Genome-wide gene-expression data from DNA-microarray technology and molecular-network data from computational text-mining have led to a paradigm shift in biological research. However, interpretation of the huge amount of data is a bottleneck. We have developed an informatics system, which we refer to as bioSpace Explorer, that can extract pathways and molecules of interest from genome-wide data and show the mutual relationships among these pathways and molecules. Differentiation of 3T3-L1 cells into adipocytes and the action of a peroxisome proliferator-activated receptor gamma (PPARγ) agonist or α-linolenic acid on this process was analyzed with bioSpace Explorer. The results suggested a biological basis for adipocyte differentiation and a strategy to enhance lipid oxidation in adipocytes. Clustered changes of molecules were apparent in the insulin, Wnt, and PPARγ signaling pathways and in the lipogenesis, lipid oxidation, and lipid transport pathways during cell differentiation. A PPARγ agonist enhanced lipid oxidation in adipocytes and α-linolenic acid gave similar results to the PPARγ agonist. An analysis of sex hormone and thyroid hormone, in addition to PPARγ signaling, suggested that these molecules are important for enhancement of lipid oxidation in adipocytes. The results indicate the utility of bioSpace Explorer for biological research on genome-wide molecular networks.

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The most important strategies in pharmacogenomics are gene expression profiling and the network analysis of human disease models. We have previously discovered novel drug target candidates in cardiovascular diseases through investigations of these pharmacogenomics. The significant induction of S100C mRNA and protein expression was detected in the rat pulmonary hypertension and myocardial infarction model. We also found increased taurine in hypoxia, a calcium-associated cytoprotective compound, to suppress the hypoxia-induced S100C gene expression and vascular remodeling. These results suggest that S100C may be one of the potential novel drug targets in hypoxic or ischemic diseases. Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage causes cerebral ischemia and infarction. Using a DNA microarray, a prominant upregulation of heme oxygenase-1 (HO-1) and heat shock protein (HSP) 72 mRNAs were observed in the basilar artery of a murine vasospasm model. Antisense HO-1 and HSP 72 oligodeoxynucleotide inhibited HO-1 and HSP 72 induction, respectively, and significantly aggravated cerebral vasospasm. Moreover, we have also developed a unique heart failure model in zebrafish and identified several candidate genes as novel drug targets. These results suggest that pharmacogenomic network analysis has the potential to bridge the gap between in vitro and in vivo studies and could define strategies for identifying novel drug targets in various cardiovascular diseases.

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The ebb and flow of cellular life depends largely on signaling pathways and networks, which are regulated by specific protein-protein interactions. These interactions often involve assembly of large signaling complexes containing many different protein kinases, protein phosphatases, their substrates, and scaffold proteins. Identification of protein complexes is the key to understanding cellular functions. One of the techniques used for the isolation of protein complexes is the affinity purification system. Inhibitors of 3-hydroxyl-3-methyglutaryl coenzyme A (HMG-CoA) reductase (i.e., statins) exert cholesterol-independent vasoprotective effects that are mediated, in part, through the activation of Akt. However, the molecular mechanism remains unknown. To elucidate the molecular mechanisms of the pleioptropic effects of statins, we searched for the binding molecule of Akt1 by using a combined mass spectrometry and affinity purification strategy. By this technique, we identified the protein-protein interactions of 23 proteins from statin-treated rat aortic endothelial cells (rAECs). Our results suggest that this approach is very effective and statin activates many Akt down-stream targets, not only endothelial nitric oxide synthase (eNOS). The methodology presented here would provide a new tool for chemical proteomics in medicinal science.

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The neuroprotective effect of schizandrin on the glutamate (Glu)-induced neuronal excitotoxicity and its potential mechanisms were investigated using primary cultures of rat cortical cells. After exposure of primary cultures of rat cortical cells to 10 μM Glu for 24 h, cortical cell cultures exhibited remarkable apoptotic death. Pretreatment of the cortical cell cultures with schizandrin (10, 100 μM) for 2 h significantly protected cortical neurons against Glu-induced excitotoxicity. The neuroprotective activity of schizandrin was the most potent at the concentration of 100 μM. Schizandrin reduced apoptotic characteristics by DAPI staining in Glu-injured cortical cell cultures. In addition, schizandrin diminished the intracellular Ca²⁺ influx, inhibited the subsequent overproduction of nitric oxide (NO), reactive oxygen species (ROS), and cytochrome c, and preserved the mitochondrial membrane potential. Furthermore, schizandrin also increased the cellular level of glutathione (GSH) and inhibited the membrane lipid peroxidation malondialdehyde (MDA). As indicated by Western blotting, schizandrin attenuated the protein level changes of procaspase-9, caspase-9, and caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Taken together, these results suggest that schizandrin protected primary cultures of rat cortical cells against Glu-induced apoptosis through a mitochondria-mediated pathway and oxidative stress.

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Ginsenoside Rb₁ (GRb₁), a major component of the traditional herb ginseng, has been reported to show a neuroprotective effect in a rodent ischemic model. The purpose of this study was to investigate effects of GRb₁ on early and delayed brain injuries in a non-human primate thromboembolic stroke model. Thromboembolic stroke was induced by occlusion of the middle cerebral artery by injection of an autologous blood clot into the left internal carotid artery. GRb₁ (300 μg/kg per day, i.v.) and vehicle were administered from 7 days before embolization to the day following embolization (total: 8 times). Neurological deficits were observed at 1, 6, and 24 h and at 2, 4, and 7 days after embolization. At 7 days after embolization, neuron damage in the peri-infarct area and core region were assessed by NeuN, TUNEL, and GFAP staining. GRb₁ improved the skeletal muscle coordination score of the neurologic deficits (median: GRb₁ vs vehicle = 10 vs 12, P<0.05). In the GRb₁ group, positive neurons expressed by NeuN staining were noted in the ischemic peri-infarct area, and TUNEL- and GFAP-positive cells significantly decreased, when compared with vehicle. These results demonstrated that GRb₁ ameliorated both early and delayed injuries in the thromboembolic stroke model in non-human primates.

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide implicated in several metabolic functions, including insulin secretion and sympathoadrenal activation. To clarify the roles of PACAP in maintenance of whole-body glucose and lipid homeostasis, the impact of the deletion of PACAP on glucose homeostasis, body weight, and adipose tissue mass was examined by comparing mice lacking the Adcyap1 gene encoding PACAP (Adcyap1^−/−) with wild-type littermate controls. Adcyap1^−/− mice showed significant hypoinsulinemia, although being normoglycemic, and lower body weight as well as reduced food intake. They also showed greatly reduced white adipose tissue mass, in which the mRNA expression of adipocyte fatty acid-binding protein (aP2), a marker of adipocyte differentiation, was decreased. Glucose and insulin tolerance tests revealed increased insulin sensitivity in Adcyap1^−/− mice. In accordance with these observations, plasma levels of resistin, an adipocytokine implicated in insulin resistance, were decreased in Adcyap1^−/− mice. After a high-fat dietary challenge for six weeks, Adcyap1^−/− mice still showed lower body weights and increased insulin sensitivity. These results indicate the crucial roles of PACAP in energy metabolism, including lipid metabolism, and in the regulation of body weight, raising the possibility that the PACAP-signaling pathway that favors energy storage could be a therapeutic target for obesity.

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Tetrahydrobiopterin (BH4) deficiency has been suggested to be an important factor in vascular endothelial dysfunction. In this study, we investigated the influence of decreased BH4 level produced by administration of 2,4-diamino-6-hydroxypyrimidine (DAHP), a specific inhibitor of the rate-limiting enzyme of BH4 synthesis, on vascular endothelial function in anesthetized rats. Wistar rats were given DAHP (0.1 – 1.0 g/kg, i.p.) or the vehicle 5 h before the experiment. Depressor responses to the endothelium-dependent vasodilator acetylcholine and the endothelium-independent vasodilator sodium nitroprusside were tested. After the experiment, blood and thoracic aorta were taken for estimating their BH4 levels and plasma concentrations of nitrite plus nitrate. DAHP produced marked decreases in BH4 levels in plasma and aorta in a dose-related manner. Baseline values for hemodynamics were not affected by DAHP. Depressor responses to acetylcholine were attenuated with the highest dose of DAHP (1.0 g/kg) but not with DAHP (0.3 g/kg), although similar decreases in BH4 levels were seen with these two doses of DAHP. Treatment with DAHP at each dose did not decrease plasma concentrations of nitrite plus nitrate. These findings suggest that a decrease in BH4 levels by acute inhibition of de novo BH4 synthesis does not necessarily cause endothelial dysfunction.

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The pharmacological action of riluzole, a drug that has been approved as a neuroprotective agent for the treatment of amyotrophic lateral sclerosis, has not yet been established. We examined the effects of riluzole on 5-hydroxytryptamine (5-HT)₃ receptors in NCB-20 neuroblastoma cells using the whole-cell voltage clamp technique combined with a fast drug application method. Co-application of riluzole (1 – 300 μM, 5 s) produced a dose-dependent reduction in peak amplitudes and in the rise slope of the currents induced by 2 μM 5-HT. In addition, 5-HT₃–mediated currents evoked by dopamine, a partial 5-HT₃–receptor agonist, were inhibited by riluzole co-application. These inhibitory effects were clearly shown at low concentrations of 5-HT. The decay time constants of the receptor desensitization and deactivation were also significantly attenuated by riluzole. G-protein inhibitors (pertussis toxin and guanosine 5'-[β-thio] diphosphate) did not completely block these inhibitory actions of riluzole. These results indicate that riluzole inhibits 5-HT₃–induced ion currents directly by slowing channel activation in NCB-20 neuroblastoma cells.

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We evaluated changes in the binding properties of sedative and non-sedative histamine H₁–receptor antagonists induced by internalization of H₁ receptors in intact human U373 MG astrocytoma cells. Internalization of H₁ receptors was induced without their degradation by treatment with 0.1 mM histamine for 30 min at 37°C, and then the intact cell binding assay was performed at 4°C. The binding properties of [³H]mepyramine, a cell-penetrating radioligand for H₁ receptors, were not changed by histamine pretreatment. Displacement curves for sedative H₁-receptor antagonists (diphenhydramine, chlorpheniramine, promethazine, ketotifen, azelastine and oxatomide) against [³H]mepyramine binding were not changed by histamine pretreatment. In contrast, the displacement curves for non-sedative H₁-receptor antagonists (mequitazine, bepotastine, olopatadine, epinastine, carebastine, desloratadine and fexofenadine) were changed by histamine pretreatment: two types of changes, i.e. a rightward shift in the monophasic curve or an increase in the proportion of the low affinity component of the biphasic curve, were prevented under hypertonic conditions, in which clathrin-mediated receptor internalization is known to be inhibited. Thus, internalization-mediated changes in the binding properties of H₁-receptor antagonists were well correlated with their sedative and non-sedative behaviors, which might confirm their permeability through the biomembrane and possibly the blood brain barrier.

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A number of patients with hyperlipidemia are prescribed 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that are concomitantly used along with the treatment of diabetes mellitus. The effects of atorvastatin and pravastatin on insulin-induced glucose uptake and the related signal transduction in 3T3L1 adipocytes were studied. 3T3L1 fibroblasts were differentiated into adipocytes, pretreated with atorvastatin or pravastatin, and then exposed to insulin. Glucose uptake and the amount of insulin signal proteins were measured. Atorvastatin significantly decreased insulin-stimulated 2-deoxyglucose uptake in 3T3L1 adipocytes associated with the prevention of translocation of GLUT4 into the plasma membrane. The amounts of Rab4 and RhoA that required lipid modification with farnesyl or geranylgeranyl pyrophosphate, in the membrane fraction were decreased by atorvastatin. Insulin-induced tyrosine phosphorylation of IRS-1 and serine/threonine phosphorylation of Akt were reduced by atorvastatin. Pravastatin did not modify these insulin-induced changes in the signal transduction. Inhibitors of the RhoA/Rho kinase system, C3 and Y27632, as well as atorvastatin reduced insulin-induced changes in signal transduction. Atorvastatin and pravastatin did not affect messenger RNA expression, protein level, and tyrosine phosphorylation of insulin receptors. In conclusion, hydrophobic atorvastatin decreases the glucose uptake by 3T3L1 adipocytes since it can enter the cell and prevents lipid modification of some proteins that are involved in the insulin signal transduction process.

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Diet is one of the most important factors that influence the risks for cardiovascular diseases. Genistein, an isoflavone found in soy, may benefit the cardiovascular system. Here, we investigated the effect of genistein on platelet-derived growth factor (PDGF)-BB–induced proliferation of primary cultured rat aortic smooth muscle cells (RASMCs). Genistein significantly inhibited 25 ng/ml PDGF-BB–induced RASMC proliferation and [³H]-thymidine incorporation into DNA at 10, 20, and 40 μM. In accordance with these findings, genistein blocked the PDGF-BB–inducible progression through G₀/G₁ to S phase of the cell cycle in synchronized cells. Western blot analysis showed that genistein not only inhibited phosphorylation of retinoblastoma protein (pRb) and expression of cyclin E, cyclin-dependent kinase (CDK) 2, and proliferating cell nuclear antigen (PCNA) protein, but also inhibited downregulation of cyclin-dependent kinase inhibitor (CKI) p27^kip1. However, genistein did not affect p21^cip1, CDK4, and cyclin D1 expression or early signal transduction through PDGF beta-receptor, extracellular signal-regulated kinases 1/2 (ERK1/2), Akt, and phospholipase C (PLC) γ1 phosphorylation. These results suggest that genistein inhibits PDGF-BB–induced RASMC proliferation via G₀/G₁ arrest in association with induction of p27^kip1, which may contribute to the beneficial effects of genistein on the cardiovascular system.

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The contribution of the T-type Ca²⁺ current to cardiac pacemaking was examined in isolated right atrial tissue from the mouse, guinea pig, and rabbit using a specific blocker, R(−)-efonidipine. At 10⁻⁶ M, R(−)-efonidipine produced negative chronotropy, which was prominent in the mouse and small but significant in the guinea pig. No effect was observed in the rabbit. Microelectrode recordings revealed that R(−)-efonidipine significantly prolongs the pacemaker (phase 4) depolarization of the sinoatrial-node action potential in the mouse and guinea pig. These results provide the first pharmacological evidence that the contribution of T-type Ca²⁺ current to cardiac pacemaking differs among experimental animal species.

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We compared the effects of the low-molecular-weight heparin (LMWH), bemiparin, and dalteparin with unfractionated heparin (UFH) on the platelet subendothelium interaction under flow conditions. All three compounds decreased the percentage of subendothelial matrix covered by platelets, although the effect of LMWH was greater than that of UFH. Subendothelial matrix covered with platelet structures greater than 100 square microns was significantly reduced by all three compounds; the effect of bemiparin tended to be greater than that of the other two heparins.

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