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1.
Pflugers Arch ; 467(2): 201-12, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24756199

RESUMEN

Stroke is the third leading cause of death in the Western world. Ischemic stroke is characterized by a rapid loss of brain function due to disturbance in the blood supply to a part of the brain. Due to fixed intracranial space, any increase in intracranial fluid volume, or progressive brain edema formation, contributes to further deterioration of the already impaired brain function. Bradykinin increases blood-brain barrier permeability and raises intracranial capillary blood pressure by arterial dilatation and venous constriction leading to brain edema formation. The aim of this paper is to summarize the recent research in the field of bradykinin function (structure, synthesis, signaling pathways, mechanism of action) followed by characterization of different types of brain edema development related to ischemic brain injury, together with the involvement of bradykinin in edema formation. Since there is currently no causal treatment addressing brain edema after ischemic stroke, specific bradykinin receptor antagonists are proposed as a possible new therapeutic approach.


Asunto(s)
Bradiquinina/metabolismo , Isquemia Encefálica/metabolismo , Edema/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Sistema de Señalización de MAP Quinasas
2.
Pflugers Arch ; 466(3): 517-27, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23982114

RESUMEN

The organic cation transporter 3 (OCT3) is a widely expressed transporter for endogenous and exogenous organic cations. Of particular interest is OCT3 expression and function in the brain, where it plays a role in serotonin clearance and influences mood and behavior. Protein kinase signaling mediates rapid modulation of cerebral processes, but little is known about acute regulation of OCT3 by protein kinases. Therefore, we cloned mouse OCT3 (mOCT3) and generated a human embryonic kidney cell line stably expressing the transporter to study transport characteristics, acute regulation by protein kinases, and interaction with psychotropic drugs. Uptake measurement was performed using the fluorescent cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+), 1 µM) as a substrate. The translational value of these findings was determined by comparing results obtained with cloned mouse and human OCT3. mOCT3-mediated transport is membrane potential dependent and pH independent. ASP(+) uptake by mOCT3 and human OCT3 (hOCT3) was efficiently inhibited by 1-methyl-4-phenylpyridinium, tetrapentylammonium (TPA(+)), corticosterone, serotonin, and histamine and by the drugs ketamine, fluoxetine, and diazepam. The half maximal inhibitory concentrations of mOCT3 and hOCT3 for TPA(+), serotonin, diazepam, and ketamine are significantly different. Diazepam is a non-transported inhibitor. Furthermore, the activities of mOCT3 and hOCT3 are acutely regulated by the p56 (lck) tyrosine kinase by decreasing their V max. Studies with freshly isolated renal proximal tubules from mOCT1/2(-/-) mice, in which mOCT3 is the only OCT present, confirmed this regulation pathway. Only the activity of hOCT3 is regulated by calmodulin. These findings suggest that even though many transport properties of mOCT3 and hOCT3 are similar, there are also species-specific aspects of OCT3 function.


Asunto(s)
Diazepam/farmacología , Fluoxetina/farmacología , Ketamina/farmacología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Psicotrópicos/farmacología , Serotonina/farmacología , 1-Metil-4-fenilpiridinio/farmacología , Animales , Células Cultivadas , Células HEK293 , Histamina/farmacología , Humanos , Transporte Iónico/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Proteínas de Transporte de Catión Orgánico/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Especificidad de la Especie
3.
Croat Med J ; 54(1): 3-11, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23444240

RESUMEN

AIM: To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). METHODS: Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. RESULTS: Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. CONCLUSION: MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema.


Asunto(s)
Isquemia Encefálica/diagnóstico por imagen , Medios de Contraste , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Microtomografía por Rayos X , Animales , Edema Encefálico/patología , Isquemia Encefálica/patología , Colorantes , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/patología , Yohexol , Ácido Yotalámico/análogos & derivados , Masculino , Ratones , Ratones Endogámicos C57BL , Coloración y Etiquetado
4.
Am J Physiol Cell Physiol ; 303(12): C1260-8, 2012 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-23054060

RESUMEN

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (V(m)) and intracellular Ca(2+) using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 µM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 µM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.


Asunto(s)
Bradiquinina/farmacología , Péptidos Natriuréticos/farmacología , Proteínas RGS/antagonistas & inhibidores , Compuestos de Boro , Bradiquinina/análogos & derivados , Antagonistas del Receptor de Bradiquinina B2 , Carbazoles/farmacología , Canales de Cloruro/antagonistas & inhibidores , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Estrenos/farmacología , Citometría de Flujo , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Potenciales de la Membrana/efectos de los fármacos , Nitrobenzoatos/farmacología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirrolidinonas/farmacología , Proteínas RGS/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tionucleótidos/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores
5.
Beilstein J Nanotechnol ; 7: 926-936, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27547609

RESUMEN

BACKGROUND: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag(®)-D-spio nanoparticles. RESULTS: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag(®)-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag(®)-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes). CONCLUSION: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies.

6.
Exp Neurol ; 284(Pt A): 1-10, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27432758

RESUMEN

Occlusion of cerebral arteries leads to ischemic stroke accompanied by subsequent brain edema. Bradykinin (BK) is involved in the formation of cerebral edema, and natriuretic peptides (NPs) potentially have beneficial effects on brain edema formation via a still unknown mechanism. The aim of this study was clarifying the mechanisms of action of NPs on BK signaling, and their interactive effects after ischemic brain injury. We used a mouse model for stroke, the middle cerebral artery (MCA) occlusion. Brain lesion and edema were measured by microcomputerized tomography volumetric measurements. To determine the effects of NPs on the BK signaling pathway in the MCAs we measured changes in vessel diameter and membrane potentials in endothelial cells. To determine the effects of NPs on BK signaling pathway in isolated astrocytes and neurons, membrane potentials and intercellular Ca2+ concentrations were measured. Urodilatin inhibited and when applied together with BK, reduced the formation of the ischemic lesion via activation of G-Protein-Signaling Protein Type 4 at the cellular (atrocities, neurons) and blood vessel (endothelial cells and isolated MCA) level as well as in in vivo experiments. The results of this study show the existence of a natural antagonist of BK in the brain, and the possible use of NPs in the treatment of stroke.

7.
Gene ; 570(1): 132-40, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26071188

RESUMEN

Krüppel-like transcription factor 8 (KLF8) is a transcription factor suggested to be involved in various cellular events, including malignant cell transformation, still its expression in the adult rodent brain remained unknown. To analyze Klf8 in the mouse brain and to identify cell types expressing it, a specific transgenic Klf8(Gt1Gaj) mouse was used. The resulting Klf8 gene-driven ß-galactosidase activity was visualized by X-gal histochemical staining of the brain sections. The obtained results were complemented by in situ RNA hybridization and immunohistochemistry. Klf8 was highly expressed throughout the adult mouse brain gray matter including the cerebral cortex, hippocampus, olfactory bulb, hypothalamus, pallidum, and striatum, but not in the cerebellum. Immunofluorescent double-labeling revealed that KLF8-immunoreactive cells were neurons, and the staining was located in their nucleus. This was the first study showing that Klf8 was highly expressed in various regions of the mouse brain and in particular in the neurons, where it was localized in the cell nuclei.


Asunto(s)
Encéfalo/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Encéfalo/citología , Núcleo Celular/metabolismo , Expresión Génica , Factores de Transcripción de Tipo Kruppel , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Factores de Transcripción/genética
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