Estradiol exerts a neuroprotective effect against focal cerebral ischemic injury through the inhibition of apoptotic signals. Phosphoprotein enriched in astrocytes 15 (PEA-15) is mainly expressed in brain that perform anti-apoptotic functions. This study investigated whether estradiol modulates the expression of PEA-15 and two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in middle cerebral artery occlusion (MCAO)-induced injury and glutamate exposure-induced neuronal cell death. Adult female rats were ovariectomized to remove endogenous estradiol and treated with vehicle or estradiol prior to MCAO. Focal cerebral ischemia was induced by MCAO and cerebral cortices were collected 24 h after MCAO. Western blot analysis indicated that estradiol prevents the MCAO-induced decrease in PEA-15, phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116). Glutamate exposure induced a reduction in PEA-15, phospho-PEA-15 (Ser 104), phospho-PEA-15 (Ser 116) in cultured neurons, whereas estradiol treatment attenuated the glutamate toxicity-induced decrease in the expression of these proteins. It has been known that phosphorylation of PEA-15 is an important step in carrying out its anti-apoptotic function. Thus, these findings suggest that the regulation of PEA-15 phosphorylation by estradiol contributes to the neuroprotective function of estradiol in ischemic brain injury.
Adult ; Animals ; Astrocytes ; Blotting, Western ; Brain ; Brain Injuries* ; Brain Ischemia ; Cell Death ; Cerebral Cortex ; Down-Regulation* ; Estradiol* ; Female ; Glutamic Acid ; Humans ; Infarction, Middle Cerebral Artery ; Neurons ; Neuroprotective Agents ; Phosphorylation ; Rats

Nitric oxide (NO) is generated by three different NO synthase (NOS) isoforms, endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS). It is known that eNOS produces NO, which exerts a protective effect, while iNOS produces NO with neurotoxic effects. Ferulic acid preserves neuronal cells against from cerebral ischemia and glutamate-induced excitotoxicity. This study confirmed the neuroprotective effect of ferulic acid and investigated the levels of three NOS isoforms in focal cerebral ischemia with or without ferulic acid. Rats were immediately treated with ferulic acid (100 mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brains tissues were collected at 24 h after the onset of occlusion. The expressions of these three isoforms in cerebral ischemia with ferulic acid were analyzed using Western blot technique. Ferulic acid treatment significantly decreases the number of TUNEL-positive cells in the cerebral cortex against MCAO injury. The levels of eNOS decreased in MCAO-operated animals, while ferulic acid treatment attenuated the MCAO-induced decrease of eNOS. However, iNOS and nNOS expression levels increased during MCAO, and ferulic acid prevented injury-induced increase of these isoforms. Thus, these findings suggest that the up- and down modulation of three isoforms by ferulic acid is associated with a neuroprotective mechanism.
Animals ; Blotting, Western ; Brain ; Brain Ischemia ; Cerebral Cortex ; Coumaric Acids ; Infarction, Middle Cerebral Artery ; Neurons ; Neuroprotective Agents ; Nitric Oxide ; Nitric Oxide Synthase ; Protein Isoforms ; Rats

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.
Animals ; Blotting, Western ; Brain ; Brain Injuries ; Brain Ischemia ; Calcium ; Cell Death ; Cerebral Cortex ; Ginkgo biloba ; Hippocalcin ; Humans ; Infarction, Middle Cerebral Artery ; Male ; Neurons ; Plant Extracts ; Rats, Sprague-Dawley

PEA-15 is a small phosphoprotein (15 kDa) that is enriched in brain astrocytes. PEA-15 acts as an important modulator of cellular function including apoptosis and signal integration. This study investigated the expression of PEA-15 in focal cerebral ischemic injury. Cerebral ischemia was surgically induced in adult male rats by middle cerebral artery occlusion (MCAO), and brains were collected 24 hr after MCAO. A proteomic approach demonstrated decreases of PEA-15 protein spots in MCAO-operated animals in comparison to sham-operated animals. Western blot analysis clearly demonstrated that MCAO induces decreases in PEA-15 levels. We previously showed that glutamate toxicity induces cell death in a hippocampus-derived cell line (HT22). Glutamate exposure induces decreases of PEA-15 levels in HT22 cells. The results of this study suggest that focal cerebral ischemia induces cell death through downregulation of PEA-15 protein.
Adult ; Animals ; Apoptosis ; Astrocytes ; Blotting, Western ; Brain ; Brain Ischemia ; Cell Death ; Cell Line ; Down-Regulation ; Glutamic Acid ; Humans ; Infarction, Middle Cerebral Artery ; Male ; Rats

PEA-15 is a small phosphoprotein (15 kDa) that is enriched in brain astrocytes. PEA-15 acts as an important modulator of cellular function including apoptosis and signal integration. This study investigated the expression of PEA-15 in focal cerebral ischemic injury. Cerebral ischemia was surgically induced in adult male rats by middle cerebral artery occlusion (MCAO), and brains were collected 24 hr after MCAO. A proteomic approach demonstrated decreases of PEA-15 protein spots in MCAO-operated animals in comparison to sham-operated animals. Western blot analysis clearly demonstrated that MCAO induces decreases in PEA-15 levels. We previously showed that glutamate toxicity induces cell death in a hippocampus-derived cell line (HT22). Glutamate exposure induces decreases of PEA-15 levels in HT22 cells. The results of this study suggest that focal cerebral ischemia induces cell death through downregulation of PEA-15 protein.
Adult ; Animals ; Apoptosis ; Astrocytes ; Blotting, Western ; Brain ; Brain Ischemia ; Cell Death ; Cell Line ; Down-Regulation ; Glutamic Acid ; Humans ; Infarction, Middle Cerebral Artery ; Male ; Rats

α-Synuclein is abundantly expressed in neuronal tissue, plays an essential role in the pathogenesis of neurodegenerative disorders, and exerts a neuroprotective effect against oxidative stress. Cerebral ischemia causes severe neurological disorders and neuronal dysfunction. In this study, we examined α-synuclein expression in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury and neuronal cells damaged by glutamate treatment. MCAO surgical operation was performed on male Sprague-Dawley rats, and brain samples were isolated 24 hours after MCAO. We confirmed neurological behavior deficit, infarction area, and histopathological changes following MCAO injury. A proteomic approach and Western blot analysis demonstrated a decrease in α-synuclein in the cerebral cortices after MCAO injury. Moreover, glutamate treatment induced neuronal cell death and decreased α-synuclein expression in a hippocampal-derived cell line in a dose-dependent manner. It is known that α-synuclein regulates neuronal survival, and low levels of α-synuclein expression result in cytotoxicity. Thus, these results suggest that cerebral ischemic injury leads to a reduction in α-synuclein and consequently causes serious brain damage.
Blotting, Western ; Brain Ischemia ; Brain* ; Cell Death ; Cell Line ; Cerebral Cortex ; Glutamic Acid ; Humans ; Infarction ; Infarction, Middle Cerebral Artery ; Male ; Nervous System Diseases ; Neurodegenerative Diseases ; Neurons ; Neuroprotective Agents ; Oxidative Stress ; Rats, Sprague-Dawley

No abstract available.
Brain* ; Tomography, Emission-Computed, Single-Photon*

The kidney and balances of fluid and volume are the basic components of bloocl pressure control, and the kidney is the primary site that initiates the hypertensive process and is affected by hypertensive vascular disease. In the kidney, the dopamine is a potent natriuretic and vasodilating agent, participat- ing in renal sodium excretion and maintenance of cardiovascular homeostasis. And the dopamine receptors in central nervous system and peripheral organs were identified by physiological, biochernical and radioligand binding techniques. Rut previous morphological and biochemical studies have been unable to characterize or determine the tissue distribution of the dopamine receptor subtypes because no selective ligands are available yet. Furthermore, the cellular distribution of the dopamine receptor subtypes in the rat kidney is not demonstrated well. In the SHR, the ability of exogenous and endogenous renal dopamine to engender a natriuresis is impaired. Since renal dopamine levels in genetic models of hypertension are not lower than their normotensive controls, the impaired intrarenal paracrine effect of dopamine in these animal models of hypertension appears to be receptor or postreceptor mediated. And renal dopamine derives mainly from renal tubular dopamine production and to a lesser extent from dopaminergic nerves. The present study utilizes imrnunohistochemistry with specific antibodies to characterize the renal distribution of dopamine receptor subtypes and recognize the role of dopamine receptor defect in the pathogenesis of hypertension in 14-week-old WKY (mean HP 108+/-5mmHg) and SHR (mean RP 174+/-7 mmHg) kidneys. Also it utilizes antibody of tyrosine hyclroxylase (TH) to recognize the site of the dopamine production mediated by TH using light microscopic immunohistochemistry. In the immunohistochemistry of the WKY kidney, dopamine D1 receptor protein is localized to glomerulus, proximal tubule, distal tubule, renal vessels, cortical and medullary collecting duct. And in the SHR kidney, dopamine D1 receptor protein is localized to glomerulus, distal tubule, renal vessels, cortical and medullary collecting duct, and juxtaglomerular apparatus (JGA). But there is no demonstrable positive reaction in the proximal tubule and weakly positive reactions in the renal arterioles of SHR compared with WKY kidney. In the immunohisto-chemistry of the WKY kidney, dopamine D1 receptor protein is localized to glomerulus, proxirnal tubule, distal tubule, renal vessels, cortical and rnedullary collecting duct. And in the SHR kidney, dopamine D2 receptor protein is localized to glomerulus, distal tubule, renal vessels, cortical and medullary collecting duct, and JGA. So, there is no demonstrable positive reaction in the proximal tubule of SHR compared with WKY. In the glomerulus of the WKY and SHR kidneys, both dopamine D1 and D2 receptors are localized. In the in situ hybridization of the WKY and SHR kidneys, dopamine D and D receptors are only demonstrated at the renal vessels. The positive reaction to TH immunohistochemistry of the WKY and SHR kidneys is only observed in the renal medulla compared with negative reaction on the renal cortex. Considering the excretion of sodium up to 65-70% with volume expansion may be mediated by dopamine D1-like receptors in the proximal tubule, our immunohistochemistry findings for the dopamine receptors may support the failure of natriuretic response in the SHR due to an abnormal dopamine receptor. Also our results rnay mean that the glornerular filtration rate is mediated by both dopamine D1 and Dz receptors comparing with the previous studies that the glomerular filtration rate was mediated by dopamine D2 receptor. I'here are some differences in the receptors expressing sites on the previous radioligand binding and pharmacologic studies, but our results suggest that at least some of the renal dopamine DA and DAz receptors correspond structurally to the central dopamine D1 and D2 receptors. Finally the result of TH immunohisto-chemistry suggests that the production of dopamine in the proximal tubule is not mediated by TH.
Animals ; Antibodies ; Arterioles ; Central Nervous System ; Dopamine* ; Filtration ; Glomerular Filtration Rate ; Homeostasis ; Hypertension ; Immunohistochemistry* ; In Situ Hybridization ; Juxtaglomerular Apparatus ; Kidney* ; Ligands ; Models, Animal ; Models, Genetic ; Natriuresis ; Rats* ; Rats, Inbred SHR* ; Receptors, Dopamine ; Receptors, Dopamine D1 ; Receptors, Dopamine D2 ; Sodium ; Tissue Distribution ; Tyrosine ; Vascular Diseases

Protein phosphatase 2A (PP2A) is a serine and threonine protein phosphatase that regulates cell cycle progression and apoptosis. PP2A is composed of various subunits. Among these subunits, subunit B plays an important role in the modulation of PP2A function in the brain. This study investigated PP2A subunit B expression levels after neuronal cell injury. Middle cerebral artery occlusions (MCAO) were surgically induced in adult male rats to induce focal cerebral ischemic injury, and brain tissues were collected 24 h after MCAO. A proteomic approach revealed reduction of PP2A subunit B protein spots in MCAO-operated animals in comparison to sham-operated animals. Western blot analysis confirmed that MCAO induces reductions in PP2A subunit B levels. Moreover, glutamate exposure induces neuronal cell death and leads to reductions of PP2A subunit B levels in a hippocampal-derived cell line. This study demonstrated the decrease of PP2A subunit B in ischemic neuronal cell injury. These results suggest that the decrease of PP2A subunit B after ischemic brain injury can mediate neuronal cell death.
Adult ; Animals ; Apoptosis ; Blotting, Western ; Brain ; Brain Injuries ; Brain Ischemia ; Cell Cycle ; Cell Death ; Cell Line ; Glutamic Acid ; Humans ; Infarction, Middle Cerebral Artery ; Male ; Middle Cerebral Artery ; Neurons ; Protein Phosphatase 2 ; Rats ; Serine ; Threonine

PURPOSE: To evaluate the antiproliferative activity of deuterium oxide (D2O) on urological cancer cells for the application of D2O in the treatment of urological cancer. MATERIALS AND METHODS: Urological cancer cell A-498 (kidney), T-24 (bladder) and DU 145 (prostate) were used in this study. The changes in cellular proliferation and the expressions of the bcl-2 and bax genes, according to changes in the D2O concentrationand exposure time were measured. The changes in cellular proliferation were measured using a hemocytometer and the MTT assay, and the changes in gene expression by Western hybridization. RESULTS: D2O had antiproliferative effects, DU-145 was most resistant and T-24 was most sensitive to D2O. The proliferation of cells in T-24, as measured by the MTT assay, showed a reduced growth rate, which was the inverse of the increased D2O concentration and exposure time. The expression of bcl-2 was reduced with increasing exposure time and D2O concentration, and that of bax was increased with increasing exposure time and D2O concentration. CONCLUSIONS: From this study, the authors believe D2O has antiproliferative effects on urological cancers, and the effect on bladder cancer cells suggests that D2O shows potential as an agent for the treatment of early small bladder cancer or the prevention of superficial bladder cancer recurrence following transurethral resection.
Cell Proliferation ; Deuterium Oxide* ; Deuterium* ; Gene Expression ; Recurrence ; Urinary Bladder Neoplasms ; Urologic Neoplasms*