Characterization of root agravitropism induced by genetic, chemical, and developmental constraints

The patterns and rates of organelle redistribution in columella (i.e., putative statocyte) cells of agravitropic agt mutants of Zea mays are not significantly different from those of columella cells in graviresponsive roots. Graviresponsive roots of Z. mays are characterized by a strongly polar movement of 45Ca2+ across the root tip from the upper to the lower side. Horizontally-oriented roots of agt mutants exhibit only a minimal polar transport of 45Ca2+. Exogenously-induced asymmetries of Ca result in curvature of agt roots toward the Ca source. A similar curvature can be induced by a Ca asymmetry in normally nongraviresponsive (i.e., lateral) roots of Phaseolus vulgaris. Similarly, root curvature can be induced by placing the roots perpendicular to an electric field. This electrotropism increased with 1) currents between 8-35 mA, and 2) time between 1-9 hr when the current is constant. Electrotropism is reduced significantly by treating roots with triiodobenzoic acid (TIBA), an inhibitor of auxin transport. These results suggest that 1) if graviperception occurs via the sedimentation of amyloplasts in columella cells, then nongraviresponsive roots apparently sense gravity as do graviresponsive roots, 2) exogenously-induced asymmetries of a gravitropic effector (i.e., Ca) can induce curvature of normally nongraviresponsive roots, 3) the gravity-induced downward movement of exogenously-applied 45Ca2+ across tips of graviresponsive roots does not occur in nongraviresponsive roots, 4) placing roots in an electrical field (i.e., one favoring the movement of ions such as Ca2+) induces root curvature, and 5) electrically-induced curvature is apparently dependent on auxin transport. These results are discussed relative to a model to account for the lack of graviresponsiveness by these roots.     

Ethylene-induced opposite redistributions of calcium and auxin are essential components in the development of tomato petiolar epinastic curvature.

Calcium has been suggested as an important mediator of gravity signaling transduction within the root cap statocyte. In a horizontally-placed root, it is redistributed in the direction of the gravity vector (i.e. it moves downward) and its redistribution is closely correlated with auxin downward movement. However, the involvement of calcium in the regulation of ethylene-induced epinasty and auxin movement is not known. In this report, we examined the involvement of calcium in lateral auxin transport during ethylene-induced epinasty in an effort to understand the relationship among calcium, auxin, and ethylene. Ethylene-induced epinasty was further stimulated by exogenously applied Ca2+, the calcium effect being the strongest among divalent cations tested. Pretreatment with NPA, an auxin transport inhibitor, negated the promotive effect of calcium ions on the petiolar epinasty. Ethylene caused redistribution/differential accumulation of 45Ca2+ toward the morphologically lower (abaxial) side of the leaf petioles, an effect opposite to that of 14C-IAA redistribution. Verapamil, a Ca2+ channel blocker, inhibited ethylene-induced epinasty, as well as the redistribution of 14C-IAA and 45Ca2+. When the petiole was inverted in the presence or absence of ethylene, the direction of 45Ca2+ differential accumulation was still toward the morphologically abaxial side of the petiole during epinastic movement regardless of gravitational direction. These results suggest that gravity-insensitive, ethylene-induced Ca2+ redistribution and accumulation toward the abaxial side are closely coupled to the adaxial auxin redistribution/accumulation and, in turn, to the petiolar epinasty.     

The polarity of statocytes and the gravisensitivity of roots are dependent on the concentration of calcium in statocytes

In order to examine a possible role of calcium in graviperception, the calcium ionophore A23187 was used to elevate the concentration of free cytoplasmic calcium in statocytes of the roots of Lepidium sativum L. After a brief incubation (30 min) in a medium that contained 10 micromoles A23187 and 5.5 micromoles CaCl2, 50% of the roots bent gravitropically during a subsequent 2 h of horizontal exposure, with an angle of curvature that varied from 5 degrees to 70 degrees. The corresponding statocytes exhibited a polar arrangement of cell organelles as did the controls. However, in statocytes from 50% of the roots which were not curved after gravistimulation a portion of the distal endoplasmic reticulum (ER) complex was displaced in the direction of gravity within 30 min of horizontal exposure. After washing of the briefly treated roots for 24 h with 1% dimethylsulfoxide the percentage of gravitropically bending roots increased to approximately 80%, but the angle of curvature amounted to only 5 degrees-10 degrees. Longer treatment (2 h) with A23187 caused a complete loss of graviresponsiveness which was accompanied by disintegration of statocyte polarity. We concluded from these results that i) calcium is involved in graviperception and ii) gravisensitivity depends on the integrity of statocytes.     

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.     

Thermodynamics of the binding of Ca2+ to porcine pancreatic phospholipase A2

The binding of Ca2+ to porcine pancreatic phospholipase A2 was studied by batch microcalorimetry. Enthalpies of binding at 25 degrees C were determined as a function of Ca2+ concentration in buffered solutions at pH 8.0 using both the Tris-HCl and Hepes-NaOH buffer systems. The calorimetric results indicate that protons are released on calcium binding and that in addition to the binding of the active-site calcium, there appears to be weak binding of a second Ca2+. Results from potentiometric titrations indicate that this proton release on binding Ca2+ arises from a change in pK of a histidine(s) functional group. The thermodynamic functions delta G0, delta H0 and delta S0 for calcium binding to phospholipase A2 have been determined. These results are compared with literature data for Ca2+ complex formation with some small molecules and also the protein troponin-C.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

The origin, quantitation, and kinetics of intracellular calcium mobilization by vasopressin and phenylephrine in hepatocytes

The addition of phenylephrine or vasopressin to isolated hepatocytes resulted in an efflux of calcium. The intracellular source of this calcium was determined by measuring the calcium released upon the sequential additions of an uncoupling agent and the Ca2+ ionophore A23187 to control and hormone-treated cells. The release promoted by these agents was used as an estimate of the calcium content of the mitochondria and endoplasmic reticulum, respectively. The validity and limitations of this method are critically evaluated. The source of the calcium mobilized by the hormones was found to depend on the intracellular calcium distribution. When the amount of total cell-releasable Ca2+ was low (less than 0.9 nmol/mg cell dry weight), the endoplasmic reticulum represented the major cellular calcium pool and was also the predominant pool mobilized by the hormone. As the cell calcium content was increased, the endoplasmic reticulum attained its maximum capacity and the mitochondria sequestered increasing amounts of calcium. Under these conditions, the hormones mobilized calcium from the mitochondria with minimal effects on the endoplasmic reticulum calcium pool. These results suggest that more than one hormone-induced Ca2+-releasing agent may be formed. Both the total amount and the rate of calcium released from the cell under the influence of hormones was independent of the cell calcium content. The appearance of hormone-releasable Ca2+ in the extracellular medium showed a lag period of 5 to 10 s, during which a rapid increase of phosphorylase activity was observed. In contrast, the mobilization of a comparable amount of calcium by carbonyl cyanide p-trifluoromethoxyphenylhydrazone showed no significant lag, but the activation of phosphorylase was slower. A kinetic analysis of the hormone-releasable Ca2+ indicated a rapid onset with a peak increase of cytosolic free Ca2+ between 5 and 10 s prior to release of Ca2+ from the cell. The results suggest that an early action of the hormone is the inhibition of the plasma membrane Ca2+ efflux pump.     

Blockage of amyloid beta peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells

Amyloid beta protein (Abeta) alters signal transduction systems, including increases in the cytosolic free calcium ([Ca2+]i) response which have pathophysiological significance in Alzheimer's disease (AD). The purposes of this study were to elucidate the mechanism involved in Abeta's effect on the Ca2+ signal and to evaluate the effect of fullerenol-1, a water-soluble hydroxyl and superoxide radical scavenger, on the Abeta-induced Ca2+ response. Both Abeta and bradykinin (BK) dose-dependently elevated [Ca2+]i in PC12 cells. Fullerenol-1, at a concentration range between 100 nM and 1 microM, dose-dependently reduced the Abeta-induced [Ca2+]i response, but did not alter the subsequent BK-mediated process. Thapsigargin, an inhibitor of Ca2+-ATPase, released Ca2+ from the internal store and diminished the BK-mediated calcium spike but did not affect the Abeta-induced Ca2+ response. In the absence of extracellular calcium, the Abeta-induced, but not BK-induced, calcium spike was completely abolished. The Ca induced by Abeta did not enter through the voltage-dependent calcium channels or ligand gated calcium channels, because the peak of Abeta-evoked Ca2+ was not significantly altered by various Ca2+ channel blockers or a NMDA receptor antagonist MK801. In addition, neither cholera toxin nor pertussis toxin altered the Abeta-induced Ca response. The results demonstrated that Abeta-stimulated [Ca2+]i increase is due to Ca influx from an extracellular source rather than from the intracellular store. Alteration of the membrane lipid structure and permeability by free radicals generated by Abeta may be a major cause of Ca -influx. Furthermore, fullerenol-1, a novel antioxidant, may provide therapeutic benefits in neurodegenerative diseases such as AD.     

Effects of Ca2+-ATPase inhibitors, ionomycin, and pharmacological modulators of ryanodine receptor on calcium release from intracellular pools and on oscillatory contractile behavior in Physarum polycephalum

Changes in calcium levels in organelles of the plasmodium of the myxomycete Physarum polycephalum were analyzed using the fluorescent calcium indicator chlortetracycline (CTC). Both the Ca2+-ATPase inhibitor 2,5;-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (100 microM) and the calcium ionophore ionomycin (1 microM) induce a significant decrease in fluorescence level (by 30%) in CTC-stained microplasmodia; this is caused by release of calcium from intracellular storage compartments. An activator of ryanodine receptors, caffeine (10-50 mM), is less effective on Ca2+ release than BHQ or ionomycin, and their inhibitor, ryanodine (100 microM), almost completely blocks the response to caffeine, but only slightly decreases the effects of BHQ or ionomycin. Procaine, another inhibitor of ryanodine receptors, at 10 mM concentration completely abolishes both the BHQ and the ionomycin responses, but 50 mM is necessary to block the effect of 25 mM caffeine. These results suggest that both the BHQ- and the ionomycin-dependent Ca2+ releases occur through the ryanodine receptor and are to be considered as calcium-induced Ca2+ release (CICR). Both the ionomycin and the BHQ responses persist in the presence of Cd2+, which blocks Ca2+ channels of the plasmalemma. In most cases, Cd2+ itself induces release of Ca2+ from the CTC-stained calcium pool; the more effective Cd2+ is, the less the following ionomycin or BHQ responses occur. This indicates that Ca2+ entry through plasmalemma plays no significant role in the ionomycin- or BHQ-evoked initiation of CICR, and that the Cd2+- and BHQ/ionomycin-depleted Ca2+ stores overlap.     

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.     

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.     

Subcellular and functional effects of quinidine, procaine amide, and lidocaine on rat myocardium.

Different antiarrhythmic agents such as quinidine, procaine amide, and lodocaine at 1 mM concentrations were found to depress the ability of an isolated perfused rat heart to generate contractile force. Quinidine, but not procaine amide or lidocaine, decreased calcium uptake by both mitochondrial and microsomal fractions at different concentrations of calcium. The mitochondrial phosphorylation rate, respiratory control index, and state 3 oxygen consumption, but not ADP:O ratio and state 4 oxygen consumption, were depressed by only quinidine. None of these agents had any effect on myofibrillar Mg2+-ATPase or Ca2+-stimulated ATPase activities. On the other hand, sarcolemmal Mg2+-ATPase and Ca2+-ATPase activities, but not Na+-K+-ATPase activity, were increased by all these drugs. The sarcolemmal adenylate cyclase (EC 4.6.1.1) activity was decreased by quinidine only. These results suggest some similarities and differences in the sites of action of quinidine, procaine amide, and lidocaine within the myocardium.     

Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production

The recently available compound quin-2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0-2 production by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin-2, the production of 0-2 was measured in conjunction with the transfer of 45Ca+2 and changes in quin-2 fluorescence upon stimulation with FNLLP. When cells were maintained in low (10 microM) extracellular calcium medium the presence of 1.5 mM quin-2 in the cytosolic space partially inhibited the rate of 0-2 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0-2 upon stimulation at rates equal to control and extended the duration of stimulated 0-2 production as well. Quin-2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0-2 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin-2 without stimulating 0-2 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin-2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0-2 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0-2 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.     

The effects of adrenalectomy on the alpha-adrenergic regulation of cytosolic free calcium in hepatocytes

We have previously published that bilateral adrenalectomy in the rat reduces the Ca2+-mediated alpha-adrenergic activation of hepatic glycogenolysis, while it increases the cellular calcium content of hepatocytes. In the experiments presented here, the concentration of cytosolic free calcium (Ca2+i) at rest and in response to epinephrine was measured in aequorin-loaded hepatocytes isolated from sham and adrenalectomized male rats. We found that in adrenalectomized rats the resting Ca2+i was elevated, the rise in Ca2+i evoked by epinephrine was reduced, and the rise in 45Ca efflux that follows such stimulation was depressed. Furthermore, the slope of the relationship between Ca2+i and calcium efflux was decreased 60% in adrenalectomized. Adrenalectomy did not change Ca2+ release from intracellular calcium pools in response to IP3 in saponin-permeabilized hepatocytes. The EC50 for inositol 1,4,5-triphosphate and the maximal Ca2+ released were similar in both sham and adrenalectomized animals. Finally, the liver calmodulin content determined by radioimmunoassay was not significantly different between sham and adrenalectomized rats. These results suggest that 1) adrenalectomy reduces calcium efflux from the hepatocyte, probably by an effect on the plasma membrane (Ca2+-Mg2+)-ATPase-dependent Ca2+ pump and thus alters cellular calcium homeostasis; 2) adrenalectomy decreases the rise in Ca2+i in response to epinephrine; 3) this decreased rise in Ca2+i is not due to defects in the intracellular Ca2+ storage and mobilization processes; and 4) the effects of adrenalectomy on cellular calcium metabolism and on alpha-adrenergic activation of glycogenolysis are not caused by a reduction in soluble calmodulin.     

Cytochemical Localization of Calcium in Cap Cells of Primary Roots of Zea mays L.

The distribution of calcium (Ca) in caps of vertically- andhorizontally-oriented roots of Zea mays was monitored to determineits possible role in root graviresponsiveness. A modificationof the antimonate precipitation procedure was used to localizeCa in situ. In vertically-oriented roots, the presumed graviperceptive(i.e., columella) cells were characterized by minimal and symmetricstaining of the plasmalemma and mitochondria. No precipitatewas present in plasmodesmata or cell walls. Within 5 min afterhorizontal reorientation, staining was associated with the portionof the cell wall adjacent to the distal end of the cell. Thisasymmetric staining persisted throughout the onset of gravicurvature.No staining of lateral cell walls of columella cells was observedat any stage of gravicurvature, suggesting that a lateral flowof Ca through the columella tissue of horizontally-orientedroots does not occur. The outermost peripheral cells of rootsoriented horizontally and vertically secrete Ca through plasmodesmata-likestructures in their cell walls. These results are discussedrelative to proposed roles of root-cap Ca in root gravicurvature. Key words: Antimonate, calcium, columella cell, peripheral cell, root gravitropism, Zea mays L.     

4',6-Diamidino-2-phenylindole, a novel conformational probe of the sarcoplasmic reticulum Ca2+ pump, and its effect on Ca2+ release

Sarcoplasmic reticulum vesicles were noncovalently labeled at micromolar concentrations with the polycationic fluorescent reagent 4',6-diamidino-2-phenylindole (DAPI), and changes in the fluorescence intensity of the membrane-bound dye associated with functions of the Ca2+ pump and Ca2+ release were investigated. It was found that 1) DAPI fluorescence changed in the [Ca2+] range in which high affinity Ca2+ binding to the Ca2+-ATPase takes place. The time course of the Ca2+-induced changes of DAPI fluorescence was essentially the mirror image of that of tryptophan fluorescence. 2) The fluorescence intensity of bound DAPI decreased upon increase of the intravesicular [Ca2+] by either ATP-dependent Ca2+ accumulation or incubation with millimolar Ca2+ in the presence of a calcium ionophore. 3) Upon induction of Ca2+ release by adding caffeine after the completion of Ca2+ uptake, DAPI fluorescence showed transient changes. Two classes of binding sites of the sarcoplasmic reticulum membrane for DAPI were clearly distinguishable: a high affinity site (Ka = 3.0 X 10(5) M-1) with a capacity of about 1 mol/mol of Ca2+-ATPase (8.0 nmol/mg of protein) and low affinity sites with about 20-fold lower affinity and 10-fold larger capacity. The partially purified Ca2+-ATPase showed similar characteristics of high affinity DAPI binding, suggesting that DAPI bound to its high affinity site on the Ca2+-ATPase monitors the enzyme conformational changes coupled with the events described above. The high affinity binding of DAPI to the enzyme led to an increase of the initial rate of Ca2+ uptake and the inhibition of Ca2+ release induced by caffeine or ionic replacement. These results suggest that the Ca2+-ATPase is involved in some steps of the Ca2+ release mechanism.     

Zinc forms complexes with higher kinetical stability than calcium, 5-F-BAPTA as a good example

Increasing interest is focused on the role of zinc in biological systems. A rapidly growing family of DNA-binding proteins contains "zinc-fingers", where zinc is bound to cysteine or histidine residues. On the other hand zinc is able to displace calcium from its binding sites and in this way it may modify calcium-mediated cellular processes. In the present report dissociation rates of Zn2(+)- and Ca2(+)-complexes with 5-F-BAPTA, a widely used NMR-active calcium indicator, have been measured by two-dimensional 19F NMR exchange spectroscopic methods. The results show that the lifetime of the Zn2(+)-complex is more than five times longer than that of the Ca2(+)-complex. The longer lifetime, when combined with a higher thermodynamical stability of the Zn2+-complex, may explain why, in some cellular processes, Zn2+ can compete with Ca2+ in spite of a presumably high [Ca2+]/[Zn2+] free ion concentration ratio.     

Effect of ethanol on the activity of the energy-dependent Ca2+-transporting system of myometrial cells

In order of estimating some regularities of ethanol direct (effectory) effect to transmembrane calcium metabolism in the myometrium the action of this substance on the energy-dependent Ca(2+)-transporting systems of the uterine myocytes subcellular structures has been studied. The systems of Mg2+, ATP-dependent Ca2+ transport regarding their sensitivity to ethanol inhibitory effect were displayed as satisfying the following sequences: endoplasmic reticulum calcium pump > plasma membrane solubilized Ca2+, Mg2+, ATP-ase > mitochondrial Ca(2+)-accumulating system = plasma membrane calcium pump. Alongside with the latter, the oxytocin-insensitive component of Mg2+, ATP-dependent Ca2+ accumulation in the endoplasmic reticulum was defined to be less resistant to inhibitory effect of ethanol if compared with the oxytocin-sensitive one. On the base of the data received some mechanisms of ethanol effectory action on the intracellular calcium homeostasis in the myometrium cells are under the discussion.     

Cytochemical localization of calcium in cap cells of primary roots of Zea mays L

The distribution of calcium (Ca) in caps of vertically- and horizontally-oriented roots of Zea mays was monitored to determine its possible role in root graviresponsiveness. A modification of the antimonate precipitation procedure was used to localize Ca in situ. In vertically-oriented roots, the presumed graviperceptive (i.e., columella) cells were characterized by minimal and symmetric staining of the plasmalemma and mitochondria. No precipitate was present in plasmodesmata or cell walls. Within 5 min after horizontal reorientation, staining was associated with the portion of the cell wall adjacent to the distal end of the cell. This asymmetric staining persisted throughout the onset of gravicurvature. No staining of lateral cell walls of columella cells was observed at any stage of gravicurvature, suggesting that a lateral flow of Ca through the columella tissue of horizontally-oriented roots does not occur. The outermost peripheral cells of roots oriented horizontally and vertically secrete Ca through plasmodesmata-like structures in their cell walls. These results are discussed relative to proposed roles of root-cap Ca in root gravicurvature.     

Calcium regulates some, but not all, aspects of light adaptation in rod photoreceptors

The role of calcium as a regulator of light adaptation in rod photoreceptors was examined by manipulation of the intracellular Ca2+ concentration through the use of the calcium ionophore A23187 and external Ca2+ buffers. These studies utilized suspensions of isolated and purified frog rod outer segments that retain their mitochondria-rich inner segments (OS-IS). Three criteria of the dark- and light-adapted flash response were characterized as a function of the Ca2+ concentration: (a) the time to peak, (b) the rate of recovery, and (c) the response amplitude or sensitivity. For all Ca2+ concentrations examined, the time to peak of the flash response was accelerated in the presence of background illumination, suggesting that mechanisms controlling this aspect of adaptation are independent of the Ca2+ concentration. The recovery kinetics of the flash response appeared to depend on the Ca2+ concentration. In 1 mM Ca2+-Ringer's and 300 nM Ca2+-Ringer's + A23187, background illumination enhanced the recovery rate of the response; however, in 10 and 100 nM Ca2+-Ringer's + A23187, the recovery rates were the same for dark- and light-adapted responses. This result implies that a critical level of Ca2+ may be necessary for background illumination to accelerate the recovery of the flash response. The sensitivity of the flash response in darkness (SDF) was dependent on the Ca2+ concentration. In 1 mM Ca2+-Ringer's SDF was 0.481 pA per bleached rhodopsin (Rh*); a background of four Rh*/s decreased SDF by half (Io). At 300 nM Ca2+ + A23187, SDF was reduced to 0.0307 pA/Rh* and Io increased to 60 Rh*/s. At 100 nM Ca2+ + A23187, SDF was reduced further to 0.0025 pA/Rh* and Io increased to 220 Rh*/s. In 10 nM Ca2+ + A23187, SDF was lowered to 0.00045 pA/Rh* and Io raised to 760 RhI/s. Using these values of SDF and Io for each respective Ca2+ concentration, the dependence of the flash sensitivity on background intensity could be described by the Weber-Fechner relation. Under low Ca2+ conditions + A23187, bright background illumination could desensitize the flash response. These results are consistent with the idea that the concentration of Ca2+ may set the absolute magnitude of response sensitivity in darkness, and that there exist mechanisms capable of adapting the photoresponse in the absence of significant changes in cytoplasmic Ca2+ concentration.