RESUMO
Cytotrophoblasts, purified from human term placentae, were cultured in the absence or presence of 8-bromo-cAMP or 8-bromo-cGMP. 8-Bromo-cAMP provoked a dose-dependent increase in the secretion of hCG and progesterone within 24 h. After 48 h, hCG secretion increased by more than 200-fold, and progesterone secretion increased nearly 5-fold. 8-Bromo-cGMP had no effect on hCG secretion. In culture in serum-supplemented medium, the mononuclear cytotrophoblasts aggregated and fused to form syncytia. This morphological transformation was not affected by 8-bromo-cAMP. Immunocytochemical studies of the alpha- and beta-subunits of hCG in control and 8-bromo-cAMP-stimulated cultures demonstrated that the cyclic nucleotide analog promoted the synthesis of both subunits in all cellular forms, including single mononuclear cells, cell aggregates, and syncytia. In serum-free medium, the cytotrophoblasts did not aggregate or form syncytia, yet they responded to 8-bromo-cAMP with an increase in hCG secretion. We conclude that the endocrine function of cytotrophoblasts can be stimulated by a cAMP-dependent mechanism which can be initiated independently of the formation of a syncytium.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Gonadotropina Coriônica/metabolismo , Progesterona/metabolismo , Trofoblastos/metabolismo , Células Cultivadas , Gonadotropina Coriônica/biossíntese , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Trofoblastos/efeitos dos fármacosRESUMO
Augmentation of selective serotonin reuptake inhibitors (SSRIs) therapy by the 5-HT(1A) receptor agent pindolol may reduce the delay between initiation of antidepressant treatment and clinical response. This hypothesis is based on the ability of pindolol to block 5-HT(1A) autoreceptors in the dorsal raphe nuclei (DRN) and to potentiate the increase in 5-HT transmission induced by SSRIs. However, placebo-controlled clinical studies of pindolol augmentation of antidepressant therapy have reported inconsistent results. Here, we evaluated the occupancy of 5-HT(1A) receptors during treatment with pindolol controlled release (CR) in nine healthy volunteers with Positron Emission Tomography and [11C]WAY 100635. Subjects were studied four times: at baseline, following one week of pindolol CR 7.5 mg/day (4 and 10 hrs post dose), and following one dose of pindolol CR 30 mg(4 hrs post dose). Occupancy of the DRN was 40 +/- 29% on scan 2, 38 +/- 26% on scan 3, and 64 +/- 15% on scan 4. The average occupancy in all other regions was significantly lower at each doses (18 +/- 5% on scan 2, 12 +/- 3% on scan 3, and 42 +/- 4% on scan 4). These results suggest that the blockade in the DRN reached in clinical studies (7.5 mg/day) might be too low and variable to consistently augment the therapeutic effect of SSRIs. However, these data indicate that pindolol exhibits in vivo selectivity for the DRN 5-HT(1A) autoreceptors. As DRN selectivity is desirable for potentiation of 5-HT function, this observation represents an important proof of concept for the development of 5-HT(1A) agents in this application.
Assuntos
Transtornos do Humor/tratamento farmacológico , Pindolol/farmacologia , Piperazinas/uso terapêutico , Piridinas/uso terapêutico , Núcleos da Rafe/metabolismo , Receptores de Serotonina/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Adulto , Antidepressivos/uso terapêutico , Encéfalo/efeitos dos fármacos , Humanos , Cinética , Imageamento por Ressonância Magnética , Masculino , Pindolol/administração & dosagem , Pindolol/sangue , Pindolol/uso terapêutico , Piperazinas/sangue , Piperazinas/farmacocinética , Piridinas/sangue , Piridinas/farmacocinética , Receptores de Neurotransmissores/fisiologia , Receptores 5-HT1 de Serotonina , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Transmissão Sináptica/fisiologia , Tomografia Computadorizada de EmissãoRESUMO
A series of xanthine sulfonamides is presented as a class of calcitonin (CT) inducers - a potentially new method for treating diseases associated with postmenopausal bone loss such as osteoporosis. We have found that certain di-n-butylxanthine sulfonamides 4 upregulate CT transcription in a CT-luciferase reporter gene assay (CT-luci) and increase the production and release of CT in a CT secretion/RIA assay (CTS). In addition, these compounds do not have potent PDE4 inhibitory activity as do the related xanthine methylene ketones such as denbufyllene (2). One compound in particular (9) shows a transcription activation ratio (TAR) of 2.1 in CT-luci, a CTS increase of 3.6-fold, and a PDE4 (phosphodiesterase type IV) IC(50) = 4.1 microM. In addition, this compound showed a statistically significant 47% trabecular bone protection in ovariectomized-induced osteopenia (OVX) rats as determined by assay when administered for 4 weeks at 30 mg/kg/day, i. p. by quantitative computed tomography (QCT). When administered p.o., compound 9 shows 50% trabecular bone protection when administered for 3 weeks at 50 mg/kg/day, i.p. This compared with salmon CT which shows 62% trabecular bone protection when administered at 50 IU/kg/day for 4 weeks.
Assuntos
Calcitonina/biossíntese , Sulfonamidas/síntese química , Xantinas/síntese química , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/tratamento farmacológico , Doenças Ósseas Metabólicas/patologia , Calcitonina/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Genes Reporter , Humanos , Luciferases/genética , Ovariectomia , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Transcrição Gênica , Xantinas/química , Xantinas/farmacologiaRESUMO
The ability of full-length human recombinant osteopontin (OPN) to support the adhesion of various alphav integrin-expressing cell lines was determined in order to characterize its integrin selectivity. The identity of this protein was assessed by cDNA sequence and mass spectroscopic analysis, and confirmed as full-length OPN. Neither the human embryonic kidney 293 cell line, which expresses the alphavbeta1 integrin, nor the human colonic adenocarcinoma HT-29 cell line, which expresses the alphavbeta5 integrin, were able to adhere to OPN; both of these cell lines are deficient in the beta3 subunit. In contrast, an alphavbeta3 integrin-expressing cell line, SK-MEL-24, was able to adhere to OPN in an arginine-glycine-aspartic acid dependent manner. In addition, this OPN-mediated cellular adhesion was completely blocked with an anti-alphavbeta3 integrin antibody (LM609), confirming that only the alphavbeta3 integrin mediated this cellular adhesion. These data demonstrate that, at least among the alphav integrins, only the alphavbeta3 is able to support cellular adhesion to osteopontin. This finding may have implications for the design of therapeutics targeting OPN-integrin interactions.
Assuntos
Adesão Celular/efeitos dos fármacos , Receptores de Vitronectina/metabolismo , Sialoglicoproteínas/farmacologia , Linhagem Celular , Matriz Extracelular/fisiologia , Células HT29 , Humanos , Oligopeptídeos/fisiologia , Osteopontina , Conformação Proteica , Receptores de Vitronectina/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sialoglicoproteínas/metabolismo , Células Tumorais CultivadasRESUMO
Interpretation of studies designed to investigate the inhibitory action of vasopressin on gastric acid secretion has proven difficult, as the in vivo models are potentially susceptible to both direct (e.g. mucosal effects) and indirect effects (e.g. changes in mucosal blood flow). In the present series of experiments we studied vasopressin inhibition of both basal and histamine-stimulated acid secretion in rat isolated gastric mucosa, a preparation which is independent of blood flow. Basal and histamine-stimulated levels of acid secretion were 2.32 +/- 0.10 (mean +/- S.E.) and 4.36 +/- 0.41 mu eqH+/h per cm2. Vasopressin inhibited both basal and histamine-stimulated acid secretion; the effect, which was maximal at 15 min post-dosing, was blocked by the specific V1 antagonist d(CH2)5Tyr(Me) AVP. No effect on acid secretion was evident with either the potent V2 agonist, dDAVP, or oxytocin, a neurohypophyseal hormone which can also affect water retention and blood pressure. These studies demonstrate that vasopressin can specifically inhibit mucosal acid secretion; the inhibitory effect is most likely mediated via a V1 vasopressin receptor subtype on the gastric mucosa.
Assuntos
Ácido Gástrico/metabolismo , Vasopressinas/farmacologia , Animais , Bucladesina/farmacologia , Depressão Química , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Histamina/farmacologia , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Preclinical studies in rodents suggest that augmentation of serotonin reuptake inhibitors (SSRIs) therapy by the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor agent pindolol might reduce the delay between initiation of treatment and antidepressant response. This hypothesis is based on the ability of pindolol to potentiate the increase in serotonin (5-HT) transmission induced by SSRIs, an effect achieved by blockade of the 5-HT(1A) autoreceptors in the dorsal raphe nuclei (DRN). However, placebo-controlled clinical studies of pindolol augmentation of antidepressant therapy have reported inconsistent results. Here, we evaluated the occupancy of 5-HT(1A) receptors following treatment with controlled release pindolol in nine healthy volunteers with positron-emission tomography (PET). Each subject was studied four times: at baseline (scan 1), following 1 week of oral administration of pindolol CR (7.5 mg/day) at peak level, 4 h after the dose (scan 2), and at 10 h following the dose (scan 3), and following one dose of pindolol CR (30 mg) (at peak level, 4 h) (scan 4). Pindolol occupancy of 5-HT(1A) receptors was evaluated in the DRN and cortical regions as the decrease in binding potential (BP) of the radiolabelled selective 5-HT(1A) antagonist [carbonyl-(11)C]WAY-100635 or [carbonyl-(11)C] N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)cyclohexa necarboxamide abbreviated as [(11)C]WAY-100635. Pindolol dose-dependently decreased [(11)C]WAY-100635 BP. Combining all the regions, occupancy was 20 +/- 8% at scan 2, 14 +/- 8% at scan 3, and 44 +/- 8% at scan 4. The results of this study suggest that at doses used in clinical studies of augmentation of the SSRI effect by pindolol (2.5 mg t.i.d.), the occupancy of 5-HT(1A) receptors is moderate and highly variable between subjects. This factor might explain the variable results obtained in clinical studies. On the other hand, at each dose tested, pindolol occupancy of 5-HT(1A) receptors was higher in the DRN compared to cortical regions, demonstrating a significant in vivo selectivity for DRN 5-HT(1A) autoreceptors relative to cortico-limbic postsynaptic receptors. This selectivity is necessary for the potentiation of 5-HT transmission, and this finding represents an important proof of concept in the development of 5-HT(1A) agents for this application. Early evaluation of new drugs with PET imaging will enable rapid screening of compounds based on DRN selectivity and more appropriate determination of doses for clinical trials.
Assuntos
Pindolol/metabolismo , Receptores de Serotonina/análise , Antagonistas da Serotonina/metabolismo , Tomografia Computadorizada de Emissão , Adulto , Química Encefálica , Humanos , Masculino , Pessoa de Meia-Idade , Núcleos da Rafe/química , Receptores 5-HT1 de SerotoninaAssuntos
Diurese/efeitos dos fármacos , Receptores de Vasopressinas/efeitos dos fármacos , Vasopressinas/antagonistas & inibidores , Animais , Arginina Vasopressina/análogos & derivados , Arginina Vasopressina/farmacologia , Ligação Competitiva , Desamino Arginina Vasopressina/farmacologia , Ratos , Ratos Endogâmicos BB , Ratos Sprague-Dawley , Especificidade da Espécie , Regulação para CimaRESUMO
Chronic administration of vasopressin [antidiuretic hormone (ADH)] antagonists has been shown to produce a paradoxical antidiuresis in both ADH-replete and ADH-deplete (diabetes insipidus) rats. The antidiuretic effect is progressive, reaching maximal levels in 4 to 5 days, and sustained, persisting for at least 24 hr after cessation of treatment. The antidiuretic profiles associated with these antagonists do not coincide with the profiles of potent ADH agonists, arginine vasopressin and 1-deamino-8-D-arginine vasopressin. To investigate the mechanism of the antidiuretic effect of ADH antagonists, male diabetes insipidus rats were administered antagonists selective for the renal [adenylate cyclase-coupled (V2)] or pressor (phosphytidyl inositol-coupled) vasopressin receptor and urine output (volume and osmolality) and renal vasopressin receptor properties (concentration and affinity) were determined and compared to rats treated with arginine vasopressin or 1-deamino-8-D-arginine vasopressin. After acute administration, only the V2-acting antagonists were antidiuretic, but were 3 orders of magnitude less potent than 1-deamino-8-D-arginine vasopressin. Following chronic administration, all of the antagonists were antidiuretic, but the level of antidiuresis achieved with the phosphytidyl inositol-coupled vasopressin receptor-selective antagonist was 2- to 3-fold lower than for analogs with V2 activity. Maximal antidiuretic effects were realized in 5 days and were apparent at 24 hr after cessation of treatment. The antidiuretic activities and potencies of the ADH antagonists appeared to be increased following chronic antagonist administration. Finally, renal vasopressin receptor concentration was significantly elevated 24 hr after cessation of antagonist treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenilil Ciclases/fisiologia , Rim/enzimologia , Receptores de Angiotensina/fisiologia , Regulação para Cima/efeitos dos fármacos , Adenilil Ciclases/efeitos dos fármacos , Antagonistas de Receptores de Angiotensina , Animais , Arginina Vasopressina/farmacologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Rim/efeitos dos fármacos , Masculino , Ratos , Ratos Brattleboro , Receptores de Angiotensina/metabolismo , Receptores de Vasopressinas , Regulação para Cima/fisiologia , Urodinâmica/efeitos dos fármacos , Vasopressinas/antagonistas & inibidores , Vasopressinas/farmacologiaRESUMO
Human syncytiotrophoblasts are derived from villous cytotrophoblasts by cell fusion. Coincident with this morphologic transformation, trophoblasts acquire specific endocrine functions, including elaboration of chorionic gonadotropin (hCG). We wondered if syncytia formation was a prerequisite for biochemical differentiation or simply was one part of the differentiation program. By growing purified human cytotrophoblasts under serum-free conditions and manipulating the culture surface, we were able to disassociate morphologic from biochemical differentiation. We have shown previously (Endocrinology 1986, 118:1567) that human cytotrophoblasts grown in the presence of fetal calf serum flatten out, aggregate, and form functional syncytiotrophoblasts in vitro over 24-96 hr. Here we demonstrate that when grown in the absence of serum, the cells do not undergo these morphologic changes, but remain as individual spherical cells. If the culture surface was precoated with fibronectin or a variety of collagens, but not albumin, the cells regained their ability to flatten, aggregate, and form syncytia. Attachment to and syncytia formation on fibronectin was blocked by the addition of the R-G-D-S-containing peptide, Gly-Arg-Gly-Asp-Ser-Pro. Attachment to and syncytia formation on type I collagen was blocked by anti-human fibronectin F(ab')2 fragments, while association with type IV collagen was not affected by this antibody, suggesting that fibronectin mediates trophoblast association with type I collagen, but not type IV. Although syncytia formation did not occur when cytotrophoblasts were cultured under serum-free conditions in the absence of ECM proteins, biochemical differentiation was not affected. These cells secreted hCG at the same rate under serum-free conditions whether they were plated on plastic only--which prevented syncytia formation--or fibronectin, laminin or, type IV collagen--which allowed syncytia formation to occur. Furthermore, cytoplasmic differentiation in the absence of syncytia formation was confirmed by performing transmission electron microscopy on cytotrophoblasts grown under serum-free conditions in the presence of 8-bromo-cAMP. We conclude that syncytia formation is not a prerequisite for biochemical differentiation, but simply part of the trophoblast differentiation program.
Assuntos
Adesão Celular , Matriz Extracelular/fisiologia , Receptores de Superfície Celular/fisiologia , Trofoblastos/fisiologia , Diferenciação Celular , Fusão Celular , Células Cultivadas , Gonadotropina Coriônica/metabolismo , Colágeno/metabolismo , Meios de Cultura , Fibronectinas/metabolismo , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Trofoblastos/citologiaRESUMO
Linear vasopressin analogs lacking a cyclic hexapeptide ring have recently been reported to possess vasopressin antagonist activity. In conscious, chronically catheterized, euhydrated Sprague-Dawley rats, we have compared the effects of two noncyclic vasopressin analogs, peptide 1 ([1-admantaneacetic acid,2-(O-ethyl)-D-tyr,4-val,6-(2-aminobutyric acid),9-arg]arginine vasopressin) and peptide 2 ([1-propionic acid, 2-(O-ethyl)-D-tyr,4-val,6-(2-aminobutyric acid),9- arg]arginine vasopressin), with a cyclic arginine vasopressin antagonist (SK&F 105494; [1-des cysteine, cyclo(2-O-ethyl-D-tyrosine,6-L-(2-amino-6,6-cyclopentamethylene suberic acid], 4-valine,7-arginine,8-D-arginine, 9-des glycine]-vasopressin). All three analogs caused a dose-dependent increase in urine flow by increasing free-water clearance without significantly changing osmotic clearance or sodium excretion, indicating true functional vasopressin antagonism. Peptides 1 and 2 were as efficacious in inducing a diuresis as SK&F 105494. The order of diuretic potency among the three analogs in vivo was the same as the order of potency determined by in vitro binding to rat renal membrane homogenates, suggesting that the analogs exerted their diuretic effect by acting at renal vasopressin receptors. Thus, noncyclic vasopressin analogs, which are easier to synthesize then cyclic structures, could provide new strategies in the design of drugs for the treatment of water balance disorders.
Assuntos
Arginina Vasopressina/análogos & derivados , Diuréticos/farmacologia , Animais , Arginina Vasopressina/antagonistas & inibidores , Arginina Vasopressina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos EndogâmicosRESUMO
Physiological regulation of receptor-effector pathways is recognized as a significant factor determining target organ selectivity and sensitivity in several hormonal systems. Whether or not physiological regulation of the renal vasopressin (V2) receptor-effector pathway participates in the control of body fluid homeostasis is unknown. We evaluated four states likely to be associated with altered sensitivities of the renal V2 receptor-effector pathway as follows: dehydration (18-h hydropenia), volume expansion, exogenous arginine vasopressin (AVP) infusion (10 ng/kg + 0.25 ng.kg-1.h-1), and cyclooxygenase blockade (indomethacin, 2 mg/kg + 2 mg.kg-1.h-1) for effects on the antidiuretic efficacies and potencies of putative V2-receptor antagonists in conscious dogs. The antidiuretic efficacies of desGly9[Pmp1-D-Tyr(Et)2Val4]AVP [Smith Kline & French (SK&F) 101926; 0.01-1,000 micrograms/kg] ranged from that of a full agonist to that of an antagonist, depending on the physiological state studied. The vasopressin antagonist potency of SK&F 101926 was increased 150-fold in association with extracellular volume expansion and decreased by blockade of renal cyclooxygenase activity. This spectrum of activities is that anticipated for a partial agonist under conditions where receptor number and/or sensitivity of receptor-effector coupling is increased or decreased, respectively. Thus volume expansion and increased circulating vasopressin concentration are associated with effective decreases, whereas hydropenia and cyclooxygenase blockade are associated with effective increases in sensitivity of the renal V2 receptor-effector pathway in the dog kidney. We conclude that the V2 receptor-effector pathway is a site of integration of physiological mechanisms participating in the control of body fluid homeostasis in conscious dogs.