Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: III. Membrane Potential

The membrane electrical potential difference was measured in cultured cells and isolated protoplasts of tobacco (Nicotiana glutinosa L.) by inserting a microelectrode into cells held fast by a suction micropipette. The potential difference (± standard deviation) for unplasmolyzed tobacco cells was −52 ± 12 millivolts, for cells in 0.3 molar mannitol, −50 ± 11 millivolts; and for cells plasmolyzed in 0.7 molar mannitol, −49 ± 12 millivolts all inside negative. The potential difference for isolated protoplasts in 0.7 molar mannitol was −49 ± 16 millivolts, inside negative. In both cultured cells and protoplasts, the addition of 0.1 millimolar KCN caused a depolarization of the membrane potential. It was concluded that plasmolysis and enzymic release of the protoplast had no significant effect on the membrane potential of cultured tobacco cells.     

The Membrane Potential of Nitella translucens

The effects of changing the external concentrations of Na, K,Ca, and Cl on the potentials of the cytoplasm and the vacuolewith respect to the bathing medium of the internodal cells ofNitella translucens have been investigated. The potential differencebetween the vacuole and the cytoplasm is practically unaffectedby the concentration changes. The observed changes of potentialdifference are therefore attributed to the boundary separatingthe cytoplasm from the medium; this boundary is possibly a plasmalemma–cellwall complex. The difference of potential between the cell walland the medium has also been measured and, in the presence ofCa, shown to be markedly sensitive only to the external Ca concentration.The results are divided into two sections: (a) for cells pretreatedin 5 mM NaCl, the subsequent experiments being carried out inCa-free media, and (b) for cells initially immersed in a standardartificial pond water containing the chlorides of Na, K, Ca.With the pretreated cells the external Na/K ratio was variedwith the total NaCl+KCl concentration kept constant at 1.1 mM.The results suggest that over a limited range of concentrationsthe cytoplasm-medium potential difference can be described byan equation similar in form to a Goldman equation but containingonly terms for Na and K, the average value of the permeabilityratio (= P_Na/P_K) being 0.27. In the presence of Ca the effectsof Na and K on the cytoplasm-medium potential difference aregreatly reduced, while the effect of Ca is relatively large.The results cannot be fitted to any form of Goldman equationcontaining terms for the major ions. The possibility of a contributionto the plasmalemma potential from electrogenic pumps is brieflydiscussed. Measurements of the Na and K content of the cytoplasmand the vacuole have been made for the pretreated cells. TheNa concentration in the cytoplasm is 37 mM and in the vacuole73 mM; the K concentration is 93 mM in the cytoplasm and 67mM in the vacuole. The Nernst potentials for both ions are comparedwith the cytoplasm-medium and cytoplasm-vacuole potential differences.This analysis shows that Na is actively transported from thecytoplasm into the medium as well as into the Vacuole; K ispumped into the cytoplasm from the medium but appears to beclose to electrochemical equilibrium across the tonoplast. ThisConfirms previously published work.     

Respiration-Dependent Membrane Hyperpolarization in Tonoplast-Free Cells of Nitella axilliformis

Cells of Nitella axilliformis were made tonoplast-free by intracellularperfusion of media containing ethyleneglycol-bis-(ß-aminoethylether)N,N'-tetraaceticacid (EGTA). When the perfusion medium contained ADP as wellas ATP, the membrane hyperpolarized in darkness in a mannersimilar to light-induced hyperpolarization. This light-independenthyperpolarization seems to be due to activation of the electrogenicion pump in the plasma membrane because the hyperpolarized valueof the membrane potential was more negative than the equilibriumpotential for K^$, the most negative ion equilibrium potentialin Nitella. The hyperpolarization was inhibited by the respiratory chaininhibitors NaCN (1 mM), antimycin A (10 µM) and rotenone(10 µM). NaCN slightly decreased the ATP concentrationin the cell perfused with medium containing 1 mM ATP and 1 mMADP; but, even after treatment with NaCN, the cell had about80% of the ATP value for the control. ^* This study is dedicated to the late Professor J. Ashida. (Received June 24, 1982; Accepted October 15, 1982)     

The Production of Reactive Oxygen Species in Intact Isolated Nerve Terminals Is Independent of the Mitochondrial Membrane Potential

Dependence on mitochondrial membrane potential (m) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H₂O₂ generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250nM) or DNP (50mM) and then the rate of H₂O₂ production was followed. Neither of the uncouplers had a significant effect on the rate of H₂O₂ production induced by rotenone or antimycin. Inhibition of the F₀F₁-ATPase by oligomycin, which also eliminates m in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H₂O₂ production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of m. In addition, we detected a significant antimycin-induced H₂O₂ production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.     

脯氨酸对黑麦原生质体膨胀势的影响

从未经抗寒锻炼的黑麦(Secale cereale cv.Puma)叶片中分离的原生质体,悬浮于山梨醇或NaGl,CaCl_2(10:1)溶液中,其可忍受的表面积增加值(TSAI_(50))为1000μm~2左右,该值不受原生质体收缩程度的影响。悬浮于脯氨酸溶液中的原生质体,其TSAI_(50)随收缩程度而改变。等渗脯氮酸溶液(0.53Osm)中的原生质体若在低渗溶液中膨胀,TSAI_(50)为1000μm~2左右,而在0.83,1.20和2.0 Osm高渗脯氨酸溶液中收缩后的原生质体再膨胀,它们的TSAI_(50)分别增加到1710±85,1873±91μm~2和2200±283μm~2。这些数值与经抗寒锻炼后的原生质体在高渗收缩后的TSAI_(50)相似。脯氨酸增加未经锻炼的原生质体膨胀势的效应发生在收缩过程中,与膨胀时的介质种类无关。     

Role of Ca2+ on Triggering Tonoplast Action Potential in Intact Nitella flexilis

Sensitive aequorin was microinjected into the cytoplasm of Nitellaflexilis to study the role of Ca²⁺ in the generation of thetonoplast action potential. The temporal relation between theincrease in cytoplasmic Ca²⁺ and the tonoplast action potentialsuggested that tonoplast action potential is triggered by anincrease in cytoplasmic Ca²⁺. This is also supported by thefact that Mn²⁺, extracellularly applied, inhibited both theincrease in cytoplasmic Ca²⁺ and the generation of the tonoplastaction potential. (Received April 2, 1997; Accepted June 7, 1997)     

The Influence of H+ on the Membrane Potential and Ion Fluxes of Nitella

The resting membrane potential of the Nitella cell is relatively insensitive to [K]_o, but behaves like a hydrogen electrode. K⁺ and Cl^- effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that P_K and P_Cl are independent of pH but are a function of membrane potential. Slope ion conductances, G_K, G_Cl, and G_Na were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H⁺ was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K⁺ equilibrium potential. In the range of external pH 5 to 6, H⁺ chord conductance was substantially equal to the membrane conductance. However, the [H]_i measured by various methods was not such as would be predicted from the [H]_o and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H⁺ extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H⁺ equilibrium potential is always maintained notwithstanding a continuous H⁺ inward current which should result from the potential difference.     

Pericyte-Like Progenitors Show High Immaturity and Engraftment Potential as Compared with Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) and pericyte progenitors (PPs) are both perivascular cells with similar multipotential properties regardless of tissue of origin. We compared the phenotype and function of the 2 cell types derived from the same bone-marrow samples but expanded in their respective media – pericyte conditions (endothelial cell growth medium 2 [EGM-2]) for PPs and standard medium (mesenchymal stem cell medium [MSM]) for MSCs. After 3 weeks of culture, whatever the expansion medium, all cells showed similar characteristics (MSC markers and adipo-osteo-chondroblastic differentiation potential), although neuronal potential was greater in EGM-2– than MSM-cultured cells. As compared with MSM-cultured MSCs, EGM-2–cultured PPs showed higher expression of the pericyte-specific antigen 3G5 than α-smooth muscle actin. In addition, EGM-2–cultured PPs showed an immature phenotype, with upregulation of stemness OCT4 and SOX2 proteins and downregulation of markers of osteoblastic, chondroblastic, adipocytic and vascular smooth muscle lineages. Despite having less effective in vitro immunosuppression capacities than standard MSCs, EGM-2–cultured PPs had higher engraftment potentials when combined with biomaterials heterotopically-transplanted in Nude mice. Furthermore, these engrafted cells generated more collagen matrix and were preferentially perivascular or lined trabeculae as compared with MSM-cultured MSCs. In conclusion, EGM-2–cultured PPs are highly immature cells with increased plasticity and engraftment potential.     

Behavior of the Plasma Membrane of Isolated Protoplasts during a Freeze-Thaw Cycle

Cryomicroscopy of protoplasts isolated from nonacclimated (NA) rye leaves (Secale cereale L. cv Puma) revealed that the predominant form of injury following cooling to the minimum temperature for 50% survival (LT₅₀) (−5°C) was expansion-induced lysis of the plasma membrane during warming and thawing of the suspending medium when the decreasing osmolality resulted in osmotic expansion of the protoplasts. When cooled to temperatures below the LT₅₀, the predominant form of injury was loss of osmotic responsiveness following cooling so that the protoplasts were osmotically inactive during warming. Only a low incidence (<10%) of expansion-induced lysis was observed in protoplasts isolated from acclimated (ACC) leaves, and the predominant form of injury following cooling to the LT₅₀ (−25°C) was loss of osmotic responsiveness. The tolerable surface area increment (TSAI) which resulted in lysis of 50% of a population (TSAI₅₀) of NA protoplasts osmotically expanded from isotonic solutions was 1122 ± 172 square micrometers. Similar values were obtained when the protoplasts were osmotically expanded from hypertonic solutions. The TSAI determined from cryomicroscopic measurements of individual NA protoplasts was similar to the TSAI₅₀ values obtained from osmotic manipulation. The TSAI₅₀ of ACC protoplasts expanded from isotonic solutions (2145 ± 235 square micrometers) was approximately double that of NA protoplasts and increased following osmotic contraction. Osmotic contractions were readily reversible upon return to isotonic solutions. During freeze-induced dehydration, endocytotic vesicles formed in NA protoplasts whereas exocytotic extrusions formed on the surface of ACC protoplasts. During osmotic expansion following thawing of the suspending medium, the endocytotic vesicles remained in the cytoplasm of NA protoplasts and the protoplasts lysed before their original volume and surface area were regained. In contrast, the exocytotic extrusions were drawn back into the surface of ACC protoplasts as the protoplasts regained their original volume and surface area.     

Oritavancin Binds to Isolated Protoplast Membranes but not Intact Protoplasts of Staphylococcus aureus

Solid-state NMR has been used to examine the binding of N′-4-[(4-fluorophenyl)benzyl)]chloroeremomycin, a fluorinated analogue of oritavancin, to isolated protoplast membranes and whole-cell sucrose-stabilized protoplasts of Staphylococcus aureus, grown in media containing [1-¹³C]glycine and l-[?-¹⁵N]lysine. Rotational-echo double-resonance NMR was used to characterize the binding by estimating internuclear distances from ¹⁹F of oritavancin to ¹³C and ¹⁵N labels of the membrane-associated peptidoglycan and to the ³¹P of the phospholipid bilayer of the membrane. In isolated protoplast membranes, both with and without 1 M sucrose added to the buffer, the nascent peptidoglycan was extended away from the membrane surface and the oritavancin hydrophobic side chain was buried deep in the exposed lipid bilayer. However, there was no N′-4-[(4-fluorophenyl)benzyl)]chloroeremomycin binding to intact sucrose-stabilized protoplasts, even though the drug bound normally to the cell walls of whole cells of S. aureus in the presence of 1 M sucrose. As shown by the proximity of peptidoglycan-bridge ¹³C labels to phosphate ³¹P, the nascent peptidoglycan of the intact protoplasts was confined to the membrane surface.     

Malate Uptake into Isolated Vacuoles from Catharanthus roseus Cells: Role of the Membrane Potential

The mechanisms involved in the transport of malate into isolated vacuoles of Catharanthus roseus (L.) cells were investigated with special reference to the effects of induced changes in membrane potential and surface charges of the tonoplast. For this purpose, thiocyanate (SCN^?), a highly permeant anion often used as a membrane potential probe, was extensively exploited. In the absence of Mg-ATP, the low accumulation ratio of ¹⁴C SCN^? could be related to the presence of negative charges at the outer surface of the tonoplast exerting a screening effect on the displacement of lipophilic anionic species. Nevertheless, malate was taken up continuously by vacuoles supporting the concept of a transport component which facilitates its transfer through the tonoplast. From experiments showing the pH dependence of malata uptake, it is suggested that the protonated form of the transporter is implicated in this process. Moreover, when the vacuoles are energized by Mg-ATP, the study of the equilibrium distribution of ¹⁴C SCN^? indicated an inside positive membrane potential difference. Advantage was taken of these results to modulate the membrane potential with high levels of thiocyanate. The data obtained demonstrate that malate uptake results from electrophoretic movement in response to the positive potential difference.     

Protoplasts of Cosmarium as a Potential Protein Source

Cosmarium turpinii, a fast-growing desmid alga, transforms into protoplasts in 4 h when incubated in a mineral medium + 0.4 M mannitol + 0.5% Cellulysin.     

Membrane Potential Measurement of Protoplasts Isolated from Vigna mungo Hypocotyl Using a Fluorescent Probe, diS-C3-(5)

Membrane potentials of protoplasts isolated from Vigna mungohypocotyl segments were measured using the fluorescent probediS-C₃-(5). The fluorescence intensity changed in response tothe external K⁺ concentration. Membrane potential was estimatedto be inside negative (–85?8 mV at 0.1 mM KCl) from theNernst equation for K₊. The membrane potential was not affectedby DCCD (50 µM) or low temperature (5?C). Addition of0.5 mM Ca₂₊ to the protoplast suspension markedly depolarizedthe membrane potential, and subsequent EDTA treatment repolarizedit to the initial level. The Ca₂₊ effect on the membrane potentialmay be due to change in the permeability ratio of Cl^–to K₊. (Received December 16, 1986; Accepted April 22, 1987)     

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.     

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P_f) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield P_f.     

Comparison of Membrane Surface Proteins of Sperm Cells and Somatic Protoplasts of Zea mays

Viable sperm cells and somatic protoplasts (leaf, callus) of Zea mays were successfully isolated and purified. The plasma membrane surface proteins of intact somatic protoplasts and sperm cells were compared after probing with N-hydroxysuccinimido-biotin (NHS-bi-otin). Horseradish peroxidase-labelled avidin (HRP-avidin) was used to detect membrane proteins after separation by SDS-PAGE and Western blot. Four protein bands characteristic of the surface membrane of sperm cells were identified varying from 48 to 78 kD, five bands of leaf protoplasts in the range of 45~78 kD, and two bands of callus protoplasts, 67 and 80 kD were detected. One protein of 48 kD was specific to the surface membrane of sperm cells and might be related to the specific roles of sperm cell physiology.     

Vasopressin Modulates Membrane Properties of Taste Cells Isolated from Bullfrogs

The effect of arginine vasopressin (AVP) on the membrane propertieswas analyzed in isolated bullfrog taste cells using a perforatedwhole-cell patch-clamp technique. AVP (100 nM) induced threekinds of responses in rod-type taste cells: appearance of inwardcurrent, inhibition of voltage ramp-induced outward currentand enhancement of the outward current. The Ca²⁺-ionophore ionomycin(3 µM) also induced inward current in taste cells. A membrane-permeablecAMP analog, 8-CPT-cAMP (0.3 mM) inhibited voltage ramp-inducedoutward current in some rod cells, but enhanced the currentin other rod cells. The results suggest that AVP may increaseeither intracellular Ca²⁺ level or cAMP level in taste cells,modulating the membrane excitability. Chem. Senses 21: 739–745,1996.