RESUMO
Correlated firing among neurons is widespread in the visual system. Neighbouring neurons, in areas from retina to cortex, tend to fire together more often than would be expected by chance. The importance of this correlated firing for encoding visual information is unclear and controversial. Here we examine its importance in the retina. We present the retina with natural stimuli and record the responses of its output cells, the ganglion cells. We then use information theoretic techniques to measure the amount of information about the stimuli that can be obtained from the cells under two conditions: when their correlated firing is taken into account, and when their correlated firing is ignored. We find that more than 90% of the information about the stimuli can be obtained from the cells when their correlated firing is ignored. This indicates that ganglion cells act largely independently to encode information, which greatly simplifies the problem of decoding their activity.
Assuntos
Células Ganglionares da Retina/fisiologia , Animais , Eletrofisiologia , Camundongos , Estimulação Luminosa , Retina/fisiologia , Transmissão Sináptica , Visão Ocular/fisiologiaRESUMO
Edge enhancement in the retina is thought to be mediated by classical center-surround antagonism, first encountered as the interactions between horizontal cells and cones. But in the salamander retina these interactions do little to enhance edges. Instead, a robust dynamic interaction between amacrine and bipolar cells appears to be responsible for a sharp edge enhancement. To demonstrate this we recorded extracellularly from a single ganglion cell and moved a flashed square, 300 micro(m) on a side, over a 1.5 x 1.0 mm2 grid at 25-micro(m) increments. Playing back all of these recordings simultaneously simulated the pattern of responses that would have been measured from an array of ganglion cells. The emerging pattern of ganglion cell activity first faithfully represented the flashed square, but after approximately 60 ms the center of the representation collapsed, leaving a representation of only the edges. We inferred that the feedback synapse from amacrine to bipolar cells at gamma-aminobutyric acid-C (GABAC) receptors mediated this effect: bicuculline and strychnine were ineffective in altering the response pattern, but in picrotoxin the center of the representation did not collapse. The GABAergic amacrine cells thought to mediate this effect have quite narrow spread of processes, so the existence of this edge-enhancing effect suggests a mechanism quite different from classical lateral inhibition, namely the delayed inhibition of a spatially expanding input pattern.
Assuntos
Neurônios/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Ambystoma , Animais , Bicuculina/farmacologia , Antagonistas de Receptores de GABA-A , Técnicas In Vitro , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Picrotoxina/farmacologia , Tempo de Reação , Receptores de Glicina/antagonistas & inibidores , Retina/citologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Ganglionares da Retina/efeitos dos fármacos , Estricnina/farmacologiaRESUMO
Heparin/heparan sulfate interacting protein (HIP) is a recently identified protein expressed by many normal epithelia and epithelial cell lines. In the present study, we examined expression and potential functions of this protein in a series of human breast cancer cells and in sections of normal and malignant human breast tissue. Four of the five breast cancer cell lines studied (MCF-7, T-47D, MDA-MB468, and BT-549) expressed HIP protein and mRNA at similar levels. In contrast, MDA-MB-231 cells failed to display reactivity with HIP-specific probes in any assay. Cell aggregation assays and cell surface antibody binding studies demonstrated that HIP was expressed on the cell surface. However, HIP expression did not correlate with the number of cell surface [3H]heparin (HP) binding sites. The K(Dapp)s for cell surface HP binding sites were similar in all breast cancer cell lines studied and ranged from 112 to 298 nM. In contrast, cell surface HP binding capacity varied greatly, ranging from 2.3 x 10(5) (MDA-MB-231 and MDA-MB-468) to 99 x 10(5) sites/cell (BT-549). All cell lines tested displayed the ability to bind to a heparan sulfate (HS)-binding synthetic peptide motif of HIP in a HP-inhibitable fashion. Binding to this motif was not inhibited by other glycosaminoglycans including hyaluronic acid, chondroitin sulfates, or keratan sulfate. Furthermore, cell binding to HIP peptide was almost completely lost when intact cells were predigested with heparinases but not chondroitinases. Cell surface HS from breast cancer cells as well as normal human breast epithelia binded to HIP peptide in a HP-inhibitable fashion, demonstrating the ability of these cell surface components to directly interact. HIP was detected in both normal breast epithelia and breast tumors in situ. It is suggested that HIP mediates aspects of HS-dependent interactions of both normal and malignant breast epithelia with other cells and extracellular matrix components.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Feminino , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Células Tumorais Cultivadas/metabolismoRESUMO
A common practice among researchers performing linkage studies is the use of equal allele frequencies as input when reporting p-values from computer linkage programs such as S.A.G.E. SIBPAL. Our results, using 5,000 sets from a uniform-prior distribution of allele frequencies, showed that such input may be problematic. Further, we found that the S.A.G.E. SIBPAL test for proportion of alleles shared identical by descent among concordantly affected sib pairs showed a greater percentage of significant p-values with decreasing parental genotype information (Table III), while the S.A.G.E. SIBPAL Haseman-Elston test produced significant p-values comparatively less frequently (Table IV).
Assuntos
Transtorno Bipolar/genética , Frequência do Gene , Ligação Genética , Marcadores Genéticos , Probabilidade , Software , Alelos , Teorema de Bayes , Feminino , Humanos , Masculino , Análise por Pareamento , Núcleo Familiar , Distribuição AleatóriaRESUMO
Uterine epithelial cells (UEC) isolated from 6-week-old CF-1 mice were immortalized using retroviral-mediated transfection of SV40 large T-antigen. One line, WEG-1, retained epithelial morphology and reacted with antibodies to cytokeratins 18, 19, laminin, fibronectin, and beta-catenin. In addition, WEG-1 cells displayed strong nuclear immunoreactivity to SV40 large T-antigen, confirming integration of the retrovirus vector and expression of this gene. WEG-1 cells were negative for nonepithelial markers such as desmin and factor 8. WEG-1 cells did not proliferate in serum-free medium; however, addition of 0.5% FBS supported proliferation to the same extent as 10% FBS. Addition of 50 ng/ml epidermal growth factor to medium containing 0.5% charcoal-stripped FBS restored proliferation comparable with 0.5% whole FBS. Epidermal growth factor or transforming growth factor-alpha (50 ng/ml), but not transforming growth factor-beta, leukemia-inhibiting factor, or fibroblast growth factor, induced the secretion of three proteins (M(r) approximately to 158K, 148K, and 36K). Comparison of protein secretions of WEG-1 cells and UEC showed shared as well as distinct bands. Like UEC, WEG-1 cells secreted PGF2a and PGE2 and expressed PG GH synthase-2. Unlike UEC, WEG-1 cells showed no apical/basal preference for either uptake of methionine or secretion of proteins. The absence of immunoreactive E-cadherin or zona occludens-1 was consistent with the absence of cell polarity in WEG-1 cells. Primary UEC, which polarize in vitro, do not support blastocyst attachment. WEG-1 cells, although not polarized in vitro, also exhibited delayed blastocyst attachment compared with nonuterine cell lines, suggesting that WEG-1 cells partially retained some aspects of UEC function relevant to embryo attachment. WEG-1 cells expressed messenger RNA for Muc-1, an UEC mucin suggested to have antiadhesive properties. Furthermore, WEG-1 cells did not display high affinity heparin binding sites, an activity associated with embryo attachment. WEG-1 cells may provide a model for studying various aspects of UEC function and murine embryo attachment.
Assuntos
Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Transformador alfa/farmacologia , Útero/citologia , Útero/metabolismo , Animais , Sequência de Bases , Biomarcadores , Divisão Celular/efeitos dos fármacos , Embrião de Mamíferos/fisiologia , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Substâncias de Crescimento/farmacologia , Células HeLa , Humanos , Metionina/farmacocinética , Camundongos , Sondas Moleculares/genética , Dados de Sequência Molecular , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/metabolismo , Proteínas/metabolismo , Útero/efeitos dos fármacosRESUMO
Previous research by Stam and Spanos suggests that if waking analgesia is followed by hypnotic analgesia, subjects refrain from maximally responding during the waking trial so they report less pain under hypnosis (i.e., a "holdback effect"). This hypothesis was re-examined using more stringent controls. Thirty-six highly susceptible subjects chosen by a combination of the Harvard Group Scale of Hypnotic Susceptibility, Form A and the Stanford Hypnotic Susceptibility Scale, Form C were randomly assigned to one of three treatment groups (waking analgesia followed by hypnotic analgesia, waking analgesia followed by waking analgesia, or hypnotic analgesia followed by waking analgesia). Each group received three 60-second immersions of cold pressor pain stimulation (baseline, Immersion 1, Immersion 2) and rated pain using a magnitude estimation and a category rating scale. The obtained results failed to support the hypotheses of a holdback effect or a "reverse-order holdback effect." Properties of within-subjects and between-subjects designs were considered in explaining the superiority of hypnotic analgesia over waking analgesia typically found in within-subjects models.
Assuntos
Hipnose Anestésica , Enquadramento Psicológico , Adulto , Feminino , Humanos , Masculino , Limiar da DorRESUMO
Our previous studies demonstrated that interleukin-1 alpha (IL-1 alpha) and soluble factors secreted by polarized uterine luminal epithelial cells (UEC) stimulate prostaglandin (PG) secretion by uterine stromal cells (USC). The present studies were aimed at determining the mechanism by which these agonists stimulate PG secretion by USC. The comoplete inhibition of IL-1 alpha- and UEC-induced PGE2 secretion by cycloheximide and actinomycin-D in the presence of a saturating concentration of arachidonic acid indicated that IL-1 alpha and UEC act to a large extent by inducing de novo expression of PG endoperoxide synthase (PGHS). Western blot analysis of membrane fractions from USC showed a 2- to 4-fold accumulation of the mitogen-inducible isoform of PGHS (PGHS-2), but not the constitutively expressed enzyme (PGHS-1), within 3 h of treatment with IL-1 alpha, UEC-conditioned medium, or serum. Inhibition of UEC-stimulated PGHS-2 expression by anti-IL-1 alpha indicated that IL-1 alpha is one factor secreted by UEC responsible for the synthesis of USC PGHS-2. Expression of PGHS-2, but not PGHS-1, was inhibited by dexamethasone. Dexamethasone also inhibited IL-1 alpha- and UEC-stimulated PGE2 secretion by USC. Immunohistochemical studies demonstrated that PGHS-2 is localized to implantation sites in newly differentiating USC at the time of blastocyst attachment, indicating a potential physiological role for PGHS-2 in early stages of mouse implantation. In contrast, PGHS-1 was localized to UEC during this period. Collectively, these results indicate that enhanced PG secretion by USC in response to IL-1 alpha and soluble factors secreted by UEC is due to selective expression of PGHS-2. In addition, the expression of PGHS-2 by USC in vivo during the periimplantation period may support PG secretion required during early stages of embryo implantation.
Assuntos
Prostaglandina-Endoperóxido Sintases/fisiologia , Útero/enzimologia , Animais , Western Blotting , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Dexametasona/farmacologia , Células Epiteliais , Epitélio/enzimologia , Feminino , Imunofluorescência , Imuno-Histoquímica , Interleucina-1/farmacologia , Isomerismo , Masculino , Camundongos , Prostaglandina-Endoperóxido Sintases/análise , Útero/citologiaRESUMO
Uterine epithelial cells (UEC) were isolated from cycling mice and cultured on Matrigel-coated nitrocellulose filters to determine their ability to secrete interleukin-1 alpha (IL-1 alpha) in response to ovarian steroids and induce prostaglandin (PG) secretion by uterine stromal cells (USC). UEC cultured in a polarized manner secreted IL-1 alpha with an 8- to 10-fold apical vs. basal preference, as determined by an enzyme-linked immunosorbent assay. There was no effect of 17 beta-estradiol, progesterone, or 17 beta-estradiol plus progesterone on IL-1 alpha secretion by UEC. The mean total IL-1 alpha secreted to the apical and basal secretory compartments over the 24-h incubation period was 0.8 +/- 0.16 and 0.07 +/- 0.05 ng/2 x 10(5) cells, respectively. Cytokine bioactivity, as determined by [3H]thymidine incorporation into D10 cells in response to UEC-conditioned medium, paralleled the pattern of IL-1 alpha secretion observed using the immunoassay. In addition to the in vitro secretion of IL-1 alpha by polarized UEC, pooled uterine fluid collected from proestrous stage mice contained IL-1 alpha at a concentration of 0.7 ng/ml, indicating that IL-1 alpha is released into the uterine lumen in vivo. Coculture with UEC or treatment with conditioned medium from either the apical or basal UEC secretory compartments induced a several-fold increase in the secretion of PGE2 and PGF2 alpha by USC. Relative to untreated USC, PGE2 was induced to a greater extent than PGF2 alpha. The addition of polyclonal anti-IL-1 alpha significantly inhibited the ability of UEC-conditioned medium and UEC coculture to induce PG secretion by USC. In addition, mouse recombinant IL-1 alpha added at a concentration similar to that secreted by UEC stimulated USC PGE2 and PGF2 alpha secretion in a manner similar to that observed with UEC coculture. Experiments designed to determine the cell type specificity of the induction of PG secretion by USC indicated that conditioned medium from a human UEC line (RL95), a rat prostate epithelial cell line (E4), and a mouse fibroblast cell line (10T1/2) induced PG secretion to an extent that paralleled their ability to induce D10 cell proliferation. The present results demonstrate the ability of UEC to secrete IL-1 alpha in a vectorial manner. Soluble products secreted by UEC are capable of stimulating PGE2 and PGF2 alpha secretion by USC, and IL-1 alpha appears to be a significant factor contributing to this effect.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Dinoprosta/metabolismo , Dinoprostona/metabolismo , Interleucina-1/metabolismo , Útero/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Epitélio/metabolismo , Feminino , Humanos , Masculino , Camundongos , Próstata , Ratos , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Útero/citologiaRESUMO
Uterine stromal (USC) and uterine epithelial (UEC) cells were isolated from immature and mature mice to determine their ability to secrete interleukin-6 (IL-6) in response to ovarian steroids, IL-1 alpha, and soluble products produced by the heterologous cell type. In addition, the effect of IL-6 on embryo attachment and outgrowth in vitro was determined. UEC cultured on nitrocellulose filter inserts in a polarized manner secreted IL-6 with a 2.5- to 5-fold apical vs. basal preference, as determined by a B9 hybridoma cell proliferation assay and enzyme-linked immunosorbent assay. The hormonal status of animals at the time uteri were removed did not influence subsequent secretion of IL-6, as UEC isolated from immature, diestrous, and estrous stage mice exhibited both a similar amount and had a similar apical preference for secretion of IL-6. The addition of 17 beta-estradiol (E) to UEC cultures markedly inhibited total IL-6 secretion, but did not affect vectorial secretion. The inhibitory effect of E on IL-6 secretion by UEC was consistent with an apparent decrease in IL-6 transcript observed by a reverse transcriptase polymerase chain reaction assay. Other transcripts detected by this assay in UEC included IL-1 alpha, but not IL-1 beta or tumor necrosis factor-alpha. Secretion of IL-6 by UEC was not stimulated by IL-1 alpha, conditioned medium from USC, or coculture with USC. USC secreted IL-6, and while this also was inhibited by E, progesterone was more effective in this regard at physiological concentrations. In addition, there was a synergistic effect of E plus progesterone on inhibition of IL-6 secretion by USC. Secretion of IL-6 by USC was stimulated by IL-1 alpha, and coculture studies demonstrated the ability of UEC to stimulate a several-fold increase in IL-6 secretion by USC. The cytokine transcripts detected in USC cultures included IL-6 and IL-1 alpha, but not IL-1 beta. Transcripts for tumor necrosis factor-alpha were present in USC only after culture with IL-1 alpha. IL-6 added to blastocysts on laminin-coated tissue culture wells resulted in a transient inhibition of the rate of blastocyst attachment and, to a greater extent, an inhibition of the rate of embryo outgrowth. In addition, IL-6 inhibited the size of embryo outgrowths at 24 and 48 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Estradiol/farmacologia , Interleucina-6/biossíntese , Progesterona/farmacologia , Útero/fisiologia , Animais , Divisão Celular , Células Cultivadas , Citocinas/genética , Diestro , Implantação do Embrião/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Estro/fisiologia , Feminino , Interleucina-1/farmacologia , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , Transcrição Gênica , Útero/citologia , Útero/efeitos dos fármacosRESUMO
Chondroitin sulfate proteoglycans (CSPGs) are the major class of proteoglycans synthesized by mouse uterine stroma in vitro (Jacobs, A. L., and Carson, D. D. (1991). J. Biol. Chem. 266, 15,464-15,473). In the present study, stromal CSPGs were isolated and examined with regard to their ability to bind to specific extracellular matrix (ECM) components. Of a variety of ECM components tested, only collagen type I formed stable complexes with stromal CSPGs in both solid phase and solution binding assays. Proteolytic digestion of the CSPGs did not affect binding and suggested that the protein cores did not participate directly in binding. Furthermore, free chondroitin sulfate polysaccharides do not compete effectively in the binding assays. Therefore, interactions with multiple CS chains and/or the higher charge density afforded by intact CSPGs appear to be required for retention by collagen type I. Intact CSPGs were examined for their ability to modulate embryo attachment and outgrowth in vitro on fibronectin- or collagen type I-coated surfaces. In both cases, intact CSPGs, but not their constituent protein cores or polysaccharides, inhibited both the rate and the extent of outgrowth formation. In addition, embryo outgrowth on stromal ECM was enhanced by predigestion with chondroitinase. Addition of exogenous CSPG markedly retarded embryo outgrowth on stromal matrix. Collectively, these data indicate that stromal cell-derived CSPGs are retained by collagen type I in the stromal interstitial ECM where these molecules may attenuate trophoblast invasive behavior.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno/metabolismo , Desenvolvimento Embrionário e Fetal , Útero/metabolismo , Animais , Cromatografia , Combinação de Medicamentos , Feminino , Fibronectinas/metabolismo , Cinética , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos , Proteoglicanas/metabolismoRESUMO
A variety of studies indicate that complex glycoproteins participate in or modulate adhesive interactions occurring during embryo implantation. In particular, proteoglycans and proteins that bind proteoglycans are involved at multiple stages of this process. Identification of these binding proteins and the molecular controls over glycoconjugate expression are required to develop a comprehensive understanding of the implantation process.
Assuntos
Implantação do Embrião/fisiologia , Glicoconjugados/fisiologia , Animais , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Células Epiteliais , Epitélio/fisiologia , Feminino , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/fisiologia , Humanos , Gravidez , Proteoglicanas/fisiologia , Útero/citologia , Útero/fisiologiaRESUMO
Chondroitin sulfate proteoglycans (CSPGs) and hyaluronate have been identified as the predominant glycoconjugates synthesized and secreted by mouse uterine stromal cells (USC) cultured in vitro. CSPGs in both the cell-associated and secreted fractions have identical characteristics with regard to anion exchange chromatographic behavior, sensitivity of the intact molecules and constituent glycosaminoglycans to a variety of chemical and enzymatic digestions, lack of interaction with hydrophobic affinity resins, and density (greater than 1.46 g/ml). Chase labeling studies indicated a metabolic half-life of cell-associated, [35S]sulfate-labeled macromolecules of 5-6 h. Once secreted, CSPGs did not appear to be degraded or endocytosed to a significant extent. In contrast, a large fraction (50%) of the cell-associated CSPGs were degraded to low Mr (less than 3000) products via a chloroquine-sensitive pathway. Studies of the kinetics of intracellular transport indicated that approximately 30 min were required for CSPG core proteins to move from the rough endoplasmic reticulum to the Golgi apparatus and 15-20 min to move from the Golgi to the cell surface, i.e. protease-accessible compartment. There was no significant lag period between the time CSPGs first arrived at the cell surface and the time at which they were first detectable in the medium. Examination of CSPG expression during USC differentiation in utero or in vitro demonstrated that these molecules were produced with similar efficiency by USC under both conditions. Collectively, these studies provide the first comprehensive description of proteoglycan production and metabolism in USC, a uterine cell type intimately involved with embryo implantation processes. Potential functions for CSPGs and hyaluronate as modulators of embryo invasive processes and uterine expansion are proposed.
Assuntos
Proteoglicanas/biossíntese , Útero/metabolismo , Animais , Transporte Biológico , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Endocitose , Retículo Endoplasmático/metabolismo , Feminino , Imunofluorescência , Complexo de Golgi/metabolismo , Técnicas In Vitro , Proteínas de Filamentos Intermediários/metabolismo , Cinética , Camundongos , Proteoglicanas/metabolismo , UltracentrifugaçãoRESUMO
Four ewes were utilized to determine the effects of prostaglandin (PG) F2 alpha, PGE2 and luteinizing hormone (LH) on activity of phospholipase C (PLC) in ovine luteal tissue. Corpora lutea were collected on d 10 post-estrus and six slices from one corpus luteum from each ewe were pre-incubated with [3H]-inositol prior to incubation with one of 6 treatments. Treatments were 1) control, 2) PGF2 alpha (100 ng/ml), 3) PGE2 (10 ng/ml), 4) LH (10 ng/ml), 5) PGF2 alpha + PGE2 and 6) PGF2 alpha + LH. Phospholipase C was determined indirectly by measuring the accumulation of [3H]-inositol mono-, bis- and tris-phosphates (IP, IP2, IP3). Effects of PGF2 alpha (0 vs. PGF2 alpha) and luteotropic treatment (0 vs. PGE2 vs. LH) and their interactions were determined by analysis of variance. There was a significant main effect of PGF2 alpha (P less than 0.01) as concentrations of IP, IP2, IP3 and total [3H]-inositol phosphates were greater in tissue slices treated with PGF2 alpha, regardless of luteotropic treatment. Within groups receiving no PGF2 alpha (1,3,4), no effect of luteotropic treatment was observed. Within groups receiving PGF2 alpha (2,5,6), LH caused a significant (P less than .05) increase in the accumulation of total [3H]-inositol phosphates. Thus, PGF2 alpha can stimulate the activity of PLC in ovine luteal tissue and LH can potentiate this effect.
Assuntos
Corpo Lúteo/enzimologia , Dinoprosta/farmacologia , Dinoprostona/farmacologia , Fosfatos de Inositol/metabolismo , Hormônio Luteinizante/farmacologia , Animais , Corpo Lúteo/efeitos dos fármacos , Feminino , Técnicas In Vitro , Inositol/metabolismo , Fosfatos de Inositol/isolamento & purificação , Cinética , OvinosRESUMO
A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.
Assuntos
Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Útero/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Compartimento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Epitélio/metabolismo , Feminino , Heparina Liase , Camundongos , Polissacarídeo-Liases/farmacologia , Fatores de Tempo , Tripsina/farmacologia , Útero/citologiaRESUMO
Uterine epithelial cells (UEC) isolated from mature mice as well as immature mice and rats were cultured on EHS matrix-coated nitrocellulose filters in order to determine their ability to secrete prostaglandin (PG) F2 alpha and PGE2 in a polarized manner. Ultrastructural analyses were performed to validate the polar nature of mouse UEC and demonstrate the presence of separate apical and basolateral plasma membrane domains. These properties included the presence of tightly juxtaposed lateral membranes, apical microvilli, and a relatively flat basal surface. Biochemical indices of polarity included the preferential (approximately 5:1) basal uptake of [35S]methionine as well as a preferential (approximately 9:1) apical secretion of protein. UEC isolated from mice during the estrous and diestrous stages of the estrous cycle did not differ in their degree of polarity, as measured by these morphological and biochemical indices. UEC of estrous and diestrous mice as well as immature mice and rats preferentially secreted PGF2 alpha to the basal medium to an approximately 4-fold greater extent than to the apical medium. PGE2 was secreted at least 10-fold less than PGF2 alpha, and a preferential basal secretion could not be demonstrated. Polarized UEC accumulated relatively large cellular pools of PGF2 alpha, while nonpolarized cells grown on matrix-coated plastic did not. This difference was reflected by the inability of an inhibitor of PG biosynthesis, indomethacin, to inhibit PGF2 alpha secretion by polarized cells during short (4-h) incubations. In contrast, this drug effectively inhibited secretion in nonpolarized cells or polarized cells incubated with indomethacin for longer (24-h) intervals. Therefore, cellular PGF2 alpha pools apparently support continued secretion of this lipid even when de novo synthesis is transiently inhibited. Preferential basal secretion of PGF2 alpha was due to the polar nature of UEC, since disruption of tight junctions with EGTA modified the basal to apical ratio of PGF2 alpha secretion to near unity. Sodium azide inhibited the secretion of PGF2 alpha, indicating that PGF2 alpha secretion was energy dependent. PGF2 alpha secretion was not coupled to protein synthesis or secretion, since cycloheximide did not inhibit this process in polarized or nonpolarized cells. These studies describe the first evidence for polarized secretion of lipid-derived hormones by epithelial cells. The preferential basal secretion of PGF2 alpha may play an important role in regulating UEC interactions with the underlying stroma.
Assuntos
Prostaglandinas/metabolismo , Útero/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Dinoprosta/metabolismo , Células Epiteliais , Epitélio/metabolismo , Epitélio/ultraestrutura , Feminino , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos , Proteínas/metabolismo , Ratos , Ratos Endogâmicos , Útero/citologia , Útero/ultraestruturaRESUMO
Progesterone was administered in pulses to 12 dairy heifers from days 17.5 to 22.5 post-estrus in order to determine its ability to modify secretion of PGF2 alpha around the time of luteolysis. Control heifers exhibited pulses of PGFM concomitant with a sharp decline in progesterone concentrations and thus these pulses were temporally associated with luteolysis. Additional pulses of PGFM were observed in heifers receiving exogenous progesterone, but these were not statistically predictable by either dose of progesterone (50 or 100 micrograms) or time of administration (3 or 6 hour intervals). However, all heifers (4/4) treated with progesterone at 3 hour intervals had additional pulses of PGFM as compared to only one heifer (1/4) treated at 6 hour intervals. When pulses of PGFM were induced by exogenous progesterone there was a substantial lag time between the initiation of progesterone treatment and their occurrence. The limited response to progesterone administration and the lack of synchrony is not consistent with an ability of exogenous progesterone to directly stimulate secretion of PGF2 alpha at the time of luteolysis.
Assuntos
Dinoprosta/análogos & derivados , Luteólise , Progesterona/farmacologia , Animais , Bovinos , Dinoprosta/metabolismo , Relação Dose-Resposta a Droga , Feminino , Progesterona/administração & dosagemRESUMO
Endogenous concentrations of testosterone increase approximately 7 d prior to estrus in cattle and goats. Inhibition of testosterone synthesis results in a delay of luteal regression in both species. The purpose of this experiment was to determine if treatment with testosterone or 5alpha-dihydrotestosterone (DHT), 2 to 6 d prior to the endogenous rise in testosterone, would result in premature luteal regression. Sixteen heifers were randomly assigned to one of three treatment groups: 1) Control (n = 6); 2) testosterone (100 mug, n = 5); or 3) DHT (100 mug, n = 5). Each heifer received a single injection of the appropriate steriod on Day 8, 9, 10, 11 or 12 post estrus. Jugular venous blood samples were collected at frequent intervals for 24 h to quantify testosterone, and then daily for 14 d to quantify progesterone. Concentrations of testosterone increased within 15 min of injection of testosterone, and reached a maximum at 30 min. Concentrations were maintained at > 2 ng/ml throughout the first 24 h after injection. Based on concentrations of progesterone, neither androgen had any effect on the lifespan of the corpus luteum or the level of luteal function.
RESUMO
Experiments were conducted to determine the role of estrogens on endogenous PGF2 alpha secretion and luteolysis following injection of cloprostenol in heifers. In Exp. 1, eight luteal-phase heifers were used to evaluate tamoxifen (T) as an estrogen antagonist. Heifers received T (35 mg i.v.) or ethanol:saline vehicle (ES) every 4 h for 44 h. All received cloprostenol (500 micrograms i.m.) immediately after the start of T or ES, and received estradiol-17 beta (500 micrograms i.m.) 12 h later. Each ES heifer had a surge of luteinizing hormone (LH) within 48 h of estradiol injection, whereas T-treated heifers did not. Estrus was observed in three ES-treated heifers, but not in T-treated heifers. In Exp. 2, 10 heifers received T (35 mg i.v.) or ES every 4 h for 64 h beginning on d 15 postestrus. Cloprostenol (500 micrograms i.m.) was injected 16 h after the start of treatment. Concentrations of LH were similar (P greater than .05) in both groups. In ES heifers, concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) increased; in T-treated heifers, PGFM remained at pre-cloprostenol levels. Luteolysis was induced in all heifers. Progesterone (P4) decreased to less than or equal to 1 ng/ml at similar (P greater than .05) rates in ES-treated and T-treated heifers. Mean concentration of P4 288 h post-cloprostenol was greater (P less than .05) in ES-treated than in T-treated heifers. Three ES-treated heifers, but no T-treated heifers, were in standing estrus. We conclude that T effectively antagonizes estrogen in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bovinos/fisiologia , Cloprostenol/farmacologia , Corpo Lúteo/efeitos dos fármacos , Luteolíticos/farmacologia , Prostaglandinas F Sintéticas/farmacologia , Prostaglandinas F/metabolismo , Tamoxifeno/farmacologia , Animais , Dinoprosta , FemininoRESUMO
Reductions in dietary fat have been recommended in the prevention of coronary heart disease. Because entrees contribute substantially to total meal fat content, we evaluated a cafeteria-based intervention for increasing the purchase rate of low-fat entrees (M = 6.83 g) relative to nonlow-fat entrees (M = 25.59 g). The intervention included a poster listing the benefits of a LF diet and the daily LF entrees (i.e., broiled or baked chicken and fish dishes). During 6 days per phase, food selections (N = 3,264) were monitored by trained observers. The intervention, which cost $80.00, produced significant increases (i.e., from 20% to 35%) in the purchase rate of LF entrees.