Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. Platelet-derived Sph-1-P seems to play an important role in vascular biology, and manipulation of Sph-1-P activities may greatly influence vascular system development or events (involving hemostasis, thrombosis, atherosclerosis, angiogenesis, and wound healing). Selective Edg antagonists may have great potential as drugs to control vascular diseases.

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Since Japan generally has a more humid and warmer climate during the grape-growing season, and hence a higher fungal disease pressure, than the major good-quality wine-grape cultivation regions of the world, grapes grown in Japan generally contain lower amounts of total phenols, especially anthocyanins. In order to make red wines rich in polyphenols but with a smooth flavour lacking astringency or bitterness, it is necessary to investigate in detail the behavior of polyphenols during fermentation and storage. For many years, we have been conducting such studies with the aim of establishing how to produce good-quality wines favored by consumers from low-polyphenol Japanese grapes. When grapes are crushed, polyphenoloxidase (PPO) oxidizes colorless diphenols to produce the corresponding quinones. o-Quinones formed enzymatically oxidize red pigments such as monomeric anthocyanins and oligomeric and polymeric pigments, thus decolorizing them. PPO can not directly oxidize these pigments. Fermentation is started by adding yeast to must, and polyphenols are extracted from seeds and skins. The maximum intensity of red coloration (red pigments) in the must is obtained within several days after the addition of yeast, but the concentration of colorless polyphenols (catechin, procyanidins, flavonols, etc.) continues to increase until the removal of the pomace (seeds and skins). In sensory evaluations, wines fermented on skins for more a few days after reaching the maximum red color intensity received the highest scores. We have produced red wines from Cabernet Sauvignon grapes annually for 22 years and stored them in an underground cellar. Color analysis of these wines showed that the total phenol content did not change appreciably for 10 years after the start of storage, whereas the total pigment content gradually decreased. Strong relationships were found in the 22 wines between antioxidant activity and total phenols, flavonoids, or red pigments. Resveratrol is a polyphenol and stilbene specific to grapes infected by the gray mold Botrytis cinerea. It is noted as an antioxidative substance that is thought to prevent LDL oxidation and/or blood platelet aggregation, and may have anti-cancer properties. Little resveratrol was found in grape juices, but its concentration in musts (wines) increased during fermentation on skins. Because grape varieties with high resveratrol productivity were found to be resistant to various fungal pathogens, and a wild grape originating from Tibet produced the a large amount of resveratrol, we have cloned the stilbene synthase gene of this wild grape for the purpose of comparing it with the corresponding genes of grapes with low resveratrol productivity.

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Specific molecular recognition characterizes individual proteins in the mediation of many biological phenomena. Specific protein-protein interactions guide the assembly of multimeric proteins, which are generally found in cell organelles, the cytosol, and cell membranes. They govern the biological system through their cooperative and coupled functions, such as signal transduction and enzyme activation, etc. Protein-nucleic acid interactions are the origin of the complex genetic regulation of transcription, replication, translation, and recombination. Antigens are recognized by the corresponding antibodies to neutralize infections. In cellular signal transduction pathways, phosphorylation of tyrosine residues provides a key of the switching mechanism. Small ligand molecules can control the action of proteins through specific protein-ligand interactions. The ligands are directly and indirectly recognized through the hydrogen bonds, the van der Waals attractions, and the hydrophobic interactions, in addition to the non-specific electrostatic interactions. These specific interactions are observed in the complex structures, yielded from either the rigid body association or the local folding mechanism. Structural approach with the aid of bioinformatics can dissect the interactions into several structural and energetic contributions by individual contacts.

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The novel drug discovery process is changing to genome-based drug discovery due to the development of Human genome project. Many technologies and methodologies have been developed for discovering disease-related genes and good drug targets. Furthermore, recent proteomics and structural genomics will contribute greatly to structure-based rational drug design in the post-genomic era.

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Recent progresses in the molecular and structural understandings in the action of K channel openers for ATP-sensitive K channels and class III anti-arrhythmic agents for HERG K channel are to be reviewed. Based on these progresses, the strategy to identify the useful drugs in these categories will be discussed.

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Importance of screening of drugs with appropriate pharmacokinetic (PK) properties in the early phase of drug development has been recognized. One of the important factors in governing the hepatic clearance of drugs is the biliary excretion following its hepatic uptake. Recent studies clarified the contribution of transporters both in the hepatic uptake and biliary excretion processes, and cDNA of the transporters have been cloned one after another. ABC transporters such as p-glycoprotein (mdr) and cMOAT/ mrp2 (canalicular multispecific organic anion transporter) have been shown to play a predominant role in the biliary excretion of xenobiotics. Recent evidence suggests the presence of multiple forms for the transporters, similar to metabolic enzymes. Clarification of these multiplicities in transport is necessary for our understanding of the physiological adaptability of the living body in terms of the detoxification of xenobiotics. We have succeeded in making transfectant of some of the transporters. In near future, the prediction of hepatobiliary excretion in human from in vitro data obtained using human hepatocytes, membrane vesicles and transfectants may be used as a tool for highthroughput screening. I also summarize our recent attempts to describe the CNS (central nervous system) disposition of xenobiotics with respect to the transport properties across the blood brain barrier and blood-CSF barrier, and to describe the efflux of some drugs and their conjugates from the intestinal epithelial cells to the lumen and blood via apical and basal membrane, respectively, focusing on the active efflux systems.

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There are eight types and subtypes of receptors for prostanoids including PGD receptor (DP), PGE receptors of EP1, EP2, EP3 and EP4 subtypes, PGF receptor (FP), PGI receptor (IP) and TXA receptor (TP). Using chimeras of the mouse IP and the mouse DP, we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, while that of mDP binds the D type of PGs alone. In the present study, we performed a more detailed chimera analysis, and narrowed the domain for the ring recognition to a region from the first transmembrane domain to the first extracellular loop. One chimera with the replacement of the second transmembrane domain and the first extracellular loop of mDP with that of mIP bound only iloprost. The amino acid substitutions in this chimera suggest that Ser50 in the first transmembrane domain of mIP confers the broad ligand recognition of mIP and that Lys75 and Leu83 in the second transmembrane domain of mDP confer the high affinity to PGD ₂ and the strict specificity of ligand binding of mDP, respectively.
Prostanoid receptors have been characterized pharmacologically by comparing the potencies of agonists or antagonists in various tissue preparations. However, these tissue preparations usually contain more than one type or subtype of these receptors, and it is well known that prostanoids and their analogues often act on several types or subtypes of receptors. Thus, it is usually difficult to evaluate the potencies of various compounds for a particular type of receptor in a tissue preparation. The liigand binding characteristics of the prostanoid receptors have also been studied biochemically by using radiolabelled prostanoids in various tissues. However, the expression levels of these receptors are low in many preparations. This hinders the accurate evaluation of their ligand binding specificities. We have cloned eight types and subtypes of the mouse prostanoid receptor. This has enabled the homogeneous expression of each type of receptor at a high expression level, and has made the systematic analysis of the ligand binding characteristics of the prostanoid receptors possible. We have examined the ligand binding specificities of mouse prostanoid receptors stably expressed in Chinese hamster ovary (CHO) cells by use of prostanoids and their analogues.

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The objective of this study was to further characterize the drug specificity for activation of atypical β-adrenoceptors. The relaxant effects of (-)-isoprenaline and (-)-noradrenaline were about 40- and 20-fold as potent as the (+)-isomers, respectively. However, the isomeric activity ratio ((+)/(-)) of isoprenaline and noradrenaline in atypical β-adrenoceptors of guinea pig gastric fundus was less than that obtained for activation of β₁- and β₂-adrenoceptors in the guinea pig atria and trachea, respectively. The β-adrenoceptor alkylating agent, (±)-pindobind (10μM), shifted the concentration-response curves of catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and β₃-adrenoceptor agonists (BRL37344 and (±)-CGP12177A) to the right by about 3-fold, respectively. (±)-Pindobind (10μM) produced greater rightward shifts of the concentration-response curves of (-)-isoprenaline in the guinea pig atria and trachea (atria, 600-fold shift, trachea, 300-fold shift). These results suggest that pharmacological properties of atypical β-adrenoceptor differ from that obtained for β₁- and β₂-adrenoceptors.

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The fruit of Nandina domestica has been used to treat asthma, whooping cough, pharyna tumor and uterine bleeding in Japan. The extract of fruit was fractionated to afford nantenine as a serotonine (5-HT) receptor blocking substance. Nantenine caused a marked leftward shift of concentration-contractile response curve for 5-HT in the aorta or renal artery without affecting contractions induced by KCl or histamine. In the fundus, the concentration-response curve for 5-HT was slightly shifted to the right by nantenine even at high concentration, suggesting that nantenine preferentially blocks the receptor subtype of 5-HT_2A but not 5-HT_2B. The 5-HT_2A receptor antagonistic activities of nantenine and its derivatives were examined using aorta. The study on the structure-activity relationship indicates that the skeleton of nantenine and nitrogen-6 in the molecule are essential to development of the high 5-HT_2A receptor antagonistic activity. It is of interest that introduction of a hydroxy group into C-4 position greatly decreases the activity. Interaction between the 5-HT_2A receptor and nantenine derivatives was examined on the basis of receptor binding and molecular modeling analysis. The difference of antiserotonergic activity among nantenine derivatives was well elucidated by both the analysis.

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We constructed a plasmid vector to have a 1.4kb insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells (VSMCs), producing a few stable transfectants. The downregulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotactic motility to the platelet derived growth factor (PDGF). However, the transfectants did not show chemotactic motility, indicating the essential role of MLCK in the motility. The role was also confirmed by observing the inhibitory effect on chemotactic motility of MLCK inhibitors, i.e., ML-9 and wortmannin. It was discussed that down-regulation of MLCK expression could be utilized to discover the drug for arterioscrelosis which prevents the proliferating VSMCs to form acterial plaques.

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We investigated effect of a neuroprotective agent, FK506 in monkey cerebral ischemia using positron emmision tomography (PET). The right middle cerebral artery (MCA) of cynomolgus monkeys was occluded for 3 hours or permanently by a transorbital device. Four consecutive PET studies were performed to assess cerebral blood flow (CBF), oxygen extraction fraction (OEF), and the cerebral metabolic rate of oxygen (CMRO₂). For investigating effect of FK506 in transient ischamic model, FK506 was injected just after the MCA occlusion. The extent of necrotic brain damage 8 hours after MCA occlusion was significantly greater in the transient model than in the permanent model. Cortical damage was greater in the transient model. The MCA occlusion decreased CBF and CMRO₂ in deep MCA territory, and increased OEF in the cortex. In the permanent MCA occlusion model, these changes continued throughout the experiment. In the transient model, the reperfusion induced post-ischemic hyperperfusion in the cortex, which showed necrotic damage at the end of the experiment. In this area, OEF and CMRO₂ were decreased by reperfusion. FK506 significantly reduced infarct volume, especially in the cortex. In PET studies, FK506 prevented the reduction of CMRO₂ during ischemia and after reperfusion, which was greater than that during ischemia. The present results suggest that reperfusion may strongly contribute to cortical brain damage. PET studies revealed a peculiar OEF and CMRO₂ decrease in the cortex immediately after reperfusion, which may be an indicator of reperfusion injury, and that reperfusion injury may be caused by excessive hyperperfusion. FK506 has a nueroprotective action, especially in reperfusion injury.

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There have been developed several types of experimental technique or visualization of renal microcirculation. Although each methodology made much contribution for pharmacological and phisiological investigations, it requires substantial artificial manipulation that might effect the renal microvascular responsiveness. To circumvent such limitation of previous ex vivo or in vitro approaches to glomerular microcirculation, we have developed a pencil lens probe CCD intravital videomicroscopic system. Acute changes in glomerular microcirculation after administration of the calcium antagonist bamidipine were also evaluated by this system.In SHR, bamidipine caused a sharper reduction in mean blood pressure compared with WKY and STZ. The Af/Ef ratio increased significantly because of the marked dilation of Af. In contrast, barnidipine dilated Ef especially, so the Af/Ef ratio was significantly reduced in STZ. Using this pencil-probe CCD videomicroscope system, the characteristic and acute changes in in vivo glomerular microcirculation was visualized in pathologic animals. The sensitivity of Af to the vasodilating activity of barnidipine was enhanced in SHR, but reduced in STZ.

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1. Mice were used to record the spinal reflex potentials and to examine the effects of some drugs upon them. In anesthetized mice, mono- and polysynaptic reflex (MSR and PSR) potentials were recorded. TRH and DOI produced transient and long-lasting increases in the MSR amplitude, respectively. Tolperisone and baclofen produced transient and long-lasting MSR depression, respectively. These results show that similar data can be obtained from mice and rats. 2. Cytosine arabinoside induced-ataxic mice (Ara-C mice) were employed as an animal model of spinocerebellar degeneration. Taltirelin, a remedy for spinocerebellar degeneration, augmented the MSR in Ara-C mice. Effects of prazosin and mazindol on the MSR suggested that the function of descending noradrenergic systems was depressed in Ara-C mice.

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The biological role of peroxisome proliferator-activated receptorγ (PPARγ) was investigated by gene targeting and case-control study of the Prol2Ala PPARγ 2 polymorphism. Homozygous PPARγ-deficient embryos died due to placental dysfunction. Heterozygous PPARγ-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. Heterozygous PPARγ-deficient mice showed overexpression and hypersecretion of leptin, which may explain these phenotypes. This study reveals a hitherto unpredicted role for PPARγ in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPARγ. A Prol2Ala polymorphism has been detected in the human PPARγ 2 gene. Since this amino acid substitution may cause a reduction in the transcriptional activity of PPARγ, this polymorphism may be associated with decreased insulin resistance and decreased risk of type 2 diabetes. To investigate this hypothesis, we performed a case-control study of the Prol2Ala PPARγ 2 polymorphism. In an obese group, subjects with A1a12 were more insulin sensitive than those without. The frequency of Ala12 was significantly lower in the diabetic group, suggesting that this polymorphism protects against type 2 diabetes. These results revealed that both in mice and humans, PPARγ is a thrifty gene mediating type 2 diabetes.

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Cardiac responses to vasoactive substances on the whole animal are due to results of their direct and indirect actions. On the other hand, the responses on the isolated heart preparation are due to the direct cardiac effects. The isolated, blood-perfused dog atrial muscle preparation is useful for pharmacologically analyzing direct cardiac effects of active substances. When the drug was applied intravenously to the donordog, the changes in the donor's blood pressure and heart rate, andisolated atrial rate and developed tension are simultaneously observed. Thus, direct and indirect cardiac effects of the drug were readily analyzed pharmacologically. In the present time, I demonstrated the effects of intravenous lidocaine, pentobarbital, nitroprusside, prostacyclin and glucagon on whole animals and isolated atria.

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The proarrhythmic effects of class III antiarrhythmic drugs and non-cardiovascular drugs which have been shown to prolong the QT interval were assessed using the three different types of canine models. The proarrhythmic action of sematilide was assessed first using the canine isolated, blood-perfused ventricular tissue preparation. Sematilide prolonged MAP₉₀ and ERP, in which the increase in the former was greater than the latter, indicating prolongation of relative refractory period (RRP=MAP₉₀ -ERP). Ventricular tachyarrhythmias were induced when extrastimulus was applied within the RRP. This arrhythmic zone increased as RRP increased Next, electrophysiological effects in vivo of dofetilide, nifekalant, cisapride, sulpiride, haloperidol and sparfloxacin were assessed using halothane-anesthetized canine model. Dofetilide and nifekalant slowed heart rate and prolonged MAP₉₀ and ERP, while no significant change was detected in other cardiovascular parameters. The other drugs also exerted similar effects to class III drugs, however they also exerted negative inotropic, dromotropic and hypotensive actions. RRP was prolonged by each drug. Finally, torsadegenic action of sematilide, nifekalant, cisapride, terfenadine, sulpiride and sparfloxacin was assessed using chronic complete atrioventricular block dogs using Holter ECG monitoring in conscious state. Oral administration of clinically relevant doses of drugs did not induce any arrhythmia, while 5-40 times higher doses induced polymorphic ventricular tachycardia. These results indicate that drug-induced prolongation and backward shift of RRP may be substrates for Torsades de Pointes.

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We evaluated a role of complement activation in pathophysiology of bronchial asthma by using two experimental models; a single and a repeated antigen challenge in rats. While actively sensitized rats against ovalbumin (OA) were intratracheally challenged by OA, lung resistance (RL) was estimated by measuring airflow and esophageal pressure under urethane anesthesia and artificial ventilation. In order to examine histological findings, lungs were obtained 6 h after the OA-challenge. Intratracheal administration of OA resulted in two phases of airway response, immediate (IAR) and late airway response (LAR). The rats stimulated by OA and C5a (ZAS; zymosan activated serum) showed a sustained elevation of RL. Such the stimulation resulted in increased and prolonged excretion of N-acetyl-leukotriene E₄ in bile, suggesting a upregulated production of cysteinyl-leukotrienes in the lungs. Next, when the rats were repeatedly exposed to OA, histological studies showed infiltration of inflammatory cells in the bronchial submucosa.. While IAR tended to decrease in proportion to times of OA challenge, LAR turned to be prominent on the third exposure to OA. Pretreatment with two types of complement inhibitor, nafamostat mesilate (Futhan) and sCR1 inhibited the amplitudes of LAR. The pretreatment with sCR1 also inhibited the cellular infiltration after the repeated inhalation of OA. The similar results were obtained by the pretreatment with a C5a receptor antagonist (hexapeptide). On the contrary, an intratracheal administration of low dose of purified rat C5a des Arg reversed the LAR reduced by the pretreatment with sCR1, but that of CINC-1 (a cytokine of IL-8 family) did not substitute for C5a des Arg in these effects. It is suggested that complement activation products, especially anaphylatoxin C5a, play an important role in the LAR induced by antigen exposure and, consequently, a C5a receptor antagonist provides a novel therapeutic intervention for the treatment of bronchial asthma.

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When the rats are exposed to immobilization stress, these rats reveal not only the negative emotional responses such as vocalization and defecation which are considered to be similar to anxiety and/or fear in humans but also increases in noradrenaline release in the extended brain regions including the hypothalamus, amygdala and locus coeruleus. These stress-induced increases in noradrenaline release in these brain regions and emotional responses were significantly attenuated by pretreatment with diazepam, a typical benzodiazepine anxiolytic, in a flumazenil, a benzodiazepine antagonist, reversible manner. Similarly, morphine and opioid peptides injected i.c.v. such as β-endorphin and Met-enkephalin also significantly attenuated stress-induced increases in noradrenaline release in these region and emotional responses in a naloxone, an opoiod antagonist, reversible manner. When both diazepam at 5 mg/kg and morphine at 6 mg/kg were administered immediately before exposure to immobilization stress for 1 h, these two drugs showed the more potent attenuating effects on stress-induced increases in noradrenaline release in these brain regions than those caued by diazepam or morphine alone. The finding suggsets that if the drug possessing affinity to both benzodiazepine and opioid receptors were discovered, the drug could be a more potent anxiolytic than benzodiazepine anxiolytics. Further, we examined the involvement of corticotropin-releasing factor (CRF) in the stress-induced increases in brain noradrenaline release. When the rast were pretreated with a-helical CRF, an antagonist of CRF, stress-induced increases in noradrenaline release in the hypothalamus, amygdala and locus coeruleus were significantly attenuated by α-helical CRF. This indicates that CRF, at least, in part, has an important role for the initiation of increases in noradrenaloine release caused by stress in the extended brain regions. The finding suggests another possibility that a CRF-antagonist could be a useful anxiolytic and or anti-stress drug.

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We investigated phosphorylation-mediated regulation of functions of monoamine transporters, which are known as main molecular targets of antidepressants. Noradrenaline (NA) uptake in PC12 cells was enhanced by activation of calmodulin-dependent protein kinases such as Ca²⁺/calmodulin-dependent kinase II (CaM kinase II) and myosin light chain kinase, probably through stimulation of translocation of NA transporter (NAT) to plasma membrane and/or direct phosphorylation of the transporter itself. Also long-term activation of CaM kinase II increased NA uptake by stimulating NAT gene expression probably through phosphorylation of transcription factors such as CREB. These findings indicate that functions of monoamine transporters may be regulated both acutely and chronically by calmodulin-dependent protein kinases. Furthermore, long-term addition of antidepressants (desipramine and nisoxetine) which selectively inhibit NA uptake decreased NAT mRNA in PC12 cells, suggesting that chronic treatment of antidepressants affects expression of monoamine transporters. Recently, partial association between polymorphism of serotonin transporter gene and neuroticism or affective disorder has been reported. Therefore, reanalysis of monoamine transporter-related neuronal functions might be needed to elucidate neurochemical pathogenesis of depression and therapeutic mechanism of antidepressants.

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It is postulated that alterations in neuronal gene expression mediated by an activation of cAMP response element binding protein (CREB) may play an important role in the pathogenesis of stress-related psychiatric disorders, such as major depression and posttraumatic stress disorder. We examined the influence of restraint stress on CREB phosphorylation in rat frontal cortex and hippocampus. Restraint stress significantly increased the levels of phospho-CREB (p-CREB) in both brain regions. In addition, the marked induction of p-CREB expression in response to acute restraint stress was found in rats vulnerable to chronic overcrowding stress. Pretreatment with FK506 significantly upregulated the induction of c-fos mRNA in response to acute stress. On the other hand, we also examined the effect of stress on the expression and the serine/threonine phosphatase activity of calcineurin to elucidate the influence of stress on the dephosphorylation of p-CREB. Whereas stress had no change in the levels of calcineurin mRNA, the activity of calcineurin was significantly upregulated. Taken together, these findings suggest that the induction of CREB phosphorylation and the changes in CRE-mediated gene expression may be, at least in part, involved in the pathogenesis of stress-related psychiatric disorders.

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We compared emotional responses (behavioral and histochemical) to contextual fear conditioning between young and aged rats. Freezing behavior right after footshock did not differ between the two groups. However, in an hour the freezing behavior as well as phospho-cAMP-responsive element-binding protein (pCREB) in CA1 area of the hippocampus significantly increased in the young rats. pCREB is considered to be involved in learning and memory. The number of pCREB positive cells did not change in other areas of the hippocampus or other brain areas in either the young or aged groups. The number of positive cells to cAMP-responsive element-binding protein did not change in either the young or aged groups. These results indicate that a transcription factor pCREB and its products in CA1 area may be responsible for the emotional memory consolidation by the contextual fear conditioning.

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In an attempt to study the pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated signaling functions in vivo, we have used gene targeting in embryonic stem cells to disrupt exon 2 of the PACAP type I receptor (PAC₁ receptor) gene, which contains the ATG translation start site and the signal peptide. Unexpectedly, active transcription of PAC₁ receptor mRNA was detected in the mutant mice, although ligand binding in the brain was greatly reduced. PAC₁ receptor exon 2^-/- mice were apparently normal and fertile. The expression study of the mutant receptor lacking the signal peptide showed that the signal peptide is required for efficient cell surface expression and N-linked glycosylation of the PAC₁ receptor. We also generated mice lacking PACAP ligand by targeted deletion of the PACAP gene exon 5. PACAP-null mice developed to adulthood at a frequency lower than expected by mendelian genetics, and were hyperactive and showed reduced anxiety. Neurochemical characterization of these mice is currently under investigation.

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