Potassium release from erythrocytes of different age rats in 9 simulated lactic acidosis

The methods are described for determination of potassium content in erythrocytes by means of a K+ selective electrode, for evaluation of changes in potassium permeability through their membranes and for detection of the Na+, K+-ATPase contribution to spontaneous and induced cation release from cells. Preincubation of erythrocytes with 5 mM lactate has no effect on the potassium release rate and content in cells of young and adult rats. Under the effect of 25 mM lactate the rate of K+ release from erythrocytes of adult animals lowers and sensitivity to ouabaine of this process stimulated by valinomycin increases. The latter effect is less pronounced in erythrocytes of young rats and is accompanied by a rise in potassium total content in cells.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

ATP synthesis in Staphylococcus aureus cells during induction of membrane potentials and proton gradient

The dynamics of ATP synthesis in Staphylococcus aureus cells was studied during membrane potential induction and K+ gradient generation in the presence of valinomycin. The starting level of intracellular ATP was 0.05 mM. Valinomycin (30 micrograms/ml) caused an increment of the intracellular ATP level up to 0.25 mM. The protonophore uncoupler, m-chlorinecarbonylcyanidephenylhydrazonium, and the H+-ATPase inhibitor, N,N'-dicyclohexylcarbodiimide, effectively suppress ATP synthesis induced by valinomycin. No ATP synthesis occurs at K+ concentration of 200 mM. The transmembrane gradient formation results in the synthesis of a smaller amount of ATP (0.10 mM).     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.     

Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.     

Intracellular accumulation of potassium and glutamate specifically enhances survival of Escherichia coli in seawater.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.     

Effect of K+ concentration on the rate of synthesis of fast-labeling proteins in the adrenal cortex

The effect of K+ concentration on protein biosynthesis and 32P-incorporation into the acid-insoluble fraction of dog and guinea pig adrenal cortex slices was studied. An increase in K+ concentration in the incubation medium from 3 to 8-11 mM induced after 15-20 min of incubation a significant stimulation of 14C-leucine incorporation into the acid-insoluble fraction of post-mitochondrial supernatant. More extensive labelling of this fraction with 32P was observed. Addition of valinomycin caused a shift in the maximum of 14C-leucine incorporation towards lower K+ concentrations. The Na+,K+-ATPase inhibitors--ouabain and strophantin K--reduced the K+-stimulated protein synthesis. These data suggest that K+ transport into the cell is necessary for the stimulating effect to be manifested. Chelation of Ca2+ strongly decreased the incorporation of 14C-leucine into proteins in the presence of 5 mM K+. However, protein labelling increased with a gradual rise in K+ concentration up to 25 mM.     

The proton electrochemical gradient in Escherichia coli cells.

The internal pH of Escherichia coli cells was estimated from the distribution of either 5,5-[14C]dimethyl-2,4-oxazolidinedione or [14C]methylamine. EDTA/valinomycin treatment of cells was employed to estimate delta psi from 86Rb+ distribution concomitant with the delta pH for calculation of delta muH. Respiring intact cells maintained an internal pH more alkaline by 0.63-0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The delta pH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN. The delta muH, established by EDTA/valinomycin-treated cells, was constant (122-129 mV) over extracellular potassium concentration of 0.01 mM-1 mM. At the lower potassium concentration delta psi (110-120 mV) was the predominant component, and at the higher concentration delta pH increased to 0.7 units (42 mV). At 150 mM potassium delta muH was reduced to 70 mV mostly due to a delta pH component of 0.89 (53 mV). The interchangeability of the delta muH components is consistent with an electronic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of delta muH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone. The highest delta pH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the delta pH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of delta pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the delta psi was almost constant over the whole range of extracellular pH values (6-8) implying electroneutral proton movement. Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.     

Calcium channels in isolated cells and strips of longitudinal muscle of rat myometrium

The properties of Ca2+ channels in strips and single muscle cells of longitudinal muscle of estrogen-dominated rat myometrium were studied under the effects of elevation of K+ concentration, the partial channel agonist Bay K 8644, and nitrendipine. In isolated strips in 0.5 mM Ca2+, Bay K 8644 (pD2 = 7.8-8.0) lowered the threshold for and enhanced the contractions in response to an elevation of K+ concentration, including the maximum response to K+ elevation alone. Bay K 8644 alone in concentrations up through 10(-6) M did not initiate contractions in 0.5 mM Ca2+ solutions. At higher concentrations (10(-5) M), Bay K 8644 behaved as an antagonist to contractions induced by elevation of K+. In isolated cells 10(-7) M Bay K 8644 enhanced the shortenings to elevated K+ and lowered the threshold K+ concentration required. Also no significant contraction occurred with 10(-7) M Bay K 8644 at normal K+ concentration. In contrast with its effect in isolated strips, no significant increase in maximum shortening (to 60 mM K+) was observed, possibly because cells without a mechanical load were maximally shortened by K+ alone. From these studies, we conclude that Ca2+ channels of isolated strips and cells of rat myometrium behave similarly and have similar properties to those of other smooth muscles in their interactions with elevation of K+, nitrendipine, and Bay K 8644.     

Asymmetric membrane expansion and modification of active and passive cation permeability of human red cells by the fluorescent probe 1-anilino-8-napththalene sulfonate.

1. The membrane perturbations induced by the interaction of the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS) with human red blood cells were studied. 2. ANS below 0.5 mM inhibits partially (20% maximum) the ouabain-insensitive Na+ and K+ influx and efflux. Above 0.5 mM ANS increases both Na+ and K+ leak fluxes. The increased cation leaks are larger for Na+ than K+. 3. The (Na+ +K+)-ATPase and ouabain-sensitive Na+ and K+ fluxes are inhibited by ANS. Ouabain-insensitive, Mg2+-dependent ATPase activity of ghosts is stimulated by [ANS] less than 0.3 mM and inhibited by [ANS] greater than 0.3 mM. 4. ANS also inhibits the Na+-dependent, ouabain-insensitive K+ influx that is inhibited by ethacrynic acid and furosemide. 5. Red cells become crenated with [ANS] less than 1 mM and sphere at [ANS] greater than 1 mM. In the former conditions hypotonic hemolysis is decreased whereas the latter increase osmotic fragility. 6. It is suggested that ANS expands the membrane asymmetrically by binding preferentially to the external membrane surface. 7. It is concluded that ANS is a general inhibitor of ion transport, particularly of those processes thought to involve facilitated-diffusion mechanisms. The increased cation leaks observed at high ANS concentrations may be related to prehemolytic membrane disruption. 8. The membrane perturbations caused by ANS are compared to those caused by other reversible inhibitors of anion exchange in red blood cells. Their possible modes of action are discussed.     

p-Chloromercuribenzenesulfonic acid stimulation of chloride-dependent sodium and potassium transport in human red blood cells

The organic mercurial p-chloromercuribenzensulfonic acid (PCMBS) reversibly increases fluxes of sodium and potassium across the human red blood cell membrane. We examined the effect of different monovalent anions on cation fluxes stimulated by PCMBS. A substantial portion of the fluxes of both cations was found to have a specific anion requirement for chloride or bromide, and was not observed when chloride was replaced by nitrate, acetate or methylsulfate. The chloride-dependent component of the cation fluxes was only observed when the cells were exposed to PCMBS concentrations of 0.5 mM or greater. Furosemide (1 mM) did not inhibit the PCMBS-stimulated cation fluxes. The observed anion specificity is directly associated with the transport process rather than PCMBS binding to the membrane. A portion of the potassium transport stimulated by PCMBS appears to involve K+-K+ exchange; however, Na+ + K+ cotransport is not stimulated by this sulfhydryl reagent.     

Stabilizing effects of cholesterol on changes in membrane permeability and potential induced in red blood cells by lysolecithin.

Effects of cholesterol on permeability of K+ ion and on change in membrane potential induced by lysolecithin were studied. Cholesterol inhibited K+ release from rabbit red blood cells treated with lysolecithin (1.25 micrograms/ml), 3.3 X 10(-6) M of cholesterol being the optimum concentration for blocking K+ release. Changes in membrane potential, monitored by changes in intensity of fluorescence of cyanine dye, were induced by lysolecithin and inhibited by cholesterol. The inhibitory action on both K+ permeability and membrane potential varied with the cholesterol concentration. The observed effects are thought to be due to membrane-stabilizing activities such as decreasing membrane fluidity and hardening the membrane at the fluid-phase transition temperature. These properties of cholesterol may have significance in relation to transformed cells (tumor cells, lymphomed cells).     

Paradoxical effects of K+ and D-600 on parathyroid hormone secretion and cytoplasmic Ca2+ in normal bovine and pathological human parathyroid cells

The effects of K+ and the Ca2+ channel blocker D-600 on parathyroid hormone (PTH) release and cytoplasmic Ca2+ activity (Ca2+i) were measured at different Ca2+ concentrations in dispersed parathyroid cells from normal cattle and from patients with hyperparathyroidism. When the extracellular Ca2+ concentration was raised within the 0.5-3.0 mM range Ca2+i increased and PTH secretion was inhibited. There was also a stimulatory effect of Ca2+ on secretion as indicated by a parallel decrease of Ca2+i and PTH release when extracellular Ca2+ was reduced to less than 25 nM. Addition of 30-50 mM K+ stimulated PTH release and lowered Ca2+i. The effect of K+ was less pronounced in the human cells with a decreased suppressability of PTH release. The Ca2+ channel blocker D-600 had no effect on Ca2+i and PTH release in the absence of extracellular Ca2+. However, at 0.5-1.0 mM Ca2+, D-600 increased Ca2+i and inhibited PTH release, whereas the opposite effects were obtained at 3.0 mM Ca2+. The transition from inhibition to stimulation occurred at a higher Ca2+ concentration in the human cells and the right-shift in the dose-effect relationship for Ca2+-inhibited PTH release tended to be normalized by D-600. It is suggested that K+ stimulates PTH release by increasing the intracellular sequestration of Ca2+ and that the reduced response in the parathyroid human cells is due to the fact that Ca2+i already is lowered. D-600 appears to have both Ca2+ agonistic and antagonistic actions in facilitating and inhibiting Ca2+ influx into the parathyroid cells at low and high concentrations of extracellular Ca2+, respectively. D-600 and related drugs are considered potentially important for the treatment of hyperparathyroidism.     

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.     

The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria. The role of potassium.

In previous studies it was found that change in the concentrations of Ca2+, H+, and HPO2-4 in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)2D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K+ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)2D-3 synthesis by isolated chick mitochondria; that the magnitnitude of this K+-dependent stimulation is enhaced by optimal concentrations of calcium (pCa = 5) and phosphate (pPi = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na+ concentration; and that the stimulatory effect of K+ is not blocked by ruthenium red, and inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH) 2D-3 synthesis. It was also found that valinomycin, a K+-specific ionophore, enhanced the sensitivity of the mitochondrial 1 alpha-hydroxylase activity to K+. In the presence of valinomycin, an increase of pK+ to 3 was sufficient to cause a significant stimulation of 1,25(OH)2D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulated the activity of the 1 alpha-hydroxylase.     

Effects of the ATP, ADP and inorganic phosphate on the transport rate of the Na+,K+-pump

(Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. In the reconstituted system the pump can be activated by adding ATP to the external medium. ATP-driven potassium extrusion by the Na+,K+-pump was studied using a voltage-sensitive dye in the presence of valinomycin. ADP strongly reduced the turnover rate of the pump with a concentration for half-maximal inhibition of cD,1/2 = 0.1 mM. cD,1/2 was found to be virtually independent of ATP concentration, indicating that the inhibition is non-competitive with respect to ATP. The non-competitive inhibition by ADP can be explained on the basis of the Post-Albers reaction cycle of the Na+,K+-pump, assuming that the main action of ADP is the reversal of the phosphorylation step. A similar 'product inhibition' was observed with inorganic phosphate, but at much higher concentrations (cP,1/2 = 14 mM).     

In vitro studies on the release of cortisol from interrenal tissue in trout (Salmo gairdneri)--II. Action of changes in extracellular electrolytes

Secretion of cortisol by the interrenal tissue of the trout Salmo gairdneri was studied in vitro by a perifusion method in relation to the effects of electrolyte concentrations in the medium. An increase in osmotic pressure (produced by adding mannitol or NaCl) induced an immediate, but brief augmentation in cortisol release. Suppression of Na+ had no effect while its reintroduction in the medium led to stimulation of hormone release. By contrast, a sharp peak was obtained whenever Cl- concentration was dropped (by 50 mM fractions). These opposite effects of Na+ and Cl- when they vary independently of each other is interpreted with regard to osmoregulation and acid-base regulation. Raising K+ even to high levels (up to 20 mM) produced no change. The absence of Ca2+ had no obvious effect while its addition induced an immediate peak of cortisol release. In addition, external Ca2+ proved necessary for the action of ACTH to occur. These results establish that cortisol release in trout may be directly affected by changes in electrolyte concentrations in the extracellular space.     

Fluid secretion by isolated Malpighian tubules of the housefly Musca domestica

The Malpighian tubules of Musca domestica secrete a fluid with a high concentration of potassium and low concentrations of sodium, calcium, magnesium and chloride compared with the isolating medium.Low secretion rates are produced by low medium potassium concentrations (< 7 mM), with low sodium concentrations (up to 5 mM) increasing secretion; higher potassium concentrations produce higher secretion rates whilst higher sodium concentrations have no further effect. Calcium and magnesium are essential for secretion.The rate of tubule secretion is inversely proportional to the osmotic pressure of the isolating medium and the osmotic pressure of the secreted fluid is slightly hyper-osmotic to the medium over a range of medium osmotic pressures.The metabolic inhibitors cyanide, iodoacetate and 2,4-dinitrophenol inhibit secretion: Cu²⁺ ions, arsenate and ouabain have no effect whereas ethacrynic acid abolishes secretion. 5-hydroxytryptamine, cycle AMP and theophylline have no effect on secretion. Sodium thiocyanate stimulates fluid secretion and increases the osmotic pressure and the concentration of sodium and chloride, but not potassium, in the secreted fluid.     

Ion activities in the lateral intercellular spaces of gallbladder epithelium transporting at low external osmolarities

The ion activities in the lateral spaces of the unilateral preparation of the gallbladder of Rana catesbiana were measured by double-barrelled ion-selective microelectrodes. The bladders were bathed in a saline solution with a low osmolarity (62 mOsm) containing, in mM: 27 Na+, 27 Cl-, 2 K+, 1 Ca++, 4 HCO3-. Working at reduced osmolarities had the advantage of an increased volume transport and of widened intercellular spaces. The reference barrel recorded an electrical potential of +2.7 mV in the spaces; they contained a solution similar to the external solution. The electrodes recorded a Na+ concentration of 27 mM, a K+ concentration of 1.7 mM, a Ca++ concentration of 0.69 mM and a Cl- concentration of 28.5 mM. In the spaces there was a lower resistance between the tip of the electrode and the serosal bath than that recorded with the tip in the lumen, and injection of fluorescent dye (11 A diameter) via the electrodes did not stain the cells. The concentrations in the secretion were similar to those in the spaces. The intracellular compartment had an apparent K+ concentration of 95 mM, and the concentrations of Na+ and Cl- were both about 5 mM. These data indicate that when the gallbladder is bathed with hypotonic solutions and is transporting fluid at approximately three or four times the normal rate, there are no significant osmotic gradients between the lumen and the lateral spaces. It is suggested that transcellular transport of water is implemented by a combination of high osmotic permeabilities across both mucosal and serosal cell membranes and low reflection coefficients (for K+ salts) at the serosal cell membranes.     

Lymphocyte membrane potential assessed with fluorescent probes

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3'-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an inophore which adds a highly K+-selective permeability membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl-, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential-the "valinomycin null point" - and under the conditions used the resting potential was approx.-60 mV. B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells. In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around -45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel. Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.