RESUMEN
Exposure to air pollution from traffic-generated sources is known to contribute to the etiology of inflammatory diseases, including cardiovascular disease (CVD) and obesity; however, the signaling pathways involved are still under investigation. Dysregulation of the renin-angiotensin system (RAS) can contribute to CVD and alter lipid storage and inflammation in adipose tissue. Our previous exposure studies revealed that traffic-generated emissions increase RAS signaling, further exacerbated by a high-fat diet. Thus, we investigated the hypothesis that exposure to engine emissions increases systemic and local adipocyte RAS signaling, promoting the expression of factors involved in CVD and obesity. Male C57BL/6 mice (6-8 wk old) were fed either a high-fat (HF, n = 16) or low-fat (LF, n = 16) diet, beginning 30d prior to exposures, and then exposed via inhalation to either filtered air (FA, controls) or a mixture of diesel engine + gasoline engine vehicle emissions (MVE: 100 µg PM/m3) via whole-body inhalation for 6 h/d, 7 d/wk, 30d. Endpoints were assessed via immunofluorescence and RT-qPCR. MVE-exposure promoted vascular adhesion factors (VCAM-1, ICAM-1) expression, monocyte/macrophage sequestration, and oxidative stress in the vasculature, associated with increased angiotensin II receptor type 1 (AT1) expression. In the kidney, MVE-exposure promoted the expression of renin, AT1, and AT2 receptors. In adipose tissue, both HF-diet and MVE-exposure mediated increased epididymal fat pad weight and adipocyte hypertrophy, associated with increased angiotensinogen and AT1 receptor expression; however, these outcomes were further exacerbated in the MVE + HF group. MVE-exposure also induced inflammation, monocyte chemoattractant protein (MCP)-1, and leptin, while reducing insulin receptor and glucose transporter, GLUT4, expression in adipose tissue. Our results indicate that MVE-exposure promotes systemic and local adipose RAS signaling, associated with increased expression of factors contributing to CVD and obesity, further exacerbated by HF diet consumption.
RESUMEN
Allosteric modulators and mutations that slow AMPAR desensitization have additional effects on deactivation and agonist potency. We investigated whether these are independent actions or the natural consequence of slowing desensitization. Effects of cyclothiazide (CTZ), trichlormethiazide (TCM), and CX614 were compared at wild-type GluR1 and "nondesensitizing" GluR1-L497Y mutant receptors by patch-clamp recording with ultrafast perfusion. CTZ, TCM, or L/Y mutation all essentially blocked GluR1 desensitization; however, the effects of L/Y mutation on deactivation and glutamate EC50 were three to five times greater than for modulators. CTZ and TCM further slowed desensitization of L/Y mutant receptors but paradoxically accelerated deactivation and increased agonist EC50. Results indicate that CTZ and TCM target deactivation and agonist potency independently of desensitization, most likely by modifying agonist dissociation (koff). Conversely, CX614 slowed desensitization and deactivation without affecting EC50 in both wild-type and L/Y receptors. The S750Q or combined L497Y-S750Q mutations abolished all CTZ and TCM actions without disrupting CX614 activity. Notably, the S/Q mutation also restored L/Y deactivation and EC50 to wild-type levels without restoring desensitization, further demonstrating that desensitization can be modulated independently of deactivation and EC50 by mutagenesis and possibly by allosteric modulators.
Asunto(s)
Benzotiadiazinas/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Activación del Canal Iónico/fisiología , Riñón/fisiología , Oxazinas/administración & dosificación , Receptores AMPA/metabolismo , Triclormetiazida/administración & dosificación , Sustitución de Aminoácidos , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Activación del Canal Iónico/efectos de los fármacos , Riñón/efectos de los fármacos , Mutagénesis Sitio-Dirigida/métodos , Receptores AMPA/efectos de los fármacos , Receptores AMPA/genética , Relación Estructura-ActividadRESUMEN
The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [3H]kainate binding and patch-clamp recording. Each of the binding site mutations was sufficient to reduce [3H]kainate binding to undetectable levels and eliminate functional responses to glutamate or kainate. As with our studies of other nonfunctional mutants (Fleck et al., 2003), immunocytochemical staining and cell-surface biotinylation studies showed that the mutant receptors were retained intracellularly and did not traffic to the cell surface. Endoglycosidase-H digests and colocalization with endoplasmic reticulum (ER) markers demonstrated that the mutant receptors are immaturely glycosylated and retained in the ER. Immunoprecipitation, native PAGE, and functional studies confirmed that the GluR6-binding site mutants are capable of multimeric assembly, indicating their retention in the ER does not result from a gross protein folding error. Together, these results confirm the role of R523, T690, and E738 directly in ligand binding to GluR6 and further support our previous report that nonfunctional GluRs are retained intracellularly by a functional checkpoint in ER quality control.
Asunto(s)
Retículo Endoplásmico/fisiología , Ligandos , Transporte de Proteínas/fisiología , Receptores de Ácido Kaínico/metabolismo , Sustitución de Aminoácidos/fisiología , Sitios de Unión/fisiología , Biotinilación/métodos , Western Blotting/métodos , Línea Celular , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Expresión Génica/genética , Ácido Glutámico/farmacología , Glicosilación/efectos de los fármacos , Humanos , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Ácido Kaínico/farmacocinética , Proteínas Luminiscentes , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/farmacología , Potenciales de la Membrana/genética , Potenciales de la Membrana/efectos de la radiación , Microscopía Confocal/métodos , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos , Mutación/fisiología , Técnicas de Placa-Clamp/métodos , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/farmacología , Unión Proteica/genética , Unión Proteica/fisiología , Ensayo de Unión Radioligante/métodos , Receptores AMPA/química , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/genética , Alineación de Secuencia/métodos , Relación Estructura-Actividad , Transfección/métodos , Tritio/farmacocinética , Receptor de Ácido Kaínico GluK2RESUMEN
On the basis that meta-chlorophenylpiperazine (mCPP; 1) is a nonselective 5-HT(2C) agonist, that benz-fused tryptamines (e.g., 5) display enhanced 5-HT(2) affinity, and that certain isotryptamines 3 reportedly bind with enhanced affinity and selectivity at 5-HT(2C) receptors, we prepared and examined a series of isotryptamine-related analogues as potentially selective 5-HT(2C) agonists. None of the compounds displayed selectivity for 5-HT(2C) versus 5-HT(2A) receptors. Detailed re-examination of a compound previously reported to display 100-fold 5-HT(2C) selectivity [i.e., S(+)-5,6-difluoro-alpha-methylisotryptamine] revealed that its selectivity versus 5-HT(2A) receptors was, at best, only 10-fold.