RESUMEN
INTRODUCTION: Trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) is a promising amino acid positron emission tomography (PET) radiotracer for visualizing prostate cancer. We previously showed that anti-FACBC is transported by amino acid transporters, especially by alanine-serine-cysteine transporter 2 (ASCT2), which is associated with tumor growth. We studied this affinity to assess the mechanism of anti-FACBC transport in prostate cancer cells. METHODS: Kinetic assays for trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid ([(14)C]FACBC) were performed in Xenopus laevis oocytes over-expressing either ASCT2 or sodium-coupled neutral amino acid transporter 2 (SNAT2), both of which are highly expressed in prostate cancer cells. We also examined the kinetics of [(14)C]FACBC uptake using mammalian cell lines over-expressing system L amino acid transporter 1 or 2 (LAT1 or LAT2). Results: ASCT2 and SNAT2 transported [14C]FACBC with MichaelisMenten kinetics Km values of 96.7 ± 45.2 µM and 196.5 ± 19.7 µM, respectively. [correted]. LAT1 and LAT2 transported [(14)C]FACBC with Michaelis-Menten Km values of 230.4 ± 184.5 µM and 738.5 ± 87.6 µM, respectively. CONCLUSIONS: Both ASCT2 and SNAT2 recognize anti-FACBC as a substrate. Anti-FACBC has higher affinity for ASCT2 than for SNAT2, LAT1, or LAT2. The ASCT2-preferential transport of anti-[(18)F]FACBC in cancer cells could be used for more effective prostate cancer imaging.
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Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/metabolismo , Sistema de Transporte de Aminoácidos A/genética , Sistema de Transporte de Aminoácidos A/metabolismo , Ácidos Carboxílicos/metabolismo , Ciclobutanos/metabolismo , Oocitos/metabolismo , Xenopus laevis/genética , Animales , Transporte Biológico , Línea Celular , Expresión Génica , Humanos , Cinética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Ratones , Antígenos de Histocompatibilidad MenorRESUMEN
A surprising shortage of information surrounds the mechanisms by which bone marrow stromal cells (BMSC) restore lost neurologic functions when transplanted into the damaged central nervous system. In the present study, we sought to elucidate whether BMSCs express the neuron-specific gamma-aminobutyric acid (GABA) receptor when transplanted into injured spinal cord. To examine this, we harvested and cultured rat femoral BMSCs. We then subjected Sprague-Dawley rats to thoracic spinal cord injury (SCI) with a pneumatic impact device. Fluorescence-labeled BMSCs (n = 7) were transplanted stereotactically or the vehicle in which these cells were cultured (n = 4) was introduced stereotactically into the rostral site of SCI at 7 days after injury. We evaluated GABA receptor function by measuring the binding potential for 125I-iomazenil (125I-IMZ) through in vitro autoradiography at 4 weeks after BMSC transplantation and simultaneously examined the fate of the transplanted BMSCs by immunocytochemistry. We found that the transplanted BMSC migrated toward the core of the injury and were densely distributed in the marginal region at 4 weeks after transplantation. BMSC transplantation significantly increased the binding potential for 125I-IMZ (p = 0.0376) and increased the number of GABA receptor-positive cells (p = 0.0077) in the marginal region of the injury site. Some of the transplanted BMSCs were positive for microtubule-associated protein-2 and the alpha1 subunit of GABA(A) receptor in the region of injury. These findings suggest that BMSCs have the potential to support the survival of neurons in the marginal region of SCI and can partly differentiate into neurons, regenerating spinal cord tissue at the site of injury.
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Células de la Médula Ósea/citología , Trasplante de Médula Ósea/fisiología , Receptores de GABA-A/metabolismo , Traumatismos de la Médula Espinal/cirugía , Animales , Autorradiografía , Células de la Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Inmunohistoquímica , Regeneración Nerviosa/fisiología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
UNLABELLED: Recent studies have indicated that bone marrow stromal cells (BMSC) have the potential to improve neurologic function when transplanted into animal models of central nervous system disorders. However, how the transplanted BMSC restore the lost neurologic function is not clear. In the present study, therefore, we aimed to elucidate whether BMSC express the neuron-specific gamma-aminobutyric acid (GABA) receptor when transplanted into brain that has been subjected to cerebral infarction. METHODS: The BMSC were harvested from green fluorescent protein-transgenic mice and were cultured. The mice were subjected to permanent middle cerebral artery occlusion. The BMSC or vehicle was transplanted into the ipsilateral striatum 7 d after the insult. Using autoradiography and fluorescence immunohistochemistry, we evaluated the binding of 125I-iomazenil and the expression of GABA receptor protein in and around the cerebral infarct 4 wk after transplantation. RESULTS: Binding of 125I-iomazenil was significantly higher in the periinfarct neocortex in the BMSC-transplanted animals than in the vehicle-transplanted animals. Likewise, the number of the GABAA receptor-positive cells was significantly higher in the periinfarct neocortex in the BMSC-transplanted animals than in the vehicle-transplanted animals. A certain subpopulation of the transplanted BMSC expressed a neuron-specific marker, microtubule-associated protein 2, and the marker protein specific for GABAA receptor in the periinfarct area. CONCLUSION: These findings suggest that BMSC may contribute to neural tissue regeneration through migrating toward the periinfarct area and acquiring the neuron-specific receptor function.
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Infarto Cerebral/metabolismo , Infarto Cerebral/cirugía , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Células del Estroma/metabolismo , Células del Estroma/trasplante , Ácido gamma-Aminobutírico/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Encéfalo/patología , Infarto Cerebral/diagnóstico por imagen , Infarto Cerebral/patología , Células Madre Hematopoyéticas/diagnóstico por imagen , Células Madre Hematopoyéticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Cintigrafía , Células del Estroma/diagnóstico por imagen , Células del Estroma/patología , Resultado del TratamientoRESUMEN
PURPOSE: Gamma-aminobutyric acid (GABA)-A/benzodiazepine receptors (BZRs) play an important inhibitory role in epileptogenesis. [123I]Iomazenil (123I-IMZ) is a specific ligand for central-type (or neuronal-type) BNRs and is available for single-photon emission computed tomography (SPECT) in brain disorders. We demonstrated alterations of central-type BZRs in human focal epilepsies and their experimental models. METHODS: We examined interictal 123I-IMZ SPECT in patients with mesial temporal lobe epilepsy (MTLE; n = 19) with hippocampal sclerosis and neocortical epilepsy with focal cortical dysplasia (NE-CD; n = 18), and compared those with magnetic resonance imaging (MRI) and 123I-IMP SPECT (for regional cerebral blood flow). We also investigated in vitro autoradiography with (123)I-IMZ at various time courses in the intraamygdala kainate, amygdala kindling, and in-utero irradiation models. RESULTS: In MTLE patients, the epileptogenic hippocampus often showed decreases in both 123I-IMZ and 123I-IMP SPECT. Consistent with those, marked reduction of 125I-IMZ binding was observed in hippocampal CA1-3 regions of the kainate model, which clearly paralleled pyramidal neuronal loss. In contrast, 125I-IMZ binding was increased in the dentate gyrus at 1 month but returned to the normal level at 3-6 months, when frequent spontaneous seizures appeared. The amygdala-kindling model demonstrated similar increases in 125I-IMZ binding in the dentate gyrus without any changes in other brain regions. In NE-CD patients, the epileptogenic foci showed decreased 123I-IMZ binding with relatively normal 123I-IMP SPECT. 125I-IMZ binding also was decreased in the cerebral cortex, hippocampus (areas CA1, 2, and 4), and caudate/putamen of the in-utero irradiation model. CONCLUSIONS: These results indicate that central-type BZRs neuroimaging is useful for detection of epileptogenic foci, but their alterations differ between epilepsy subtypes and time-courses.
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Epilepsias Parciales/fisiopatología , Flumazenil/análogos & derivados , Receptores de GABA-A/fisiología , Adulto , Amígdala del Cerebelo/fisiología , Animales , Autorradiografía , Corteza Cerebral/anomalías , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/fisiopatología , Giro Dentado/diagnóstico por imagen , Giro Dentado/embriología , Giro Dentado/fisiopatología , Modelos Animales de Enfermedad , Epilepsias Parciales/diagnóstico por imagen , Epilepsias Parciales/etiología , Epilepsia del Lóbulo Temporal/diagnóstico por imagen , Epilepsia del Lóbulo Temporal/etiología , Epilepsia del Lóbulo Temporal/fisiopatología , Femenino , Hipocampo/irrigación sanguínea , Hipocampo/diagnóstico por imagen , Hipocampo/fisiopatología , Humanos , Radioisótopos de Yodo , Ácido Kaínico , Excitación Neurológica/fisiología , Imagen por Resonancia Magnética , Masculino , Neocórtex/irrigación sanguínea , Neocórtex/fisiopatología , Ratas , Flujo Sanguíneo Regional , Tomografía Computarizada de Emisión de Fotón Único/estadística & datos numéricosRESUMEN
[(123)I]Iomazenil (IMZ) is a specific ligand for central-type benzodiazepine receptors (BZRs) and is available for single photon emission computed tomography (SPECT) to detect epileptogenic foci. We have recently demonstrated time-dependent alterations of [(125)I]IMZ binding in the rat kainate model of temporal lobe epilepsy. Quantitative evaluation of central-type benzodiazepine receptors with [(125)I]Iomazenil in experimental epileptogenesis. I. The rat kainate model of temporal lobe epilepsy. In the present study, we investigated regional changes in central-type BZRs in the cortical dysplasia (CD) model of epilepsy in rats. Pregnant rats were irradiated at day 17 of gestation with 1.2 Gy to produce CD in their pups, and in vitro autoradiography with [(125)I]IMZ was performed at 8 weeks after birth. Intact rats at the same age were used as controls. [(125)I]IMZ binding was significantly decreased in various cortical regions of the in utero irradiated rats, including the bilateral frontal cortex (down to 92-93% of control), cingulate cortex (91-92%), hippocampal areas CA1 (95%), CA2 (94-95%) and CA4 (95-96%), and caudate/putamen (90-94%). In addition, amygdala-kindling was significantly facilitated in the CD model, especially during the late phase of kindling, suggesting seizure susceptibility of this model. These results may replicate the clinical usefulness of central-type BZRs neuroimaging for detection of human epileptogenic CD and indicate dysfunction of GABA-A/BZR-mediated inhibition responsible for the seizure susceptibility.
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Anticonvulsivantes , Corteza Cerebral/anomalías , Epilepsia/patología , Flumazenil/análogos & derivados , Receptores de GABA-A/efectos de los fármacos , Anomalías Inducidas por Radiación/patología , Amígdala del Cerebelo/fisiología , Animales , Autorradiografía , Corteza Cerebral/patología , Epilepsia/etiología , Femenino , Hipocampo/patología , Excitación Neurológica/fisiología , Tamaño de los Órganos , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismoRESUMEN
This study aimed at quantitatively evaluating hippocampal central-type benzodiazepine receptors (BZRs) in the kainate model of temporal lobe epilepsy (TLE) by in vitro autoradiography (ARG) using [(125)I] Iomazenil (IMZ) specific ligand for central-type BZRs. Kainate (1 microg/0.5 microl) was injected into the left amygdala to induce limbic status epilepticus. One, three, or six months after injection, in vitro ARG with [(125)I] IMZ and cell counts were performed in the hippocampal CA1-4 regions and dentate gyrus ipsilateral to the kainate injection site, and were compared with the vehicle-injected control group. In all kainate-treated rats, clear pyramidal neuron loss was observed in left hippocampal areas CA1-4. Compared with the control group, progressive reduction of [(125)I] IMZ binding was also observed. This resulted in a marked binding decrease paralleling pyramidal neuron loss in hippocampal areas CA1 (down to 83% of control), CA2 (76%), CA3 (75%), and CA4 (90%) at 6 months after kainate administration. Conversely, [(125)I] IMZ binding significantly increased in the dentate gyrus (up to 106% of control) at 1 month, but returned to nearly normal at 3-6 months. These results suggest that central-type BZR neuroimaging is useful in detecting hippocampal sclerosis in the mesial TLE, though central BZR alterations differ depending on hippocampal subfields and post-seizure time-courses.