Hydrogen sulfide (H₂S) is an important signaling molecule in various mammalian cells and tissues. H₂S is synthesized from L-cysteine and regulates several cellular and physiological phenomena (vasorelaxation, hormone secretion, and apoptosis) and multicellular events (neuromodulation and inflammatory responses). H₂S can be produced in pancreatic β-cells by cystathionine β-synthase (CBS) or cystathionine γ-lyase (CSE). H₂S inhibits insulin release and regulates β-cell survival. We found that glucose stimulation increased CSE expression at transcript and protein levels in mouse pancreatic islets. We also found that H₂S protects β-cells that were chronically exposed to high glucose from apoptotic cell death. Loss of β-cell mass and failures of β-cell function are important in the pathogenesis and/or progression of diabetes mellitus; therefore, molecular analyses of the mechanisms of H₂S production and its protective effects on β-cells may lead to new insights into diabetes mellitus.

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Several lines of evidence suggest that cholinergic deficits may contribute to the pathophysiology of psychiatric disorders as well as Alzheimer’s disease. There is growing clinical evidence that galantamine, currently used for the treatment of Alzheimer’s disease, may improve cognitive dysfunction and psychiatric illness in schizophrenia, major depression, bipolar disorder, and alcohol abuse. Since galantamine is a rather weak acetylcholinesterase inhibitor, but has additional allosteric potentiating effects at nicotinic receptors, it affects not only cholinergic transmission but also other neurotransmitter systems such as monoamines, glutamate, and γ-aminobutyric acid (GABA) through its allosteric mechanism. It is likely that these effects may result in more beneficial effects. To understand the underlying mechanism for the clinical effectiveness of galantamine, neuropharmacological studies have been performed in animal models of several psychiatric disorders. These studies suggest that not only the nicotinic receptor–modulating properties but also the muscarinic receptor activation contribute to the antipsychotic effect and improvement of cognitive dysfunction by galantamine. This review summaries the current status on the pharmacology of galantamine, focusing on its effect on neurotransmitter release and pharmacological studies in animal models of psychiatric disorders.

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Vascular endothelial cells (VECs) secrete tissue plasminogen activator (tPA) in an active form and thus its facilitated secretion directly enhances fibrinolytic activity. We have recently demonstrated its unique secretory dynamics in GFP-tagged tPA expressing VECs using total internal reflection fluorescence microscopy. tPA-GFP appeared to remain on the cell surface after secretion. Studies using a domain-deleted mutant of tPA-GFP suggested that its binding to the cell surface was heavy-chain dependent. PA inhibitor-1 (PAI-1) facilitated dissociation of tPA-GFP by forming a high molecular weight complex. Lack of dissociation from the cell surface of catalytically inactive mutant tPA-GFP, which does not complex with PAI-1, supported PAI-1 dependence of the disappearance of tPA from the VEC surface. To confirm the possibility that retained active tPA modified cell surface fibrinolytic activity, we analyzed binding of Alexa Fluor 568–labeled plasminogen (plg-568) in tPA-GFP expressing cells. Plg-568 appeared to accumulate at tPA-GFP–retained spots as well as in pericellular/matrix adhesive areas. Either modification of the active site or deletion of the tPA-GFP heavy chain resulted in decreased accumulation of plg-568. Prolonged retention appeared essential for tPA to effectively express and amplify fibrinolytic activity on VECs, which may also be responsible for development of deleterious effects in the case of stroke.

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Previous studies have shown that the risk of intracranial hemorrhage (ICH) associated with the treatment of ischemic stroke is mainly attributable to antithrombotic agents. On the basis of clinical trials, only tissue-type plasminogen activator (t-PA) has been approved for treating acute ischemic strokes, but delayed treatment with t-PA is associated with the risk of cerebral hemorrhagic transformation of ischemic stroke. t-PA converts plasminogen to plasmin, which participates primarily in clot lysis via fibrin degradation and, to some extent, in tissue remodeling via degradation of various extracellular matrix proteins, either directly or via activation of matrix metalloproteinases (MMPs). MMPs mediate the pathogenesis of ischemic-stroke-associated ICH by causing the disruption of vasculature. In particular, the binding of t-PA with one of its receptors leads to the activation of low-density lipoprotein receptor–related protein (LRP), which in turn results in the release of MMP-3 by endothelial cells. LRP production is reported to be upregulated in endothelial cells exposed to ischemia, and elevated LRP levels have been implicated in the increased ICH risk associated with delayed t-PA treatment. This implies that the t-PA / LRP / MMP-3 pathway may be a suitable target for developing strategies to improve the therapeutic efficacy of t-PA in acute ischemic stroke.

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Stroke is the second cause of mortality worldwide, and intravenous administration of tissue plasminogen activator (t-PA) within 3 h of symptom onset is the only treatment proven effective for re-establishment of cerebral blood flow following acute ischemic stroke. However, its widespread application remains limited by its narrow therapeutic time window and the related risks of intracranial hemorrhage. On the other hand, in patients with atherothrombotic risk, antiplatelet agents are widely used to decrease the risk of occlusive arterial events. All of these drugs are used during coronary interventions and in the medical management of acute coronary syndromes. In contrast, only aspirin, cilostazol, and thienopyridine derivatives (ticlopidine and clopidogrel) are used in the long-term prevention of cerebrovascular events in patients with risk of recurrence. In this paper, we introduce recent clinical findings on antiplatelet therapies for secondary prevention after ischemic stroke and describe basic research that has focused on cerebrovascular protection by cilostazol, which has a unique pharmacological profile.

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Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with several reported pharmacological actions. We have assessed the protective action of GA on iron-induced neuronal cell damage by employing the PC12 cell line and primary culture of rat cortical neurons (PCRCN). A strong protection by GA, assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay, was revealed, with IC₅₀ values <1 μM. GA also inhibited Fe³⁺–ascorbate reduction, iron-induced oxidative degradation of 2-deoxiribose, and iron-induced lipid peroxidation in rat brain homogenate, as well as stimulated oxygen consumption by Fe²⁺ autoxidation. Absorption spectra and cyclic voltammograms of GA–Fe²⁺/Fe³⁺ complexes suggest the formation of a transient charge transfer complex between Fe²⁺ and GA, accelerating Fe²⁺ oxidation. The more stable Fe³⁺ complex with GA would be unable to participate in Fenton-Haber Weiss-type reactions and the propagation phase of lipid peroxidation. The results show a potential of GA against neuronal diseases associated with iron-induced oxidative stress.

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Mosapride citrate (mosapride), a prokinetic agent with 5-HT₄–receptor agonistic activity, is known to enhance gastric emptying and alleviate symptoms in patients with functional dyspepsia (FD). As hyperalgesia and delayed gastric emptying play an important role in the pathogenesis of FD, we used in this study balloon gastric distension to enable abdominal muscle contractions and characterized the visceromotor response (VMR) to such distension in conscious rats. We also investigated the effects of mosapride on gastric distension–induced VMR in the same model. Mosapride (3 – 10 mg/kg, p.o.) dose-dependently inhibited gastric distension–induced VMR in rats. However, itopride even at 100 mg/kg failed to inhibit gastric distension–induced VMR in rats. Additionally, a major metabolite M1 of mosapride, which possesses 5-HT₃–receptor antagonistic activity, inhibited gastric distension–induced VMR. The inhibitory effect of mosapride on gastric distension–induced visceral pain was partially, but significantly inhibited by SB-207266, a selective 5-HT₄–receptor antagonist. This study shows that mosapride inhibits gastric distension–induced VMR in conscious rats. The inhibitory effect of mosapride is mediated via activation of 5-HT₄ receptors and blockage of 5-HT₃ receptors by a mosapride metabolite. This finding indicates that mosapride may be useful in alleviating FD-associated gastrointestinal symptoms via increase in pain threshold.

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Dextromethorphan (DEX) is a widely used non-opioid antitussive. However, the precise site of action and its mechanism were not fully understood. We examined the effects of DEX on AMPA receptor–mediated glutamatergic transmission in the nucleus tractus solitarius (NTS) of guinea pigs. Excitatory postsynaptic currents (evoked EPSCs: eEPSCs) were evoked in the second-order neurons by electrical stimulation of the tractus solitarius. DEX reversibly decreased the eEPSC amplitude in a concentration-dependent manner. The DEX-induced inhibition of eEPSC was accompanied by an increased paired-pulse ratio. Miniature EPSCs (mEPSCs) were also recorded in the presence of Cd²⁺ or tetrodotoxin. DEX decreased the frequency of mEPSCs without affecting their amplitude. Topically applied AMPA provoked an inward current in the neurons, which was unchanged during the perfusion of DEX. BD1047, a σ-1–receptor antagonist, did not block the inhibitory effect of DEX on the eEPSCs, but antagonized the inhibition of eEPSCs induced by SKF-10047, a σ-1 agonist. Haloperidol, a σ-1 and -2 receptor ligand, had no influence on the inhibitory action of DEX. These results suggest that DEX inhibits glutamate release from the presynaptic terminals projecting to the second-order NTS neurons, but this effect of DEX is not mediated by the activation of σ receptors.

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SSH-BM-I was synthesized from tryptamine by using a newly developed synthetic method, and it has structural similarity to bromomelatonin. Recently, it had been reported that SSH-BM-I increases osteoblasts in scales of gold fish. However, the effect of SSH-BM-I on osteoblast differentiation in mammalian cells has not yet been examined. Therefore, this study examined the effect of SSH-BM-I on osteoblast differentiation in mesenchymal progenitor-like cells and mature osteoblast-like cells. SSH-BM-I enhanced terminal osteoblast differentiation, as indicated by mineralization, which was accompanied by upregulation of the osteogenic marker genes bone sialoprotein (BSP) and osteocalcin (OC). However, in mesenchymal progenitor ROB-C26 cultures, no mineralized nodules were observed regardless of SSH-BM-I treatment, although BMP-2 was able to induce nodule formation in these cells. Furthermore, BMP-2–induced nodule formation was suppressed by SSH-BM-I treatment in ROB-C26 cultures. We further investigated the impact of the timing and duration of SSH-BM-I treatment on osteoblast differentiation. The effect of SSH-BM-I treatment on osteoblast differentiation of ROB-C26 in the presence of BMP-2 switches from negative to positive sometime between day 6 and 9, because SSH-BM-I treatment enhanced the formation of mineralized nodules when it was started on day 9, but suppressed nodule formation when it was started at day 6 or earlier. These results suggest that the stimulatory effects of SSH-BM-I on the formation of mineralized nodules depend on the degree of cell differentiation.

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Some class I antiarrhythmic drugs induce a sporadic hypoglycemia by producing insulin secretion via inhibition of ATP-sensitive K⁺ (K_ATP) channels of pancreatic β-cells. It remains undetermined whether amiodarone produces insulin secretion by inhibiting K_ATP channels. In this study, effects of amiodarone on K_ATP channels, L-type Ca²⁺ channel, membrane potential, and insulin secretion were examined and compared with those of quinidine in a β-cell line (MIN6). Amiodarone as well as quinidine inhibited the openings of the K_ATP channel in a concentration-dependent manner without affecting its unitary amplitude in inside-out membrane patches of single MIN6 cells, and the IC₅₀ values were 0.24 and 4.9 μM, respectively. The L-type Ca²⁺ current was also inhibited by amiodarone as well as quinidine in a concentration-dependent manner. Although glibenclamide (0.1 μM) or quinidine (10 μM) significantly potentiated the insulin secretion from MIN6 cells, amiodarone (1 – 30 μM) failed to increase insulin secretion. Amiodarone (30 μM) and nifedipine (10 μM) significantly inhibited the increase in insulin secretion produced by 0.1 μM glibenclamide. Amiodarone (30 μM) produced a gradual decrease of the membrane potential, but did not produce repetitive electrical activity in MIN6 cells. Glibenclamide (1 μM) produced a slow depolarization, followed by spiking activity which was inhibited by 30 μM amiodarone. Thus, amiodarone is unlikely to produce hypoglycemia in spite of potent inhibitory action on K_ATP channels in insulin-secreting cells, possibly due to its Ca²⁺ channel–blocking action.

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In small cell lung carcinoma (SCLC), acetylcholine (ACh) is synthesized and secreted, and it acts as an autocrine growth factor through activation of its receptors, muscarinic receptor (mAChR) and nicotinic receptor (nAChR). Alteration of tumor growth by blockade of M₃ mAChR in a human SCLC cell line, NCI-H82, was investigated in the present study. We used a highly selective M₃ muscarinic antagonist, N-(2-[3-([3R]-1-(cyclohexylmethyl)-3-piperidinyl]methylamino)-3-oxopropyl]amino-2-oxoethyl)-3,3,3-triphenyl-propioamide (J-115311). Our results show that J-115311 inhibited the increased intracellular calcium elicited by carbachol, a muscarinic agonist, in SCLC cells. J-115311 also inhibited SCLC cell growth in vitro. In a mouse orthotopic xenograft model, J-115311 dose-dependently reduced tumor growth when NCI-H82 cells were inoculated into the upper left lobe of the lung. These findings indicate that blockade of M₃ mAChR can suppress tumor growth in SCLC, suggesting the potential therapeutic utility of M₃ muscarinic antagonists as anti-cancer agents.

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NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II enzyme, plays an important role in the detoxification or chemoprotection of carcinogens, and induction of this enzyme is a target for the prevention of carcinogenesis. Natural stilbenoids have potential cancer chemopreventive activities, potentially through affecting NQO1 activity. Along this line, several stilbenoids were evaluated to procure more potent compounds for inducing NQO1 activity in cultured murine Hepa 1c1c7 cells. As a result, we found that 3,5-dimethoxy-trans-stilbene (DMS) possesses potent NQO1 induction activity through up-regulation of both protein and mRNA expression of NQO1 as determined by Western blot and reverse transcription–polymerase chain reaction analysis, respectively. DMS also increased protein expression of heme oxygenase-1 (HO-1), another phase II enzyme. This induction of NQO1 and HO-1 by DMS was closely related to the regulation of nuclear factor E2–related factor 2 (Nrf2). The translocation and activation of Nrf2 by DMS was also involved in the modulation of the upstream signal transduction molecule, protein kinase C δ. These findings suggest that DMS might have a cancer chemopreventive activity by inducing detoxifying enzymes such as NQO1 and HO-1.

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In the present study, we evaluated the antioxidant effects of a pepsin-treated novel collagen peptide (P-NCP) on reactive oxygen species (ROS) such as hydroxyl radical (HO^•), superoxide anion radical (O₂^•–), and singlet oxygen (¹O₂), and the effects on cell viability after ultraviolet ray (UV) irradiation of human fibroblasts. We confirmed, using electron spin resonance, that P-NCP directly inhibited HO^• and ¹O₂. Furthermore, addition of P-NCP to fibroblasts inhibited cell death induced by UVA (400 – 315 nm) irradiation in a dose-dependent manner. In addition, the antioxidant effect on ¹O₂ was observed in the peptide fractions rich in Gly, Pro, Hyp, Glu, Ala, and Arg. We found that Gly, Hyp, Glu, and Ala directly scavenged ¹O₂. These results indicated that a peptide sequence including Gly, Hyp, Glu, and Ala could play a key role in the antioxidant effects of P-NCP on ¹O₂. It was suggested that P-NCP can inhibit photo-aging related to ROS owing to its antioxidant effects.

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Hepatic ATP-binding cassette transporter A1 (ABCA1) plays a key role in high-density lipoprotein (HDL) production by apolipoprotein A-I (ApoA-I) lipidation. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, increase ABCA1 mRNA levels in hepatoma cell lines, but their mechanism of action is not yet clear. We investigated how statins increase ABCA1 in rat hepatoma McARH7777 cells. Pitavastatin, atorvastatin, and simvastatin increased total ABCA1 mRNA levels, whereas pravastatin had no effect. Pitavastatin also increased ABCA1 protein. Hepatic ABCA1 expression in rats is regulated by both liver X receptor (LXR) and sterol regulatory element–binding protein (SREBP2) pathways. Pitavastatin repressed peripheral type ABCA1 mRNA levels and its LXR-driven promoter, but activated the liver-type SREBP-driven promoter, and eventually increased total ABCA1 mRNA expression. Furthermore, pitavastatin increased peroxisome proliferator–activated receptor α (PPARα) and its downstream gene expression. Knockdown of PPARα attenuated the increase in ABCA1 protein, indicating that pitavastatin increased ABCA1 protein via PPARα activation, although it repressed LXR activation. Furthermore, the degradation of ABCA1 protein was retarded in pitavastatin-treated cells. These data suggest that pitavastatin increases ABCA1 protein expression by dual mechanisms: SREBP2-mediated mRNA transcription and PPARα-mediated ABCA1 protein stabilization, but not by the PPAR–LXR–ABCA1 pathway.
[Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10241FP]

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Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135–143). Myosin ATPase activity measurements indicate that the 26 – 41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not β-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells.

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To elucidate the role of Na⁺/Ca²⁺ exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3′,4′-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho–ROCK signal transduction pathways in neurite outgrowth.

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The chronic atrioventricular block (CAVB) dog has been widely used as an in vivo proarrhythmia model. mRNA levels of K⁺ and Ca²⁺ channels in the isolated ventricular tissues from normal and CAVB dogs were assayed using a real-time PCR. The mRNA levels of KvLQT1 and MiRP1 were significantly less in the CAVB heart compared with those in the intact heart, whereas no significant difference was detected in the mRNA levels of other K⁺- or Ca²⁺-channel subunits. Adaptation against chronic bradycardia–related pathophysiology may have decreased the mRNA levels of cardiac K⁺ channels, which may partly explain the arrhythmogenic property of this model.

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