Heart failure is a major cause of death in developed countries, and the development of an epoch-making cure is desired from the viewpoint for improving the quality of life and reducing the medical cost of the patient. The importance of neurohumoral factors, such as angiotensin (Ang) II and catecholamine, for the progression of heart failure has been supported by a variety of evidence. These agonists stimulate seven transmembrane-spanning receptors that are coupled to heterotrimeric GTP-binding proteins (G proteins). Using specific pharmacological tools to assess the involvement of G protein signaling pathways, we have revealed that α subunit of G_q (Gα_q) activates Ca²⁺-dependent hypertrophic signaling through diacylglycerol-activated transient receptor potential canonical (TRPC) channels (TRPC3 and TRPC6: TRPC3/6). In contrast, activation of Gα₁₂ family proteins in cardiomyocytes confers pressure overload–induced cardiac fibrosis via stimulation of purinergic P2Y₆ receptors induced by extracellular nucleotides released from cardiomyocytes. In fact, direct or indirect inhibition of TRPC3/6 or P2Y₆ receptors attenuates pressure overload–induced cardiac dysfunction. These findings will provide a new insight into the molecular mechanisms underlying pathogenesis of heart failure.

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In the brain of Alzheimer’s disease patients, down-regulation of both cholinergic and glutamatergic systems have been found and is thought to play an important role in impairment of cognition, learning, and memory. Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as a cognitive-enhancing effect. The present study was undertaken to elucidate mechanisms underlying the action of nefiracetam on glutamatergic receptors and intracellular protein kinases. N-Methyl-D-aspartate (NMDA)-evoked currents were recorded from rat cortical neurons in long-term cultured primary neurons using the whole-cell patch-clamp technique. NMDA-evoked currents were greatly and reversibly potentiated by bath application of nefiracetam, resulting in a bell-shaped dose–response curve. The maximum potentiation of 170% relative to the control was produced at 10 nM. Treatment with an inhibitor of the glycine binding site of the NMDA receptor, 7-chlorokynurenic acid, at 1 μM prevented augmentation of NMDA-evoked currents by nefiracetam. In rat hippocampal CA1 slices, field excitatory postsynaptic potentials were recorded by stimulation of Schaffer collateral/commissural pathways. Nefiracetam treatment significantly enhanced long-term potentiation (LTP) with the same bell-shaped dose–response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with calcium/calmodulin-dependent protein kinase II (CaMKII) activation with concomitant increase in phosphorylation of AMPA-type glutamate receptor subunit 1 (GluA1) (Ser-831) as a postsynaptic CaMKII substrate. In conclusion, nefiracetam enhances NMDA-receptor function through stimulation of its glycine binding site and nefiracetam-induced CaMKII activation likely contributes to improvement of cognition, learning, and memory.

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We aimed to examine the rate of thrombotic events after discontinuation of one year clopidogrel therapy in patients with implanted coronary stent, and to determine platelet aggregability by multiple electrode analyzer after cessation of clopidogrel. This prospective, multicenter study enrolled 200 patients subjected to coronary stent implantation and treated with aspirin + clopidogrel one year after the stent placement. Platelet aggregation was measured using 3 agonists [adenosine diphosphate with PGE₁ (ADPHS), arachidonic-acid (ASPI), and thrombin receptor activating peptide (TRAP)] on the day of cessation of clopidogrel and at 10, 45, and 90 days after clopidogrel was stopped. Two thrombotic events were registered during the 6-months follow up (one ischemic stroke and one myocardial infarction; incidence of 1%). The mean values of ADP + PGE₁- and ASPI-induced aggregation 10 – 90 days after the cessation of clopidogrel were significantly higher than values obtained before the termination of the drug (P < 0.001, all). Cessation of clopidogrel did not influence the TRAP-induced aggregation, which reached the plateau in all measurements. In conclusion, the incidence of thrombotic events after the cessation of one-year clopidogrel treatment might be lower than expected in patients with implanted coronary stent.

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The thienopyridine derivative ticlopidine (TCL) is an inhibitor of adenosine diphosphate-induced platelet aggregation. Combination therapy with a thienopyridine derivative and aspirin is standard after coronary stenting, although more hemorrhagic complications occur with the combination therapy than with aspirin alone. A proton pump inhibitor (PPI) is required for prevention or treatment of upper gastrointestinal bleeding in such cases. We examined the effects of PPIs [omeprazole (OPZ) and rabeprazole (RPZ)] on TCL metabolism using pooled human liver microsomes prepared from various human liver blocks and 12 individual human liver microsomes. We calculated the K_i values of each PPI for TCL metabolic activity and compared the inhibitory effect of each PPI on TCL metabolism. The K_i values of OPZ and RPZ were 1.4 and 12.7 μM, respectively. The inhibitory effect of OPZ (78.6 ± 0.05%) was significantly greater than that of RPZ (24.2 ± 0.05%) (P < 0.001). Interestingly, a negative correlation existed between the inhibitory effect of OPZ and CYP2C19 activity (r = −0.909, P < 0.001). These results suggest that the inhibitory effect of OPZ is more potent than that of RPZ in vitro. In conclusion, RPZ appears preferable when administering TCL, aspirin, and a PPI in combination.

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A better understanding of the neurochemical mechanisms mediating the aversive consequences of drug withdrawal is important for understanding drug addiction. We previously demonstrated that the inhibitory effect of glutamate receptor antagonists on the conditioned place aversion (CPA) induced by naloxone-precipitated withdrawal after a single morphine exposure could be blocked by dopamine receptor antagonists. Thus, a glutamatergic–dopaminergic interaction may participate in this phenomenon. The current study was undertaken to further characterize this interaction by employing both D₁ (SCH 23390) and D₂ (raclopride and eticlopride) dopamine receptor antagonists. The influence of these antagonists on the attenuation of CPA by MK-801 (NMDA receptor antagonist), GYKI 52466 (AMPA receptor antagonist), and MCPG (metabotropic glutamate receptor antagonist) was determined in rats receiving a single dose of morphine. The dopamine antagonists showed either a significant reversal or a tendency to reverse the effects of MK-801 on CPA. The effect of GYKI 52466 was also attenuated by the blockade of either D₁ or D₂ receptors. The effect of MCPG, however, was only blocked by D₂ antagonists and not by the D₁ antagonist SCH 23390. These results add evidence to the hypothesis that a glutamatergic–dopaminergic interaction may be involved in the CPA induced by naloxone-precipitated withdrawal following a single morphine exposure and suggest that both D₁ and D₂ dopamine receptor signaling mechanisms play a role in mediating the aversive aspects of acute dependence.

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Sneezing and persistent itching of the nasal mucosa are distressing symptoms of allergic rhinitis (AR). Recent studies have revealed that hyperinnervation of sensory neurons in the nasal turbinate is one of the underlying causes of sneezing and itching. Since Semaphorin-3A (Sema3A) has been previously shown to restrict innervation of sensory neurons, it is presumed that reduced Sema3A expression in the nasal mucosa might contribute to the hypersensitivity. Analysis of the mouse model of ovalbumin-sensitized AR demonstrated a decreased expression of Sema3A in the nasal epithelium, which was accompanied by an increased nerve fiber density in the lamina propria of the turbinate. In rescue experiments, intranasal administration of recombinant Sema3A in the AR model mice alleviated sneezing and nasal rubbing symptoms. In addition, histological examinations also revealed that nerve fiber density was decreased in the lamina propria of the Sema3A-treated nasal turbinate. These results suggest that the nasal hypersensitivity of AR may be attributed to reduction of Sema3A expression and intranasal administration of Sema3A may provide a novel approach to alleviate the allergic symptoms for AR treatment.

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Ecto-5′-nucleotidase (NT5E), a predominant enzyme that produces extracellular adenosine from AMP, plays an important role in a variety of physiological and pathophysiological processes. This study was performed to identify agents that affect NT5E activity using C6 glioma cells. When cells were incubated with sodium nitroprusside (SNP), phorbol 12-myristate 13-acetate, forskolin, lipopolysaccharide, or interferon-γ, only SNP inhibited NT5E activity in a time- and concentration-dependent manner (IC₅₀ = 1.2 μM). The inhibitory effect of SNP was long-lasting even after SNP washout; and its action was not mimicked by nitric oxide generating agents, 8-bromo cyclic GMP, ferricyanide, ferrocyanide, or sodium cyanide. SNP did not change NT5E mRNA level or membrane surface protein expression. Similar to SNP, Fe²⁺ inhibited NT5E activity, but to a lesser extent. Although Fe²⁺ is known to increase oxidative stress, Fe²⁺-mediated oxidative stress was not involved in SNP inhibition of NT5E because the inhibition of NT5E by SNP was not affected by superoxide dismutase and catalase. In contrast, addition of Zn²⁺, an essential metal co-factor of NT5E activity, prevented SNP from inhibiting NT5E. These results suggest that SNP disrupts a critical Zn²⁺-dependent enzyme activity and might be useful as a pharmacological tool for inhibiting NT5E.

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Chlorpromazine (CPZ), the first widely used phenothiazine tranquilizer, is shown to inhibit the action of intracellular calmodulin (CaM) and bone resorption in vivo and in vitro. In this study, CPZ (0.63 – 10 μM) dose-dependently inhibited the formation of tartrate-resistant acid phosphatase (TRAP) staining–positive osteoclast-like cells in mouse bone marrow cells (BMCs) treated with 1α,25(OH)₂D₃ (10 nM) or soluble receptor activator of nuclear factor-κB ligand (s-RANKL) (20 ng/ml). Expressions of mRNA for the nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation; dendritic cell-specific transmembrane protein (DC-STAMP), an essential protein for cell–cell fusion; and characteristic markers of osteoclasts such as TRAP, cathepsin K, carbonic anhydrase II, and calcitonin receptor in BMCs were up-regulated by s-RANKL and decreased by the addition of CPZ (5 μM) or the selective CaM antagonist W7, but not the inactive analog W5. The general CaM kinase (CaMK) inhibitor KN-93 and CaM-dependent phosphatase calcineurin inhibitor FK-506 also inhibited s-RANKL–induced osteoclastogenesis. Phenothiazines such as CPZ, trifluoperazine (TFPZ), and promethazine (PMZ) inhibited s-RANKL–induced osteoclast-like cell formation in mouse BMCs. Osteoclastogenesis inhibitory effects decreased in the order of TFPZ, CPZ, PMZ, depending on their anti-CaM potency. These findings suggest that CPZ inhibits RANKL-induced osteoclastogenesis by its anti-CaM action.

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Trypsin-treated β-lactoglobulin significantly decreased the glucose level after an oral glucose tolerance test using mice. We performed the present study to identify the active peptide inhibiting dipeptidyl peptidase-4 from trypsin-treated β-lactoglobulin. Trypsin-treated β-lactoglobulin showed a concentration-dependent inhibition for dipeptidyl peptidase-4, with an IC₅₀ value of 210 μM, although non-treated β-lactoglobulin showed no significant effect in the in vitro assay. The active peptide was isolated from trypsin-treated β-lactoglobulin and identified as the hexapeptide Val-Ala-Gly-Thr-Trp-Tyr (β-lactoglobulin f15-20). This hexapeptide also exhibited a concentration-dependent inhibitory effect and IC₅₀ value was 174 μM, suggesting that this hexapeptide is almost totally responsible for the DPP-4 inhibitory activity of trypsin-treated β-lactoglobulin.

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T-817MA (1-{3-[2-(1-benzothiophen-5-yl)ethoxy]propyl}azetidin-3-ol maleate), a neurotrophic compound newly synthesized for the treatment of Alzheimer’s disease, has been found to reduce oxidative stress and exert neuroprotective effects. By assessing the auditory functioning and observing the cochlear sensory epithelia, we investigated whether T-817MA protects the cochlea from inner ear barotrauma in guinea pigs treated with rapidly intense pressure change. Sustained oral administration of T-817MA significantly reduced the extent of auditory threshold shifts and outer hair cell loss, indicating that T-817MA attenuates the intense pressure–induced cochlear damage that accompanies inner ear barotrauma via antioxidative activity.

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