Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-α regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm²) on TNF-α-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-α activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-α activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-α. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

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Mitogen-activated protein kinases (MAP kinases), including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38, play a central role in cellular responses by various stress stimuli such as cell proliferation, apoptosis, migration, or gene expression. Furthermore, activator protein-1 (AP-1), a transcription factor which can be activated by MAP kinases, also is involved in a variety of celllar responses, as well as MAP kinases. MAP kinases and AP-1 are significantly activated in vascular tissues by hypertension, angiotensin II, or balloon injury. We have made dominant negative mutants of MAP kinases or c-Jun, to specifically inhibit in vivo activation of MAP kinases or AP-1. Vascular gene transfer of each dominant negative mutant of MAP kinases or c-Jun prevents intimal hyperplasia after balloon injury, which is associated with the inhibition of smooth muscle cell proliferation in the intima and the media and probably also associated with inhibition of smooth muscle cell migration. However, in vitro findings on cultured vascular smooth muscle cells suggest that the molecular mechanism underlying inhibition of intimal hyperplasia may be different among each dominant negative mutant of MAP kinases and c-Jun. MAP kinases and c-Jun seem to be the promising therapeutic target for vascular remodeling.

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Endothelial dysfunction is associated with pathological vascular conditions including atherosclerosis, hypertension, and diabetes. The oxidatively modified form of low-density lipoprotein (LDL) is recognized as a major cause of endothelial dysfunction in atherogenesis. As the receptor for oxidized LDL in endothelial cells, we have identified the lectin-like oxidized LDL receptor-1 (LOX-1). LOX-1 is up-regulated by products of oxidative stresses and the molecules that induce oxidative stresses. Activation of LOX-1 induces the generation of reactive oxygen species and decreases NO released from endothelial cells. LOX-1 activation further induces the expression of endothelin-1, AT₁ receptor, and cell adhesion molecules. Together with these properties, LOX-1 works as an adhesion molecule for activated platelets and neutrophils. Thus, LOX-1, within the close relationships between oxidative stress generation and response, enhances functional changes in endothelial cells that are relevant to the disturbed vascular homeostasis under pathological settings.

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Although insulin-resistant states have been associated with endothelial dysfunction due to increased vascular oxidative stress, the underlying mechanisms are pooly understood. Recent experimental evidence suggests that tetrahydrobiopterin (BH₄), the natural and essential cofactor of NO synthases (NOS), plays a crucial role not only in increasing the rate of NO generation by NOS but also in controlling the formation of superoxide anion (O₂⁻) in endothelial cells. Because insulin resistance has been suggested to be a significant contributing factor in the development of abnormal pteridine metabolism and endothelial dysfunction, we investivated pteridine content and NO/O₂⁻ production with the use of isolated thoracic aortas obtained from fructose-induced insulin-resistant rats. Under insulin-resistant conditions where BH₄ levels are suboptimal, the production of O₂⁻ by NOS leads to endothelial dysfunction. Furthermore, oral supplementation of BH₄ restores endothelial function and relieved oxidative tissue damage, at least in part, through activation of endothelial NOS (eNOS) in the aorta of insulin-resistant rats. These results indicate that insulin resistance may be a pathogenic factor for endothelial dysfunction through impaired eNOS activity and increased oxidative breakdown of NO due to enhanced formation of O₂⁻, which are caused by relative deficiency of BH₄ in vascular endothelial cells.

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Atherosclerosis and restenosis after percutaneous coronary interventions have become major issues in public health in Western countries. Recent studies have revealed that inflammation plays an important role in pathogenesis of cardiovascular diseases. Vascular injury may involve an inflammatory response, which accelerates the recruitment and activation of monocytes through monocyte chemoattractant protein-1 (MCP-1). MCP-1 expression has been shown to be increased in atherosclerotic lesions and balloon injured arteries. Recently, we have devised a new strategy for anti-MCP-1 gene therapy by transfecting mutant MCP-1 gene into skeletal muscle. This mutant MCP-1 has been shown to work as a dominant-negative inhibitor of MCP-1. We here demonstrate that this strategy limited progression of pre-existing atherosclerotic lesions and improved the lesion composition into a more stable phenotype in the hypercholesterolemic mice. This strategy also suppressed monocyte infiltration/activation in the injured site and markedly inhibited restenotic changes (neointimal hyperplasia) in the carotid artery in rabbits, rats, and monkeys after balloon injury or stent implantation. Therefore, MCP-1-mediated monocyte infiltration is essential in the development of restenotic changes as well as atherosclerosis progression. MCP-1 can be a practical therapeutic target for human restenosis and atherosclerosis.

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Toki-shakuyaku-san (TJ-23), a Japanese traditional herbal medicine, has a long history in Asia for the treatment of neurodegenerative, immune, and airway diseases. However, the effect of TJ-23 on heart function has not been elucidated. This study was designed to examine the effect of TJ-23 on ventricular contractile function at the single cardiomyocyte level. Ventricular cardiomyocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), and maximal velocity of shortening/relengthening (±dL/dt). TJ-23 (10⁻⁸ – 10⁻⁵ mg/ml) exhibited significant augmentation in PS, with a maximal response of 27.2%. TJ-23 at 10⁻⁷ – 10⁻⁵ mg/ml also increased ±dL/dt, shortened TR₉₀, while had no effect on TPS. Pretreatment with the Na⁺-K⁺-ATPase inhibitor ouabain (1 μM), removal of extracellular sodium from contractile buffer (which inhibits Na⁺/Ca²⁺ exchanger), or both concurrently abolished the positive effect of TJ-23 in cell shortening without inhibiting the baseline cell shortening. This study demonstrated a direct cardiac stimulatory action of TJ-23 at the cardiomyocyte level, which may be related to, at least in part, a Na⁺/K⁺-ATPase and/or Na⁺/Ca²⁺ exchanger-dependent mechanism.

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The purpose of this study was to investigate the possible mechanisms of Salvia miltiorrhiza (Sm) in carbon tetrachloride (CCl₄)-induced acute hepatotoxicity in rats. Male Wistar rats received a single dose of CCl₄ (2 ml/kg in corn oil, intraperitoneally). Three hours after CCl₄ intoxication, rats received either Sm (100 mg/kg) or silymarin (100 mg/kg) by gastrogavage twice a day for 2 consecutive days. CCl₄-induced liver damage was shown by significant elevation of serum aminotransferase levels. Additionally, a significant decrease was observed in hepatic microsomal P450 2E1 protein content and hepatic concentrations of antioxidant enzymes. In contrast, rats given both Sm and silymarin supplement had less elevation of serum aminotransferase concentrations associated with less severe lobular damage of hepatocytes than rats receiving CCl₄ alone. Sm administration restored the reduction of hepatic microsomal P450 2E1 protein content as well as inducing an increase in hepatic glutathione concentration. On the other hand, administration of silymarin resulted in an elevation of hepatic superoxide dismutase levels. Moreover, both Sm and silymarin treatment inhibited the elevation of hepatic inducible nitric oxide (iNOS) protein content and nitrite concentration in liver homogenate 24 h after CCl₄ intoxication. We concluded that administration of Sm is effective in amelioration of CCl₄-induced hepatotoxicity. This effect may be due to its ability to decrease the metabolic activation of CCl₄ by an increase in P450 2E1 protein content and its antioxidant activity associated with less increase in hepatic iNOS protein content.

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The effects of pimobendan, a Ca²⁺ sensitizer with inhibitory action against cyclic-GMP-inhibited phosphodiesterase (PDE-III), on catecholamine (CA) secretion were studied in bovine adrenal chromaffin cells. In intact cells, pimobendan (10 – 100 μM) inhibited CA secretion stimulated by acetylcholine (10 and 30 μM) and 1,1-dimethyl-4-phenyl-piperazinium (DMPP) (3 and 10 μM), but facilitated CA secretion stimulated by high K⁺ (30 mM), histamine (3 μM), and angiotensin-II (3 μM). Histamine and angiotensin-II had no effect on CA secretion in Ca²⁺-free medium. The inhibition or facilitation by pimobendan of the stimulation-evoked CA secretion was not affected by H-89 (1 μM) and H-8 (30 μM), inhibitors of cyclic-AMP-dependent protein kinase. Milrinone (10 and 30 μM) and amrinone (100 and 300 μM), inhibitors of PDE-III, did not affect the stimulation-evoked CA secretion. In β-escin-permeabilized cells, pimobendan (10 – 100 μM) did not affect CA secretion stimulated by Ca²⁺ (0.1 – 10 μM) in the presence and absence of MgATP (2 mM). These results indicate that pimobendan has dual effects, inhibition and facilitation, on CA secretion. The inhibition may be due to an inhibitory action on nicotinic receptors and the facilitation may be due to a facilitatory action on stimulation-induced Ca²⁺ influx. Neither Ca²⁺ sensitizing nor PDE-III inhibiting actions seem to be related to these effects.

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An enterohemorrhagic Escherichia coli (EHEC) O157 oral infection murine model was established to examine the potentiating activity of drugs on mucosal immune responses. Groups of ICR mice inoculated intragastrically with 10¹¹ CFU/kg EHEC O157 showed chronic intestinal infection with the pathogen that persisted over 3 weeks and resulted in the synthesis of relatively high levels of antigen specific fecal IgA antibody. Intraperitoneal administration of 80 NU/kg Neurotropin, an immunopotentiator, augmented the antigen specific mucosal immune responses to EHEC O157. On the other hand, FK506 clearly suppressed the response. To further document the augmenting effect of Neurotropin on mucosal immune responses, mice were immunized intranasally with a mixture of ovalbumin and cholera toxin. Co-administration of 80 NU/kg Neurotropin significantly potentiated the synthesis of fecal IgA and serum IgG antibodies. These results suggest that Neurotropin has potential as a mucosal adjuvant to promote secretory IgA antibody production and that the mice model of oral infection with EHEC O157 is useful for immunopharmacological studies of bacterial infection-defensive mucosal immune responses.

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Modulation by taurine of the pacemaking activity and the underlying ionic currents, especially a hyperpolarization-activated inward current (I_f) and a sustained inward current (I_ST), in rat sino-atrial (SA) nodal cells was investigated at different pCa levels using a patch-clamp technique. Increasing pCa levels from 10 to 6 stimulated the spontaneous activity and simultaneously increased the I_f. Application of taurine depressed more strongly the spontaneous activity at higher pCa levels. At all pCa levels, however, taurine (20 mM) increased the I_f by 60.1 ± 1.7% (n = 8, P<0.001) at pCa 10 and by 48.0 ± 1.4% (n = 8, P<0.01) at pCa 7. At pCa 7, taurine (10 and 20 mM) decreased the sustained inward current (I_ST) by 13.3 ± 1.1% (n = 5, P<0.05) and by 38.1 ± 2.4% (n = 5, P<0.01), respectively. Taurine (20 mM) inhibited the L-type Ca²⁺ current (I_CaL) by 35.8 ± 2.5% (n = 8, P<0.01), whereas taurine enhanced the T-type Ca²⁺ current (I_CaT) by 29.3 ± 2.9% (n = 8, P<0.05). Also, taurine at pCa 7 decreased the delayed rectifier K⁺ current; taurine at 20 mM inhibited the rapidly activated K⁺ current (I_Kr) by 55.6 ± 3.3% (n = 6, P<0.001), but not the slowly activated K⁺ current (I_Ks). Taurine often elicited dysrhythmias, dependent on taurine’s concentrations and pCa levels. These results indicate that taurine causes a negative chronotropic effect due to the inhibitions of the pacemaking ionic currents such as I_CaL, I_Kr and I_ST, and suggest that the I_f and I_CaT currents make a minor contribution to pacemaking activity in rat SA nodal cells.

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The effects of ZnSO₄ on mitochondrial monoamine oxidase (MAO) activity in monkey brain were compared with those in rat and rabbit, in vitro. After preincubation at 25°C for 20 min with 1 μM ZnSO₄, MAO-A activity in monkey brain was about 50% using serotonin (5-HT) as a substrate, and the inhibition was proportional to the concentration of ZnSO₄. However, ZnSO₄ had no effect on MAO-B activity in monkey brain using β-phenylethylamine (β-PEA) as a substrate. The inhibition by ZnSO₄ of MAO-A activity was competitive and reversible. CdSO₄ also inhibits MAO-A, but not MAO-B in monkey brain mitochondria. ZnSO₄ did not inhibit either MAO-A or MAO-B activity in rat and rabbit brain mitochondria. These results indicate that the inhibiting action of Zn²⁺ differs depending on animal species. In monkey brain mitochondria, MAO-A was highly sensitive to Zn²⁺ and MAO-B was less sensitive. These results also suggest that Zn²⁺ may regulate the level of catecholamine content in monkey brain.

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Arc (activity-regulated cytoskeleton-associated protein) gene is one of the neuron-specific immediate early genes induced by neural activity. The regulation of Arc gene expression is unknown. We found that Arc mRNA is expressed constitutively in L929 cells, a mouse fibroblast cell line, and was, not transiently, increased by the calcium ionophore A23187. To address the induction of Arc mRNA by A23187, we isolated the mouse Arc gene and found that it consists of three exons, with the first exon including the whole coding region. We then constructed luciferase reporters fused with various 5' flanking regions of the mouse Arc gene. The reporter activities were not enhanced by A23187 in the tested regions up to about −9500 bp. As it is reported that protein synthesis is inhibited in by A23187, we treated L929 cells with a protein synthesis inhibitor, cycloheximide (CHX). The increase of Arc mRNA was induced by CHX alone in a calcium-independent manner and was comparable to that by A23187. No additive effect of A23187 was observed on the increase by CHX, whereas the additive effect was seen in PC12 cells. These results suggest that the inhibition of protein synthesis is a crucial factor for the accumulation of Arc mRNA in L929 cells.

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We examined the role of hydrogen peroxide production in cyclosporine A (CsA)-induced LLC-PK1 injury. After exposure to CsA (0.1 μM – 100 μM), cytotoxicity assessed by lactate dehydrogenase release to the media increased dose-dependently. LLC-PK1 cells produced hydrogen peroxide, visualized by 2,7-dichlorodihydrofluorescein assay by the treatment with 100 μM CsA, that was blocked by the treatment with catalase. The cytotoxicity of CsA significantly decreased either by the treatment with catalase, mannitol, or deferoxamine, but not with superoxide dismutase. These results suggest the role of hydrogen peroxide as the source of hydroxyl radical, which mainly contributes to CsA-induced LLC-PK1 injury.

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Serum prolactin levels were investigated in 41 patients with schizophrenia who were receiving clinically effective doses of perospirone or risperidone for more than 4 weeks. In order to determine baseline prolactin levels, blood samples were obtained in the morning, 10 – 14 h after antipsychotic medication. Median levels were within normal limits in both female and male patients receiving perospirone, while risperidone induced significant elevation. These results suggest that in contrast to risperidone, where baseline prolactin levels were elevated 5.3-fold in female and 4.2-fold in male patients, baseline prolactin levels are not elevated after treatment with perospirone. However, these results should be cautiously interpreted, because drug-by-time interaction has previously been reported in antipsychotic-induced hyperprolactinemia.

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We examined whether azelastine would inhibit itch-associated responses of mice to mosquito allergy. Repeated injections of mosquito salivary gland extract increased scratching and sensory nerve activity. Azelastine inhibited the increased scratching and nerve activity, while terfenadine was without effects. Dexamethasone did not affect the increased scratching. Azelastine suppressed high K⁺-induced increase in intracellular free Ca²⁺ in primary cultures of mouse sensory neurons. Direct inhibition by azelastine of sensory neurons may be at least involved in the anti-pruritic effect of azelastine. Histamine, substance P, and leukotriene B₄ may not play a key role in the itching of mosquito allergy.

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