Acutely dissociated cell bodies of mouse Purkinje neurons spontaneously fired action potentials at approximately 50 Hz (25 degrees C). To directly measure the ionic currents underlying spontaneous activity, we voltage-clamped the cells using prerecorded spontaneous action potentials (spike trains) as voltage commands and used ionic substitution and selective blockers to isolate individual currents. The largest current flowing during the interspike interval was tetrodotoxin-sensitive sodium current (approximately -50 pA between -65 and -60 mV). Although the neurons had large voltage-dependent calcium currents, the net current blocked by cobalt substitution for calcium was outward at all times during spike trains. Thus, the electrical effect of calcium current is apparently dominated by rapidly activated calcium-dependent potassium currents. Under current clamp, all cells continued firing spontaneously (though approximately 30% more slowly) after block of T-type calcium current by mibefradil, and most cells continued to fire after block of all calcium current by cobalt substitution. Although the neurons possessed hyperpolarization-activated cation current (Ih), little current flowed during spike trains, and block by 1 mM cesium had no effect on firing frequency. The outward potassium currents underlying the repolarization of the spikes were completely blocked by 1 mM TEA. These currents deactivated quickly (<1 msec) after each spike. We conclude that the spontaneous firing of Purkinje neuron cell bodies depends mainly on tetrodotoxin-sensitive sodium current flowing between spikes. The high firing rate is promoted by large potassium currents that repolarize the cell rapidly and deactivate quickly, thus preventing strong hyperpolarization and restoring a high input resistance for subsequent depolarization. (+info)
Cyanide poisoning: pathophysiology and treatment recommendations.
(2/2206)
This paper aims to assess and compare currently available antidotes for cyanide poisoning. Such evaluation, however, is difficult. Thus, extrapolation from the results of animal studies has potential pitfalls, as significant inter-species differences in response may exist, and these experiments often involve administration of toxin and antidote almost simultaneously, rather than incorporating a more realistic time delay before initiation of treatment. Direct inference from human case reports is also problematic; either because of uncertainties over the exposure levels involved (and hence the likely outcome without treatment), or because of difficulties in identifying the specific contribution of a particular antidote within the overall treatment regimen. Certainly an effort to compare the relative efficacy of cyanide antidotes produces equivocal findings, with no single regimen clearly standing out. Indeed, factors such as the risks of antidote toxicity to various individuals and other practical issues, may be more important considerations. There is therefore no single treatment regimen which is best for all situations. Besides individual risk factors for antidote toxicity, the nature of the exposure and hence its likely severity, the evolving clinical features and the number of persons involved and their proximity to hospital facilities, all need to be considered. Clinically mild poisoning may be treated by rest, oxygen and amyl nitrite. Intravenous antidotes are indicated for moderate poisoning. Where the diagnosis is uncertain, sodium thiosulphate may be the first choice. With severe poisoning, an additional agent is required. Given the various risks with methaemoglobin formers or with unselective use of kelocyanor, hydroxocobalamin may be preferred from a purely risk-benefit perspective. However the former alternatives will likely remain important. (+info)
Source of inappropriate receptive fields in cortical somatotopic maps from rats that sustained neonatal forelimb removal.
(3/2206)
Previously this laboratory demonstrated that forelimb removal at birth in rats results in the invasion of the cuneate nucleus by sciatic nerve axons and the development of cuneothalamic cells with receptive fields that include both the forelimb-stump and the hindlimb. However, unit-cluster recordings from primary somatosensory cortex (SI) of these animals revealed few sites in the forelimb-stump representation where responses to hindlimb stimulation also could be recorded. Recently we reported that hindlimb inputs to the SI forelimb-stump representation are suppressed functionally in neonatally amputated rats and that GABAergic inhibition is involved in this process. The present study was undertaken to assess the role that intracortical projections from the SI hindlimb representation may play in the functional reorganization of the SI forelimb-stump field in these animals. The SI forelimb-stump representation was mapped during gamma-aminobutyric acid (GABA)-receptor blockade, both before and after electrolytic destruction of the SI hindlimb representation. Analysis of eight amputated rats showed that 75.8% of 264 stump recording sites possessed hindlimb receptive fields before destruction of the SI hindlimb. After the lesions, significantly fewer sites (13.2% of 197) were responsive to hindlimb stimulation (P < 0.0001). Electrolytic destruction of the SI lower-jaw representation in four additional control rats with neonatal forelimb amputation did not significantly reduce the percentage of hindlimb-responsive sites in the SI stump field during GABA-receptor blockade (P = 0.98). Similar results were obtained from three manipulated rats in which the SI hindlimb representation was silenced temporarily with a local cobalt chloride injection. Analysis of response latencies to sciatic nerve stimulation in the hindlimb and forelimb-stump representations suggested that the intracortical pathway(s) mediating the hindlimb responses in the forelimb-stump field may be polysynaptic. The mean latency to sciatic nerve stimulation at responsive sites in the GABA-receptor blocked SI stump representation of neonatally amputated rats was significantly longer than that for recording sites in the hindlimb representation [26.3 +/- 8.1 (SD) ms vs. 10.8 +/- 2.4 ms, respectively, P < 0.0001]. These results suggest that hindlimb input to the SI forelimb-stump representation detected in GABA-blocked cortices of neonatally forelimb amputated rats originates primarily from the SI hindlimb representation. (+info)
Regulation of interleukin-8 expression by reduced oxygen pressure in human glioblastoma.
(4/2206)
Oxygen deprivation is an important biological feature of tumor growth. We previously showed that in glioma, anoxia increases expression of IL-8, a chemokine and angiogenic factor. Here, we analysed for the first time the biochemical mechanisms inducing the IL-8 gene upon anoxia in glioma cells, and showed that they differ from those inducing the VEGF gene. Both genes are induced in biologically and genetically heterogenous glioblastoma cell lines (LN-229, LN-Z308, U87MG, T98G), whereas, in gliosarcoma cells (D247MG), only the VEGF gene is induced. The kinetics of IL-8 and VEGF mRNA inductions differ in these cells and reoxygenation experiments showed that the induction is due to the anoxic stress per se. Furthermore, in LN-229 and LN-Z308 cell lines actinomycin D, DRB and nuclear run-on experiments showed that anoxia stimulates increased transcription of both genes. Electromobility shift assays show increased protein binding to the AP-1 site on the IL-8 promoter following anoxia treatment. Finally, in situ hybridization on glioblastoma sections shows that the in vivo expression patterns of IL-8 and VEGF genes overlap, but are not identical. Since intratumoral augmentation of IL-8 and VEGF secretion, following microenvironmental decreases in oxygen pressure, may promote angiogenesis, further definition of these pathways is essential to appropriately target them for antitumoral therapy. (+info)
Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve.
(5/2206)
1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization. (+info)
The distribution of neurons expressing calcium-permeable AMPA receptors in the superficial laminae of the spinal cord dorsal horn.
(6/2206)
The superficial dorsal horn is a major site of termination of nociceptive primary afferents. Fast excitatory synaptic transmission in this region is mediated mainly by release of glutamate onto postsynaptic AMPA and NMDA receptors. NMDA receptors are known to be Ca2+-permeable and to provide synaptically localized Ca2+ signals that mediate short-term and long-term changes in synaptic strength. Less well known is a subpopulation of AMPA receptors that is Ca2+-permeable and has been shown to be synaptically localized on dorsal horn neurons in culture (Gu et al., 1996) and expressed by dorsal horn neurons in situ (Nagy et al., 1994; Engelman et al., 1997). We used kainate-induced cobalt uptake as a functional marker of neurons expressing Ca2+-permeable AMPA receptors and combined this with markers of nociceptive primary afferents in the postnatal rat dorsal horn. We have shown that cobalt-positive neurons are located in lamina I and outer lamina II, a region strongly innervated by nociceptors. These cobalt-positive neurons colocalize with afferents labeled by LD2, and with the most dorsal region of capsaicin-sensitive and IB4- and LA4-positive afferents. In contrast, inner lamina II has a sparser distribution of cobalt-positive neurons. Some lamina I neurons expressing the NK1 receptor, the receptor for substance P, are also cobalt positive. These neurons are likely to be projection neurons in the nociceptive pathway. On the basis of all of these observations, we propose that Ca2+-permeable AMPA receptors are localized to mediate transmission of nociceptive information. (+info)
EPR characterization of axial bond in metal center of native and cobalt-substituted guanylate cyclase.
(7/2206)
The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g perpendicular;1 component and a g;1 component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of alpha-subunits of Co2+-hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3- elicited the activation of the ferric enzyme by yielding five-coordinate high spin N3- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3- binding. (+info)
Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling.
(8/2206)
It has been proposed that cells sense hypoxia by a heme protein, which transmits a signal that activates the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), thereby inducing a number of physiologically relevant genes such as erythropoietin (Epo). We have investigated the mechanism by which two heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen sensing and signaling. Two concentrations of CO (10 and 80%) suppressed the activation of HIF-1 and induction of Epo mRNA by hypoxia in a dose-dependent manner. In contrast, CO had no effect on the induction of HIF-1 activity and Epo expression by either cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane coefficient, approximately 0.5). Parallel experiments were done with 100 microM sodium nitroprusside, a nitric oxide donor. Both NO and CO inhibited HIF-1 DNA binding by abrogating hypoxia-induced accumulation of HIF-1alpha protein. Moreover, both NO and CO specifically targeted the internal oxygen-dependent degradation domain of HIF-1alpha, and also repressed the C-terminal transactivation domain of HIF-1alpha. Thus, NO and CO act proximally, presumably as heme ligands binding to the oxygen sensor, whereas desferrioxamine and perhaps cobalt appear to act at a site downstream. (+info)