Adrenalectomy Potentiates Immediate Early Gene Expression in Rat Brain

Administration of kainate or pentylenetetrazole increased c-fos, c-jun, junB, and junD mRNA levels in rat brain in a dose-dependent manner. Kainate increased these mRNA levels predominantly in the hippocampus, and pentylenetetrazole was more effective in the cortex. Adrenalectomy (3 days) was used to eliminate endogenous glucocorticoid hormones. Adrenalectomy significantly potentiated kainate-induced increases, compared with increases caused by kainate (4 mg/kg) alone, in the hippocampal mRNA levels of c-fos and junB by 6.5-fold and of junD by twofold and tended to augment c-jun mRNA. Corticosterone administration blocked the potentiated stimulation of these mRNA levels caused by adrenalectomy. Adrenalectomy also significantly increased pentylenetetrazole-induced levels of c-fos mRNA in the cortex. These results demonstrate that glucocorticoids modulate immediate early gene expression in the brain, raising the possibility that this interaction contributes to interneuronal and interindividual differences in responses to stimuli and to the effects of stress- or disease-induced changes in glucocorticoid concentrations.     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.     

NMDA and Non-NMDA Receptor Gene Expression Following Global Brain Ischemia in Rats: Effect of NMDA and Non-NMDA Receptor Antagonists

Abstract: Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous sludy, we have shown that expression of GIuR2, the kainate/a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca' permeability, is preferentially reduced in CA1 at a time point proceeding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), protects CAI neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor anltagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDARI. In ischemic rats treated with saline, GIuR2 and (31uR3 mRNAs were markedly reduced in CAI but were unchanged in CA3 or dentate gyrus. GluRl and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modificatian of GIuR2 expression changes     

N-Methyl-D-aspartate receptor-dependent and -independent cytotoxic effects of Dictyostelium discoideum differentiation-inducing factor-1 on rat cortical neurons

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (small lipophilic hormone) that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. Recent studies have revealed that DIF-1 inhibits growth and induces the differentiation of mammalian tumor cells. The present study examines the effects of DIF-1 on rat cortical neurons in primary culture. We found that DIF-1 induced rapid neuronal cell death. The release of lactate dehydrogenase (LDH), as an indicator of cell death, increased dose-dependently with DIF-1. The release of LDH was inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists MK801 and AP5, suggesting that the NMDA receptor is involved in the induction of cell death by DIF-1. However, glutamate cytotoxicity could not explain the entire action of DIF-1 on neurons because the estimated concentration of glutamate around DIF-1-treated neurons was below 50 microM and DIF-1 caused more severe cell death than 500 microM glutamate. We discovered that another portion of DIF-1 cytotoxicity is independent of the NMDA receptor; that is, coaddition of DIF-1 and MK801 induced dendritic beading and increased expression of the immediate early genes c-fos and zif/268. These results indicate that DIF-1 induces rapid cell death via both NMDA receptor-dependent and -independent pathways in rat cortical neurons.     

鸡视网膜谷氨酸受体和GABA受体在两栖类卵母细胞中的表达

本工作利用两栖类卵母细胞作为功能表达系统,对鸡视网膜中的谷氨酸受体和ＧＡＢＡ受体的类型和基本性质进行了研究。在注射鸡视网膜ｍＲＮＡ的卵母细胞上,谷氨酸受体有明显的表达。Ｌ－Ｇｌｕ及其类似物ＫＡ,ＡＭＰＡ,ＱＡ都毫无例外地能诱导卵母细胞产生快速平滑的去极化电流,而ＮＭＤＡ,Ｌ－ＡＰ４,ＡＣＰＤ以及天冬氨酸不能诱导明显的电流反应。并且ＡＭＰＡ,ＱＡ对ＫＡ反应存在一定的抑制作用,提示ＡＭＰＡ,ＱＡ可能与ＫＡ作用于同一受体。抑制性氨基酸ＧＡＢＡ的受体被证明大部分为ＧＡＢＡＡ亚型,但有小部分的ＧＡＢＡ反应不能为荷包牡丹碱（ｂｉｃｕｃｕｌｌｉｎｅ）所阻断。     

Stimulation of Muscarinic Receptors Induces Expression of Individual fos and jun Genes Through Different Transduction Pathways

Abstract: The transduction pathways coupling muscarinic receptors to induction of fos and jun genes were investigated in neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced expression of c- fos , fosB , c- jun , junB , and junD . This effect was abolished by pretreatment with atropine, indicating an involvement of muscarinic receptors. These genes were also induced by activation of protein kinase C with phorbol ester or by elevating the intracellular Ca²⁺ concentration with a Ca²⁺ ionophore. The Ca²⁺ effect was inhibited by KN-62, suggesting an induction through Ca²⁺/calmodulin-dependent kinase II. Inhibition of protein kinase C with GF109203X suppressed the carbachol-stimulated increase in mRNA levels of c- fos , fosB , and junB by ∼70% but had only minor effects on the expression of c- jun and junD . On the other hand, preincubation with KN-62 attenuated the carbachol-induced increase in c- jun and junD expression by 70% but had no effect on c- fos , fosB , and junB mRNA levels. Simultaneous inhibition of both protein kinase C and Ca²⁺/calmodulin-dependent kinase II completely abolished the carbachol-stimulated expression of c- jun and junD , but c- fos , fosB , and junB were still expressed to a certain extent under this condition. Comparison of the inhibitory effects of GF109203X and Gö 6976 suggests the involvement of classical protein kinase C isozymes in muscarinic receptor-stimulated expression of fos and jun genes. These results demonstrate that the muscarinic receptor-induced expression of individual fos and jun genes is regulated via different pathways, primarily protein kinase C or Ca²⁺/calmodulin-dependent kinase II.     

Glutamate induces formation of free radicals in rat brain synaptosomes

The influence of glutamate and agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes was investigated using the fluorescent dye DCFDA. Glutamate at concentrations of 100 μM and 1 mM increased the production of reactive oxygen species. This phenomenon was eliminated by removing calcium from the incubation medium. Addition of NMDA (100 μM) or kainate (100 μM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.     

Mechanical stretch and progesterone differentially regulate activator protein-1 transcription factors in primary rat myometrial smooth muscle cells

During pregnancy, stretch of the uterus, imposed by the growing fetus, is an important signal for the induction of genes involved in the onset of labor. In this study, the expression of activator protein-1 (AP-1) family mRNAs in response to in vitro stretch was investigated in myometrial cells. Rat primary myometrial smooth muscle cells were plated onto collagen I-coated Flex I culture plates and subjected to 25% static stretch on day 4 of culture. Static stretch induced an increase in the expression of c-fos, fosB, fra-1, c-jun, and junB. The expression of both c-fos and junB was maximally induced at 30 min by static stretch. The peak induction for fosB and c-jun occurred at 1 h, whereas the peak of fra-1 induction occurred between 1 and 2 h after application of stretch. Treatment of myometrial cells with progesterone (100 nM, 400 nM, 1 microM) for 1 or 6 h before the application of static stretch did not affect the magnitude of the c-fos response. However, 24 h of progesterone exposure reduced the magnitude of c-fos and fosB stretch induction at both the 400 nM and 1 microM doses. These data indicate that several members of the AP-1 family are stretch-responsive genes in myometrial smooth muscle cells. This response can be attenuated by pretreatment with progesterone; however, the requirement for longer pretreatment times suggests that the inhibitory actions of progesterone do not occur through a direct action of the progesterone receptor within the promoter regions of AP-1 genes.     

Growth conditions differentially affect the constitutive expression of primary response genes in cultured cereballar granule cells

Cultured cerebellar granule cells underwent apoptotic degeneration when grown in medium containing 10 instead of 25 mM K⁺. Knowing that apoptosis is associated with changes in the expression of primary response genes, we have measured c-fos, zif/268, and c-jun mRNA levels during maturation of cultured granule cells grown in 10 or 25 mM K⁺. The constitutive expression of c-fos and zif/268 was differentially regulated by extracellular K⁺ concentration at 5 days of maturation in vitro (DIV), when cells grown under suboptimal conditions (i.e. in 10 mM K⁺) are committed to degenerate. At this stage, c-fos mRNA levels were detectable only in cultures grown in 25 mM K⁺, whereas zif/268 mRNA levels were dramatically elevated in cultures grown in 10 mM K⁺. This provides one of the few conditions in which c-fos and zif/268 are differentially regulated in nerve cells. Substantial changes in c-jun, or -actin mRNA levels were detectable only at 7 DIV, when the percentage of apoptotic cells had already reached a plateau in ultures grown in 10 mM K⁺. We speculate that changes in the expression of zif/268 are important in the gene program associated with the induction of apoptosis by trophic deprivation in cultured neurons.Special issue dedicated to Dr. Robert Balázs