[Cyanide, in And Then There Were None, Sparkling Cyanide].

And Then There Were None and Sparkling Cyanide, two of Agatha Christie's famous novels describe potassium cyanide-induced deaths. Cyanide, a tasteless, odorless, strongly alkaline poison is a powerful gastrointestinal irritant, following oral ingestion. It reacts with hydrochloric acid in the gastric juice to produce hydrogen cyanide gas, which is absorbed and inhibits the mitochondrial electron transfer system and consequently suppresses adenosine triphosphate (ATP) production. Therefore, the central nervous system, which consumes a large amount of ATP, is first affected and symptoms of poisoning manifest as dizziness, disorientation, coma, and convulsions. The orally lethal dose is approximately 300 mg.

Statin protects endothelial nitric oxide synthase activity in hypoxia-induced pulmonary hypertension.

OBJECTIVE: We investigated the effects of fluvastatin on hypoxia-induced (1 to 3 weeks, 10% O2) pulmonary hypertension with focus on endothelial nitric oxide synthase (eNOS) activity. METHODS AND RESULTS: Oral fluvastatin treatment (1 mg/kg daily) prevented the causing and progression of pulmonary hypertension as determined by the right ventricular pressure, right ventricular hypertrophy, and muscularization of pulmonary artery. We also revealed that fluvastatin treatments prevented the hypoxia-induced decrease in cGMP production in the rat lung and restored the endothelium-dependent relaxation in the pulmonary artery. We revealed that this beneficial effect was not dependent on the increase in eNOS mRNA or protein expression, but was dependent on the inhibition of the eNOS-tight coupling with caveolin-1, the eNOS dissociation from heat shock protein 90, and the decrease in eNOS Ser1177-phosphorylation induced by hypoxia. Furthermore, in a whole-mount immunostaining the hypoxia-induced eNOS protein condensation with caveolin-1 of pulmonary endothelial cells was restored by the fluvastatin-treatment. CONCLUSIONS: These results suggest that the fluvastatin exerts beneficial effects on chronic hypoxia-induced pulmonary hypertension by protecting against the eNOS activity at the post-transcriptional level.

Vascular endothelium has a local anti-adenovirus vector system and glucocorticoid optimizes its gene transduction.

OBJECTIVE: Although adenovirus is a powerful tool for vascular research and therapy, endothelial impairment after infection has been reported. We investigated the mechanisms of this impairment and the effect of dexamethasone (DEX) on gene transfer into the vascular endothelial cells. METHODS AND RESULTS: Beta-galactosidase gene encoding adenovirus vector (beta-gal-Ad) (7.5 x 10(8) plaque-forming units/mL) transduced beta-gal into the rabbit organ-cultured pulmonary endothelium, followed by an apoptosis and an impairment of endothelium-dependent relaxation (EDR). Endothelial cell infected by beta-gal-Ad expressed proinflammatory genes mRNAs and suppressed endothelial nitric oxide synthase (eNOS) mRNA. Treatment with DEX dramatically increased beta-gal protein expression in the endothelium, attenuated beta-gal-Ad-induced apoptosis, and prevented the impairment of EDR. DEX also suppressed the mRNAs expressions of proinflammatory genes and recovered eNOS mRNA expression in organ-cultured vascular endothelium. In addition, we confirmed the DEX's beneficial effects in an endothelial cell line (in vitro) and rat femoral artery (in vivo) experiments. CONCLUSIONS: These results suggest that adenovirus vector induces host-immune responses and apoptosis in vascular endothelial cells. DEX is found to be a useful and potent tool to prevent the Ad-induced impairments of the endothelium and to optimize gene expression efficiency by adenovirus vector at the protein translation level in both in vitro and in vivo experiments.

Mechanisms responsible for the in vitro relaxation of a novel dibenzothiepine derivative (NSU-242) on tracheal and vascular smooth muscles.

In our previous general screening experiments, we found that NSU-242, a dibenzothiepine derivative (1-10 mg/kg), inhibited antigen-induced immediate asthmatic response in actively sensitized guinea pigs in a dose-dependent manner. The purpose of the present study was to assess the mechanism of the relaxing effect of NSU-242 on smooth muscle contractions in isolated smooth muscle tissues of the porcine trachea and rat aorta. NSU-242 administration resulted in a concentration-dependent inhibition of the tracheal-tissue contractions induced by carbachol and high K(+) and the aortic-tissue contractions induced by norepinephrine and high K(+). The IC(50) values of these inhibitions were 1-10 microM, and there was no selectivity for the type of stimulation. In tracheal tissue, the relaxations were accompanied by neither changes in cAMP nor changes in cGMP. Carbachol (1 microM) and high K(+) (59.2 mM) increased myosin light chain (MLC) phosphorylation in the trachea, and NSU-242 (3-30 microM) had no effect on the level of MLC phosphorylation. Furthermore, NSU-242 (300 microM) had no effect on contractions in membrane-permeabilized tracheal tissue. FITC-phalloidin staining of the actin fiber in cultured vascular smooth muscle cells (A7r5) indicated that NSU-242 (10-100 microM) altered the configuration of actin stress fiber in the cytosol. However, unlike cytochalasin D, NSU-242 did not inhibit actin polymerization as assessed by in vitro assay. These results suggest that NSU-242 inhibits smooth muscle contractions without any effect on the Ca(2+)-dependent MLC phosphorylation. NSU-242 may uncouple the force generated by the activated actomyosin interaction, possibly by modifying the actin assembly in smooth muscle cells without a direct effect on actin molecules.

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells.

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcepsilonRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500-5,000 ng/ml) alone increased cytosolic Ca2+ level ([Ca2+]i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca2+]i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5-50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca2+]i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca2+]i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca2+]i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca2+ signaling and degranulation.

Decrease in activity of smooth muscle L-type Ca2+ channels and its reversal by NF-kappaB inhibitors in Crohn's colitis model.

We investigated the mechanisms of dysmotility of the colonic circular muscle of the Crohn's disease rat model. Contractions induced by KCl, carbachol, and Bay K 8644 were decreased in circular smooth muscles isolated from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat colon. However, the absolute force and Ca2+ sensitivity of contractile proteins were not affected as assessed in alpha-toxin permeabilized smooth muscle. The current density of the L-type Ca2+ channel in circular smooth muscle cells was significantly decreased in the TNBS-treated colonic cells. However, expressions of the L-type Ca2+ channel mRNA and protein did not differ between control and TNBS-treated preparations. Pretreatment with the NF-kappaB inhibitors pyrrolidinedithiocarbamate and sulfasalazine partially recovered the decreased contractility and current density of the L-type Ca2+ channel by TNBS treatment. These results suggest that the decrease in the contraction of circular smooth muscle isolated from TNBS-induced colitis rat colon, which may be related to gut dysmotility in Crohn's disease, is attributable to the decreased activity of the L-type Ca2+ channel. The dysfunction of the L-type Ca2+ channel may be mediated by NF-kappaB-dependent pathways.

Increased locomotor activity, increased food and water intake and decreased PVN neurons in H1 calponin gene-deficient mice.

Calponin (h1 or basic) is an actin-binding protein that is expressed abundantly in smooth muscle. Our previous study using h1 calponin-null mutant mice demonstrated that h1 calponin inhibits the shortening velocity of smooth muscle contraction without significantly affecting the amplitude of force production. Furthermore, early onset of osteogenesis and increased bone formation have been reported in mutated mice. In the present study, we examined the effect of h1 calponin depletion on the metabolism and behavior of mice and found that the mutated mice showed increased locomotor activity, as well as increased intake of food and water, associated with the decreased number of neurons in the paraventricular nucleus of the hypothalamus (PVN).

FcepsilonRI cross-linking-induced actin assembly mediates calcium signalling in RBL-2H3 mast cells.

1. To determine the role of actin assembly in the Ca(2+) signalling of mast cells activated by cross-linking of FcepsilonRI, we examined the effects of cytochalasin D, an inhibitor of actin polymerization. 2. In the RBL-2H3 cells, F-actin content was increased by sensitization with anti-dinitrophenol (DNP) IgE. In these cells, cytochalasin D induced oscillatory increases in cytosolic Ca(2+) ([Ca(2+)](i)); these increase were inhibited by jasplakinolide, a stabilizer of actin filaments. 3. In the IgE-sensitized RBL-2H3 cells, DNP-human serum albumin (DNP-HSA) augmented actin assembly. DNP-HSA also increased the production of IP(3), [Ca(2+)](i) and degranulation. Cytochalasin D enhanced all of these DNP-HSA-induced effects. 4. In a Ca(2+)-free solution, DNP-HSA induced a transient increase in [Ca(2+)](i), and this increase was accelerated by cytochalasin D. After cessation of the DNP-HSA-induced Ca(2+) release, the re-addition of Ca(2+) induced a sustained increase in [Ca(2+)](i) through capacitative Ca(2+) entry (CCE), and this increase was enhanced by cytochalasin D. 5 The effect of cytochalasin D in enhancing the CCE activity was prevented by xestospongin C. 6. In contrast, neither the Ca(2+) release nor the CCE activation that was induced by thapsigargin was affected by cytochalasin D. 7. These results suggest that actin de-polymerization stimulates the FcepsilonRI-mediated signalling to augment the release of Ca(2+) from the endoplasmic reticulum in RBL-2H3 cells.

Xestospongin C, a novel blocker of IP3 receptor, attenuates the increase in cytosolic calcium level and degranulation that is induced by antigen in RBL-2H3 mast cells.

1. We evaluated the role of the cross-linking of Fc epsilon RI-mediated inositol 1,4,5-triphosphate (IP(3)) in the increase in cytosolic Ca(2+) level ([Ca(2+)](i)) using xestospongin C, a selective membrane permeable blocker of IP(3) receptor, in RBL-2H3 mast cells. 2. In the cells sensitized with anti-dinitrophenol (DNP) IgE, DNP-human serum albumin (DNP-HSA) and thapsigargin induced degranulation of beta-hexosaminidase and a sustained increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited both of these changes that were induced by DNP-HSA without changing those induced by thapsigargin. 3. In the absence of external Ca(2+), DNP-HSA induced a transient increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited this increase in [Ca(2+)](i). 4. In the cells permeabilized with beta-escin, the application of IP(3) decreased Ca(2+) in the endoplasmic reticulum (ER) as evaluated by mag-fura-2. Xestospongin C (3 - 10 microM) inhibited the effect of IP(3). 5. After the depletion of Ca(2+) stores due to stimulation with DNP-HSA or thapsigargin, the addition of Ca(2+) induced capacitative calcium entry (CCE). Xestospongin C (3 - 10 microM) inhibited the DNP-HSA-induced CCE, whereas it did not affect the thapsigargin-induced CCE. 6. These results suggest that Fc epsilon RI-mediated generation of IP(3) contributes to Ca(2+) release not only in the initial phase but also in the sustained phase of the increase in [Ca(2+)](i), resulting in prolonged Ca(2+) depletion in the ER. The ER Ca(2+) depletion may subsequently activate CCE to achieve a continuous [Ca(2+)](i) increase, which is necessary for degranulation in the RBL-2H3 mast cells. Xestospongin C may inhibit Ca(2+) release and consequently may attenuate degranulation.