胡萝卜及其愈伤组织细胞质Ca~(2 )水平分析的研究

为测定植物细胞质内[Ca~(2 )]_i,对胡萝卜(Daucus carota var.sativa DC.)原生质体制备介质做了改进,并在正常生理条件下,用温和的、非损伤性的方法将Ca~(2 )荧光指示剂indo-1 K~ 和fura-2 K~ 导入该原生质体,能很好地标记细胞质内的游离Ca~(2 )。在此基础上,用显微荧光光度单波法测定被标记原生质体单个细胞胞质[Ca~(2 )]_i。结果表明:被indo-1 K~ 标记的胡萝卜及其愈伤组织的原生质体[Ca~(2 )]_i分别为88.3nmol/L和263.0nmol/L;fura-2 K~ 标记的分别为99.9nmol/L和255.5nmol/L。由此可见,脱分化的、处在细胞周期中的愈伤组织细胞质中[Ca~(2 )]_i远高于分化了的、处于静息态的胡萝卜细胞。此外,为了确认测量的可靠性,对两种Ca~(2 )荧光指示剂分别做了体外校正,证明其线性相关。     

Measurement of cytoplasmic calcium in aleurone protoplasts using indo-1 and fura-2

Previous attempts to measure cytoplasmic Ca2+ in plant cells using the new generation of fluorescent probes, indo-1 and fura-2, have been unsuccessful. We investigated the use of indo-1 and fura-2 to measure cytoplasmic Ca2+ in barley aleurone protoplasts and found that indo-1 could be successfully used when it was loaded into protoplasts in the Ca2+-sensitive form. The acetoxymethyl esters of both dyes accumulated in aleurone protoplasts, but fura-2 was sequestered in the vacuole and indo-1 was not adequately hydrolyzed. We developed a non-disruptive method for loading the Ca2+-sensitive form of indo-1 into aleurone protoplasts in mildly acidic solutions. Using this approach, protoplasts accumulate indo-1 in a pH-dependent manner. The accumulated dye is Ca2+-sensitive, it is not sequestered in vacuoles or the endomembrane system, and it is not rapidly secreted. Fluorescence from indo-1 in individual cells was quenched by Mn2+ in the presence of digitonin. We estimate the cytoplasmic Ca2+ concentration in aleurone protoplasts to be approximately 250 nM. The Ca2+ ionophore, ionomycin does not induce changes in the fluorescence of protoplasts loaded with indo-1, but fluorescence changes could be induced by changes in extracellular Ca2+ in the presence of digitonin. We conclude that the strategy of loading indo-1 at acidic pH provides a useful means of measuring cytoplasmic Ca2+ in the barley aleurone that may also be applicable to other types of plant cells.     

The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators

Fluorescent calcium indicators fluo-3, fura-2 and indo-1, and fluorescent magnesium indicators mag-fura-2 (FURAPTRA) and mag-indo-1 were evaluated for the effects of pH on their association and dissociation rates, ion selectivity and thermodynamic properties. Calcium indicator affinities for Ca and Mg were reduced and the discrimination between Ca and Mg decreased in fura-2 and indo-1 at acidic pH. Alterations in apparent dissociation constants were caused primarily by reduced association rates. Magnesium indicators did not show these changes. The enthalphies of the calcium indicators' Ca complex were 1-3 kcal/mole and magnesium indicators' Mg complex were 7-9 kcal/mole. The potential effects of a biexponential dissociation rate of fluo-3 and of Ca interactions with magnesium indicators were examined.     

Demonstration of Na+-dependent Ca2+ efflux using low concentrations of fluorometric Ca2+ probes

The effects of different concentrations of the fluorometric Ca2+ probes, fura-2 and indo-1, on Ca2+ transients in cultured rat aortic smooth muscle cells were examined. When stimulated with the agonists, angiotensin II and arginine vasopressin, cells incubated with low concentrations of fura-2 or indo-1 (less than 1 microM) produced Ca2+ transients characterized by a small increase followed by a dramatic decrease in fluorescence below the original baseline. This effect of agonists was concentration-dependent, reversible, and blocked by receptor antagonists. In contrast to the agonists, stimulation of Ca2+ transients with depolarizing concentrations of K+ or with caffeine did not produce decreases in fluorescence and Ca2+ levels at any loading concentration of probe. The decrease in Ca2+ observed with agonists was dependent on the presence of extracellular Na+. These data suggest that under certain loading conditions, fluorescent Ca2+ indicators measure agonist-stimulated Ca2+ efflux mediated by a Na+/Ca2+ exchange mechanism.     

Activation of protein kinase C assists insulin producing cells in recovery from raised cytoplasmic Ca2+ by stimulating Ca2+ efflux

The effects of protein kinase C (PKC) activation on the cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in clonal insulin-producing RINm5F cells, using the fluorescent Ca2+ indicators quin-2 and fura-2. Both under basal and stimulatory conditions PKC activation lowered [Ca2+]i in these cells by promoting an active extrusion of Ca2+ to the extracellular space. PKC activation therefore assists insulin-producing cells in recovery from raised [Ca2+]i. Such an effect might be part of the signal regulating the insulin secretory process.     

Na+/Ca2+ exchange in isolated smooth muscle cells demonstrated by the fluorescent calcium indicator fura-2

Fura-2, a novel fluorescent indicator of cytoplasmic calcium concentrations ([Ca2+i]), was 'loaded' into smooth muscle cells isolated from guinea pig taenia coli. Resting cells maintained a stable [Ca2+i] of 107 +/- 26 nM (n = 13), which could be perturbed with ionomycin. [Ca2+i] was elevated by stimulation of the cells with carbachol or 50 mM KCl. Reduction of the plasmalemmal Na+ concentration gradient by inhibition of the Na+/K+-ATPase with ouabain markedly elevated [Ca2+i]; this elevation was dependent on extracellular Ca2+. [Ca2+i] was also increased by replacement of the extracellular Na+ with an organic cation.     

Use of a microfluorometric method for measuring free calcium in the cytoplasm of isolated cultured rat cardiomyocytes

Dual wavelength microfluorometry was used to measure the cytoplasmic free calcium concentration [( Ca2+]in) in single cultured cells from ventricular myocytes of neonatal rats loaded with the indicator fura-2. At 2.5 nmol/l extracellular Ca2+ in the resting cells [Ca2+]in was between 80 and 110 nmol/l. Sometimes, spontaneous low-frequency (approximately 0.1 Hz) [Ca2+]in oscillations were observed. High-potassium depolarization led to a Ca2+-antagonists-sensitive rise of [Ca2+]in. Both caffeine++ (5-10 mmol/l) and thymol (lmmol/l) initialized transient increase of [Ca2+]in. Mechanisms of [Ca2+]in homeostasis in heart muscle cells were discussed.     

Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes.

The distribution of intracellular free calcium ions ([Ca2+]i) was measured in pollen tubes of Lilium longiflorum using video imaging microscopy and the calcium sensitive indicators fura-2 and quin-2. The mean [Ca2+]i in growing pollen tubes measured with fura-2 shows a maximum of 1.7 to 2.6 microM in the tube tip and decreases almost exponentially to 60 to 100 nM at 100 microns behind the tip. Using quin-2, the maximum [Ca2+]i was also found in the tube tip but with a lower Ca2+ concentration, namely 1 microM. Addition of the calcium channel blocker La3+ caused a decrease of the [Ca2+]i maximum in the tube tip, indicating a heterogeneous distribution of Ca2+ channels along the plasma membrane of pollen tubes. The [Ca2+]i increased after addition of vanadate or compound 48/80. This suggests an involvement of a calmodulin-dependent Ca2+ pump in generation of the Ca2+ gradient in lily pollen tubes. The high [Ca2+]i found in the tube tip with fura-2 seems to indicate the real Ca2+ concentration and is probably responsible for vesicle fusion, fragmentation of actin filaments, and inhibition of cytoplasmic streaming.     

Glucose-stimulated efflux of indo-1 from pancreatic beta-cells is reduced by probenecid

Indo-1 loaded pancreatic beta-cells, isolated from obese hyperglycaemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), efflux of indicator and insulin release. In the absence of glucose there was a continuous efflux of indo-1 which increased upon stimulation with 20 mM of the sugar. The anion exchange inhibitor probenecid reduced both basal efflux of indo-1 and prevented that promoted by glucose. Measurements of [Ca2+]i and insulin release revealed similar results as previously reported with quin-2 and fura-2. Furthermore, probenecid did not influence the [Ca2+]i responses. It is thus possible to reduce efflux of indo-1 probenecid and thereby improve the measurements of [Ca2+]i in pancreatic beta-cells.     

New ratiometric fluorescent calcium indicators with moderately attenuated binding affinities

Mono-halogenated derivatives of the calcium indicators fura-2 and indo-1 were synthesized and their spectroscopic properties evaluated. Halogenation ortho or para to the bridging oxygen in the BAPTA nucleus had a more pronounced weakening effect on binding affinity than in the meta position in the fura derivatives. Two new excitation ratioable fluorescent calcium indicators, benzothiaza-1 and 2, were also synthesized. Kd values of 400 nM to 5.3 microM [Ca2+] were observed in these families of new probes.     

A novel tumour promoter, thapsigargin, transiently increases cytoplasmic free Ca2+ without generation of inositol phosphates in NG115-401L neuronal cells.

Thapsigargin, a sesquiterpene lactone with potent irritant and tumour-promoting activities, stimulates a rapid (within 15 s) transient increase in intracellular [Ca2+] in the NG115-401L neural cell line, as measured by the fluorescent indicator dye fura-2. This increase in cytoplasmic free [Ca2+] is concentration-dependent (ED50 around 20 nM) and occurs in the absence of extracellular Ca2+. Activation of NG115-401L cells by the inflammatory peptide bradykinin generates inositol phosphates, which parallel increases in intracellular [Ca2+]. However, the rise in cytoplasmic [Ca2+] stimulated by thapsigargin occurs in the absence of detectable production of inositol phosphates. Thapsigargin is unlike phorboid tumour promoters in that it has no action on two non-invasive indicators of phorbol stimulation of these cells, i.e. [3H]choline metabolite production and rise in intracellular pH. These data suggest that thapsigargin releases Ca2+ from an intracellular store by a novel mechanism, independent of the hydrolysis of phosphoinositides and concomitant activation of protein kinase C. Thus thapsigargin may provide a valuable tool for the analysis of intracellular signalling mechanisms.     

Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2.

We studied intracellular binding and possible compartmentalization of the fluorescent Ca2+ indicators, indo-1 and fura-2, in single mammalian cardiac ventricular cells that had been loaded with indo-1 and fura-2 by exposure to the acetoxymethylester form of the indicators (indo-1/AM and fura-2/AM). Techniques similar to those used in experiments on fluorescence recovery after photobleaching (FRAP) were used. It was assumed that reversible binding in myoplasm would be evident as slowed recovery of fluorescence after photobleaching, and that irreversible binding of the indicators to immobile myoplasmic sites (or "compartmentalization" in organelles) would be evident as incomplete recovery. Through the use of a mask, one half of a cell was exposed to high-intensity ultraviolet (UV) light to bleach the indo-1 or fura-2 in only that part of the cell. Upon removal of the mask and termination of the high-intensity UV illumination, fluorescence recovered in the bleached half of the cell, indicating diffusion of indo-1 and fura-2. Mathematical modeling of the diffusional redistribution of the indicators indicated that in these cells the apparent diffusion coefficient for indo-1 is 1.57 x 10(-7) cm2 s-1 (SD 0.48 x 10(-7) cm2 s-1; n = 5 cells, 21 degrees C), and for fura-2 is 3.19 x 10(-7) cm2 s-1 (SD 1.85 x 10(-7) cm2 s-1; n = 6 cells, 21 degrees C). These values are approximately 6 and 3, respectively, times smaller than those expected for free diffusion in the myoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2.

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.     

Purification of gonadotropes and intracellular free calcium oscillation. Effects of gonadotropin-releasing hormone and interleukin 6

The intracellular free calcium concentration ([Ca2+]i) in single gonadotropes was measured with a calcium-sensitive fluorescent dye indo-1 or fura-2 and a digital imaging fluorescence microscopic system to determine how interleukin-6 (IL-6) increases release of gonadotropins. IL-6 induced an increase in the basal [Ca2+]i or the amplitude of spontaneous oscillation of [Ca2+]i in gonadotropes in a mixed population. Gonadotropin-releasing hormone (Gn-RH) induced a biphasic increase in [Ca2+]i, a transient increase, and then a prolonged increase. These effects were inhibited by the absence of extracellular calcium or pretreatment with calcium channel blockers, cobalt or nifedipine. Next, purified gonadotropes were prepared by fluorescence-activated cell sorting and argon laser treatment of the cells. Gonadotropes labeled with anti-luteinizing hormone antibody were sorted by fluorescence-activated cell sorting and then cultured as monolayers for 24-48 h. In this way, gonadotropes were concentrated from 5-10% to 70-85% from whole pituitary cells. After relabeling with anti-luteinizing hormone antibody, 100% purified gonadotropes were obtained by killing other types of cells with an argon laser. Gonadotropin-releasing hormone induced almost the same responses of [Ca2+]i in the purified cell population as in the mixed cell population, but IL-6 did not affect [Ca2+]i in the purified gonadotropes. These results suggest that IL-6 affects calcium mobilization in gonadotropes indirectly via paracrine pathways.     

Microfluorometric analysis of Cl- permeability and its relation to oscillatory Ca2+ signalling in glucose-stimulated pancreatic beta-cells

The cytoplasmic concentrations of Cl-([Cl-]i) and Ca2+ ([Ca2+]i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic beta-cells isolated from ob/ob mice. Steady-state [Cl-]i in unstimulated beta-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl- into beta-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl-]i either in the absence or presence of furosemide inhibition of Na+, K+, 2 Cl- co-transport. Glucose-induced oscillations of [Ca2+]i were transformed into sustained elevation in the presence of 4,4' diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). A similar effect was noted when replacing 25% of extracellular Cl- with the more easily permeating anions SCN-, I-, NO3- or Br-. It is concluded that glucose stimulation of the beta-cells is coupled to an increase in their Cl- permeability and that the oscillatory Ca2+ signalling is critically dependent on transmembrane Cl- fluxes.     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

Assessment and validation of a microinjection method for kinetic analysis of [Ca2+]i in individual cells undergoing apoptosis

Normal and malignant epithelial cells are induced to undergo apoptosis by a large variety of mechanistically diverse agents. Regardless of inducing agent, apoptosis characteristically occurs asynchronously within a population of epithelial cells over a period of 12-96 h and is associated with permeability and enzymatic perturbations. Pre-loading of cells with acetoxymethyl esters (AM) derivatives of fluorescent Ca2+ indicators (i.e. fura-2, indo-1, fluo-3) by passive diffusion allows longitudinal kinetic analysis of acute [Ca2+] changes subsequent to exposure to apoptosis inducing agents. Using prostate cancer cell lines, however, it is demonstrated that dye leakage and compartmentalization into organelles limit such passive loading to longitudinal [Ca2+]i measurements of < 2 h. Post-loading of cells exposed to the apoptosis inducing agent for several hours is also inaccurate owing to decreased loading efficiency and de-esterification of the probes resulting in increased production of fluorescent Ca(2+)-insensitive dye species. To accurately measure kinetics of [Ca2+]i changes longitudinally in individual cells undergoing apoptosis, cells were microinjected with fura dextran and maintained in a physiologic environment. [Ca2+] and morphological changes characteristic of apoptosis were then followed simultaneously in individual cells over several days following exposure to the apoptosis inducing agent.     

Effect of cyclosporin A on the membrane potential and Ca2+ level of human lymphoid cell lines and mouse thymocytes

The effect of the immunosuppressive cyclosporin A (CsA) on the cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential of human B and T lymphoblastoid cells and mouse thymocytes was studied in order to reveal some features of the early stage of drug-cell interaction. Cytosolic free Ca2+ concentration of the cells was measured by spectrofluorimetry using indo-1 and quin2 fluorescent calcium indicators. Membrane potential was monitored in a flow cytometer with oxonol dye. CsA applied at 2-20 micrograms/ml final concentrations caused a dose-dependent, rapid, transient rise of [Ca2+]i in all cell types. This effect could be blocked by chelating the extracellular Ca2+ with EGTA but was not sensitive to Ca2+ channel blockers verapamil and nifedipine or K+ channel blocker 4-aminopyridine. A possible explanation for the calcium mobilizing effect of CsA is an ionophore-like mode of action at the cell membrane level. Besides directly interfering with mitogenic signals, the elevation of [Ca2+]i could be responsible for an initial hyperpolarization observed in CsA-treated T lymphocytes. This hyperpolarization, however, was not detectable in B lymphoblastoid cells. A further difference between B and T cells was the diverse pattern of depolarization following CsA treatment. This variance in the behaviour of T and B lymphocytes and the diversity of membrane transport systems in its background could account for the different final outcome of the drug-cell interaction.     

A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes.

We have measured the distribution of cytoplasmic calcium in lily pollen tubes by microinjecting them with indo-1 and performing fluorescence ratio image analysis on them. All of the 16 tubes that were growing at the time of the calcium measurements showed a gradient of [Ca2+]i in the tip region, with Ca2+ being 1.25 to 3.32 times higher at the distal end in 15 cases and more than 5 times higher in one case. The extent of the gradient ranged from 22 to 65 microns. Most of the 15 nongrowing tubes either had no gradient or had lower Ca2+ in the tip region. While we have confirmed a previous report that lily pollen tubes can be loaded with the membrane-permeable acetoxymethyl ester forms of calcium indicators, the dyes loaded in this way are visibly partitioned into organelles and this method of loading is, therefore, not useful for the measurement of [Ca2+]i. Iontophoresis of the dye free acids into tubes produces a more uniform and diffuse fluorescence which does not appear to partition into organelles. Indo-1 remains in the pollen tubes longer than fura-2. The correlation between growth and the [Ca2+]i gradient in the apical portion of the pollen tube is discussed in relation to previous reports that have suggested that such a gradient should exist during polarized growth.     

Generation of H2O2 is regulated by cytoplasmic free calcium in cultured porcine thyroid cells

Effects of calcium ionophore A23187 and BAY-K-8644, a calcium channel agonist, on cytoplasmic free calcium ([Ca2+]i) and H2O2 generation were studied in cultured porcine thyroid cells. We monitored continuously the effects of A23187 and BAY-K-8644 on [Ca2+]i and H2O2 generation, using the intracellularly trapped fluorescent Ca2+ indicator, fura-2, and homovanillic acid, respectively. A23187 and BAY-K-8644 induce an immediate increase in [Ca2+]i and H2O2 generation. The A23187- and BAY-K-8644-induced [Ca2+]i responses and H2O2 generation occur immediately, reach a maximum within several seconds, and then slowly decline. The minimum doses of A23187 or BAY-K-8644 to increase [Ca2+]i stimulate H2O2 generation. H2O2 generation is regulated by [Ca2+]i.