RESUMO
Dental occlusion requires harmonious development of teeth, jaws, and other elements of the craniofacial complex, which are regulated by environmental and genetic factors. We performed the first genome-wide association study (GWAS) on dental development (DD) using the Demirjian radiographic method. Radiographic assessments from participants of the Generation R Study (primary study population, N1 = 2,793; mean age of 9.8 y) were correlated with ~30 million genetic variants while adjusting for age, sex, and genomic principal components (proxy for population stratification). Variants associated with DD at genome-wide significant level (P < 5 × 10-8) mapped to 16q12.2 (IRX5) (lead variant rs3922616, B = 0.16; P = 2.2 × 10-8). We used Fisher's combined probability tests weighted by sample size to perform a meta-analysis (N = 14,805) combining radiographic DD at a mean age of 9.8 y from Generation R with data from a previous GWAS (N2 = 12,012) on number of teeth (NT) in infants used as proxy of DD at a mean age of 9.8 y (including the ALSPAC and NFBC1966). This GWAS meta-analysis revealed 3 novel loci mapping to 7p15.3 (IGF2BP3: P = 3.2 × 10-8), 14q13.3 (PAX9: P = 1.9 × 10-8), and 16q12.2 (IRX5: P = 1.2 × 10-9) and validated 8 previously reported NT loci. A polygenic allele score constructed from these 11 loci was associated with radiographic DD in an independent Generation R set of children (N = 703; B = 0.05, P = 0.004). Furthermore, profiling of the identified genes across an atlas of murine and human stem cells observed expression in the cells involved in the formation of bone and/or dental tissues (>0.3 frequency per kilobase of transcript per million mapped reads), likely reflecting functional specialization. Our findings provide biological insight into the polygenic architecture of the pediatric dental maturation process.
Assuntos
Estudo de Associação Genômica Ampla , Dente , Lactente , Humanos , Criança , Animais , Camundongos , Alelos , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Loci GênicosRESUMO
(1) The rate of palmitate oxidation in the 7800 C1 Morris hepatoma cells was about 60% of the activity observed in hepatocytes. The stimulatory effect of glucagon in hepatocytes was not observed in the hepatoma cells. The rate of fatty acid synthesis from [2-14C]acetate in the hepatoma cells was 1/20 of the activity in hepatocytes. The conversion of [2-14C]acetate to cholesterol was not different in the two kinds of cell. (2) Acetyl-CoA carboxylase and fatty acid synthetase were significantly decreased in the hepatoma cells. The hepatoma cells had, however, raised activities of malate dehydrogenase (decarboxylating), and glucose-6-phosphate and 6-phosphogluconate dehydrogenases. (3) The activities of the enzymes were not affected by different concentrations of glucose or palmitate in the culture medium. Insulin, dexamethasone, triiothyronine and glucagon had no effect on the enzyme activities. This is in contrast to the adaptation of the peroxisomal beta-oxidation system, which is induced by fatty acids and modified by hormones.
Assuntos
Oxirredutases do Álcool/metabolismo , Metabolismo dos Lipídeos , Células Tumorais Cultivadas/enzimologia , Acetil-CoA Carboxilase/metabolismo , Animais , Colesterol/biossíntese , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Glucose/metabolismo , Hormônios/farmacologia , Técnicas In Vitro , Fígado/metabolismo , Masculino , Oxirredução , Palmitatos/metabolismo , Ratos , Ratos EndogâmicosRESUMO
(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obtained with approx. 13 nM dexamethasone. About 80% growth inhibition was obtained with 250 nM of the hormone and the growth rate was normalized on cessation of treatment. (2) These hepatoma cells contain dexamethasone receptors with equilibrium dissociation constant of 0.24 nM and a capacity of 24 fmol/mg cell protein. Treatment of the cells with insulin did not change these dexamethasone binding properties. Binding experiments showed that 2, 10 and 100% of the receptors were occupied when the cells were incubated with 1 nM, 7 nM and 250 nM dexamethasone, respectively. (3) Insulin completely counteracted the growth inhibition by dexamethasone and antagonized the induction of peroxisomal acyl-CoA oxidase and tyrosine aminotransferase caused by the glucocorticoid. (4) Micro-flow fluorometry showed that the cultures had a major diploid DNA stem line and a minor tetraploid stem line. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. Dexamethasone reduced the proportion of cells in S phase and of tetraploid cells. Insulin partly reversed the action of dexamethasone in S phase, but prevented the reduction in tetraploid cells caused by dexamethasone. (5) The mitotic rate was significantly reduced by dexamethasone and this effect was reversed by insulin. (6) Continuous [3H]methyl-thymidine labelling showed a growth fraction of unity in all treatment groups. (7) It is concluded that dexamethasone induces growth inhibition by reducing the G1-S transition. Insulin is able to counteract this effect and increase the rate of DNA synthesis.
Assuntos
Dexametasona/farmacologia , Insulina/farmacologia , Neoplasias Hepáticas Experimentais/patologia , Células Tumorais Cultivadas/citologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , DNA de Neoplasias/análise , DNA de Neoplasias/biossíntese , Dexametasona/antagonistas & inibidores , Dexametasona/metabolismo , Enzimas/metabolismo , Cinética , Ratos , Receptores de Glucocorticoides/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
(1) The relation between the effects of the sulfur-substituted fatty acid analogue, tetradecylthioacetic acid (TTA), dexamethasone and insulin on enzyme induction and growth rate was studied in Morris hepatoma 7800 C1 cells in culture. (2) The activities of the cynanide-insensitive palmitoyl-CoA oxidase and palmitoyl-CoA hydrolase were induced about 2-fold by 50 microM TTA after 72 h of treatment. Catalase was less induced and NADPH-cytochrome-c2 reductase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase were unaffected by the fatty acid analogue. (3) Dexamethasone (250 nM) induced the same enzymes as did TTA, but was a less efficient than 50 microM TTA. However, in combination their effects were more than additive, resulting in 4-7-fold increases. (4) Insulin (400 nM) counteracted the inductive effects of both TTA and dexamethasone on all enzymes except for lactate dehydrogenase, which was induced by the combination of all three compounds. (5) TTA inhibited the growth rate of the cells, and this effect was potentiated by dexamethasone and counteracted by insulin. (6) The enzyme inductions were similar in exponential and plateau phases of growth, indicating that these processes were independently affected by the three compounds.
Assuntos
Carcinoma Hepatocelular/enzimologia , Dexametasona/farmacologia , Insulina/farmacologia , Microcorpos/enzimologia , Sulfetos/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas , Masculino , Microcorpos/efeitos dos fármacos , Oxirredução , Oxirredutases/metabolismo , Palmitoil-CoA Hidrolase/metabolismo , Ratos , Ratos EndogâmicosRESUMO
In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.
Assuntos
24,25-Di-Hidroxivitamina D 3/farmacologia , Adenilil Ciclases/metabolismo , Fosfatase Alcalina/metabolismo , Calcitriol/farmacologia , Osteoblastos/enzimologia , Hormônio Paratireóideo/farmacologia , Animais , Calcitonina/farmacologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/biossíntese , Guanilil Imidodifosfato/farmacologia , Técnicas In Vitro , Ratos , Células Tumorais CultivadasRESUMO
The effects of tetradecylthioacetic acid (TTA) (50 microM), dexamethasone (0.25 microM) and insulin (0.4 microM) on induction of peroxisomal acyl-CoA oxidase activity and mRNA levels were studied in short term cultures of Morris 7800C1 and MH1C1 hepatoma cells and of rat hepatocytes. Dexamethasone and TTA resulted in parallel increases in the enzyme activity and the steady state mRNA content in the hepatoma cells. Combination of dexamethasone and TTA resulted in a synergistic and parallel stimulation of both the enzyme activity and the mRNA levels up to 11-12-fold and maximal changes were observed after 14 days of treatment. Semiquantitative immunoblot analyses of acyl-CoA oxidase were in concordance with enzyme and mRNA results. Insulin counteracted the inductive effects of dexamethasone and TTA on all parameters. The half-life of the acyl-CoA oxidase mRNA increased after treatment with the 3-thia fatty acid (t1/2 = 10.0 h +/- 0.4) compared to control (t1/2 = 5.9 h +/- 0.3). However, in combination with dexamethasone there was no further increase in the mRNA stability (t1/2 = 8.0 h +/- 0.3). Southern blot analysis did not reveal any changes on the oxidase gene level in any treatment group. TTA alone or in combination with dexamethasone did not affect the expression of either the glucocorticoid receptor or the peroxisomal proliferator acting receptor (PPAR) steady state mRNA levels. In cultured hepatocytes the acyl-CoA oxidase was modified in similar manner by these treatments, but the changes were less marked. We suggest that the changes in peroxisomal acyl-CoA oxidase activity in hepatoma cells are due to a major effect on the level of mRNA, involving both transcriptional effects and message stabilization.
Assuntos
Dexametasona/farmacologia , Insulina/farmacologia , Microcorpos/enzimologia , Oxirredutases/biossíntese , Sulfetos/farmacologia , Acil-CoA Oxidase , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Indução Enzimática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Microcorpos/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , RNA Mensageiro/metabolismo , Ratos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the alpha-subunits of the stimulatory and inhibitory G-proteins of AC (Gs alpha and G(i)-2 alpha) in cultured prolactin-producing rat pituitary adenoma cells (GH3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6-48 h) with ionomycin (1 microM) or 1-oleoyl-2-acetylglycerol (OAG; 1 microM) showed that ionomycin regulated Gs alpha levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered Gs alpha levels by more than 50% at all time-points. G(i)-2 alpha levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane Gs protein alpha-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of 'cross-talk' between the PLC- and AC-dependent signalling pathways.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Linhagem Celular , Diglicerídeos/farmacologia , Ionomicina/farmacologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Ratos , Transdução de Sinais , Hormônio Liberador de Tireotropina/farmacologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
MicroRNAs (miRNAs) are an abundant and conserved class of small RNAs, which play important regulatory functions by interacting with the 3' untranslated region (UTR) of target mRNAs. Through this mechanism, miR-223 was shown to regulate genes involved in mammalian haematopoiesis, both in physiological and pathological contexts. MiR-223 is essential for normal myelopoiesis in mammals, promoting granulocyte, osteoclast and megakaryocyte differentiation and suppressing erythropoiesis. However, there is a general lack of knowledge regarding miR-223 function in other vertebrates, which could help to clarify its role in other processes, such as development. In this work, we explored the functional conservation of miR-223 using zebrafish as a model. We show that miR-223 gene structure and genomic context have been maintained between human and zebrafish. In addition, we identified 22 novel sequences of miR-223 precursor and demonstrate that it contains domains highly conserved among vertebrates, suggesting function preservation throughout evolution. Furthermore, collected evidences show that miR-223 expression is highly correlated with haematopoietic events and osteoclastogenesis throughout zebrafish development. In adults, expression of miR-223 in zebrafish tissues mimics the distribution in mice, with high levels found in the major fish haematopoietic organ, the head kidney. These results suggest a conservation of miR-223 role in haematopoiesis, and osteoclastogenesis between zebrafish and human. Accordingly, validated targets of miR-223 in mammalian models were investigated and defined as putative targets in zebrafish, by in silico and gene expression analysis. Our data compiles critical evidence showing that miR-223, a highly conserved miRNA, appears to have kept similar regulatory functions throughout evolution.
Assuntos
MicroRNAs/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Clonagem Molecular , Evolução Molecular , Hematopoese , Humanos , Mamíferos/genética , Mamíferos/metabolismo , MicroRNAs/metabolismo , Osteoclastos/citologia , FilogeniaRESUMO
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5' end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10(-10)-10(-9) M) of human PTH 1-84 [hPTH(1-84)] and hPT(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.
Assuntos
Lâmina de Crescimento/metabolismo , Proteínas de Grupo de Alta Mobilidade/biossíntese , Osteoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Proteínas/metabolismo , Transativadores/biossíntese , Animais , Desenvolvimento Ósseo , Regulação da Expressão Gênica , Lâmina de Crescimento/embriologia , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Camundongos , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Fatores de Transcrição SOXC , Transativadores/genética , Fatores de TranscriçãoRESUMO
We have produced in yeast two human parathyroid hormone (hPTH) analogs with amino-terminal deletions, hPTH(3-84) and hPTH(4-84), employing the mating factor alpha (MF alpha) expression system. The authenticity of the polypeptides was demonstrated by amino-terminal analysis, amino acid composition, and molecular mass analysis. In cells (LLC-PK1) transfected with the human PTH/parathyroid hormone-related protein (PTHrP) receptor, using [125I-Tyr36]chickenPTHrP(1-36)NH2 as radioligand, binding studies revealed dissociation constants at equilibrium (Kd) for hPTH(3-84) and hPTH(4-84) of 4.7 and 8.0 nM, respectively, only slightly higher than natural recombinant hPTH(1-84) Kd = 2.3 nM). In comparison, [Nle8,18,Tyr34]bovinePTH(3-34)NH2 and [Tyr36]cPTHrP(1-36)NH2 showed equal Kd's of 1.9 nM. Neither of the N-terminally deleted hPTH analogs showed any detectable stimulation of cAMP production in the cells at concentrations below 20 nM. At supersaturated concentrations (500 nM) with receptor occupancy of more than 95% these hPTH analogs revealed about 15% rest agonism compared with that of hPTH(1-84). hPTH(1-84) and [Tyr36]cPTHrP(1-36)NH2 showed an equal half maximal cyclic adenosine monophosphate (cAMP) stimulation of about 0.8 and 0.7 nM, respectively. The hPTH analogs did not show any ability to antagonize cellular cAMP production induced by either hPTH or [Tyr36]cPTHrP(1-36)NH2. [Nle8,18,Tyr34]bPTH(3-34)NH2 did also not antagonize cAMP stimulation by hPTH, but inhibited [Tyr36]cPTHrP(1-36)NH2-induced cAMP production by 40% when present at a 1000 M excess. These distinct results related to PTH and PTHrP from different species are important to consider in experiments evaluating potential hPTH or PTHrP antagonism, and employment of a hPTH/PTHrP receptor model is a requirement.
Assuntos
AMP Cíclico/metabolismo , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Hormônios Paratireóideos/metabolismo , Aminoácidos/análise , Animais , Ligação Competitiva , Bovinos , Galinhas , Cromatografia Líquida de Alta Pressão , Humanos , Ligantes , Fator de Acasalamento , Modelos Químicos , Hormônio Paratireóideo/genética , Fragmentos de Peptídeos/genética , Peptídeos/metabolismo , Feromônios/metabolismo , Ensaio Radioligante , Proteínas Recombinantes/análise , Saccharomyces cerevisiae , TransfecçãoRESUMO
Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA 3' splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1-139) and PTHrP(1-141) in both cell lines. PTHrP(1-139) mRNAs predominated in TPXM cells and PTHrP(1-173) mRNAs were only detected in TPXM cells. Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA. In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 microM forskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells. The different responsiveness to agents activating PKA- and PKC-dependent pathways may depend on the cellular state of differentiation, or alternatively, cancer cell line-specific defects. Our data demonstrating distinct differences in mRNA species and the amounts of PTHrP produced by the two cell lines as compared with roughly equivalent overall mRNA levels may suggest that post-transcriptional mechanisms play an important role in limiting the production of intracellular and secreted PTHrPs in human osteoblastic cells.
Assuntos
Proteínas de Neoplasias/fisiologia , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Hormônio Paratireóideo/fisiologia , Proteínas/fisiologia , Colforsina/farmacologia , Éxons , Humanos , Proteínas de Neoplasias/genética , Osteoblastos/citologia , Osteossarcoma/patologia , Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo , Proteínas/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
The effects of prostaglandins E1, E2, F1alpha, and F2alpha on prolactin and growth hormone synthesis and secretion were studied in cultured rat pituitary cells. Total extracellular accumulation of prolactin was measured by radioimmunoassay. When hormone synthesis was studied, the cells were cultured in the presence of [3H]leucine for the last hour of each treatment period. The intra- and extracellular radioactive hormones were determined in the same microsample by a specific and quantitative immunoprecipitation method, employing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The results show that all prostaglandins stimulated the extracellular accumulation of prolactin at 3-30 nM. Prostaglandin E1 was more effective in stimulating extracellular accumulation of prolactin than prostaglandin E2 and the F compounds. Prostaglandin F2alpha at 3 nM doubled the rate of prolactin synthesis, but had no effect on growth hormone or total cell protein synthesis after 24 h of treatment. The first effect of prostaglandin F2alpha was observed after 5 h of incubation, and the time-course of effect on prolactin synthesis was similar to that of thyrotropin-releasing hormone.
Assuntos
Hormônio do Crescimento/biossíntese , Hipófise/metabolismo , Prolactina/biossíntese , Prostaglandinas E/farmacologia , Prostaglandinas F/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Hormônio do Crescimento/metabolismo , Hipófise/citologia , Prolactina/metabolismo , Biossíntese de Proteínas , Ratos , Estimulação Química , Hormônio Liberador de Tireotropina/farmacologiaRESUMO
A clonal strain of rat pituitary tumor cells (GH3) was used to study the effects of different sex steroids on the production of prolactin (PRL). Hormone production was measured by radioimmunoassay and expressed as the amount of hormone which accumulated in the medium of monolayer cultures during 24 h. The stimulatory effect of 17beta-estradiol (10(-11)M-10(-6)M) on PRL production was significant after 4 days and the maximum effect (300% of control cultures) was observed at 10(-8)M after 10 days of treatment. After removal of added 17beta-estradiol, the production of PRL returned to control levels in 5 days. Progesterone (10(-11)M-10(-6)M) caused a dose-related decrease in PRL production reaching 60% of control values at 10(-6)M. Testerone (10(-6)M) stimulated the production of PRL (130% of controls), whereas 5alpha-dihydrotestosterone (10(-6)M) had a small effect (107% of controls) which was not always reproducible. None of the sex steroids affected cell growth. Progesterone (10(-6)M) inhibited the stimulatory effect of 17beta-estradiol (10(-8)M) on PRL production. The effect of 17beta-estradiol (10(-8)M) and thyrotropin releasing hormone (TRH) (3 X 10(-7) M) was addititive, while no additional stimulatory effect was observed when 17beta-estradiol (10(-8)M) was combined with testosterone (10(-6)M). If the properties of the GH3 cells are analogous to those of normal lactotropes, the sex steroids may alter PRL production at the pituitary level, an influence that may be further modulated by TRH.
Assuntos
Hormônios Esteroides Gonadais/farmacologia , Neoplasias Hipofisárias/metabolismo , Prolactina/metabolismo , Linhagem Celular , Di-Hidrotestosterona/farmacologia , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Progesterona/farmacologia , Testosterona/farmacologia , Hormônio Liberador de Tireotropina/farmacologia , Fatores de TempoRESUMO
A specific and sensitive immunoprecipitation method for measurements of biosynthesized radioactive prolactin and growth hormone is described. Antisera to rat prolactin and growth hormone were developed in the rabbit and monkey, respectively. The specificity of the immune sera was assessed by polyacylamide gel electrophoresis of the dissolved immunoprecipitates. The two antisera showed cross-reactions with the nonhomologous hormone of less than 1%. Separation of tritium-labelled prolactin and growth hormone by immunoprecipitation, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate was shown to be 95-57% complete. When both hormones were measured in the same microsample by sequential immunoprecipitation, the reaction was 97% complete for determination of intra- and extracellular prolactin and extracellular growth hormone, but 85% complete for determination of intracellular growth hormone. This method has been used to characterize the basal synthesis and secretion of prolactin and growth hormone in three different but related, pituitary cell strains. Radioactive prolactin and growth hormone was obtained from monolayer cultures when the cells were grown in the presence of [3H]L-leucine. The rate of prolactin synthesis and extracellular accumulation was higher than that of growth hormone in a cell strain which produced both hormones. In these cells prolactin synthesis represents 1-5%, and growth hormone 0.1-0.6% of total protein synthesis.
Assuntos
Hipófise/metabolismo , Prolactina/biossíntese , Animais , Linhagem Celular , Reações Cruzadas , Hormônio do Crescimento/imunologia , Hormônio do Crescimento/metabolismo , Cinética , Leucina/metabolismo , Hipófise/citologia , Testes de Precipitina , Prolactina/imunologia , Prolactina/metabolismo , Biossíntese de Proteínas , RatosRESUMO
Estrogens stimulate PRL synthesis in cultured GH3 cells, which are clonal strains derived from the rat pituitary gland. This model system was used to study the mechanism by which 17 beta-estradiol (E2) enters target cells. The cellular uptake of [3H]E2 was rapid at 37 C and reached half-maximal values within 10-15 sec. Maximal uptake was observed in less than 2 min at 37 C. The initial rates of E2 uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]E2 in intact cells and the binding to cytosol studied at different temperatures resulted in linear Arrhenius plots, and the energies of activation were 39.0 and 33.5 kJ mol-1 degree-1, respectively. Purified GH3 cells membrane fractions, which showed specific binding sites for [3H]TRH, displayed the same maximal binding of [3H]E2 in the absence or presence of cold hormone. The amount of membrane-associated [3H]E2 increased linearly with temperature and extra-cellular hormone concentration. The effect of temperature on binding of E2 to membrane fractions occurred gradually without phase transitions and was not saturable. We suggest that the mechanism by which E2 is taken up by target cells at physiological temperature involves instantaneous dissolution in the cell membrane from where it diffuses passively into the cell. E2 binds thereafter to specific receptors in an energy-dependent step.
Assuntos
Estradiol/metabolismo , Hipófise/efeitos dos fármacos , Neoplasias Hipofisárias/metabolismo , Prolactina/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Dietilestilbestrol/metabolismo , Cinética , Matemática , Hipófise/metabolismo , Ratos , Temperatura , Hormônio Liberador de Tireotropina/metabolismoRESUMO
Thyrotropin-releasing hormone (TRH) has 3 effects on clonal strains of rat pituitary cells in culture (GH-cells). Two long-term effects of TRH on GH-cells, which are measurable after 3 h or longer, have been previously reported; these are an increase in prolactin synthesis and a decrease in growth hormone production. We report here that TRH also stimulates the rapid release of stored intracellular prolactin. We have investigated the role of cyclic AMP as a possible mediator of the effects of TRH on GH-cells. Cyclic AMP concentrations are higher in cells treated with TRH compared with paired controls; a maximum difference of greater than 150% of control values is detected at 15 min if the incubation is performed in serum-free medium in the presence of 1 mM theophylline. The concentration of TRH required to give half-maximum increases in both prolactin release and cyclic AMP accumulation is 0.3 nM; half-maximal increases in prolactin synthesis occur at 3 nM TRH. Exogenous cyclic AMP (1 mM) causes only a slight increase in prolactin release; 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (1 mM) do not cause significant release. Phosphodiesterase inhibitors (0.3 mM theophylline, 0.03 mM isobutyl-methylxanthine) increase prolactin release but their effects on hormone synthesis are more complicated. Isobutylmethylxanthine, 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (0.4 MM) increase prolactin synthesis, but do not significantly affect growth hormone synthesis. Theophylline increases the synthesis of both hormones. Dibutyryl cyclic AMP (0.5 mM or more) increases prolactin release and both growth hormone and prolactin synthesis, but equivalent amounts of sodium butyrate have the same effects. We conclude that in GH-cells under carefully defined experimental conditions: 1) TRH causes an increase in intracellular cyclic AMP concentrations; 2) the increase in endogenous cyclic AMP and the effects of phosphodiesterase inhibitors are consistent with a model with cyclic AMP as a mediator of the effects of TRH on prolactin release; however, they do not prove this model, because the interpretation of these results depends on assumptions which may not all be valid; and 3) none of the analogs of cyclic AMP or the phosphodiesterase inhibitors tested mimic the decrease in growth hormone production caused by TRH.
Assuntos
AMP Cíclico/fisiologia , Hormônio do Crescimento/biossíntese , Adeno-Hipófise/metabolismo , Hipófise/metabolismo , Prolactina/metabolismo , Hormônio Liberador de Tireotropina/farmacologia , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Inibidores de Fosfodiesterase , Prolactina/biossíntese , Ratos , Estimulação QuímicaRESUMO
A full-length cDNA encoding human parathyroid hormone (hPTH) containing the prepro region was cloned into Bombyx mori baculovirus under the control of the polyhedrin promoter and polyadenylation sequences. After transfection and generation of the recombinant baculovirus, hPTH production was examined in silkworm larvae and BmN cell cultures. The larvae synthesized and efficiently secreted the correctly processed and authentic hPTH (9.4 kDa) with no sign of internal degradation. In BmN cells, the major secreted form was the correctly sized protein, but small amounts of degraded hPTH could also be detected in the medium by immunoblotting. Unlike the situation in larvae, prepro-hPTH could also be demonstrated intracellularly in BmN cells. The concentration of hPTH in the larval hemolymph was about 70 mg/l, as compared to approx. 55 micrograms/l in the medium per 7.5 x 10(6) cells. Recombinant hPTH (re-hPTH) from the hemolymph was purified by reverse-phase HPLC and subjected to chemical and biological analyses. The authenticity of the purified re-hPTH was confirmed by N-terminal sequencing, amino acid composition and a mass of 9425 Da, close to the theoretical value. The hormone showed high-affinity receptor binding and full biological potency in increasing cellular cAMP.
Assuntos
Hormônio Paratireóideo/biossíntese , Animais , Baculoviridae , Bombyx/genética , Células Cultivadas , AMP Cíclico/metabolismo , Expressão Gênica , Vetores Genéticos , Hemolinfa/citologia , Humanos , Larva , Hormônio Paratireóideo/química , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Ensaio Radioligante , Ratos , Proteínas Recombinantes/químicaRESUMO
Longitudinal changes in the serum concentration of calcium, phosphate, alkaline phosphatase, PTH, calcitonin and the renal handling of calcium and phosphate were studied in 19 normal women of the same age before and after the menopause. Significant increase in serum calcium, phosphate and calcitonin and urine calcium/creatinine and TmPO4/GF were shown to precede the premenopause. After cessation of the menstruation, no statistically significant further changes were observed in these variables. Changes in PTH were not observed neither during the premenopausal nor the post-menopausal period. Alkaline phosphatase increased in the postmenopausal period suggesting an increase in bone turn-over.
Assuntos
Cálcio/metabolismo , Menopausa/metabolismo , Feminino , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Fosfatos/metabolismoRESUMO
Renal osteodystrophy with increased bone resorption is a major clinical problem in patients with chronic renal failure. Previous reports have shown that treatment with 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) may result in decreased bone resorption. The present study addresses basic mechanisms for the action of 24,25(OH)2D3 in bone of patients with elevated serum parathyroid hormone (PTH) levels due to chronic renal disease. Twenty-four patients 56 +/- 17 years old (mean +/- SE) with chronic kidney disease in the predialytic state (serum creatinine > 150 mumol/l) and elevated serum midregion PTH > 1.2 micrograms/l were randomly assigned to oral treatment with either 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) (0.25-0.50 microgram/day), 24,25(OH)2D3 (daily dose of 15 micrograms), or a combination of the two vitamin D3 analogs. The control group received calcium carbonate (maximal dosage of 1 g x 3). Selected variables in serum and urine as well as hormone sensitive adenylate cyclase (AC) in iliac crest biopsies were assessed before treatment and during follow-up after two and six months. Serum levels of 1,25(OH)2D3 and 24,25(OH)2D3 were significantly (P < 0.05) increased after two and six months in the respective treatment groups. Net bone PTH-enhanced AC (PTH-AC) fell abruptly (P < 0.01) after two months of treatment and was nearly abolished (P < 0.01) after six months with 24,25(OH)2D3 given alone or in combination with 1,25(OH)2D3. An inverse relationship (r = -0.57, P < 0.05, n = 48) between net PTH-AC in bone and serum levels of 24,25(OH)2D3 was demonstrated. In all groups, serum total calcium (s-Ca) was maintained within normal range.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
24,25-Di-Hidroxivitamina D 3/farmacologia , Adenilil Ciclases/metabolismo , Calcitriol/farmacologia , Ílio/efeitos dos fármacos , Hormônio Paratireóideo/sangue , Uremia/enzimologia , 24,25-Di-Hidroxivitamina D 3/sangue , Adulto , Idoso , Reabsorção Óssea/prevenção & controle , Calcitriol/sangue , Carbonato de Cálcio/farmacologia , Feminino , Humanos , Ílio/enzimologia , Falência Renal Crônica/sangue , Masculino , Pessoa de Meia-Idade , Uremia/sangue , Uremia/complicaçõesRESUMO
In GH(1)2C1 rat pituitary cells treated with 5-azacytidine, the stimulatory effects exerted by vasoactive intestinal peptide (VIP), the GTP analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p), 12-O-tetradecanoyl phorbol 13-acetate, cholera toxin and pertussis toxin on the membrane-bound adenylyl cyclase were almost completely abolished. The corresponding inhibitory effect of somatostatin was increased. Alterations in adenylyl cyclase responsiveness began at the end of the drug treatment, and were most pronounced on day 5 after removal of 5-azacytidine. The cells subsequently and completely recovered after 10 days in the absence of the drug. Measurements of cholera toxin- and VIP-enhanced cyclic AMP levels in intact cells confirmed these results, and VIP appeared to have no stimulatory effect on GH secretion after 5-azacytidine treatment. Down-regulation of G alpha s RNA also occurred on day 5 after cessation of drug treatment. ADP-ribosylation subsequent to stimulation with pertussis toxin was markedly increased, indicating an enhancement of G alpha i and/or G alpha o. Furthermore, both basal and Gpp(NH)p-stimulated phospholipase C activities were augmented by pre-exposure to 5-azacytidine. Treatment of GH(1)2C1 rat pituitary tumour cells with 5-azacytidine therefore causes a marked but temporary increase in the ratio of G alpha i/G alpha s protein levels.