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1.
The protective effect of ADP on unspecific Ca2+ release and collapse of the transmembrane potential was analyzed in mitochondria from kidneys of rats. The presence of ADP in the incubation mixture prevents Ca2+ leakage and collapse of in sucrose-containing medium, but fails to do so in KCl medium. The effect of the adenine nucleotide in sucrose media correlates with an increase in the level of reduced pyridine nucleotides; the increase was due to a stimulatory effect on the activity of glutamic dehydrogenase. It also was observed that in KCl media, in the presence and in the absence of ADP the rate of NADH oxidation through the respiratory chain was higher than in sucrose; in this latter medium a high level of reduced pyridine nucleotides was found, in comparison to KCl media. It is proposed that the role of ADP is to increase glutamic dehydrogenase activity and in consequence to provoke a higher rate of formation of NADH which in turn controls Ca2+ release. 相似文献
2.
Kimberly M. Broekemeier Randy J. Krebsbach Douglas R. Pfeiffer 《Molecular and cellular biochemistry》1994,139(1):33-40
Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca2+ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca2+ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I50 values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca2+ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP
carbonyl cyanide p-chlorophenylhydrazone
- CSA
cyclosporin A
- Hepes
4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid 相似文献
3.
Mitochondria in Ca2+ Signaling and Apoptosis 总被引:8,自引:0,他引:8
Soraya S. Smaili Yi-Te Hsu Richard J. Youle James T. Russell 《Journal of bioenergetics and biomembranes》2000,32(1):35-46
Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injuryand programmed cell death; mitochondria play a pivotal role in the regulation of such cytosolicCa2+ ([Ca2+]c) signals. Mitochondria are endowed with multiple Ca2+ transport mechanismsby which they take up and release Ca2+ across their inner membrane. These transport processesfunction to regulate local and global [Ca2+]c, thereby regulating a number of Ca2+-sensitivecellular mechanisms. The permeability transition pore (PTP) forms the major Ca2+ effluxpathway from mitochondria. In addition, Ca2+ efflux from the mitochondrial matrix occursby the reversal of the uniporter and through the inner membrane Na+/Ca2+ exchanger. Duringcellular Ca2+ overload, mitochondria take up [Ca2+]c, which, in turn, induces opening of PTP,disruption of mitochondrial membrane potential (m) and cell death. In apoptosis signaling,collapse of ;m and cytochrome c release from mitochondria occur followed by activationof caspases, DNA fragmentation, and cell death. Translocation of Bax, an apoptotic signalingprotein from the cytosol to the mitochondrial membrane, is another step during thisapoptosis-signaling pathway. The role of permeability transition in the context of cell death in relationto Bcl-2 family of proteins is discussed. 相似文献
4.
Support from the National Institutes of Health and the American Heart Association is gratefully acknowledged. 相似文献
5.
Anantamongkol U Takemura H Suthiphongchai T Krishnamra N Horio Y 《Biochemical and biophysical research communications》2007,352(2):537-542
Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk. 相似文献
6.
Ca2+ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca2+ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca2+ ATPase, the enzyme that participates in the vectorial translocation of Ca2+ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca2+ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis. 相似文献
7.
Evidence has accrued during the past two decades that mitochondrial Ca2+ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca2+ transport systems might be able to relay the changes of cytosolic Ca2+ concentration ([Ca2+]c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca2+ concentration ([Ca2+]m) become essential. In this article, we review the results from recent studies attempting to measure [Ca2+]m in living cells. In addition, the significance of each study is discussed. 相似文献
8.
Marianne S. Jurkowitz Gerald P. Brierley 《Journal of bioenergetics and biomembranes》1982,14(5-6):435-449
A rapid loss of accumulated Ca2+ is produced by addition of H+ to isolated heart mitochondria. The H+-dependent Ca+ efflux requires that either (a) the NAD(P)H pool of the mitochondrion be oxidized, or (b) the endogenous adenine nucleotides be depleted. The loss of Ca2+ is accompanied by swelling and loss of endogenous Mg2–. The rate of H+-dependent Ca2+ efflux depends on the amount of Ca2+ and Pi taken up and the extent of the pH drop imposed. In the absence of ruthenium red the H+-induced Ca2+-efflux is partially offset by a spontaneous re-accumulation of released Ca2+. The H+-induced Ca2+ efflux is inhibited when the Pi transporter is blocked withN-ethylmaleimide, is strongly opposed by oligomycin and exogenous adenine nucleotides (particularly ADP), and inhibited by nupercaine. The H+-dependent Ca2+ efflux is decreased markedly when Na+ replaces the K+ of the suspending medium or when the exogenous K+/H+ exchanger nigericin is present. These results suggest that the H+-dependent loss of accumulated Ca2+ results from relatively nonspecific changes in membrane permeability and is not a reflection of a Ca2+/H+ exchange reaction. 相似文献
9.
Cefaratti C 《Molecular and cellular biochemistry》2007,295(1-2):241-247
Isolated hepatocytes release 2–3 nmol Mg2+/mg protein or ~10% of the total cellular Mg2+ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During
Mg2+ release, a quantitatively similar amount of Ca2+ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg2+:1Ca2+. Calcium induced Mg2+ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg2+:1Ca2+ exchange ratio. The uptake of Ca2+ for the release of Mg2+ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium
(TPP+) electrode or 3H-TPP+. Collapsing the Δψ by high concentrations of TPP+ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca2+-induced Mg2+ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca2+ uptake and Mg2+ release. These data demonstrate the operation of an electroneutral Ca2+/Mg2+ exchanger which represents a novel pathway for Ca2+ accumulation in liver cells following adrenergic receptor stimulation.
This work was supported by National Institutes of Health Grant HL 18708. 相似文献
10.
Naranjan S. Dhalla Vincenzo Panagia Naoki Makino Robert E. Beamish 《Molecular and cellular biochemistry》1988,82(1-2):75-79
In order to identify defects in Na+-Ca2+ exchange and Ca2+-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na+-dependent Ca2+ uptake, Na+-induced Ca2+ release, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na+-induced Ca2+-release. Similar results were obtained in Ca2+-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca2+ following a 5 min perfusion with Ca2+-free medium. Although a 2 min reperfusion of the Ca2+-free perfused hearts depressed sarcolemmal Ca2+-pump activities without any changes in Na+-induced Ca2+-release, Na+-dependent Ca2+ uptake was increased. These results indicate that alterations in the sarcolemmal Ca2+-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca2+ overload. 相似文献
11.
Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton. 相似文献
12.
The steady-state levels of Ca2+ within the endoplasmic reticulum (ER) and the transport of 45Ca2+ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca2+-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca2+ level in the ER was measured using the Ca2+-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca2+ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of 45Ca2+ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA3) and Ca2+. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of 45Ca2+ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that 45Ca2+ transport was into the ER. The sensitivity of 45Ca2+ transport to inhibitors and the Km of 45Ca2+ uptake for ATP and Ca2+ transport in the microsomal fraction of barley aleurone cells. The rate of 45Ca2+ transport is stimulated several-fold by treatment with GA3. This effect of GA3 is mediated principally by an effect on the activity of the Ca2+ transporter rather than on the amount of ER.Abbreviations CCR
cytochrome-c reductase
- DCCD
dicyclohexylcarbodiimide
- EGTA
ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- ER
endoplasmic reticulum
- FCCP
carbonylcyanide p-trifluoromethoxyphenyl hydrazone
- GA3
gibberellic acid
- IDPase
inosine diphosphatase
- Mon
monensin 相似文献
13.
《Molecular membrane biology》2013,30(3):167-180
A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5′-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca2+ transport activity (half-maximal inhibition at ~10 μM vanadate, corresponding to ~12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca2+ transport is noncompetitive with respect to added Ca2+ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca2+ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems. 相似文献
14.
Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves 总被引:3,自引:0,他引:3
Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(K
m
(Ca2+)=0.4 m) and ATP(K
m
(ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl– or NO
3
–
. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl– was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO
4
3–
which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl–-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl–-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl–-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves. 相似文献
15.
Effects of endotoxin administration on the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca2+ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the Vmax for ATP and for Ca2+ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca2+ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca2+ transport, but the Mg2+-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca2+. Additional experiments show that the Ca2+ sensitivity of the Ca2+-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca2+ pumping without affecting Ca2+-ATPase activity. Since sarcolemmal ATP-dependent Ca2+ transport plays an important role in the regulation of cytosolic Ca2+ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca2+ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock. 相似文献
16.
A high affinity Ca2+/Mg2+ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca2+ (1 M) believed to approximate the range of Ca2+ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca2+-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca2+-stimulated ATP hydrolysis was characterized by a reduction inV
max for Ca2+. Levorphanol pretreatment reduced the Hill coefficient (HN) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca2+ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca2+/Mg2+ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca2+-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca2+-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV
max for both Ca2+ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca2+-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca2+/Mg2+ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca2+ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca2+ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca2+ following inhibition of Ca2+/Mg2+ ATPase may underlie some of the pharmacological effects of opiate drugs. 相似文献
17.
We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca(2+) transport in vivo and in vitro. When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl(2), the matrix free Ca(2+) concentration is 200 nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca(2+) and promptly increases the value to 1.2 microM. Elevated Ca(2+) concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca(2+) when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca(2+) release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502-40509]. When a respiratory substrate is not present, Ca(2+) enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca(2+) and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K(1/2) value for Ca(2+) that is near to 900 nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca(2+) in yeast mitochondria is a function of the matrix Ca(2+) content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca(2+) transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca(2+) in yeast. 相似文献
18.
Kevin K. W. Wang Basil D. Roufogalis Antonio Villalobo 《The Journal of membrane biology》1989,112(3):233-245
Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca2+ transport function of the Ca2+-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca2+ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca2+/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca2+-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca2+ translocation function of the Ca2+-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain. 相似文献
19.
Summary Using Ca2+- and K+-selective microelectrodes, the cytosolic free Ca2+ and K+ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca2+ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca2+ concentration were observed in the submicromolar ranges. During the Ca2+ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca2+ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca2+ concentration down to 10–7
m or by applying a Ca2+ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca2+-activated K+ channel, sizable Ca2+ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K+ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K+ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca2+ level, which, in turn, periodically stimulate Ca2+-activated K+ channels. 相似文献
20.
Summary Different cytochemical methods were employed to demonstrate the existence of Ca2+-binding sites (Ca2+-bs) at the membranes of barley root tip cells, involving addition of CaCl2 (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca2+-dependent ATPase activity. Addition of 10 mM CaCl2 to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl2 electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl2) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca2+-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion. 相似文献