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Nitric oxide (NO) donors generally relax vascular preparations through cGMP-mediated mechanisms. Relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and coronary arteries to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) is almost eliminated by inhibition of soluble guanylate cyclase activation with 10 [micro]M 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), whereas only a modest inhibition of relaxation is observed under hypoxia (P[O.sub.2] = 8-10 Torr). This effect of hypoxia is independent of the contractile agent used and is also observed with NO gas. ODQ eliminated SNAP-induced increases in cGMP under hypoxia in BPA. cGMP-independent relaxation of BPA to SNAP was not attenuated by inhibition of [K.sup.+] channels (10 mM tetraethylammonium), myosin light chain phosphatase (0.5 [micro]M microcystin-LR), or adenylate cyclase (4 [micro]M 2',5'-dideoxyadenosine). SNAP relaxed BPA contracted with serotonin under [Ca.sup.2+]-free conditions in the presence of hypoxia and ODQ, and contraction to [Ca.sup.2+] readdition was also attenuated. The sarcoplasmic reticulum [Ca.sup.2+]-reuptake inhibitor cyclopiazonic acid (0.2 mM) attenuated SNAp-mediated relaxation of BPA in the presence of ODQ. Thus hypoxic conditions appear to promote a cGMP-independent relaxation of BPA to NO by enhancing sarcoplasmic reticulum [Ca.sup.2+] reuptake. calcium; guanylate cyclase; nitrovasodilator

Abstract Background Deterioration of peroxisomal β-oxidation activity causes an accumulation of very long chain saturated fatty acids (VLCSFA) in various organs. We have recently reported that the levels of VLCSFA in the plasma and/or membranes of blood cells were significantly higher in patients with metabolic syndrome and in patients with coronary artery disease than the controls. The aim of the present study is to investigate the effect of VLCSFA accumulation on inflammatory and oxidative responses in VLCSFA-accumulated macrophages derived from X-linked adrenoleukodystrophy (X-ALD) protein (ALDP)-deficient mice. Results Elevated levels of VLCSFA were confirmed in macrophages from ALDP-deficient mice. The levels of nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), intracellular reactive oxygen species (ROS), and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interluekin-6 (IL-6), and interleukin-12p70 (IL-12p70), were significantly higher in macrophages from ALDP-deficient mice than in those from wild-type mice. The inducible NO synthase (iNOS) mRNA expression also showed an increase in macrophages from ALDP-deficient mice. Conclusion These results suggested that VLCSFA accumulation in macrophages may contribute to the pathogenesis of inflammatory diseases through the enhancement of inflammatory and oxidative responses.

Iesaki, Takafumi, Sachin A. Gupte, Pawel M. Kaminski, and Michael S. Wolin. Inhibition of guanylate cyclase stimulation by NO and bovine arterial relaxation to peroxynitrite and [H.sub.2][O.sub.2]. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H978-H985, 1999.--The inhibitor of soluble guanylate cyclase (sGC) stimulation by nitric oxide (NO), 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), was examined for its effects on the prolonged relaxation of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to peroxynitrite ([ONOO.sup.-]) and on [H.sub.2][O.sub.2]-elicited relaxation and sGC stimulation. Our previous studies suggest that [ONOO.sup.-] causes a prolonged relaxation of BPA by regenerating NO an that a 2-min exposure of BCA or BPA to 50 nM NO causes an [ONOO.sup.-]-elicited relaxation. The relaxation of [K.sup.+]-precontracted BCA to 50 nM NO or 100 [micro]M [ONOO.sup.-] was essentially eliminated by 10 [micro]M ODQ. ODQ also eliminated relaxation to 0.1 nM-10 [micro]M of NO donor S-nitroso-N-acetyl-penicillamine (SNAP), but it did not alter relaxation to 1-300 [micro]M [H.sub.2] [O.sub.2]. Similar responses were also observed in BPA. ODQ did not increase lucigenin-detectable superoxide production in BCA, and it did not alter luminol-detectable endogenous [ONOO.sup.-] formation observed during a 2-min exposure of BCA to 50 nM NO. In addition, ODQ did not affect tissue release of NO after 2 min exposure of BCA to 50 nM NO. The activity of sGC in BPA homogenate that is stimulated by endogenous [H.sub.2][O.sub.2] was not altered by ODQ, whereas sGC activity in the presence of 10 [micro]M SNAP (+fungal catalase) was reduced by ODQ. Thus relaxation of [K.sup.+]-precontracted BCA and BPA to [ONOO.sup.-] appears to be completely mediated by NO stimulation of sGC, whereas the actions of ODQ suggest that NO is not involved in [H.sub.2][O.sub.2]-elicited relaxation and sGC stimulation. This study did not detect evidence for the participation of additional mechanisms potentially activated by [ONOO.sup.-] in the responses studied. hydrogen peroxide; nitric oxide; redox signalling

Perlecan (HSPG2), a basement membrane-type heparan sulfate proteoglycan, has been implicated in the development of aortic tissue. However, its role in the development and maintenance of the aortic wall remains unknown. Perlecan-deficient mice (Hspg2−/−-Tg: Perl KO) have been found to show a high frequency (15–35%) of aortic dissection (AD). Herein, an analysis of the aortic wall of Perl KO mice revealed that perlecan deficiency caused thinner and partially torn elastic lamina. Compared to the control aortic tissue, perlecan-deficient aortic tissue showed a significant decrease in desmosine content and an increase in soluble tropoelastin levels, implying the presence of immature elastic fibers in Perl KO mice. Furthermore, the reduced expression of the smooth muscle cell contractile proteins actin and myosin in perlecan-deficient aortic tissue may explain the risk of AD. This study showed that a deficiency in perlecan, which is localized along the elastic lamina and at the interface between elastin and fibrillin-1, increased the risk of AD, largely due to the immaturity of extracellular matrix in the aortic tissue. Overall, we proposed a new model of AD that considers the deficiency of extracellular molecule perlecan as a risk factor. [ABSTRACT FROM AUTHOR]

Objective In vascular strips, hydrogen peroxide (H2O2) relaxes α1-adrenergic agonist-induced but not high-K+-induced contractions. The aim of this study was to explore H2O2-induced changes in [Ca2+]i of vascular smooth muscle and to elucidate the mechanisms of action of H2O2. Methods Isolated rabbit aortic strips were isometrically contracted with high-K+ (64.7 mM) or phenylephrine (PE, 0.3 μM). The effects of 300 μM H2O2 on [Ca2+]i of endothelium-denuded vascular smooth muscle and tension were determined simultaneously by the fura-2 method. Changes in [Ca2+](were expressed as percentages of high-K+-induced values measured at the beginning of the experiments. In another series of experiments, the relaxant effect of 300 μM H2O2 was examined in high-K+ (20 mM)-induced contraction in the presence of the protein kinase C activator, phorbol 12,13-dibutyrate (PDBu). Results Hydrogen peroxide caused a reversible rise in [Ca2+]i of vascular smooth muscle under both resting conditions and in the precontracted state. During high-K+-induced contraction, H2O2 further increased [Ca2+]i by 26.6(s.e.m. 1.7)% accompanied by a small increase in tension of 6.5(1.9)% of high-K+-induced tension. By contrast, during PE-induced contraction, although H2O2 caused a comparable additional increase in [Ca2+]i (26.4(4.7)%), muscle tension fell by 28.9(2.2)% of the steady-state PE-induced tension. Hydrogen peroxide had a relaxant effect on augumented high-K+-induced contraction in which Ca2+ sensitivity of the contractile apparatus was elevated by PDBu. Conclusions In spite of its effect of increasing [Ca2+]i of vascular smooth muscle, hydrogen peroxide causes relaxation of endothelium-denuded, PE-precontracted rabbit aorta. The mechanism is probably through suppression of agonist-induced augmentation of Ca2+ sensitivity of the contractile apparatus. [ABSTRACT FROM PUBLISHER]

Objective: The aims were to investigate the effects of H2O2 on arterial contractions induced by vasoactive amine agonists and a high concentration of potassium ions (high K+) in vitro and to explore the possible underlying mechanism(s) involved. Methods: Isometric tension of rabbit isolated aortic strips was measured and the effects of pretreatment with H2O2 on contractions induced by phenylephrine and high K+ were compared. The effects of H2O2 on precontracted strips were determined in the presence and absence of the aortic endothelium and compared with those of acetylcholine. Results: The tension developed in response to an agonist was expressed as a percentage of the contraction induced by high K+ (64.7 mM) supervision. Pretreatment with 300 μM H2O2 reduced the mean phenylephrine (0.3 μM) induced contraction from 96.2(SEM 1.4) to 61.8(2.8)%; the effect was stable and reversed by washing out the H2O2. Hydrogen peroxide relaxed phenylphrine precontracted strips with and without endothelium but it showed no relaxant effect when the strips were precontracted by high K+, whereas acetylcholine (1 μM) induced transient relaxation of high K+ precontracted strips by 27.8(2.9)%. The relaxant effect of H2O2 was not affected by pretreatment with indomethacin (a cyclo-oxygenase inhibitor), desferrioxamine (a hydroxyl radical scavenger), or diphenylphenylenediamine (a lipophilic antioxidant). Conclusions: H2O2 inhibits vasoactive amine induced contractions of the vascular smooth muscle of rabbit aorta in vitro without affecting voltage dependent Ca2+ influx or contractile machinery. The mechanism responsible for its inhibitory effects may be related to impairments of the cellular signalling reactions initiated by the agonists.Cardiovascular Research 1994;28:963-968 [ABSTRACT FROM PUBLISHER]

Abstract: The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca2+ levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca2+]o on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca2+]o enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca2+]o induces an increase in [Ca2+]i. An intracellular Ca2+ chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca2+]o in BMSCs. These data suggest a new role for extracellular Ca2+ in the bone marrow stroma: increased [Ca2+]o induces an increase in [Ca2+]i levels, which in turn enhances the accumulation of adipocytes under certain conditions. [Copyright &y& Elsevier]

Abstract: Inositol 1,4,5-trisphosphate receptor (IP3R) plays a crucial role in generating Ca2+ signaling and three subtypes of IP3R have been identified. In spite of a high degree of similarity among these subtypes, their effects on spatio-temporal Ca2+ patterns are specific and diverse; therefore the physiological significance of the differential expression levels of IP3R subtypes in various tissues remains unknown. Here, we examined the relative contribution of the specific subtype of IP3Rs to the agonist-induced Ca2+ signaling and contraction in IP3R-deficient vascular smooth muscle cells and found that IP3R1 deficient cells exclusively showed less sensitivity to the agonist, compared to those from the other genotypes. We also found that IP3R1 dominantly expressed in vascular aortae on a consistent basis, and that phenylephrine (PE)-induced aortic muscle contraction was reduced specifically in IP3R1-deficient aortae. Taken together, we concluded that IP3R1 plays a predominant role in the function of the vascular smooth muscle in vivo. [Copyright &y& Elsevier]

Aims: To clarify the role of Fc receptors (FcR) for immunoglobulin in endothelial dysfunction induced by hypercholesterolaemia, we evaluated the effect of deletion of the FcR gamma chain on endothelium-dependent relaxation and oxidative stress after 10 weeks on a high-fat diet in FcR gamma(-/-) mice compared with that in wild-type mice., Methods and Results: Plasma cholesterol levels of those on the high-fat diet were significantly increased compared with those on the normal chow diet in both groups of mice. Endothelium-dependent relaxation of the aortic ring with acetylcholine in wild-type mice was significantly reduced by the high-fat diet (ED(50): 0.22 vs. 0.43 nM, P < 0.002), whereas the relaxation in FcR gamma(-/-) mice was not inhibited (ED(50): 0.22 vs. 0.23 nM, NS). Furthermore, superoxide detection by dihydroethidium-derived fluorescence and immunohistochemical staining of p22phox expression were significantly increased in wild-type mice fed on the high-fat diet, while these oxidative stresses in FcR gamma(-/-) mice were not enhanced by the high-fat diet. Oil Red O-staining showed no significant lipid accumulation at the aortic sinus in both groups of mice., Conclusion: This study demonstrates that the deletion of the FcR gamma chain preserves the endothelial function and attenuates oxidative stress affected by hypercholesterolaemia in FcR gamma(-/-) mice. These results indicate that FcR may play the pivotal role in endothelial dysfunction through oxidative stress induced by hypercholesterolaemia.