RESUMEN
Alcoholic hepatitis is a serious form of liver damage. Inflammation is a key factor in alcoholic hepatitis and plays a key role in the progression of alcoholic liver disease. Adenosine receptor A2B (A2BAR) is a member of the adenosine receptor family and generally considered to be a negative regulator of the inflammatory response. We found that A2BAR was the most highly expressed adenosine receptor in ETOH-fed mouse liver tissue and was also highly expressed in primary Kupffer cells and ETOH-induced RAW264.7 cells. In addition, injection of BAY 60-6583 stimulated A2BAR, induced upregulation of the expression levels of cAMP, and reduced ETOH-induced steatosis and inflammation in mice. At the same time, knockdown of A2BAR in vitro increased the inflammatory response in RAW264.7 cells triggered by ETOH. After knockdown of A2BAR in vitro, the release of the inflammatory cytokines IL-6, IL-1ß and TNF-α was increased. After overexpression of A2BAR in vitro, the cAMP level was significantly increased, PKA expression was increased, the expression of phosphorylated proteins in the NF-kB signal transduction pathway was significantly affected, and the expression of the key phosphorylated protein p-P65 was decreased. However, after the simultaneous overexpression of A2BAR and inhibition of PKA, the expression of the key phosphorylated protein p-P65 was still significantly decreased. In addition, after the expression of A2BAR increased or decreased in RAW264.7 cells, AML-12 cells were cultured in the supernatant of RAW264.7 cells stimulated by ETOH, and the apoptosis rate was significantly changed by flow cytometry. These results suggest that A2BAR can reduce alcoholic steatohepatitis by upregulating cAMP levels and negatively regulating the NF-kB pathway. Overall, these findings suggest the significance of A2BAR-mediated inflammation in alcoholic liver disease.
Asunto(s)
Hepatitis Alcohólica/tratamiento farmacológico , Macrófagos del Hígado/efectos de los fármacos , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Receptor de Adenosina A2B/uso terapéutico , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BLAsunto(s)
Analgésicos Opioides/farmacología , Desarrollo Infantil/efectos de los fármacos , Tolerancia a Medicamentos/fisiología , Morfina/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Síndrome de Abstinencia a Sustancias/diagnóstico , Analgésicos Opioides/efectos adversos , Humanos , Recién Nacido , Metadona/administración & dosificación , Morfina/efectos adversos , Proteína Quinasa C/efectos de los fármacos , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/prevención & controlRESUMEN
8-Cl-cyclic adenosine monophosphate (8-Cl-cAMP) has been known to induce growth inhibition and differentiation in a variety of cancer cells by differential modulation of protein kinase A isozymes. To understand the anticancer activity of 8-Cl-cAMP further, we investigated the effect of 8-Cl-cAMP on apoptosis in human cancer cells. Most of the tested human cancer cells exhibited apoptosis upon treatment with 8-Cl-cAMP, albeit with different sensitivity. Among them, SH-SY5Y neuroblastoma cells and HL60 leukemic cells showed the most extensive apoptosis. The effect of 8-Cl-cAMP was not reproduced by other cAMP analogues or cAMP-elevating agents, showing that the effect of 8-Cl-cAMP was not caused by simple activation of protein kinase A (PKA). However, competition experiments showed that the binding of 8-Cl-cAMP to the cAMP receptor was essential for the induction of apoptosis. After the treatment of 8-Cl-cAMP, cells initially accumulated at the S and G2/M phases of the cell cycle and then apoptosis began to occur among the population of cells at the S/G2/M cell cycle phases, indicating that the 8-Cl-cAMP-induced apoptosis is closely related to cell cycle control. In support of this assumption, 8-Cl-cAMP-induced apoptosis was blocked by concomitant treatment with mimosine, which blocks the cell cycle at early S phase. Interestingly, 8-Cl-cAMP did not induce apoptosis in primary cultured normal cells and non-transformed cell lines, showing that 8-Cl-cAMP-induced apoptosis is specific to transformed cells. Taken together, our results show that the induction of apoptosis is one of the mechanisms through which 8-Cl-cAMP exerts anticancer activity.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/toxicidad , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Ciclo Celular/fisiología , Células 3T3 , Animales , Neoplasias de la Mama , Células CHO , Ciclo Celular/efectos de los fármacos , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Fase G2 , Células HL-60 , Células HeLa , Humanos , Células K562 , Ratones , Mitosis , Neuroblastoma , Neoplasias Ováricas , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/fisiología , Fase S , Células Tumorales CultivadasRESUMEN
Studies at the basic and clinical levels demonstrate that neuronal atrophy and cell death occur in response to stress and in the brains of depressed patients. Although the mechanisms have yet to be fully elucidated, progress has been made in characterizing the signal transduction cascades that control neuronal atrophy and programmed cell death and that may be involved in the action of antidepressant treatment. These pathways include the cyclic adenosine monophosphate and neurotrophic factor signal transduction cascades. It is notable that these same pathways have been demonstrated to play a pivotal role in cellular models of neural plasticity. This overlap of plasticity and cell survival pathways, together with studies demonstrating that neuronal activity enhances cell survival, suggests that neuronal atrophy and death could result from a disruption of the mechanisms underlying neural plasticity. The role of these pathways and failure of neuronal plasticity in stress-related mood disorders are discussed.
Asunto(s)
Trastornos del Humor , Plasticidad Neuronal/fisiología , Neuronas/patología , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Atrofia/etiología , Atrofia/genética , Atrofia/patología , Factor Neurotrófico Derivado del Encéfalo/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Genes bcl-2/efectos de los fármacos , Genes bcl-2/genética , Humanos , Trastornos del Humor/tratamiento farmacológico , Trastornos del Humor/etiología , Trastornos del Humor/metabolismo , Plasticidad Neuronal/genética , Neuronas/metabolismo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estrés Psicológico/psicología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The physiologic basis for opioid tolerance has been elucidated, resulting in a better understanding of this problem. As a result of this ongoing effort, patients exhibiting opioid tolerance can now expect better pain management both in the postoperative period and during the course of their treatment. This article outlines the pathophysiology of opioid tolerance and a practical clinical approach to this problem.
Asunto(s)
Analgésicos Opioides/farmacología , Manejo del Dolor , Dolor/fisiopatología , Tolerancia a Medicamentos/fisiología , Humanos , Cuidados Intraoperatorios , Cuidados Posoperatorios , Proteína Quinasa C/efectos de los fármacos , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
Accumulating evidence suggested that signal transduction cascade including protein phosphorylation is implicated in the neurochemical action of antidepressant agents. Clinical data indicated that moclobemide, a short acting and reversible inhibitor of monoamino oxidase type. A, is an effective antidepressant medication. However, little is known about the intracellular effects of this compound. Thus, in the present study we assessed the binding of cAMP to cAMP-dependent protein kinase (PKA) in rat cerebral cortex following short and long-term administration of moclobemide. The results showed that 21 days of treatment with moclobemide significantly increased the specific [32P]-cAMP covalent binding into the soluble 52-54 kDa cAMP-receptor. This effect was not seen following 1, 5 and 12 days of treatment. These findings suggest that PKA could be implicated in the biochemical effects of moclobemide.
Asunto(s)
Antidepresivos/farmacología , Benzamidas/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Inhibidores de la Monoaminooxidasa/farmacología , Animales , Corteza Cerebral/efectos de los fármacos , AMP Cíclico/metabolismo , Masculino , Moclobemida , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de AMP Cíclico/efectos de los fármacosRESUMEN
cAMP receptor protein (CRP) regulates expression of a number of genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two structural domains. The biological activation of CRP upon cAMP binding may involve the subunit realignment as well as reorientation between the domains within each subunit. In order to study the interactions between the subunits or domains, we performed stopped-flow measurements of the guanidine hydrochloride (GuHCI)-induced denaturation of CRP. The changes in CRP structure induced by GuHCl were monitored using both intrinsic Trp fluorescence as well as the fluorescence of an extrinsic probe, 8-anilino-1-Naphthalenesulfonic acid (ANS). Results of CRP denaturation using Trp fluorescence detection are consistent with a two-step model [Malecki, and Wasylewski, (1997), Eur. J. Biochem. 243, 660], where the dissociation of dimer into subunits is followed by the monomer unfolding. The denaturation of CRP monitored by ANS fluorescence reveals the existence of two additional processes. One occurs before the dissociation of CRP into subunits, whereas the second takes place after the dissociation, but prior to proper subunit unfolding. These additional processes suggest that CRP denaturation is described by a more complicated mechanism than a simple three-state equilibrium and may involve additional changes in both inter- and intrasubunit interactions. We also report the effect of cAMP on the kinetics of CRP subunit unfolding and refolding.
Asunto(s)
Escherichia coli/química , Guanidina/farmacología , Receptores de AMP Cíclico/química , Receptores de AMP Cíclico/metabolismo , Espectrometría de Fluorescencia/métodos , Naftalenosulfonatos de Anilina/química , Naftalenosulfonatos de Anilina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Dimerización , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Guanidina/química , Guanidina/metabolismo , Cinética , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Receptores de AMP Cíclico/efectos de los fármacosRESUMEN
OBJECTIVES: This study tested the hypothesis that metoprolol restores the reduction of the inotropic effect of the cyclic adenosine monophosphate (cAMP)-phosphodiesterase inhibitor milrinone, which is cAMP dependent but beta-adrenoceptor independent. BACKGROUND: Treatment with beta-adrenergic blocking agents has been shown to lessen symptoms and improve submaximal exercise performance and left ventricular ejection fraction in patients with heart failure. Restoration of the number of down-regulated beta-adrenoceptors has been suggested to be one mechanism of beta-blocker effectiveness. However, the reversal of postreceptor events, namely, an increase in inhibitory G-protein alpha-subunit concentrations, could also play a role. METHODS: Fifteen patients with heart failure due to dilated cardiomyopathy (left ventricular ejection fraction 24.6 +/- 1.5% [mean +/- SD], New York Heart Association functional class II or III) were treated with metoprolol (maximal dose 50 mg three times daily) for 6 months. Before and after metoprolol treatment, inotropic responses to milrinone (5 to 10 micrograms/kg body weight per min) were measured echocardiographically. For comparison, responses to milrinone were determined under control conditions and after accelerated application of 150 mg of metoprolol to inactivate beta-adrenoceptors in subjects with normal left ventricular function. RESULTS: In subjects with normal left ventricular function, treatment with metoprolol did not alter the increase in fractional shortening or pressure/dimension ratio of circumferential fiber shortening after application of milrinone. In patients with heart failure, treatment with metoprolol significantly increased left ventricular ejection fraction, fractional shortening and submaximal exercise tolerance and reduced heart rate, plasma norepinephrine concentrations and functional class. After metoprolol treatment, milrinone increased fractional shortening but had no effect before beta-blocker treatment. CONCLUSIONS: Milrinone increases inotropic performance independently of beta-adrenoceptors in vivo. Metoprolol treatment restores the blunted inotropic response to milrinone in patients with heart failure, indicating that postreceptor events (e.g., increase in inhibitory G-protein) are favorably influenced. This mechanism could contribute to the beneficial effects observed in the study patients and represents an important mechanism of how beta-blocker treatment influences the performance of the failing heart.
Asunto(s)
Antagonistas Adrenérgicos beta/uso terapéutico , Cardiotónicos/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Metoprolol/uso terapéutico , Inhibidores de Fosfodiesterasa/uso terapéutico , Piridonas/uso terapéutico , Adulto , Anciano , Cardiomiopatía Dilatada/complicaciones , Sinergismo Farmacológico , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/efectos de los fármacos , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/etiología , Humanos , Masculino , Persona de Mediana Edad , Milrinona , Contracción Miocárdica/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores de AMP Cíclico/efectos de los fármacos , Volumen Sistólico/efectos de los fármacos , UltrasonografíaRESUMEN
The G alpha 2-subunit of Dictyostelium discoideum is essential to the initial stage of the cell's developmental life cycle. In response to the extracellular chemoattractant, cAMP, G alpha 2 is activated and transiently phosphorylated on serine-113 [Chen et al. (1994): J Biol Chem 269:20925-20930]. The role of G alpha 2 phosphorylation remains elusive; cells expressing the S113A, nonphosphorylated mutation of G alpha 2 appear to proceed through the developmental phase normally. To gain insight into the function of G alpha 2 phosphorylation, the conditions for G alpha 2 phosphorylation were examined using a variety of alpha-subunit point mutations and chimeras. Mutations that block the G protein activation cycle prior to or at the hydrolysis of GTP (G alpha 2-S45A, G alpha 2-G207A, and G alpha 2-Q208L) preclude G alpha 2 phosphorylation in vivo. Phosphorylation of the G alpha 2-Q208L mutation does however occur in an in vitro phosphorylation assay. It appears that G alpha 2 phosphorylation, shown previously in vivo to require the cAMP receptor, also requires signaling through the G2 pathway. Results from the in vitro assay suggest that the substrate for phosphorylation is the alpha-subunit monomer.
Asunto(s)
Dictyostelium/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/fisiología , Animales , Dictyostelium/genética , Dictyostelium/fisiología , Proteínas de Unión al GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Mutagénesis Sitio-Dirigida , Fenotipo , Fosforilación/efectos de los fármacos , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Recent studies have begun to characterize the actions of stress and antidepressant treatments beyond the neurotransmitter and receptor level. This work has demonstrated that long-term antidepressant treatments result in the sustained activation of the cyclic adenosine 3',5'-monophosphate system in specific brain regions, including the increased function and expression of the transcription factor cyclic adenosine monophosphate response element-binding protein. The activated cyclic adenosine 3',5'-monophosphate system leads to the regulation of specific target genes, including the increased expression of brain-derived neurotrophic factor in certain populations of neurons in the hippocampus and cerebral cortex. The importance of these changes is highlighted by the discovery that stress can decrease the expression of brain-derived neurotrophic factor and lead to atrophy of these same populations of stress-vulnerable hippocampal neurons. The possibility that the decreased size and impaired function of these neurons may be involved in depression is supported by recent clinical imaging studies, which demonstrate a decreased volume of certain brain structures. These findings constitute the framework for an updated molecular and cellular hypothesis of depression, which posits that stress-induced vulnerability and the therapeutic action of antidepressant treatments occur via intracellular mechanisms that decrease or increase, respectively, neurotrophic factors necessary for the survival and function of particular neurons. This hypothesis also explains how stress and other types of neuronal insult can lead to depression in vulnerable individuals and it outlines novel targets for the rational design of fundamentally new therapeutic agents.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Trastorno Depresivo/fisiopatología , Factores de Crecimiento Nervioso/fisiología , Animales , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/genética , Diseño de Fármacos , Hipocampo/fisiopatología , Humanos , Leucina Zippers/fisiología , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/fisiología , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/fisiología , Restricción Física , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estrés Psicológico/fisiopatologíaRESUMEN
cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped-flow fluorescence spectroscopy at 20 degrees C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three-state model: (CRP native)2<=> 2 (CRP native)<=>2 (CRP denatured). The faster process, characterized by the relaxation time tau 2 = 80 +/- 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time tau t = 1.9 +/- 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is delta G dis degrees = 46.9 +/- 2.5 kJ/mol and for monomer unfolding delta G unf degrees = 30.9 +/- 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 +/- 0.3 degrees C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21 degrees C is equal to 30.5 +/- 0.4 kJ/mol and the apparent specific heat change is equal to delta Cp, app = 10.7 +/- 0.7 kJ mol-1 K-1. The predicted values of cold denaturation midpoint is equal to tau G = -18.8 +/- 1.5 degrees C and for high-temperature transition tau G = 63.1 +/- 1.5 degrees C. The predicted midpoint of high-temperature unfolding transition is about the same as determined experimentally.
Asunto(s)
Escherichia coli/metabolismo , Pliegue de Proteína , Receptores de AMP Cíclico/química , Receptores de AMP Cíclico/metabolismo , Dicroismo Circular , Estabilidad de Medicamentos , Escherichia coli/química , Guanidina , Guanidinas/farmacología , Cinética , Desnaturalización Proteica , Receptores de AMP Cíclico/efectos de los fármacos , TermodinámicaRESUMEN
There is great interest in utilizing butyrate as a chemotherapeutic agent. To elucidate its mechanism of action, the effect of butyrate on cAMP receptor protein kinase (PKA) activity in young adult mouse colon (YAMC) cells isolated from transgenic mice bearing a temperature sensitive mutation of the SV40 large T antigen gene was investigated. Conditionally immortalized cultures were plated at the permissive temperature (33 degrees C) or growth arrested by incubation at the nonpermissive temperature (39 degrees C). In addition, cells were incubated at 33 degrees C with or without 1 mmol/L butyrate for 24 h. Butyrate treatment reduced cell proliferation by 28% and enhanced apoptosis by 350% compared with cultures not exposed to butyrate. The PKA type I/II isozyme activity ratio was lower (P < 0.05) in cells incubated with butyrate. The relative level of PKA I isozyme was higher in proliferating cells at 33 degrees C (63% of total PKA), while the relative level of PKA II was higher in nonproliferating cells undergoing apoptosis at 39 degrees C (59% of total PKA). Neither incubation conditions (33 vs. 39 degrees C) nor butyrate treatment altered total PKA activity. When YAMC cells were incubated with 8-CI-cAMP, an activator of PKA II, growth was markedly inhibited in cells at both temperatures. Consistent with in vitro data, increased PKA I isozyme levels were associated with dysregulated growth in vivo. Specifically, the relative level of PKA I isozyme was three- to fivefold higher in rat colonic tumors compared with normal nontransformed colonic mucosa. These data indicate that the biological effects of butyrate on colonocyte proliferation and apoptosis are associated with changes in PKA isozyme-dependent signal transduction, and the YAMC cell line is a relevant model to examine the molecular mechanisms by which dietary-derived factors affect relative cancer risk.
Asunto(s)
Butiratos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Receptores de AMP Cíclico/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Ácido Butírico , División Celular/efectos de los fármacos , Línea Celular , Colon/efectos de los fármacos , Colon/metabolismo , Ratones , Ratones Transgénicos , RatasRESUMEN
Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called conditioned medium factor (CMF) reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form of the cAMP receptor and the loss of ligand binding, which is the first step of ligand-induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess approximately 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a 10-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF binding; disruption of the cAR1 gene abolishes both cAMP and CMF binding. In wild-type cells, downregulation of cAR1 with high levels of cAMP also downregulates CMF binding, and CMF similarly downregulates cAMP and CMF binding. This suggests that the cAMP binding and CMF binding are closely linked. Binding of approximately 200 molecules of CMF to starved cells affects the affinity of the majority of the cAR1 cAMP receptors within 2 min, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cARs. In cells lacking the G-protein beta subunit, cAMP induces a loss of cAMP binding, but not CMF binding, while CMF induces a reduction of CMF binding without affecting cAMP binding, suggesting that the linkage of the cell density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G-protein.
Asunto(s)
Arginasa/genética , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/fisiología , Dictyostelium/fisiología , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética , Proteínas Protozoarias , Transducción de Señal/fisiología , Animales , Moléculas de Adhesión Celular/farmacología , Comunicación Celular/fisiología , Recuento de Células , AMP Cíclico/farmacología , Dictyostelium/química , Dictyostelium/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores de Superficie Celular/fisiología , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/fisiologíaRESUMEN
We have previously demonstrated that nitric oxide (NO)-releasing compounds inhibit the differentiation and aggregation of D. discoideum cells (Tao et al., FEBS Lett. 314:49, 1992). In the present study, we demonstrate that treatment of intact cells with NO-releasing compounds inhibits their production of cAMP. This occurred even though the developmental expression of the known components necessary for proper cAMP signalling was unaffected. The inhibitory effects of NO-releasing compounds on cell aggregation were reversed by stimulating cells with pulses of cAMP. In response to an applied cAMP pulse, NO-treated cells displayed a normal signal relay response, indicating that receptor-activated adenylate cyclase activity was not inhibited by NO. This also argues that the processes of desensitization/resensitization occur normally in NO-treated cells. The data indicate that the developmental expression of the components of the chemotactic signalling occurs independently of cAMP production and that the activity of the adenylate cyclase may be regulated by cAMP/ cAMP-receptor independent pathway. These findings indicate both a new mechanism for the regulation of adenylate cyclase in D. discoideum and a novel means by which NO can function to alter cellular processes.
Asunto(s)
Adenilil Ciclasas/metabolismo , AMP Cíclico/antagonistas & inhibidores , Dictyostelium/efectos de los fármacos , Óxido Nítrico/fisiología , Receptores de AMP Cíclico/efectos de los fármacos , Sistemas de Mensajero Secundario/efectos de los fármacos , Animales , Quimiotaxis/efectos de los fármacos , AMP Cíclico/metabolismo , Dictyostelium/fisiología , Activación Enzimática , Proteínas Fúngicas/metabolismo , Molsidomina/análogos & derivados , Molsidomina/farmacología , Nitroprusiato/farmacología , Periodicidad , Receptores de AMP Cíclico/fisiología , Tasa de Secreción/efectos de los fármacosRESUMEN
Multiple signal transduction pathways within a single cell may share common components. In particular, seven different transmembrane helix receptors may activate identical pathways by interacting with the same G-proteins. Dictyostelium cells respond to cAMP using one such receptor, cAR1, coupled by a typical heterotrimeric G-protein to intracellular effectors. However, cells in which the gene for cAR1 has been deleted are unexpectedly still able to respond to cAMP. This implies either that certain responses are mediated by a different receptor than cAR1, or alternatively that a second, partially redundant receptor shares some of the functions of cAR1. We have examined the dose response and ligand specificity of one response, cAMP relay, and the dose response of another, cyclic GMP synthesis. In each case, the EC50 was approximately 100-fold higher and the maximal response was smaller in car1- than wild-type cells. These data indicate that cAR1 normally mediates responses to cAMP. The ligand specificity suggests that the responses seen in car1- mutants are mediated by a second receptor, cAR3. To test this hypothesis, we constructed a cell line containing deletions of both cAR1 and cAR3 genes. As predicted, these lines are totally insensitive to cAMP. We conclude that the functions of the cAR1 and cAR3 receptors are partially redundant and that both interact with the same heterotrimeric G-protein to mediate these and other responses.
Asunto(s)
Dictyostelium/metabolismo , Receptores de AMP Cíclico/metabolismo , Adenilil Ciclasas/metabolismo , Animales , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Proteínas de Unión al GTP/metabolismo , Eliminación de Gen , Genes Fúngicos , Genes Protozoarios , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/genética , Transducción de Señal , Tionucleótidos/farmacologíaRESUMEN
Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Proteínas Quinasas/efectos de los fármacos , ARN Mensajero/análisis , Receptores de AMP Cíclico/efectos de los fármacos , Secuencia de Aminoácidos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Regulación hacia Abajo , Humanos , Datos de Secuencia Molecular , Neuroblastoma/enzimología , Neuroblastoma/patología , Oligonucleótidos Antisentido/química , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/química , Receptores de AMP Cíclico/metabolismo , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/patología , Células Tumorales CultivadasRESUMEN
The phosphorylation of the cardiac sodium channel by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A leads to its inactivation. It was shown that extracellular cAMP can also modulate the sodium channel of rat, guinea pig, and frog ventricular myocytes in a rapid (less than 50 milliseconds), reversible, and dose-dependent manner. The decrease in the sodium current was accompanied by a 10- to 15-millivolt shift in the steady-state availability of the sodium channel toward more negative potentials and was inhibited by guanosine-5'-O-(2-thiodiphosphate) or pertussis toxin, suggesting that the extracellular modulation of the sodium channel by cAMP is mediated by a membrane-delimited mechanism that includes a pertussis toxin-sensitive G protein.
Asunto(s)
AMP Cíclico/farmacología , Corazón/fisiología , Receptores de AMP Cíclico/fisiología , Canales de Sodio/fisiología , Animales , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Cobayas , Corazón/efectos de los fármacos , Isoproterenol/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Toxina del Pertussis , Rana pipiens , Ratas , Receptores de AMP Cíclico/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Tionucleótidos/farmacología , Factores de Virulencia de Bordetella/farmacologíaAsunto(s)
Arteriosclerosis/metabolismo , LDL-Colesterol/sangre , Hipolipemiantes/uso terapéutico , Animales , Antibacterianos/uso terapéutico , Arteriosclerosis/tratamiento farmacológico , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Fosfatidilinositoles/metabolismo , Conejos , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/metabolismoRESUMEN
This study was undertaken to evaluate the cyclic adenosine monophosphate (cAMP) binding proteins in the cerebral cortex of rat after short- and long-term administration with antidepressants. Prolonged treatment with different antidepressants that inhibit serotonin or norepinephrine uptake such as fluoxetine and the (+) enantiomer of oxaprotiline, respectively, was able to induce an increase in the photoactivated incorporation of 8-N3-[32P]cAMP into a protein band with apparent molecular weight of 52,000 in both soluble and crude microtubule fraction. On the contrary, chronic treatment with the (-) enantiomer of oxaprotiline, which does not affect monoamine uptake, failed to produce this effect. Moreover, no changes were observed after acute or in vitro addition of antidepressants, suggesting that modification in the cAMP binding may be related to adaptive changes elicited by prolonged antidepressants treatment. In conclusion, our studies indicate that the cAMP binding protein associated with the crude microtubule fraction could be an intracellular target for the action of antidepressant drugs.
Asunto(s)
Corteza Cerebral/metabolismo , Fluoxetina/farmacología , Maprotilina/análogos & derivados , Norepinefrina/metabolismo , Receptores de AMP Cíclico/metabolismo , Antagonistas de la Serotonina/farmacología , Marcadores de Afinidad/metabolismo , Animales , Azidas/metabolismo , Corteza Cerebral/efectos de los fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Electroforesis en Gel de Poliacrilamida , Masculino , Maprotilina/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Peso Molecular , Norepinefrina/antagonistas & inhibidores , Ratas , Ratas Endogámicas , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/aislamiento & purificación , EstereoisomerismoRESUMEN
In developing Dictyostelium discoideum cells, binding of cyclic AMP to the chemotactic receptor has been shown to oscillate. These oscillations represent cycles of activation, adaptation and deadaptation of the cyclic AMP receptor system. We studied the molecular basis of these oscillatory changes in cyclic AMP receptor binding. We developed a rapid method of lysing cells during the course of the oscillations. This method guaranteed good preservation of ligand binding properties of the cyclic AMP receptor. We found that oscillations in cyclic AMP binding resulted from changes in receptor affinity. The total number of receptors did not significantly change during oscillations. Our experiments also showed that both GTP and GDP abolished oscillations in receptor binding completely, presumably by acting via a G protein. The guanine nucleotides reduced the affinity of the receptor at all time-points of the oscillation cycle to the minimal, i.e. adapted, level. We conclude that the cyclic process of activation, adaptation and de-adaptation in D. discoideum, at cyclic AMP receptor level, involves changes in receptor-G protein interaction. During adaptation, the affinity of the cyclic AMP receptor decreases and the receptor becomes insensitive to guanine nucleotides.