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1.
Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a ''Ca2+ wave''. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.  相似文献   

2.
In order to evaluate the contribution of pinocytosis to basal (no agonist) and lanthanide-insensitive store-activated Ca2+ inflow in freshly-isolated rat hepatocytes, the uptake of extracellular fluid by pinocytosis was measured at 20°C and used to predict the amount of extracellular Ca2+ taken up by pinocytosis. This was compared with the measured rate of Ca2+ uptake in the basal state, and with the measured lanthanide-insensitive component of divalent cation uptake stimulated by 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the smooth endoplasmic reticulum (Ca2+ + Mg2+)ATP-ase. Fluid uptake by pinocytosis was measured using [14C]sucrose. In hepatocytes incubated at 20°C, DBHQ increased the initial rate of sucrose uptake by about 35%. The data for sucrose uptake were used to calculate the volume of extracellular fluid taken up by pinocytosis which, in turn, was used to predict the amount of extracellular Ca 2+ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20°C by measuring the uptake of 45Ca2+. The degree of stimulation of Ca2+ inflow by DBHQ and the lanthanide-insensitive component of DBHQ-stimulated divalent cation inflow were determined by measuring the rate of Mn2+-induced quenching of intracellular quin-2 in the absence of an agonist, and in the presence of DBHQ or DBHQ plus Gd3+. It was calculated that the process of pinocytosis accounts for at least 15% of Ca2+ uptake in the basal (no agonist) state, and for about 10% of DBHQ-stimulated lanthanide-insensitive Ca2+ uptake. It is concluded that in isolated hepatocytes (i) the release of Ca2+ from intracellular stores stimulates pinocytosis and (ii) the process of pinocytosis can account for a substantial proportion of basal Ca2+ inflow and a small proportion of DBHQ-stimulated lanthanide-insensitive Ca2+ inflow.Abbreviations RACC receptor-activated Ca2+ channel - DBHQ 2,5-di-tert-butylhydroquinone - [Ca2+] intracellular free Ca2+ concentration  相似文献   

3.
Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca2+-ATPase, ATP-dependent Ca2+ transport and Na+/Ca2+ exchange have been studied with special emphasis on the relative transport capacities of the two Ca2+ transport systems. The kinetic parameters of Ca2+-ATPase activity in digitonin-treated membranes are:K m =0.11 m Ca2+ andV max=81±4 nmol Pi/min·mg protein at 37°C. ATP-dependent Ca2+ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca2+/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca2+ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca2+ transport, a stoichiometry of 0.7 mol Ca2+ transported per mol ATP is found for the Ca2+-ATPase. In the presence of 75mm Na+ in the incubation medium ATP-dependent Ca2+ uptake is inhibited 22%. When Na+ is present at 5mm an extra Ca2+ accumulation is observed which amounts to 15% of the ATP-dependent Ca2+ transport rate. This extra Ca2+ accumulation induced by low Na+ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na+–K+)-ATPase generates a Na+ gradient favorable for Ca2+ accumulation via the Na+/Ca2+ exchanger. In the absence of ATP, a Na+ gradient-dependent Ca2+ uptake is measured which rate amounts to 5% of the ATP-dependent Ca2+ transport capacity. The Na+ gradient-dependent Ca2+ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na+/Ca2+ exchange system for Ca2+ is between 0.1 and 0.2 m Ca2+, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca2+ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca2+-ATPase system exceeds the capacity of the Na+/Ca2+ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca2+ homeostasis of rat kidney cortex cells.  相似文献   

4.
The cell-wall lysin in gametes from Chlamydomonas reinhardii which under normal mating conditions is activated by flagellar cell contact was found to be susceptible to stimulation by the antibiotic ionophore A 23187 provided that CA2+ was included in the medium. Ionophore-induced release of the cell-wall lysin did not deend on the mating type or the gametic state of the cells. Vegetative cells which normally do not exhibit any mating capacity reacted with cell-wall lysis like gametes stimulated by cell contact.Ionophore-dependent Ca2+-transfer across the cell membranes generated a signal for cell-wall lysis only in cells with intact flagella. Deflagellated cells did not respond to A 23187 before regeneration of the amputated organelles. Another indication for a possible role of flagella in Ca2+-mediated cell-wall lysis was obtained from a conditional flagellar-assembly mutant of C. reinhardii which had been isolated and described by Huang et al. (1977). Upon shift-up the mutant strain immediately became unresponsive to A 23187 and Ca2+ but regained susceptibility soon after being retransferred to permissive conditions (20°C).  相似文献   

5.
A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans.  相似文献   

6.
Divalent cation (Mn2+, Ca2+) entry into rat parotid acinar cells is stimulated by the release of Ca2+ from the internal agonist-sensitive Ca2+ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn2+ influx into internal Ca2+ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca2+-free medium for 10 min) and passive 45Ca2+ influx in basolateral membrane vesicles (BLMV). Mn2+ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn2+ entry appeared to be inactivated since it was not increased by raising extracellular [Mn2+] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn2+ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q10=1.7) at 21–37°C to 25 kcal/mol (Q10=3.0) at 21-8°C. Under the same conditions, Mn2+ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E a of 7.8 kcal/mol (Q10=1.5) and 6.2 kcal/mol (Q10 < 1.5), respectively. These data suggest that depletion-activated Mn2+ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn2+ entry into unstimulated acini.As in intact acini, Ca2+ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca2+ influx in BLMV at 37°C demonstrated the presence of two Ca2+ influx components: a saturable component, with K Ca =279 ± 43 m, Vmax = 3.38 ± 0.4 nmol Ca2+/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but Vmax decreased by 61% to 1.3 ± 0.4 nmol Ca2+/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca2+ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca2+ uptake at 37°C there was no significant loss of Ca2+ influx. These data suggest that the temperature sensitive high affinity Ca2+ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca2+ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work.  相似文献   

7.
Cardiac plasma membrane Ca2+/Mg2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca2+ or Mg2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca2+/Mg2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca2+/Mg2+ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca2+-pump ATPase or sarcolemmal Ca2+-pump ATPase. On the other hand, the cardiac Ca2+/Mg2+ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca2+-pump ATPase or plasma membrane Ca2+-pump ATPase. Furthermore, the immune serum inhibited the Ca2+/Mg2+ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca2+/Mg2+ ecto-ATPase antibodies indicated the localization of Ca2+/Mg2+ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca2+/Mg2+ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca2+/Mg2+ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.  相似文献   

8.
The present studies were conducted to investigate the mechanisms underlying the 1,25-dihydroxycholecalciferol (1,25(OH)2D3)-induced increase in intracellular Ca2+ ([Ca2+] i ) in individual CaCo-2 cells. In the presence of 2mm Ca2+, 1,25(OH)2D3-induced a rapid transient rise in [Ca2+] i in Fura-2-loaded cells in a concentration-dependent manner, which decreased, but did not return to baseline levels. In Ca2+-free buffer, this hormone still induced a transient rise in [Ca2+] i , although of lower magnitude, but [Ca2+] i then subsequently fell to baseline. In addition, 1,25(OH)2D3 also rapidly induced45Ca uptake by these cells, indicating that the sustained rise in [Ca2+] i was due to Ca2+ entry. In Mn2+-containing solutions, 1,25(OH)2D3 increased the rate of Mn2+ influx which was temporally preceded by an increase in [Ca2+] i . The sustained rise in [Ca2+] i was inhibited in the presence of external La3+ (0.5mm). 1,25(OH)2D3 did not increase Ba2+ entry into the cells. Moreover, neither high external K+ (75mm), nor the addition of Bay K 8644 (1 μm), an L-type, voltage-dependent Ca2+ channel agonist, alone or in combination, were found to increase [Ca2+] i , 1,25(OH)2D3 did, however, increase intracellular Na+ in the absence, but not in the presence of 2mm Ca2+, as assessed by the sodium-sensitive dye, sodium-binding benzofuran isophthalate. These data, therefore, indicate that CaCo-2 cells do not express L-type, voltage-dependent Ca2+ channels. 1,25(OH)2D3 does appear to activate a La3+-inhibitable, cation influx pathway in CaCo-2 cells.  相似文献   

9.
Ursula Meindl 《Protoplasma》1982,110(2):143-146
Summary Developing cells ofMicrasterias denticulata Bréb. show a characteristic fluorescence of the plasma membrane (or cortical protoplasm) after treatment with chlorotetracycline (CTC), which is known to be an indicator for membrane-bound Ca2+. Depending on the stage of development the fluorescing sites of the young half cell are distributed in a specific pattern which corresponds to cell pattern formation. Therefore growth and thus cytomorphogenesis inMicrasterias seem to be mediated by a patterned accumulation of Ca2+ at the periphery of the differentiating cell. Participation of Ca2+ in a membrane-recognition process responsible for local vesicle incorporation is discussed.  相似文献   

10.
In isolated scale melanophores ofLabeo rohita the melanosome aggregating effect of K+ was inhibited in Ca2+ deprived medium. Moreover, the Ca2+-antagonists, verapamil and lanthanum inhibited the action of K+ in concentration dependent manner. The elevation of extracellular Ca2+ could compromise the verapamil induced inhibition in a concentration dependent manner. The cation Ca2+ appeared to be required only for K+ -induced aggregation and not melanosome aggregationper se, as in this fish adrenaline and melanin concentrating hormone effectively caused aggregation of melanosomes in Ca2+ free medium  相似文献   

11.
There is evidence for a role of increased cytoplasmic Ca2+ in the stomatal closure induced by abscisic acid (ABA), but two points of controversy remain the subject of vigorous debate—the universality of Ca2+ as a component of the signaling chain, and the source of the increased Ca2+, whether influx across the plasmalemma, or release from internal stores. We have addressed these questions by patch-clamp studies on guard cell protoplasts of Vicia faba, assessing the effects of ABA in the presence and absence of external Ca2+, and of internal Ca2+ buffers to control levels of cytoplasmic Ca2+. We show that ABA-induced reduction of the K+ inward rectifier can occur in the absence of external Ca2+, but is abolished when Ca2+ buffers are present inside the cell. Thus, some minimum level of cytoplasmic Ca2+ is a necessary component of the signaling chain by which ABA decreases the K+ inward rectifier in stomatal guard cells, thus preventing stomatal opening. Release of Ca2+ from internal stores is capable of mediating the response, in the absence of any Ca2+ influx from the extracellular medium. The work also shows that enhancement of the K+ outward rectifier by ABA is Ca2+ independent, and that other signaling mechanisms must be involved. A role for internal pH, as suggested by H.R. Irving, C.A. Gehring and R.W. Parish (Proc. Natl. Acad. Sci. USA 89:1790–1794, 1990) and M.R. Blatt (J. Gen. Physiol. 99:615–644, 1992), is an attractive working hypothesis.  相似文献   

12.
Hubert Felle 《Planta》1988,176(2):248-255
In cells of Zea mays (root hairs, coleoptiles) and Riccia fluitans (rhizoids, thalli) intracellular Ca2+ and pH have been measured with double-barrelled microelectrodes. Free Ca2+ activities of 109–187 nM (Riccia rhizoids), 94–160 nM (Riccia thalli), 145–231 nM (Zea root hairs), 84–143 nM (Zea coleoptiles) were found, and therefore identified as cytoplasmic. In a few cases (Riccia rhizoids), free Ca2+ was in the lower millimolar range (2.3±0.8 mM). A change in external Ca2+ from 0.1 to 10 mM caused an initial and short transient increase in cytoplasmic free Ca2+ which finally levelled off at about 0.2 pCa unit below the control, whereas in the presence of cyanide the Ca2+ activity returned to the control level. It is suggested that this behaviour is indicative of active cellular Ca2+ regulation, and since it is energy-dependent, may involve a Ca2+-ATPase. Acidification of the cytoplasmic pH and alkalinization of the vacuolar pH lead to a simultaneous increase in cytoplasmic free Ca2+, while alkalinization of pHc decreased the Ca2+ activity. Since this is true for such remote organisms as Riccia and Zea, it may be concluded that regulation of cytoplasmic pH and free Ca2+ are interrelated. It is further concluded that double-barrelled microelectrodes are useful tools for investigations of intracellular ion activities in plant cells.Symbols and abbreviations m, m membrane potential difference, changes thereof - PVC polyvinylchloride  相似文献   

13.
Y. Iwadate  K. Katoh  H. Asai  M. Kikuyama 《Protoplasma》1997,200(3-4):117-127
Summary The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca2+ level in each cell. However, these putative increases in cytosolic Ca2+ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca2+ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca2+ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca2+ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca2+ rise inDidinium, theParamecium also showed an increase in cytosolic Ca2+. This is the first simultaneous recording of changes in the Ca2+ level during a predator-prey interaction in ciliates. The possible roles of these Ca2+ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.Abbreviations AED aminoethyldextran - Pi inorganic phosphate - FITC fluorescein isothiocyanate  相似文献   

14.
Summary In order to demonstrate the presence of a Ca2+-activated Cl-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca2+ concentration was modified by internal perfusion. An increase in the Ca2+ concentration caused a large Cl efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca2+ concentration was about 4.0 m. Neither the Cl efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca2+ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca2+-dependent Cl-channel.  相似文献   

15.
Tip-growing organisms maintain an apparently essential tip-high gradient of cytoplasmic Ca2+. In the oomycete Saprolegnia ferax, in pollen tubes and root hairs, the gradient is produced by a tip-localized Ca2+ influx from the external medium. Such a gradient is normally dispensable for Neurospora crassa hyphae, which may maintain their Ca2+ gradient by some form of internal recycling. We localized Ca2+ in N. crassa hyphae at the ultrastructural level using two techniques (a) electron spectroscopic imaging of freeze-dried hyphae and (b) pyroantimoniate precipitation. The results of both methods support the presence of Ca2+ in the wall vesicles and Golgi body equivalents, providing a plausible mechanism for the generation and maintenance of the gradient by Ca2+ shuttling in vesicles to the apex, without exogenous Ca2+ influx. Ca2+ sequestration into the vesicles seems to be dependent on Ca2+–ATPases since cyclopiazonic acid, a specific inhibitor of Ca2+ pumps, eliminated all Ca2+ deposits from the vesicles of N. crassa.  相似文献   

16.
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   

17.
Y. Tominaga  M. Tazawa 《Protoplasma》1981,109(1-2):103-111
Summary The effect of the intracellular concentration of Ca2+ on the cytoplasmic streaming of tonoplast-free cells ofChara australis was studied by intracellular perfusion. The perfusion media contained 1 mM Mg · ATP. Both cell ends were cut and left open. Media of different Ca2+ concentrations were perfused through the cell and the rate of the cytoplasmic streaming just after perfusion was measured. The critical concentration of Ca2+ for inhibiting the streaming was 5 × 10–4M, which was substantially higher than that found earlier byWilliamson (1975) andHayama et al. (1979). Recovery from the inhibition occurred, though not completely, by removing Ca2+.In tonoplast-free cells the Ca2+ sensitivity differed according to the culture conditions. Cells cultured indoors exhibited a higher sensitivity than those cultured outdoors. Theformer cells contained granule-rich endoplasm aggregates after loss of the tonoplast, while the latter cells did no such aggregates. The aggregates were fixed to the cortical gel with a high dosage of Ca2+ and freed by removing it.  相似文献   

18.
Summary The flow of calcium ions from the stigma to germinating pollen was studied by autoradiography in Primula officinalis (dry stigma) and Ruscus aculeatus (wet stigma). 45Ca2+ ions were observed to be taken up by the pistils from an agar medium and then transported intracellularly to both the stigmal cells and the stigmal exudate. The 45Ca2+ present in the stigma was taken up by the germinating pollen grains.  相似文献   

19.
Measurements of Ca2+ influx and [Ca2+]i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca2+ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca2+]i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca2+]i levels were dependent on extracellular Ca2+. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca2+ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca2+ channels. The trivalent cation La3+, a non-specific blocker of plasma membrane Ca2+ channels, eliminated the rPTTH-stimulated increase of [Ca2+]i levels in PG cells and so did amiloride, an inhibitor of T-type Ca2+ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca2+]i levels, which was also dependent on extracellular Ca2+ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca2+ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca2+]i levels and one agent’s mediated increase of [Ca2+]i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca2+ release from inositol 1,4,5 trisphosphate (IP3)-sensitive Ca2+ stores, blocked the rPTTH-stimulated increases of [Ca2+]i levels, suggesting an involvement of IP3 in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca2+]i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca2+]i levels, filling state of IP3-sensitive intracellular Ca2+ stores and the PTTH-receptor’s-mediated Ca2+ influx.  相似文献   

20.
The changes in cytosolic Ca2+ levels play important roles in the signal transduction pathways of many environmental and developmental stimuli in plants and animals. We demonstrated that the increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) of Arabidopsis thaliana leaf cells was induced by exogenous application of jasmonic acid (JA). The elevation of [Ca2+]cyt was detected within 1 min after JA treatment by the fluorescence intensity using laser scanning confocal microscopy, and the elevated level of fluorescence was maintained during measuring time. With pretreatment of nifedipine (Nif), a nonpermeable L-type channel blocker, the fluorescence of [Ca2+]cyt induced by JA was inhibited in a dose-dependent manner. In contrast, verapamil, another L-type channel blocker, had no significant effect. Furthermore, Nif repressed JA-induced gene expression of JR1 but verapamil did not. JA-induced gene expression could be mimicked by higher concentration of extracellular Ca2+. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an antagonist of calmodulin (CaM), blocked the JA induction of JR1 expression while W-5 [N-(6-aminohexyl)-1-naphthalenesulfonamide], its inactive antagonist, had no apparent effect. These data provide the evidence that the influx of extracellular Ca2+ through Nif sensitive plasma membrane Ca2+ channel may be responsible for JA-induced elevation of [Ca2+]cyt and downstream gene expression, CaM may be also involved in JA signaling pathway.  相似文献   

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