- Email: [email protected]
Induction of TRE- and CRE-binding activities in cultured mouse cerebellar granule cells stimulated via glutamate receptors
S- l-5
LOCALIZATION OF PROTEIN KINASE C SUBSPECIES AND ITS FUNCTIONAL ROLES IN THE RAT BRAIN. CHIKAKO TANAKA AND NAOAKI SAITO,
Department of Pharrnacologv. Kobe University School of Medicine. Kusunoki-cho 7-5- 1. Chuo-ku, Kobe 650. Japan Protein kinase C (PKC) is a calcium-activated and phospholipid-dependent
protein kinase
and the activation of this enzyme has been linked to various neuronal functions such as the regulation of neurotransmitter receptors, ion channels, secretion, gene expression, neuronal plasticity and neuronal death. PKC is a member of a large family consisting of at least eight subspecies of PKC, a, l31,l3II, y, 6, E, < and q with closely related structures. We localized each PKC subspecies in the rat brain by in situ hybridization and by light and electron microscopic immunohistochemistry. All subspecies was predominantly expressed in the neuron but showed distinct distribution. In the limited areas, each PKC subspecies was co-localized with a single neurotransmitter, while in the other areas, multiple PKC subspecies were co-expressed in a neuron. The intracellular localization of PKC subspecies was distinctly different from each other. The a - , PI - and l3II-PKCs were predominantly found in the postsynaptic component such as perikaryon and dendrite, while the E-PKC was mainly present in presynaptic component such as axon and synaptic terminals. On the basis of these findings, the roles of each PKC subspecies on various synapse-related proteins such as receptors and transporters were examined.
INDUCTION OF TRE- AND CRE-BINDING ACTIVITIES IN CULTURED MOUSE CEREBELLAR GRANULE CELLS STIMULATED VIA GLUTAMATE RECEPTORS. MASAAKI TSUDA. Deoartment of Microbioloav. Facultv of Pharmaceutical Sciences. Okavama Universitv . Tsushima-naka. Okavama 700, s-1-6
&X2! In order to understand the relationship between the synaptic transmission and gene expression, we have been investigating the genetical responses of mouse cerebellar granule cells stimulated via glutamate receptors in primary culture, focusing on the expression of immediate early genes encoding transcriptional factors and some realizator genes which might contribute to the long-term changes of synaptic transmission. By use of nuclear mini-extracts prepared from cultured cerebellar granule cells in a gel-shift assay, exogenous NMDA (N-methyl-D-aspartate) or kainate was shown to increase both TRE (TPA responsrve element)and CRE (cyclic-AMP responsive element)-binding activity specifically through NMDA or non-NMDA receptors. The increase of TRE-binding activity appeared to be caused by the c-fas induction followed by de novo synthesis of c-Fos proteins. The inductions of TRE-binding activities caused by both NMDA and kainate were dependent upon the influx of extracellular Ca *+ into the cells. Competition and proteolytic bandshift experiments revealed that the increases in TRE- and CRE-binding activities were both mediated by the same DNA-binding complexes whose binding affinity to the CRE was higher than that to the TRE. c-Fos or Fos-related proteins were involved in the DNA-binding complexes formed on not only the TRE but also the CRE. Thus, the stimulation of cerebellar granule cells with NMDA or kainate could induce the TRE-binding activity cross-binding to the CRE. Recently, we have developed a method to prepare the nuclear mini-extracts from small punches of brain slices (approximately 1-3 mg tissue weight). Using this method, we have found that intraperitoneal administration of NMDA or kainate caused the increase in not only the TRE- but also the CRE-binding activity in the subregions of mouse hippocampus in accompany with seizures. We are now trying to characterize this type of DNA-binding complexes expressed in brain and to know its relationship with the expression of realizator genes, such as growth factor and receptor genes, which might be regulated by an activation of glutamate receptors.
LOCALIZATION OF PROTEIN KINASE C SUBSPECIES AND ITS FUNCTIONAL ROLES IN THE RAT BRAIN. CHIKAKO TANAKA AND NAOAKI SAITO,
Department of Pharrnacologv. Kobe University School of Medicine. Kusunoki-cho 7-5- 1. Chuo-ku, Kobe 650. Japan Protein kinase C (PKC) is a calcium-activated and phospholipid-dependent
protein kinase
and the activation of this enzyme has been linked to various neuronal functions such as the regulation of neurotransmitter receptors, ion channels, secretion, gene expression, neuronal plasticity and neuronal death. PKC is a member of a large family consisting of at least eight subspecies of PKC, a, l31,l3II, y, 6, E, < and q with closely related structures. We localized each PKC subspecies in the rat brain by in situ hybridization and by light and electron microscopic immunohistochemistry. All subspecies was predominantly expressed in the neuron but showed distinct distribution. In the limited areas, each PKC subspecies was co-localized with a single neurotransmitter, while in the other areas, multiple PKC subspecies were co-expressed in a neuron. The intracellular localization of PKC subspecies was distinctly different from each other. The a - , PI - and l3II-PKCs were predominantly found in the postsynaptic component such as perikaryon and dendrite, while the E-PKC was mainly present in presynaptic component such as axon and synaptic terminals. On the basis of these findings, the roles of each PKC subspecies on various synapse-related proteins such as receptors and transporters were examined.
INDUCTION OF TRE- AND CRE-BINDING ACTIVITIES IN CULTURED MOUSE CEREBELLAR GRANULE CELLS STIMULATED VIA GLUTAMATE RECEPTORS. MASAAKI TSUDA. Deoartment of Microbioloav. Facultv of Pharmaceutical Sciences. Okavama Universitv . Tsushima-naka. Okavama 700, s-1-6
&X2! In order to understand the relationship between the synaptic transmission and gene expression, we have been investigating the genetical responses of mouse cerebellar granule cells stimulated via glutamate receptors in primary culture, focusing on the expression of immediate early genes encoding transcriptional factors and some realizator genes which might contribute to the long-term changes of synaptic transmission. By use of nuclear mini-extracts prepared from cultured cerebellar granule cells in a gel-shift assay, exogenous NMDA (N-methyl-D-aspartate) or kainate was shown to increase both TRE (TPA responsrve element)and CRE (cyclic-AMP responsive element)-binding activity specifically through NMDA or non-NMDA receptors. The increase of TRE-binding activity appeared to be caused by the c-fas induction followed by de novo synthesis of c-Fos proteins. The inductions of TRE-binding activities caused by both NMDA and kainate were dependent upon the influx of extracellular Ca *+ into the cells. Competition and proteolytic bandshift experiments revealed that the increases in TRE- and CRE-binding activities were both mediated by the same DNA-binding complexes whose binding affinity to the CRE was higher than that to the TRE. c-Fos or Fos-related proteins were involved in the DNA-binding complexes formed on not only the TRE but also the CRE. Thus, the stimulation of cerebellar granule cells with NMDA or kainate could induce the TRE-binding activity cross-binding to the CRE. Recently, we have developed a method to prepare the nuclear mini-extracts from small punches of brain slices (approximately 1-3 mg tissue weight). Using this method, we have found that intraperitoneal administration of NMDA or kainate caused the increase in not only the TRE- but also the CRE-binding activity in the subregions of mouse hippocampus in accompany with seizures. We are now trying to characterize this type of DNA-binding complexes expressed in brain and to know its relationship with the expression of realizator genes, such as growth factor and receptor genes, which might be regulated by an activation of glutamate receptors.