Osteogenic potential of murine periosteum for critical-size cranial defects.

Tissue engineering of bone has combined bespoke scaffolds and osteoinductive factors to maintain functional osteoprogenitor cells, and the periosteum has been confirmed as a satisfactory source of osteoblasts. Suitable matrices have been identified that support cell proliferation and differentiation, including demineralised bone matrix (both compatible and osteoinductive) and acellular human dermis. We have evaluated the osteogenic potential of an osteogenic unit, developed by combining periosteum, demineralised bone matrix, and acellular human dermis, in rodents with critical-size cranial defects. Briefly, remnants from the superior maxillary periosteum were used to harvest cells, which were characterised by flow cytometry and reverse retrotranscriptase-polymerase chain reaction (RT-PCR). Cells were cultured into the osteogenic unit and assessed for viability before being implanted into 3 rodents, These were compared with the control group (n=3) after three months. Histological analyses were made after staining with haematoxylin and eosin and Von Kossa, and immunostaining, and confirmed viable cells that stained for CD90, CD73, CD166, runt-related transcription factor, osteopontin, and collagen type I in the experimental group, while in the control group there was only connective tissue on the edges of the bone in the injury zone. We conclude that osteogenic unit constructs have the osteogenic and regenerative potential for use in engineering bone tissue.

Reconstrucción auricular con implantes osteointegrados en un solo tiempo en pacientes seleccionados / Auricular reconstruction with osseointegrated implants in a single procedure in selected cases

Nuestro objetivo es demostrar que el uso de materiales de osteointegración permite lograr una fijación adecuada de una prótesis auricular con un grado mínimo de morbilidad en niños desde 6 años de edad, sin antecedente de reconstrucción, así como en casos en los que otros métodos reconstructivos han fracasado Presentamos la experiencia en reconstrucción auricular con implantes osteointegrados del Instituto Nacional de Pediatría de México D F. Entre Enero del 2007 a Noviembre del 2009. Todos los pacientes fueron seleccionados desde los 6 años, pero principalmente aquellos en los que otras técnicas no habían tenido resultados adecuados desde el punto de vista estético y aquellos en los que la reconstrucción se inició de manera tardía (entre los 12 y los 17 años), lo que hace más complicado el uso de tejido autólogo y supone además menos tiempo para terminar la reconstrucción auricular antes de que cumplan los 18 años. Algunos de los casos que presentamos han sido tratados desde el nacimiento hasta los 17 años e incluimos pacientes en los que los resultados estéticos con tejido autólogo, con implantes de Medpore® o con expansión tisular, no han sido satisfactorios o casos en los que se han usado dos o más técnicas reconstructivas para un mismo paciente, presentando alta morbilidad y resultados estéticos inaceptables. También casos de niños de 9 años de edad como media en los que se practica este procedimiento de primera intención. Todos han tenido un excelente resultado estético, con una satisfacción del 100% por parte de los pacientes. (..) (AU)

Our goal is to demonstrate that the use of Osseo integrated materials allows us to achieve a proper fixation of auricular prosthesis with a minimum degree of morbidity in children from 6 years without antecedent of reconstruction and in cases in which other reconstructive methods have failed We introduce the experience of auricular reconstruction with osseointegrated implants in the National Institute of Pediatrics, México DF, from January 2007 to November 2009. Patients are selected from the age of 6, but mainly those in which other techniques have not had adequate results from the aesthetic point of view, and in those in which reconstruction started in late way (between 12-17 years), using more complicated autologous tissue and with less time to finish the reconstructive procedures before the age of 18 years. We present auricular malformations reconstructed in our hospital; some of these children had been treated from birth for this cause (up to 17years); patients in which the aesthetic results with autologous tissue, Medpore® implants or tissue expansion have not been satisfactory or cases in which two or more techniques have been used for the same patient, presenting high morbidity and unacceptable esthetic results. Also, patients 9years old who suffered this procedure as first intention. For us all of these(..) (AU)

En paladar hendido, ¿es indispensable seccionarla úvula por la mitad? Implicaciones anatómicas y fisiológicas para una uvuloplastia diferente / In cleft palate is indispensable to section the uvula for one half? Anatomical and phisiological implications for a different uvuloplasty

La función del paladar depende de la acción sincronizada del músculo constrictor superior de la faringe y de los músculos del paladar, en especial del elevador del velo del paladar y el de la úvula que se contra en contra la pared posterior de la faringe en situación normal. Proponemos una técnica de uvuloplastia en paladar hendido mediante la cual resecamos la mitad más hipotrófica de la úvula hendida y dejamos la mitad más parecida a lo normal unida al paladar blando, en lugar de resecarla mitad de cada una y unirlas finalmente en la línea media. Presentamos un total de 936 casos, recogidos entre 1995 y2006, en los que practicamos resección de una hemiúvula dejándola de aspecto más natural, con sutura en el paladar blando contra lateral de la hemiúvula no resecada. La media de edad de los pacientes del grupo fue de 14meses (entre los 6 y los 20 meses). Las complicaciones (4%),fueron: hemorragia 3% y dehiscencia de la sutura 1%.Pensamos que el aspecto natural brinda apoyo a los padres de los pacientes operados, ya que al observar la presencia de la úvula, se motivan y acuden mejor a las citas con el rehabilitador del lenguaje; la presencia de una hemiúvula no interfiere con la función del paladar para el habla (AU)

The role of the palate depends on the synchronous action of the superior constrictor of the pharynx and the muscles of the palate, specially the levator muscle of the soft palate and the uvula, that contract against the posterior pharyngeal wall. We propose an uvuloplasty in cleft palate where half hypotrophic part of cleft uvula is resected, lefting the most similar half to normality joined to the soft palate, rather than resecting half of each one and putting them together in the midline. We present a total of 936 cases, collected between 1995 and 2006. The surgical procedure was resection of an hemiuvula, leaving the more natural looking sutured to the soft palate of unresected hemiuvula. The mean age of our patients was 14 months (6 to 20months). Complications (4%) were: bleeding 3% and wound dehiscence 1%.We believe that natural look supports the parents who observe the presence of the uvula and so, are motivated to attend appointments for language rehabilitation; the presence of an hemiuvula not interfere with function palate for talking (AU)

Palatoplastia con incisiones mínimas. Proposición de una técnica y revisión de la literatura / Palatoplasty with minimal incisions. Technique proposal and literature review

Existen numerosas técnica quirúrgicas para la reparación de las fisuras del paladar, desde colgajos uni obipediculados hasta Z-plastias, sin que se haya llegado a encontrar la técnica ideal. Proponemos una técnica quirúrgica con incisiones mínimas a fin de obtener mejores resultados y menor morbilidad, con menor número de complicaciones, tanto tempranas como tardías. Desde 1998 hemos empleado la técnica de palatoplastia con incisiones mínimas en un total de 336niños, 195 mujeres (58%) y 141 varones (42%), con una media de edad de 18 meses (de 6 a 36 meses),logrando disminuir el riesgo de hemorragia y fibrosis, así como el tiempo quirúrgico y la estancia hospitalaria. No se presentaron casos de sangrado postoperatorio, el porcentaje de dehiscencia de la herida y de fístulas secundarias fue del 5% y 323 casos (el 96%) fueron intervenidos en régimen ambulatorio. Concluimos que se trata de una técnica fácil de realizar, con un porcentaje de fístulas menor al referido en la literatura y en general, con una menor morbilidad (AU)

There are several surgical techniques described for cleft palate reparing, from uni or bipediculated flapsto Z-plastias, without finding the ideal one. We propose a new technique with minimal incisions, to get better results, less morbidity and less early or delayed complications. Since 1998 we have used the technique of palatoplasty with minimal incisions in 336 children, 195(58%) girls and 141 (42%) boys, with an average age of 18 months (6 to 36 months), diminishing risk of bleed and fibrosis, surgical time and reducing hospitalisation. In the postoperatory, did not appear bleed, the percentage of wound open and secondary fistula was 5% and 332 cases (96%) were handled like ambulatory procedures. As a conclusion, we present an easy surgical technique, with an smaller reported percentage of fistula and less morbidity (AU)

Técnica de cierre vertical en labio hendido. Reporte de 837 casos y revisión de la literatura / Vertical closing in lip cleft. Report of 837 cases and review of the literature

Ninguna otra deformidad congénita tiene el potencial de alterar la forma facial tan perceptiblemente como lo hace un labio y paladar hendido, en donde el tercio medio de la cara se modifica y supone un gran desafío para el cirujano plástico. Proponemos el cierre vertical de la piel tras la miorrafia, formando una línea y tratando de dar apariencia a la columna del filtrum que no se formó en el labio hendido. Presentamos el resultado en niños operados en el Servicio de Cirugía Plástica del Instituto Nacional de Pediatría de México DF desde enero de 1998 hasta diciembre de2006, en total 837 pacientes. La técnica quirúrgica utilizada consistió en el cierre de labio hendido en forma vertical. Del total de pacientes, 310 fueron mujeres y 527 varones; el 13% (n=109) presentaban hendidura bilateral. El 95%de los pacientes (n= 795) tuvieron un resultado estético adecuado; no se presentaron complicaciones postoperatorias inmediatas y sólo en un paciente se produjo dehiscencia de la herida. Como ventajas de la técnica podemos citar el que produce una línea cicatricial que parece la columna del filtrum hendido, el que conlleva una disección anatómica de la piel(líneas de máxima tensión) y el que evita las incisiones que cruzan el filtrum o el ala nasal, con lo que la cicatriz resultante es menos visible (AU)

No other congenital deformity has the potential to alter the face form as perceivably as it makes a cleft lip and palate. The middle third of the face is altered and it represents a great challenge for plastic surgeon. We propose the vertical closing of the skin after miorraphy forming a line and trying to appear like the filtrum column that was not formed in the cleft lip. We report the result in children operated on our Service of Plastic Surgery at the Instituto Nacional de Salud, México DF, from january 1998 to december 2006, total 837patients. Surgical technique consisted of the closing of cleft lip in vertical way. Total number of 837 patients was composed by 310 female and 527 male, 13% (n=109) presenting bilateral cleft. As a result, 95% of the patients (n=795) had a suitable aesthetic result; it did not appear immediate postoperative complications and only a patient reported wound deshicience. We consider as advantages of the technique: a scar line that seems the column of the cleaved filtrum, an anatomical dissection of the skin (lines of tension)and the fact that we can avoid those incisions that cross the filtrum or the nasal ala, with less visible scar (AU)

Human granulosa-lutein cells express functional EP1 and EP2 prostaglandin receptors.

Prostaglandin E(2) (PGE(2)) exerts mainly luteotrophic effects in the corpus luteum. In other tissues, PGE(2) acts via specific PGE(2) receptor subtypes including EP1, which modulates intracellular calcium ([Ca(2+)](i)) and EP2, which is coupled to cyclic AMP (cAMP) generation. We have therefore investigated the presence of functional EP1 and EP2 receptors using human granulosa-lutein (GL) cells. Reverse-transcription PCR revealed that GL cells expressed mRNA transcripts encoding both EP1 and EP2 receptors. When GL cells were challenged with ligands that can bind to both receptor subtypes (PGE(2) and 16,16 dimethyl PGE(2)) or exclusively to EP2 (butaprost), both cAMP formation and progesterone synthesis were stimulated. Furthermore, the cAMP response to these agonists could be significantly blocked by an EP1/2 antagonist AH6809 but not by an EP1-selective antagonist SC19220. Exposure of GL cells to 16,16-dm PGE(2) transiently raised [Ca(2+)](i) levels, which could be prevented by both AH6809 and SC19220. We therefore conclude that human GL cells express functional EP1 and EP2 receptors.

Overview of current research in parturition.

The mechanism of human parturition is not understood and further research into this important physiological process is needed. Preterm labour remains a major cause of perinatal mortality and morbidity and there is controversy about the effectiveness of current tocolytic agents. In some species, notably the sheep, parturition is preceded by an activation of the fetal hypothalamic-pituitary-adrenal axis. However, in primates this axis has a supportive, rather than essential, role. A fall in maternal progesterone levels is a prerequisite for parturition in most mammals and this takes place either through increased conversion of progesterone to oestrogens in the placenta, or through the demise of the corpus luteum of pregnancy, depending on the species. In primates and guinea-pigs parturition occurs without an apparent fall in maternal progesterone levels. Gene targeting experiments in mice have demonstrated the critical role of prostaglandin FP receptors, necessary to mediate the luteolytic effect of PGF(2alpha) before parturition. Prostaglandin synthesis is required for the onset and progress of labour as demonstrated by experiments with cPLA(2)- and PGHS-1-deficient mice. The importance of local tissue conversion of progesterone to reduced androgens in the regulation of cervical ripening has been demonstrated in 5alpha-reductase-deficient mice. The chronic and ubiquitous gene inactivation obtained with conventional methods has disadvantages, in that it may allow the activation of compensating pathways, making the interpretation of results difficult. This problem may be overcome by using pulsed and tissue-selective gene knockout strategies. The study of human parturition is complicated by the lack of access to direct experimentation, whereas the endocrine differences between species make it difficult to extrapolate animal data to humans. However, the development of genomic/proteomic technologies that allow the simultaneous screening of thousands of genes and gene products in small samples of tissue, and new methods to study the biochemistry of receptors and proteins involved in smooth muscle physiology promise new insights into the control of human labour. Nevertheless, the integration of rapidly expanding knowledge into a complete understanding of the roles of the mother and the fetus in the initiation of parturition, and the development of selective medication for the effective management of preterm labour remain an arduous challenge for the next decade. Experimental Physiology (2001) 86.2, 213-222.

Uterine quiescence: the role of cyclic AMP.

It is accepted that whilst hormones such as oxytocin, vasopressin and prostaglandin F2alpha induce myometrial contractions, essentially via an elevation of intracellular calcium, other ligands, such as beta-adrenoceptor agonists, calcitonin gene-related peptide, and prostaglandin E2, promote uterine quiescence via their ability to increase intracellular cyclic AMP levels. At present, the exact factors initiating human parturition remain unknown, and labour may occur due to a loss of uterine quiescence, an increase in uterine contractility, or a combination of both. Whilst many studies have aimed to understand the mechanisms underlying uterine contractility there is a relative paucity of data regarding myometrial relaxation. We have verified the presence of mRNA encoding adenylyl cyclase (AC) isoforms I, II, III, V, VI, VII, VIII and IX in both non-pregnant and pregnant human myometrium, and in isolated myometrial cells maintained in cell culture. Furthermore, by means of immunoblotting and immunocytochemistry, we have demonstrated the expression of these isoforms as membrane-associated AC proteins, and identified changes in individual AC isoform expression during gestation. These findings illustrate the diversity of potential cAMP generating pathways in human myometrium, and the complexity of the signal transduction systems underlying uterine quiescence. Experimental Physiology (2001) 86.2, 265-272.

Corticotrophin releasing hormone: its potential for a role in human myometrium.

Aside from its role as a hypothalamic stress hormone, corticotrophin releasing hormone (CRH) is also a placental hormone, at least in primates. Although the function of placentally derived CRH remains to be fully elucidated, elevated CRH levels have been associated with premature labour, suggesting that the hormone may be involved in regulating the duration of pregnancy. Indeed, pregnant human myometrium expresses functional CRH receptors (CRH R1 and CRH R2 subtypes) thought to signal predominantly via the second messenger cAMP. Thus, like other cAMP-producing hormones in the myometrium such as beta(2) agonists, CRH may play a part in maintaining uterine quiescence. However, several of the CRH receptor isoforms identified to date have a reduced ability to activate adenylate cyclase, raising the question as to whether they are linked to other signal transduction pathways. Here, we discuss critically the evidence for the peptide's role in regulating contractility, both directly at the myometrium and indirectly via the fetal membranes and decidua. The possibility of a role in myometrial growth modulation is also described. Experimental Physiology (2001) 86.2, 273-281.

Desensitisation of the oxytocin receptor and other G-protein coupled receptors in the human myometrium.

G-protein coupled receptors (GPCRs) are key to maintaining uterine quiescence and inducing phasic contractions at term. However, the biochemical mechanisms whereby uterine GPCRs are desensitised and re-sensitised during these physiological conditions are unknown. For example, the number of oxytocin receptors (OTRs) on uterine myocytes decrease significantly after the addition of oxytocin. Therefore, further understanding of the desensitisation/re-sensitisation of the OTR and other uterine GPCRs during pregnancy may provide a target for more efficient tocolytic drugs and more selective ways to modulate uterine activity. Here, we briefly review some of the mechanisms that may be involved during OTR and other GPCR desensitisation. Experimental Physiology (2001) 86.2, 303-312.

Myosin light chain kinase and the onset of labour in humans.

Myosin light chain kinase (MLCK) is essential for myometrial contractions induced by calcium-mobilizing agonists. From the gene of vertebrate smooth muscle/non-muscle MLCK there are at least four proteins expressed. We have found that both a > 200 and a 137 kDa MLCK are equally expressed in human non-pregnant (NP) and term pregnant (P) uterine smooth muscle and confirmed that 19 kDa telokin (TK) is only expressed in P myometrium. In addition, we have observed that a MLCK immunogen at approximately 60 kDa is only expressed in NP myometrium, suggesting that its expression is inhibited during normal pregnancy in a hormonally dependent manner. However, when we compared pregnant myometrium from patients delivered preterm (PT) (< 34 weeks gestation), but not in labour (NIL), with PT patients in labour (IL) we found that PT(IL) samples expressed the approximately 60 kDa MLCK immunogen and thus displayed a NP phenotype whereas PT(NIL) samples did not express the protein and retained a pregnant phenotype. We hypothesize that the novel approximately 60 kDa MLCK immunogen contributes to the aberrent contractility associated with preterm labour. Experimental Physiology (2001) 86.2, 313-318.

Characterization of adenylyl cyclases in cultured human granulosa cells.

Granulosa cells play an essential role in follicular development and formation of corpora lutea. Many functions of granulosa-lutein cells are controlled by activation of G protein-coupled receptors and the formation of cyclic AMP (cAMP) by adenylyl cyclase. There are at least nine mammalian adenylyl cyclase isoenzymes, which show different sensitivities towards other signalling systems. The aim of this study was to identify the types of adenylyl cyclase present in human granulosa cells and to investigate its functional regulation by G proteins, calcium and the protein kinase C and A pathways. Granulosa cells were obtained from women undergoing IVF. The cells were maintained in primary culture and they consistently expressed mRNA coding for adenylyl cyclase I, III, VI, VII and IX. The signals for adenylyl cyclase V and VIII were more variable among patients and there was no signal for adenylyl cyclase II. The expression of multiple adenylyl cyclase proteins was confirmed by immunochemistry with subtype-specific antibodies. The formation of cAMP in cultured cells was stimulated many times by hCG (EC(50) value 4.2 iu ml(-1)) and by prostaglandin E(2) (PGE(2); EC(50) = 0.75 micromol l(-1)) in a concentration-dependent manner, thus confirming the presence of receptors coupled positively to G(s). The diterpene forskolin, which stimulates all isoforms of adenylyl cyclase except for adenylyl cyclase IX, increased cAMP formation to higher levels than hCG or PGE(2). The strong stimulation by forskolin indicates that adenylyl cyclase IX is unlikely to be the major source of cyclase activity in these cells. Basal and forskolin- or PGE(2)-stimulated adenylyl cyclase activity was amplified 1.5-2.0 times by phorbol-12,13-dibutyrate, indicating that protein kinase C-sensitive enzymes (for example, adenylyl cyclase types IV, V, VI or VII) may be active in the cells. In contrast, hCG-stimulated activity was inhibited (76 +/- 6%) by phorbol ester. Stimulation of G(i) with the alpha-adrenoceptor agonist clonidine inhibited hCG-induced cyclase activity. This finding indicates that adenylyl cyclase II and IV subtypes, which are stimulated by betagamma subunits released from G(i), are not predominant. Increases in intracellular free calcium concentrations by the ionophore A23187, the calcium-ATPase inhibitor thapsigargin or by fluprostenol, a selective prostanoid FP receptor agonist, which is known to open calcium channels in granulosa cells, or removal of calcium by EGTA, had no significant effects on basal or forskolin-stimulated formation of cAMP. These results indicate that subtypes adenylyl cyclase I, III and VIII, which are activated by calcium, and adenylyl cyclase V and VI, which are inhibited by calcium, are not dominant isoforms in granulosa-lutein cells. The protein kinase A inhibitor H89 had no effects on formation of cAMP; this finding rules out the involvement of adenylyl cyclase V and VI subtypes, which are subjected to negative feedback by protein kinase A. These results indicate that adenylyl cyclase VII is the dominant functional isoenzyme in human granulosa-lutein cells.

Fetal surfactant as a source of arachidonate in human amniotic fluid.

The factors responsible for the onset of labor in women are not well understood but it is clear that parturition is associated with increased production of prostanoids and release of arachidonic acid by intrauterine tissues. Pulmonary surfactant is secreted from the fetal lung into the amniotic fluid where its concentration increases toward term. In this paper we have shown that the ability of fetal surfactant to stimulate prostaglandin production by amnion cells is greatly enhanced by pre-incubating surfactant with amniotic fluid. This is due to the release of fatty acids, including arachidonate, from the lipids of fetal surfactant by the sequential action of phospholipase C and diglyceride lipase. Thus, in addition to providing the amnion with a source of arachidonate derived from the intracellular transfer of arachidonate from surfactant phosphatidylcholine to phosphatidylethanolamine and phosphatidylinositol in amnion cells, fetal surfactant also contributes to the pool of free arachidonate in amniotic fluid.

Ovarian response to repeated controlled stimulation in in-vitro fertilization cycles in patients with ovarian endometriosis.

In-vitro fertilization (IVF) is an effective infertility treatment for women with endometriosis, but most women need to undergo several cycles of treatment to become pregnant. This case-control study was designed to assess how consistently women with ovarian endometriosis respond to ovarian stimulation in consecutive treatment cycles compared to women with tubal infertility. We compared outcome measures in 40 women with a history of surgically confirmed ovarian endometriosis and 80 women with tubal infertility, all of whom had at least three IVF treatment cycles. The groups were matched for age and early follicular follicle stimulating hormone (FSH) concentration at their first IVF cycle. Outcome measures included number of follicles, number of oocytes, peak oestradiol concentration and number of FSH ampoules required per follicle. Cumulative pregnancy and live birth rates were calculated in both groups. The ovarian endometriosis group had a significantly poorer ovarian response and required significantly more ampoules of FSH per cycle, a difference that became greater with each subsequent cycle. However, cumulative pregnancy (63.3 versus 62.6% by fifth cycle) and live birth (46.8 versus 50.9% by fifth cycle) rates were similar in both groups. In conclusion, despite decreased ovarian response to FSH, ovarian endometriosis does not decrease the chances of successful IVF treatment.

Extremes of body mass do not adversely affect the outcome of superovulation and in-vitro fertilization.

The effect of extremes of body mass on ovulation is well recognized by clinicians. However, the effect of obesity and extreme underweight on the outcome of in-vitro fertilization (IVF) cycles has received relatively little attention. In a retrospective nested case-control study we examined the effect of the extremes of body mass index (BMI) on IVF-embryo transfer outcome at a university-based IVF unit. A total of 333 patients were included in the study; 76 obese patients (BMI > 27.9) with 152 controls, and 35 underweight patients (BMI < 19) with 70 controls. The patients were matched with their controls in age +/- 1 year, day 3 follicle stimulating hormone (FSH) concentration, daily dose of gonadotrophin (+/- 37.25 IU), gonadotrophin preparation and the year of treatment. The following parameters were compared between the study and control groups: duration of administration and dose of gonadotrophin, number of follicles aspirated, number of eggs, fertilization rate, number of embryos, serum oestradiol concentration on human chorionic gonadotrophin (HCG) day (peak oestradiol), clinical pregnancy rate, implantation rate, miscarriage rate, and incidence of ovarian hyperstimulation syndrome. Apart from a significantly lower peak oestradiol concentration (P = 0.009) in the obese patients, they and the underweight patients were not significantly different from their normal controls. The extremes of body mass index do not adversely affect the outcome of IVF-embryo transfer treatment. However, the obese patients had lower peak oestradiol concentrations than their normal controls despite receiving similar gonadotrophin doses.

Desensitization of oxytocin receptors in human myometrium.

In the present study, we investigated the possible mechanisms by which oxytocin might regulate oxytocin receptor (OTR) density. Exposure of cultured myometrial cells to oxytocin for a prolonged period caused desensitization: the steady-state level of oxytocin binding was 210 x 10(3) binding sites/cell, but this was time-dependently reduced to 20.1 x 10(3) sites/cell by exposing the cells to oxytocin for up to 20 h. In contrast, Western blotting data showed that the total amount of OTR protein was not affected by oxytocin treatment for up to 24 h. Flow cytometry experiments demonstrated that OTRs were not internalized during this treatment. However, RNase protection assays and Northern analysis showed that in cultured myometrial cells OTR mRNA was reduced by oxytocin treatment to reach a new low steady-state concentration. Analysis of this mRNA in myometrial biopsies from 17 patients undergoing emergency Caesarean section showed how it decreased with advancing labour. Samples obtained after 12 h of labour contained approximately 50 times less OTR mRNA than samples obtained from patients in labour for less than 12 h. We speculate that this decrease in OTR mRNA represents in-vivo OTR desensitization.

Prostaglandin E receptors in myometrial cells.

Prostaglandins (PGs) exert their effects via binding to specific cell surface receptors and influencing second messenger systems through G-proteins. PGE2 may interact with at least four receptor subtypes (EP1, EP2, EP3, EP4), each showing different pharmacological profiles. The second messengers calcium, inositol phosphates (InsPs) and cyclic nucleotides play decisive roles in uterine contractility. The question in this investigation was, which EP receptors, G-proteins and second messenger systems transmit PGE2 induced signals in human myometrium. We have measured changes in InsPs and cAMP formation and also in intracellular calcium concentration ([Ca2+]i) induced by PGE2 and receptor subtype selective analogues in cultured human myometrial cells. PGE2 increased cAMP level and this effect was shared by the EP2 receptor subtype selective agonist Butaprost and by Misoprostol (EP3 > EP2 > EP1). Sulprostone (EP3 > EP1) did not stimulate adenylyl cyclase activity per se, but inhibited forskolin-stimulated adenylyl cyclase in a pertussis toxin (PT) sensitive way. PGE2, GR63799X (EP3 selective), Sulprostone and Misoprostol activated phospholipase-C (PLC), this effect was resistant to PT treatment. PGE2 also elevated [Ca2+]i from the resting level of 60-90 nM up to 350 nM. Low concentrations (1-300 nM) of PGE2 increased [Ca2+]i without PLC activation. The selective EP1 inhibitor AH6809, Nifedipine, Verapamil and PT treatment inhibited this effect of PGE2. In cultured human myometrial cells PGE2 interacts with EP1 receptors, which elevate [Ca2+]i independently from PLC, but involving a Gi protein and plasmamembrane calcium channels; EP2 receptors which stimulate adenylyl cyclase; EP3A receptors, which inhibit adenylyl cyclase activity through Gi activation and EP3D receptors which activate PLC through a PT-insensitive pathway and also elevate [Ca2+]i.

Prostaglandin E2 activates phospholipase C and elevates intracellular calcium in cultured myometrial cells: involvement of EP1 and EP3 receptor subtypes.

PGE2 is a powerful modulator of uterine contractility, but there is uncertainty as to which receptor subtypes (EP1, EP2, EP3, or EP4), G proteins, and second messenger systems are activated by PGE2 in myometrium. Here we show that in cultured human myometrial cells, PGE2 (1-100 microM) activates phospholipase C (PLC) up to 500% over the control level and elevates intracellular calcium ([Ca2+]i) from the resting level of 60-90 nM up to 350 nM in a concentration-dependent manner. Stimulation by the receptor subtype-selective analogs GR63799X (EP3), sulprostone (EP3 > EP1), and misoprostol (EP3 > EP2 > EP1) indicates that these effects are transmitted through EP3 receptors. Both effects are resistant to pertussis toxin (PT). Lower concentrations of PGE2 (1-300 nM) increase [Ca2+]i via a PT-sensitive pathway, without PLC activation. This [Ca2+]i increase occurs after an inverse dose-related delay and is inhibited by the selective EP1 antagonist AH6809 and calcium channel blockers. By comparison, oxytocin stimulates PLC up to 1000% over the control level and elevates [Ca2+]i up to 800 nM in a concentration-dependent manner without any measurable delay; both effects are partly sensitive to PT. These data provide functional evidence for the presence of different stimulatory mechanisms for PGE2 in myometrium: 1) a low affinity receptor (probably EP3D) that activates PLC through a PT-insensitive pathway; and 2) a high affinity receptor (probably EP1), independent from PLC and involving a PT-sensitive G protein (G(i)?). Both pathways lead to elevation of [Ca2+]i.

The presence of NK3 tachykinin receptors on rat uterus.

The NK3 agonist, senktide, induced a potent contraction of rat uterus in the presence of tetrodotoxin, atropine and indomethacin, or the tachykinin receptor antagonists L-659877 and [D-Pro4,D-Trp7,9,10]substance P (4-11). Additional contractile and radioligand binding studies with receptor selective agonists and antagonists confirmed the presence of NK3 receptors and also revealed the presence of NK1 and NK2 receptors. The rat uterus is the second peripheral tissue in which a post-synaptic, non-neuronal NK3 receptor has been identified.