RESUMEN
Mechanisms of regulatory cell volume increase following cell shrinkage include accumulation of organic osmolytes such as betaine, taurine, sorbitol, glycerophosphorylcholine (GPC) and myo-inositol. Myo-inositol is taken up by the sodium-myo-inositol-transporter SMIT1 (SLC5A3) expressed in a wide variety of cell types. Hypertonicity induces the transcription of the SMIT1 gene upon binding of the transcription factor tonicity enhancer binding protein (TonEBP) to tonicity responsive enhancers (TonE) in the SMIT1 promoter region. However, little is known about post-translational regulation of the carrier protein. In this study we show that SMIT1 is modulated by the serum- and glucocorticoid-inducible kinase SGK1, a protein genomically up-regulated by hypertonicity. As demonstrated by two-electrode voltage-clamp in the Xenopus oocyte expression system, SMIT1-mediated myo-inositol-induced currents are up-regulated by coexpression of wild type SGK1 and constitutively active (S422D)SGK1 but not by inactive (K127N)SGK1. The increase in SMIT1 activity is due to an elevated cell surface expression of the carrier while its kinetic properties remain unaffected. According to the decay of SMIT1 activity in the presence of brefeldin A, SGK1 stabilizes the SMIT1 protein in the plasma membrane. The SGK isoforms SGK2, SGK3 and the closely related protein kinase B (PKB) are similarly capable of activating SMIT1 activity. SMIT1-mediated currents are decreased by coexpression of the ubiquitin-ligase Nedd4-2, an effect counteracted by additional coexpression of SGK1. In conclusion, the present observations disclose SGK isoforms and protein kinase B as novel regulators of SMIT1 activity.
Asunto(s)
Tamaño de la Célula , Proteínas Inmediatas-Precoces/metabolismo , Oocitos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Simportadores/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Células Cultivadas , Presión Osmótica , Regulación hacia Arriba/fisiología , Xenopus laevisRESUMEN
Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Canales de Calcio/genética , Epitelio/metabolismo , Humanos , Ratones , Oocitos/enzimología , Oocitos/metabolismo , Dominios PDZ , Técnicas de Placa-Clamp , Fosfoproteínas/metabolismo , Isoformas de Proteínas/metabolismo , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/metabolismo , Canales Catiónicos TRPV/genética , XenopusRESUMEN
Mutations in GJB2, the gene encoding for the Gap Junction protein Connexin 26 (Cx26), have been established as the major cause of hereditary, non-syndromic hearing impairment (HI). We report here the identification of a novel point mutation in GJB2, c.40A>G [p.N14D], detected in compound heterozygosity with the c.35delG mutation in two brothers with moderate non-syndromic sensorineural HI. The mother who carried one wildtype and a p.N14D allele displayed normal hearing. The mutation leads to substitution of the neutral amino acid asparagine (N) by the negatively charged aspartic acid (D) at amino acid number 14, a position that is conserved among Cx26 of different organisms and among many other connexin isoforms. To investigate the impact of this mutation on protein function, Cx26 activity was measured by depolarization activated hemichannel conductance in non-coupled Xenopus laevis oocytes. Oocytes injected with the p.N14D mutant cRNA showed strongly reduced currents compared to wildtype. Coinjection of wildtype and mutant cRNA at equimolar levels restored the conductive properties supporting the recessive character of this mutation. Total Cx26 protein expression and cell surface abundance examined by western blotting and by quantitative immunoassays revealed that the hemichannel was properly synthesized but not integrated into the plasma membrane. In this study we have shown that the GJB2 mutation p.N14D is associated with recessively inherited HI and exhibits a defective phenotype due to diminished expression at the cell surface.
Asunto(s)
Conexinas/genética , Pérdida Auditiva/genética , Mutación , Transporte de Proteínas/fisiología , Animales , Antígenos de Superficie/genética , Membrana Celular/fisiología , Niño , Preescolar , Clonación Molecular , Conexina 26 , Conexinas/metabolismo , Análisis Mutacional de ADN , Uniones Comunicantes/fisiología , Expresión Génica , Pérdida Auditiva/etiología , Humanos , Técnicas In Vitro , Masculino , Oocitos/metabolismo , Linaje , Xenopus laevisRESUMEN
Mutations in GJB2, which encodes the gap junction protein connexin 26 (Cx26), are one of the major causes for inherited and sporadic nonsyndromic hearing impairment. This study aimed to functionally characterize more frequent GJB2 mutations identified in patients showing nonsyndromic hearing impairment. Following injection of wild type and mutated cRNA in Xenopus oocytes, Cx26 hemichannel activity was measured by depolarization activated conductance in noncoupled oocytes. All mutants showed a partially or completely defective phenotype, except (V27I)Cx26, a polymorphism tested as positive control. Coexpression of wild type and mutant Cx26 injected at equimolar levels revealed that p.M34T, p.V37I and p.I82M, but not p.G59V, p.L90P, p.R127H and p.R143W exert a dominant inhibitory effect. When coexpressed with Cx30, a connexin partially colocalized with Cx26 in the cochlea, all mutants had a dominant behavior. This study provides data that might be important for the improvement of genetic diagnosis and counseling for patients with hearing impairment.
Asunto(s)
Conexinas/genética , Uniones Comunicantes/metabolismo , Predisposición Genética a la Enfermedad/genética , Pérdida Auditiva Sensorineural/genética , Mutación/fisiología , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Cóclea/metabolismo , Cóclea/fisiopatología , Conexina 26 , Conexina 30 , Conexinas/química , Conexinas/metabolismo , Células Epiteliales/metabolismo , Retroalimentación Fisiológica/genética , Femenino , Uniones Comunicantes/genética , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/fisiopatología , Humanos , Potenciales de la Membrana/genética , Oocitos , Técnicas de Placa-Clamp , Potasio/metabolismo , Estructura Terciaria de Proteína/fisiología , Xenopus laevisRESUMEN
The serum- and glucocorticoid-inducible kinase SGK1 is an ubiquitously expressed kinase with the ability to regulate a variety of transport systems. Recent observations point to a role of SGK1 in the regulation of diverse physiological functions such as epithelial transport and cardiac and neuronal excitability. At least partially through its effect on transport, SGK1 contributes to a number of pathophysiological conditions including metabolic syndrome and fibrosing disease.
Asunto(s)
Transporte Biológico/fisiología , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Activación Enzimática , Proteínas Inmediatas-Precoces , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na(+) channel by phosphorylating the ubiquitin ligase Nedd4-2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na(+)-P(i)) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4-2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4-2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current (I(P)). Exposure to 3 mM phosphate induces an I(P) in NaPi IIb-expressing oocytes. Coinjection of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2, significantly downregulates I(P), whereas the coinjection of (S422D)SGK1 markedly stimulates I(P) and even fully reverses the effect of Nedd4-2 on I(P). The effect of (S422D)SGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active (T308D,S473D)PKB, or inactive (K127N)SGK1. Moreover, (S422D)SGK1 and SGK3 phosphorylate Nedd4-2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4-2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.
Asunto(s)
Proteínas Portadoras/fisiología , Íleon/metabolismo , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/fisiología , Simportadores/metabolismo , Animales , Células Cultivadas , Enterocitos/metabolismo , Humanos , Íleon/citología , Proteínas Inmediatas-Precoces , Proteínas de la Membrana , Ratones , Técnicas de Placa-Clamp , Fosforilación , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb , Xenopus laevisRESUMEN
A hypertonic environment, as it prevails in renal medulla or in hyperosmolar states such as hyperglycemia of diabetes mellitus, has been shown to impair the immune response, thus facilitating the development of infection. The present experiments were performed to test whether hypertonicity influences activation of T lymphocytes. To this end, peripheral blood lymphocytes (PBL) of cytomegalovirus (CMV)-positive donors were stimulated by human leukocyte antigen (HLA)-A2-restricted CMV epitope NLVPMVATV to produce interferon (IFN)-gamma at varying extracellular osmolarity. As a result, increasing extracellular osmolarity during exposure to the CMV antigen indeed decreased IFN-gamma formation. Addition of NaCl was more effective than urea. A 50% inhibition was observed at 350 mosM by addition of NaCl. The combined application of the Ca(2+) ionophore ionomycin (1 microg/ml) and the phorbol ester phorbol 12-myristate 13-acetate (PMA; 5 microg/ml) stimulated IFN-gamma production, an effect again reversed by hyperosmolarity. Moreover, hyperosmolarity abrogated the stimulating effect of ionomycin (1 microg/ml) and PMA (5 microg/ml) on the transcription factors activator protein (AP)-1, nuclear factor of activated T cells (NFAT), and NF-kappaB but not Sp1. In conclusion, osmotic cell shrinkage blunts the stimulatory action of antigen exposure on IFN-gamma production, an effect explained at least partially by suppression of transcription factor activation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Linfocitos T/citología , Apoptosis/fisiología , Tamaño de la Célula/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Concentración Osmolar , Ósmosis/fisiología , Linfocitos T/fisiologíaRESUMEN
Sympathetic neurons depend on NGF binding to TrkA for their survival during vertebrate development. NGF deprivation initiates a transcription-dependent apoptotic response, which is suggested to require activation of the transcription factor c-Jun. Similarly, apoptosis can also be induced by selective activation of the p75 neurotrophin receptor. The transcriptional dependency of p75-mediated cell death has not been determined; however, c-Jun NH2-terminal kinase has been implicated as an essential component. Because the c-jun-null mutation is early embryonic lethal, thereby hindering a genetic analysis, we used the Cre-lox system to conditionally delete this gene. Sympathetic neurons isolated from postnatal day 1 c-jun-floxed mice were infected with an adenovirus expressing Cre recombinase or GFP and analyzed for their dependence on NGF for survival. Cre immunopositive neurons survived NGF withdrawal, whereas those expressing GFP or those uninfected underwent apoptosis within 48 h, as determined by DAPI staining. In contrast, brain-derived neurotrophic factor (BDNF) binding to p75 resulted in an equivalent level of apoptosis in neurons expressing Cre, GFP, and uninfected cells. Nevertheless, cycloheximide treatment prevented BDNF-mediated apoptosis. These results indicate that whereas c-jun is required for apoptosis in sympathetic neurons on NGF withdrawal, an alternate signaling pathway must be induced on p75 activation.
Asunto(s)
Apoptosis/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factor de Crecimiento Nervioso/deficiencia , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-jun/deficiencia , Receptor de Factor de Crecimiento Nervioso/metabolismo , Ganglio Cervical Superior/embriología , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Células Cultivadas , Cicloheximida/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Indicadores y Reactivos , Integrasas/genética , Proteínas Luminiscentes , Ratones , Ratones Noqueados , Mutación/efectos de los fármacos , Mutación/fisiología , Factor de Crecimiento Nervioso/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-jun/genética , Receptor de Factor de Crecimiento Nervioso/efectos de los fármacos , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/crecimiento & desarrollo , Transfección , Proteínas Virales/genéticaRESUMEN
Strong evidence indicated that spermatic nerves are involved in the regulation of testosterone secretion. Our previous work showed that the inferior spermatic nerves play a more significant role than the superior ones in the regulation of testosterone secretion. However, it is unknown whether traditional neurotransmitters are involved in this regulation. In order to evaluate this point, the present experiments were carried out in an in vitro system where an isolated testis-spermatic nerve plexus preparation was incubated in two separate containers, one for the testis and the other for the nerve plexus and both interconnected by the inferior spermatic nerves. Both tissues were maintained in the same environmental conditions except for the neurotransmitter treatment, applied only to the nerve plexus. Acetylcholine can significantly inhibit the secretion of testosterone until the end of incubation. The present experiments suggest that the secretion of testosterone could be regulated, at least in part, by acetylcholine through the inferior spermatic nerves.
Asunto(s)
Acetilcolina/fisiología , Testículo/inervación , Testosterona/metabolismo , Animales , Técnicas In Vitro , Masculino , Nervios Periféricos/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
EAAC1-mediated glutamate transport concentrates glutamate across plasma membranes of brain neurons and epithelia. In brain, EAAC1 provides a presynaptic uptake mechanism to terminate the excitatory action of released glutamate and to keep its extracellular concentration below toxic levels. Here we report the effect of well known anxiolytic compounds, benzodiazepines, on glutamate transport in EAAC1-stably transfected Chinese hamster ovary (CHO) cells and in EAAC1-expressing Xenopus laevis oocytes. Functional properties of EAAC1 agreed well with already reported characteristics of the neuronal high-affinity glutamate transporter (Km D-Asp,CHO cells: 2.23+/-0.15 microM; Km D-Asp,oocytes: 17.01+/-3.42 microM). In both expression systems, low drug concentrations (10-100 microM) activated substrate uptake (up to 200% of control), whereas concentrations in the millimolar range inhibited (up to 50%). Furthermore, the activation was more pronounced at low substrate concentrations (1 microM), and the inhibition was attenuated. The activity of other sodium cotransporters such as the sodium/D-glucose cotransporter SGLT1, stably transfected in CHO cells, was not affected by benzodiazepines. In electrophysiological studies, these drugs also failed to change the membrane potential of EAAC1-expressing Xenopus laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Ansiolíticos/farmacología , Proteínas Portadoras/efectos de los fármacos , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/efectos de los fármacos , Simportadores , Animales , Sitios de Unión , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clozapina/farmacología , Cricetinae , Cricetulus , Diazepam/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Transporte Iónico , Lorazepam/farmacología , Medazepam/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oocitos , Oxazepam/farmacología , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Sodio/metabolismo , Xenopus laevisRESUMEN
In the nervous system, glutamate is an excitatory aminoacid which at higher concentrations has been implicated in a number of disorders. Glutamate is stored in presynaptic vesicles and is released by calcium-dependent exocytosis. After its action on ionotropic receptors (iGluR, related to ionic channels) or metabotropic receptors (mGluR, related to metabolic formation of second messengers), glutamate can be removed from the synaptic cleft through two processes: re-uptake back into pre-synaptic terminals or diffusion out of synaptic cleft for uptake by glial cells. This is achieved by glutamate transporters. In pre-synaptic terminals, glutamate is packed into the specialized secretory vesicles by means of a specific vesicular transporter. The level of glutamate available for neurosecretion is regulated by the vesicular transport activity. In order to achieve a proper concentration of the neurotransmitter in synaptic vesicles, glutamate must be synthesized. Glutamine is obtained in astroglial cells from the glutamate reuptaken, and as it has no neurotransmitter activity, it is the metabolite which regenerates glutamate in neurones (glutamate-glutamine cycle). Moreover, glutamate is also obtained from glucose by an intermediate of TCA cycle. In this paper we want to introduce some aspects of glutamate biosynthesis and release: glutamate receptors, neurotransmitter uptake by the glutamate transporters and neurotransmitter inactivation and new formation by metabolism.