Phil Ok Koh

Estradiol prevents the focal cerebral ischemic injury-induced decrease of forkhead transcription factors phosphorylation

Estradiol prevents neuronal cell death through the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the activation of Akt and its downstream targets, including forkhead transcription factors FKHR and FHKRL1. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Estradiol administration significantly reduced infarct volume and decreased the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser473, pFKHR at Ser256, and pFKHRL1 at Thr32 using Western blot analysis and immunohistochemistry. Estradiol prevents the injury-induced decrease of pAkt, pFKHR, and pFKHRL1. Further, in the presence of estradiol, the interaction of pFKHRL1 and 14-3-3 increased, compared to that of oil-treated animals. Our findings suggest that estradiol plays a potent protective role against brain injury and that Akt activation and FKHR phosphorylation by estradiol mediated these protective effects.

Protective effect of pyruvate against ethanol-induced apoptotic neurodegeneration in the developing rat brain

Exposure to alcohol during the early stages of brain development can lead to neurological disorders in the CNS. Apoptotic neurodegeneration due to ethanol exposure is a main feature of alcoholism. Exposure of developing animals to alcohol (during the growth spurt period in particular) elicits apoptotic neuronal death and causes fetal alcohol effects (FAE) or fetal alcohol syndrome (FAS). A single episode of ethanol intoxication (at 5 g/kg) in a seven-day-old developing rat can activate the apoptotic cascade, leading to widespread neuronal death in the brain. In the present study, we investigated the potential protective effect of pyruvate against ethanol-induced neuroapoptosis. After 4h, a single dose of ethanol induced upregulation of Bax, release of mitochondrial cytochrome-c into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1), all of which promote apoptosis. These effects were all reversed by co-treatment with pyruvate at a well-tolerated dosage (1000 mg/kg). Histopathology performed at 24 and 48 h with Fluoro-Jade-B and cresyl violet stains showed that pyruvate significantly reduced the number of dead cells in the cerebral cortex, hippocampus and thalamus. Immunohistochemical analysis at 24h confirmed that ethanol-induced cell death is both apoptotic and inhibited by pyruvate. These findings suggest that pyruvate treatment attenuates ethanol-induced neuronal cell loss in the developing rat brain and holds promise as a safe therapeutic and neuroprotective agent in the treatment of neurodegenerative disorders in newborns and infants.

Estradiol prevents the injury-induced decrease of Akt activation and Bad phosphorylation.

Estradiol prevents neuronal cell death through the inhibition of apoptotic signals and the activation of cell survival signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the activation of Akt and its downstream targets, including Bad, Bcl-x(L), and 14-3-3. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser473 and Bad at Ser136 using Western blot analysis. Estradiol prevents the injury-induced decrease of pAkt, pBad, and Bcl-x(L). Further, in the presence of estradiol, the interaction of pBad and 14-3-3 increased, compared to that of oil-treated animals. Our findings suggest that estradiol prevents cell death due to brain injury and that Akt activation and Bad phosphorylation by estradiol mediated these protective effects.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type I A receptor mRNAs in granulosa cells of preovulatory follicles of the rat ovary.

Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and known to stimulate the production of cAMP in anterior pituitary cells. In the recent report, the expression of PACAP was detected in preovulatory follicles, and treatment with PACAP stimulated the production of progesterone and prostaglandin E2 through the action of AC and PLC pathways in the ovary. PACAP binds to three type receptors. Type I A receptor is coupled to adenylate cyclase (AC) and phospholipase C (PLC) pathways, while type I B and type II receptors are only coupled to AC. Thus, the present study aimed to evaluate the temporal expression of PACAP and its type I A receptor mRNAs in the rat ovary after treatment with pregnant mares serum gonadotropin and human chorionic gonadotropin (hCG).

Estradiol prevents the injury-induced decrease of 90 ribosomal S6 kinase (p90RSK) and Bad phosphorylation

Estradiol prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the activation of Raf-MEK-ERK and its downstream targets, including 90 ribosomal S6 kinase (p90RSK) and Bad. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the positive cells of TUNEL staining in the cerebral cortex. Estradiol prevents the injury-induced decrease of Raf-1, MEK1/2, and ERK1/2 phosphorylation. Also, it inhibits the injury-induced decrease of p90RSK and Bad phosphorylation. Further, in the presence of estradiol, the interaction of phospho-Bad and 14-3-3 increased, compared with that of oil-treated animals. Our findings suggest that estradiol prevents cell death due to brain injury and that Raf-MEK-ERK cascade activation and its downstream targets, p90RSK, Bad phosphorylation by estradiol mediated these protective effects.

Estradiol prevents the injury-induced decrease of Akt/glycogen synthase kinase 3β phosphorylation

Estradiol prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta (GSK3beta). Adult female rats were ovariectomized and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Estradiol administration significantly reduced infarct volume and decreased the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser(473) and GSK3beta at Ser(9) using Western blot analysis and immunohistochemistry. Estradiol prevented the injury-induced decrease of pAkt and pGSK3beta. Furthermore, pretreatment with estradiol decreased glutamate toxicity-induced cell death in a hippocampal cell line (HT22). Also, estradiol prevented the glutamate toxicity-induced decrease of pAkt and pGSK3beta in HT22 cells. Our findings suggest that estradiol plays a potent protective role against brain injury and that phosphorylation of Akt and GSK3beta by estradiol mediated these protective effects.

Ethanol modulates GABAB receptor expression in cortex and hippocampus of the adult rat brain

Using in situ hybridization, RNase protection assay and Western blot, we studied the effects of ethanol on the expression levels of GABA B receptor mRNA and protein in the cortex and hippocampus from adult rat brain. The results showed that ethanol significantly increased GABA B1 and GABA B2 receptor protein expression in the cortex, whereas only GABA B2 was increased in the hippocampus. GABA B receptor agonist baclofen could partially reverse the effect of ethanol. Further studies of the mRNA levels defined that GABA B1 mRNA levels were significantly increased in the hippocampus, with no significant changes of GABA B2 mRNA levels. Moreover, GABA B1 and GABA B2 receptor mRNA levels were increased on 3-week ethanol treatment. Finally, GABA B agonist baclofen and antagonist phaclofen showed significant decreasing effects on GABA B1 receptor mRNA levels in the cortex, but not in the hippocampus. These results were further confirmed by in situ hybridization. Thus, the present results showed the effects of ethanol on GABA B receptors in the cortex and hippocampus, implying the possible role of GABA B receptor in ethanol effects. The effects of GABA B receptor agonist and antagonist suggested that the possible mechanisms underlying that GABA B receptor modulated the behavioral effect induced by ethanol.

The localization of dopamine D2 receptor mRNA in the human placenta and the anti-angiogenic effect of apomorphine in the chorioallantoic membrane.

While there are some reports indicating that dopamine (DA) and D2-like receptors (Rc) are detected in human placenta, there is little evidence available regarding the function of DA or the precise localization of its receptors in this organ. In the present study, we confirmed the placental expression of DA D2 Rc transcripts by Northern blot analysis. Using in situ hybridization, we also first revealed that DA D2 Rc mRNA was expressed in cytotrophoblasts, syncytial trophoblasts, vascular endothelial cells, Hafbauer cells, and fibroblasts in the chorionic villi of the human placenta. The expression sites of DA D2 Rc mRNA led us to suspect other functions of DA in the placenta besides the regulation of human placental lactogen. Since the cells expressing DA D2 Rc mRNA are related to proliferation and remodeling of placental tissue, we tried to evaluate a possible involvement of DA in the regulation of placental angiogenesis. To this end, we used the chicken chorioallantoic membrane (CAM) assay. In CAM assay, apomorphine, a potent nonselective agonist of DA, has an anti-angiogenic effect. These results suggest that DA may regulate the vascularization of human placenta through its receptors present in the chorionic villi.

Gingko biloba extract (EGb 761) attenuates ischemic brain injury-induced reduction in Ca2+ sensor protein hippocalcin

Gingko biloba extract 761 (EGb 761) protects neuronal cells from ischemic brain injury via a number of neuroprotective mechanisms. Hippocalcin is a calcium sensor protein that regulates intracellular calcium concentrations and apoptotic cell death. We investigated whether EGb 761 regulates hippocalcin expression in cerebral ischemia. Male Sprague-Dawley rats were treated with vehicle or EGb 761 (100 mg/kg) prior to middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 h after MCAO. A proteomic approach demonstrated reduction in hippocalcin expression in vehicle-treated animals during MCAO, whereas EGb 761 treatment prevented injury-induced decreases in hippocalcin expression. RT-PCR and Western blot analyses indicated that EGb 761 attenuates injury-induced decrease in hippocalcin. These results suggest that the maintenance of hippocalcin during cerebral ischemia contributes to the neuroprotective role of EGb 761.

Modulation by the GABAB receptor siRNA of ethanol-mediated PKA-α, CaMKII, and p-CREB intracellular signaling in prenatal rat hippocampal neurons

Fetal alcohol syndrome (FAS) is a developmental neuropathology resulting from in utero exposure to ethanol; many of ethanols effects are likely to be mediated by the neurotransmitter γ-aminobutyric acid (GABA). We studied modulation of the neurotransmitter receptor GABABR and its capacity for intracellular signal transduction under conditions of ethanol treatment (ET) and RNA interference to investigate a potential role for GABA signaling in FAS. ET increased GABAB1R protein levels, but decreased protein kinase A-α (PKA-α), calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of cAMP-response element binding protein (p-CREB), in cultured hippocampal neurons harvested at gestation day 17.5. To elucidate GABAB1R response to ethanol, we observed the effects of a GABABR agonist and antagonist in pharmacotherapy for ethanol abuse. Baclofen increased GABABR, CaMKII and p-CREB levels, whereas phaclofen decreased GABABR, CaMKII and p-CREB levels except PKA-α. Furthermore, when GABAB1R was knocked down by siRNA treatment, CaMKII and p-CREB levels were reduced upon ET. We speculate that stimulation of GABAB1R activity by ET can modulate CaMKII and p-CREB signaling to detrimental effect on fetal brain development.