The waveform of the myocardial action potential (AP) triggering contraction differs among the species, developmental stage, and pathological state. The species difference in heart rate, which inversely correlates with body size, originates in the ion-channel mechanisms responsible for diastolic depolarization of the sinoatrial node. In some cases, such as the chronically AV-blocked dog and 11- to 13-day chick embryo, the repolarization reserve is decreased making the heart useful for drug evaluation. The degree of dependence of contraction on sarcoplasmic reticulum (SR) function increases during development. The large SR dependence and short AP of the adult mouse and rat support their rapid contraction under high heart rate. The function of the Na⁺/Ca²⁺ exchanger is affected by AP waveform and ion concentrations; its major role is Ca²⁺ extrusion, but under pathological conditions such as ischemia-reperfusion, it allows Ca²⁺ influx and leads to myocardial injury, including loss of mitochondrial function. The role of mitochondria in ATP supply is less in the fetus where glycolysis plays a greater role. The pharmacological properties of the myocardium are affected by all of these factors and also by autonomic innervation and the hormonal status. Such comprehensive understanding is indispensable for the development of novel therapeutic strategies.

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Regulation of cardiac ion channels by sex hormones accounts for gender differences in susceptibility to arrhythmias associated with QT prolongation (TdP). Women are more prone to develop TdP than men with either congenital or acquired long-QT syndrome. The risk of drug-induced TdP varies during the menstrual cycle, suggesting that dynamic changes in levels of ovarian steroids, estradiol and progesterone, have cyclical effects on cardiac repolarization. Although increasing evidence suggests that the mechanism of this involves effects of female hormones on cardiac repolarization, it has not been completely clarified. In addition to well-characterized transcriptional regulation of cardiac ion channels and their modifiers through nuclear hormone receptors, we recently reported that physiological levels of female hormones modify functions of cardiac ion channels in mammalian hearts. In this review, we introduce our recent findings showing that physiological levels of the two ovarian steroids have opposite effects on cardiac repolarization. These findings may explain the dynamic changes in risk of arrhythmia in women during the menstrual cycle and around delivery, and they provide clues to avoiding potentially lethal arrhythmias associated with QT prolongation.

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Mitochondrial ATP-sensitive K⁺ (mitoK_ATP) and Ca²⁺-activated K⁺ (mitoK_Ca) channels exist in cardiac myocytes, and they play key roles in cardioprotection. We have recently reported that K⁺ influx through mitoK_ATP or mitoK_Ca channels occurs independently of each other and confers cardioprotection in a similar manner. Activation of mitoK_ATP channel is augmented by protein kinase C (PKC), whereas mitoK_Ca channel is activated by protein kinase A (PKA). However, phosphatidylinositol 3-kinase (PI3-K) is linked to neither mitoK_ATP nor mitoK_Ca channels. We have demonstrated that bioactive substances modulate the opening of mitoK_ATP channels via a PKC-dependent pathway or opening of mitoK_Ca channels via a PKA-dependent pathway and thereby protecting the heart from ischemia/reperfusion injury. Several endogenous substances such as adenosine and bradykinin can reduce infarct size by activation of mitoK_ATP channels in a PKC-dependent manner. Adrenomedullin, a potent vasodilator peptide, potentiates the opening of mitoK_Ca channels by PKA activation. Treatment with adrenomedullin prior to ischemia results in the reduction of infarct size via a PKA-mediated activation of mitoK_Ca channels. Thus, some endogenous substances confer cardioprotection via PKA- or PKC-mediated activation of mitoK_ATP or mitoK_Ca channels.

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The heart is abundantly innervated, and the nervous system precisely controls the function of this organ. The density of cardiac innervation is altered in diseased hearts, which can lead to unbalanced neural activation and lethal arrhythmia. For example, diabetic sensory neuropathy causes silent myocardial ischemia, characterized by loss of pain perception during myocardial ischemia, and it is a major cause of sudden cardiac death in diabetes mellitus. Despite the clinical importance of cardiac innervation, the mechanisms underlying the control of this process remain poorly understood. We demonstrate that cardiac innervation is determined by the balance between neural chemoattractants and chemorepellents within the heart. Nerve growth factor (NGF), a potent chemoattractant, is synthesized abundantly by cardiomyocytes, and is induced by the upregulation of endothelin-1 during development. By comparison, the neural chemorepellent Sema3a is expressed at high levels in the subendocardium in the early stage of embryogenesis and is downregulated as development progresses, leading to epicardial-to-endocardial transmural sympathetic innervation patterning. We also show that the downregulation of cardiac NGF is a cause of diabetic neuropathy and that NGF supplementation prevents silent myocardial ischemia in diabetes mellitus. Both Sema3a-targeted and Sema3a-overexpressing mice display sudden cardiac death or lethal arrhythmias due to disruption of innervation patterning. The present review focuses on the regulatory mechanisms controlling cardiac innervation and the critical roles of these processes in cardiac performance.

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Cyclic AMP (cAMP) is known to play a major role in regulating cardiac function. Difference in adenylyl cyclase (AC) isoforms is a potential mechanism by which the cAMP signal, a common second messenger signal, can be regulated in a tissue-specific manner. However, the physiological significance of expressing multiple AC isoforms in a tissue and how each specific isoform regulates the cAMP signal remains poorly understood. In a genetically engineered mouse model in which the expression of the type 5 AC is knocked out (AC5KO), we identified the attenuation of autonomic regulation and calcium-mediated inhibition of cardiac function. We also identified that disruption of type 5 AC preserves cardiac function in response to chronic pressure-overload and catecholamine stress, at least in part, through the inhibition of cardiac apoptosis, which plays a major role in the development of heart failure. The protection against both apoptosis and development of cardiac dysfunction induced by left ventricular pressure overload in AC5KO makes this molecule potentially important for developing future pharmacotherapy, where suppressing the activity of type 5 AC, and not the entire β-adrenergic signaling (β-AR) signaling pathway, may have an advantage over the current β-AR–blockade therapy in the treatment of heart failure.

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Despite of the huge socio-economic burden, stroke still represents an unmet therapeutic need. Researchers failed to reproduce preclinical efficacy in subsequent clinical development. To bridge this translation failure, the Stroke Therapy Academic Industry Round Table (STAIR) has suggested a rigorous, robust, and detailed preclinical evaluation in at least 2 species and multiple cerebral ischemia models to avoid the clinical failure. Considering these recommendations, in the present study, we have investigated the effects of pioglitazone in global model cerebral ischemic-reperfusion (IR) injury in gerbils. Global cerebral IR injury, produced by bilateral carotid artery occlusion for 5 min, was characterized by neurological deficits, hyperlocomotion, and neurodegeneration in the hippocampal CA1 region. Global ischemia was also associated with oxidative stress and DNA fragmentation as evident from increased malondialdehyde (MDA) levels and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive cells. Global cerebral IR injury associated neurological damage was significantly attenuated by pioglitazone pretreatment as evident from reduction in neurological symptoms, hyperlocomotion, and CA1 hippocampal neuronal damage in IR-challenged gerbils. Pioglitazone pretreatment also attenuated the oxidative stress and DNA fragmentation after cerebral IR injury. Pioglitazone post-treatment has also significantly reduced the CA1 hippocampal neuronal damage and DNA fragmentation after cerebral IR injury in IR-challenged gerbils. This study demonstrates the neuroprotective activity of pioglitazone in global cerebral IR injury and its neuroprotective effects may be attributed to reduction in oxidative stress and DNA fragmentation.

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The roots of Panax notoginseng (PN) are commonly used as a therapeutic agent to stop hemorrhage and as a tonic to promote health in traditional Korean medicine. Stroke triggers an inflammatory response that not only plays a central role in the pathogenesis of cerebral ischemia, but also induces secondary damage. This study was designed to investigate the neuroprotective effects of the methanol extract of PN on the infarct volume induced by middle cerebral artery occlusion (MCAO) (90-min occlusion and 24-h reperfusion) in rat brains. The PN extract (50 mg/kg, i.p.) was administered 2 h after the onset of MCAO. The PN-treated groups had a reduction in infarct volume by 23.82 ± 8.9%. In the PN extract–treated groups, the microglial density was significantly decreased in the peri-infarct region; the underlying mechanism was inhibition of inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, via blocking of the NF-κB pathway. Furthermore, in vitro studies showed that the PN extract significantly reduced the production of iNOS-derived NO and COX-2–derived prostaglandin E₂ through the regulation of gene transcription levels in primary microglia and BV-2 cells. These results suggest that anti-inflammatory and microglial activation inhibitory effects of the PN extract may contribute to its neuroprotective effects in brain ischemia.

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Polycyclic aromatic hydrocarbons (PAHs) and dioxins are ubiquitous environmental pollutants and activate the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. It has been reported that testosterone represses 2,3,7,8-tetrachlorodibenzo-p-dioxin–induced transcription of the cytochrome P450 (CYP) 1A1 gene in LNCaP cells. In this study, we investigated the mechanism for the repression of 3-methylcholanthrene (3MC)-induced transcription of AhR-regulated genes, CYP1A1, CYP1A2, CYP1B1, and AhR repressor (AhRR), by 5α-dihydroteststerone (DHT) in LNCaP and T47D cells, which are androgen receptor (AR)- and AhR-positive. Real-time PCR analysis showed that DHT repressed 3MC-induced mRNA expression of the CYP1 family and AhRR genes. DHT repressed 3MC-induced luciferase activity in an AhR response element–driven luciferase reporter assay in LNCaP and T47D cells. The inhibitory effect of DHT was abolished by knockdown of AR protein with siRNA. The protein levels of AhR and AhR nuclear translocator (Arnt), the AhR-dimerizing partner, were not affected by DHT. Co-immunoprecipitation assay showed that DHT significantly facilitated the complex formation between AR and AhR in 3MC-treated cells. These results suggest that complex formation between activated AR and AhR plays an important role in the suppression of 3MC-induced transcription of CYP1 family genes by DHT.

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This study investigated the involvement of tryptase and proteinase-activated receptor (PAR) subtypes in spontaneous scratching, an itch-associated behavior, in NC mice. This strain of mice showed chronic atopy-like dermatitis and severe spontaneous scratching, when kept a long time in a conventional environment. The trypsin-like serine proteinase inhibitor nafamostat mesilate (1 – 10 mg/kg) dose-dependently inhibited spontaneous scratching in mice with dermatitis. The activity of tryptase was increased in the lesional skin, which was inhibited by nafamostat at a dose inhibiting spontaneous scratching. Enzyme histochemistry revealed the marked increase of toluidine blue–stained cells, probably mast cells, with tryptase activity in the dermis of the lesional skin. Intravenous injection of anti-PAR₂ antibody suppressed spontaneous scratching of mice with dermatitis. Intradermal injection of the PAR₂-activating peptide SLIGRL-NH₂, but not PAR₁, ₃, ₄-activating peptides, elicited scratching at doses of 10 – 100 nmol/site in healthy mice. PAR₂-immunoreactivity was observed in the epidermal keratinocytes in healthy and dermatitis mice. These results suggest that PAR₂ and serine proteinase(s), mainly tryptase, are involved in the itch of chronic dermatitis.

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It has been demonstrated that treatment of hyperactive mice with psychostimulants produced a calming effect depending on serotonergic neurotransmission. Our previous study also showed that hyperactivity in mice lacking pituitary adenylate cyclase–activating polypeptide (PACAP) was ameliorated by amphetamine in a serotonin (5-HT)_1A-dependent manner and that amphetamine calmed wild-type mice given the 5-HT_1A agonist 8-OH-DPAT. Here, we examined if 5-HT_1A–mediated pathways can be a determinant of the action of other psychostimulants as well as the non-stimulant atomoxetine by examining locomotor activity in mice co-administered with the 5-HT_1A agonist osemozotan. Co-administration of osemozotan with either methamphetamine or amphetamine was not only antihyperkinetic, but also decreased locomotion to below basal levels. In contrast, osemozotan just nullified methylphenidate-induced hyperactivity. The non-stimulant atomoxetine did not induce hyperactivity, but co-administration of atomoxetine with osemozotan produced a calming effect. The adjunctive effect of osemozotan added to the psychostimulants was blocked by the 5-HT_1A antagonist WAY-100635 at a low dose (0.1 mg/kg), suggesting the involvement of a presynaptic 5-HT_1A–mediated mechanism. However, WAY-100635 (up to 1 mg/kg) did not block the effect of atomoxetine plus osemozotan. The present results may provide insights into the therapeutic mechanisms of the psychostimulants and atomoxetine for hyperkinetic disorders.

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We determined the action mechanism of cordycepin, a major bioactive component of Cordyceps militaris, on responses of rat aortic smooth muscle cells (RASMCs) and on vascular disorders, especially neointimal formation. Cordycepin inhibited platelet-derived growth factor-BB (PDGF-BB)-induced RASMCs migration and proliferation in a dose-dependent manner. However, pre-treatment with N^ω-nitro-L-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor, and 1,3-dipropyl-8-sulphophenylxanthine (DPSPX), an A₁/A₂ adenosine–receptor antagonist, abolished the inhibitory role of cordycepin. Cordycepin suppressed the phosphorylation of p38 mitogen–activated protein kinase (p38 MAPK) and heat shock protein 27 (Hsp27), but not that of extracellular signal-regulated kinase (ERK) 1/2 in RASMCs stimulated by PDGF-BB. The production of reactive oxygen species (ROS), O₂⁻ and H₂O₂, induced by PDGF-BB was abolished by the treatment of cordycepin. Moreover, the sprout outgrowth of aortic rings by PDGF-BB was inhibited by cordycepin. In vivo neointimal formation evoked by balloon-injury was significantly attenuated by the administration of cordycepin. These results demonstrate that cordycepin may exert inhibitory effects on PDGF-BB–induced migration and proliferation via interfering with adenosine receptor–mediated NOS pathways, thus resulting in the attenuation of neointima formation. In conclusion, cordycepin may be a potent, promising anti-atherosclerosis agent.

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Tacrolimus (FK506) has been used as a therapeutic drug beneficial for the treatment of rheumatoid arthritis in humans. In this study, we investigated the effects of FK506 on cellular differentiation in cultured chondrogenic cells. Culture with FK506 led to a significant and concentration-dependent increase in Alcian blue staining for matrix proteoglycan at 0.1 to 1,000 ng/ml, but not in alkaline phosphatase (ALP) activity, in ATDC5 cells, a mouse pre-chondrogenic cell line, cultured for 7 to 28 days, while the non-steroidal anti-inflammatory drug indomethacin significantly decreased Alcian blue staining in a concentration-dependent manner, without altering ALP activity. FK506 significantly increased the expression of mRNA for both type II and type X collagen, but not for osteopontin, in ATDC5 cells. Similar promotion was seen in chondrogenic differentiation in both mouse metatarsals and chondrocytes cultured with FK506. However, FK506 failed to significantly affect transcriptional activity of the reporter construct for either sry-type HMG box 9 (Sox9) or runt-related transcription factor-2 (Runx2), which are both transcription factors responsible for chondrocytic maturation as a master regulator. These results suggest that FK506 may predominantly promote cellular differentiation into proliferating chondrocytes through a mechanism not relevant to the transactivation by either Sox9 or Runx2 in chondrogenic cells.

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In the present study, we investigated the neuroprotective effects of Kangen-karyu (KGK) in a repeated cerebral ischemia model (2 × 10 min, 1-h interval). A 21-day pre- and post-ischemic treatment with KGK (10 – 300 mg/kg) and aspirin (5 mg/kg) improved the spatial memory impairment and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia. However, a 7-day post-ischemic treatment with KGK did not attenuate the spatial memory impairment and neuronal death in this model. To determine the mechanism of action of KGK, we investigated the effects of a 14-day pre-ischemic treatment with KGK on cerebral blood flow in the hippocampal area of the repeated cerebral ischemia model using laser Doppler flowmetry. The 14-day pre-ischemic treatment with KGK increased the cerebral blood flow during reperfusion. These results suggest that a 21-day pre- and post-ischemic treatment with KGK can protect against brain damage caused by cerebral ischemia by increasing the cerebral blood flow in the hippocampal area.

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Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type α cytosolic phospholipase A₂ (cPLA₂α)] and L929-cPLAα–siRNA cells (L929 cells treated with small interference RNA for cPLA₂α). Treatment with pharmacological inhibitors of secretory and Ca²⁺-independent PLA₂s decreased the DMB-mC11S–induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA₂ activity and its toxicity in cells.
[Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP]

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Ataxic movement, the common major symptom of spinocerebellar atrophy, has been considered to involve impaired glutamatergic excitatory neurotransmission in the cerebellum. Considering the therapeutic importance of ataxia control, we assessed the effectiveness of increasing the extracellular concentration of glycine by administering it exogenously or via blockade of glycine transporter 1, using its selective inhibitors sarcosine and N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS), for amelioration of motor ataxia in a mouse model of spinocerebellar atrophy developing after neonatal treatment with cytosine β-D-arabinofuranoside. Intracerebroventricular (i.c.v.) injection of sarcosine (3, 10, and 30 μg) and NFPS (0.01 and 0.03 μg) reduced the number of falls without affecting spontaneous motor activity, and therefore the falling index [(number of falls / spontaneous motor activity) × 100], and dose-dependently ameliorated ataxic movements. Similar effects were observed upon i.c.v. injection of D-serine (1 and 10 μg), an agonist of the glycine-recognition site of the N-methyl-D-aspartate (NMDA) receptor. However, exogenously injected glycine (1, 3, and 10 μg, i.c.v.) only weakly ameliorated the ataxic movements at 3 μg. These results suggest the therapeutic relevance of GlyT1 inhibitors for amelioration of motor ataxia in spinocerebellar atrophy by increasing the endogenous concentration of glycine near the glycine-recognition site of the NMDA receptor.

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The present study was undertaken to clarify the effects of anti-dementia drugs on sleep pattern in rats. Electrodes were chronically implanted into the frontal cortex and the dorsal neck muscle of rats for the electroencephalogram (EEG) and electromyogram (EMG), respectively. EEG and EMG were recorded with an electroencephalograph. SleepSigh ver. 2.0 was used for analysis of the sleep–wake state. Total times of waking, non-rapid eye movement (non-REM) sleep, and rapid eye movement (REM) sleep were measured from 10:30 to 16:30. Galantamine had no significant influence on the sleep pattern. On the other hand, donepezil and memantine showed significant increases in sleep latency and total waking time and a decrease in total non-REM sleep time. Furthermore, memantine decreased total REM sleep time. To investigate the characteristics of non-REM sleep in detail, non-REM sleep was classified as stage 1, 2, or 3 according to the depth of sleep. Different from donepezil and galantamine, memantine significantly decreased stage 1 and increased stage 3 in non-REM sleep. From these findings, it can be concluded that galantamine caused no sleep disturbance, different from donepezil and memantine.

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Ca²⁺ sensitizers are cardiotonic agents that directly increase the Ca²⁺ sensitivity of cardiac myofilament. To find a novel Ca²⁺ sensitizer, we have screened a group of phenolic compounds by examining their effects on the Ca²⁺-dependent force generation in cardiac muscle fibers. We found that propyl gallate, a strong antioxidant, increased the Ca²⁺ sensitivity of cardiac myofilament in a dose-dependent and reversible manner. The present study indicates that propyl gallate is a novel type of Ca²⁺ sensitizer with antioxidant activity, which might be more beneficial for the treatment of congestive heart failure associated with oxidative stress than existing Ca²⁺ sensitizers.

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A single dose by gavage of bilobalide (30 mg/kg) was found to produce a time-dependent induction of hepatic cytochrome P450 (CYP) enzyme activity and protein expression in rats. An RT-PCR study further showed that mRNA expression of CYP2B was maximal at 6 h. Plasma and liver bilobalide concentration in rats following administration of Ginkgo biloba extract equivalent to bilobalide of approximately 40 mg/kg showed a similar response to that exhibited by mRNA expression. These findings suggest that bilobalide markedly induced hepatic CYPs, but the induction could be mitigated due to rapid elimination from the liver.

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Although a loss-of-function type mutation was identified in familial Parkinson’s disease PARK7, the wild-type of DJ-1 is known to act as an oxidative stress sensor in neuronal cells. Recently, we found a DJ-1 modulator UCP0054278 by in silico virtual screening. In this study, we determined the neuroprotective effects of UCP0054278 against focal ischemia-induced neurodegeneration in rats. Hydrogen peroxide–induced cell death and the production of reactive oxygen species were significantly inhibited by UCP0054278 in normal SH-SY5Y cells, but not in DJ-1–knockdown cells. These results suggest that UCP0054278 interacts with endogenous DJ-1 and then exhibits antioxidant and neuroprotective responses.

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This study examined in mice whether limaprost, a prostaglandin E₁ analog, would relieve allodynia induced by chemotherapeutic agents. Single intraperitoneal injections of paclitaxel, oxaliplatin, and vincristine sulfate induced and gradually increased mechanical allodynia. Repeated administration of limaprost alfadex inhibited the late, but not early, phase of mechanical allodynia induced by paclitaxel and oxaliplatin, but not vincristine. Paclitaxel and oxaliplatin, but not vincristine, gradually decreased peripheral blood flow, which was prevented by limaprost. These results suggest that limaprost is effective against mechanical allodynia induced by paclitaxel and oxaliplatin, which may be due to inhibition of the decrease in peripheral blood flow.

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