Block of Na+,K+-ATPase and induction of hybrid death by 4-aminopyridine in cultured cortical neurons.
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K(+) channel blockers such as 4-aminopyridine (4-AP) can be toxic to neurons; the cellular mechanism underlying the toxicity, however, is obscure. In cultured mouse cortical neurons, we tested the hypothesis that the toxic effect of 4-AP might result from inhibiting the Na(+),K(+)-ATPase (Na(+),K(+)-pump) and thereafter induction of a hybrid death of concomitant apoptosis and necrosis. The Na(+),K(+)-pump activity, monitored as whole-cell membrane currents, was markedly blocked by 4-AP in concentration- and voltage-dependent manners in low millimolar ranges. At similar concentrations, 4-AP induced a neuronal death sensitive to attenuation by the caspase inhibitor Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) or Ca(2+) chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Electron microscopy confirmed hybrid ultrastructural features of coexisting apoptotic and necrotic components in same cells. We suggest that 4-AP is a potent antagonist of the Na(+),K(+)-ATPase and an inducer of the hybrid death of central neurons. (+info)
Organic cation uptake in vitro by the rabbit iris-ciliary body, renal cortex, and choroid plexus.
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The uptake in vitro of radioactively labeled test substances was studied in tissues from albino rabbits. Choroid plexus, slices of outer renal cortex, and iris-ciliary body were incubated in a K-rich medium containing one of the cations 14C-Emepronium (Cetiprin), 14C-tetraethylammonium, 14C-choline, or 125I-o-iodobenzyltrimethylammonium and sometimes the anions 131I-o-iodohippurate and 125I-iodipamide. Choroid plexus and renal cortex accumulated all test substances, some to very high tissue-medium ratios. The iris-ciliary body preparation accumulated the anions well but the organic cations only weakly. The only convincing uptake was that of Emepronium. The affinity of this uptake system seemed to be similar to that in the kidney, half-saturating around 10(-4)M Emepronium. (+info)
Ionic selectivity of the sodium channel of frog skeletal muscle.
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The ionic selectivity of the Na channel to a variety of metal and organic cations is studied in frog semitendinosus muscle. Na channel currents are measured under voltage clamp conditions in fibers bathed in solutions with all Na+ replaced by a test ion. Permeability ratios are calculated from measured reversal potentials using the Goldman-Hodgkin-Katz equation. The permeability sequence was Na+ approximately Li+ approximately hydroxylammonium greater than hydrazinium greater than ammonium greater than guanidinium greater than K+ greater than aminoguanidinium in the ratios 1:0.96:0.94:0.31:0.11:0.093:0.048:0.031. No inward currents were observed for Ca++, methylammonium, methylguanidinium, tetraethylammonium, and tetramethylammonium. The results are consistent with the Hille model of the Na channel selectivity filter of the node of Ranvier and suggest that the selectivity filter of the two channels is the same. (+info)
Role of endothelium-derived relaxing factors in the renal response to vasoactive agents in hypothyroid rats.
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This study analyzed the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in the abnormal renal vascular reactivity of hypothyroid rats. Renal responses to vasoconstrictors [VC: phenylephrine (PHE) and ANG II] and vasodilators [VD: ACh, sodium nitroprusside (SNP), and papaverine (PV)] were studied in kidneys from control and hypothyroid rats under normal conditions and after NO or EDHF blockade. NO was blocked by the administration of Nomega-nitro-l-arginine methyl ester (l-NAME) and EDHF by the administration of tetraethylammonium (TEA) or by an increased extracellular K+. The response to VC was also evaluated after endothelium removal. Hypothyroid kidneys showed reduced responsiveness to PHE and a normal response to ANG II. l-NAME and TEA administration produced an increased sensitivity to PHE and to ANG II in control preparations. l-NAME also increased the response to PHE in hypothyroid kidneys, but the differences between control and hypothyroid kidneys were maintained. TEA administration did not change the response to either VC in hypothyroid preparations. In endothelium-removed preparations, TEA was unable to increase pressor responsiveness to VC. Hypothyroid kidneys showed reduced responsiveness to ACh and SNP and normal response to PV. The differences between hypothyroid and control preparations in the responses to ACh and SNP were maintained after l-NAME or increased K+. In conclusion, this study shows that 1) the attenuated response to PHE in hypothyroidism is not related to an increased production of endothelium-derived relaxing factors NO and EDHF; 2) the response to VC in hypothyroid preparations is insensitive to EDHF blockade; and 3) hypothyroid preparations have a reduced reactivity to the NO donor, and NO-independent vasodilatation remains unaffected. (+info)
Further studies of the potential-dependence of the sodium-induced membrane current in snail neurones.
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1. The potential-dependence of the membrane current induced by intracellular injections of sodium ions was studied on giant neurones of the snail Helix pomatia. This current decreases with membrane hyperpolarization at room temperature and can be reversed at sufficiently negative holding potentials. The same injections at 7 degrees C, as well as injections of lithium or potassium ions do not induce membrane currents and do not increase membrane conductance. 2. An increase in the amount of injected sodium changes the potential-dependence of the induced membrane currents. Small injections (about 1 muC) induce a current that does not depend upon the membrane potential. Further increase in the injection size not only increases the induced current but also enhances its potential-dependence and often reveals the existence of a reversal potential. The latter reaches -60 to -65 mV with large sodium injections. 3. An increase in extracellular potassium concentration from 4 to 8 mM shifts the reversal potential 17 mV in the depolarizing direction, and a decrease from 4 to 2 mM shifts it 14 mV in the hyperpolarizing direction. Replacement of potassium by rubidium or elimination of sodium ions from the outside solution, does not affect the induced current or its potential-dependence. 4. The coefficient of electrogenicity (the ratio between the amount of charge transferred by the sodium-induced membrane current and the amount brought into the cell during the injection) increases with an increase in the injection size if the membrane potential is clamped near the resting potential level. This relation is reversed when the holding potential is -80 mV. The reversal takes place at holding potentials near -60 mV. 5. 10 mM TEA does not affect the induced current and its potential-dependence. 6. It is suggested that the potential-dependence of the sodium-induced membrane current is a result of a specific increase in the membrane potassium conductance that is coupled with high activity of the sodium pump. (+info)
Effects of high-cholesterol diet and parallel exercise training on the vascular function of rabbit aortas: a time course study.
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It is plausible to assume that exercise training, when applied early enough, can completely correct atherosclerotic defects. Using rabbit aortic specimens, we examined the effects of chronic exercise and high-cholesterol diet feeding on vascular function for different time periods. Male New Zealand White rabbits were divided into four groups: the normal diet groups with or without exercise training and the high-cholesterol diet groups with or without exercise training. Animals in high-cholesterol diet groups were fed 2% cholesterol rabbit chow for 2, 4, or 6 wk. Those in exercise training groups ran on a treadmill at 0.88 km/h for up to 40 min/day, 5 days/wk for the same period of time as the diet feeding. Thoracic aortas were isolated for functional and immunohistochemical analyses. We found that 1). although high-cholesterol diet feeding (>or=2 wk) elevated serum cholesterol levels and impaired acetylcholine-evoked vasorelaxation, only the latter effect was reversed by exercise training; 2). the effects of diet and exercise on acetylcholine-evoked vasorelaxation were mainly due to altered release of nitric oxide and endothelium-derived hyperpolarizing factor; and 3). diet feeding for 4 or 6 wk caused significant lipid deposition and expression of P-selectin, VCAM-1, monocyte chemoattractant protein-1, and inducible nitric oxide synthase, which were largely reduced by exercise training. In conclusion, parallel exercise training almost completely reverses the early-stage endothelial dysfunction caused by high-cholesterol diet feeding. (+info)
Action potentials in chick atria. Ontogenetic changes in the dependence of tetrodotoxin-resistant action potentials on calcium, strontium, barium.
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Action potentials were recorded from chick embryo atrial muscle cells bathed in Tyrode's solution. Tetrodotoxin (TTX), 3.1 muM, was added to block the early, transient, Na+-dependent conductance system. Rectangular stimuli were used to evoke action potentials the peak amplitude (Ep) of which depend on the external concentration of divalent cations, [Me2+]0. The relationship between Ep and [Me2+]0 shifted to the right with increasing age. For example, the slope of Ep was 33 +/- 2,22 +/- 1 and 11 +/- 3 mV per 10-fold change in [Ca2+]0 on the 9th, 12th, and 18th incubation days, respectively. In solutions with reduced [Ca2+]0, Ep increased when Ba2+ or Sr2+ was added to the bath. The potency of Me2+ in generating action potentials was Ba2+ greater than Sr2+ greater than Ca2+ and this sequence did not change during development. Action potential amplitude, which was reduced in 18-day preparations, was increased by isoproterenol (increased Ca2+ conductance, gCa2+) and by tetraethylammonium (TEA) ion (decreased K+ conductance, gK). The results show that (1) Me2+-dependent action potentials support membrane excitation in chick atrial cells treated with TTX, and (2) the ability of Me2+ to support action potentials decreases during ontogenesis. We conclude from these experiments that the ontogenetically related decrease in Me2+-induced action potentials is the result of a reduction in gMe2+/gK+ during stimulation. (+info)
Three components in the light-induced current of the Limulus ventral photoreceptor.
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1. Light-induced currents were measured in Limulus ventral nerve photoreceptors using a two-electrode voltage clamp. Three kinetically distinct components in the light-induced current could be distinguished by varying the light adaptation state of the photoreceptor and the intensity of the stimulus light. 2. The components could be partly separated by choosing appropriate stimulus intensities and dark adaptation time. Thus the properties of the components could be separately studied. The first component is the first to recover after a light adaptation, appears temporally first in the light-induced response, has the lowest activation threshold and is the smallest. The second component needs a longer time to recover after an adapting illumination and its kinetics differ from that of the other components. Applying a bright stimulus to a dark-adapted cell a third component can be observed late in the response. 3. The time to peak of the first and the third components depended on the stimulus intensity, but not on the dark adaptation time. The time to peak of the second component became shorter the longer the dark adaptation time. For a constant adaptation state the time to the maximum of component 2 was independent, but those of components 1 and 3 were dependent on the membrane voltage. 4. To exclude the possibility of the contribution of voltage-gated currents, light-activated currents were measured at clamp potentials more negative than -50 mV after adding 4-aminopyridine into the bath solution or injecting tetraethyl-ammonium chloride into the cell. The properties of the three components remained unchanged under these conditions. 5. The I-V curve of the first component was flat at negative membrane potentials and had a strong outward rectification at positive membrane potentials. The I-V curve of component 3 showed a negative resistance at potentials more negative than about -30 mV. In contrast, the I-V curve for the second component was always nearly linear. 6. No membrane potential was found where the light-induced current was zero. Instead, current traces close to the reversal potential showed a complex waveform indicating different reversal potentials for the three components. 7. The results indicate that the current components are caused by three different populations of light-sensitive channels. The different activations, deactivations and recovery kinetics of the components suggest that the three types of channels are activated by distinct intracellular transmitters. (+info)