Nitric oxide (NO) is produced in almost all tissues and organs, exerting multiple biological actions under both physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS): neuronal, inducible, and endothelial NOSs. Due to the substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. To address this point, we have successfully developed mice in which all three NOS genes are completely disrupted. NOS expression and activities were totally absent in the triply n/i/eNOS^−/− mice before and after treatment with lipopolysaccharide. While the triply n/i/eNOS^−/− mice were viable, their survival and fertility rates were markedly reduced as compared with wild-type mice. The phenotypes of those mice that we first noticed were polyuria, polydipsia, and renal unresponsiveness to vasopressin, characteristics consistent with nephrogenic diabetes insipidus. We subsequently observed that in those mice, arteriosclerosis is spontaneously developed with a clustering of cardiovascular risk factors. These results provide the first evidence that the systemic deletion of all three NOSs causes a variety of cardiovascular diseases in mice, demonstrating a critical role of the endogenous NOSs system in maintaining cardiovascular homeostasis.

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The aim of the present article is to review the cardioprotective properties of cannabinoids, with an emphasis on the signaling pathways involved. Cannabinoids have been reported to protect against ischemia in rat isolated hearts, as well as in rats and mice in vivo. Although these effects have been observed mostly with a pre-treatment of a cannabinoid, we report that the selective CB₂-receptor agonist JWH133 is able to reduce infarct size when administered either before ischemia, during the entire ischemic period, or just upon reperfusion. Little is known about the signaling pathways involved in these cardioprotective effects. Likely candidates include protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) since they are activated during ischemia-reperfusion and contribute to the protective effect ischemic preconditioning. The use of pharmacological inhibitors suggests that PKC, p38 MAPK, and p42/p44 MAPK (ERK1/2) contribute to the protective effect of cannabinoids. In addition, perfusion with JWH133 in healthy hearts caused an increase in both p38 MAPK phosphorylation level and activity, whereas the CB₁-receptor agonist ACEA was associated with an increase in the phosphorylation status of both ERK1 and ERK2 without any change in activity. During ischemia, both agonists doubled p38 MAPK activity, whereas ERK1/2 phosphorylation level and activity during reperfusion were enhanced only by the CB₁-receptor agonist. Finally, although nitric oxide (NO) was shown to exert both pro and anti-apoptotic effects on cardiomyocytes, with an apparently controversial effect on myocardial survival, our data suggest that NO may contribute to the cardioprotective effect of some cannabinoids.

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We obtained a new mutant of the β₁-adrenergic receptor (β₁-AR) by point mutations that can constitutively activate β₁-AR. Aspartate104 of the β₁-AR in the 2nd transmembrane was replaced with alanine. The β₁-AR mutant expressed in human embryonic kidney (HEK)-293 cells displayed high level of constitutive activity with respect to wild-type (P<0.05), which could be partially inhibited by some beta-blockers. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the β₁-AR as well as the mechanism of action of beta-blockers.

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To elucidate the involvement of cyclooxygenase (COX) in degradation of aggregated protein in diabetic glomeruli, we used streptozotocin (STZ)-induced diabetic mice and aggregated bovine serum albumin (a-BSA) as a model protein. There was a higher deposition of a-BSA in diabetic glomeruli compared to normal glomeruli 18 h after a-BSA injection at 4 and 8 weeks after STZ. Degradation of a-BSA was confirmed using isolated glomeruli. Diabetic glomeruli produced prostaglandin E₂ (PGE₂) more than normal glomeruli in the basal level at 8 weeks. a-BSA caused further increase of PGE₂ production in normal glomeruli, but not in diabetic glomeruli. Niflimic acid, a selective COX-2 inhibitor, reduced PGE₂ production of normal glomeruli in the a-BSA loading group, but not that in the control group. In diabetic glomeruli, niflimic acid reduced PGE₂ production in both the control group and a-BSA loading group. In normal glomeruli, a-BSA increased expressions of both COX-2 mRNA and protein. However, in diabetic glomeruli, a-BSA increased COX-2 mRNA expression but not COX-2 protein expression. These results suggest that retarded degradation of aggregated protein in diabetic glomeruli is associated with lack of further expression of COX-2 protein and further production of PGE₂ in response to aggregated protein.

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Prostaglandin E₂-receptor subtypes, EP₁, EP₂, EP₃, and EP₄, are present in the kidney. The aim of this study was to elucidate the anti-nephritic effect of an EP₄-receptor agonist on an experimental nephritic model. Mice were injected i.v. with anti-glomerulus antiserum to induce nephritis. Nephritic glomeruli generated more prostaglandin E₂ (2.6 and 0.7 ng) and less cyclic AMP than normal glomeruli (11 and 26 pmol). The production of cyclic AMP in nephritic glomeruli increased 67% in response to AE1-329, an EP₄ agonist, at 10⁻⁵ M. Nephritic glomeruli expressed a lesser amount of mRNA of prostaglandin E₂-receptor subtypes as compared with normal glomeruli. AE1-329 was administered s.c. at 100 μg/kg per day for 3 weeks. AE1-329 suppressed the increase in creatinine and cholesterol compared to those in the control nephritic mice. AE1-329-treated nephritic mice had less crescentic glomeruli and less deposition of rabbit IgG (anti-glomerular basement membrane antibody) in glomeruli than the control mice. AE1-329 prevented the development of glomerulonephritis. These findings suggest that EP₄-receptor agonists are a promising drug to prevent the development of glomerulonephritis.

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Mutations producing constitutively active G-protein coupled receptors have been found in the pathophysiology of several diseases, implying that inverse agonists at the constitutively active receptors may have preferred therapeutic applications. Because of the involvement of 5-HT_2A receptors in mediating many cardiovascular diseases, constitutively active mutants of the 5-HT_2A receptor may be responsible for the disease states. Thus, the purpose of the present study was to investigate the inverse agonist activity of sarpogrelate, a selective 5-HT_2A-receptor antagonist, and its active metabolite, M-1; and we compared their activities with those of other 5-HT_2A-receptor antagonists such as ritanserin, ketanserin, and cyproheptadine. Using a constitutively active mutant (C322K) of the human 5-HT_2A receptor, we demonstrated that like other 5-HT_2A-receptor antagonists, sarpogrelate acts as a potent inverse agonist by significantly reducing basal inositol phosphate levels. However, there were no significant differences between sarpogrelate and other 5-HT_2A-receptor antagonists for their inverse agonist activity. Compared with the wild type receptor, mutant receptor displayed significantly higher affinity for 5-HT and lower affinity for sarpogrelate. These results indicate that stabilization of the inactive conformation of the 5-HT_2A receptor may be a key component of the mechanism of action of sarpogrelate.

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Lig-8, a lignophenol derivative from bamboo lignin, potently suppresses oxidative stress-induced apoptosis. Here, we first examined in vitro whether lig-8 protects against neuronal damage induced by oxygen-glucose deprivation (OGD) followed by reoxygenation, tunicamycin [endoplasmic reticulum (ER)-stress inducer], or PSI (proteasome inhibitor). In pheochromocytoma (PC12) cell cultures, lig-8 (1 to 30 μM) concentration-dependently inhibited OGD- and tunicamycin (2 μg/ml)-induced cell deaths (significant at ≥3 μM and ≥1 μM, respectively). In human neuroblastoma (SH-SY5Y) cell culture, the PSI-induced apoptotic cell death and fusion protein accumulation (revealing reduced proteasome activity) was inhibited by lig-8 (30 μM). On the other hand, lig-8 at 30 μM alone did not affect any proteasome activity under resting conditions. In vivo, lig-8 (0.1 nmol/eye) reduced intravitreal N-methyl-D-aspartate (NMDA, 20 nmol)-induced retinal damage (decreases in retinal ganglion cells and inner plexiform layer thickness). Hence, lig-8 protects, partly by inhibiting excessive ER-stress, against neuronal damage in vitro and in vivo.

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We investigated the effects of 4-(N-{1-[2-(4-cyanophenyl)ethyl]-4-hydroxypiperidin-4-ylmethyl}-N-methylamino)benzoic acid monohydrochloride (M58996), a novel analgesic, on persistent and neuropathic pain in rats. In the formalin test, oral M58996 (0.3 – 10 mg/kg) reduced nociceptive behaviors only in the late phase. In the neuropathic pain model, oral M58996 (1 – 10 mg/kg) attenuated mechanical allodynia and heat hyperalgesia in the nerve-injured paw without affecting normal responses of the uninjured paw. High doses (10 – 100 mg/kg) of oral M58996 did not influence normal motor function. Thus, M58996 had a wide dose range showing antinociceptive, antiallodynic, and antihyperalgesic effects without motor dysfunction. In addition, we studied the possible mechanisms involved in the M58996-induced antinociception. The antinociceptive effect of M58996 was reversed by intrathecal pertussis toxin, an inhibitor of the inhibitory- and other-GTP-binding protein (G_i/o protein), but not by subcutaneous naloxone, an opioid-receptor antagonist. This effect was also reversed by intracerebroventricular or intrathecal tropisetron, a 5-hydroxytryptamine₃ (5-HT₃)-receptor antagonist, and intraperitoneal bicuculline, a γ-aminobutyric acid_A (GABA_A)-receptor antagonist. These results suggest that M58996 produces its antinociceptive effect by a pertussis toxin-sensitive G protein mechanism. In addition, the GABA released by the activation of supraspinal and/or spinal 5-HT₃ receptors is likely to contribute to the M58996-induced antinociception.

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Antipsychotic drugs induce weight gain and metabolic abnormalities. Recently, the role of adipocytokines secreted from adipocytes in the development of metabolic syndrome has received attention. The aim of this study was to investigate the effects of chlorpromazine (Cp) on body weight, glucose, lipid metabolism, and adipocytokine secretion in rats fed sucrose. Wistar rats received 15% sucrose (Suc group), 15% sucrose and Cp at 7.5 mg/kg per day (Suc + Cp group), or Cp alone (Cp group) in water for 10 weeks. Fasting glucose levels in the Suc and Suc + Cp groups were significantly higher than in the control (Cont) group. Fasting insulin levels in the Suc, Suc + Cp, and Cp groups were also significantly higher than in the Cont group. The adiponectin level in the Suc group was significantly higher than in the Cont group, although the adiponectin level in the Suc + Cp group was not. Furthermore, the plasma tumor necrosis factor (TNF)-α level in the Suc + Cp group was significantly higher than in the Suc group. These data suggest that Cp inhibits the compensatory response of adiponectin with respect to obesity due to increased expression of plasma TNF-α level. Cp may exert more harmful effects on the glucose level and insulin resistance than on other factors, which may be one of the mechanisms responsible for the metabolic syndrome induced by antipsychotic agents.

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Previous reports revealed up-regulation of L-type high voltage-gated calcium channels (HVCCs) in mouse brains with ethanol physical dependence. We investigated mechanisms of enhancement of L-type HVCC function using mouse cerebrocortical neurons exposed to 50 mM ethanol for 3 days and the brains of mouse physically dependent on ethanol. Ethanol facilitated 30 mM KCl-stimulated ⁴⁵Ca²⁺ influx in dose- and duration-dependent manners, which was abolished by nifedipine, an inhibitor specific to L-type HVCCs, but not by inhibitors for other types of HVCCs. Increase in [³H]PN200-110 binding to the particulate fractions from the ethanol-treated neurons was due to increased B_max value with no changes in K_d value. Western blot analysis showed the increased expression of α1C, α1D, and α2/δ1 subunits with decreased β4 subunit expression and no changes in expressions of α1A, α1B, α1F, and α2 subunits. A similar pattern of the changes in the expression of these subunits of L-type HVCCs were observed in the cerebral cortex from mouse with ethanol physical dependence. These results indicate that sustained ethanol exposure to the neurons induces up-regulation of L-type HVCCs, which is due to increased expressions of α1C, α1D, and α2/δ1 subunits, and produces no alterations in P/Q- and N-type HVCC functions.

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Soluble guanylyl cyclase (sGC) is a target enzyme for endogenous nitric oxide (NO), and it converts GTP to cyclic GMP (guanosine 3',5'-cyclic monophosphate) as part of a cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, and inhibition of platelet aggregation. Here we examine a representative of the novel class sCG activators, A-778935 ((±)-cis-3-[2-(2,2-dimethyl-propylsulfanyl)-pyridin-3-yl]-N-(3-hydroxy-cyclohexyl)-acrylamide). A-778935 activated sGC synergistically with sodium nitroprusside (SNP) over a wide range of concentration, inducing up to 420-fold activation. A specific inhibitor of sGC, ODQ (1H-[1,2,4]-oxadiazolo[4,3-α]quinoxalin-1-one), did not block basal sGC activity, but competitively inhibited the activation by A-778935. A-778935, with or without SNP, did not activate heme-deficient sGC, indicating that the activation of sGC by A-778935 is fully heme-dependent. A-778935 increased intracellular cGMP level dose-dependently in smooth muscle cells. In the presence of 1 μM SNP, a lower concentration of A-778935 increased cGMP than A-778935 alone, and the cGMP concentration reached the same level at 100 μM of A-778935. A-778935 relaxed cavernosum tissue strips in a dose-dependent manner; and in the presence of 1 μM SNP, A-778935 relaxed the strips more potently, shifting the dose-response curve to the left. This novel activator of sGC may have potential efficacy for the treatment of a variety of disorders associated with reduced NO signaling.

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Effects of a new lipophilic L-type calcium channel blocker, azelnidipine, on the expression of molecular components of the renin-angiotensin-aldosterone system (RAAS) were assessed. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of diabetes with hypertension, and their lean littermates, Long-Evans Tokushima Otsuka (LETO) rats, were treated with azelnidipine for 2 weeks. The renal cortical mineralocorticoid receptor mRNA in OLETF was higher than in LETO, but was suppressed (P<0.05) by azelnidipine. Renal cortical angiotensin-converting enzyme mRNA of OLETF was lower than that of LETO rats, and it was further suppressed by azelnidipine (P<0.05). Azelnidipine can down-regulate the gene expression of molecular components of RAAS.

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DJ-1 has multiple functions and its dysfunction may be linked to the onset of familial Parkinson’s disease PARK7. However, the function and distribution of DJ-1 is unclear. In this study, we determined DJ-1 distribution and change after intranigral injection of 6-hydroxydopamine (6-OHDA). Although distribution of DJ-1 immunoreactivity was not changed in cerebral cortex and striatum, 6-OHDA caused increase of DJ-1 in the particulate fraction and decrease in the cytosolic fraction in substantia nigra. At that time, DJ-1 shifted to acid forms. These results suggest that distributional changes, translocation, and acidic shift of DJ-1 may be compensatory responses to protect against 6-OHDA-induced oxidative stress.

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The effects of 2,4,4-trimethyl-3-(15-hydroxypentadecyl)-2-cyclohexen-1-one (tCFA15) on diabetic hypoalgesia and neuropathic hyperalgesia were examined. Treatments of streptozotocin (STZ)-pretreated mice with tCFA15 (8 – 40 mg/kg, i.p.) for 7 days significantly reversed the depressed inflammatory nociceptive licking response in the formalin test. In addition, similar drug treatments and dosing in 7-day postoperative neuropathic pain model rats (prepared by the method of Bennett and Xie) yielded a similarly favorable outcome by significantly reversing decreased nociceptive thresholds in the paw pressure test. These results suggest that tCFA15 may have the potential to normalize sensory nerve abnormalities induced in experimental diabetes and nerve injury.

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