According to our previous studies together with others, GnRH, a hypothalamic decapeptide, has been known to be a major regulator for LH release and its subunit biosynthesis in anterior pituitary gonadotropes. But the precise mechanisms by which GnRH exerts stimulatory effects on LH release and its subunit biosynthesis have not been clearly understood. In the present study we examined the effect of GnRH on protein kinase C (PKC) activity and intracellular cAMP content in cultured anterior pituitary cells of rat to clarify whether PKC or cAMP are involved in GnRH action. Moreover, we examined the effects of staurosporine (ST), a PKC inhibitor and 2',3'-dideoxyadenosine (2',3'-DDA), an adenylate cyclase inhibitor, on LH release and steady state LH beta subunit mRNA levels in cultured anterior pituitary cells of rat. PKC activity was rapidly increased within 30 min after GnRH treatment whereas intracellular cAMP level was elevated 18 h after GnRH treatment. ST significantly inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner, showing an half maximal response at 50 nM ST. 2',3'-DDA inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner in pituitary cells. From these results, it is suggested that GnRH stimulates LH beta subunit mRNA level as well as LH release in anterior pituitary cells and this GnRH action might be mediated by PKC activation and cAMP stimulation.
Adenylate Cyclase/*antagonists & inhibitors ; Alkaloids/*pharmacology ; Animal ; Cells, Cultured ; Cyclic AMP/metabolism ; Dideoxyadenosine/*pharmacology ; Female ; Gonadorelin/*pharmacology ; Luteinizing Hormone/*biosynthesis/*metabolism ; Pituitary Gland, Anterior/*drug effects/metabolism ; Protein Kinase C/*antagonists & inhibitors/metabolism ; Rats ; Rats, Sprague-Dawley ; Staurosporine ; Support, Non-U.S. Gov't

The structural complexity and heterogeneity of cerebral cortex have been obvious since the earliest days of light microscopy. In fact, if there is one word that captures many of the key attributes of cortical structures, it is diversity. Neurodevelopmental approach is the one of the effective ways to understand complicated structures of cerebral cortex. In this experiment, as a first step to clone the genes related with development of cerebral cortex, the developmentally differentially expressed genes were cloned from developing rat brain with ordered differential display PCR(ODD-PCR). Novel genes were screened from these cloned genes by sequencing and sequence analysis with blast search program. The developmental expression patterns of novel genes in the cerebral cortex were investigated with in situ hybridization histochemistry on the developing and adult rat brain sections. Among the forty one developmentally differentially expressed cDNA bands, amplified with InEGA primer and TEAC primer by ODD PCR, twenty novel genes were screened by sequencing and sequence analysis with blast search program. Through the investigation of developmental expression pattern with in situ hybridization histochemistry, specific expression of five novel genes in the developing rat cerebral cortex was identified. 20-E14-2 was highly expressed in the cerebral cortex during the period of neurogenesis. The expression of 20-E20-1, 20-E20-6b, and 20-P0-5 was relatively well matched with neuronal cell migration in the cerebral cortex. And the strong expression of 20-P0-8b was observed in the neuronal cells of cerebral cortex during the period of syanptogenesis. From these results, it may be suspected that the five novel genes play roles in the development of cerebral cortex.
Adult ; Animals ; Brain ; Cell Movement ; Cerebral Cortex* ; Clone Cells* ; Cloning, Organism* ; DNA, Complementary ; Humans ; In Situ Hybridization ; Microscopy ; Neurogenesis ; Neurons ; Polymerase Chain Reaction ; Population Characteristics ; Rats* ; Sequence Analysis

The mRNA expression of protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon and zeta) in the rat nervous system was investigated with in situ hybridization histochemistry. In the central nervous system of rat, each PKC isozyme mRNAs was expressed in isozyme-specific pattern. PKC alpha mRNA was highly expressed in the olfactory bulb, piriform cortex, hippocampus, substantia nigra compacta, and inferior olive. The expression of PKC beta was highest in the olfactory tubercle, piriform cortex, caudate putamen, accumbens nucleus, neocortex, hippocampus, basolateral amygdaloid nucleus, pontine nucleus, and cerebellum. PKC gamma mRNA was distributed in the caudate putamen, hippocampus and cerebellum and PKC delta was expressed in the thalamus. PKC epsilon had widespread distribution, with relatively high levels in the anterior olfactory nucleus, olfactory tubercle, tinea tecta, piriform cortex, dorsal lateral septal nucleus, neocortex, hippocampus and cerebellum. PKC zeta had widespread and low expression. The spacially differential expression of PKC isozymes (alpha, beta, gamma, delta, epsilon and zeta) suggests that each PKC isozyme may be related with specific cellular function in the nervous system.
Animals ; Brain* ; Central Nervous System ; Cerebellum ; Gene Expression ; Hippocampus ; In Situ Hybridization ; Isoenzymes ; Neocortex ; Nervous System ; Olea ; Olfactory Bulb ; Olfactory Pathways ; Protein Kinase C* ; Protein Kinase C-epsilon ; Protein Kinases* ; Putamen ; Rats* ; RNA, Messenger ; Septal Nuclei ; Substantia Nigra ; Thalamus ; Tinea

Although the induction of various members of hsp (heat shock protein) gene family has been linked to the resistance to apoptosis by a range of diverse stress stimuli, detail information has not been available yet as to the temporal and spatial expression patterns of various hsp genes after traumatic brain injury. In the present study, using a lateral fluid percussion (FP) injury as a model of traumatic brain injury, expression profiles of stress induced hsp genes were comparatively evaluated in the adult rat brain by in situ hybridization (ISH). We found that the temporal and regional expression patterns between the hsp70 superfamily members, hsp110 and hsp70, and the small hsp member, hsp25 are distinct. While the hsp110 and hsp70 expression was observed as early as 1 hr after injury and maximally induced at 3 hr after injury, the hsp25 expression appeared 6 hr after injury and the expression sustained until 6 days after the injury. Moreover, the expression of hsp110 and hsp70 was localized primarily in the impact site, that of the small hsp25 was observed throughout the ipsilateral cortical area in the distant regions remote from the impact site as well as in the impact site following injury. These results suggest that the sequential and combinatorial manipulation of various hsp genes can be exploited in reducing acute and delayed post-traumatic apoptosis and associated neurological dysfunction.
Adult ; Animals ; Apoptosis ; Brain Injuries* ; Brain* ; Heat-Shock Proteins* ; Hot Temperature* ; Humans ; In Situ Hybridization ; Percussion* ; Rats ; Shock

In this study, S59158, a gene of GLAST (L-Glutamate/L-Aspartate transporter), was cloned by ordered differential display PCR with developing rat brains. The mRNA expression of this gene in the developing rat brain and the effect of kainic acid (KA), glutamate analogue, on this gene were investigated with in situ hybridization histochemistry. The expression of S59158 was restricted to nervous system and observed from E12 (embryonic day 12), peaked at E20, and gradually decreased to adult level. In embryos, S59158 was prominently expressed in the subventricular zones throughout the brain. After birth, strong expression was observed in the purkinje cell layer of cerebellum and moderate level of expression was observed in the subventricular zone, olfactory bulb, hippocampal formation, and cerebral cortex. In the KA treated rat brains, the expression of S59158 was significantly increased in dentate gyrus, hippocampus, and cerebral cortex. From these results, it may be suspected that S59158 is related to the development of the brain and is induced by increased extracellular glutamate level.
Adult ; Amino Acid Transport System X-AG ; Animals ; Brain ; Cerebellum* ; Cerebral Cortex ; Clone Cells* ; Cloning, Organism* ; Dentate Gyrus ; Embryonic Structures ; Genes, vif ; Glutamic Acid ; Hippocampus ; Humans ; In Situ Hybridization ; Kainic Acid ; Nervous System ; Olfactory Bulb ; Parturition ; Polymerase Chain Reaction ; Rats* ; RNA, Messenger