Vitamin C protects against ethanol and PTZ-induced apoptotic neurodegeneration in prenatal rat hippocampal neurons.

Exposure to alcohol during brain development may cause a neurological syndrome called fetal alcohol syndrome, characterized by pre- and postnatal growth deficiencies, craniofacial anomalies, and evidence of CNS dysfunction. The objective of this study was to evaluate pentylenetetrazol (PTZ) and ethanol effects on Bax, Bcl-2 expression, which further induced activation of caspase-3, release of cytochrome-c from mitochondria, and to observe the protective effects of vitamin C (vit-C) against PTZ and ethanol-induced apoptotic neurodegeneration in primary-cultured neuronal cells at gestational day 17.5. Apoptotic neurodegeneration and neuroprotective effect of vit-C were measured by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay, Western blot analysis, which further conformed by the measurement of mitochondrial membrane potential using JC-1 detection kit and immunofluorescence analysis. The results showed that PTZ and ethanol produced extensive Bax-dependent caspase-9 and caspase-3 activation and caused neuronal apoptosis. Furthermore, the cotreatment of vit-C along with ethanol and PTZ showed significantly decreased expression of Bax, caspase-9, caspase-3, cytochrome-c, and significantly increased expression of antiapoptotic Bcl-2 protein when compared with control group. Our findings indicate that PTZ and ethanol activate an intrinsic apoptotic death program in neurons that is likely to contribute to the neuropathologic effects in fetal alcohol exposure, and vit-C can prevent some of the deleterious effects of PTZ and ethanol on the developing brain. The available experimental evidence and the safety of vit-C in pregnancy suggest the experimental use of ascorbic acid as a new and effective protective agent ethanol and PTZ-induced apoptotic neurodegeneration.

Proteomic identification of proteins differentially expressed by nicotinamide in focal cerebral ischemic injury.

Nicotinamide exerts a potent neuroprotective effect against ischemia-induced brain injury. We identified proteins that were differentially expressed by nicotinamide treatment in ischemic brain injury. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Adult male Sprague-Dawley rats were treated with vehicle or nicotinamide (500 mg/kg) 2 h after the onset of MCAO. Brains were collected 24 h after MCAO and cerebral cortex regions were isolated. Protein spots with different intensities between vehicle- and nicotinamide-treated groups were detected using two-dimensional gel electrophoresis and identified by mass spectrometry. Among these proteins, γ-enolase, protein phosphatase 2A (PP2A) subunit B, and peroxiredoxin-2 (Prx-2) were significantly decreased in the vehicle-treated group compared to the nicotinamide-treated group. These identified proteins mediate cell differentiation and stabilization, and play a role as antioxidant enzymes. In contrast, 60 kDa heat shock protein (Hsp 60) was significantly increased in vehicle-treated animals, while nicotinamide prevented the injury-induced increase of this protein. These results suggest that nicotinamide mediates neuroprotective effects by up- and down-regulation of various specific proteins.

DNA methylation contributes to the tissue-specific expression of the rPL-Iv gene.

To understand the tissue-specific expression of the rat placental lactogen-I variant (rPL-Iv) gene, we investigated the methylation pattern of the 5'-flanking region of this gene in various rat tissues. We report that the 5'-flanking region of the rPL-Iv gene was hypomethylated in placenta that expressed the gene and hypermethylated in those tissues that did not express the gene. Moreover, the intron region of the rPL-Iv gene was hypomethylated in the placenta, but hypermethylated in the liver, kidney and pituitary. Although there are 5 CpG sites and the density of CpG dinucleotide is lower within 2 kb of the rPL-Iv 5'-flanking region, the methylated promoter reporter gene produced strong repression in the transcriptional activity of the gene. In addition, the 5'-flanking and intron regions of the rPL-Iv gene were hypomethylated on day 12 of gestation, and the methylation pattern in the placenta remained unchanged from mid-pregnancy until term. The entire genomic region of the rPL-Iv gene might be hypermethylated in tissues other than the placenta, within which its methylated status repress expression of the placenta-specific rPL-Iv gene. Interestingly, the methylation status of the intron region of the rPL-Iv in proliferating Rcho-1 cells was changed to the unmethylated status on day 8 and 12 of differentiation of Rcho-1 cells. These results demonstrate that demethylation in the rPL-Iv upstream region was induced at an early stage of placental development, and once the 5'-flanking region of the rPL-Iv had been demethylated, its status on the rPL-Iv genomic region was continued during pregnancy. Taken together, these results suggest that DNA methylation is responsible for the silencing of tissue-specific genes in non-expressing cells, while defined combinations of trophoblast factors dictate the expression of unmethylated rPL-Iv gene in placenta trophoblast cells.

D1 dopamine receptor regulation of microtubule-associated protein-2 phosphorylation in developing cerebral cortical neurons.

This study addresses the hypothesis that the previously described capacity of D1 dopamine receptors (D1Rs) to regulate dendritic growth in developing cortical neurons may involve alterations in the phosphorylation state of microtubule-associated protein-2 (MAP2). The changes in phosphorylation of this protein are known to affect its ability to stabilize the dendritic cytoskeleton. The study involved two systems: primary cultures of mouse cortical neurons grown in the presence of the D1R agonists, SKF82958 or A77636, and the cortex of neonatal transgenic mice overexpressing the D1A subtype of D1R. In both models, a decrease in dendritic extension corresponded with an elevation in MAP2 phosphorylation. This phosphorylation occurred on all three amino acid residues examined in this study: serine, threonine, and tyrosine. In cultured cortical neurons, D1R stimulation-induced increase in MAP2 phosphorylation was blocked by the protein kinase A (PKA) inhibitor, H-89, and mimicked by the PKA activator, S(p)-cAMPS. This indicates that D1Rs modulate MAP2 phosphorylation through PKA-associated intracellular signaling pathways. We also observed that the elevations in MAP2 phosphorylation in neuronal cultures in the presence of D1R agonists (or S(p)-cAMPS) were maintained for a prolonged time (up to at least 96 hr). Moreover, MAP2 phosphorylation underwent a substantial increase between 24 and 72 hr of exposure to these drugs. Our findings are consistent with the idea that D1Rs can modulate growth and maintenance of dendrites in developing cortical cells by regulating the phosphorylation of MAP2. In addition, our observations suggest that MAP2 phosphorylation by long-term activation of D1Rs (and PKA) can be divided into two phases: the initial approximately 24-hr-long phase of a relatively weak elevation in phosphorylation and the delayed phase of a much more robust phosphorylation increase taking place during the next approximately 48 hr.

Receptor crosstalk protein, calcyon, regulates affinity state of dopamine D1 receptors.

The recently cloned protein, calcyon, potentiates crosstalk between G(s)-coupled dopamine D1 receptors and heterologous G(q/11)-coupled receptors allowing dopamine D1 receptors to stimulate intracellular Ca(2+) release, in addition to cAMP production. This crosstalk also requires the participating G(q/11)-coupled receptors to be primed by their agonists. We examined the ability of calcyon and priming to regulate the affinity of dopamine D1 receptors for its ligands. Receptor binding assays were performed on HEK293 cell membrane preparations expressing dopamine D1 receptors either alone or in combination with calcyon. Co-expression of dopamine D1 receptor and calcyon affected neither the affinity of this receptor for antagonists nor the affinity of agonist binding to this receptor high and low-affinity states. However, the presence of calcyon dramatically decreased the proportion of the high-affinity dopamine D1 receptor agonist binding sites. This decrease was reversed by carbachol, which primes the receptor crosstalk by stimulating endogenous G(q/11)-coupled muscarinic receptors. Our findings suggest that calcyon regulates the ability of dopamine D1 receptors to achieve the high-affinity state for agonists, in a manner that depends on priming of receptor crosstalk.

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.

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 E(2) 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.

Localization of dopamine D1 and D2 receptor mRNAs in the rat systemic and pulmonary vasculatures.

The present study was designed to evaluate the expression of dopamine D1 and D2 receptor mRNAs in systemic and pulmonary vasculatures. Using specific antisense riboprobes for dopamine D1 and D2 receptor cDNAs, in situ hybridization histochemistry was performed in the aorta, common carotid artery, vertebral artery, pulmonary artery, and superior vena cava of the adult male Sprague Dawley rat. In the case of the aorta, common carotid artery, and vertebral artery, dopamine D1 receptor mRNAs localized mainly in the smooth muscle cells of the tunica media. However, the signals of dopamine D2 receptor mRNAs were found in the endothelium and subendothelial layer of tunica intima, and interstitial cells of tunica adventitia. In the case of the pulmonary artery, signals of dopamine D1 receptor mRNAs were detected within the tunica intima, media, and adventitia. Expression of D2 receptor mRNAs was detected in the walls of small blood vessels within the tunica adventitia of the pulmonary artery. There were no detectable signals of dopamine D1 and D2 receptor mRNAs in the vein. The uneven distribution of dopamine D1 and D2 receptor mRNAs in the rat systemic vasculatures and pulmonary artery suggests that dopamine differentially regulates the vasodilation of the systemic and pulmonary arteries through the differential stimulation of dopamine D1 and D2 receptor.