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1.
Abstract. It is suggested that increased levels of free cytosolic calcium ([Ca2+]cyt) may serve as the primary physiological transducer of chilling injury in plants. Numerous similarities between the effects of [Ca2+]cyt-raising treatments on plants and the effects of chilling temperatures on chilling-sensitive (CS) plants are noted. It is proposed that chilling temperatures may lead to increases in [Ca2+]cyt in CS plant cells by reducing the rate at which they exclude Ca2+ from their cytosol and that rapid cooling (coldshock) may cause rapid increases in [Ca2+]cyt due to the activation of voltage-dependent cation channels. Chill-induced increases in [Ca2+]cyt in the cells of CS plants may reflect either an inherent inability of such plants to maintain homeostatic levels of Ca2+ at low temperatures or a stress-induced reaction which has evolved to enable such cells to cope more effectively with the short-term hardships imposed by cold. Previous proposals concerning the physiological transduction of chilling injury are also discussed. It is argued that there is little evidence to suggest that the immediate effects of low temperatures on CS cells include either decreases in ATP levels, general increases in the passive permeability of membranes, or increased rates of fermentation.  相似文献   

2.
Abstract: Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca2+ concentration ([Ca2+]i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca2+ influx brought about by K+ depolarization. Similar effects were obtained with the application of SS analogues, such as d -Trp8-SS, d -Trp8- d -Cys14-SS, CGP-23996, and SMS-201995. In addition, treatment with cyclo-SS, a SS antagonist, did not alter [Ca2+]i. Experiments with selective blockers of different voltage-dependent Ca2+ channels, such as methoxyverapamil (D600) and Ω-conotoxin GVIA, demonstrated that the effects of SS on [Ca2+]i were mediated by voltage-dependent Ca2+ channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activity by altering Ca2+ influx.  相似文献   

3.
Free cytosolic Ca2+ ([Ca2+]cyt) is an ubiquitous second messenger in plant cell signaling, and [Ca2+]cyt elevation is associated with Ca2+-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca2+ channels and their regulation remains limited in planta . A type of voltage-dependent Ca2+-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba2+ and Ca2+, and their activities can be inhibited by micromolar Gd3+. The unitary conductance and the reversal potential of the channels depend on the Ca2+ or Ba2+ gradients across the plasma membrane. The inward whole-cell Ca2+ (Ba2+) current, as well as the unitary current amplitude and NPo of the single Ca2+ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.  相似文献   

4.
Abstract: We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca2+ concentration ([Ca]in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]in was dependent on external Ca2+, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca2+ channel blockers. ATP was effective in increasing [Ca]in in the presence of a desensitizing concentration of nicotine (100 µ M ), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na+ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na+ influx through P2 purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca2+ influx through voltage-dependent Ca2+ channels. However, ATP still caused a small increase in [Ca]in under Na+-free conditions, and this [Ca]in increase was little affected by Ca2+ channel blockers. ATP also increased Mn2+ influx under Na+-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca2+ but also to Mn2+, in addition to monovalent cations.  相似文献   

5.
In freshwater (FW) rainbow trout Oncorhynchus mykiss of spontaneously low plasma calcium concentrations ([Ca2+]pl), plasma melatonin at night was significantly lower than that measured in FW fish with the highest [Ca2+]pl. In brackish water adapted rainbow trout with originally high [Ca2+]pl, plasma melatonin concentration at night was elevated. In cannulated flounder Platichthys flesus , night plasma melatonin increases (ΔMel) corresponded to [Ca2+]pl. It is postulated that in physiological steady-state conditions, melatonin synthesis capacity is coupled to free calcium concentration in plasma of O. mykiss and P. flesus .  相似文献   

6.
Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na+ ([Na+]i) and intracellular Ca2+ ([Ca2+]i) concentrations, and basal glutamate release of synaptosomes. Both H2O2 and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na+]i over an incubation period of 15 min. A steady rise of the [Ca2+]i due to peroxides was also observed that was external Ca2+ dependent and detected only at an inwardly directed Ca2+ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H2O2. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.  相似文献   

7.
Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.  相似文献   

8.
Abstract: The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high-K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin-sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.  相似文献   

9.
Abstract: Hypoxia (5% O2) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca2+ concentration ([Ca2+]i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca2+. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca2+]i increase, showing that the Ca2+ influx was attributable to L- and N-type voltage-dependent Ca2+ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O2). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K+ current. Among the K+ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K+ channels, inhibited the hypoxia-induced [Ca2+]i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K+ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K+ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.  相似文献   

10.
Protoplasts were isolated from leaves of tomato seedlings ( Lycopersicon esculentum Mill., cv. Marmande) at the 2nd to 4th true leaf stage and were loaded with the calcium binding tetra[acetoxymethyl+] ester of the fluorescent stilbene chromophore, Fura 2. Although the loading efficiency of the dye in these protoplasts was low, many protoplasts loaded only in the cytosol were always obtained. Changes in the cytosolic calcium concentration ([Ca2+]cyt) were determined in single protoplasts in a temperature-controlled perfusion chamber by use of fluorescence photometry microscopy after excitation at 340 and 380 nm. When the protoplasts were subjected to chilling temperatures (10–15°C) by a circulating solution, the [Ca2+]cyt increased in 64% of the analysed protoplasts. Depending on the initial resting level of [Ca2+]cyt, three main types of kinetics were obtained in these protoplasts: (1) In 21% of the protoplasts, [Ca2+]cyt increased to a maximum within 10–20 s from the start of temperature decrease, followed by a fast decrease; (2) in 11% of the protoplasts, the [Ca2+]cyt both increased and decreased somewhat slower; and (3) in 32% a constant increase of [Ca2+]cyt was obtained 1 min after the start of temperature decrease.  相似文献   

11.
Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca2+]i were increased by 0.3–100 µ M cyclothiazide, with an EC50 value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca2+]i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca2+]i to both kainate and AMPA in the absence of extracellular Na+, and these Na+-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC50 value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg2+]i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.  相似文献   

12.
Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca2+ medium; whereas in the presence of Ca2+, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca2+ ([Ca2+]c). The plateau component of the increase can be inhibited additively by the L-type Ca2+ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca2+]c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca2+ dependent for the first 6–10 min after the evoked increase in [Ca2+]c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.  相似文献   

13.
Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca2+, and the demonstration of an increase in phospholipase A2 (PLA2) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA2 in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca2+]i triggered by Et-1, whereas valinomycin, which causes K+ efflux, not only caused a rapid rise in [Ca2+]i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA2 in this cell type requires calcium influx dependent on K+ efflux.  相似文献   

14.
Abstract: Inositol phosphate accumulation on carbachol stimulation of rat cerebellar granule cells shows a marked dependence on factors affecting cytosolic Ca2+ concentration ([Ca2+]c). After 5 min, potassium depolarisation caused a modest accumulation of inositol phosphates but augmented the response to carbachol by a factor of 2–3. These effects of potassium were dependent on an extracellular source of calcium and could be partially blocked by specific (nifedipine) and nonspecific (verapamil) calcium channel blockers. Measurements of [Ca2+]c under a range of stimulatory conditions demonstrated a close correlation between the elevation of [Ca2+]c and agonist-stimulated phospholipase C (PLC) activity. The maximal potentiation of carbachol-stimulated inositol phosphate accumulation was achieved using 20 m M KCl, which increased [Ca2+]c from ∼20 to ∼75 n M , indicating the involvement of relatively low threshold Ca2+ channels and the high sensitivity of the relevant PLC to small changes in [Ca2+]c. By contrast, increases in [Ca2+]c induced by the Ca2+ ionophore ionomycin were associated with more modest and less potent effects on agonist-stimulated PLC. These results demonstrate a cooperative interaction between a receptor/G protein-regulated PLC and voltage-stimulated elevations of [Ca2+]c, which may function to integrate ionotropic and metabotropic signalling mechanisms in cerebellar granule cells.  相似文献   

15.
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   

16.
In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca2+]i (the intracellular free Ca2+ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca2+]i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca2+]i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca2+-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca2+]i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca2+-independent reaction downstream of Ca2+-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.  相似文献   

17.
Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca2+]i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca2+]i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP3) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca2+]i rise, though it did not affect agonist-induced IP3 generation. However, the addition of atropine at plateau phases of CCh-induced [Ca2+]i rise and IP3 production caused a rapid decline to the basal levels and then restored the [Ca2+]i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP3 production. However, the [Ca2+]i rise induced by both agonists in the presence or absence of extracellular Ca2+ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP3-sensitive Ca2+ stores are shared by signal pathways from both receptors.  相似文献   

18.
Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+]i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca2+ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.  相似文献   

19.
Vitamin D3 at low concentration (10−9 M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [3H]-leucine incorporation into protein (2 h), and increased their labelling with 45Ca2+ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D3 stimulation of root 45Ca2+ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10−5JW) induced similar changes both in root elongation and 45Ca2+ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca2+ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca2+ transport.  相似文献   

20.
Abstract: Bradykinin (BK) receptor and P2-purinergic receptor are known to be coupled to phospholipase C (PLC) in PC12 cells. To study the interaction between these two PLC-linked receptors, the presence of both receptors on individual cells was demonstrated by sequential Ca2+ spikes caused by BK and ATP in a single fura-2-loaded cell. BK- and ATP-induced catecholamine (CA) secretions were desensitized within 5 min. However, in the sequential experiment, the BK-induced homologous desensitization of CA secretion did not block the ATP-induced secretion, and vice versa. Each agonist-induced an increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular free Ca2+ concentration also led to homologous desensitization. However, there was no heterologous desensitization between the two agonists. When the cells were treated with both BK and ATP simultaneously, the amounts of CA secretion, IP3 production, internal Ca2+ mobilization, and Ca2+ influx were all additive. We also found that both IP3-induced Ca2+ release from intracellular Ca2+ stores and Ca2+ influx from extracellular space were able to release [3H]norepinephrine, and the secretion induced by both agonists was exactly additive in the absence or presence of extracellular Ca2+. The data suggest that the CA secretions caused by BK or ATP may have separate secretory pathways even though they activate identical second messenger pathways.  相似文献   

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