Thermal membrane potential across charged membranes in NaCl-NH4Cl and LiCl-NH4Cl solutions

Measurements of the thermal membrane potential across cation exchange membranes were carried out by using aqueous solutions containing two 1-1 electrolytes, with an anion in common. The same solution was used on both sides of the membrane. In all cases a good linear relationship was observed between the thermal membrane potential Δψ and the temperature difference ΔT (in the range ΔT = ± 10°C). Assuming that the activity of one cation is equal to that of another cation in the solutions and the sum of transport numbers of cations is unity, the plot of Δψ/ΔT vs logarithmic activity of one cation is linear with a slope of R/F. These experimental results aie in agreement with a theory presented previously. From the analysis of thermal membrane potential in mixtures of electrolytes it is obtained that the cross coefficient of cation-cation interaction in membranes is negative and about 6 to 9% of the main coefficient.     

Changes in membrane potential during calcium ion influx and efflux across the mitochondrial membrane.

1. A depolarisation of the membrane of rat liver mitochondria, as measured with the safranine method, is seen during Ca2+ uptake. The depolarisation is followed by a slow repolarisation, the rate of which can be increased by the addition of EGTA or phosphate. 2. Plots relating the initial rate of calcium ion (Ca2+) uptake and the decrease in membrane potential (delta psi) to the Ca2+ concentration show a half-maximal change at less than 10 micron Ca2+ and a saturation above 20 micron Ca2+. 3. Plots relating the initial rate of Ca2+ uptake to delta psi are linear. 4. Addition of Ca2+ chelators, nitriloacetate or EGTA, to deenergized mitochondria equilibrated with Ca2+ causes a polarisation of the mitochondrial membrane due to a diffusion potential created by electrogenic Ca2+ efflux. 5. If the extent of the response induced by different nitriloacetate concentrations is plotted against the expected membrane potential a linear plot is obtained up to 70 mV with a slope corresponding to two-times the extent of the response induced by valinomycin in the presence of different potassium ion gradients. This suggests that the Ca2+ ion is transferred across the membrane with one net positive charge in present conditions.     

Electrolyte transport across rabbit late proximal colon in vitro

The second part of rabbit proximal colon was investigated in vitro under short circuit conditions. Unidirectional sodium and chloride fluxes were measured during the soft faeces period and during the hard faeces period. Rabbit late proximal colon has a potential difference (psi mS) of 4 mV, a tissue conductance (GT) of 10-11 mS/cm2 and a short circuit current (Isc) of 1.5 mueq/cm2 X hr. Under control conditions sodium (2.65 mueq/cm2 X hr) and chloride (0.67 mueq/cm2 X hr) are absorbed. Ouabain abolished psi ms,Isc and the net sodium flux totally, whereas 0.1 mM amiloride only slightly decreased the net sodium flux. No differences in electrical properties and Na,Cl-fluxes were found between the faeces periods. Removal of sodium abolished psi ms and Isc totally, and a high potassium solution depolarized the preparation (psi ms = 0). A linear current-voltage relation characterizes the tissue as an ohmic resistor between -40 and +50 mV, and reveals a slope conductance of 14 mS/cm2 under KCl conditions. We conclude that the transport functions under in vitro conditions differ markedly from the in vivo situation, and that the diurnal differences of electrolyte transport in vivo occur mainly by the involvement of ionic gradients.     

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes ofNecturus small intestine

Intracellular electrical potentials and K activity, (K)c, were determined simultaneously in Necturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (psi mc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of psi mc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine. In the absence of alanine, psi mc averaged -32 mV and (K)c averaged 67 mM. When a steady state was achieved in the presence of alanine these values averaged -24 mV and 50 mM, respectively. The steady-state electrochemical potential differences for K across the basolateral membrane in the absence and presence of alanine did not differ significantly. Inasmuch as the rate of transcellular active Na transport or "pump activity" was increased two- to threefold in the presence of alanine, it follows that, if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease in rs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in "pump activity". This "homocellular" regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and, (ii) repolarize psi mc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.     

Validation of a Noninvasive Method to Measure Brain Temperature In Vivo Using 1H NMR Spectroscopy

Abstract: The goal of this study was to evaluate the potential of using the difference between the ¹H NMR frequencies of water and N -acetylaspartic acid (NAA) to measure brain temperature noninvasively. All water-suppressed and non-water-suppressed ¹H NMR spectra were obtained at a field strength of 4.7 T using a surface coil. Experiments performed on model solutions revealed a decrease in the difference between NMR frequencies for NAA and water as a linear function of increasing temperature from 14 to 45°C. Changing pH in the range 5.5–7.6 produced no discernible trends for concurrent changes in the slope and intercept of the linear relationship. There were minor changes in slope and intercept for solutions containing 80 or 100 mg of protein/ml versus no protein, but these changes were not considered to be of sufficient magnitude to deter the use of this approach to measure brain temperature. The protein content of swine cerebral cortex was found to remain constant from newborn to 1 month old (78 ± 12 mg/g; n = 41). Therefore, data collected for the model solution containing 80 mg of protein/ml were used as a calibration curve to calculate brain temperature in eight swine during control, hypothermia, ischemia, postischemia, or death, over a temperature range of 23–40°C. A plot of 61 temperatures determined from ¹H NMR versus temperatures measured from an optical fiber probe sensor implanted 1 cm into the cerebral cortex showed excellent linear agreement (slope = 1.00 ± 0.03, r ² = 0.96). We conclude that ¹H NMR spectroscopy presents a practical means of making noninvasive measurements of brain temperature with an accuracy of better than ± 1°C.     

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles

The temperature dependence of sodium-dependent and sodium-independent d-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent d-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15°C. The sodium-independent uptake of d-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35–5°C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4°C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.     

The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase.

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.     

Mechanism of proline transport in Escherichia coli K12. I. Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles

The stoichiometric coupling mechanism of the membrane potential (delta psi) in the reaction of H+/proline symport was investigated kinetically, using cytoplasmic membrane vesicles of the proline carrier-overproducing strain of Escherichia coli MinS/ pLC4 -45. When a delta psi was imposed across the cytoplasmic membrane by respiration, the Michaelis constant of transport (Kt) was lowered to about 1 microM, which was 2 orders of magnitude smaller than that of passive influx and efflux, and the maximum velocity (Vmax) was concomitantly enhanced as an exponential function of delta psi. Thermodynamically, the carrier translocated proline with a stoichiometry of 2 mol of protons versus 1 mol of substrate when driven by a delta psi at pH 8.0. Data on the delta psi dependence of Vmax of proline transport could be explained quantitatively by the Geck-Heinz hypothesis (Geck, P., and Heinz, E. (1976) Biochim, Biophys. Acta 443, 49-63). A symmetrical model of the 2H+/proline symport via formation of a carrier/H+/substrate (CH+H+S) intermediate is proposed. In this model, the effect of delta psi on the Kt was resolved as stimulation of formation of a transport intermediate, whereas the effect of delta psi on the Vmax was explained by enhancement of translocation of loaded carriers between the two sides of the membrane.     

Normal and anomalous potential responses due to K+ changes in bullfrog antrum

The effect of changing the K+ concentration in the bathing media was studied in the bullfrog antrum. Usually increasing K+ on the nutrient side in standard Cl- -containing and Cl- -free solutions decreased the transmucosal potential difference (nutrient became more negative) - a normal effect. Similar results were obtained on the secretory side. Moreover, for K+ changes on the nutrient side in Cl- media, a plot of magnitude of delta V vs. log [K+] was linear for [K+] greater than 20 mM with slope of 27 mV per 10-fold change in [K+]. However, after bathing the mucosa in Cl- media with zero K+ for about 20 min, elevating the nutrient [K+] to 4 mM increased the potential difference (V) by 4.8 mV in 5 min and repeating the same sequence increased V by 6.9 mV in 5 min - both anomalous effects. Beyond 20 mM K+ the response was normal. In SO2-4 media, an anomalous potential difference of about 1 mV was obtained for changes from 0 to 3 or 6 mM nutrient K+. Ouabain (1 X 10(-3) M) in the nutrient solution abolished the anomalous response in Cl- and SO2-4 media. The normal response is attributed to passive, conductance pathways and the anomalous response because of the effect of ouabain, to a (Na+ + K+)-ATPase pump on the nutrient-facing membrane in which more Na+ than K+ ions are transported per cycle.     

Thermoosmosis through charged membranes

Thermoosmosis through oxidized collodion and collodion-sulfonated polystyrene interpolymer membranes has been observed in KCl solutions of various concentrations. The effective temperature difference acting for thermoosmosis was determined by measuring the thermal membrane potential appearing on both sides of membrane. It was found that the velocity of thermoosmosis is proportional to the effective temperature difference and the proportionality constant (thermoosmotic coefficient) is a function of electrolyte concentration. The dependence of the thermoosmotic coefficient of charged membranes on the electrolyte concentration is found to have a characteristic feature.     

Increase in membrane permeability of electrolytes and betacyanin in beet root disc by fenitrothion

Effect of fenitrothion (phosphorothioic acid, 0,0-dimethyl 0-4-nitro-m-tolyl ester), an organophosphorous insecticide, on membrane permeability employing the leakage of betacyanin and electrolytes as the criteria were studied in beet root(Beta vulgaris) discs. The leakage of both betacyanin and electrolytes increased with increasing concentrations (10–150 ppm) of fenitrothion in the incubation medium. At 0.33 mM the increase in electrolyte leakage was approximately linear for the first 6h, while the increase in betacyanin leakage started with a lag of about 2 h. Long term incubation (24 h) showed a biphasic nature (in the semilog plot) for the increase in betacyanin leakage, while the increase in electrolyte leakage appeared more complex. In the control sample, the Arrhenius plots (25–50°C) of leakage showed a break at 40°C. In treated samples no break was observed, but the slope decreased (for both electrolyte and betacyanin leakage) as compared to the respective slopes in the control in the temperature region greater than 40°C. The results are discussed in terms of the possible effect of the insecticide on the active transport in plant membranes     

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.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

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.     

Electrophysiology of flounder intestinal mucosa. II. Relation of the electrical potential profile to coupled NaCl absorption

We characterized the hyperpolarization of the electrical potential profile of flounder intestinal cells that accompanies inhibition of NaCl cotransport. Several observations indicate that hyperpolarization of psi a and psi b (delta psi a,b) results from inhibition of NaCl entry across the apical membrane: (a) the response was elicited by replacement of mucosal solution Cl or Na by nontransported ions, and (b) mucosal bumetanide or serosal cGMP, inhibitors of NaCl influx, elicited delta psi a,b and decreased the transepithelial potential (psi t) in parallel. Regardless of initial values, psi a and psi b approached the equilibrium potential for K (EK) so that in the steady state following inhibition of NaCl entry, psi a approximately equal to psi b approximately equal to ECl approximately equal to EK. Bumetanide decreased cell Cl activity (aClc) toward equilibrium levels. Bumetanide and cGMP decreased the fractional apical membrane resistance (fRa), increased the slope of the relation of psi a to [K]m, and decreased cellular conductance (Gc) by approximately 85%, which indicates a marked increase in basolateral membrane conductance (Gb). Since the basolateral membrane normally shows a high conductance to Cl, a direct relation between apical salt entry and GClb is suggested by these findings. As judged by the response to bumetanide or ion replacement in the presence of mucosal Ba, inhibition of Na/K/Cl co-transport alone is not sufficient to elicit delta psi a,b. This suggests the presence of a parallel NaCl co-transport mechanism that may be activated when Na/K/Cl co-transport is compromised. The delta psi a,b response to reduced apical NaCl entry would assist in maintaining the driving force for Na-coupled amino acid uptake across the apical membrane as luminal [NaCl] falls during absorption.     

Respiration and germination rates of tomato seeds at suboptimal temperatures and reduced water potentials

Suboptimal temperature (T) affects germination rates (reciprocalof time to radicle emergence) on a thermal time basis; thatis, the T in excess of a base or minimum temperature multipliedby the time to a given per cent germination [t_g) is a constant.Respiration rates are also sensitive to T, and proportionalrelationships are often found between respiration rates andgermination rates. Reduced water potential () delays seed germinationon a hydrotime basis (i.e. the in excess of a base water potentialmultiplied by t_g is a constant). It was tested whether respirationrates prior to radicle emergence vary in proportion to T and as expected from the thermal and hydrotime models. Respirationrates (C0₂ evolution) of cold-tolerant, rapidly germinating(PI 341988) and cold-sensitive, more slowly germinating (T5)tomato [Lycopersicon esculentum Mill.) seeds were evaluatedover a range of T and conditions. For both genotypes, respirationrates until the beginning of radicle emergence were relatedto T on a thermal time basis and increased approximately linearlywith above -2.0 MPa, consistent with the hydrotime model. Respirationrates were uniquely related to germination rates, regardlessof whether germination timing was affected by T, , or genotype.However, germination timing was unaffected when respirationrates were manipulated by varying 0₂ partial pressure. Thus,while both germination and respiration rates vary with T and consistent with thermal and hydrotime models of biologicaltime, respiration rates per se were not the limiting factorin germination timing of tomato seeds. Key words: Lycopersicon esculentum Mill., tomato, germination, respiration, temperature, water potential     

Electrochemical potential of protons in vesicles reconstituted from purified, proton-translocating adenosine triphosphatase.

Measurements were made of the difference in the electrochemical potential of protons (delta-mu H+) across the membrane of vesicles restituted from the ATPase complex (TF0.F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (delta psi) and pH difference across the membrane (delta pH), respectively. In the presence of Tris buffer the maximal delta psi ans no delta pH were produced, while in the presence of the permeant anion NO-3 the maximal delta pH and a low delta psi were produced by the addition of ATP. When thATP concentration was 0.24 mm, the delta psi was 140-150 mV (positive inside) in Tris buffer, and the delta pH was 2.9-3.5 units (acidic inside) in the presence of NO-3. Addition of a saturating amount of ATP produced somewhat larger delta psi and delta pH values, and the delta -muH+attained was about 310mV. By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4-5 during ATP hydrolysis. The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

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.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.     

A membrane potential-sensitive dye for vascular smooth muscle cells assays

Changes in membrane potential of rat aorta smooth muscle cells were investigated using the bis-oxonol sensitive probe DIBAC2(3). We compared the changes in membrane potential induced by a high external KCl concentration in aorta smooth muscle cells from normotensive 2 kidney (2K) and from renal hypertensive 2 kidney-1 clip (2K-1C) rats. The spectral properties of the membrane potential were first characterized in aqueous buffers and in cultured smooth muscle cells from 2K and 2K-1C rat aortas. Fluorescence emission and the images were recorded using a laser scanning confocal microscope. The relationship between fluorescence intensity (FI) and membrane potential (psi(m)) as a function of the increasing extracellular KCl concentration was linear in the 5-40 mmol/L KCl range in both 2K and 2K-1C rat aorta cells. Cell membranes from 2K-1C rat aorta cells were more depolarized (-55 mV) than 2K rat aorta cells (-65 mV). The results show that in 2K-1C aorta cells only 10 mmol/L KCl was needed to induce complete membrane depolarization while in 2K cells 40 mmol/L KCl was needed to induce a similar effect. This study clearly shows that the method is suitable to measure the membrane potential in cultured smooth muscle cells.