RESUMEN
In this clinical trial, we investigated the blood glucose (BG)-lowering effects of 30, 60 and 90 mg dextromethorphan (DXM) as well as 100 mg sitagliptin alone versus combinations of DXM and sitagliptin during an oral glucose tolerance test (OGTT) in 20 men with T2DM. The combination of 60 mg DXM plus 100 mg sitagliptin was observed to have the strongest effect in the OGTT. It lowered maximum BG concentrations and increased the baseline-adjusted area under the curve for serum insulin concentrations in the first 30 min of the OGTT (mean ± standard deviation 240 ± 47 mg/dl and 8.1 ± 6.1 mU/l/h, respectively) to a significantly larger extent than did 100 mg sitagliptin alone (254 ± 50 mg/dl and 5.8 ± 2.5 mU/l/h, respectively; p < 0.05) and placebo (272 ± 49 mg/dl and 3.9 ± 3.0 mU/l/h, respectively; p < 0.001). All study drugs were well tolerated, alone and in combination, without serious adverse events or hypoglycaemia. Long-term clinical trials are now warranted to investigate the potential of the combination of 30 or 60 mg DXM and dipeptidyl peptidase-4 inhibitors in the treatment of individuals with T2DM, in particular as preclinical studies have identified the ß-cell protective properties of DXM.
Asunto(s)
Glucemia/efectos de los fármacos , Dextrometorfano/administración & dosificación , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores de la Dipeptidil-Peptidasa IV/administración & dosificación , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Insulina/sangre , Fosfato de Sitagliptina/administración & dosificación , Anciano , Estudios Cruzados , Diabetes Mellitus Tipo 2/sangre , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Quimioterapia Combinada , Prueba de Tolerancia a la Glucosa , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/administración & dosificación , Masculino , Persona de Mediana EdadRESUMEN
Hepatic encephalopathy (HE) in liver cirrhosis is a clinical manifestation of a low-grade cerebral oedema, which is exacerbated in response to ammonia and other precipitating factors. This low-grade cerebral oedema is accompanied by an increased production of reactive oxygen and nitrogen oxide species (ROS/RNOS), which trigger multiple protein and RNA modifications, thereby affecting brain function. The action of ammonia, inflammatory cytokines, benzodiazepines and hyponatraemia integrates at the level of astrocyte swelling and oxidative stress. This explains why heterogenous clinical conditions can precipitate HE episodes. Oxidised RNA species, which are formed in response to oxidative stress, also participate in local postsynaptic protein synthesis in neurons, which is required for memory formation. Although the functional consequences of RNA oxidation in this context remain to be established, these findings bear a potential biochemical explanation for the multiple alterations of neurotransmitter receptor systems and of synaptic plasticity. Such changes may in part also underlie the pathologically altered oscillatory networks in the brain of HE patients in vivo, as detected by magnetencephalography. These disturbances of oscillatory networks, which in part are triggered by hypothalamic structures, can explain the motor and cognitive deficits in patients with HE. Current therapeutic strategies aim at the elimination of precipitating factors. The potential of therapies targeting downstream pathophysiological events in HE has not yet been explored, but offers novel potential sites of therapeutic intervention.
Asunto(s)
Edema Encefálico/complicaciones , Encefalopatía Hepática/etiología , Astrocitos/patología , Encefalopatía Hepática/patología , Encefalopatía Hepática/fisiopatología , Humanos , Cirrosis Hepática/complicaciones , Oxidación-Reducción , Estrés Oxidativo , ARN/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Background. Glutamine synthetase (GS) is ubiquitously expressed in the human and plays a major role for many metabolic pathways. However, little is known about its role during the fetal period. Methods. Cultured skin fibroblasts derived from an aborted fetus deficient in GS activity due to a R324C exchange as well as fetal and mature controls were used to determine the level of GS-expression, apoptosis, and proliferation in presence or absence of exogenous glutamine. Results. Glutamine synthetase can be found at early gestational stages. Loss of GS activity either inherited or induced through l-methionine sulfoximine leads to an upregulation of the GS protein but not of the GS mRNA and results in a significant drop in the proliferation rate but has no effect on apoptosis. Exogenous glutamine does not influence the rate of apoptosis but increases proliferation rates of the fetal but not the mature fibroblasts. Conclusion. GS can be found during early human fetal stages when it displays a significant effect on cell proliferation.
Asunto(s)
Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glutamato-Amoníaco Ligasa/fisiología , Piel/embriología , Apoptosis , Cationes , Proliferación Celular , Supervivencia Celular , Medios de Cultivo/metabolismo , Genotipo , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/metabolismo , Humanos , Modelos Biológicos , Factores de TiempoRESUMEN
5-Aminolevulinate (ALA) photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. ALA is applied as a precursor of porphyrins serving as endogenous photosensitizers. Irradiation of HaCaT cells preincubated with 1 mM ALA for 24 h with red light of 570-750 nm at a dose of 4.5 J/cm2 leads to a 6-fold elevation of cellular c-Jun N-terminal kinase activity; phosphorylation of p38 mitogen-activated protein kinase (MAPK) is enhanced to a similar extent. In contrast, neither activation nor increased phosphorylation of the extracellular stimulus-regulated kinase MAPKs is detected. p38 is also phosphorylated by ALA-PDT in the human melanoma cell lines Bro and SkMel-23, applying doses that lead to 80-95% cell death after 24 h. Hence, the effects of ALA-PDT on MAPKs are similar to stresses like UV irradiation or exposure to hydrogen peroxide with respect to activation of JNK and p38 MAPKs. They are different, however, in that extracellular stimulus-regulated kinase activity is not raised by ALA-PDT. Of the 830 pmol porphyrins/mg protein that were present at 24 h in HaCaT cells, 99 pmol/mg were intracellular. When extracellular porphyrins had been removed by washing, p38 responses were retained. Thus, intracellular porphyrins synthesized from ALA are sufficient to elicit activation of p38 on photosensitization.
Asunto(s)
Ácido Aminolevulínico/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Fotoquimioterapia , Piel/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Activación Enzimática , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Queratinocitos , Melanoma , Fosforilación , Piel/enzimología , Piel/efectos de la radiación , Neoplasias Cutáneas , Células Tumorales Cultivadas , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The influence of aniso-osmolarity on the activity of the MAP kinases Erk-1 and Erk-2 was studied in C6 glioma cells. Hypo-osmotic treatment (205 mosmol/l) led to an increased activity of Erk-1 and Erk-2 within 3 min, which became maximal at 10 min and returned to basal level within 120 min. In contrast, Erk activity was reduced under hyper-osmotic conditions (405 mosmol/l), compared to the normo-osmotic control (305 mosmol/l). Erk activation was accompanied by a mobility shift of Raf-1. Hypo-osmotic exposure increased the cytosolic Ca2+ concentration ([Ca2+]i). Absence of extracellular Ca2+ largely abolished the [Ca2+]i response to hypo-osmolarity, whereas Erk activation following hypo-osmotic stimulation remained unaffected, suggesting a Ca2+ independence of the osmosignalling pathway to the MAP kinases. Both the Ca2+ response as well as the Erk activation following hypo-osmotic exposure were maintained in the presence of the phospholipase C inhibitor U73122. Application of 8-CPT cAMP, forskolin/isobutylmethylxanthine or isoproterenol blocked Erk activation following hypo-osmotic treatment of the cells, suggesting a role of the Ras/Raf pathway upstream from Erk-1 and Erk-2. Protein kinase C (PKC) is unlikely to play a role in the hypo-osmolarity- induced signalling towards MAP kinases, as revealed by inhibition of PKC with Go6850. Inhibition of pertussis- or cholera toxin-sensitive G-proteins as well as inhibition of tyrosine kinases with genistein and of PI3 kinase by wortmannin had no effect on the Erk response to hypo-osmolarity. It is concluded that osmosignalling in C6 glioma cells differs upstream of the MAP kinases from that observed in primary rat astrocytes, H4IIE rat hepatoma cells and isolated rat hepatocytes.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Calcio/metabolismo , Glioma/patología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Concentración Osmolar , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Ratas , Células Tumorales CultivadasRESUMEN
The influence of anisoosmolarity on NF-kappaB binding activity was studied in H4IIE rat hepatoma cells. Hypoosmolarity induced a sustained NF-kappaB binding activity whereas the hyperosmotic NF-kappaB response was only minor. Hypoosmotic NF-kappaB activation was accompanied by degradation of the inhibitory IkappaB-alpha. Protein kinase C, PI(3)-kinase, reactive oxygen intermediates and the proteasome apparently participate in mediating the hypoosmotic effect on NF-kappaB. Hypoosmolarity plus PMA induced, amplified and prolonged IkappaB-alpha degradation and NF-kappaB binding activity. Transforming growth factor beta-induced apoptosis was diminished by hypoosmolarity. However, this anti-apoptotic effect was probably not related to NF-kappaB activation.
Asunto(s)
FN-kappa B/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis , Carcinoma Hepatocelular , Caspasa 3 , Caspasas/metabolismo , Inhibidores Enzimáticos/farmacología , Neoplasias Hepáticas , FN-kappa B/química , Concentración Osmolar , Inhibidores de las Quinasa Fosfoinosítidos-3 , Poli(ADP-Ribosa) Polimerasas , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Acetato de Tetradecanoilforbol , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales CultivadasAsunto(s)
Astrocitos/metabolismo , Hígado/metabolismo , Transducción de Señal/fisiología , Animales , Carcinoma Hepatocelular/patología , Glioma/patología , Encefalopatía Hepática/patología , Encefalopatía Hepática/fisiopatología , Humanos , Hígado/citología , Concentración Osmolar , Células Tumorales CultivadasRESUMEN
Alterations of cell volume induced by either aniso-osmotic environments or under the influence of hormones, concentrative amino acid uptake and oxidative stress were recognized as an independent signal contributing to the regulation of metabolism and gene expression. The regulation of cell function by hydration changes requires structures, which register fluctuations of cell hydration (osmosensing) and thereby activate intracellular signalling pathways towards effector sites (osmosignalling). Meanwhile, it is well established that osmosensing and signalling integrate into the overall context of hormone- and nutrient-induced signal transduction. Recent evidence suggests integrins to play a major role in osmosensing and signalling due to hepatocyte swelling. This review focuses on the role of integrins in sensing of hepatocyte swelling as triggered by hypo-osmolarity, glutamine and insulin and the relevance of integrin-dependent osmosignalling for inhibition of autophagic proteolysis, stimulation of canalicular bile acid excretion and regulatory volume decrease.
Asunto(s)
Hepatocitos/metabolismo , Integrinas/metabolismo , Transducción de Señal/fisiología , Animales , Autofagia , Ácidos y Sales Biliares/metabolismo , Tamaño de la Célula , Glutamina/metabolismo , Humanos , Insulina/metabolismo , Equilibrio HidroelectrolíticoRESUMEN
Insulin- and amino acid-induced signalling by the mammalian target of rapamycin (mTOR) involves hyperphosphorylation of the p70 ribosomal S6 protein kinase (p70S6-kinase) and the eukaryotic initiation factor 4E (eIF4E) binding protein 4E-BP1 and contributes to regulation of protein metabolism. This review considers the impact of cell hydration on mTOR-dependent signalling. Although hypoosmotic hepatocyte swelling in some instances activates p70S6-kinase, the hypoosmolarity-induced proteolysis inhibition in perfused rat liver is insensitive to mTOR inhibition by rapamycin. Likewise, swelling-dependent proteolysis inhibition by insulin and swelling-independent proteolysis inhibition by leucine, a potent activator of p70S6-kinase and 4E-BP1 hyperphosphorylation, in perfused rat liver is insensitive to rapamycin, indicating that at least rapamycin-sensitive mTOR signalling is not involved. Hyperosmotic dehydration in different cell types produces inactivation of signalling components around mTOR, thereby attenuating insulin-induced glucose uptake, glycogen synthesis, and lipogenesis in adipocytes, and MAP-kinase phosphatase MKP-1 expression in hepatoma cells. Direct inactivation of mTOR, stimulation of the AMP-activated protein kinase, and the destabilization of individual proteins may impair mTOR signalling under dehydrating conditions. Further investigation of the crosstalk between the mTOR pathway(s) and hyperosmotic signalling will improve our understanding about the contribution of cell hydration changes in health and disease and will provide further rationale for fluid therapy of insulin-resistant states.
Asunto(s)
Adipocitos/metabolismo , Hígado/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Equilibrio Hidroelectrolítico/fisiología , Aminoácidos/metabolismo , Animales , Tamaño de la Célula , Insulina/metabolismo , Fosforilación , Ratas , Serina-Treonina Quinasas TORRESUMEN
The predominant integral membrane protein of the CNS myelin of amphibia, reptiles, birds and mammals is proteolipid protein (PLP) and P0, the main glycoprotein in PNS myelin. Alternative splicing of the transcripts of the single genes of PLP and myelin basic protein (MBP) is the underlying mechanism by which the isoforms of the two main proteins of the myelin membrane arise. DM20 is an isoform of PLP in mammalian, avian and reptilian myelin. It does not occur in the CNS myelin of amphibia. DM20 lacks an extended hydrophilic sequence exposed on the extracytoplasmic surface of the lipid bilayer as a result of the usage of a cryptic donor splice site within exon III. We report about comparative studies on PLP and its DM20 isoform on the protein and DNA level of frog, chicken, rat CNS and the P0-related IP proteins of the CNS of trout. Chemical cleavage at tryptophan residues with N-chlorosuccinimide yields identical patterns of PLP peptides which refers to a high conservation between amphibia, birds and mammals and is totally different from the cleavage pattern of hydrophobic myelin proteins IP-1 and IP-2 of trout CNS and that of P0 of rat PNS. The N-terminal 19 amino-acid residues of IP-1 of trout CNS- and P0 of frog PNS myelin were sequenced and proved to be homologous on one hand with the P0 analogue of CNS of the shark, a cartilage fish, and on the other hand with P0 protein of PNS of birds and mammals. The complete amino-acid sequence of chicken CNS PLP was derived from its cDNA. Coding and noncoding segments of the PLP gene of frog were sequenced: there is a high degree of conservation between amphibian and mammalian PLP within the hydrophobic domains. Numerous mutations were found within the part of exon III encoding the hydrophilic domain. Base exchanges within the putative splice site in exon III explain the absence of DM20 in the protein pattern of amphibia CNS myelin. This result is being discussed in view of the membrane organization and the function of PLP.
Asunto(s)
Evolución Biológica , Sistema Nervioso Central/fisiología , Proteínas de la Mielina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Moléculas de Adhesión Celular Neuronal/genética , Exones , Humanos , Datos de Secuencia Molecular , Proteína Básica de Mielina/genética , Proteína P0 de la Mielina , Proteína Proteolipídica de la Mielina , Vaina de Mielina/fisiología , Conformación Proteica , Empalme del ARN , Ratas , Homología de Secuencia de Ácido Nucleico , Tiburones , Trucha , Xenopus laevisRESUMEN
Changes in cell hydration are critically important for the signalling towards metabolic responses to hormones, substrates and reactive oxygen intermediates. In liver insulin-induced cell swelling is due to a net K(+)-uptake resulting from the concerted activation of Na(+)/K(+)/2Cl(-) cotransport, Na(+)/H(+) exchange and the Na(+)/K(+)-ATPase. Insulin-induced swelling is essential for generating the antiproteolytic response to the hormone, which depends on activation of the MAP-kinase p38. Recent investigations show, that cell swelling induced by either hypoosmolarity or insulin triggers the activation of signalling cascades. Cell swelling by insulin is Ptdins-3-kinase mediated and contributes to the activation of Erk- and p38-type MAP-kinases. Conditions dehydrating insulin target tissues such as hyperosmolarity or amino acid deprivation are frequently associated with insulin resistance. In liver, hyperosmolarity impairs the Ptdins-3-kinase-dependent K(+) uptake and cell swelling in response to insulin, leading to resistance of MAP-kinases and proteolysis to regulation by insulin. Likewise, a reduction of insulin-induced swelling by the loop diuretics furosemide and bumetanide cause insulin resistance shown by the levels of cell swelling, MAP-kinase activation and proteolysis control. Blockage of the cell volume response to insulin may be the common denominator in dehydration-induced insulin resistance found in clinical settings such as sepsis, burn injury and diabetes mellitus.
Asunto(s)
Insulina/metabolismo , Transducción de Señal , Agua/metabolismo , AnimalesRESUMEN
The hepatocellular hydration state, i.e. liver cell volume, is a dynamic parameter, which changes within minutes in response to alterations in the environmental or hormonal milieu. These changes in cell hydration act as a signal which modifies metabolism and gene expression due to complex alterations in protein phosphorylation. The role of cellular hydration as an important determinant of liver cell function and gene expression may shed a new light not only on liver physiology but also on liver pathophysiology.
Asunto(s)
Hígado/citología , Hígado/fisiología , Animales , Bilis/metabolismo , Agua Corporal/metabolismo , Humanos , Proteínas/metabolismo , Transducción de Señal , Replicación ViralRESUMEN
The influence of astrocyte swelling on the cytosolic free calcium concentration [Ca2+]i was studied at the single cell level. Sudden exposure of normo-osmotically (305 mosmol/l) cultured astrocytes to hypo-osmotic medium induced a biphasic increase in cytosolic calcium with an initial peak followed by a sustained plateau. The response was osmolarity dependent and was maximal at 205 mosmol/l with respect to [Ca2+]i and the percentage of responding cells. Other modes of astrocyte swelling [gradual adjustment of hypo-osmolarity, normo-osmotic exposure of hyper-osmotic (405 mosmol/l) maintained cells] produced a much weaker [Ca2+]i response. Change from 405 to 205 mosmol/l, however, resulted in the entire peak and an increased plateau. Experiments with Ca(2+)-free medium and after pretreatment with BAPTA-AM, thapsigargin, phorbol myristate acetate, or nimodipine revealed that the peak mainly resulted from depletion of intracellular Ca2+ stores, whereas the plateau was probably due to capacitative Ca2+ entry and Ca2+ influx independent of store depletion including a nimodipin-sensitive component. Prior depletion of ryanodine-, bradykinin- or ATP-sensitive stores revealed that the initial hypo-osmolarity-induced Ca(2+)-release was from a Ca2+ pool also affected by ATP and bradykinin, but not by ryanodine. The recent finding, that the hypo-osmolarity-induced [Ca2+]i response was completely maintained if phospholipase C-mediated phosphatidylinositol hydrolysis was blocked, suggests that hypo-osmolarity may exert an inositol (1,4,5) triphosphate-independent access to these stores.
Asunto(s)
Astrocitos/fisiología , Encéfalo/fisiología , Calcio/metabolismo , Soluciones Hipotónicas , Adenosina Trifosfato/farmacología , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Bradiquinina/farmacología , Células Cultivadas , Quelantes/farmacología , Medios de Cultivo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Flunarizina/farmacología , Cinética , Nimodipina/farmacología , Ratas , Ratas Wistar , Rianodina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacologíaRESUMEN
Hepatic metabolism and gene expression are among the factors controlled by the cellular hydration state, which changes within minutes in response to aniso-osmotic environments, cumulative substrate uptake, oxidative stress and under the influence of hormones such as insulin. The signalling events coupling cell-volume changes to altered cell function were studied in H4IIE rat hepatoma cells. Hypo-osmotic cell swelling resulted within 1 min in a tyrosine kinase-mediated activation of the extracellular signal-regulated protein kinases Erk-1 and Erk-2, which was independent of protein kinase C and cytosolic calcium. Activation of mitogen-activated protein kinases was followed by an increased phosphorylation of c-Jun, which may explain our recently reported finding of an about 5-fold increase in c-jun mRNA level in response to cell swelling. Pretreatment of cells with pertussis or cholera toxin abolished the swelling-induced activation of Erk-1 and Erk-2, suggesting the involvement of G-proteins. Thus, a signal-transduction pathway resembling growth factor signalling is activated already by osmotic water shifts across the plasma membrane, thereby providing a new perspective for adaption of cell function to alterations of the environment.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Proteínas Tirosina Quinasas/metabolismo , Animales , Calcio/metabolismo , Tamaño de la Célula , Toxina del Cólera/farmacología , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Genisteína , Isoflavonas/farmacología , Neoplasias Hepáticas Experimentales/enzimología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Presión Osmótica , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/metabolismo , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The influence of osmolarity and compatible organic osmolytes on the phosphorylation of the MAP-kinases Erk-1 and Erk-2 and on the expression of taurine transporter (TAUT) and lipopolysaccharide (LPS)-induced nitric oxide synthetase (iNOS) was studied in RAW 264.7 mouse macrophages. Hypoosmolarity (205 mosmol/l) but not hyperosmolarity (405 mosmol/l) or challenge of the cells with betaine or taurine increased phosphorylation of Erk-1 and Erk-2. Hypoosmotic Erk-phosphorylation was blocked by the MEK-inhibitor PD098059 but was resistant to depletion of extracellular calcium and to inhibition of PLC, PKC, erbstatin-sensitive tyrosine kinases and elevation of intracellular cAMP. Hyperosmolarity stimulated Na+-dependent taurine uptake and led to an increase of TAUT mRNA levels, whereas hypoosmotic exposure diminished both and induced a rapid efflux of the osmolyte from taurine-preloaded cells. The hyperosmotic elevation of TAUT mRNA levels was antagonized upon addition of taurine but not of betaine or myo-inositol. Hyperosmolarity increased the LPS-induced iNOS expression at the mRNA and the protein level. This was suppressed by betaine but not by taurine or myo-inositol. The osmotic regulation of taurine transport and iNOS expression appeared independent of the MEK-Erk pathway and the p38MAPK.
Asunto(s)
Macrófagos/metabolismo , Proteínas de Transporte de Membrana , Proteínas Quinasas Activadas por Mitógenos , Óxido Nítrico Sintasa/metabolismo , Animales , Betaína/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Inositol/farmacología , Lipopolisacáridos/farmacología , Lipotrópicos/farmacología , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Concentración Osmolar , Fosforilación , Taurina/metabolismo , Taurina/farmacologíaRESUMEN
The effects of hypo- and hyper-osmotic shock on endogenous MAP-kinase activities and MKP-1 and c-jun mRNA levels were studied in H4IIE rat hepatoma cells. In presence of vanadate hypo-osmolarity stimulated a rapid and sustained activation of MAP-kinases (Erk-2, JNK-2 and p38). In the absence of vanadate a hypo-osmotic MAP-kinase response was not detectable. Hyper-osmolarity stimulated a delayed and transient MAP-kinase activation and vanadate was not required for its detection. Vanadate, however, amplified the hyper-osmotic MAP-kinase stimulation. c-jun and MKP-1 mRNA levels were maximal after 0.5-1 h of hypo-osmotic exposure and returned towards basal levels within 2 h, whereas the hyper-osmotic induction of c-jun and MKP-1 mRNA was delayed. Vanadate was not required for the aniso-osmotic effects on MKP-1 and c-jun mRNA levels. Whereas the hyper-osmolarity-induced c-jun mRNA accumulation returned towards basal levels within 8 h, MKP-1 mRNA was still highly expressed at this time point. The role of MAP-kinases for the induction of aniso-osmolarity-induced gene expression and the potential importance of MKP-1 for termination of aniso-osmotic MAP-kinase activation are discussed.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Transducción de Señal , Vanadatos/metabolismo , Animales , Northern Blotting , Regulación Neoplásica de la Expresión Génica , Presión Osmótica , Ratas , Células Tumorales CultivadasRESUMEN
The action of hyperosmotic stress on the MAP kinase phosphatase MKP-1 mRNA expression was studied in H4IIE rat hepatoma cells. Hyperosmotic (405 mosmol/L) challenge of the cells led to a transient expression of MKP-1 mRNA, which was maximal after 6-8 h and disappeared completely after 24 h. Hyperosmotic MKP-1 mRNA induction was preceded by a transient activation of the MAP kinases Erk-1, Erk-2, and JNK-2, which were not prerequisite for MKP-1 mRNA accumulation. However, the hyperosmolarity-induced MKP-1 mRNA expression was sensitive to antioxidants and to inhibition of p38 by SB203580. A reduced sensitivity of Erk-1/Erk-2 to other stimuli was found after prolonged hyperosmotic exposure. The data are consistent with a hyperosmolarity-induced MKP-1 expression via reactive oxygen intermediates and p38, which may participate in the termination of MAP kinase activation and contribute to desensitization of the MAP kinases after prolonged hyperosmotic exposure of the cells.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos , Neoplasias Hepáticas Experimentales/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Fosfoproteínas Fosfatasas , Proteínas Tirosina Fosfatasas/biosíntesis , Solución Salina Hipertónica , Animales , Antioxidantes/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Fosfatasa 1 de Especificidad Dual , Activación Enzimática , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Expresión Génica , Imidazoles/farmacología , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/genética , Cinética , MAP Quinasa Quinasa 4 , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/genética , Piridinas/farmacología , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The influence of cell hydration on the heat shock response was investigated in H4IIE hepatoma cells at the levels of HSP70 expression, MAP kinase activation, induction of c-jun and the MAP kinase phosphatase MKP-1, heat resistance, and development of tolerance/sensitization to arsenite after a priming heat treatment. Induction of HSP70, MKP-1, and c-jun by heat was delayed, but more pronounced or sustained, under hyperosmotic conditions compared with normo- and hypo-osmotically exposed cells. Anisosmolarity per se was ineffective to induce HSP70; some expression of the mRNAs for MKP-1 and c-jun in response to hyperosmolarity was found, but was small compared with the response to heat. Heat-induced activation of JNK-1 was increased under hyperosmotic conditions and more sustained than the JNK-activity induced by hyperosmolarity at 37 degrees C. A prominent Erk-2 activation was found immediately after heat shock under hypo- and normo-osmotic conditions, but Erk-2 activation was weak in hyperosmolarity-exposed cells. Despite anisosmotic alterations of the heat shock response at the molecular level, the heat resistance of H4IIE cells toward heat shock was not affected by ambient osmolarity. However, an osmolarity-dependent sensitization to arsenite was induced by a priming heat shock. The osmodependence of the H4IIE cell response to heat differs from that recently found in primary rat hepatocytes. The data are discussed in terms of cellular adaption mechanisms and their physiological relevance.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Neoplasias Hepáticas Experimentales/fisiopatología , Proteínas Quinasas Activadas por Mitógenos , Fosfoproteínas Fosfatasas , Proteínas Proto-Oncogénicas c-jun/genética , Animales , Fosfatasa 1 de Especificidad Dual , Inducción Enzimática , Genes jun , Calor , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Concentración Osmolar , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , RatasRESUMEN
Insulin-induced cell swelling was recently suggested to reflect an independent signal for metabolic insulin effects such as inhibition of hepatic proteolysis, which is transmitted at the level of autophagosome formation via p38MAPK activation [Häussinger et al., Gastroenterology 116 (1999), 921-935]. Here, the role of insulin-induced cell swelling in the overall context of insulin signalling towards proteolysis inhibition was studied in perfused rat liver. Loop diuretics and hyperosmolarity, which impair insulin-stimulated cell swelling, strongly blunt Erk-2 and p38MAPK activation as well as proteolysis inhibition by insulin, but are without effect on insulin-induced tyrosine phosphorylation of IR-beta and IRS-1. Inhibitors of phosphatidylinositol-3-kinase (PI3-kinase) also block insulin-induced cell swelling, MAP kinase activation and proteolysis inhibition, but the antiproteolytic response to hypoosmolarity remains unaffected. We suggest that PI3-kinase-mediated cell swelling induced by insulin is required to amplify the insulin signal to MAP kinases and thus proteolysis regulation. The perturbation of insulin-induced cell swelling may be of pathophysiological relevance for the development of insulin resistance in clinical situations associated with hyperosmotic dehydration and loop diuretic treatment.
Asunto(s)
Diuréticos/farmacología , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Hígado/metabolismo , Animales , Western Blotting , Diuresis/efectos de los fármacos , Diuresis/fisiología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Insulina/farmacología , Insulina/fisiología , Hígado/efectos de los fármacos , Hígado/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Concentración Osmolar , Inhibidores de las Quinasa Fosfoinosítidos-3 , Potasio/metabolismo , Ratas , Ratas Wistar , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
In perfused rat liver, hypoosmotic exposure (225 mosmol/L) leads to a volume-regulatory decrease by release of K(+), Cl(-) and HCO(3)(-) through Ba(2+)-, DIDS- and quinidine-sensitive ion channels. The underlying signal transduction mechanisms, however, are unknown. As hypoosmotic hepatocyte swelling leads to a rapid activation of extracellular signal regulated kinases (Erks) and of p38(MAPK), the role of mitogen-activated protein kinases (MAPK) and PI-3-kinase in mediating the RVD in perfused rat liver was studied. The presence of the MEK inhibitor PD 098 059, which blocks the hypoosmotic activation of Erks, had no effect on the extent and time course of cell volume regulatory K(+) efflux. However, inhibitors of p38(MAPK) such as SB 203 580 and PD 169 316, but not their inactive analogue SB 202 474, significantly delayed and diminished the volume-regulatory K(+) efflux. Accordingly, in presence of these p38(MAPK) inhibitors, the hepatocytes remained in a more swollen state after completion of RVD. Inhibition of hypoosmotic Erk activation by pertussis or cholera toxin, erbstatin or genistein had no effect on RVD by hypoosmolarity. Likewise, neither inhibition of PI-3-kinase by wortmannin or LY 294 002 nor inhibition of S 6 phosphorylation by rapamycin nor protein kinase inhibition by H-7, H-89 or KT 5823 led to a significant change of RVD upon hypoosmolarity. The amount and time course of K(+) release by oxidative stress upon addition of t-BOOH or H(2)O(2) remained unaffected by inhibition of p38(MAPK) by SB 203 580, suggesting a specific inhibition of RVD-dependent K(+) release by this inhibitor. The findings suggest that swelling-induced activation of p38(MAPK), but not of Erks and PI-3-kinase, is involved in RVD in liver, whereas p38(MAPK) is apparently not involved in the net K(+) release induced by oxidative stress.