Characterization of a heme c nitrite reductase from a non-ammonifying microorganism, Desulfovibrio vulgaris Hildenborough.
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A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 micromol NH(4)(+)/min/mg and 1.0 micromol H(2)/min/mg. The enzyme was studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E(0)' of +160 mV (12% of total absorption), -5 mV (38% of total absorption), -110 mV (38% of total absorption) and -210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme-heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed. (+info)
Differences in the calcium-handling response of isolated rat and guinea-pig cardiomyocytes to metabolic inhibition: implications for cell damage.
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Species differences in response to hypoxic damage have been observed in studies using whole hearts. The aims of this study were to determine whether (i) species differences in response to simulated hypoxia could be detected at the level of the single myocyte, and (ii) there were any interspecies differences in the Ca2+ handling properties of the cells. Ventricular myocytes were isolated from hearts of adult rats and guinea-pigs and electrically stimulated on the stage of a fluorescence microscope. Cell length was measured using an edge-tracking device, and total intracellular [Ca2+] ([Ca2+]i) determined using indo-1. Cells were exposed to metabolic inhibition (MI) (2.5 mM NaCN and no glucose) to simulate hypoxia followed by washout of CN and re-addition of glucose ('reperfusion'). Following exposure to MI, rat cells underwent rigor contracture in 18.8+/-0.8 min (n = 80 cells), whereas the time was longer for guinea-pig cells (32.9+/-1.2 min, n = 83) (P<0.001). If cells were reperfused after 1-5 min in rigor, then rat cells showed improved morphological recovery compared with guinea-pig cells (P< 0.05); thereafter recovery decreased with increasing time spent in rigor, and was similar in both groups. In indo-1 loaded cells, [Ca2+]i was significantly increased in cells from both species at the end of MI; however, the actual increase was much higher in guinea-pig cells. Upon reperfusion, [Ca2+]i recovered fully in rat cells, but in guinea-pig cells there was no significant decrease. The restoration of [Ca2+]i to normal levels in rat cells following MI was associated with improved contractile recovery compared with guinea-pig cells. We conclude that rat cells are more resistant to effects of MI than are guinea-pig cells; this may be related to species differences in Ca2+ handling during and following exposure to MI. (+info)
State influences on ventral medullary surface and physiological responses to sodium cyanide challenges.
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Intravenous sodium cyanide (NaCN) administration lowers ventral medullary surface (VMS) activity in anesthetized cats. Sleep states modify spontaneous and blood pressure-evoked VMS activity and may alter VMS responses to chemoreceptor input. We studied VMS activation during peripheral chemoreceptor stimulation by intravenous NaCN using optical procedures in six cats instrumented for recording sleep physiology during sham saline and control site trials. Images of scattered 660-nm light were collected at 50 frames/s with an optical device after 80-100 microg total bolus intravenous NaCN delivery during waking and sleep states. Cyanide elicited an initial ventilatory decline, followed by large inspiratory efforts and an increase in respiratory rate, except in rapid eye movement sleep, in which an initial breathing increase occurred. NaCN evoked a pronounced decrease in VMS activity in all states; control sites and sham injections showed little effect. The activity decline was faster in rapid eye movement sleep, and the activity nadir occurred later in waking. Sleep states alter the time course but not the extent of decline in VMS activity. (+info)
Various nitric oxide donors protect chick embryonic neurons from cyanide-induced apoptosis.
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The discovery of numerous biochemical effects of cyanide not directly related to the inhibition of the respiratory chain, including the involvement of apoptosis, has challenged the basis of traditional antidote treatment, which primarily depends on nitrite-induced conversion of hemoglobin into methemoglobin, releasing the blockade of cytochrome c oxidase by high-affinity binding of cyanide as cyanmethemoglobin. The fact that amyl nitrite has antidotal effects not related to methemoglobin formation has unfolded new mechanism of actions of nitrites including release of nitric oxide (NO). In this study, we characterized the effect of various NO donor compounds on cyanide-induced cell death in cultured chick embryonic neurons. Apoptosis was induced by treating the neuronal cultures with 1 mM NaCN for 1 h, followed by a cyanide-free incubation period of 23 h. Using this treatment protocol, we showed that cyanide-induced apoptosis was blocked in the presence of the different NO donors sodium nitroprusside, S-nitrosoglutathione, S-nitroso-N-acetylpenicillamin, nitroglycerin, 3-morpholinosydnonimine, and diethylamine nitric oxide, indicating independence of the redox-related species of NO released. The effect was confirmed to be mediated by NO, since exhausted NO donors did not afford protection, and the mechanism likely involved chemical modification of thiol groups, since the effect was completely reversed by dithiothreitol. Furthermore, NMDA antagonists protected against cyanide-induced cell death, whereas inhibitors of nitric oxide synthase increased cyanide-induced apoptotic damage, indicating a protective effect of endogenously generated NO, at least in cell cultures. (+info)
Oxygen-sensing persistent sodium channels in rat hippocampus.
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1. Persistent sodium channel activity was recorded before and during hypoxia from cell-attached and inside-out patches obtained from cultured hippocampal neurons at a pipette potential (Vp) of +30 mV. Average mean current (IU) of these channels was very low under normoxic conditions and was similar in cell-attached and excised inside-out patches (-0.018 +/- 0.010 and -0.025 +/- 0.008 pA, respectively, n = 24). 2. Hypoxia increased the activity of persistent sodium channels in 10 cell-attached patches (IU increased from -0. 026 +/- 0.016 pA in control to -0.156 +/- 0.034 pA during hypoxia, n = 4, P = 0.013). The increased persistent sodium channel activity was most prominent at a VP between +70 and +30 mV (membrane potential, Vm = -70 to -30 mV) and could be blocked by lidocaine, TTX or R56865 (n = 5). Sodium cyanide (NaCN, 5 mM; 0.5-5 min) increased persistent sodium channel activity in cell-attached patches (n = 3) in a similar manner. 3. Hypoxia also increased sodium channel activity in inside-out patches from hippocampal neurons. Within 2-4 min of exposure to hypoxia, I had increased 9-fold to -0. 18 +/- 0.04 pA (n = 21, P = 0.001). Sodium channel activity increased further with longer exposures to hypoxia. 4. The hypoxia-induced sodium channel activity in inside-out patches could be inhibited by exposure to 10-100 microM lidocaine applied via the bath solution (I = -0.03 +/- 0.01 pA, n = 8) or by perfusion of the pipette tip with 1 microM TTX (I = -0.01 +/- 0.01 pA, n = 3). 5. The reducing agent dithiothreitol (DTT, 2-5 mM) rapidly abolished the increase in sodium channel activity caused by hypoxia in excised patches (I = -0.01 +/- 0.01 pA, n = 4). Similarly, reduced glutathione (GSH, 5-20 mM) also reversed the hypoxia-induced increase in sodium channel activity (IU = -0.02 +/- 0.02 pA, n = 5). 6. These results suggest that persistent sodium channels in neurons can sense O2 levels in excised patches of plasma membrane. Hypoxia triggers an increase in sodium channel activity. The redox reaction involved in increasing the sodium channel activity probably occurs in an auxiliary regulatory protein, co-localized in the plasma membrane. (+info)
A brainstem area mediating cerebrovascular and EEG responses to hypoxic excitation of rostral ventrolateral medulla in rat.
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We sought to identify the medullary relay area mediating the elevations of regional cerebral blood flow (rCBF) and synchronization of the electroencephalogram (EEG) in the rat cerebral cortex elicited by hypoxic excitation of reticulospinal sympathoexcitatory neurons of the rostral ventrolateral medulla (RVLM ). In anaesthetized spinalized rats electrical stimulation of RVLM elevated rCBF (laser-Doppler flowmetry) by 31 +/- 6 %, reduced cerebrovascular resistance (CVR) by 26 +/- 8 %, and synchronized the EEG, increasing the power of the 5-6 Hz band by 98 +/- 25 %. Stimulation of a contiguous caudal region, the medullary cerebral vasodilator area (MCVA), had comparable effects which, like responses of RVLM, were replicated by microinjection of L-glutamate (5 nmol, 20 nl). Microinjection of NaCN (300 pmol in 20 nl) elevated rCBF (17 +/- 5 %) and synchronized the EEG from RVLM, but not MCVA, while nicotine (1.2 nmol in 40 nl) increased rCBF by 13 +/- 5 % and synchronized the EEG from MCVA. In intact rats nicotine lowered arterial pressure only from MCVA (101 +/- 3 to 52 +/- 9 mmHg). Bilateral electrolytic lesions of MCVA significantly reduced, by over 59 %, elevations in rCBF and, by 78 %, changes in EEG evoked from RVLM. Bilateral electrolytic lesions of RVLM did not affect responses from MCVA. Anterograde tracing with BDA demonstrated that RVLM and MCVA are interconnected. The MCVA is a nicotine-sensitive region of the medulla that relays signals elicited by excitation of oxygen-sensitive reticulospinal neurons in RVLM to reflexively elevate rCBF and slow the EEG as part of the oxygen-conserving (diving) reflex initiated in these neurons by hypoxia or ischaemia. (+info)
Parasympathetic innervation of canine tracheal smooth muscle.
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To investigate whether efferent parasympathetic fibers to the tracheal smooth muscle course through the pararecurrent nerve rather than the recurrent or the superior laryngeal nerve, we stimulated all three nerves in anesthetized dogs. We also recorded the pararecurrent nerve activity response to bronchoconstrictor stimuli and compared it with pressure changes inside a saline-filled cuff of an endotracheal tube. Electrical stimulation (30 s, 100 Hz, 0.1 ms, 10 mA) increased tracheal cuff pressure by 21.0 +/- 3.2 and 1.3 +/- 0.7 cmH(2)O for the pararecurrent and the recurrent laryngeal nerve, respectively. Stimulation of the superior laryngeal nerve increased tracheal cuff pressure before, but not after, sectioning of the ramus anastomoticus, which connects it to the pararecurrent nerve. Intravenous administration of sodium cyanide increased pararecurrent nerve activity by 208 +/- 51% and tracheal cuff pressure by 14.4 +/- 3.5 cmH(2)O. Elevation of end-tidal PCO(2) to 50 Torr increased pararecurrent nerve activity by 49 +/- 19% and tracheal cuff pressure by 8.4 +/- 3.6 cmH(2)O. Further elevation to 60 Torr increased pararecurrent nerve activity by 101 +/- 33% and tracheal cuff pressure by 11.3 +/- 2.9 cmH(2)O. These results lead us to the conclusion that parasympathetic efferent fibers reach the smooth muscle of the canine trachea via the pararecurrent nerve. (+info)
Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C.
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When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells. (+info)