RESUMO
In vertebrates, SLC22A13 is an evolutionarily conserved transport protein of the plasma membrane. In humans and rat, it is principally expressed in the kidney. The precise localization and physiological function are unknown. In the present study, immunohistochemistry revealed that expression of SLC22A13 is confined to the basolateral membrane of type A intercalated cells in rat kidney. Double-staining confirmed that SLC22A13 co-localizes with anion exchanger 1. LC-MS difference shading showed that heterologous expression of human and rat SLC22A13 in HEK (human embryonic kidney)-293 cells stimulates efflux of guanidinosuccinate, aspartate, glutamate and taurine. Time courses of uptake of [3H]aspartate and [3H]glutamate revealed that SLC22A13 counteracted endogenous uptake. By contrast, OAT2 (organic anion transporter 2), a bidirectional glutamate transporter, increased accumulation of [3H]glutamate. Thus SLC22A13 catalyses unidirectional efflux. Velocity of efflux of standard amino acids was measured by LC-MS/MS. Expression of SLC22A13 strongly stimulated efflux of aspartate, taurine and glutamate. When the intracellular concentrations of aspartate and taurine were increased by pre-incubation, velocities of efflux increased linearly. We propose that in type A intercalated cells, SLC22A13 compensates luminal exit of protons by mediating the basolateral expulsion of the anions aspartate and glutamate. In this context, unidirectional efflux is essential to avoid anion re-entering. Loss of SLC22A13 function could cause distal tubular acidosis.
Assuntos
Ácido Aspártico/metabolismo , Células Epiteliais/metabolismo , Ácido Glutâmico/metabolismo , Túbulos Renais Coletores/metabolismo , Transportadores de Ânions Orgânicos/biossíntese , Animais , Catálise , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos/genética , Transporte Proteico/fisiologia , RatosRESUMO
We report here the generation and pharmacological characterization of two novel PDE 4B1 and PDE 4D3 reporter cell lines. Intracellular cAMP levels are monitored in these cells by a cAMP-sensitive biosensor. We used the recombinant PDE 4B1 and PDE 4D3 reporter cell lines to characterize the cellular effects of various competitive and allosteric PDE 4 inhibitors. In addition, we compared the cellular activity of these PDE 4 inhibitors with the in vitro inhibition of full-length PDE 4D3 and a truncated enzyme comprising the PDE 4D3 catalytic domain. Two different groups of PDE 4 inhibitors could be identified. The first group, including competitive inhibitors like roflumilast, cilomilast and piclamilast, shows similar in vitro activity on full-length and truncated PDE 4D3 and comparably low cellular activity. The second group, including the allosteric inhibitors PMNPQ, D159153, and D159404, shows much better inhibition of full-length versus truncated PDE 4D3. In addition, these compounds show high cellular activity. Our data obtained with the prototype PDE 4 inhibitor rolipram show that rolipram has properties intermediate between the two groups. The results imply that these novel PDE 4 reporter cell lines are well-suited for the characterization of the cellular activity of PDE 4 inhibitors and may also support a better understanding of the complex PDE 4 pharmacology.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Aminopiridinas/farmacologia , Animais , Benzamidas/farmacologia , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ácidos Cicloexanocarboxílicos/farmacologia , Ciclopropanos/farmacologia , Humanos , Modelos Biológicos , Nitrilas/farmacologia , Piridinas/farmacologia , Receptores Adrenérgicos beta 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca(2+)-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Progesterona/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Proteínas Reguladoras de Apoptose , Células COS , Sobrevivência Celular , Chlorocebus aethiops , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Grupo de Alta Mobilidade , Humanos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Progesterona/genética , Transativadores , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Elevated plasma levels of C-reactive protein (CRP), the prototype acute-phase protein (APP), are predictive for future cardiovascular events. Controversial evidence suggests that CRP may play a causal role in cardiovascular disease. CRP synthesis inhibition is a potential approach for reducing cardiovascular mortality. We show here that endogenous and plant-derived inhibitors of the Na(+)/K(+)-ATPase, i.e. the cardiac glycosides ouabain and digitoxin, inhibit IL-1beta- and IL-6-induced APP expression in human hepatoma cells and primary human hepatocytes (PHH) at nanomolar concentrations. Inhibition is demonstrated on transcriptional and on protein level. The molecular target of cardiac glycosides, i.e. the alpha1 subunit of the Na(+)/K(+)-ATPase, is strongly expressed in human hepatocytes. Inhibition of APP synthesis correlates with the potency of cardiac glycosides at the Na(+)/K(+)-ATPase. The trigger for APP expression inhibition is an increase in intracellular calcium since the calcium ionophore calcimycin is also active. Qualified specificity of oubain for hepatocellular APP synthesis inhibition is demonstrated by lack of effectivity on IL-1beta-induced IL-6 release from primary human coronary artery smooth muscle cells. The inhibitory activity of cardiac glycosides on CRP expression may have important implications for the treatment of cardiovascular disease. Cardiac glycosides may be used for CRP synthesis inhibition in the future.
Assuntos
Proteína C-Reativa/antagonistas & inibidores , Glicosídeos Cardíacos/farmacologia , Hepatócitos/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Adulto , Proteína C-Reativa/biossíntese , Proteína C-Reativa/genética , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Digitoxina/farmacologia , Feminino , Hepatócitos/metabolismo , Humanos , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Masculino , Pessoa de Meia-Idade , Ouabaína/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for neuronal cell death by oxidative stress. In this model, extracellular glutamate blocks cystine uptake via the glutamate/cystine antiporter system x(c)(), eventually leading to depletion of the antioxidant glutathione and cell death. We used subtractive suppression hybridization and a screening procedure using various HT22 sublines to identify transcripts relevantly upregulated in resistance to oxidative glutamate toxicity. One of these coded for a novel protein of 3440 amino acids comprising a superoxide dismutase (SOD) motif, which we named TIGR for "transcript increased in glutamate resistance." TIGR is mainly expressed in the nervous system in cortical pyramidal and hippocampal neurons. Intracellularly, TIGR colocalizes with catalase, strongly suggesting a peroxisomal localization. Overexpression of TIGR but not of a mutant lacking two conserved histidine residues in the SOD motif increased SOD activity and protected against oxidative stress in mammalian cells, but had no direct SOD activity in yeast. We conclude that this novel giant peroxisomal protein is implicated in resistance to oxidative stress. Despite the presence of a SOD motif, which is necessary for protection in mammalian cells, the protein is not a functional SOD, but might be involved in SOD activity.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Peroxissomos/metabolismo , Superóxido Dismutase/metabolismo , Animais , Células Cultivadas , Clonagem Molecular , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Ácido Glutâmico/toxicidade , Glicoproteínas/genética , Camundongos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) receptors and their respective ligands play important roles in cardiovascular (patho-)physiology. Functional expression of ADM and CGRP receptors requires the presence of the calcitonin receptor-like receptor (CRLR) together with receptor-activity-modifying proteins (RAMPs). We have characterized the expression patterns of CRLR and RAMP1 to RAMP3 in human cardiovascular-related tissues by quantitative polymerase chain reaction. We could identify high expression levels of CRLR, RAMP1, and RAMP2 in human heart and various blood vessels. RAMP3 expression in these tissues, however, was detectable at significantly lower levels. In addition, we describe here a novel, aequorin luminescence-based G protein-coupled receptor reporter assay that enables the real-time detection of receptor activation in living cells. In the assay system, intracellular cAMP levels are monitored with high sensitivity by using a modified, heteromultimeric cyclic nucleotide-gated channel mediating calcium influx. G(q)-coupled receptor activation is detected via aequorin luminescence stimulated by calcium release from intracellular stores. Using this novel reporter assay, we established and characterized stable ADM1 and CGRP1 receptor cell lines. The peptide ligands ADM, CGRP1, and CGRP2 were characterized as potent agonists at their respective receptors. In contrast, intermedin acted as a weak agonist on both receptors and showed only partial agonism on the ADM1 receptor. Agonist activities were effectively antagonized by the receptor antagonists ADM(22-52) and CGRP(8-37). Various vasoactive ADM fragments were also characterized but showed no activity on the ADM1 receptor cell line. In addition, luminescence signal kinetics after activation of G(s)- and G(q)-coupled receptors were found to be markedly different.
Assuntos
Adrenomedulina/metabolismo , Genes Reporter , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Adrenomedulina/genética , Animais , Vasos Sanguíneos/metabolismo , Células CHO , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Proteína Semelhante a Receptor de Calcitonina , Linhagem Celular , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rim/metabolismo , Cinética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Miocárdio/metabolismo , Hormônios Peptídicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 Modificadora da Atividade de Receptores , Proteína 2 Modificadora da Atividade de Receptores , Proteína 3 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/antagonistas & inibidores , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismoRESUMO
GPR39 is a constitutively active orphan G-protein-coupled receptor capable of increasing serum response element-mediated transcription. We found GPR39 to be up-regulated in a hippocampal cell line resistant against diverse stimulators of cell death and show that its overexpression protects against oxidative and endoplasmic reticulum stress, as well as against direct activation of the caspase cascade by Bax overexpression. In contrast, silencing GPR39 rendered cells more susceptible to cell death. An array analysis of transcripts induced by GPR39 revealed up-regulation of RGS16 (inhibitor of G-protein signaling 16), which suggested coupling to Galpha(13) and induction of serum response element-mediated transcription by the small GTPase RhoA. In line with this, co-expression of GPR39 with RGS16, dominant-negative RhoA, or serum response factor abolished cell protection, whereas overexpression of the serum response factor protected from cell death. Further downstream the signaling cascade, GPR39 overexpression leads to increased secretion of the cytoprotective pigment epithelium-derived growth factor (PEDF). Medium conditioned by cells overexpressing GPR39 contained 4-fold more PEDF, and when stripped off it lost most but not all of its protective properties. We conclude that GPR39 is a novel inhibitor of cell death, which might represent a therapeutic target with implications for processes involving apoptosis and endoplasmic reticulum stress like cancer, ischemia/reperfusion injury, and neurodegenerative disease.
Assuntos
Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Fatores de Crescimento Neural/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Serpinas/metabolismo , Animais , Meios de Cultivo Condicionados/farmacologia , Retículo Endoplasmático/metabolismo , Inativação Gênica , Humanos , Camundongos , Modelos Biológicos , Proteínas RGS/metabolismo , Tunicamicina/farmacologia , Proteína X Associada a bcl-2/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Recently, we have identified the ergothioneine (ET) transporter ETT (gene symbol SLC22A4). Much interest in human ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic inflammatory diseases. ETT was originally designated a multispecific novel organic cation transporter (OCTN1). Here we reinvestigated, based on stably transfected 293 cells and with ET as reference substrate, uptake of quinidine, verapamil, and pyrilamine. ETT from human robustly catalyzed transport of ET (68micfrol/(minmgprotein)), but no transport of organic cations was discernible. With ET as substrate, ETT was relatively resistant to inhibition by selected drugs; the most potent inhibitor was verapamil (K(i)=11micromol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, efficiency of ETT-mediated transport of methimazole (K(i)=7.5mmol/l) was 130-fold lower, and transport of hercynine (K(i)=1.4mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high affinity, sodium-driven uptake of ET very similar to ETT from human. Additional real-time PCR experiments based on 16 human tissues revealed ETT mRNA levels considerably lower than in bone marrow. Our experiments establish that ETT is highly specific for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high affinity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific molecular marker of intracellular ET activity.