RESUMO
To improve culture system for in vitro maturation (IVM) of porcine oocytes, ghrelin, leptin or growth hormone (GH), at concentration of 0, 0.5, 5, 50 and 500 ng/ml were added to the porcine follicular fluid (pFF)-supplemented medium NCSU23, and their effects on the maturation and cytoskeletal distribution of the oocytes with or without cumulus cells were compared. In the cumulus-denuded oocytes, no significant changes were noted in the maturation rate by different hormone treatments due to a marked decline in the controls. Maturation of the cumulus intact oocytes was moderately interfered by ghrelin (0.5-50 ng/ml, p < 0.01), but not significantly affected by leptin and GH. Distribution density of the cytoplasmic microtubules was decreased significantly by addition of ghrelin (by approximately 30% in 50-500 ng/ml, p < 0.01), whereas no remarkable effect was noted by leptin supplementation. High concentration (500 ng/ml) of ghrelin or leptin decreased significantly the cytoplasmic microfilaments in density (by 43% and 38%, p < 0.01, respectively). GH did not affect cytoskeletal distribution. The results suggest, in the culture system using pFF-supplemented medium that (i) ghrelin may have some inhibitory effect on the organization of microtubules and microfilaments, probably being a factor in lowered maturation rate and (ii) the addition of higher concentration of leptin may decrease microfilaments in density with no effect on meiotic maturation of the porcine oocytes.
Assuntos
Técnicas de Cultura de Células/veterinária , Grelina/farmacologia , Leptina/farmacologia , Meiose/efeitos dos fármacos , Oócitos/fisiologia , Suínos/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Meios de Cultura , FemininoRESUMO
We have recently demonstrated that salsolinol (SAL), a dopamine (DA)-derived compound, is present in the posterior pituitary gland and is able to stimulate the release of prolactin (PRL) in ruminants. The aim of the present study was to clarify the effect that the interaction of SAL with thyrotropin-releasing hormone (TRH) or DA has on the secretion of PRL in ruminants. A single intravenous (i.v.) injection of SAL (5mg/kg body weight (b.w.)), TRH (1microg/kg b.w.), and SAL plus TRH significantly stimulated the release of PRL in goats (P<0.05). The cumulative response curve (area under the curve: AUC) during 120min was 1.53 and 1.47 times greater after the injection of SAL plus TRH than either SAL or TRH alone, respectively (P<0.05). A single i.v. injection of sulpiride (a DA receptor antagonist, 0.1mg/kg b.w.), sulpiride plus SAL (5mg/kg b.w.), and sulpiride plus TRH (1microg/kg b.w.) significantly stimulated the release of PRL in goats (P<0.05). The AUC of PRL during 120min was 2.12 and 1.78 times greater after the injection of sulpiride plus TRH than either sulpiride alone or sulpiride plus SAL, respectively (P<0.05). In cultured bovine anterior pituitary (AP) cells, SAL (10(-6)M), TRH (10(-8)M), and SAL plus TRH significantly increased the release of PRL (P<0.05), but the additive effect of SAL and TRH detected in vivo was not observed in vitro. In contrast, DA (10(-6)M) inhibited the TRH-, as well as SAL-induced PRL release in vitro. All together, these results clearly show that SAL can stimulate the release of PRL in ruminants. Furthermore, they also demonstrate that the additive effect of SAL and TRH on the release of PRL detected in vivo may not be mediated at the level of the AP, but that DA can overcome their releasing activity both in vivo and in vitro, confirming the dominant role of DA in the inhibitory regulation of PRL secretion in ruminants.
Assuntos
Bovinos/fisiologia , Dopamina/administração & dosagem , Cabras/fisiologia , Isoquinolinas/administração & dosagem , Prolactina/metabolismo , Hormônio Liberador de Tireotropina/administração & dosagem , Animais , Células Cultivadas , Antagonistas de Dopamina/administração & dosagem , Interações Medicamentosas , Feminino , Injeções Intravenosas , Masculino , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Sulpirida/administração & dosagemRESUMO
The aims of the present study were to determine whether salsolinol (SAL), a dopamine-related compound, is present in the bovine posterior pituitary (PP) gland, and to clarify the effect of SAL on the secretion of prolactin (PRL) in ruminants. SAL was detected in extract of bovine PP gland using high-pressure liquid chromatography with electrochemical detection (HPLC-EC). A single intravenous (i.v.) injection of SAL (5 and 10mg/kg body weight) significantly and dose-dependently stimulated the release of PRL in goats (P<0.05). Plasma PRL levels reached a peak 10min after the injection, then gradually returned to basal values in 60-80min. The PRL-releasing pattern was similar to that in response to sulpiride (a dopamine receptor antagonist). The intracerebroventricular (i.c.v.) injection of 1mg of SAL had no significant effect on the release of PRL in calves, however, 5mg significantly stimulated the release (P<0.05) with peak values reached 30-40min after the injection. Moreover, SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 10(-6) and 10(-5)M, compared to control cells (P<0.05). Taken together, our data clearly show that SAL is present in extract of the PP gland of ruminants, and has PRL-releasing activity both in vivo and in vitro. Therefore, this endogenous compound is a strong candidate for the factor having PRL-releasing activity that has been previously detected in extract of the bovine PP gland.
Assuntos
Bovinos/fisiologia , Cabras/fisiologia , Isoquinolinas/metabolismo , Neuro-Hipófise/fisiologia , Prolactina/metabolismo , Animais , Antagonistas de Dopamina/farmacologia , Feminino , Isoquinolinas/farmacologia , Masculino , Adeno-Hipófise/fisiologia , Neuro-Hipófise/metabolismo , Prolactina/sangue , Sulpirida/farmacologiaRESUMO
Kisspeptins are peptide hormones encoded by the KiSS-1 gene and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. However, peripheral administration, as well as central administration, of kisspeptin stimulates luteinizing hormone (LH) secretion in some mammalian species. In order to evaluate the direct effects of kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) on LH secretion from bovine and porcine anterior pituitary (AP) cells, LH-releasing effects of kisspeptin-10 on AP cells were compared with GnRH in vitro. The AP cells were prepared from 1-month-old intact male calves, 8-month-old castrated male calves, or 6-month-old barrows, and then the cells were incubated for 2h with the peptides. The 1000 nM and 10,000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from the bovine AP cells (P<0.05). The 100 nM and 1000 nM, but not lower concentrations, of kisspeptin-10 significantly stimulated LH secretion from porcine AP cells (P<0.05). As 10nM of GnRH strongly stimulated LH secretion from all AP cells tested in this study, the present results suggest that kisspeptin-10 has a direct, but weak, stimulating effect on LH secretion in bovine and porcine AP cells. The present study is the first to examine the direct actions of kisspeptin on the bovine and porcine pituitary gland as far as we know. Kisspeptin might have other actions on the pituitary because the pituitary has multiple roles.
Assuntos
Bovinos/fisiologia , Hormônio Luteinizante/metabolismo , Oligopeptídeos/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Suínos/fisiologia , Animais , Células Cultivadas , Kisspeptinas , Masculino , Adeno-Hipófise/citologiaRESUMO
Kisspeptins are peptide hormones encoded by the KiSS-1 gene, and act as the principal positive regulator of the reproductive axis by directly stimulating gonadotropin-releasing hormone (GnRH) neuron activity. We recently observed that kisspeptin-10 (the minimal kisspeptin sequence necessary for receptor activation) also has a direct stimulating effect on luteinizing hormone (LH) secretion in bovine anterior pituitary (AP) cells. In the present study, we evaluated the direct effect of kisspeptin-10 on the secretion of other pituitary hormones, growth hormone (GH) and prolactin (PRL), from bovine AP cells. The AP cells, which were prepared from 1- or 8-month-old male calves, were incubated for 2h with the peptides. Kisspeptin-10 at 100 nM (P<0.05), 1000 nM (P<0.01) and 10,000 nM (P<0.01), but not at 10 nM, significantly stimulated GH secretion from the AP cells of 1-month-old calves, while in 8-month-old calves it was significantly (P<0.05) stimulated at 1000 nM (P<0.01) and 10,000 nM (P<0.01), but not at 10nM and 100 nM. The response of GH to 100 nM (P<0.01), 1000 nM (P<0.05) and 10,000 nM (P<0.01) kisspeptin-10 in the AP cells of 1-month-old calves was significantly greater than in those of 8-month-old calves. All tested doses of kisspeptin-10 had no effect on PRL secretion from AP cells of 1-month-old calves. However, 1000 nM (P<0.05) and 10,000 nM (P<0.01), but not lower concentrations, of kisspeptin-10 significantly stimulated PRL secretion from the AP cells of 8-month-old calves. The present study is, as far as we know, the first to examine the direct actions of kisspeptin on the secretion of GH and PRL from the bovine pituitary gland. Further studies are necessary to evaluate the importance of multiple actions of kisspeptin on the pituitary of various animals in vivo.
Assuntos
Hormônio do Crescimento/metabolismo , Oligopeptídeos/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Animais , Bovinos , Células Cultivadas , Kisspeptinas , Masculino , Adeno-Hipófise/citologia , Taxa Secretória/efeitos dos fármacosRESUMO
N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.
Assuntos
Ácidos Araquidônicos/antagonistas & inibidores , Plaquetas/metabolismo , Cálcio/sangue , Colágeno/antagonistas & inibidores , Etilmaleimida/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Animais , Ácido Araquidônico , Ácidos Araquidônicos/sangue , Transporte Biológico/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Calcimicina/farmacologia , Membrana Celular/metabolismo , Citoplasma/metabolismo , Lipídeos de Membrana/sangue , Ácidos Fosfatídicos/sangue , Fosfolipídeos/sangue , Coelhos , Trombina/farmacologiaRESUMO
cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.
Assuntos
Plaquetas/fisiologia , Ácidos Graxos Insaturados/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Tromboxano B2/sangue , Animais , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Citosol/metabolismo , Técnicas In Vitro , Cinética , Masculino , Fluidez de Membrana , Ácidos Fosfatídicos/biossíntese , Ácidos Fosfatídicos/sangue , Fosfolipases A/sangue , Fosfolipases A2 , Coelhos , Trombina/fisiologia , Fosfolipases Tipo C/sangueRESUMO
The action of phosphatidic acid generated from membrane phospholipids on phospholipase A2 activation in rabbit platelets was investigated. When [3H]arachidonic acid-labelled platelets were treated with phorbol 12-myristate 13-acetate (PMA) and the membranes isolated from the cells incubated at 37 degrees C with 50 microM CaCl2 and 50 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), both phosphatidic acid production and arachidonic acid liberation increased in PMA- and GTP gamma S-concentration-dependent manners. Ethanol dose-dependently inhibited these responses, accompanied by the formation of phosphatidylethanol. Since propranolol, an inhibitor of phosphatidic acid phosphohydrolase, had no influence on the production of phosphatidic acid, the arachidonic acid liberated does not appear to be derived from diacylglycerol which may be produced from phosphatidic acid through the action of this enzyme. In another approach, treatment of [3H]arachidonic acid-labelled membranes with phospholipase D from Streptomyces chromofuscus induced arachidonic acid liberation as well as phosphatidic acid formation in time- and dose-dependent manners. The former response was suppressed by p-bromophenacyl bromide, a phospholipase A2 inhibitor. These results suggest that phosphatidic acid derived from membrane phospholipids potentiates phospholipase A2 activation and contributes to the amplification of platelet activation.
Assuntos
Ácido Araquidônico/metabolismo , Plaquetas/metabolismo , Lipídeos de Membrana/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipídeos/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Etanol/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Fosfolipase D/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , Coelhos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.
Assuntos
Ácidos Araquidônicos/farmacologia , Plaquetas/metabolismo , Endoperóxidos de Prostaglandina/sangue , Receptores de Prostaglandina/metabolismo , Tromboxano A2/sangue , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Ácido Araquidônico , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Humanos , Masculino , Fluidez de Membrana/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária , Endoperóxidos Sintéticos de Prostaglandinas/metabolismo , Endoperóxidos Sintéticos de Prostaglandinas/farmacologia , Receptores de Prostaglandina/efeitos dos fármacosRESUMO
Cytochrome P-450, designated as P-450-MK2, was purified to an electrophoretic homogeneity from polychlorinated biphenyl (PCB)-treated female crab-eating monkeys. P-450-MK2 catalyzed nifedipine and nilvadipine oxidations, at a rate comparable to human P-450-HM1. The N-terminal amino acid sequence of P-450-MK2 was highly homologous to those of P-450-HM1 and NF 25. The antibodies to P-450-HM1 recognized P-450-MK2 and effectively inhibited the activity of testosterone 6 beta-hydroxylase in monkey liver microsomes. These results suggest that a form of cytochrome P-450 corresponding to human P-450-HM1 or P-450NF which belongs to the P450 III gene family is also present in liver microsomes of crab-eating monkeys.
Assuntos
Sistema Enzimático do Citocromo P-450/isolamento & purificação , Macaca fascicularis/fisiologia , Macaca/fisiologia , Microssomos Hepáticos/enzimologia , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Biotransformação , Dados de Sequência Molecular , Peso Molecular , Bifenilos Policlorados/farmacologiaRESUMO
The expression of basic fibroblast growth factor (bFGF) and its receptor, the high-affinity type I basic fibroblast growth factor receptor (FGFR-1): were immunohistologically studied in tissues specimens from 167 patients with a pulmonary adenocarcinoma. Of the 167 specimens, 82 (49%) expressed bFGF and 104 (62%) expressed FGFR-1, bFGF and FGFR-1 were simultaneously expressed in 72 (43%). It was also found that many patients who showed intensely positive staining for bFGF were also positive for FGFR-1, and that the expression of bFGF or FGFR-1 or both was associated with p-stage, T and N factors. The overall prognosis was significantly poorer in the bFGF-positive or FGFR-1-positive patients than in negative patients (P < 0.01). The prognosis was also significantly poorer in all patients positive for both bFGF and FGFR-1 than in those negative for both (P < 0.01); this was also true for stage I patients (P < 0.05). Multivariate analysis showed that bFGF had a significant affect on prognosis, whereas FGFR-1 did not. As FGFR-1 is significantly linked with the bFGF expression, it may be that FGFR-1 interferes with the bFGF effect on survival. These findings suggest that bFGF and FGFR-1 play important roles in tumour progression, and that bFGF expression may be a useful prognostic marker for pulmonary adenocarcinomas.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Prognóstico , Taxa de SobrevidaRESUMO
N-[(Benzoyloxy)alkyl]-1,2,3,4-tetrahydro-2-naphthylamine derivatives were synthesized from 1,2,3,4-tetrahydro-2-naphthylamines and evaluated for their spasmolytic. Some of these compounds showed a nerve-selective effect on colon rather than stomach in anesthetized dogs and were found to be equal to or more active than the reference drug (mebeverine). The biological data have indicated some structure-activity relationships. Among these compounds, N-ethyl-N-[6-(3,4-dimethoxybenzoyl)oxy]hexyl]-1,2,3,4-tetrahydro-6-methoxy-2- napththylamine hydrochloride (63) was found to be the most active spasmolytic agent.
Assuntos
Naftalenos/síntese química , Parassimpatolíticos/síntese química , Tetra-Hidronaftalenos/síntese química , Animais , Fenômenos Químicos , Química , Colo/efeitos dos fármacos , Cães , Estimulação Elétrica , Feminino , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Estômago/efeitos dos fármacos , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/farmacologia , Nervo Vago/fisiologiaRESUMO
Concentration of Tc-99m(Sn)-N-pyridoxyl-5-methyltryptophan (Tc-99m PMT), a biliary agent, in hepatic tumors was studied with delayed hepatobiliary imaging in 23 patients with histologically verified hepatocellular carcinomas. All 23 showed filling defects on liver images obtained with Tc-99m tin colloid. In the images taken 5 hr after Tc-99m PMT injection, ten cases showed increased uptake in the carcinoma, six nearly normal uptake, and seven decreased uptake. In those showing the increased uptake of Tc-99m PMT in the tumor, the ratio of the radioactivity in the lesion to that in the adjacent liver parenchyma (T/L ratio) increased progressively with time for 5 hr after injection. These results indicate that delayed Tc-99m PMT images, obtained 5 hr after injection, are useful in assessment of uptake of the radioactivity by hepatocellular carcinoma.
Assuntos
Sistema Biliar/diagnóstico por imagem , Carcinoma Hepatocelular/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Fígado/diagnóstico por imagem , Compostos de Organotecnécio , Piridoxal/análogos & derivados , Tecnécio , Triptofano/análogos & derivados , Avaliação de Medicamentos , Humanos , Injeções Intravenosas , Cintilografia , Fatores de TempoRESUMO
The effect of biscoclaurine (bisbenzylisoquinoline) alkaloids on phospholipase A2 and C activation in signal transduction system of rabbit platelet was studied. Isotetrandrine, cepharanthine and berbamine inhibited the aggregation induced by collagen but not by other stimuli such as thrombin and arachidonic acid, while tetrandrine equally inhibited the aggregation by any of these agonists. All these four alkaloids suppressed arachidonic acid liberation in response to collagen or thrombin, but not diacylglycerol formation and increase in cytoplasmic Ca2+ concentration in response to thrombin or arachidonic acid. In saponin-permeabilized platelets, they also suppressed arachidonic acid liberation induced by an addition of both GTP gamma S and Ca2+, whereas the liberation induced by an addition of Ca2+ alone was not prevented by them. These data suggest that isotetrandrine, cepharanthine and berbamine have a rather specific potency to suppress the phospholipase A2 activation by a mechanism other than direct inhibition of the enzyme or interference with the ligand-receptor interaction. They seem, at least in part, to exert the effect on the GTP-binding protein-phospholipase A2 complex in the platelet signal transduction system. In contrast, tetrandrine appears to inhibit a step following an increase in cytosolic free Ca2+ concentration in the agonist-induced signal transduction system, in addition to suppressing the phospholipase A2 activation.
Assuntos
Alcaloides/farmacologia , Benzilisoquinolinas , Plaquetas/efeitos dos fármacos , Fosfolipases A/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Animais , Plaquetas/enzimologia , Cálcio/farmacologia , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Coelhos , Transdução de Sinais/efeitos dos fármacosRESUMO
The basic fibroblast growth factor (bFGF), the transforming growth factor (TGF)-beta 1, and the vascular endothelial growth factor (VEGF) expressions have been studied individually and in combinations with the factor 8-related antigen (F8RA) in relation to the microvascular density and prognosis of a pulmonary adenocarcinoma. The findings have revealed that the bFGF, TGF-beta 1 and VEGF expressions all correlated with microvascular density and the prognosis. A reduced expression of all three angiogenic factors correlated with-less tumor angiogenesis and a better prognosis. A multivariate analysis of the three prognostic factors also revealed that the the bFGF expression correlated with survival. These findings indicate that bFGF, TGF-beta 1, and VEGF expressions play important roles in tumor angiogenesis, and that the bFGF expression is a useful prognostic marker for assessing the outcome bf a pulmonary adenocarcinoma.
RESUMO
Implications of P-450 in human hepatic disorders were immunohistochemically examined. We first confirmed that an antibody against P-450-HM1, an isozyme of cytochrome P-450 which was purified from human livers at autopsy, detects only P-450 on immunoblots. In a study of 79 consecutive autopsied livers using the avidin-biotin-peroxidase complex method, the antibody reacted strongly with fetal hepatocytes, the reaction being more intense in the left lobe than in the right lobe. In normal livers, immunoreactivity was confined to centrilobular hepatocytes, decreasing in the periportal zone. Enhanced expression was occasionally found in scattered hepatocytes and in hepatocytes surrounding sublobular veins; this enhancement was related to longterm steroid therapy. No specific induction was observed in patients with toxic hepatitis. In patients with fibrosis, cirrhosis, or regenerative nodules, however, P-450-positive hepatocytes were observed in the periportal and middle zones as well as in the central zone. In contrast, hepatocellular carcinomas were devoid of P-450 immunoreactivity. These results suggest that P-450-HM1, which is abundant in the fetal liver, is reexpressed in regenerating hepatocytes but not in cancers.
Assuntos
Sistema Enzimático do Citocromo P-450/imunologia , Hepatopatias/patologia , Fígado/patologia , Especificidade de Anticorpos , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Imuno-Histoquímica , Fígado/imunologia , Fígado/metabolismo , Hepatopatias/imunologia , Hepatopatias/metabolismoRESUMO
The role of phosphatidic acid (PA) in the signal transduction system of platelets was studied using 1-stearoyl 2-arachidonoyl PA (PASA). When PASA was added to rabbit platelets, aggregation occurred. BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase, as well as p-bromophenacyl bromide and mepacrine, inhibitors of phospholipase A2, inhibited the aggregation induced by low concentrations of PASA, but not that induced by high concentrations. PASA also stimulated, in a dose-dependent manner, arachidonic acid liberation, lysophosphatidylcholine and diacylglycerol formation, and mobilization of intracellular Ca2+; all of which were dependent on the presence of Ca2+ in the outer medium. The arachidonic acid liberation was inhibited by p-bromophenacyl bromide or mepacrine, while diacylglycerol formation by low concentrations of PASA was inhibited by BW755C. With platelet membrane fractions or with the platelets made permeable to Ca2+ by pretreatment with ionomycin, PASA caused arachidonic acid liberation in the presence of Ca2+. Furthermore, PASA enhanced the activity of phospholipase A2 partially purified from platelet cytosol acting on 1-palmitoyl-2-[14C]arachidonoyl-glycerophosphoethanolamine. These results provide evidence that PASA preferentially potentiates the activation of phospholipase A2 in cooperation with Ca2+, suggesting that PA acts as a positive feedback regulator to potentiate the activation of phospholipase A2 and contributes to the amplification of platelet activation.
Assuntos
Plaquetas/enzimologia , Ácidos Fosfatídicos/farmacologia , Fosfolipases A/sangue , Agregação Plaquetária/efeitos dos fármacos , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina/farmacologia , Acetofenonas/farmacologia , Animais , Citosol/enzimologia , Ativação Enzimática , Cinética , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Quinacrina/farmacologia , CoelhosRESUMO
The modulating effect of sphingosine on the metabolism of inositol phospholipids was investigated using rabbit platelets. When [3H]arachidonic acid- or [3H]inositol-labeled platelets were incubated at 37 degrees C with sphingosine, the radioactivity of the phosphatidylinositol (PI) fraction obtained on TLC decreased time-dependently up to 5 min, and phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) increased concomitantly, though neither arachidonic acid nor 1,2-diacylglycerol was formed. The effect of sphingosine was dose-dependent, the maximum effect being observed at 20 microM. Treatment with a sphingosine derivative, sphingosine-1-phosphate (Sph-1-P) or N-hexanoyl-sphingosine (C6-ceramide), did not result in an increase in PIP. The increased radioactivity of PIP with sphingosine was attributable to an increase in phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate. Furthermore, wortmannin, an inhibitor of PI 3-kinase, did not affect the modulating effect of sphingosine at 100 nM, at which the enzyme is known to be completely inhibited. The activity of PI 4-kinase in the platelet lysate was increased by sphingosine but not by Sph-1-P. These results suggest that sphingosine enhances the activity of PI 4-kinase and thereby contributes to the regulation of inositol phospholipid metabolism.
Assuntos
Plaquetas/enzimologia , Lisofosfolipídeos , Fosfotransferases (Aceptor do Grupo Álcool)/sangue , Esfingosina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-Fosfatidilinositol 4-Quinase , Androstadienos/farmacologia , Animais , Ácido Araquidônico/sangue , Ceramidas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fosfatidilinositol 3-Quinases , Fosfatidilinositóis/sangue , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Coelhos , Esfingosina/análogos & derivados , Estaurosporina/farmacologia , WortmaninaRESUMO
Treatment of rabbit platelets for 1 min with 10-15 microM sphingosine enhanced arachidonic acid liberation after stimulation with U46619, although sphingosine or U46619 alone elicited little liberation of the lipid. Thrombin-induced arachidonic acid liberation was not influenced by sphingosine up to 10 microM, and was suppressed at concentrations higher than 20 microM. Sphingosine also promoted lysophosphatidylcholine formation in response to U46619, indicating that sphingosine caused an increase in hydrolytic action of phospholipase A2 (PLA2). The enhancing effect of sphingosine on arachidonic acid liberation decreased with increases in pretreatment time, accompanied by the conversion of sphingosine to sphingosine-1-phosphate. Of various sphingosine derivatives examined, sphingosine-1-phosphate, N,N-dimethylsphingosine, and N-acetylsphingosine (C2-ceramide) showed little enhancing effect on arachidonic acid liberation. Sphingosine increased cytosolic PLA2 activity in response to U46619 with enhancement of mitogen-activated protein kinase activity. These results indicate that sphingosine potentiates the transduction process of stimulus-PLA2 activation, resulting in enhancement of arachidonic acid liberation.
Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Ácido Araquidônico/sangue , Plaquetas/enzimologia , Fosfolipases A/sangue , Esfingosina/farmacologia , Animais , Biotransformação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/sangue , Citosol/efeitos dos fármacos , Citosol/enzimologia , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Fosfolipases A2 , Coelhos , Esfingosina/análogos & derivados , Esfingosina/farmacocinética , Trombina/farmacologiaRESUMO
To study the involvement of sphingolipids in glycerophospholipid metabolism, the contribution of ceramide to the activation of group IV cytosolic phospholipase A2 (cPLA2) was investigated in platelets using cell-permeable C6-ceramide (N-hexanoylsphingosine). The addition of ceramide led to potentiation of thrombin-induced activation of cPLA2 and mitogen-activated protein kinase (MAPK) as well as arachidonic acid release and lysophosphatidylcholine formation. However, ceramide by itself did not induce any response. The arachidonic acid release due to the synergistic action of ceramide and thrombin was inhibited by PD98059, a MAPK kinase inhibitor. Ceramide also stimulated thrombin-induced protein kinase C (PKC) activation, but ceramide by itself failed to do so. Furthermore, ceramide synergistically enhanced diacylglycerol (DAG) formation and Ca2+ mobilization with thrombin, and also DAG formation with Ca2+-ionophore A23187. The DAG formation in response to ceramide with thrombin or A23187, as well as arachidonic acid release with thrombin were completely inhibited by U73122, a phospholipase C (PLC) inhibitor. These results suggest that ceramide triggers PLC activation through its synergistic action with thrombin, and subsequently potentiates the sequential PKC-MAPK cascade-cPLA2 pathway, thus resulting in enhancement of arachidonic acid release.