Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+-activated K+ channels

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.     

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H(+)-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Delta(psi)) generated by the Schizosaccharomyces pombe plasma membrane H(+)-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Delta(psi) because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Delta(psi) at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H(+)-ATPase generates Delta(psi) of about 160 mV on the vesicle membrane. The generated Delta(psi) was stable at least over tens of minutes. An influence of the H(+) membrane permeability on the Delta(psi) buildup was demonstrated by manipulating the H(+) permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H(+)-ATPase-mediated Delta(psi) buildup.     

Plasma membrane potential of murine erythroleukemia cells: approach to measurement and evidence for cell-density dependence

The plasmamembrane potential (delta psi p) of murine erythroleukemia (MEL) cells has been determined by measuring the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) across the plasmamembrane. TPP+ accumulation within the cells (experimental accumulation ratio, AR exp) was measured by adding 2 microM TPP+ directly to the culture flasks, avoiding any other perturbation of the experimental system. The mitochondrial contribution to AR exp, evaluated by adding carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or 2,4-dinitrophenol (DNP), was apparently negligible in standard cultures, AR exp being substantially the same in either the absence or presence of these uncouplers. However, the addition of oligomycin produced a strong AR exp enhancement, which was abolished by FCCP, suggesting that MEL cell mitochondria are in state 3. The aspecific TPP+ binding was estimated by a new mathematical approach worked out to fit AR exp values measured in the presence of valinomycin at various extracellular K+ concentrations and plotted against the ratio of intracellular to extracellular K+ concentration ([K+]i/[K+]e). This binding was found to be close to zero in MEL cells. By applying the Nernst equation directly to AR exp values, delta psi p of these cells was then measured; this potential varying from -65 mV to -16 mV (inside negative) is inversely related to the cell density on the culture surface on which the cells sediment (cells/cm2; CD). The dependence of delta psi p on CD is practically eliminated by valinomycin and appears to be related to a cell interaction with the culture surface of the flasks, suggesting that in the immediate environment of MEL cells one or more factors are produced that modulate the K+ plasma membrane permeability (PK).     

Differentiation Between Alterations in Plasma and Mitochondrial Membrane Potentials in Synaptosomes Using a Carbocyanine Dye

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (psi p and psi m, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 microM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. psi p was calculated to be -54 mV and was not altered significantly by prior depolarization of psi m with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and psi p was not altered by depolarization of psi m. Valinomycin, a K+ ionophore, caused a psi p-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized psi p by about 5 mV. The pore-forming antibiotic gramicidin depolarized both psi p and psi m maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of psi p). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on psi p and psi m.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the membrane potential on the passive transport of Ca2+ in fractions of vesicles of the myometrium sarcolemma

Using a potential-sensitive fluorescent probe diS-C3-(5), the formation of the membrane (K+-diffusion) potential, delta psi, in the myometrium sarcolemmal vesicular fraction was demonstrated. The magnitude of this potential corresponds to that calculated according to the Nernst equation, is time-stable (characteristic dissociation time--3-5 min) and temperature-dependent and is generated upon the substitution of the anion (Cl- for gluconate-) and the compensating cation (Na+ for Tris+, choline+). The change in delta psi from -61 to 0 mV leads to the activation of passive Ca2+ efflux from the vesicles (with choline+ as the compensating cation in the dilution medium). At the same value of the potential, i. e., -61 mV, the substitution of choline in the dilution medium for Na+ or Li+ stimulates the passive release of Ca2+. Co2+, Mn2+ and D-600 suppress this process by 15-20% in depolarized vesicles which points to the inhibition of Ca2+ release with an alteration of the membrane potential value from 0 to -61 mV (20%). The potential-dependent component of passive Ca2+ transport is characterized by saturation with the substrate (Km = 0.5 mM). The dependence of Ca2+ flux release from the sarcolemmal vesicles on the membrane potential value (-60-+27 mV) is bell-shaped and qualitatively relative to the volt-amper characteristics of the steady state Ca2+ flux in single smooth muscle cells. Analysis of experimental results revealed that the potential-dependent component of passive Ca2+ transport in myometrium sarcolemmal vesicles is determined by the non-activated Ca2+ conductivity of plasma membrane.     

The Intracellular pH of Clostridium paradoxum, an Anaerobic, Alkaliphilic, and Thermophilic Bacterium

When the extracellular pH was increased from 7.6 to 9.8, Clostridium paradoxum, a novel alkalithermophile, increased its pH gradient across the cell membrane ((Delta)pH, pH(infin) - pH(infout)) by as much as 1.3 U. At higher pH values (>10.0), the (Delta)pH and membrane potential ((Delta)(psi)) eventually declined, and the intracellular pH increased significantly. Growth ceased when the extracellular pH was greater than 10.2 and the intracellular pH increased to above 9.8. The membrane potential increased to 110 (plusmn) 8.6 mV at pH 9.1, but the total proton motive force ((Delta)p) declined from about 65 mV at pH 7.6 to 25 mV at pH 9.8. Between the extracellular pH of 8.0 and 10.3, the intracellular ATP concentration was around 1 mM and decreased at lower and higher pH values concomitantly with a decrease in growth rate.     

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.     

Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

Uptake of Ca2+ driven by the membrane potential in energy-depleted yeast cells

The time-course of 45Ca2+ influx into yeast cells was measured under non-steady-state conditions obtained by preincubating the cells in a Ca2+-free medium containing glucose and buffer. Two components were distinguished: a saturable component which reached a steady-state after about 40 s of 45Ca2+ uptake and a linear increase in cellular 45Ca2+ starting after 60-90 s. Using differential extraction methods it was determined that after 20 s of uptake, 45Ca2+ was localized in the cytoplasmic pool and in bound form with no 45Ca2+ in the vacuole. After 3 min most of the cellular 45Ca2+ was concentrated in the vacuole and in bound form. The initial rate of 45Ca2+ uptake under non-steady-state conditions thus measured 45Ca2+ transport across the plasma membrane without interference by vacuolar uptake. The effect of membrane potential (delta psi) on this transport was investigated in cells depleted of ATP. A high delta psi was produced by preincubating the cells with trifluoperazine (TFP) and subsequently washing the cells free from TFP. Substantial 45Ca2+ influx was measured in the absence of metabolic energy in cells with a high delta psi. Below a threshold value of -69.5 mV the logarithms of the initial rate of 45Ca2+ influx and of the steady-state level of the first component were linear with respect to delta psi. It is suggested that 45Ca2+ influx across the plasma membrane is mediated by channels which open when delta psi is below a threshold value. The results indicated that Ca2+ influx across the plasma membrane was driven electrophoretically by delta psi.     

Factors determining the plasma-membrane potential of lymphocytes.

1. Lymphocytes from pig mesenteric lymph node have low permeability to K+ (Rb+), Na+ and Cl-. None of these ions is in Nernst equilibrium with the plasma-membrane potential (delta psi p). 2. delta psi p can be calculated from the transmembrane distribution of the permeant cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to abolish uptake into intracellular mitochondria. In normal culture medium delta psi p is 56 mV. 3. A similar potential is found in T-enriched pig cells and in mouse thymocytes. 4. The contribution of electrogenic (Na+ + K+)ATPase to delta psi p is about 7 mV. 5. The remainder of the lymphocyte delta psi p is a polyionic potential set up by K+ and Cl- with a permeability coefficient for Cl- of similar magnitude to that for K+.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

Modulation of plasma membrane Ca2+ pump by membrane potential in cultured vascular smooth muscle cells

We examined the effect of membrane potential (Em) on the activity of the plasma membrane Ca2+ pump in cultured rat aortic smooth muscle cells (VSMCs). Inside-negative K+ diffusion potential higher or lower than the resting Em (-46 mV) was artificially imposed on VSMCs with various concentrations of extracellular K+ (K+o) and 1 microM valinomycin. We found that the recovery phase of the intracellular Ca2+ transient elicited with 1 microM ionomycin was accelerated by depolarizing Em, whereas it was retarded by hyperpolarizing Em. The rate of extracellular Na+ (Na+o)-independent 45Ca2+ efflux from VSMCs stimulated with 1 microM ionomycin increased almost linearly with a change in Em from -98 to -3 mV. This effect of Em was abolished by extracellularly added LaCl3 or a combination of high pH (pH 8.8) and high Mg2+ (20 mM), conditions that presumably inhibit the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., & Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Intracellular contents of Na+ and K+ and intracellular pH, on the other hand, were not influenced by the change in Em under the conditions used. These results indicate that alteration in Em can modulate the intracellular Ca2+ concentration in intact VSMCs by changing the rate of Ca2+ extrusion by the plasma membrane Ca2+ pump. The data strongly suggest that the plasma membrane Ca2+ pump in VSMCs is electrogenic.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. Na+-dependent K+ accumulation

We have studied some features of K+ accumulation by glycolysing Mycoplasma mycoides var. Capri cells. We report that when Na+ is absent from the external medium, K+ accumulates up to the level predicted by the amplitude of the transmembrane electrical potential, delta psi m, measured by Rb+ and methyltriphenylphosphonium cation (TPMP+) distribution. Therefore, under these experimental conditions, the coupling mechanism of K+ uptake consists of a delta psi m-driven uniport. More important, when Na+ is present in the external medium, the level of K+ accumulation by glycolysing Mycoplasma cells is far too steep to be equilibrium with delta psi m (-120 mV for delta muK+ compared with -90mV for delta muRb+ or delta muTPMP+). Our results clearly indicate the presence in Mycoplasma of an active K+-transport system specifically stimulated by Na+. Furthermore, by controlling the amplitude of the energy-dependent delta muH+, we obtain strong evidence that this specific Na+-stimulated K+ transport is modulated by the transmembrane electrical potential. Finally, we show that ATP is consumed when such a transport system is in activity.     

Ion-dependent generation of the electrochemical proton gradient delta muH+ in reconstituted plasma membrane vesicles from the yeast Metschnikowia reukaufii

Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.     

Enthalpy changes in the formation of the proton electrochemical potential and its components

Enthalpy changes in the formation of a proton electrochemical potential (Delta mu H+) and its components, DeltapH (proton gradient) and Deltapsi (electrical potential), across two types of E. coli membrane vesicles were investigated. Flow dialysis experiments showed that in 0.1 M KPi, pH 6.6, E. coli GR19N membrane vesicles coupled with d-lactate exhibited 57 mV for DeltapH, 70 mV for Deltapsi, and 127 mV for Delta mu H+. Microcalorimetric measurements revealed that the corresponding enthalpy changes (DeltaH(pH), DeltaH(psi) and DeltaHm) were 3.5, 3.3 and 6.9 kcal/mole, respectively. Moreover, in E. coli ML 308-225 membrane vesicles across which 120mV of Delta mu H+ was generated, values of DeltaH(pH) and DeltaH(psi) were determined as 7.0 and 6.6 kcal/mole, as compared with the previously reported 14.1 kcal/mole for DeltaH(m). Comparisons of these enthalpy data revealed that component enthalpies (DeltaH(pH) and DeltaH(psi)) essentially added up to the total enthalpy (DeltaHm), providing a self-consistent test for the obtained data. In both membranes, the ratio ofDeltaH(psi) to Deltapsi was comparable to that of DeltaH(pH) to DeltapH in the formation of Delta mu H+. These observations indicated that the process of the movement of H+ across the membranes was the major contributor to the observed energetic changes. Moreover, the enthalpy change in the formation of Delta mu H+ was compared with the membranes derived from GR19N and ML 308-225 and coupled with NADH and d-lactate. The results were discussed in terms of trans-membrane phenomena.     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.     

Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.