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1.
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   

2.
The role of calmodulin (CaM) in gibberellic acid (GA3)-stimulated Ca2+ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca2+ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA3 and Ca2+ compared with those isolated from layers incubated in Ca2+ alone. However, the level of uptake from Ca2+-treated tissue could be stimulated to that of the GA3-treated cells by applying exogenous CaM which increased the V max of the Ca2+ transporter approximately threefold. Calcium uptake in ER from GA3-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA3-treated tissue was unaffected. Treatment with GA3 also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca2+ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca2+ uptake into the ER induced by GA3 may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca2+-dependent α-amylase synthesis in the lumen of the ER.  相似文献   

3.
Russell L. Jones 《Planta》1980,150(1):70-81
Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA3) and H2O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA3, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg2+ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc-1 while that from layers incubated in GA3 for 7.5–18 h has a density of 1.11–1.12 g cc-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg2+ the second peak of activity has a density of 1.12 g cc-1 in GA3-treated tissue and 1.13–1.14 g cc-1 in H2O-treated tissue. With high-Mg2+ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA3 gibberellic acid  相似文献   

4.
Verkhratsky  A.  Solovyova  N. 《Neurophysiology》2002,34(2-3):112-117
For many years, the endoplasmic reticulum (ER) was considered to be involved in rapid signalling events due to its ability to serve as a dynamic calcium store capable of accumulating large amounts of Ca2+ ions and of releasing them in response to physiological stimulation. Recent data significantly increased the importance of the ER as a signalling organelle, by demonstrating that the ER is associated with specific pathways regulating long-lasting adaptive processes and controlling cell survival. The ER lumen is enriched by enzymatic systems involved in protein synthesis and correcting post-translational folding of these proteins. The processes of post-translational protein processing are controlled by a class of specific enzymes known as chaperones, which in turn are regulated by the free Ca2+ concentration within the ER lumen ([Ca2+]L). At the same time, a high [Ca2+]L determines the ability of the ER to generate cytosolic Ca2+ signals. Thus, the ER is able to produce signals interacting within different temporal domains. Fast ER signals result from Ca2+ release via specific Ca2+-release channels and from rapid movements of Ca2+ ions within the ER lumen (calcium tunneling). Long-lasting signals involve Ca2+-dependent regulation of chaperones with subsequent changes in protein processing and synthesis. Any malfunctions in the ER Ca2+ homeostasis result in accumulation of unfolded proteins, which in turn activates several signalling systems aimed at appropriate compensatory responses or (in the case of severe ER dysregulation) in cellular pathology and death (ER stress responses). Thus, the Ca2+ ion emerges as a messenger molecule, which integrates various signals within the ER: fluctuations of the [Ca2+]L induced by signals originating at the level of the plasmalemma (i.e., Ca2+ entry or activation of the metabotropic receptors) regulate in turn protein synthesis and processing via generating secondary signalling events between the ER and the nucleus.  相似文献   

5.
E. A. C. MacRobbie 《Planta》1989,178(2):231-241
The influx of 45Ca into isolated guard cells of Commelina communis L. has been measured, using short uptake times, and washing in ice-cold La3+-containing solutions to remove extracellular tracer after the loading period. Over 0.5–4 min the uptake was linear with time, through the origin. Over 20–200M external Ca2+ the influx measured with 10–20 mM external KCl was in the range 0.3–2.3 pmol·cm-2·s-1 (on the basis of estimated guard-cell area); with only 1 mM KCl externally the 45Ca influx was significantly reduced, in the range 0.3–1.1 pmol·cm-2·s-1 for external Ca2+ of 50–100 M. The results indicate that the Ca-channel is voltage-sensitive, opening with depolarisation. No consistent effect of the addition of abscisic acid could be found. In different experiments, on the addition of 0.1 mM abscisic acid the Ca2+ influx was sometimes stimulated by 28–79%, was sometimes unaffected, and was sometimes inhibited by 16–29%. The results rule out a long-lasting stimulation of 45Ca influx by ABA, but they do not rule out a transient stimulation followed by inhibition, perphaps as a consequence of down-regulation of Ca2+ influx by increasing cytoplasmic Ca2+. The hypothesis that ABA may act via an action on Ca2+ influx, increasing cytoplasmic Ca2+, with consequent effects on voltage-dependent and Ca2+-dependent ion channels in both plasmalemma and tonoplast, is neither proved nor disproved by these results.Abbreviations ABA abscisic acid - Cao, Ko external Ca and K concentrations  相似文献   

6.
Douglas S. Bush 《Planta》1996,199(1):89-99
Gibberellins (GAs) control a wide range of physiological functions in plants from germination to flowering. The cellular mechanisms by which gibberellic acid (GA3) acts have been most extensively studied in the cereal aleurone. In this tissue, alterations in cellular calcium are known to be important for the primary response to GA, which is the production and secretion of hydrolytic enzymes. The extent to which cytosolic Ca2+ mediates the early events in GA action, however, is not known. In order to address this question, changes in cytosolic Ca2+ in wheat (Triticum aestivum L. cv. Inia) aleurone cells that occur rapidly after treatment with GA were characterized. In addition, GA-induced changes were compared with changes induced by three environmental stimuli that are known to modify the GA response: osmotic stress, salt (NaCl), and hypoxia. The Ca2+-sensitive dye fluo-3 was used to photometrically measure cytosolic Ca2+. It was found that GA3 induced a steady-state increase in cytosolic Ca2+ of 100–500 nM. This increase was initiated within a few minutes of treatment with GA and was fully developed after 30–90 min. The changes in cytosolic Ca2+ that were induced by GA were distinct from those induced by mannitol, NaCl, or hypoxia. Mannitol caused a steady-state decrease whereas NaCl and hypoxia both increased cytosolic Ca2+. In the case of NaCl this increase was transient but for hypoxia the increase was prolonged as long as hypoxic conditions were maintained. Gibberellin-induced changes in cytosolic Ca2+ were not induced by the inactive GA, GA8, nor did the GA-insensitive wheat mutant, D6899, respond to active GA3 with altered cytosolic Ca2+. It is concluded that changes in cytosolic Ca2+ are an early and integral part of the GA response in aleurone cells. The data also indicate, however, that changes in Ca2+ are not sufficient, by themselves, to induce the GA response of aleurone cells.Abbreviations AM acetoxymethyl ester - GA gibberellin - GA3 gibberellic acid - Mes 2-[N-morpholino]ethanesulfonic acid - PM plasma membrane The author is very grateful to Dr. T-h. D. Ho for his gift of D6899 grain and to Dr. R. Hooley for supplying the inactive GA8. This work was supported by National Science Foundation Grant DCB-9206692.  相似文献   

7.
J. A. Napier  J. M. Chapman  M. Black 《Planta》1989,179(2):156-164
Aleurone tissue of mature wheat (Triticum aestivum L. cv. Sappo) grains make novel polypeptides in response to abscisic acid (ABA), but only in the presence of Ca2+. Effects of ABA plus Ca2+ include up- and down-modulation of other polypeptides. The ABA-induced polypeptides appear not to be the 21-kilodalton (kDa) amylase inhibitor which has been reported to be ABA-inducible in barley.Aleurone tissue from developing grains of different ages failed to respond to ABA plus Ca2+ in any way. Endogenous ABA levels were determined by monoclonal radioimmunoassay in developing, mature, and sensitised developing tissues. The ABA level rose to a maximum at 35 days post anthesis but was not detectable in mature cells. Developing layers sensitised to gibberellic acid (GA) showed decreased levels of ABA, similar to those in mature tissue, concurrent with acquired responsiveness to GA in respect of its induction of -amylase. However, these sensitised cells still remained non-responsive to added ABA in terms of modulation of polypeptide pattern, though they did respond to ABA in the blocking of GA-induced -amylase production. The role of protein phosphorylation in signal transduction was examined. The implications of these findings are discussed with reference to the role of ABA in developing and mature aleurone cells.Abbreviations ABA Abscisic acid - dpa days post anthesis - GA3 Gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid  相似文献   

8.
The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA3). Using [35S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA3 was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA3-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA3 treatment and two were found after incubation without GA3. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA3 gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis  相似文献   

9.
Gibberellic acid (GA3) stimulates K+ efflux from the barley (Hordeum vulgare L. cv. Himalaya) aleurone. We investigated the mechanism of K+ flux across the plasma membrane of aleurone protoplasts using patch-clamp techniques. Potassium-ion currents, measured over the entire surface of the protoplast plasma membrane, were induced when the electrochemical gradient for K+ was inward (into the cytoplasm). The magnitude and voltage-dependence of this inward current were the same in protoplasts treated with GA3 and in control protoplasts (no GA3). Inward currents activated by negative shifts in the membrane potential (EM) from the Nernst potential for K+ (EK) showed membrane conductance to be a function of the electrochemical gradient (i.e. EM-EK). Single-channel influx currents of K+ were recorded in small patches of the plasma membrane. These channels had a single-channel conductance of 5–10 pS with 100 mM K+ on the inside and 10 mM K+ on the outside of the plasma membrane. Single-channel currents, like whole-cell currents, were the same in protoplasts treated with GA3 and control protoplasts. Voltage-gated efflux currents were found only in protoplasts tha thad been incubated without GA3. We conclude that K+ influx in the aleurone is mediated by channels and these membrane proteins are not greatly effected by GA3.Abbreviations and symbols FK Nernst potential for K+ - EM membrane potential - Erev reversal potential - GA3 gibberellic acid - Ki concentration of K+ inside the cell - Ko concentration of K+ outside the cell - R gas constant - S conductance (siemens) - T temperature (oK) - i ionic activity coefficient for internal (cytoplasmic) solution - o ionic activity coefficient for external medium  相似文献   

10.
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   

11.
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   

12.
13.
Summary Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in a Ca2+-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3-diethyl-2,2-thiadicarbocyanine (Di–S–C2(5)) to monitor inside positive or inside negative membrane, potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca2+-ATPase, while negative potential disrupts preformed Ca2+-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca2+-ATPase.  相似文献   

14.
Summary The relationship between the external Ca2+ concentrations [Ca2+]0 and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca2+]0. Increasing [Ca2+]0 caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La3+ solution. [Mg2+]0 depressed the membrane electrical tolerance which was supposed to be due to competition with Ca2+ at the Ca2+ binding site of the membrane. Such a depressive effect of Mg2+ was almost masked when the [Ca2+]0/[Mg2+]0 ratio was roughly beyond 2.  相似文献   

15.
The effects of gibberellic acid (GA3) and calcium ions on the production of α-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA3 or Ca2+ show qualitative and quantitative changes in hydrolase production following incubation in either GA3 or Ca2+ or both. Incubation in H2O or Ca2+ results in the production of low levels of α-amylase or acid phosphatase. The addition of GA3 to the incubation medium causes a 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of Ca2+ at 10 millimolar causes a further 8- to 9-fold increase in α-amylase release and a 75% increase in phosphatase release. Production of α-amylase isoenzymes is also modified by the levels of GA3 and Ca2+ in the incubation medium. α-Amylase 2 is produced under all conditions of incubation, while α-amylase 1 appears only when layers are incubated in GA3 or GA3 plus Ca2+. The synthesis of α-amylases 3 and 4 requires the presence of both GA3 and Ca2+ in the incubation medium. Laurell rocket immuno-electrophoresis shows that two distinct groups of α-amylase antigens are present in incubation media of aleurone layers incubated with both GA3 and Ca2+, while only one group of antigens is found in media of layers incubated in GA3 alone. Strontium ions can be substituted for Ca2+ in increasing hydrolase production, although higher concentrations of Sr2+ are required for maximal response. We conclude that GA3 is required for the production of α-amylase 1 and that both GA3 and either Ca2+ or Sr2+ are required for the production of isoenzymes 3 and 4 of barley aleurone α-amylase.  相似文献   

16.
The ability of the Ca2+-Mg2+ ATPase pump of skeletal SR to produce and maintain a Ca2+ gradient was studied as a function of the ATP/ADP/Pi ratio. The internal free Ca2+ concentration [Ca2+]i was monitored by changes in fluorescence of CTC. Increasing ADP concentrations in the medium reduce the maximal [Ca2+]i concentration achieved. The inclusion or the omission of 4×10–4 M Pi or doubling the absolute ATP and ADP concentrations at a constant ATP/ADP ratio does not affect the level obtained. The level depends primarily on the ATP/ADP ratio. The [Ca2+] concentration shows a 1.5 power dependence on the ATP/ADP ratio. Further, [Ca2+]i achieved at steady state does not depend on whether the pump had been working in the forward or the reverse direction prior to testing. Analysis shows that the levels of Ca2+ achieved are much lower than the levels predicted thermodynamically under the assumption of ideal coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2:1. Under this condition the osmotic energy of the [Ca2+]i/[Ca2+]o ratio was shown to be 48% as large as the free energy of hydrolysis of ATP, giving an overall thermodynamic efficiency of 48%. Analysis shows that maximal steady-state uptake is determined by the balance between the rates of uptake by the pump and rates of leak processes (intrinsic or extrinsic to the pump). Comparison with other studies shows that the [Ca2+]i achieved results in trans-inhibition of the pump by tying up the Ca2+ translocator in the inwardly oriented phosphorylated form. The absence of an effect of Pi can be taken as evidence that the dissociation of Ca2+ from the inwardly oriented translocator on the phosphoylated enzyme must precede the dephosphorylation of the enzyme.  相似文献   

17.
Previous results with potato tuber discs showed that a treatment with abscisic acid stimulated K+ uptake. In this investigation, we determine the relationship between increased K' uptake and H+extrusion, and Ca2+ fluxes by treating tissues with specific Ca2+ channel blocker (La3+), calmodulin (CaM) inhibitors (chlorpromazine and W7), and with Ca2+ ionophore (A23187). K+ uptake increased with increasing external pH whether tissues were treated with ABA or not. Treatment of tissues with La3+ inhibited K+ uptake, whereas CaM inhibitors have no effect. By contrast ABA and A23187 produced a synergistic effect, suggesting that ABA may act in part, on K+ uptake, like a Ca2+ agonist, in accord with Huddart's hypothesis. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

18.
Treatments designed to influence abscisic acid (ABA) or gibberellin (GA) concentrations were applied to developing tassels of maize (Zea mays L.) plants in different environments or to anthers in culture to determine the effect on formation of embryo-like structures (ELS). Production of ELS was significantly affected in certain environments when ABA, GA3, ancymidol, or fluridone solutions were pipetted into whorls of field-grown plants approximately 3 days before tassel harvest. In 1996 anthers from 10 M ancymidol-treated plants were most responsive, producing 35 ELS/100 anthers and 50 M GA3-treated plants were least responsive, producing 12 ELS/100 anthers. In 1997 under hotter, drier conditions, anthers from 50 M GA3-treated plants were most responsive, producing 20 ELS/100 anthers and those from 50 M ABA-treated plants were least responsive, producing 2.4 ELS/100 anthers. Anthers from growth chamber plants were significantly more responsive when grown in a 16-h than a 12-h photoperiod. With the 16-h photoperiod the response was significantly greater with a 250 M ABA whorl treatment. With the 12-h photoperiod there was no significant effect from whorl treatments. Modification of the culture medium with added ABA, GA3, ancymidol, or fluridone was generally ineffective, except in 1997 when the response was significantly higher with 1 M ABA added to the culture medium. The results suggest that the maize anther culture response may be influenced by environmental conditions that interact with ABA and GA treatments to donor plants during tassel development.  相似文献   

19.
Summary We have measured Ca2+ uptake and Ca2+ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca2+ uptake into cells was monitored with a Ca2+ electrode, whereas Ca2+ uptake into membrane vesicles was measured with45Ca2+. Using inhibitors of known action, such as the H+ ATPase inhibitors NBD-Cl and NEM, the Ca2+ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP3) and its analog inositol 1,4,5-trisphosphorothioate (IPS3), we could functionally differentiate two non-mitochondrial Ca2+ pools. Ca2+ uptake into the IP3-sensitive Ca2+ pool (IsCaP) occurs by a MgATP-dependent Ca2+ uptake mechanism that exchanges Ca2+ for H+ ions. In the absence of ATP Ca2+ uptake can occur to some extent at the expense of an H+ gradient that is established by a vacuolar-type MgATP-dependent H+ pump present in the same organelle. The other Ca2+ pool takes up Ca2+ by a vanadate-sensitive Ca2+ ATPase and is insensitive to IP3 (IisCaP). The IsCaP is filled at higher Ca2+ concentrations (10–6 mol/liter) which may occur during stimulation. The low steady-state [Ca2+] of 10–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca2+ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.  相似文献   

20.
Summary Activators of protein kinase C (PKC) stimulate Na transport (J Na) across frog skin. We have examined the effect of Ca2+ on PKC stimulation ofJ Na. Both the phorbol ester 12-O-tetradecanoylglycerol (DiC8) were used as PKC activators. Blocking Ca2+ entry into the cytosol (either from external or internal stores) reduced the subsequent natriferic effect of the PKC activators. This negative interaction did not simply reflect saturation of activation of the apical Na+ channels, since the stimulations produced by blocking Ca2+ entry and adding cyclic AMP were simply additive.The Ca2+ dependence of the natriferic effect could have reflected either a direct action of cytosolic Ca2+ on PKC or an indirect action on the final receptor site (the Na+ channel). To distinguish between these possibilities, the TPA- and phospholipid-dependent kinase activity of broken-cell preparations was assayed. The kinase activity was not stimulated by physiological levels of Ca2+, and in fact was inhibited at millimolar concentrations of Ca2+.We conclude that the effects of Ca2+ on the natriferic response to PKC activators are indirect. Reducing cytosolic uptake of Ca2+ may have stimulated Na+ transport by a chemical modification of the apical channels observed in other tight epithelia. The usual stimulation of Na+ transport produced by PKC activators in frog skin may reflect the operation of a nonconventional form of PKC. This enzyme is Ca2+ independent and seems related to thenPKC or PKC observed in other systems.  相似文献   

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