Effects of Octylguanidine on Cell Permeability and Other Protoplasmic Properties of Allium cepa Epidermal Cells

Effects of octylguanidine (OG) were studied on the permeability of cells of the adaxial epidermis of Allium cepa bulb scales to water and methyl urea and on the protoplast surface. Interference of OG with the Ca²⁺ and Al³⁺ action on the cell surface was also investigated.     

Al3+-Ca2+ interactions in aluminum rhizotoxicity

Several mineral rhizotoxicities, including those induced by Al³⁺, H⁺, and Na⁺, can be relieved by elevated Ca²⁺ in the rooting medium. This leads to the hypothesis that the toxic cations displace Ca²⁺ from transport channels or surface ligands that must be occupied by Ca²⁺ in order for root elongation to occur. In this study with wheat (Triticum aestivum L.) seedlings, we have determined, in the case of Al³⁺, that (i) Ca²⁺, Mg²⁺, and Sr²⁺ are equally ameliorative, (ii) that root elongation does not increase as Ca²⁺ replaces Mg²⁺ or Sr²⁺ in the rooting media, and (iii) that rhizotoxicity is a function solely of Al³⁺ activity at the root-cell membrane surface as computed by a Gouy-Chapman-Stern model. The rhizotoxicity was indifferent to the computed membrane-surface Ca²⁺ activity. The rhizotoxicity induced by high levels of tris(ethylenediamine)cobaltic ion (TEC³⁺), in contrast to Al³⁺, was specifically relieved by Ca²⁺ at the membrane surface. The rhizotoxicity induced by H⁺ exhibited a weak specific response to Ca²⁺ at the membrane surface. We conclude that the Ca²⁺-displacement hypothesis fails in the case of Al³⁺ rhizotoxicity and that amelioration by cations (including monovalent cations) occurs because of decreased membrane-surface negativity and the consequent decrease in the membrane-surface activity of Al³⁺. However, TEC³⁺, but not Al³⁺, may be toxic because it inhibits Ca²⁺ uptake. The nature of the specific H⁺-Ca²⁺ interaction is uncertain.Abbreviations {Al³⁺ }₀ chemical activity of Al³⁺ at the root-cell membrane surface - {Al³⁺ }_E chemical activity of Al³⁺ in the external rooting medium - E₀ electrical potential at the root-cell membrane surface - HXM²⁺ hexamethonium ion - TEC³⁺ tris(ethylenediamine)cobaltic ion     

Influence of calcium and magnesium on ethylene production by apple tissue slices

The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1-acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca²⁺, Mg²⁺, and Ca²⁺ plus Mg²⁺. Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca²⁺ in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane.     

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

Characteristics of glutamate dehydrogenase in mitochondria prepared from corn shoots

The amination of α-ketoglutarate (α-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca²⁺. The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca²⁺ in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca²⁺ had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca²⁺.

About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca²⁺ had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca²⁺. In a simulated in vitro system using concentrations of 6.4 millimolar NAD, 0.21 millimolar NADH, 5 millimolar α-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca²⁺, 10 and 50 millimolar NH₄⁺, and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.

     

Passive transport of Ca2+ ions through lipid bilayers imaged by widefield second harmonic microscopy

In biology, release of Ca²⁺ ions in the cytosol is essential to trigger or control many cell functions. Calcium signaling acutely depends on lipid membrane permeability to Ca²⁺. For proper understanding of membrane permeability to Ca²⁺, both membrane hydration and the structure of the hydrophobic core must be taken into account. Here, we vary the hydrophobic core of bilayer membranes and observe different types of behavior in high-throughput wide-field second harmonic imaging. Ca²⁺ translocation is observed through mono-unsaturated (DOPC:DOPA) membranes, reduced upon the addition of cholesterol, and completely inhibited for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion-induced transient pores, which requires nonequilibrium membrane restructuring. This results in different rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.     

Incorporation of [C]Glucose into Cell Wall Polysaccharides of Cotton Roots: Effects of NaCl and CaCl(2)

Cotton (Gossypium hirsutum L. cv Acala SJ-2) seedlings were grown in nutrient solutions with four combinations of NaCl (0.1 and 150 millimolar) and CaCl₂ (1 and 10 millimolar) for 7 days, and then exposed to [¹⁴C]glucose for 5 hours. Uptake and incorporation of [¹⁴C]glucose into various cell wall fractions of the root tips were determined. At 1 millimolar Ca²⁺, treatment with 150 millimolar NaCl slightly stimulated uptake but considerably inhibited glucose incorporation into noncellulosic and cellulosic polysaccharides. Supplemental Ca²⁺ did not affect incorporation of glucose into the noncellulosic fraction (regardless of NaCl treatment) but completely alleviated the inhibitory effect of NaCl on glucose incorporation into cellulose. We suggest that high Na⁺ concentrations reduce synthesis of cellulose in cotton roots via disturbance of plasma membrane integrity and that supplemental Ca²⁺ counteracts this effect. The effects on cellulose biosynthesis are proposed to be related to Ca²⁺ displacement from the plasma membrane.     

Ca2+ control of electrolyte permeability in plasma membrane vesicles from cat pancreas

Summary The influence of Ca²⁺ and other cations on electrolyte permeability has been studied in isolated membrane vesicles from cat pancreas.Ca²⁺ in the micromolar to millimolar concentration range, as well as Mg²⁺, Sr²⁺, Mn²⁺ and La³⁺ at a tested concentration of 10^–4 m, increased Na⁺ permeability when applied at the vesicle inside. When added to the vesicle outside, however, they decreased Na⁺ permeability. Ba²⁺ was effective from the outside but not from the vesicle inside.When Ca²⁺ was present at both sides of the membrane, Na⁺ efflux was not affected as compared to that in the absence of Ca²⁺. Monovalent cations such as Rb⁺, Cs⁺, K⁺, Tris⁺ and choline⁺ decreased Na⁺ permeability when present at the vesicle outside at a concentration range of 10 to 100mm. Increasing Na⁺ concentrations from 10 to 100mm at the vesicle inside increased Na⁺ permeability.The temperature dependence of Na⁺ efflux revealed that the activation energy increased in the lower temperature range (0 to 10°C) when Ca²⁺ was present at the outside or at both sides, but not when present at the vesicle inside only or in the absence of Ca²⁺.The results suggest that the Ca²⁺ outside effect is due to binding of calcium to negatively charged phospholipids with a consequent reduction of both fluidity and Na⁺ permeability of the membrane. The Ca²⁺-inside effect most likely involves interaction with proteins with consequent increase in Na⁺ permeability.The data are consistent with current hypotheses on secretagogue-induced fluid secretion in acinar cells of the pancreas according to which secretagogues elicit NaCl and fluid secretion by liberating Ca²⁺ from cellular membranes and by stimulating Ca²⁺ influx into the cell. The increased intracellular Ca²⁺ concentration in turn increases the contraluminal Na⁺ permeability which leads to NaCl influx. The luminal sodium pump finally transports Na⁺ ions into the lumen.     

Release of Sucrose from Vicia faba L. Leaf Discs

The release of sucrose from leaf discs of Vicia faba L. to a bathing medium was studied for evidence of a relationship between this release and mesophyll export of photosynthate in vivo. Sucrose was released specifically over hexoses and represented over 85% of total photosynthate released. The sucrose appeared to be derived from the mesophyll tissue directly and release did not require concurrent photosynthesis. The data indicated two separate channels for sucrose release. The first was sensitive to inhibition by 1 millimolar p-chloromercuribenzenesulfonic acid and the second was promoted by lowering the Ca²⁺ concentration below 0.1 millimolar. Flow through both channels was about equal when tissue that had been actively photosynthesizing for several hours was used. The rate of release was not dependent on the extracellular pH, but was inhibited by 10 micromolar carbonylcyanide p-trifluromethoxyphenylhydrazone. Lowering the Ca²⁺ concentration below 0.1 millimolar or raising the K⁺ concentration above 100 millimolar stimulated sucrose release. The stimulation by high K⁺ was not reversed by adding Ca²⁺. The data supported the postulate that Ca²⁺ removal or K⁺ addition changed the permeability of the mesophyll plasma membrane to sucrose.     

On simultaneous transport of water and solute through plant cell membranes: Evidence for the absence of solvent drag effect and insensitivity of the reflection coefficient

The simultaneous efflux of tritiated water and ¹⁴C labelled ethanol from inner epidermal cells of the bulb scale of Allium cepa was measured with a specially designed efflux chamber. It was found that water and ethanol moved essentially independently. Rates of efflux of tritiated water and ¹⁴C ethanol were essentially the same in the presence or absence of a simultaneous influx of water. Using the same technique the efflux of tritiated water from the epidermal cells was measured during a simultaneous flow of nonlabelled ethanol. When tritiated water and ethanol moved in opposite directions, the water permeability values became slightly reduced depending upon the concentration of ethanol. When ethanol and tritiated water moved in the same direction, however, no effect on water permeability values could be detected. These results are best explained by the molecular theory of diffusion across lipid bilayer membranes, and are consistent with the above findings of lack of interaction between water and ethanol as they are transported across the cell membrane. In another study, the solute permeability coefficients (K_s) for non-electrolytes such as urea and methyl urea were measured by plasmolyzing the epidermal cells and transferring them to equimolal solutions of urea and methyl urea. This method was also used to measure the reflection coefficient (σ) for these nonelectrolytes. The K_s values for methyl urea were 16 times greater than the ones for urea. The values of σ for both of these solutes, however, were very close to 1. Using the K_s data available in the literature for the subepidermal cells of the Pisum sativum stem basis, the σ values were calculated for malonamide, glycerol, methyl urea, ethyl urea, dimethyl urea, and formamide. Again the K_s values for these nonelectrolytes varied by several orders of magnitude, whereas all σ values were found to be close to 1. These findings point out that σ is an insensitive parameter and that K_s, the solute permeability constant, has to be used for characterizing solute transport through the membrane. The present study shows that fast (e.g. ethanol, formamide) as well as slowly permeating molecules do not interact with water as they are transported across the cell membrane. Aqueous pores for the simultaneous transport of water and solutes, therefore, are absent in the plant cell membranes investigated here.     

A Voltage-Dependent Ca<Superscript>2+</Superscript> Influx Pathway Regulates the Ca<Superscript>2+</Superscript>-Dependent Cl<Superscript>−</Superscript> Conductance of Renal IMCD-3 Cells

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca²⁺-dependent Cl⁻ current (I_CLCA). Here we report that I_CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al³⁺-sensitive, voltage-dependent, Ca²⁺ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), I_CLCA was found to be inactive and resting currents were predominantly K⁺ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I_CLCA (T _0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in I_CLCA (T _0.5 ~ 100 s). The activation of I_CLCA by depolarization was reduced by lowering extracellular Ca²⁺ and completely inhibited by buffering cytosolic Ca²⁺ with EGTA, suggesting a role for Ca²⁺ influx in the activation of I_CLCA. However, raising bulk cytosolic Ca²⁺ at −60 mV did not produce sustained I_CLCA activity. Therefore I_CLCA is dependent on both an increase in intracellular Ca²⁺ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca²⁺ influx pathway that displays equal permeability to Ca²⁺ and Ba²⁺ ions and that is blocked by extracellular Al³⁺ and La³⁺. Furthermore, Al³⁺ completely and reversibly inhibited depolarization-induced activation of I_CLCA, thereby directly linking Ca²⁺ influx to activation of I_CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I_CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Minocycline chelates Ca<Superscript>2+</Superscript>, binds to membranes,and depolarizes mitochondria by formation of Ca<Superscript>2+</Superscript>-dependent ion channels

Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca²⁺-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca²⁺ ions. Secondly, minocycline, in a Ca²⁺-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca²⁺, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca²⁺-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.     

Ca2+- and phospholipid-binding properties ofTorpedo electric organ calelectrin

The Ca²⁺-regulated lipid-binding properties of the H- and L-forms of calelectrin present in the electric organ ofTorpedo marmorata have been compared. Binding of H-calelectrin required Ca²⁺ in millimolar concentrations, whereas that of L-calelectrin occurred in the micromolar range. Dissociation of H-calelectrin previously bound to lipids in the presence of 2 mM Ca²⁺ took place only when the Ca²⁺ concentration was reduced to micromolar concentrations. Binding was most effective to acidic phospholipids such as phosphatidylserine. Both forms of calelectrin promoted the aggregation of membrane vesicles in the presence of Ca²⁺, Mg²⁺, Na⁺ and K⁺ inhibited the Ca²⁺-induced binding to phospholipid, decreasing in effectiveness in that order. Binding was also inhibited by high pH. The surface activity and hdyrophobicity index showed that H-calelectrin is a hydrophilic molecule. It may represent a less active, more highly phosphorylated ‘down-regulated’ form of L-calelectrin. The role of calcium in H-calelectrin binding to lipid appeared to be consistent with the formation of a ternary complex of the protein, an acidic lipid and Ca²⁺, rather than with a direct interaction of lipid with hydrophobic sequences in H-calelectrin whose accessibility is Ca²⁺-regulated.     

A possible mechanism and sequence of events that lead to the Al3+-induced [Ca2+]cyt transients and inhibition of root growth

Fluorescence resonance energy transfer (FRET)-sensitized emission imaging of Arabidopsis thaliana roots expressing the yellow cameleon 3.60 calcium (Ca²⁺) reporter showed that the concentration of calcium in the cytosol ([Ca²⁺]_cyt) increased upon aluminum ion (Al³⁺) treatment in root cells from the transition zone within seconds. The Al³⁺-induced [Ca²⁺]_cyt transients were biphasic and were modified by Ca²⁺ channel blockers and by an antagonist of neuronal glutamate receptors, 2-amino-5-phosphonopentanoate (AP-5), and by the anion channel blocker, 5-nitro-2-(3′-phenylpropyl-amino) benzoate (NPPB). The [Ca²⁺]_cyt transients were not uniquely associated with Al³⁺ toxicity mechanisms since lanthanum (La³⁺) and gadolinium (Gd³⁺) also elicited [Ca²⁺]_cyt transients that were similar to those induced by Al³⁺. Here a testable model that describes a possible mechanism and sequence of events that lead to the Al³⁺-induced [Ca²⁺]_cyt transients and inhibition of root growth is proposed. This model can be applied to study also the signal-response coupling of the trivalent ions La³⁺ and Gd³⁺.Key words: aluminum toxicity, Al³⁺ transport, Ca²⁺ signaling, fluorescence resonance energy transfer (FRET), yellow cameleonAluminum (Al) is a naturally occurring component of soil particles and is the third most abundant element in the earth''s crust.¹ In acidic soils, Al dissolves in the soil solution and different ionic Al species form.^2,3 The most toxic Al species in acidic soils is ionic Al, Al³⁺.⁴ Al³⁺ toxicity stems from its interference with a plethora of cellular processes that control plant growth and development.^3,5–7The interactions between calcium (Ca²⁺) and Al³⁺ are well documented in the literature. One of the toxic effects of Al³⁺ on plant growth and development has been ascribed to the disruption of Ca²⁺ homeostasis by Al³⁺.^8,9 The fact that Al³⁺ inhibits Ca²⁺ uptake by roots,¹⁰ blocks voltage-regulated Ca²⁺ channels,^11,12 and affects the concentration of Ca²⁺ in the cytosol ([Ca²⁺]_cyt)^13–18 support this view. Ca²⁺ alleviates Al³⁺ toxicity^19–22 perhaps by inhibiting Al³⁺ accumulation in the roots and cells.^23,24Rincón-Zachary et al.¹⁸ using fluorescence resonance energy transfer (FRET)-sensitized emission to image Arabidopsis thaliana roots expressing the yellow cameleon 3.60 Ca²⁺ reporter demonstrated increases in the concentration of free Ca²⁺ in the cytosol ([Ca²⁺]_cyt) within seconds of Al³⁺ application. Al³⁺ induced distinct [Ca²⁺]_cyt signatures in cells from the different developmental root regions-meristem, elongation and maturation zones. The [Ca²⁺]_cyt signature in the transition zone, which is the most Al-sensitive root region,²⁵ was biphasic and was modified by treatments that chelate external Ca²⁺ (EGTA), block Ca²⁺ entry through the plasma membrane (verapamil), by an antagonist of neuronal glutamate receptors, 2-amino-5-phosphonopentanoate (AP-5), and by the anion channel blocker, 5-nitro-2-(3′-phenylpropyl-amino) benzoate (NPPB). All of these agents affected the first peak of the Al³⁺-induced [Ca²⁺]_cyt signature by reducing its magnitude or abolishing it. These results support the notion that Al³⁺ interacts with different types of plasma membrane Ca²⁺ channels, causing them to open. Al³⁺-induced [Ca²⁺]_cyt transients were also observed in the Arabidopsis Al-resistant and Al-sensitive mutants alr104 and als3, respectively. In addition, the trivalent ions lanthanum (La³⁺) and gadolinium (Gd³⁺) evoked [Ca²⁺]_cyt signatures in the transition zone of the wild-type Arabidopsis and of the alr104 and als3 roots similar to those elicited by Al³⁺. Hence the authors concluded that the observed [Ca²⁺]_cyt transients were not uniquely associated with Al³⁺ toxicity mechanisms. Al³⁺, La³⁺ and Gd³⁺ appear to elicit the same Ca²⁺ signaling pathway.I would like to propose a testable model that describes the possible sequence of events during Ca²⁺ signaling triggered by trivalent ions using Al³⁺ as a prototype (Fig. 1). (1) Al³⁺ causes Ca²⁺ channels in cells of the root transition zone to open allowing Ca²⁺ influx into the cells. (2) [Ca²⁺]_cyt rises producing the first peak of the biphasic [Ca²⁺]_cyt signature. (3) Increased [Ca²⁺]_cyt activates internal Ca²⁺ channels located in membranes of internal Ca²⁺ stores such as the vacuole, ER, mitochondria or plastids producing the second peak of the [Ca²⁺]_cyt signature. Ca²⁺-induced Ca²⁺ release from internal stores has been described in plant cells.²⁶ (4) Al³⁺ may permeate plasma membrane Ca²⁺ and non-selective cation channels and interact with internal Ca²⁺ channels allowing Ca²⁺ to be released into the cytosol, contributing to the rise in [Ca²⁺]_cyt. In this context, supporting data come from unpublished results (Leblanc J and Rincón-Zachary M) that show Al³⁺ transport across plasma membrane (PM) vesicles isolated from 5 mm wheat (Triticum aestivum) root tips by aqueous two-phase partitioning²⁷ (Fig. 2). In this experiment isolated PM vesicles were loaded with the fluorescent histochemical aluminum indicator morin (2′, 3′, 4′, 5, 7-pentahydroxyflavone) for 30 min at room temperature and then centrifuged at 100,000 xg for 15 min at 4°C and the pellet was washed twice to remove excess morin. The PM vesicles (25 µg protein mL⁻¹) were then incubated in a 2 mL buffer (250 mM sucrose, 50 mM K₂SO⁴, 1 mM DTT, 5 mM MES-Tris [pH 7.0]) containing different concentrations of Al³⁺ for 10 min at room temperature. Al³⁺uptake by the PM vesicles was monitored by fluorometry (excitation at 420 nm; emission at 475 nm). The results show that PM vesicles isolated from the Al-sensitive wheat cultivar Scout 66 root tips are more permeable to Al³⁺ than those isolated from the Al-tolerant cultivar Atlas 66 (Fig. 2A). In this experiment, the relationship between the rate of Al³⁺ uptake and the Al³⁺ concentration in the solution was linear for both Scout 66 (Y = 0.114X + 0.741, R² = 0.99) and Atlas 66 (Y = 0.108X + 0.193, R² = 0.98) PM vesicles. In addition, Leblanc²⁸ showed that compounds known to block Ca²⁺ channels inhibited Al³⁺ uptake by plasma membrane vesicles (Fig. 2B; Leblanc J and Rincón-Zachary M, unpublished data). La³⁺, verapamil and nifedipine were very effective in inhibiting Al³⁺ uptake by plasma membrane vesicles: 5 µM La³⁺ and 1 mM nifedipine caused 67% and 73% inhibition, respectively, and 1 mM verapamil completely abolished the Al³⁺ uptake by the vesicles. Thus, it is feasible that Al³⁺ permeates non-selective cation channels or/and Ca²⁺ channels. (5) Lastly, the overall [Ca²⁺]_cyt elevation could set off mechanisms that inhibit root growth (e.g., callose synthesis and its deposition in the cell wall, disruption of the cytoskeleton organization, formation of reactive oxygen species, etc.). Testing these hypotheses is underway.Open in a separate windowFigure 1A model that describes a possible mechanism and sequence of events that lead to the [Ca²⁺]_cyt transients and inhibition of root growth. (1) Al³⁺ interacts with Ca²⁺ channels in the plasma membrane of root cells in the root transition zone. The Ca²⁺channels open and external Ca²⁺ enters the cytosol. (2) [Ca²⁺]_cyt rises producing the first peak of the biphasic [Ca²⁺]_cyt signature. (3) Increased [Ca²⁺]_cyt activates internal Ca²⁺ channels located in membranes of internal Ca²⁺ stores (e.g., tonoplast, ER, mitochondria or plastids) producing the second peak of the [Ca²⁺]_cyt signature. (4) Al³⁺ permeates the PM through Ca²⁺- and non-selective cation channels. (5) Al³⁺ opens internal Ca²⁺ channels in the tonoplast, ER, mitochondria or plastids and as a result more Ca²⁺ is released into the cytosol. (6) The overall [Ca²⁺]_cyt elevation stimulates mechanisms that inhibit root growth.Open in a separate windowFigure 2Al³⁺ uptake by PM vesicles isolated from 5 mm root tips of both the Al-sensitive cultivar Scout 66 and the Al-tolerant cultivar Atlas 66. (A) Rate of Al³⁺ uptake by PM vesicles incubated in increasing concentrations of Al³⁺. The PM vesicles from the Al sensitive cultivar Scout 66 were more permeable to Al³⁺ than those of the Al-tolerant cultivar Atlas 66. The values are means ± SD. Rates of Al³⁺ uptake are expressed in Fluorescence Intensity Units (FIU) mg⁻¹ protein min⁻¹. (B) Effect of Ca²⁺ channel blockers on the rate of Al³⁺ uptake by PM vesicles s percent of the control. All Ca²⁺ channel blockers tested inhibited the rate Al³⁺ uptake by the PM vesicles in both cultivars. The accumulation of Al³⁺ in the PM vesicles was monitored by measuring the fluorescence emitted by the Al-morin complex as described in the text. Both experiments were repeated three times in triplicate (n = 9). The PM vesicles were pooled from multiple independent membrane isolations in order to obtain enough membrane protein for the assays.     

Effects of NaCl and CaCl(2) on Cell Enlargement and Cell Production in Cotton Roots

In many crop species, supplemental Ca²⁺ alleviates the inhibition of growth typical of exposure to salt stress. In hydroponically grown cotton seedlings (Gossypium hirsutum L. cv Acala SJ-2), both length and weight of the primary root were enhanced by moderate salinities (25 to 100 millimolar NaCl) in the presence of 10 millimolar Ca²⁺, but the roots became thinner. Anatomical analysis showed that the cortical cells of these roots were longer and narrower than those of the control plants, while cortical cells of roots grown at the same salinities but in the presence of only 0.4 millimolar Ca²⁺ became shorter and more nearly isodiametrical. Cell volume, however, was not affected by salinities up to 200 millimolar NaCl at either 0.4 or 10 millimolar Ca²⁺. Our observations suggest Ca²⁺-dependent effects of salinity on the cytoskeleton. The rate of cell production declined with increasing salinity at 0.4 millimolar Ca²⁺ but at 10 millimolar Ca²⁺ was not affected by salinities up to 150 millimolar NaCl.     

Microsomal membrane structure and function subsequent to calcium activation of an endogenous phospholipase

An endogenous system in the membranes of rat liver endoplasmic reticulum is capable upon Ca²⁺ activation of considerable disruption of normal structure and function. Phosphatidylethanolamine (PE) and to a lesser extent phosphatidylcholine (PC) are degraded to hydrophilic products. This lipid loss is greater at an alkaline pH, preferentially utilizes millimolar Ca²⁺ rather than Mg²⁺ ions, and is inhibited by KCl. Diethyl ether has no effect on the rate of loss of PE or PC, and the Ca²⁺ ionophore A23187 does not lower the Ca²⁺ requirement. Phospholipids are most likely lost from the membranes in a two-step process. Lysophospholipids generated in the first, Ca²⁺-dependent step are removed by an endogenous lysophospholipase demonstrated by the hydrolysis of either added lyso PE or lysophospholipids generated from endogenous substrates by Naja naja phospholipase A₂. The depletion of microsomal membrane phospholipid is accompanied by a loss of glucose 6-phosphatase and of cytochrome P-450. The latter is not associated with any change in total heme content. Polyacrylamide gel electrophoresis showed no difference between the pattern or relative amounts of solubilized membrane proteins before or after depletion of membrane phospholipid. It is concluded that activation of an endogenous phospholipase by Ca²⁺ can result in significant depletion of PE and PC that is accompanied by considerable disruption of membrane function. The significance of this system with respect to the maintenance of cell integrity and its possible role in cell injury are discussed.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.     

Displacement of Ca2+ by Na+ from the Plasmalemma of Root Cells : A Primary Response to Salt Stress?

A microfluorometric assay using chlorotetracycline (CTC) as a probe for membrane-associated Ca²⁺ in intact cotton (Gossypium hirsutum L. cv Acala SJ-2) root hairs indicated displacement of Ca²⁺ by Na⁺ from membrane sites with increasing levels of NaCl (0 to 250 millimolar). K⁺(⁸⁶Rb) efflux increased dramatically at high salinity. An increase in external Ca²⁺ concentration (10 millimolar) mitigated both responses. Other cations and mannitol, which did not affect Ca²⁺-CTC chelation properties, were found to have no effect on Ca²⁺-CTC fluorescence, indicating a Na⁺-specific effect. Reduction of Ca²⁺-CTC fluorescence by ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid, which does not cross membranes, provided an indication that reduction by Na⁺ of Ca²⁺-CTC fluorescence may be occurring primarily at the plasmalemma. The findings support prior proposals that Ca²⁺ protects membranes from adverse effects of Na⁺ thereby maintaining membrane integrity and minimizing leakage of cytosolic K⁺.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.