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Intracellular calcium overload is a critical pathophysiological factor in ischemic injury. Hippocalcin is a neuronal calcium sensor protein that buffers intracellular calcium levels and protects cells from apoptotic stimuli. Ferulic acid exerts a neuroprotective effect in cerebral ischemia through its anti-oxidant and anti-inflammation activity. This study investigated whether ferulic acid contributes to hippocalcin expression during cerebral ischemia and glutamate exposure-induced neuronal cell death. Rats were immediately treated with vehicle or ferulic acid (100 mg/kg, i.v.) after middle cerebral artery occlusion (MCAO). Brain tissues were collected 24 h after MCAO and followed by assessment of cerebral infarct. Ferulic acid reduced MCAO-induced infarct regions. A proteomics approach elucidated a decrease in hippocalcin in MCAO-operated animals, ferulic acid attenuates the injury-induced decrease in hippocalcin expression. Reverse transcription-polymerase chain reaction and Western blot analyses confirmed that ferulic acid prevents the injury-induced decrease in hippocalcin. In cultured HT22 hippocampal cells, glutamate exposure increased the intracellular Ca(2+) levels, whereas ferulic acid attenuated this increase. Moreover, ferulic acid attenuated the glutamate toxicity-induced decrease in hippocalcin expression. These findings can suggest the possibility that ferulic acid exerts a neuroprotective effect through modulating hippocalcine expression and regulating intracellular calcium levels.

Melatonin is an antioxidant that has neuroprotective functions in ischemic brain injury. Protein phosphatase 2A (PP2A) is a serine and threonine phosphatase that modulates cell metabolism and cell survival. This study investigated whether melatonin modulates PP2A subunit B in focal cerebral ischemia and glutamate toxicity-induced neuronal cell death in a rat model. Middle cerebral artery occlusion (MCAO) was performed to induce permanent cerebral ischemic injury. Adult male rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortex tissues were collected 24 hr after MCAO. A proteomic approach elucidated the decrease in PP2A subunit B in MCAO-operated animals. Melatonin treatment attenuated injury-induced reductions in PP2A subunit B levels. Western blot analyses indicated that melatonin prevents injury-induced decrease in PP2A subunit B levels. In neuronal cells, glutamate toxicity induced a lowering of PP2A subunit B, while melatonin treatment attenuated the glutamate exposure-induced decreases in PP2A subunit B. These results suggest that the maintenance of PP2A subunit B by melatonin in ischemic injury is critical to the neuroprotective function of melatonin during neuronal cell damage.

Melatonin functions as a free-radical scavenger and has a neuroprotective effect against ischemic brain damage. PEA-15 (phosphoprotein enriched in astrocytes 15) regulates various cellular processes including cell proliferation and apoptosis. In this study, we investigated whether melatonin regulates the levels of PEA-15 and the two phosphorylated forms of PEA-15 (Ser 104 and Ser 116) in a middle cerebral artery occlusion (MCAO)-induced injury model and neuronal cells exposed to glutamate. Adult male rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortex tissues were collected 24 h after MCAO. PEA-15 levels after ischemic brain injury were monitored using a proteomic approach. Melatonin pretreatment prevented the ischemic injury-induced reduction in PEA-15 levels. Moreover, Western blot analysis demonstrated that melatonin attenuated the ischemic injury-induced reduction in PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116) levels. Neuronal cells exposed to glutamate showed decreased expression of PEA-15, phospho-PEA-15 (Ser 104), and phospho-PEA-15 (Ser 116), while melatonin pretreatment prevented the glutamate toxicity-induced decreases in the levels of these proteins. The reduction in the levels of phospho-PEA-15 proteins indicates the inhibition of anti-apoptotic function of PEA-15. Together, in vivo and in vitro results suggest that melatonin protects neurons against ischemic injury by maintaining levels of phospho-PEA-15 proteins.

Melatonin is a potent scavenger of reactive oxygen species and a strong antioxidant. Melatonin exerts protective effects against damage by the enhancing the Akt signal pathway, thus regulating apoptotic cell death. Akt phosphorylates pro-apoptotic proteins such as Bad and FoxO1 and inhibits the pro-apoptotic functions of these proteins. This study investigated the protective effects of melatonin through Akt and its downstream targets, Bad and FoxO1, in hepatic ischemia-reperfusion (I/R) damage. Adult mice were subjected to 1 h of hepatic ischemia and 3 h of reperfusion. Hepatic ischemia was induced by occlusions of the hepatic artery, portal vein, and bile duct. Melatonin (10 mg/kg, i.p.) or vehicle was administrated 15 min prior to ischemia and just before reperfusion. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in I/R group than in sham-operated group. Melatonin attenuated increases in these levels. Moreover, melatonin attenuates injury-induced increases in positive TUNEL staining in hepatic tissues. Hepatic I/R injury induced reductions in the Akt up-stream target, PDK1 phosphorylation. The levels of phospho-Akt, phospho-Bad, and phospho-FoxO1 were decreased in vehicle-treated animals. However, melatonin prevented hepatic I/R injury-induced decreases in these proteins levels. Moreover, the interaction levels between phospho-Bad and 14-3-3 and between phospho-FoxO1 and 14-3-3 are reduced in vehicle-treated animals, and melatonin attenuated decreases in the binding levels of these proteins. 14-3-3 exerts an anti-apoptotic function by sequestration of Bad and FoxO1. These findings suggest that melatonin exerts protective effects in case of hepatic I/R damage by maintaining the binding of phospho-Bad and 14-3-3 and the binding of phospho-FoxO1 and 14-3-3, thus preventing activation of apoptotic cell death.

Hepatic ischemia-reperfusion (I-R) injury induces hepatic dysfunction or failure. Melatonin is a potent free radical scavenger and a strong antioxidant. Although many studies have demonstrated the protective effect of melatonin in hepatic injury, the molecular mechanisms of this protection are unclear. We identified specific proteins that are differentially expressed by melatonin treatment in hepatic I-R injury. Adult mice were subjected to 1 hr of ischemia and 3 hr of reperfusion. Animals were treated with vehicle or melatonin (10 mg/kg, i.p.) 15 min prior to ischemia and just before reperfusion. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in I-R group than in sham-operated group, and these increases were reduced by melatonin treatment. Proteins that were differentially expressed following melatonin treatment during hepatic I-R injury were detected using two-dimensional gel electrophoresis. Hepatic I-R injury induced down-regulation of glyoxalase I, glutaredoxin-3, spermidine synthase, proteasome subunit beta type-4, and dynamin like protein-1 (DLP-1). However, melatonin prevented the reductions in these proteins induced by I-R injury. Among the identified proteins, we focused on DLP-1, which is essential for the maintenance of mitochondrial and endoplasmic reticulum morphology. Western blot analysis confirmed that melatonin prevents the hepatic I-R injury-induced decrease in DLP-1. These results suggest that melatonin protects hepatic cells against hepatic I-R injury and that its protective effects involve the regulation of specific proteins.

Dopamine D1 receptors have critical neuromodulatory influences on the working memory functions of the prefrontal cortex, a brain region affected in many neuropsychiatric disorders. When D1 receptor agents are administered to rats or monkeys performing working memory tasks, an "inverted U" dose/response function is typically observed, whereby either too little or too much D1 receptor stimulation impairs working memory. There are two subtypes of D1 receptors, the D1A and the D1B (also known as the D1 and D5, respectively), but the relative contributions of these subtypes to prefrontal cortical function are not known, as there are no pharmacological agents that can distinguish between these receptors. Thus, genetically altered mice are needed to address this question. However, it is not known whether the mouse prefrontal cortex contains both D1A and D1B receptor subtypes, nor is it known whether mice will exhibit responses to D1 receptor agonists similar to those seen in rats and monkeys. The current study examined these issues by immunostaining the mouse brain with specific antibodies directed at the D1A and D1B receptor subtypes and by assessing the effects of increasing doses of a D1 receptor agonist, SKF81297, on spatial working memory performance in mice. Results indicate that mice are generally similar to monkeys and rats, expressing both D1A and D1B receptors in the prefrontal cortex and exhibiting an inverted "U" dose/response curve when administered SKF81297.

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 mare's serum gonadotropin and human chorionic gonadotropin (hCG). Northern blot analysis showed that PACAP transcripts were transiently expressed from 3–9 hr after hCG treatment, reaching a maximum at 6 hr. During these time points, PACAP mRNAs were specifically and strongly expressed in granulosa cells and cumulus cells of large preovulatory follicles and interstitial glandular cells. Type I A receptor mRNAs were also transiently expressed in granulosa cells of large preovulatory follicles from 3–9 hr after hCG treatment. PACAP and its type I A receptor mRNAs were expressed in the same preovulatory follicles. These results demonstrate that PACAP acts as an autoregulator or pararegulator through type I A receptor in granulosa cells and cumulus cells of large preovulatory follicles. Thus, we suggest that PACAP may have a critical role in granulosa cells of preovulatory follicles for the preparation of ovulation. Mol. Reprod. Dev. 55:379–386, 2000. © 2000 Wiley-Liss, Inc.