Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium.
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In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01-10 micrometers), in the presence of indomethacin (2.8 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation (n = 5). In pressurized arteries, ACh (10 microM), applied intraluminally in the presence of indomethacin (2.8 microM) and L-NAME (100 microM), dilated both PE-stimulated (0.3-0.5 microM; n = 5) and myogenic tone (n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect ACh-induced dilatations of either PE-stimulated (n = 5) or myogenic tone (n = 3). This is the first demonstration that ChTX and apamin act selectively on the endothelium to block EDHF-mediated relaxation. (+info)
Endothelium-dependent hyperpolarization in resting and depolarized mammary and coronary arteries of guinea-pigs.
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1. The membrane potential responses in guinea-pig coronary and mammary arteries attributable to endothelium-derived nitric oxide (NO), prostaglandin (PG) and hyperpolarizing factor (EDHF), and to exogenous NO and the prostacyclin analogue, iloprost, were compared at rest and when depolarized with the thromboxane analogue, U46619. 2. In the coronary artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization attributable to NO and a PG with similar pD2s at rest and in the presence of U46619. However, in depolarized tissues, the pD2 of the response attributed to EDHF required a 10 fold lower concentration of ACh compared with at rest. 3. In the mammary artery, lower concentrations of ACh were required to evoke NO- and EDHF-dependent hyperpolarizations in depolarized mammary artery compared with at rest, while PG-dependent hyperpolarization did not occur until the concentration of ACh was increased some 10 fold both at rest and in U46619. 4. The smooth muscle of the coronary artery of guinea-pigs was some 4 fold more sensitive to exogenous NO and iloprost than was the mammary artery. 5. In conclusion, the membrane potential response in arteries at rest, that is, in the absence of constrictor, may be extrapolated to events in the presence of constrictor when NO and PG are under study. However, the sensitivity to ACh and the magnitude of the hyperpolarization attributed to EDHF obtained in tissues at rest may underestimate these parameters in depolarized tissues. (+info)
Effects of Aspergillus fumigatus culture filtrate on antifungal activity of human phagocytes in vitro.
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BACKGROUND: Aspergillus fumigatus can colonise the airways and the lungs with localised underlying conditions and occasionally invade the surrounding lung tissues even in subjects without systemic predisposing factors, presumably by escaping the local host defences. The aim of this study was to investigate the effects of A fumigatus culture filtrate (ACF) on the activities of human phagocytes--inhibition of germination of A fumigatus spores by alveolar macrophages (AMs) and hyphal damage by polymorphonuclear leucocytes (PMNs)--which are the critical host defences against A fumigatus. METHODS: Spores were incubated with AMs at a ratio of 1:1 in a medium containing different concentrations of ACF for 10 hours at 37 degrees C. Spore germination was visualised with light microscopy and the inhibition rate was calculated. The percentage of hyphal damage caused by PMNs pretreated with various concentrations of ACF was measured by a colorimetric tetrazolium metabolic assay. RESULTS: The inhibition rate of spore germination by AMs cultured with medium alone (control) was 90 (0.8)% whereas that by AMs cultured with the medium containing 10% ACF was significantly (p < 0.05) reduced to 41.7 (4.6)%. ACF suppressed the inhibition of spore germination in a dose dependent manner without altering the phagocytosing activity against the spores. The percentage of hyphal damage caused by PMNs pretreated with medium-199 (control) was 78.1 (2.3)% compared with 65.3 (2.8)% when PMNs were pretreated with 50% ACF (p < 0.05). CONCLUSIONS: A fumigatus releases biologically active substance(s) which suppress the inhibition of spore germination by AMs and also suppress PMN mediated hyphal damage, and thus may contribute to the pathogenicity of this fungus. (+info)
Inhibition of the production of endothelium-derived hyperpolarizing factor by cannabinoid receptor agonists.
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1. The endogenous cannabinoid, anandamide, has been reported to induce an 'endothelium-derived hyperpolarizing factor (EDHF)-like' relaxation in vitro. We therefore investigated the effects of cannabinoid CB1 receptor agonists; HU 210, delta9-tetrahydrocannabinol (delta9-THC) and anandamide, and a CB1 antagonist/inverse agonist, SR 141716A, on nitric oxide (NO) and EDHF-mediated relaxation in precontracted rings of porcine coronary, rabbit carotid and mesenteric arteries. 2. In rings of mesenteric artery HU 210 and delta9-THC induced endothelium- and cyclo-oxygenase-independent relaxations which were sensitive to SR 141716A. Anandamide (0.03-30 microM) induced a slowly developing, endothelium-independent relaxation which was abolished by diclofenac and was therefore mediated by cyclo-oxygenase product(s). None of the CB1 agonists tested affected the tone of precontracted rings of rabbit carotid or porcine coronary artery. 3. In endothelium-intact segments, HU 210, delta9-THC and anandamide did not affect NO-mediated responses but under conditions of continuous NO synthase/cyclo-oxygenase blockade, significantly inhibited acetylcholine and bradykinin-induced relaxations which are attributed to the production of EDHF. The effects of HU 210 and delta9-THC were not observed when experiments were performed in the presence of SR 141716A suggesting the involvement of the CB1 receptor. 4. In a patch clamp bioassay of EDHF production, HU 210 decreased the EDHF-mediated hyperpolarization of detector smooth muscle cells when applied to the donor segment but was without effect on the membrane potential of detector cells. The inhibition of EDHF production was unrelated to alterations in Ca2+ -signalling or cytochrome P450 activity. 5. These results suggest that the activation of endothelial CB1 receptors appears to be negatively coupled to the production of EDHF. (+info)
Proinflammatory mediators chronically downregulate the formation of the endothelium-derived hyperpolarizing factor in arteries via a nitric oxide/cyclic GMP-dependent mechanism.
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BACKGROUND: Endothelium-dependent dilator responses mediated by NO and endothelium-derived hyperpolarizing factor (EDHF) are altered in arteriosclerosis and sepsis. The possibility that proinflammatory mediators that stimulate the expression of inducible NO synthase (NOS II) affect the generation of EDHF was examined in isolated arteries. METHODS AND RESULTS: Under combined blockade of NOS and cyclooxygenase, EDHF-mediated relaxation elicited by several agonists was significantly attenuated in rabbit carotid and porcine coronary arteries exposed to cytokines and lipopolysaccharide. The blunted relaxation was coincident with NOS II expression and was prevented by inhibition of NOS II as well as of global protein synthesis. The NO donor CAS 1609 and 8-bromo-cGMP mimicked the proinflammatory mediator effect. In contrast, long-term blockade of endothelial NO generation increased the relaxation in carotid but not in coronary arteries. Proinflammatory mediators reduced the synthesis of EDHF assessed as hyperpolarization of vascular smooth muscle cells elicited by the effluent from bradykinin-stimulated coronary arteries. Proinflammatory mediators induced NOS II expression in cultured endothelial cells and decreased the expression of cytochrome P450 enzymes, which are the most probable candidates for the synthesis of EDHF. CONCLUSIONS: Proinflammatory mediators inhibit the formation of EDHF in isolated arteries. This impairment is coincident with NOS II expression in the arterial wall and seems to be mediated through the induced generation of NO, which downregulates the putative EDHF-forming enzyme. Thus, a decreased formation of EDHF may contribute to the endothelial dysfunction in arteriosclerosis and sepsis. (+info)
A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis.
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Treatment of HL-60 cells with staurosporine (STS) induced mitochondrial cytochrome c efflux into the cytosol, which was followed by caspase-3 activation and apoptosis. Consistent with these observations, in vitro experiments demonstrated that, except for cytochrome c, the cytosol of HL-60 cells contained sufficient amounts of all factors required for caspase-3 activation. In contrast, treatment of HCW-2 cells (an apoptotic-resistant HL-60 subclone) with STS failed to induce significant amounts of mitochondrial cytochrome c efflux, caspase-3 activation, and apoptosis. In vitro assays strongly suggested that a lack of cytochrome c in the cytosol was the primary limiting factor for caspase-3 activation in HCW-2 cells. To explore the mechanism which regulates mitochondrial cytochrome c efflux, we developed an in vitro assay which showed that cytosolic extracts from STS-treated, but not untreated, HL-60 cells contained an activity, which we designated 'CIF' (cytochrome c-efflux inducing factor), which rapidly induced cytochrome c efflux from HL-60 mitochondria. In contrast, there was no detectable CIF activity in STS-treated HCW-2 cells although the mitochondria from HCW-2 cells were responsive to the CIF activity from STS-treated HL-60 cells. These experiments have identified a novel activity, CIF, which is required for cytochrome c efflux and they indicate that the absence of CIF is the biochemical explanation for the impaired ability of HCW-2 cells to activate caspase-3 and undergo apoptosis. (+info)
Involvement of CB1 cannabinoid receptors in the EDHF-dependent vasorelaxation in rabbits.
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1. It was recently suggested that an endogenous cannabinoid could represent an endothelium-derived hyperpolarizing factor (EDHF). The aim of the present study was to clarify whether CB1 cannabinoid receptors are involved in the nitric oxide (NO)- and prostanoid-independent vasodilation produced by acetylcholine in rabbits. 2. Pithed rabbits received indomethacin. Noradrenaline was infused to raise blood pressure, and vasodilation was elicited by bolus injections of acetylcholine. The NO-synthase inhibitor Nomega-nitro-L-arginine methylester inhibited the acetylcholine-evoked vasodilation by about 40%. The remaining vasodilation was unaffected by the CB1 cannabinoid receptor antagonist SR141716A, but was inhibited by the potassium channel blocker tetraethylammonium. In addition, the mixed CB1/CB2 cannabinoid receptor agonist WIN55212-2 did not elicit vasodilation. 3. No CB1 cannabinoid receptors were involved in the prostanoid- and NO-independent vasodilation produced by acetylcholine. An exogenous cannabinoid also did not cause vasodilation. Therefore, it is unlikely that an endogenous cannabinoid serves as an EDHF acting at smooth muscle CB1 cannabinoid receptors in the rabbit. (+info)
Relationship between NaF- and thapsigargin-induced endothelium-dependent hyperpolarization in rat mesenteric artery.
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1. In isolated rat mesenteric artery with endothelium, NaF caused slowly developing hyperpolarization. The hyperpolarizing effect was unchanged in the presence of N(G)-nitro-L-arginine (L-NOARG) and indomethacin, but was markedly reduced by high K+. In Ca2+ -free medium or in the presence of Ni2+, NaF failed to produce hyperpolarization. 2. NaF-induced hyperpolarization was substantially unaffected by deferoxamine, an Al3+ chelator, okadaic acid and calyculin A, phosphatase inhibitors, and preincubation with pertussis toxin, suggesting that neither the action of fluoroaluminates as a G protein activator nor inhibition of phosphatase activity contributes to the hyperpolarizing effect. 3. The selective inhibitors of the Ca2+ -pump ATPase of endoplasmic reticulum, thapsigargin and cyclopiazonic acid, elicited hyperpolarization, whose properties were very similar to those of NaF. When intracellular Ca2+ stores had been depleted with these inhibitors, NaF no longer generated hyperpolarization. 4. In Ca2+ -free medium, NaF (or thapsigargin) caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells, and subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i. 5. In arterial rings precontracted with phenylephrine, NaF produced endothelium-dependent relaxation followed by sustained contraction even in the presence of L-NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid. 6. These results indicate that NaF causes endothelium-dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid. (+info)