growth factor (IGF) binding protein (BP)-1 production by cultured human decidual cells was investigated. METHOD OF THE STUDY: Decidual cells were cultured for 24, 48, or 96 hr in medium supplemented with 5% ACA-containing or 5% control serum and increasing concentrations of insulin (1-10 micrograms/mL) or IGF-1 (10-100 ng/mL). RESULTS: No significant increase in prolactin production was observed after addition of increasing doses of insulin and IGF-I in the presence of ACA-containing serum, while a dose-dependent stimulation was seen with control serum. Time-dependent prolactin accumulation was also reduced when cells were cultured in the former conditions. IGF BP-1 release was not affected by insulin and IGF-I in the presence of both sera. However, lower IGF BP-1 levels and a less pronounced time-dependent accumulation were observed in the presence of ACA-positive serum. CONCLUSIONS: Our data suggest that ACAs affect cellular transduction mechanisms regulating critical events, such as decidual cell differentiation. These cellular dysfunctions might be relevant in the induction of some obstetric disorders typical of this syndrome.
Subject(s)
Antibodies, Anticardiolipin/pharmacology , Decidua/metabolism , Insulin-Like Growth Factor Binding Protein 1/biosynthesis , Prolactin/biosynthesis , Cell Count , Cell Differentiation , Cell Survival , Cells, Cultured , Female , Humans , Insulin/metabolism , Insulin/pharmacology , Insulin-Like Growth Factor I/pharmacology , PregnancyABSTRACT
PROBLEM: Several recent data suggest an involvement of endothelin (ET)-1, a powerful vasoconstrictor peptide, in reproductive function. This study was designed to investigate the presence and role of ET-1 in human corpus luteum. METHOD OF STUDY: Purified luteal cells were incubated for different times with ET-1 or ET-3 alone or associated with human chorionic gonadotropin. In another set of experiments cells were treated with ET-1 and BQ485, an ET-A receptor antagonist, or with phorbol 12-myristate-13 acetate (PMA), an activator of protein kinase C. RESULTS: ET-1 reduced both basal and human chorionic gonadotropin-induced progesterone production at all examined times, similarly PMA inhibited basal progesterone synthesis. BQ485 prevented the inhibitory effect of ET-1, while no effect was observed with ET-3. Finally, ET-1 mRNA was detected in the luteal cells. CONCLUSION: ET-1 is expressed by human luteal cells and reduces basal and human chorionic gonadotropin-induced progesterone synthesis through the ET-A receptors and the protein kinase C pathway. Conversely, ET-3 does not affect luteal steroidogenesis.
Subject(s)
Corpus Luteum/physiology , Endothelin-1/biosynthesis , Endothelin-1/physiology , Adult , Azepines/pharmacology , Cells, Cultured , Chorionic Gonadotropin/pharmacology , Corpus Luteum/cytology , Corpus Luteum/drug effects , Endothelin Receptor Antagonists , Endothelin-1/pharmacology , Endothelin-3/pharmacology , Female , Humans , Oligopeptides/pharmacology , Progesterone/biosynthesis , Receptor, Endothelin A , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Endothelin-1 (ET-1) is a peptide classically produced by endothelial cells and known for its powerful vasoconstrictor activity. However, recent data suggest an involvement of ET-1 also in reproductive function. This study was designed to examine the possible presence and role of ET-1 in human luteal cells. Purified luteal cells were incubated for different times with ET-1 (10(-9)-10(-6) M) or ET-3 (10(-9)-10(-6)) alone or associated with human chorionic gonadotrophin (HCG) (100 ng/ml). Both basal and HCG-induced progesterone production were significantly reduced by ET-1 at all examined times whereas preincubation of luteal cells with BQ485 (10(-9)-10(-6) M), an ET-A receptor antagonist, prevented the inhibitory effect of ET-1. Conversely, no effect on progesterone synthesis was observed when ET-3 was added to the cultures. Luteal cells were then incubated for 24 h with phorbol 12-myristate-13 acetate (PMA) (100 ng/ml), an activator of protein kinase C. Inhibition of progesterone synthesis by PMA was similar to that induced by ET-1 alone. This study demonstrates that ET-1 negatively affects, at physiological concentrations, basal and HCG-induced progesterone synthesis. These effects seem to be exerted through the ET-A receptors and the protein kinase C pathway. Conversely, ET-3 was not able to influence human luteal steroidogenesis.
Subject(s)
Chorionic Gonadotropin/pharmacology , Endothelin-1/pharmacology , Endothelin-3/pharmacology , Luteal Cells/metabolism , Progesterone/biosynthesis , Cells, Cultured , Drug Interactions , Female , HumansABSTRACT
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel bioactive peptide isolated from ovine hypothalamus. Recently, its presence and action have been demonstrated also in peripheral tissues such as testis and ovary. On the basis of sequence similarity, PACAP is included in the vasoactive intestinal peptide (VIP)/glucagon/secretin/growth hormone-releasing factor (GRF) family of neuropeptides. Because both VIP and GRF stimulate oocyte maturation in the rat ovary, we wanted to evaluate whether PACAP also could influence this process. Granulosa cells and follicle-enclosed, cumulus-enclosed, and denuded oocytes were obtained from immature eCG-treated rats. The addition of PACAP-38 significantly accelerated meiotic maturation in follicle- and cumulus-enclosed oocytes from treated rats and in follicle enclosed-oocytes from immature untreated rats, while VIP was effective only on follicle-enclosed oocytes. Interestingly, when used on denuded oocytes, PACAP was able to directly affect the meiotic process. In fact, the neuropeptide delayed oocyte maturation by maintaining elevated levels of intracellular cAMP. Our results clearly demonstrate an involvement of PACAP in oocyte meiotic maturation. Furthermore, for the first time, a direct effect of a peptide on the oocytes has been shown. Moreover, the differences in the action of PACAP and VIP on granulosa cells and oocytes suggest the presence of PACAP type I receptors on both cell types. Our results, along with the data demonstrating the presence of the peptide in the ovary, strongly suggest a potential relevance of PACAP in ovarian physiology.
Subject(s)
Meiosis/drug effects , Neuropeptides/pharmacology , Oocytes/drug effects , Oocytes/growth & development , Ovarian Follicle/physiology , Animals , Cells, Cultured , Cyclic AMP/metabolism , Female , Follicle Stimulating Hormone/pharmacology , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Oocytes/metabolism , Ovarian Follicle/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide , Rats , Rats, Sprague-Dawley , Stimulation, Chemical , Vasoactive Intestinal Peptide/pharmacologyABSTRACT
OBJECTIVE: To examine the possible effect of growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide on basal and hCG-stimulated P production by human luteal cells. DESIGN: Cultures of human luteal cells from the early and midluteal phase. SETTING: All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Università Cattolica, a public care center. PATIENT(S): Ten nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders, such as leiomyomatosis. INTERVENTION(S): Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURE(S): Luteal cells were incubated with GHRH, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide with or without hCG at different concentrations. RESULT(S): Pituitary adenylate cyclase-activating peptide stimulated P production in a dose- and time-dependent manner, whereas GHRH and vasoactive intestinal peptide did not affect luteal steroidogenesis. None of the three peptides were found to synergize with hCG. CONCLUSION(S): Pituitary adenylate cyclase-activating peptide can influence human luteal steroidogenesis.
Subject(s)
Corpus Luteum Hormones/biosynthesis , Corpus Luteum/metabolism , Growth Hormone-Releasing Hormone/physiology , Neuropeptides/physiology , Vasoactive Intestinal Peptide/physiology , Adult , Cells, Cultured , Corpus Luteum/cytology , Female , Humans , Hysterectomy , Middle Aged , Pituitary Adenylate Cyclase-Activating PolypeptideABSTRACT
OBJECTIVE: To examine the possible direct effect of insulin-like growth factor (IGF)-I and IGF-II on basal and hCG-stimulated P production by cultured human luteal cells. The possible role of IGF-I as mediator of GH action on luteal steroidogenesis also was investigated. DESIGN: Cultures of human luteal cells from early and midluteal phase. SETTING: All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Universita Cattolica, a public care center. PATIENTS: Eight nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders such as leiomyomatosis. INTERVENTIONS: Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURES: Luteal cells were incubated with IGF-I or IGF-II with or without hCG at different concentrations. Growth hormone also was used alone and with an anti-IGF-I-antibody. RESULTS: We found that IGF-I and IGF-II were able to stimulate directly the P production at all used concentrations and that both of them significantly amplified the steroidogenic hCG effect. Finally, IGF-I was shown to mediate the positive GH action on P synthesis.