Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σ_e is smaller than that of the cytosol σ_i. The stretching force vanishes when σ_e is equal to or larger than σ_i. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.). Received: 15 May 2001/Revised: 20 July 2001     

The Effect of Electrical Deformation Forces on the Electropermeabilization of Erythrocyte Membranes in Low- and High-Conductivity Media

Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks' in the plasma membrane generated by electric breakdown. Received: 12 December 1997/Revised: 13 March 1998     

Electric Field Pulses Can Induce Apoptosis

Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane. Here we demonstrate that application of supercritical field pulses between 4.5 and 8.1 kV/cm strength and 40 μsec duration induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage of the poly(ADP ribose) polymerase. Apoptosis induction did not depend on the presence of any particular electrolyte in the extracellular medium. However, no apoptosis was observed in solutions without a minimum amount of salt. Apoptotic DNA fragmentation was prevented by the caspase inhibitor zVAD. Received: 16 December 1998/Revised: 24 February 1999     

Role of Nitrite, a Nitric Oxide Derivative, in K-Cl Cotransport Activation of Low-Potassium Sheep Red Blood Cells

K-Cl cotransport (COT) is the coupled movement of K and Cl, present in most cells, associated with regulatory volume decrease, susceptible to oxidation and functionally overexpressed in sickle cell anemia. The aim of this study was to characterize the effect of the oxidant nitrite (NO₂ ⁻) on K-Cl COT. NO₂ ⁻ is a stable metabolic end product of the short-lived highly reactive free radical nitric oxide (NO), an oxidant and modulator of ion channels, and a vasodilator. In some systems, the response to NO₂ ⁻ is identical to that of NO. We hypothesized that NO₂ ⁻ activates K-Cl COT. Low potassium (LK) sheep red blood cells (SRBCs) were used as a model. The effect of various concentrations (10⁻⁶ to 10⁻¹ m) of NaNO₂ was studied on K efflux in hypotonic Cl and NO₃ media, Cl-dependent K efflux (K-Cl COT), glutathione (GSH), and methemoglobin (MetHb) formation. In support of our hypothesis, K efflux and K-Cl COT were stimulated by increasing concentrations of NaNO₂. Stimulation of K efflux was dependent upon external Cl and exhibited a lag phase, consistent with activation of K-Cl COT through a regulatory mechanism. Exposure of LK SRBCs to NaNO₂ decreased GSH, an effect characteristic of a thiol-oxidizing agent, and induced MetHb formation. K-Cl COT activity was positively correlated with Methb formation. N-ethyl-maleimide (NEM), a potent activator of K-Cl COT, was used to assess the mechanism of NO₂ ⁻ action. The results suggest that NEM and NO₂ ⁻ utilize at least one common pathway for K-Cl COT activation. Since NaNO₂ is also a well known vasodilator, the present findings suggest a role of K-Cl COT in vasodilation. Received: 15 January 1998/Revised: 3 September 1998     

Activation of K-Cl Cotransport by Mild Warming in Guinea Pig Red Cells

Unidirectional, ouabain-insensitive K⁺ influx rose steeply with warming at temperatures above 37°C in guinea pig erythrocytes incubated in isotonic medium. The only component of ouabain-insensitive K⁺ influx to show the same steep rise was K-Cl cotransport (Q₁₀ of 10 between 37 and 41°C); Na-K-Cl cotransport remained constant or declined and residual K⁺ influx in hypertonic medium with ouabain and bumetanide rose only gradually. Similar results were obtained for unidirectional K⁺ efflux. Thermal activation of K-Cl cotransport-mediated K⁺ influx was fully dependent on the presence of chloride in the medium; none occurred with nitrate replacing chloride. The increase of K⁺ influx through K-Cl cotransport from 37 to 41°C was blocked by calyculin A, a phosphatase inhibitor. The Q₁₀ of K-Cl cotransport fully activated by hydroxylamine and hypotonicity was about 2. The time course of K⁺ entry showed an immediate transition to a higher rate when cells were instantly warmed from 37 to 41°C, but there was a 7-min time lag in returning to a lower rate when cells were cooled from 41 to 37°C. These results indicate that the steepness of the response of K-Cl cotransport to mild warming is due to altered regulation of the transporter. Total unidirectional K⁺ influx was equal to total unidirectional K⁺ efflux at 37–45°C, but K⁺ influx exceeded K⁺ efflux at 41°C when K-Cl cotransport was inhibited by calyculin or prevented by hypertonic incubation. The net loss of K⁺ that results from the thermal activation of isosomotic K-Cl cotransport reported here would offset a tendency for cell swelling that could arise with warming through an imbalance of pump and leak for Na⁺ or for K⁺. Received: 1 November 1997/Revised: 5 March 1998     

Red Blood Cells of a Transgenic Mouse Expressing High Levels of Human Hemoglobin S Exhibit Deoxy-stimulated Cation Flux

+ and Na⁺ transport in RBCs from control mice (C57Bl/6J) and a transgenic (α^Hβ^S[β^MDD]) mouse line that expresses high levels of human α^H and β^S-chains and has a small percent dense cells but does not exhibit anemia. In transgenic mouse RBCs (n= 5) under oxygenated conditions, K⁺ efflux was 0.22 ± 0.01 mmol/L cell × min and Na⁺ influx was 0.17 ± 0.02 mmol/L cell × min. Both fluxes were stimulated by 10 min deoxygenation in transgenic but not in control mice. The deoxy-stimulated K⁺ efflux from transgenic mouse RBCs was about 55% inhibited by 5 nm charybdotoxin (CTX), a blocker of the calcium activated K⁺-channel. To compare the fluxes between human and mouse RBCs, we measured the area of mouse RBCs and normalized values to area per liter of cells. The deoxy-simulated CTX-sensitive K⁺ efflux was larger than the CTX-sensitive K⁺ efflux observed in RBCs from SS patients. These results suggest that in transgenic mice, deoxygenation increases cytosolic Ca²⁺ to levels which open Ca²⁺-activated K⁺ channels. The presence of these channels was confirmed in both control and transgenic mice by clamping intracellular Ca²⁺ at 10 μm with the ionophore A23187 and measuring Ca²⁺-activated K⁺ efflux. Both types of mouse had similar maximal rates of CTX-sensitive, Ca²⁺-activated K⁺ efflux that were similar to those in human SS cells. The capacity of the mouse red cell membrane to regulate cytosolic Ca²⁺ levels was examined by measurements of the maximal rate of calmodulin activated Ca²⁺-ATPase activity. This activity was 3-fold greater than that observed in human RBCs thus indicating that mouse RBC membranes have more capacity to regulate cytosolic Ca²⁺ levels. In summary, transgenic mouse RBCs exhibit larger values of deoxy-stimulated K⁺ efflux and Na⁺ influx when compared to human SS cells. They have a similar Ca²⁺-activated K⁺ channel activity to human SS cells while expressing a very high Ca²⁺ pump activity. These properties may contribute to the smaller percent of very dense cells and to the lack of adult anemia in this animal model. Received: 23 October/Revised: 15 May 1997     

5-Lipoxygenase Metabolites of Arachidonic Acid Regulate Volume Decrease by Mudpuppy Red Blood Cells

We examined whether metabolites of arachidonic acid (AA) regulate K⁺ efflux during regulatory volume decrease (RVD) by mudpuppy red blood cells (RBCs). Volume regulation was inhibited by the phospholipase A₂ antagonists mepacrine (10 μm) and ONO-RS-082 (10 μm); the inhibitory effect of ONO-RS-082 was reversed by gramicidin (5 μm). Eicosatetraynoic acid (ETYA, 100 μm), a general antagonist of AA metabolism, also blocked RVD. In addition, volume regulation was inhibited by the lipoxygenase pathway antagonist nordihydroguaiaretic acid (NDGA, 10 μm), the 5 lipoxygenase antagonists AA-861 (5 μm) and curcumin (20 μm), and by the 5-lipoxygenase activating protein inhibitor L-655,298 (5 μm). Inhibition by all four of these agents was reversed with gramicidin. In contrast, the 12- and 15-lipoxygenase pathway inhibitor ethyl-3,4-dihydroxy-benzylidene-cyanoacetate (EDBCA, 1 μm) and the cytochrome P-450 monooxygenase pathway blocker ketoconazole (20 μm) had no effect. On the other hand, the cyclooxygenase pathway inhibitor aspirin (100 μm) slightly enhanced RVD. Consistent with these findings, a K⁺-selective whole cell conductance responsible for K⁺ efflux during cell swelling was inhibited by ONO-RS-082 (10 μm), NDGA (10 μm), AA-861 (5 μm), curcumin (20 μm), and l-655,298 (5 μm). In contrast, EDBCA (1 μm), ketoconazole (20 μm), and indomethacin (10 μm) did not block this whole cell conductance. These results indicate that a channel mediating K⁺ loss during RVD is regulated by a 5-lipoxygenase metabolite of arachidonic acid. Received: 12 December 1996/Revised: 28 February 1997     

Uptake of L-leucine by Trout Red Blood Cells and Peripheral Lymphocytes

The uptake of l-leucine by trout red blood cells and peripheral lymphocytes has been analyzed. The present study shows two functionally different Na⁺-independent systems for apolar branched-chain amino acids. They are designated as L systems because they share some properties with the mammalian L system. The carrier present in red blood cells has low K _m values, is trans-stimulable and not stereospecific for leucine uptake; on the other hand, the system present in lymphocytes is stereospecific for leucine uptake and trans-inhibitable. Both carriers are pH sensitive in a similar fashion at low pHs, but there are important differences at higher pH values (above neutrality). These properties are compared with these of the asc systems previously reported in these cells. Received: 2 June 1995/Revised: 7 March 1996     

Protein Kinase C and Regulatory Volume Decrease in Mudpuppy Red Blood Cells

This study examined whether protein kinase C (PKC) stimulates K⁺ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K⁺ efflux (gramicidin is a cationophore that was used to ensure a high K⁺ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K⁺ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K⁺ loss during RVD is regulated, at least in part, by protein kinase C. Received: 20 January 1998/Revised: 2 September 1998     

Mechanically Induced Calcium Movements in Astrocytes, Bovine Aortic Endothelial Cells and C6 Glioma Cells

Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca²⁺. It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca²⁺ is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca²⁺. In these three cell types, no influx of ions is required for Ca²⁺ elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca²⁺. The Ca²⁺ response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP₃-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn²⁺ influx pathway. This Mn²⁺ pathway may be mechanosensitive channels, Ca²⁺-activated cation channels or depletion-activated Ca²⁺ channels. Received: 7 July 1999/Revised: 12 November 1999     

Electrorotation of Isolated Generative and Vegetative Cells, and of Intact Pollen Grains of Lilium longiflorum

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m⁻¹. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques. Received: 9 June 1997/Revised: 4 August 1997     

Kinetics of Chloride-Bicarbonate Exchange Across the Human Red Blood Cell Membrane

We had previously shown that an influx of extracellular Ca²⁺ (Ca²⁺ _e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca²⁺ _e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca<sup>2+</sup>] <sub>e</sub> ≥ 5 × 10<sup>−6</sup> m, while normally a [Ca<sup>2+</sup>] <sub>e</sub> = 0.5 mm is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca<sup>2+</sup>] <sub>e</sub> even beyond levels usually available to cells. Quenching of [Ca<sup>2+</sup>] <sub>e</sub> by EGTA application to levels of resting [Ca<sup>2+</sup>] <sub>i</sub> or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca<sup>2+</sup> <sub>e</sub> . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca<sup>2+</sup>-mobilization from subplasmalemmal pools (``alveolar sacs') and, as a superimposed step, a Ca²⁺-influx, since exocytotic membrane fusion can occur at [Ca²⁺] _e even slightly below resting [Ca²⁺] _i . The other important conclusion is that increasing [Ca²⁺] _e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca²⁺] _e also drives detachment of ``ghosts' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca²⁺, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle. Received: 27 September 1996/Revised: 11 February 1997     

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders,and from Several Other Mammalian Species

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO₃ media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO₃) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO₃ (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity. Received: 5 June 1996/Revised: 4 October 1996     

Influence of Hypo-osmolality on the Activity of Short-Chain Neutral Amino Acid Carriers in Trout (Salmo trutta) Red Blood Cells

The present study shows that in trout red blood cells the activity of some amino acid carriers, not directly involved in cell volume regulation, is affected by external osmolality. Glycine uptake has been used as the experimental approach because it was shown previously that it is effected by different carriers, namely the Na⁺-dependent ASC and Gly systems, as well as the Na⁺-independent asc and L systems. An increase in the uptake through the Gly system and the two Na⁺-independent carriers was found, while the ASC system appeared to be downregulated. Those systems whose activities were increased by hypo-osmolality did not share the mechanism by which this increase was obtained. Thus, the Gly system was sensitive to intracellular ionic changes, while the Na⁺- independent systems were mechanically stimulated, as assessed by the iso-osmotic swelling caused by ammonium chloride. On the other hand, a volume-sensitive transporter may be present in trout red blood cells, which is involved in the swelling-induced glycine movement, as can be deduced from the effect of some inhibitors such as pyridoxal phosphate, DIDS (4,4′-diisothiocyanate-stilbene-2,2′-disulfonic acid) and quinine. Received: 12 February 1996/Revised: 9 September 1996     

Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin

Dephosphorylation of Ca²⁺ channels by the Ca²⁺-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca²⁺-dependent inactivation of Ca²⁺ current in rat pituitary tumor (GH₃) cells. Although recent evidence favors an inactivation mechanism involving direct binding of Ca²⁺ to the channel protein, the alternative ``calcineurin hypothesis' has not been critically tested using the specific calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH₃ cells. To determine if calcineurin plays a part in the voltage- and/or Ca²⁺-dependent components of dihydropyridine-sensitive Ca²⁺ current decay, we rapidly altered the intracellular Ca²⁺ buffering capacity of GH₃ cells by flash photolysis of DM-nitrophen, a high affinity Ca²⁺ chelator. Flash photolysis induced a highly reproducible increase in the extent of Ca²⁺ current inactivation in a two-pulse voltage protocol with Ca²⁺ as the charge carrier, but had no effect when Ba²⁺ was substituted for Ca²⁺. Despite confirmation of the abundance of calcineurin in the GH₃ cells by biochemical assays, acute application of CsA or FK506 after photolysis had no effect on Ca²⁺-dependent inactivation of Ca²⁺ current, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK506 or CsA did not inhibit Ca²⁺-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca²⁺-dependent inactivation after photolysis. The results provide strong evidence against Ca²⁺-dependent dephosphorylation as the mechanism of Ca²⁺ current inactivation in GH₃ cells, but support the alternative idea that Ca²⁺-dependent inactivation reflects a direct effect of intracellular Ca²⁺ on channel gating. Received: 12 August 1996/Revised: 21 October 1996     

Effect of Ferriprotoporphyrin IX and Non-heme Iron on the Ca2+ Pump of Intact Human Red Cells

Previous studies have shown that ferriprotoporphyrin IX (FP) and non-heme iron have a marked inhibitory effect on the Ca²⁺-Mg²⁺-ATPase activity of isolated red cell membranes, the biochemical counterpart of the plasma membrane Ca²⁺ pump (PMCA). High levels of membrane-bound FP and non-heme iron have been found in abnormal red cells such as sickle cells and malaria-infected red cells, associated with a reduced life span. It was important to establish whether sublytic concentrations of FP and non-heme iron would also inhibit the PMCA in normal red cells, to assess the possible role of these agents in the altered Ca²⁺ homeostasis of abnormal cells. Active Ca²⁺ extrusion by the plasma membrane Ca²⁺ pump was measured in intact red cells that had been briefly preloaded with Ca²⁺ by means of the ionophore A23187. The FP and nonheme iron concentrations used in this study were within the range of those applied to the isolated red cell membrane preparations. The results showed that FP caused a marginal inhibition (∼20%) of pump-mediated Ca²⁺ extrusion and that non-heme iron induced a slight stimulation of the Ca²⁺ efflux (11–20%), in contrast to the marked inhibitory effects on the Ca²⁺-Mg²⁺-ATPase of isolated membranes. Thus, FP and non-heme iron are unlikely to play a significant role in the altered Ca²⁺ homeostasis of abnormal red cells. Received: 22 November 1999/Revised: 29 February 2000     

Effects of Thiol-Modifying Agents on a K(Ca2+) Channel of Intermediate Conductance in Bovine Aortic Endothelial Cells

Ca²⁺-activated K⁺ channels (K(Ca²⁺)) constitute key regulators of the endothelial cell electrophysiological response to InsP₃-mobilizing agonists. Inside-out and outside-out patch clamp experiments were thus undertaken to determine if the gating properties of a voltage-insensitive K(Ca²⁺) channel of intermediate conductance present in bovine aortic endothelial (BAE) cells could be modified by specific sulfhydryl (SH) oxidative and/or reducing reagents. The results obtained first indicate that cytosolic application of hydrophilic oxidative reagents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (0.2 to 5 mm) or [(O-carboxyphenyl)thio]ethyl mercury sodium salt (thimerosal) (0.5 to 5 mm) reduces gradually the K(Ca²⁺) channel activity with no modification of the channel unitary conductance. The inhibitory action of DTNB (1 to 5 mm) or thimerosal (1 to 5 mm) was not reserved following withdrawal of the oxidative agents, but channel activity could partly be restored by the addition of the SH group reducing agents dithiothreitol (DTT) (5 mm) or reduced glutathione (GSH) (5 mm) in 53% and 50% of the inside-out experiments performed with DTNB and thimerosal respectively. Similar results were obtained using H₂O₂ at concentrations ranging from 500 μm to 10 mm as oxidative reagent. In contrast, the lipid soluble oxidative agent 4,4′-dithiodipyridine (4-PDS) (1 mm) appeared in inside-out experiments less potent than DTNB and thimerosal at inhibiting the K(Ca²⁺) channel activity, suggesting that the critical SH groups involved in channel gating are localized at the inner face of the cell membrane. This conclusion was further substantiated by a series of outside-out patch clamp experiments which showed that DTNB (5 mm) and thimerosal (5 mm) were unable to inhibit the K(Ca²⁺) channel activity when applied to the external surface of the excised membrane. Finally, no significant changes of the gating properties of the K(Ca²⁺) channel were observed in inside-out experiments where the SH group reducing agents DTT and GSH were applied immediately following membrane excision. However, the application of either GSH or DTT was found to partly restore channel activity in experiments where the K(Ca²⁺) channels showed significant rundown. Received: 7 August 1996/Revised: 20 February 1997     

Activation of Divalent Cation Influx into S. cerevisiae Cells by Hypotonic Downshift

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca²⁺ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca²⁺ accumulation 6.7-fold. Hypotonic downshift-induced Ca²⁺ accumulation and steady-state Ca²⁺ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg²⁺. The Ca²⁺-influx pathway responsible for hypotonic-induced Ca²⁺ influx may account for about 10–35% of Ca²⁺ accumulation by cells growing in YPD. Ca²⁺ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd²⁺ suggesting that mutants resistant to acute Cd²⁺ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997     

Separate Determination of the Electrical Properties of the Tonoplast and the Plasmalemma of the Giant-Celled Alga Valonia utricularis: Vacuolar Perfusion of Turgescent Cells with Nystatin and Other Agents

In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 μm of the H⁺-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to −22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10⁻² to 40.6 × 10⁻²Ωm². The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81–94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 × 10⁻² Fm⁻² compared to 0.77 × 10⁻² Fm⁻² of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system. Received: 22 October 1996/Revised: 16 January 1997     

Hydrophobic and Hydrophilic Radio-Iodination, Crosslinking, and Differential Extraction of Cell Surface Proteins in Paramecium tetraurelia Cells

We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens (``immoblization' or ``i-AGs'). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane proteins were recognized particularly with lipophilic 5-[¹²⁵I]-iodonaphthalene-1-azide (INA) labeling which reportedly ``sees' integral proteins and cytoplasmic cell membrane-associated proteins. With INA (+DTT), bands of ≤55 kDa were similar as with hydrophilic iodogen (+DTT), but instead of large size bands including i-AGs, a group of 122, 104 and 94 kDa appeared. Several bands of the non i-AG type are compatible with integral (possibly oligomeric) or associated proteins of the cell membrane of established molecular identity, as we discuss. In summary, we can discriminate between i-AGs and some functionally important minor cell membrane components. Our methodical approach might be relevant also for an analysis of some related protozoan parasites. Received: 5 April 1999/Revised: 19 July 1999