RESUMO
Current frontline therapies have improved overall survival in acute promyelocytic leukemia (APL) patients to exceptional rates; however, relapse is still a problem among high-risk and old patients. Therefore, the development of better and safer therapies continues to be a goal in the treatment of this disease. In the present work, we examined three different pathways that hinder cell death in the APL cell line NB4, shedding light on the mechanisms that underlie resistance to apoptosis in these cells and that might help provide them with a proliferative advantage. We found that the proteasome inhibitor MG-132 specifically induces in NB4 cells an Nrf2-mediated antioxidant response which counteracts mitochondria-dependent apoptosis induced by the lipophilic cation dequalinium. More importantly, we also demonstrated that high basal autophagy levels and the gain-of-function of mutant p53 are intrinsic mechanisms of resistance to apoptosis in this cell line. According to our results, the pharmacological inhibition of autophagy and p53 mutants are useful tools to explore resistance to apoptosis in APL and other types of cancer and could be the bases of new therapeutic approaches that improve the efficiency and allow dose reduction of the current treatments.
Assuntos
Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dequalínio/administração & dosagem , Dequalínio/farmacologia , Relação Dose-Resposta a Droga , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leupeptinas/administração & dosagem , Leupeptinas/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mitochondria play central roles in diverse physiological and pathological conditions associated with cell survival and death. Delocalized lipophilic cations, such as dequalinium (DQA), are accumulated in cancer cells attracted by the highly negative mitochondrial transmembrane potential of these cells. DQA showed a potent anticancer activity in cells from different malignancies. Here, we report the effect of DQA on PC-3 prostate cancer cells. Incubation with DQA at concentrations between 1.5 and 100 microM from 24 to 48 h decreases cell viability. The decrease in cell viability together with a loss of mitochondrial transmembrane potential induced an increase in reactive oxygen species production and cell death via caspase-3 dependent apoptotic pathway. QA was shown to cause moderate to strong cell death in a time and concentration dependent manner, causing a most advantageous effect at a concentration of 10 microM applied for a long 48 h time period, which might be a consequence of the kinetics of intracellular DQA accumulation in mitochondria, but also of the mechanisms of DQA-induced cell death. This data shows DQA as a promising agent against the human prostate cancer PC-3 cell line, activating the caspase-3 dependent apoptotic pathway. This fact might be beneficial for possible future applications in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Dequalínio/farmacologia , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Estresse Oxidativo , Neoplasias da Próstata/metabolismo , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Male rats were given 10% (w/v) ethanol in drinking fluid during the first week, 15% (w/v) during the second week, 20% (w/v) during the third, and 25% (w/v) during the fourth week, at the end of which they were kept on 25% (w/v) ethanol drinking water for 3 weeks. Some animals were then allowed the withdrawal of ethanol for a period of 2 weeks or 7 weeks. No significant differences were seen for the basal and forskolin (FK)-stimulated adenylate cyclase (AC) enzyme activities in the pancreatic acinar membranes of ethanol-treated and ethanol withdrawal rats as compared to the control group. Chronic ethanol ingestion resulted in an attenuation of somatostatin(SS)-inhibited FK-stimulated AC in rat pancreatic acinar membranes. The ability of the stable GTP analogue 5'-guanylylimidodiphosphate (Gpp[NH]p) to inhibit FK-stimulated AC activity was also decreased in pancreatic acinar membranes from ethanol-treated rats. Gpp[NH]p was a much less potent inhibitor of SS binding in the pancreatic acinar membranes from chronic ethanol-treated animals than in those from controls, suggesting a change of Gi. A significant reduction in the number of 125I-Tyr11-SS receptors was observed after ethanol ingestion, when compared with control values. Two weeks after the replacement of the ethanol solution by water, the ethanol effect on the parameters cited above persisted. At week 7 of withdrawal, these parameters reached the level of water controls. Ethanol administration did not affect either the number or the affinity of secretin receptors as compared to control values which suggests that the change in SS binding is not a non-specific effect. Neither chronic ethanol consumption nor withdrawal affected somatostatin-like immunoreactivity (SSLI). These results suggest that the attenuated inhibition of AC by SS in pancreatic acinar membranes from ethanol-treated rats and ethanol withdrawal (2 weeks) rats may be caused by decreases in both Gi activity and in the number of SS receptors. Alternatively, an uncoupling of SS receptors from Gi and/or a decrease in the level of functional Gi may result in both a decrease in apparent Bmax for SS binding and in SS-mediated inhibition of AC. Since SS has been suggested to be an inhibitor of basal and cholecystokinin (CCK)- and/or secretin-stimulated exocrine pancreatic secretion, it is tempting to speculate that the impairment of the SS receptor/effector system seen in the present study can participate in the increase of basal pancreatic exocrine secretion described after chronic ethanol consumption.
Assuntos
Inibidores de Adenilil Ciclases , Etanol/farmacologia , Pâncreas/enzimologia , Somatostatina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva , Colforsina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanilil Imidodifosfato/farmacologia , Masculino , Pâncreas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Somatostatina/metabolismo , Secretina/metabolismo , Somatostatina/metabolismo , Tirosina/metabolismoRESUMO
The present experiments were devoted to analyzing the hypothesis that somatostatin (SS) could modulate glomerular filtration rate by interacting with mesangial cells. Studies were performed in cultured human mesangial cells, passages 3-5. Radioligand experiments demonstrated the presence in the cells of two kinds of receptors, with high (dissociation constant 14 pM. Number of sites: 426 fmol/mg) and low (dissociation constant 56 pM. Number of sites: 20, 111 fmol/mg) affinity. SS prevented in a dose-dependent manner the reduction in planar cell surface area induced by 100 nM Angiotensin II (AII). This effect was not inhibited by the blockade of the vasorelaxing prostaglandins (indomethacin, 10 microM), nitric oxide (L-N-methyl-arginine, 0.2 mM), adenylate cyclase (2,5'-dideoxyadenosine, 0.1 mM), or guanylate cyclase (Methylene blue, 30 microM; LY-83583, 10 microM), but it was potentiated by zaprinast, an inhibitor of the cyclic GMP (cGMP)-specific phosphodiesterase. SS also blocked the increase in myosin light chain phosphorylation induced by AII. SS increased cGMP synthesis by cultured human mesangial cells, an effect that seemed to be dependent on the stimulation of a particulate guanylate cyclase. Preincubation of the cells with pertussis toxin (0.5 microgram/ml) inhibited the effect of SS on the AII-dependent changes in planar cell surface area, as well as the SS-dependent cGMP stimulation. In summary, these results demonstrate the ability of SS to relax cultured human mesangial cells, thus supporting a role for this peptide in the regulation of the glomerular filtration rate. The SS-dependent mesangial cell relaxation may be due to changes in the intracellular concentrations of cGMP, as a consequence of the activation of a particulate guanylate cyclase.
Assuntos
Mesângio Glomerular/efeitos dos fármacos , Somatostatina/farmacologia , Angiotensina II/antagonistas & inibidores , Angiotensina II/farmacologia , Células Cultivadas , Taxa de Filtração Glomerular/efeitos dos fármacos , Mesângio Glomerular/citologia , Humanos , Octreotida/análogos & derivados , Octreotida/metabolismo , Testes de PrecipitinaRESUMO
Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.
Assuntos
Pâncreas/enzimologia , Proteína Quinase C/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Isoenzimas/metabolismo , Lactação , Pâncreas/patologia , Ratos , Ratos WistarRESUMO
Numerous reports in both humans and animals have confirmed that benzodiazepines produce amnesia; however, mechanisms mediating this effect are not clear. In view of the important role of brain somatostatin (SRIF) in the cognitive function of rats, this study sought to determine if the benzodiazepine, diazepam, alters somatostatinergic system in the rat frontoparietal cortex. Intraperitoneal (i.p.) administration of diazepam (5 mg/kg/day) to male Wistar rats (200-250 g) for 3 or 7 days decreased the number of SRIF receptors (26 and 37%, respectively) in synaptosomes from the frontoparietal cortex, without influencing their apparent affinity. This decrease in the tracer binding was not attributable to a direct effect of diazepam on SRIF receptors, because no decrease of SRIF binding was induced by a large concentration of diazepam (10(-4) M) when the drug was added to a preparation of synaptosomes from frontoparietal cortex of untreated rats. To determine if the effect of diazepam on SRIF binding is related to the binding of diazepam to its recognition site on the GABA(A) receptor, a benzodiazepine antagonist, 2-phenylpyrazolo[3,4-c]quinolin-3(5H)-one (CGS 8216) was administered before the diazepam injection. Pretreatment with CGS 8216 (20 mg/kg/day, i.p.) blocked completely the diazepam-induced decrease in the number of SRIF receptors. CGS 8216 alone had no observable effect. The decrease in the number of 125I-Tyr11-SRIF receptor induced by diazepam was accompanied by a decrease in the effect of SRIF, after 15 seconds of stimulation, on inositol 1,4, 5-trisphosphate (IP3) mass accumulation in the rat frontoparietal cortex at 3 (64%) or 7 days (59%) after its administration. Diazepam alone had no observable effect on mass accumulation of IP3. After 14 days of daily diazepam injections, the levels of binding of 125I-Tyr11-SRIF in the frontoparietal cortex returned to control values, coinciding with the tolerance that develops to this benzodiazepine agonists when administered chronically. The decrease in IP3 levels was still observed after 14 days (57%) diazepam administration. Diazepam and CGS 8216 did not affect SRIF-like immunoreactivity levels in the frontoparietal cortex at the three time intervals studied (3, 7 or 14 days). The alteration of frontoparietal cortex SRIF receptor-effector system after 3 or 7 days of diazepam treatment suggests that somatostatinergic neurotransmission plays a role in the mechanism of diazepam action on memory.
Assuntos
Diazepam/administração & dosagem , Lobo Frontal/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Lobo Parietal/efeitos dos fármacos , Somatostatina/metabolismo , Animais , Ansiolíticos/administração & dosagem , Ansiolíticos/antagonistas & inibidores , Ligação Competitiva/efeitos dos fármacos , Diazepam/antagonistas & inibidores , Esquema de Medicação , Lobo Frontal/metabolismo , Antagonistas de Receptores de GABA-A , Radioisótopos do Iodo , Masculino , Lobo Parietal/metabolismo , Pirazóis/farmacologia , Ratos , Ratos Wistar , Somatostatina/farmacologia , Sinaptossomos/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
The cholinergic regulation of phospholipase D activity was studied in SH-SY5Y human neuroblastoma cells with phosphatidylethanol formation as a specific marker for the enzyme activity. The muscarinic antagonists, hexahydrosiladifenidol and pirenzepine, inhibited carbachol-induced phosphatidylethanol formation in a concentration-dependent manner and the inhibitory constants indicated that muscarinic M1 receptors are responsible for the major part of the phospholipase D activation. The mechanism of receptor-mediated phospholipase D activation varies between different cell types and receptors. In SH-SY5Y cells, the carbachol-induced phospholipase D activity was inhibited by protein kinase C inhibitors. Since both phospholipases D and C are activated by muscarinic stimulation in SH-SY5Y cells, most of the phospholipase D activation is probably secondary to the protein kinase C activation that follows phospholipase C-mediated increase in diacylglycerols. Other kinases may be involved in the regulation since also a tyrosine kinase inhibitor decreased the phosphatidylethanol formation. Stimulation of G-protein(s) and increase in the intracellular Ca2+ concentration activated phospholipase D and may be additional mechanisms for the muscarinic regulation of phospholipase D in SH-SY5Y cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, increased the carbachol-induced formation of phosphatidic acid at the expense of 1,2-diacylglycerol. This indicates that phospholipase D contributes to the formation of 1,2-diacylglycerol after carbachol stimulation in SH-SY5Y cells.
Assuntos
Carbacol/farmacologia , Neuroblastoma/metabolismo , Fosfolipase D/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Relação Dose-Resposta a Droga , HumanosRESUMO
The present study investigates the effects of the administration of an intracerebroventricular (i.c.v.) dose of 500 micrograms/rat of the neuroleptic (-) sulpiride on somatostatin-like immunoreactivity (SSLI) levels, 125I-Tyr11-SS binding to its specific receptors, SS-modulated adenylyl cyclase (AC) activity and the pertussis toxin (PTX) substrates measured by toxin-catalysed ADP ribosylation of the alpha-subunits from G-proteins. (-) Sulpiride significantly decreased the SSLI levels in the frontoparietal cortex at 30 min but was without effect on the SSLI concentration in the striatum. This decrease had disappeared within 24 hr. The administration of (-) sulpiride produced a significant increase in the number of 125I-Tyr11-SS receptors and a significant reduction in their affinity at 30 min after injection in the striatum without affecting the frontoparietal cortex. The effects of the (-) sulpiride injection had disappeared after 24 hr. This change in SS binding was not due to a direct effect of (-) sulpiride on these receptors since no effect on binding was produced by high concentrations of (-) sulpiride (10(-5) M) when added in vitro. No significant differences were seen in either brain region for the basal or the forskolin (FK)-stimulated AC enzyme activities in the control and (-) sulpiride groups. In the (-) sulpiride group, the capacity of SS to inhibit FK-stimulated AC in the frontoparietal cortex was significantly higher than in the control group with no significant difference in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenilil Ciclases/metabolismo , Encéfalo/enzimologia , Receptores de Somatostatina/efeitos dos fármacos , Somatostatina/metabolismo , Sulpirida/farmacologia , Adenosina Difosfato Ribose/metabolismo , Toxina Adenilato Ciclase , Inibidores de Adenilil Ciclases , Animais , Encéfalo/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Colforsina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Injeções Intraventriculares , Masculino , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Toxina Pertussis , Radioimunoensaio , Ratos , Ratos Wistar , Somatostatina/imunologia , Sulpirida/imunologia , Sulpirida/metabolismo , Sinaptossomos/metabolismo , Fatores de Virulência de Bordetella/metabolismoRESUMO
A recent study carried out by our group demonstrated that exogenous dopamine increases the somatostatin (SS) receptor-effector system in the rat striatum. The present study examined the participation of the D1- and D2-dopaminergic systems in the modulation of the rat striatal SS receptor-effector system by use of the D1-receptor agonist and antagonist SKF 38393 and SCH 23390, respectively, and the D2-receptor agonist and antagonist bromocriptine and raclopride, respectively. In view of the rapid onset of dopamine action, the effect of dopaminergic agents on the SS mechanism of action were studied 3 h after their administration. SKF 38393 (4 mg/kg i.p.) or bromocriptine (2 mg/kg i.p.) administered to male Wistar rats increased the number of 125I-Tyr3-SMS receptors in the striatum (52 and 30%, respectively) without changing the affinity constant. The effect of SKF 38393 on 125I-Tyr3-SMS binding was antagonized by the D1-specific antagonist SCH 23390 (0.25 mg/kg i.p.) whereas the effect of bromocriptine was abolished by the D2-specific antagonist raclopride (5 mg/kg i.p.). No change in binding was produced when SKF 38393 or bromocriptine were added directly to the incubation medium. The acute systemic administration of SCH 23390 or raclopride alone had no effect on the binding of 125I-Tyr3-SMS to its receptors. The increase of the number of 125I-Tyr3-SMS receptor induced by SKF 38393 or bromocriptine was accompanied by an increase in the capacity of SMS 201-995 to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity when compared to the control groups. In addition, the effect of SMS 201-995 on the mass accumulation of inositol 1,4,5-trisphosphate (IP3) was investigated. SKF 38393 as well as bromocriptine increased the capacity of SMS 201-995 to accumulate IP3 in the rat striatum although this effect was only statistically significant in the case of SKF 38393. These results suggest that the activation of D1 and D2 receptors increases the activity of the SS receptor-effector system, the effect being greater in the case of D1 receptors. These findings are consistent with a functional interaction between dopamine and SS in the rat striatum.
Assuntos
Adenilil Ciclases/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Somatostatina/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Wistar , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D2/efeitos dos fármacos , Fatores de TempoRESUMO
It has been reported that ingestion of an ammonium-containing diet produces hyperammonemia without encephalopathy, thus permitting the study of the specific effects of ammonia toxicity. The present study investigated the rat cerebral somatostatinergic system using this experimental model of hyperammonemia. Wistar rats were fed a high ammonia diet prepared by mixing a standard diet with ammonium acetate (20% w/w); in addition, 5 mM of ammonium acetate was added to their water supply. Control rats were fed with a standard diet. The animals were sacrificed at 3, 7 and 15 days of ammonia ingestion. Ammonia levels in blood had increased approximately 3-fold at 7 days of ammonia ingestion. These changes were associated with a significant decrease in the specific binding of somatostatin (SS) to putative receptors sites in the frontoparietal cortex and hippocampus at 7 and 15 days after starting the high ammonia diet. Scatchard analysis shows that the decrease in SS binding resulted from a decrease in the number of available SS receptors rather than a change in receptor affinity. No changes in the somatostatin-like immunoreactivity content (SSLI) were detected in either brain area at the three study times. These results suggest that hyperammonemia alone can affect the rat brain somatostatinergic system. However, the animal model of hyperammonemia used here is insufficient to produce encephalopathy despite the significant increase in serum ammonia.
Assuntos
Amônia/toxicidade , Encéfalo/efeitos dos fármacos , Dieta/efeitos adversos , Somatostatina/metabolismo , Sinaptossomos/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Avaliação Pré-Clínica de Medicamentos , Lobo Frontal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Modelos Lineares , Lobo Parietal/efeitos dos fármacos , Peptídeos/análise , Ratos , Ratos Wistar , Valores de Referência , Sinaptossomos/metabolismoRESUMO
Previous results from our group showed that hyperammonemia decreases the number of somatostatin (SS) receptors and that benzodiazepine receptors might regulate the number of SS receptors in rat brain. These findings together with the supersensitivity of benzodiazepine receptors in the hyperammonemic rat brain suggest that benzodiazepine receptors might mediate the effect of hyperammonemia on SS receptors. To assess this hypothesis we tested whether 2-phenylpyrazolo[3,4-c]-quinolin-3(5H)-one (CGS 8216), a benzodiazepine antagonist, prevented the effect of ammonium acetate on rat brain SS receptors. Administration of ammonium acetate (5 mmol/kg, i.p.) for 7 days did not affect the levels of somatostatin-like immunoreactivity but decreased the number of SS receptors in synaptosomes from the frontoparietal cortex and hippocampus without affecting their apparent affinity. This decrease could be blocked by the concomitant administration of CGS 8216 (10 mg/kg, i.p.). The benzodiazepine antagonist alone had no observable effect on the somatostatinergic system. These results suggested that the effect of hyperammonemia on SS receptors could be mediated, at least in part, through the benzodiazepine receptors.
Assuntos
Encéfalo/efeitos dos fármacos , Antagonistas GABAérgicos/farmacologia , Peptídeos/metabolismo , Pirazóis/farmacologia , Receptores de Somatostatina/efeitos dos fármacos , Somatostatina/metabolismo , Amônia/sangue , Animais , Encéfalo/metabolismo , Ratos , Ratos Wistar , Receptores de Somatostatina/metabolismoRESUMO
The formation of the abnormal phospholipid phosphatidylethanol (PEth) was studied in hippocampus, cerebellum and cerebrum of rat brain after intraperitoneal ethanol administration. Prior to analysis by high performance thin layer chromatography PEth was purified. After one injection, PEth levels reached a maximum after 2 h and remained detectable for 14-24 h in all three regions. Repeated injections led to additional accumulation. Maximum in vivo levels of 30-50 nmol/g wet wt. were reached.
Assuntos
Química Encefálica/efeitos dos fármacos , Etanol/farmacologia , Glicerofosfolipídeos , Ácidos Fosfatídicos/metabolismo , Animais , Cromatografia em Camada Fina , Masculino , Peso Molecular , Palmitatos/metabolismo , Ácidos Fosfatídicos/biossíntese , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Adenylate cyclase activity in pancreatic acinar cell membranes was determined in rats that had undergone a treatment with pentagastrin (250 micrograms/kg, intraperitoneal three times daily) for 1 week or that had undergone small bowel resection (90%) and were sacrified at 2 weeks, 1 month and 6 months after intervention. Both treatments are potent stimulators of pancreatic acinar cell proliferation. Adenylate cyclase activity was similar under basal conditions and after the diterpene forskolin stimulation in pancreatic acinar membranes from all groups studied. The ability of low concentrations of the stable GTP analogue, 5'-guanylylimidodiphosphate (Gpp[NH]p) to inhibit forskolin-stimulated adenylate cyclase activity was decreased in pancreatic acinar membranes from enterectomized rats at 2 weeks and 1 month after the operation and returned to control values at 6 months after enterectomy. Stimulation of adenylate cyclase by high concentration of Gpp[NH]p or by secretin (10(-8) M) was higher in both pancreatic hyperplasia conditions as compared with control animals. These findings suggest that the coupling efficiency of the Gs protein to adenylate cyclase from pancreatic acinar membranes is enhanced without any alterations in the catalytic activity of the enzyme during pancreatic proliferation. In addition, it is possible that the highly regulated pancreatic acinar adenylate cyclase activity may be necessary to regulate pancreatic acinar cell proliferation.
Assuntos
Adenilil Ciclases/metabolismo , Pâncreas/enzimologia , Pâncreas/patologia , Animais , AMP Cíclico/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Guanilil Imidodifosfato/farmacologia , Hiperplasia , Masculino , Ratos , Ratos WistarRESUMO
Hydrogen peroxide (H(2)O(2)) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H(2)O(2) are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca(2+)) from intracellular stores or influx of extracellular Ca(2+). One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP(3)) mobilizes intracellular Ca(2+) by binding to a family of receptors (IP(3)Rs) on the endoplasmic-sarcoplasmic reticulum that act as ligand-gated Ca(2+) channels. IP(3)Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP(3)R content. In this study we show that IP(3)R(1) and IP(3)R(3) are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H(2)O(2), through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP(3)R by H(2)O(2) is accompanied by a reduction in calcium efflux induced by IP(3) in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP(3)R by activation of NADPH oxidase and that preincubation with H(2)O(2) decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP(3) receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H(2)O(2). Altogether, these results suggest that H(2)O(2) mediates IP(3)R down-regulation via proteasome activity.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Angiotensina II/farmacologia , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Although alterations in adenylate cyclase (AC) activity and somatostatin (SRIF) receptor density have been reported in Alzheimer's disease, the effects of amyloid beta-peptide (Abeta) on these parameters in the hippocampus are unknown. Our aim was to investigate whether the peptide fragment Abeta(25-35) can affect the somatostatinergic system in the rat hippocampus. Hence, Abeta(25-35) was injected intracerebroventricularly (i.c.v.) to Wistar rats in a single dose or infused via an osmotic minipump connected to a cannula implanted in the right lateral ventricle during 14 days. The animals were decapitated 7 or 14 days after the single injection and 14 days after chronic infusion of the peptide. Chronic i.c.v. infusion of Abeta(25-35) decreased SRIF-like immunoreactive content without modifying the SRIF receptor density, SRIF receptor expression, or the Gialpha(1), Gialpha(2), and Gialpha(3) protein levels in the hippocampus. This treatment, however, caused a decrease in basal and forskolin-stimulated AC activity as well as in the capacity of SRIF to inhibit AC activity. Furthermore, the protein levels of the neural-specific AC type I were significantly decreased in the hippocampus of the treated rats, whereas an increase in the levels of AC V/VI was found, with no alterations in type VIII AC. A single i.c.v. dose of Abeta(25-35) exerted no effect on SRIF content or SRIF receptors but induced a slight decrease in forskolin-stimulated AC activity and its inhibition by SRIF. Because chronic Abeta(25-35) infusion impairs learning and memory whereas SRIF facilitates these functions, the alterations described here might be physiologically important given the decreased cognitive behavior previously reported in Abeta-treated rats.
Assuntos
Adenilil Ciclases/efeitos dos fármacos , Peptídeos beta-Amiloides/administração & dosagem , Hipocampo/efeitos dos fármacos , Fragmentos de Peptídeos/administração & dosagem , Somatostatina/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Western Blotting , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Hipocampo/metabolismo , Injeções Intraventriculares , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Somatostatina/efeitos dos fármacos , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismoRESUMO
The regulation of phospholipase D was studied in human neuroblastoma cells using phosphatidylethanol as a marker of the enzyme activity. Carbachol induced phospholipase D activity in SH-SY5Y cells. Muscarinic antagonists inhibited the response with potencies suggesting that muscarinic M1 receptors are responsible for the activation. In permeabilized SH-SY5Y cells, both the carbachol- and GTP gamma S-induced Peth formation was inhibited by GDP beta S, indicating that both responses are mediated via a G-protein. The protein kinase C inhibitors, bisindolylmaleimide and staurosporine significantly inhibited the carbachol-induced Peth formation whereas H7 had no effect. Thus, the cholinergic activation of phospholipase D in SH-SY5Y cells is probably mediated via a direct receptor-G-protein coupling but an involvement of protein kinase C cannot be excluded. Calmidazolium, a calmodulin antagonist, induced an increase in phosphatidylethanol formation in both SH-SY5Y and IMR-32 cells. This effect was inhibited by genistein and tyrphostin, indicating a tyrosine kinase dependent pathway for phospholipase D activation in neuroblastoma cells.
Assuntos
Neuroblastoma/metabolismo , Fosfolipase D/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Humanos , Antagonistas Muscarínicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Since melatonin (N-acetyl-5-methoxytryptamine) decreases locomotor activity and rearing and increases grooming behavior in a similar manner as somatostatin (SRIF), we examined if melatonin could induce these changes through somatostatinergic neurotransmission in the rat frontoparietal cortex. Male Wistar rats (200-250 g) received a single injection of melatonin (25 microg/kg per day) subcutaneously (s.c.) and were sacrificed 5 hr later. Melatonin treatment increased the number of 125I-Tyr11-SRIF receptors in frontoparietal cortical membranes without any changes in the dissociation constant (Kd). The capacity of SRIF to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity was increased in melatonin-treated rats as compared to the control animals. Melatonin administration also induced a lower AC activity, both under basal conditions and after stimulation of the enzyme via stimulatory guanine nucleotide-binding proteins (Gs), or directly with FK. Functional inhibitory guanine nucleotide-binding protein (Gi) activity was increased in frontoparietal cortical membranes from melatonin-treated rats when compared to controls. Western blot analyzes showed that melatonin administration did not alter the amount of the Gialpha1, or Gialpha3 subunits, but reduced Gialpha2 levels in frontoparietal cortical membranes. No significant changes in SRIF-like immunoreactivity content and SRIF mRNA levels were detected in this brain area after melatonin treatment. Administration of the melatonin receptor antagonist luzindole (10 mg/kg, s.c.) 30 min before melatonin injection did not change the melatonin-induced effects on the SRIF receptor effector system. In conclusion, the present results show that acute melatonin administration increases the activity of the SRIF receptor effector system and decreases Gialpha2 levels in the rat frontoparietal cortex. In addition, the coupling of Gs to AC is disturbed by melatonin.
Assuntos
Lobo Frontal/efeitos dos fármacos , Melatonina/farmacologia , Lobo Parietal/efeitos dos fármacos , Receptores de Somatostatina/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Lobo Frontal/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Cinética , Masculino , Lobo Parietal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Somatostatina/metabolismo , Somatostatina/genética , Somatostatina/metabolismoRESUMO
In the last years, reactive oxygen species (ROS) have been proposed as mediators of proliferative/hypertrophic responses to angiotensin II (Ang II), both in vivo and in vitro. However, the hypothesis that the Ang II-dependent cell contraction could be mediated by ROS, particularly H2O2, has not been tested. Present experiments were devoted to test this hypothesis and to analyze the possible mechanisms involved. Catalase (CAT) prevented the increased myosin light chain phosphorylation and the decreased planar cell surface area (PCSA) induced by 1 microM Ang II in cultured rat vascular smooth muscle cells (VSMC). This preventive effect of CAT was also detected when 1 microM platelet-activating factor (PAF) was used as a contractile agonist instead of Ang II. Similar results were found when using horseradish peroxidase as an H2O2 scavenger or cultured rat mesangial cells. In vascular smooth muscle cells, CAT modified neither the binding of labeled Ang II nor the Ang II-induced inositol 1,4,5-trisphosphate (IP3) synthesis. However, it completely abolished the Ang II-dependent calcium peak, in a dose-dependent fashion. CAT-loaded cells (increased intracellular CAT concentration over 3-fold) did not show either a decreased PCSA or an increased intracellular calcium concentration after Ang II treatment. Ang II stimulated the H2O2 synthesis by cultured cells, and the presence of CAT in the extracellular compartment significantly diminished the Ang II-dependent increased intracellular H2O2 concentration. The physiological importance of these findings was tested in rat thoracic aortic rings: CAT prevented the contraction elicited by Ang II. In summary, present experiments point to H2O2 as a critical intracellular metabolite in the regulation of cell contraction.