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931.
I. I. Pottosin P. R. Andjus D. Vučelić G. N. Berestovsky 《The Journal of membrane biology》1993,136(2):113-124
We studied the effects of H2O/D2O substitution on the permeation and gating of the large conductance Ca2+-activated K+ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K+>Rb+≫Li+, Na+, Cs+ and Cl−. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D2O as compared to H2O. The blockade of channel conductance by cytosolic Ca2+ weakened in D2O as a result of a decrease in zero voltage Ca2+ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D2O primarily due to the change in Ca2+ binding to the channel during the activation step. The Hill coefficient for Ca2+ binding was 3 in D2O and around 1 in H2O. The values of the Ca2+ binding constant in the open channel conformation were 0.6 and 6 μm in H2O and D2O, respectively, while the binding in the closed conformation was much less affected by D2O. The H2O/D2O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large
as 60 mV with 1mm internal Ca2+. 相似文献
932.
Bo Skaaning Jensen Flemming Jessen Else K. Hoffmann 《The Journal of membrane biology》1993,131(3):161-178
Summary Net Cl– uptake as well as unidirectional36Cl influx during regulatory volume increase (RVI) require external K+. Half-maximal rate of bumetanide-sensitive36Cl uptake is attained at about 3.3mm external K+. The bumetanide-sensitive K+ influx found during RVI is strongly dependent on both Na+ and Cl–. The bumetanide-sensitive unidirectional Na+ influx during RVI is dependent on K+ as well as on Cl–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na+, K+ and Cl–. In the presence of ouabain and Ba+ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na+, 0.8 K+, 2.0 Cl– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K+ and Cl– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na+, K+, 2Cl– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K+ influx is activated. During hypotonic conditions a small bumetanide-sensitive K+ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca2+ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K+ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na+, K+, Cl– cotransport involves both Ca2+/calmodulin and protein kinase C. 相似文献
933.
Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A2 is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD4. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD4 seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl– channels, i.e., via a depolarization of the cell membrane. 相似文献
934.
Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2 总被引:1,自引:0,他引:1
Vicente Felipo Eugenio Grau María-Dolores Miñana Santiago Grisolía 《Journal of neurochemistry》1993,60(5):1626-1630
Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule-associated protein 2 (MAP-2). Activation of the N -methyl- d -aspartate (NMDA) receptor dephosphorylates MAP-2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP-2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP-2. Also, the amount of MAP-2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP-2 was decreased by ∼75% 1-2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP-2 was prevented completely by injection of 2 mg/kg MK-801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l -Carnitine, which protects rats against ammonia toxicity, also prevented MAP-2 degradation. Because activation of the NMDA receptor increases [Ca2+ ]i , we determined whether rat brain contains a Ca2+ -dependent protease that selectively degrades MAP-2. We show that there is a cytosolic Ca2+ -dependent protease that degrades MAP-2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca2+ ]i , which activates calpain I. This, in turn, selectively degrades MAP-2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed. 相似文献
935.
M. V. Catania Z. Hollingsworth J. B. Penney A. B. Young 《Journal of neurochemistry》1993,60(1):236-245
Abstract: Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A2 (PLA2) on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA2 pretreatment induced a significant increase in α-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [3H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [3H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [3H]glutamate binding site were also increased by PLA2 pretreatment. [3H]Kainate binding and NMDA-sensitive [3H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA2 effect was reversed by EGTA, the PLA2 inhibitor p-bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA2. Arachidonic acid failed to mimic the PLA2 effect on metabotropic binding. These results indicate that PLA2 can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS. 相似文献
936.
Yoichi Tamai Hisako Kojima Shintaro Saito† Kikue Uchida Rie Kitajima Hiroshi Komatsu Tsuneo Moriya‡ 《Journal of neurochemistry》1993,60(5):1854-1863
Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed. 相似文献
937.
Ronnitte S. Badar-Goffer Nicola M. Thatcher Peter G. Morris Herman S. Bachelard 《Journal of neurochemistry》1993,61(6):2207-2214
(1) The energy state and free intracellular calcium concentration ([Ca2±i) of super-fused cortical slices were measured in moderate hypoxia (~65 μM O2), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using 19F and 31P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca2±i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca2±, and a decrease in PCr level to ~25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca2±i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca2±i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca2±i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult. 相似文献
938.
Peter B. Simpson R. A. John Challiss Stefan R. Nahorski 《Journal of neurochemistry》1993,61(2):760-763
Abstract: The [Ca2+ ]1 of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K+ level), which both activate Ca2+ influx. The possibility that these stimuli activate Ca2+ -induced Ca2+ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca2+ ]1 response to K+ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca2+ ], response to K+ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K+ -evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K+ . The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K+ - and NMDA-induced elevation of [Ca2+ ]1 appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca2+ pools. 相似文献
939.
Coupling Among Energy Failure, Loss of Ion Homeostasis, and Phospholipase A2 and C Activation During Ischemia 总被引:1,自引:1,他引:0
Ken-ichiro Katsura‡ Elena B. Rodriguez de Turco† Jaroslava Folbergrov᧠Nicolas G. Bazan† Bo K. Siesjö 《Journal of neurochemistry》1993,61(5):1677-1684
Abstract— The objective of the present experiments was to correlate changes in cellular energy metabolism, dissipative ion fluxes, and lipolysis during the first 90 s of ischemia and, hence, to establish whether phospholipase A2 or phospholipase C is responsible for the early accumulation of phospholipid hydrolysis products. Ischemia was induced for 15–90 s in rats, extracellular K+ (K+ e ) was recorded, and neocortex was frozen in situ for measurements of labile tissue metabolites, free fatty acids, and diacylglycerides. Ischemia of 15-and 30-s duration gave rise to a decrease in phosphocreatine concentration and a decline in the ATP/free ADP ratio. Although these changes were accompanied by an activation of K+ conductances, there were no changes in free fatty acids until after 60s, when free arachidonic acid accumulated. An increase in other free fatty acids and in total diacylglyceride content did not occur until after anoxic depolarization. The results demonstrate that the early functional changes, such as activation of K+ conductances, are unrelated to changes in lipids or lipid mediators. They furthermore suggest that the initial lipolysis occurs via both phospholipase A2 and phospholipase C, which are activated when membrane depolarization leads to influx of calcium into cells. 相似文献
940.
Allen R. Rhoads Rabin Parui Ngoc-Diep Vu Robert Cadogan Paul D. Wagner 《Journal of neurochemistry》1993,61(5):1657-1666
Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2 -type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca2+ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg2+ , and this inhibition was overcome by the addition of excess ATP suggesting that ATP4- was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12- O -tetradecanoylphorbol 13-acetate. The stimulatory effects of 12- O -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α-32 P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP-γS and BzATP but not GTP blocked labeling of the proteins by [32 P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P2 receptor responsible for ATP-stimulated secretion in these cells. 相似文献