[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.

Inhibition of actin polymerization by marine toxin pectenotoxin-2.

Pectenotoxin-2 (PCTX-2) is one of the polyether macrolide toxins isolated from scallops involved in diarrheic shellfish poisoning via actin depolymerization. In the present study, we examined the bioactive mechanism of PCTX-2 in smooth muscle cells and clarify mode of action of the PCTX-2-induced actin depolymerization using purified skeletal actin. PCTX-2 (300 nM-3 µM) non-selectively inhibited vascular smooth muscle contractions elicited by high K+ or phenylephrine in a dose-dependent manner. However, elevated cytosolic Ca2+ and myosin light chain phosphorylation stimulated by high K+ were only slightly inhibited by PCTX-2. By monitoring the fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit both the velocity and degree of actin polymerization. The critical concentration of G-actin was linearly increased in accordance with the concentration of PCTX-2, indicating sequestration of G-actin with 1 to 1 ratio. The kinetics of F-actin depolymerization by dilution assay indicated that PCTX-2 does not sever F-actin. Transmission electron microscopic and confocal microscopic observations demonstrated that PCTX-2 selectively depolymerized filamentous actin without affecting tublin. In conclusion, PCTX-2 is a potent natural actin depolymerizer which sequesters G-actin without severing F-actin.

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.

Dexamethasone prevents impairment of endothelium-dependent relaxation in arteries cultured with fetal bovine serum.

In the present study, we assessed the effects of dexamethasone on fetal bovine serum-induced dysfunction of mesenteric endothelial cells using an organ culture procedure. In rabbit mesenteric arteries cultured in the presence of 10% fetal bovine serum for 7 days, the endothelium-dependent, nitric oxide (NO)-mediated relaxations caused by substance P and ionomycin were decreased as compared to those in non-treated arteries. Dexamethasone (3 microM) inhibited the proliferative stimuli-induced endothelial dysfunction without affecting the contractility or NO susceptibility of smooth muscle cells. Cross-sectioned hematoxylin-eosin staining and whole-mount CD31 staining indicated that chronic proliferative stimulation induced detachment of endothelial cells from the tunica intima in some regions, and also caused thickening of the arterial wall and shortening of the internal diameter. Endothelial NO synthesis (eNOS) mRNA expression was also decreased by the treatment with fetal bovine serum. The dexamethasone treatment did not inhibit the smooth muscle hypertrophy, but it inhibited the peeling of endothelial cells and recovered the eNOS mRNA expression. These results suggest that DEX ameliorate the impairments of arterial relaxation induced by proliferative stimuli and that these beneficial effects may be mediated by maintaining the adhesion of endothelial cells to the vascular wall and/or by recovering eNOS mRNA expression.

Dexamethasone blocks hypoxia-induced endothelial dysfunction in organ-cultured pulmonary arteries.

We assessed the effects of dexamethasone (DEX) on hypoxia-induced dysfunction of the pulmonary endothelium using organ-cultured rabbit intrapulmonary arteries; 3-microM DEX inhibited the 7-day hypoxia (5% oxygen)-induced impairments of endothelial-dependent relaxation, cGMP accumulation, and increase in intracellular Ca(2+) level under substance P-stimulated conditions. Treatment with DEX over the final 3 days of the 7-day hypoxic exposure period also restored the decreased endothelium-dependent relaxation. Although chronic hypoxia did not change the mRNA expression of endothelial nitric oxide synthase (eNOS), 3 microM of DEX increased eNOS mRNA expression in both the hypoxic and normoxic (20% oxygen) pulmonary endothelium. On the other hand, eNOS protein expression was not changed in any of the arteries. We next assessed the effects of DEX on the eNOS activation pathway. Chronic hypoxia impaired eNOS phosphorylation and Akt phosphorylation under both the nonstimulated and substance P-stimulated conditions, and 3-microM DEX restored these phosphorylations. Morphologic study revealed that 3-microM DEX inhibited chronic hypoxia-induced atrophy of endothelial cells and eNOS protein condensation into plasma membranes. These results suggest that DEX exerts beneficial effects on chronic hypoxia-induced impairments of nitric oxide-mediated arterial relaxation by increasing eNOS mRNA expression and inhibiting hypoxia-induced impairments in eNOS activation pathway with atrophy of endothelial cells.

Role of actin microfilaments in canine distemper virus replication in vero cells.

Several studies have indicated that viruses require a specific cytoskeletal structure for replication in host cells. In this study, we examined the role of actin fiber in the replication of canine distemper virus (CDV), belonging to the Morbillivirus genus of the family Paramyxoviridae. For this purpose, we used two actin depolymerizing agents, cytochalasin-D (C-D) and mycalolide-B (ML-B). In Vero cells, C-D disrupted actin fibers distributed in the cytosol, but peripheral actin fibers remained intact. On the other hand, ML-B completely disrupted the actin fibers distributed in both areas. Treatment of Vero cells with C-D or ML-B inhibited the replication of CDV. Double staining of CDV-infected Vero cells with antibody to N-protein and rhodamine-phalloidin revealed the presence of N-protein in mid-cytoplasm. However, the N-protein was specifically localized at the submembrane region in the presence of C-D, whereas it was clustered in the presence of ML-B. Viral mRNA levels of N- and H-proteins were rather increased by treatment with C-D or ML-B. The treatment with ML-B strongly inhibited N-protein expression, whereas C-D only slightly inhibited N-protein expression. These results suggest that actin microfilaments distributed in the cytoplasm and on the membrane region in host cells may have a different role in the process of CDV replication.

Mechanism of human urotensin II-induced contraction in rat aorta.

Urotensin II induced sustained contraction with an EC(50) value of 2.29 +/- 0.12 nM in rat aorta. Urotensin II (100 nM) transiently increased cytosolic Ca(2+) level ([Ca(2+)](i)), followed by a small sustained phase superimposed with rhythmic oscillatory change. In the presence of verapamil and La(3+), the [Ca(2+)](i) oscillation was completely inhibited, although a small transient increase in [Ca(2+)](i) remained. The urotensin II-induced contraction was also partially inhibited by verapamil and La(3+). Combined application of verapamil, La(3+), and thapsigargin completely inhibited the increase in [Ca(2+)](i) with only partial inhibition of the contraction elicited by urotensin II. Urotensin II increased myosin light chain (MLC) phosphorylation to a level greater than that induced by 72.7 mM KCl (high K(+)). Pretreatment with Go6983 (PKC inhibitor), U0126 (MEK inhibitor), or SB203580 (p38MARK inhibitor) partially inhibited the urotensin II-induced contraction with no effects on the high K(+)-induced contractions. Wortmannin (MLC kinase inhibitor) only partially inhibited urotensin II-induced contraction, although it completely inhibited the high K(+)-induced contraction. These results suggest that urotensin II-induced contraction is mediated by the Ca(2+)/calmodulin/MLC kinase system and modulated by the Ca(2+) sensitization mechanisms to increase MLC phosphorylation. In addition, activations of PKC, p38MAPK, and ERK1/2 modulate the contractility mediated by urotensin II in rat aorta.

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.

Actin filaments play an essential role for transport of nascent HIV-1 proteins in host cells.

To investigate the role of actin filaments (F-actin) for human immunodeficiency virus type 1 (HIV-1) production in host cells, the effect of mycalolide B that is a novel actin-depolymerizing marine toxin was examined. Mycalolide B blocked the production of HIV-1 from primary infected T-lymphoblastoid and clonically infected monocytoid cells in a concentration-dependent manner. In the presence of 10 microM of mycalolide B, F-actins were disorganized and mostly disappeared in the host cells, and viral envelope- and capsid-proteins did not reach the plasma membrane, but were distributed in the cytoplasm forming aggregates. In electron micrographs, no HIV-1 virions were detected on the cell surface, but many lysosome-like vesicles containing electron dense granules were observed in the cytoplasm, implying that mycalolide B did not disturb the synthesis of viral proteins, but rather inhibited their transport processes of HIV-1 in the host cells.

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.

Difference in signal transduction mechanisms involved in 5-hydroxytryptamine- and U46619-induced vasoconstrictions.

In order to elucidate the signal transduction pathways of vascular smooth muscle contractions induced by stimulation of receptors for 5-hydroxytryptamine (5-HT) and thromboxane A2 (TXA2), both of which are released from activated platelets, we examined whether protein kinases, such as tyrosine kinase, p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC), are involved in the contraction produced by either 5-HT or U46619 (an analog of TXA2) in the rat aorta. Both 5-HT and U46619 induced sustained contractions, which were markedly reduced in the absence of extracellular Ca2+. Verapamil (a L-type Ca2+ channel blocker) markedly inhibited the contractile response to 5-HT, while the U46619-induced contraction was only slightly inhibited by verapamil. Both contractile responses to 5-HT and U46619 were significantly inhibited by calphostin C (a PKC inhibitor). On the other hand, both genistein (5 microM, a tyrosine kinase inhibitor) and SB203580 (a p38 MAPK inhibitor) significantly inhibited 5-HT-induced contractions but had little effects on the contractions induced by U46619. These results suggest that the signal transduction mechanisms involved in the contractions mediated via 5-HT and TXA2 receptors are different as follows. Both the tyrosine kinase and p38 MAPK pathways are involved in 5-HT contraction but not in TXA2 contraction, while both contractions are strongly dependent on transplasmalemmal Ca2+ entry. The contractile responses to both 5-HT and TXA2 involve voltage-dependent Ca2+ channels and PKC.

Up-regulation of Rnd1 during pregnancy serves as a negative-feedback control for Ca2+ sensitization of contractile elements in rat myometrium.

We investigated the role of Rnd1, a member of the small GTP-binding Rho protein family, in the change in Ca(2+) sensitivity of contractile element in rat myometrium at estrus, gestation, and postpartum stages. In the permeabilized muscles, GTPgammaS or carbachol with GTP increased Ca(2+) sensitivity of contractile force in non-pregnant myometrium at the estrus stage, whereas these stimuli were ineffective in pregnant myometrium at day 21. After postpartum, the reduced Ca(2+) sensitization was recovered. Semi-quantitative RT-PCR analysis indicated that the expressions of RhoA, ROCKI, and ROCKII were not significantly different between non-pregnant and pregnant myometria. In contrast, the expression of Rnd1 was increased during the course of pregnancy, reaching a maximal at day 21, and rapidly declined after the delivery. On the other hand, Ca(2+) sensitization of contractile elements was decreased during the progress in gestation. These results suggest that Rnd1 may have an important role as a negative-feedback control of uterine contraction during gestation through the inhibition of RhoA-mediated increase in the Ca(2+) sensitivity of contractile elements.

Possible role of the protein kinase C/CPI-17 pathway in the augmented contraction of human myometrium after gestation.

1. Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 microm) induced sustained contractions with no increase in [Ca2+]i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser19. 2. The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 microM). LY333531 (1 microM), a specific inhibitor of PKC beta, also inhibited the PDBu-induced contraction in the pregnant myometrium. 3. In the pregnant myometrium permeabilized with alpha-toxin, PDBu increased the contractions induced at fixed Ca2+ concentration (0.3 microM) both in nonpregnant and pregnant myometria, indicating Ca2+ sensitization of contractile elements. 4. Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (alpha, beta, gamma), novel PKC (delta, epsilon, theta), and atypical PKC (zeta but not iota and lambda). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of beta isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. 5. CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. 6. These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the beta PKC isoform and CPI-17.

Chronic treatment with interleukin-1beta attenuates contractions by decreasing the activities of CPI-17 and MYPT-1 in intestinal smooth muscle.

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that plays a central role in inflammatory bowel disease (IBD). In order to elucidate the mechanism of motility disorders frequently observed in IBD, we investigated the long term effects of IL-1beta on rat ileal smooth muscle contractility by using an organ culture system. When ileal smooth muscle strips were cultured with IL-1beta (10 ng/ml), contractions elicited by high K+ and carbachol were inhibited in a time-dependent manner. IL-1beta more strongly inhibited the carbachol-induced contractions than high K+ with decreasing myosin light chain phosphorylation. In the alpha-toxin-permeabilized ileal muscle, carbachol with GTP or guanosine 5'-3-O-(thio)triphosphate increased the Ca2+ sensitivity of contractile elements, and this G protein-coupled Ca2+ sensitization was significantly reduced in the IL-1beta-treated ileum. Among the functional proteins involved in the smooth muscle Ca2+ sensitization, CPI-17 expression was significantly reduced after the culture with IL-1beta, whereas the expressions of RhoA, ROCK-I, ROCK-II, MYPT-1, myosin light chain kinase, and myosin phosphatase (PP1) were unchanged. The phosphorylation level of CPI-17 by carbachol was low in accordance with the decrease in CPI-17 expression due to IL-1beta treatment. In contrast, constitutively phosphorylated MYPT-1 was also decreased in the IL-1beta-treated muscles. These results suggest that long term treatment with IL-1beta decreases either CPI-17 expression or MYPT-1 phosphorylation, which may result in an increase in myosin phosphatase activity to reduce force generation. Based on these findings, we consider IL-1beta to be an important mediator of gastrointestinal motility disorders in IBD, and CPI-17 and MYPT-1 are key molecules in the decreased smooth muscle contractility due to IL-1beta.

Positive inotropic effect of endothelin-1 in the neonatal mouse right ventricle.

In neonatal mouse right ventricles, endothelin-1 (ET-1, 1-300 nM) induced a dose-dependent increase in twitch contractions and the dose-response curve was shifted to the right by BQ-123 (10 microM), an endothelin ET(A) receptor antagonist. The ET-1 (100 nM)-induced positive inotropy was accompanied by an increase in [Ca(2+)](i) transients without any change in the [Ca(2+)](i)-force relationship. Ryanodine (1 microM) partially decreased the [Ca(2+)](i) transients and contractile force, but did not affect the ET-1 (100 nM)-induced positive inotropy. Reduction of [Na(+)](o) elicited an increase in contractile force, and this effect was significantly inhibited by KB-R7943 (30 microM), an inhibitor of the Na(+)-Ca(2+) exchanger. KB-R7943 (30 microM) almost completely suppressed the positive inotropic effect of ET-1. Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (100 nM) decreased the contractile force, an effect which was suppressed by bisindolylmaleimide I (3 microM). On the other hand, the ET-1-induced positive inotropic effect was unaffected by bisindolylmaleimide I (3 microM). These results suggest that the positive inotropic effect of ET-1 in neonatal mouse right ventricles is caused by the increase in [Ca(2+)](i) transients through activation of the endothelin ET(A) receptor and the increase in Ca(2+) influx via the Na(+)-Ca(2+) exchanger during an action potential. Furthermore, the ET-1-induced positive inotropy is independent of the effects of PKC, which makes it distinct from the ET-1-mediated pathways reported for cardiac tissues in other species.