Monitoring of the mitochondrial and plasma membrane potentials in human fibroblasts by tetraphenylphosphonium ion distribution

The lipophilic cation tetraphenylphosphonium (TPP⁺) is accumulated by human skin fibroblasts across both the plasma and mitochondrial membranes. We show here that TPP⁺ uptake is indeed greatly decreased under conditions leading to de-energization of mitochondria. The TPP⁺ accumulation in the presence of the proton ionophore FCCP has been used for determination of the plasma membrane potential across the plasma membrane, after correction for potential-independent binding of TPP⁺ to cellular components. Following this procedure, a value of 75 mV has been obtained. Through the amount of TPP⁺ released by FCCP treatment, an estimate of thein situ mitochondrial membrane potential has been made. Furthermore, we report that the mitochondrial component of TPP⁺ accumulation decreases with aging of fibroblast cultures.Abbreviations _m membrane potential across thein situ mitochondria - _p membrane potential across the plasma membrane - TPP⁺ tetraphenylphosphonium - HEPES N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone     

Potassium accumulation in growing Methanobacterium thermoautotrophicum and its relation to the electrochemical proton gradient

Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K⁺]_{intracellular}/[K⁺]_{extracellular}) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (¹⁴C) tetraphenylphosphonium (TPP⁺). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of ¹²C-TTP⁺ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K⁺ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP⁺ Tetra phenylphosphonium bromide - DTE Dithioerythritol - TCS 3,5,3,4-Tetrachlorosalycylanilide     

Competitive reactions of a ruthenium arene anticancer complex with histidine, cytochrome c and an oligonucleotide

The ruthenium arene anticancer complex [(⁶-bip)Ru(en)Cl][PF₆] (1) (bip is biphenyl, en is ethylenediamine) reacted slowly with the amino acid L-histidine (L-His) in aqueous solution at 310 K. Two L-His adducts of 1 were separated by high-performance liquid chromatography and identified by electrospray ionisation mass spectrometry and NMR: an imidazole N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺, and an N-bound complex [(⁶-bip)Ru(en)(N–L-His)]²⁺. At 310 K, after 24 h only about 22% of complex 1 (2 mM) reacted with L-His, and of the unreacted 1, 59% had hydrolysed. In the presence of 100 mM NaCl, approximately 90% of 1 remained unreacted. In aqueous solution or triethylammonium acetate (TEAA) buffer (pH 7.6), ¹⁵N-labelled 1 reacted with cytochrome c to give two monoruthenated protein adducts. The reaction reached equilibrium within 2 h by which time approximately 50% of cytochrome c was ruthenated. On the basis of [¹H, ¹⁵N] NMR data, one adduct may have Ru bound to the N-terminus, and the other to a carboxylate group on the protein. In TEAA buffer and at 310 K, more than 90% of the 14-mer oligonucleotide d(TATGTACCATGTAT) reacted with 2 mol Eq of 1 to give rise to monoruthenated and diruthenated oligonucleotide adducts. The presence of cytochrome c (1 mol Eq) or L-His (4 mol Eq) had little effect on the course of the reaction with the oligonucleotide. In cells, DNA (or RNA) may be a favoured reaction site for this Ru anticancer complex.Electronic supplementary material is available for this article at .

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Properties of an anion/H+ contransport system in L1210 cells that utilizes phthalate as a nonphysiological substrate

Summary [¹⁴C]Phthalate is transported into L1210 cells via two separate routes, an anion exchange system whose primary substrates are folate compounds, and a second less active system which is sensitive to bromosulfophthalein. When the principal uptake component was blocked by a specific irreversible inhibitor of this system, the remaining route (at pH 7.4) appeared to be saturable and was inhibited by several anions in addition to bromosulfophthalein (K _i =2 m), including 8-anilino-1-naphthalein sulfonate (K _i =25 m), unlabeled phthalate (K _i =500 m), and chloride (K _i =3500 m). A pronounced effect by pH was also observed. Influx and total uptake of phthalate both increased progressively with decreasing pH and reached values that were 20-fold higher at pH 6.0, compared with pH 7.4. This pH-dependent increase could be blocked, however, by the addition of compounds (nigericin and carbonylcyanidem-chlorophenylhydrazone) which, in combination, collapse proton gradients. Phthalate efflux was relatively insensitive to changes in extracellular pH but could be inhibited (up to 90%) by bromosulfophthalein. Several other anions also inhibited efflux, but to a lesser extent, while chloride, phthalate, lactate, glycolate and acetate enhanced efflux up to 1.8-fold. Efflux also increased at pH 6.0, but not at pH 7.5, upon addition of nigericin and carbonylcyanidem-chlorophenylhydrazone. These results suggest that phthalate is a nonphysiological substrate for a carrier system which mediates transport via an anion/H⁺ symport mechanism. This system is not the lactate/H⁺ symport carrier of L1210 cells since: (A) phthalate and lactate influx were inhibited to differing degrees by various anions; and (B) lactic anhydride inhibited the influx and efflux of lactate but had no effect on the transmembrane movement of phthalate. The specificity of this system suggests that its primary anion substrate may be chloride.     

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

The influence of potassium and sodium ions on the negative pasteur effect in Brettanomyces claussenii custers

Summary The present article deals with the negative Pasteur effect in Brettanomyces claussenii Custers, i.e. with the inhibition of the alcoholic fermentation under anaerobic conditions and its stimulation in presence of atmospheric oxygen.As distinct from the negative Pasteur effect in resting cells of Saccharomyces species the effect in Br. claussenii is not specific for cell suspensions prepared with succinic acid-succinate buffer but may at Ph 4.5–4.6 in addition be demonstrated in resting cells suspended in distilled water or phosphate buffer as well as in buffer solutions of a great number of organic acids and their alkali salts, e. g. in acetic acid-acetate, propionic acid-propionate, oxalic acid-oxalate, malonic acid-malonate, fumaric acid-fumarate, malic acid-malate, d-tartaric acid-tartrate, and citric acid-citrate mixtures.The aerobic fermentation of glucose by resting cells of Br. claussenii is quite sensitive to potassium and sodium ions. In all systems examined, except the succinic acid-succinate buffer and the buffer solutions inhibiting the fermentation completely or practically completely, the rate of the aerobic fermentation is considerably increased on increasing the concentration of the potassium ions and decreasing that of the sodium ions. Under anaerobic conditions the alcoholic fermentation is insusceptible to the ions mentioned.Because of the fact that the influence of the potassium ions or of the K⁺/Na⁺ ratio upon the rate of fermentation is comparatively large under aerobic conditions but small or even negligible under anaerobic conditions, the magnitude of the negative Pasteur effect will under the proper conditions be determined by the potassium ion concentration or by the ratio between the concentrations of the potassium and sodium ions. The negative Pasteur effect obtained in a buffer of an acid and its potassium salt may be considerably larger than that observed in a buffer of the same acid and its sodium salt. In solutions containing a mixture of the potassium and sodium salts in addition to an acid the magnitude of the negative Pasteur effect will increase with increasing potassium ion concentration and decreasing sodium ion concentration at constant total molar concentration of the alkali ions.To Professor Dr. E. G. Pringsheim with best wishes on his 80th birthday.     

Effects of Na+, H+, and Cl− on alanine transport by lobster hepatopancreatic brush border membrane vesicles

Summary Epithelial brush border membrane vesicles (BBMV) of lobster hepatopancreas were formed by a magnesium precipitation technique previously described (Ahearn et al. 1985).³H-l-alanine transport by these vesicles was sodium and potassium insensitive, in contrast to a strong Na-dependency exhibited by³H-d-glucose transport. Initial alanine entry rates (15 s uptake) were stimulated and transient alanine uptake overshoots were observed when external pH was acidic (e. g. pH 4.0, 5.0 or 6.0) and a Cl gradient was imposed across the vesicular wall; at pH_o=7.4 alanine uptake was reduced in rate and hyperbolic in character. Alanine uptake from an acidic extravesicular environment in the absence of Cl responded to a transmembrane electrical potential difference created by an outwardly-directed, valinomycin-induced, potassium diffusion potential, suggesting that the alanine molecule alone carried sufficient charge under these conditions to respond to the electrical gradient. External 5.0 mMl-lysine andl-serine similarly inhibited the influx and overshoot properties of 0.05 mM³H-l-alanine uptake, whereas 5.0 mMl-leucine had virtually no effect. Trans-stimulation of alanine initial uptake rates and an enhancement of alanine accumulation against a concentration gradient were observed by vesicles preloaded with 1 mMl-lysine, but not by vesicles lacking amino acids or those containing 1 mMl-leucine orl-serine.³H-l-alanine influx from acidic external environments in the presence of a Cl gradient occurred by a combination of carrier-mediated transfer and apparent diffusion. Decreasing pH_o from 6.0 to 4.0 elevated alanineK _t from 0.55 to 2.64 mM, while alanineJ _M increased from 55 to 550 pmol/mg protein· 15 s. Apparent diffusional permeability of the membranes to alanine under these conditions increased slightly. These results suggest, but do not conclusively prove, that alanine transport across BBMV of lobster hepatopancreas may occur by way of a classical y+ transprot protein at acidic pH. The extent of this transport is determined by the magnitude of the transmembrane chloride gradient which serves as a powerful driving force for cationic amino acids in this tissue.     

Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP

Summary The ratio of valinomycin-mediated unidirectional K⁺ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K⁺ net efflux and⁴²K influx. The driving force for the net efflux (V _m –E _K ⁺) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K⁺ net efflux has been calculated from the K⁺ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K⁺ transport from unrestricted electrodiffusion, when protonophore is present.In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 m and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.     

Ammonium-stimulated,sodium-dependent uptake of glutamine in Bacillus pasteurii

The uptake of glutamine was studied in Bacillus pasteurii DSM 33. Only one uptake system was detected in the concentration range studied (between 1 and 100 M glutamine) which exhibited Michaelis-Menten saturation kinetics, with an apparent K _t of 10.7 (±3.5) M glutamine. The uptake was sodium-dependent (apparent K _t=0.2 mM Na⁺); none of several monovalent cations tested was able to replace sodium in the uptake reaction. Ionophores interfering with proton, sodium or potassium gradients across membranes strongly inhibited uptake of glutamine. Low uptake rates correlating with low potassium content and an acidic cytoplasm were measured in cells grown at high ammonium¹ concentrations. Ammonium and other permeant amines as well as potassium stimulated the uptake reaction in these cells, leading to an increase of up to 100-fold in V _max without affecting the affinity of the uptake system. In cells grown at low concentrations of ammonium, an alkaline cytoplasm and both high glutamine uptake activities and potassium content were measured; the uptake reaction was not further stimulated by permeant amines or potassium in such cells. Growth of the strain was inhibited by Tris at high concentrations; this inhibition was relieved by the addition of increasing amounts of ammonium.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide This work is dedicated to Prof. Dr. H. Kaltwasser on the occasion of his 60th birthday     

Role of the sodium pump and the background K+ channel in passive K+(Rb+) uptake by isolated cardiac sarcolemmal vesicles

Summary A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of⁸⁶Rb⁺ by the preparation was measured in the presence of an opposing K⁺ gradient using a rapid ion exchange technique. At low extravesicular Rb⁺ concentrations, at least 50% of the uptake was blocked by addition of 1mm ouabain to the assay medium. Orthovanadate (50 m), ADP (2.5mm), or Mg (1mm) were also partial inhibitors of Rb⁺ uptake under these conditions, and produced a complete block of Rb⁺ influx in the presence of 1mm ouabain. When⁸⁶Rb⁺ was used as a tracer of extravesicular K⁺ (Rb ₀ ⁺ 40 m K ₀ ⁺ =0.1–5mm) a distinct uptake pathway emerged, as detected by its inhibition by 1mm Ba²⁺ (K _0.5=20 m). At a constant internal K⁺ concentration (K _in ⁺ =50mm) the magnitude of the Ba²⁺-sensitive K⁺ uptake was found to depend on K ₀ ⁺ in a manner that closely resembles the K⁺ concentration dependence of the background K⁺ conductance (I _Kl) observed electrophysiologically in intact cardiac cells. We conclude that K⁺ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (¹⁵NH₄)₂SO₄ ranging from 10 to 200 M NH ₄ ⁺ with and without 200 M K(⁸⁶Rb)Cl or to K(⁸⁶Rb)Cl ranging from 10 to 200 M K⁺ with and without 200 M NH ₄ ⁺ as (¹⁵NH₄)₂SO₄. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H⁺ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V _max and increases in K_m of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium

Summary Internal perfusion ofHelix neurons with a solution containing potassium aspartate, MgCl₂, ATP, and HEPES causes the calcium-activated potassium current (I _K(Ca)) evoked by depolarizing voltage steps to decrease with time. When internal free Ca⁺⁺ is strongly buffered to 10^–7 m by including 0.5mm EGTA and 0.225mm CaCl₂ in the internal solution,I _K(Ca) remains constant for up to 3 hours of perfusion. In cells whereI _K(Ca) is small at the start of perfusion, perfusion with the strongly buffered 10^–7 m free Ca⁺⁺ solution produces increases inI _K(Ca) which ultimately saturate. In cells perfused with solutions buffered to 10^–6 m free Ca⁺⁺,I _K(Ca) is low and does not change with perfusion. These results lead us to conclude thatI _K(Ca) is stable in perfusedHelix neurons and that the apparent loss ofI _K(Ca) seen initially with perfusion is due to accumulation of cytoplasmic calcium. Since the calcium current (I _Ca) provides the Ca⁺⁺ which activatesI _K(Ca) during a depolarizing pulse,I _Ca is also stable in perfused cells when free intracellular Ca⁺⁺ is buffered.Perfusion with 1 m calmodulin (CaM) produces no effect onI _K(Ca) with either 10^–7 or 10^–6 m free internal calcium. Inhibiting endogenous CaM by including 50 m trifluoperazine (TFP) in both the bath and the internal perfusion solution also produces no effect onI _K(Ca) with 10^–7 m free internal calciu. It is concluded that CaM plays no role inI _K(Ca) activation.     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Transport of leucine in the cyanobacterium Anabaena variabilis

The amino acid leucine was transported by the cyanobacterium Anabaena variabilis. The K _m for transport was 10.8 M; the V _max was 8.7 nmoles min^–1 mg^–1 chlorophyll a. Transport of leucine was energy dependent: uptake of leucine was inhibited in the dark, and by DCMU and cyanide. Transport was neither dependent on nor enhanced by Na⁺. Prior growth of cells with leucine did not repress transport of [¹⁴C]-leucine. Alanine, glycine, valine, and methionine were strong competitive inhibitors of leucine uptake; serine, threonine, isoleucine, norleucine, and d-alanine competitively inhibited to a lesser degree. Other amino acids or amino acid analogues, including d-leucine, -aminoisobutyrate, and d-serine did not inhibit the transport of leucine.Abbreviations Chl a chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TES N-tris(hydroxymethyl)-2-aminoethane-sulfonic acid - TCA trichloroacetic acid - Tris N-tris(hydroxymethyl)aminoethane     

Analysis of glutamate receptors in primary cultured neurons from fetal rat forebrain

In order to further analyze the development of glutamatergic pathways in neuronal cells, the expression of excitatory amino acid receptors was studied in a model of neurons in primary culture by measuring the specific binding of L-[³H]glutamate under various incubation conditions in 8-day-old intact living neurons isolated from the embryonic rat forebrain, as well as in membrane preparations from these cultures and from newborn rat forebrain. In addition, the receptor responsiveness to glutamate was assessed by studying the uptake of tetraphenylphosphonium (TPP⁺) which reflects membrane polarization. In the presence of a potent inhibitor of glutamate uptake, the radioligand bound to a total number of sites of 36.7 pmol/mg protein in intact cells incubated in a Tris buffer containing Na⁺, Ca²⁺, and Cl^–, with a Kd around 2 M. In the absence of the above ions, [³H]glutamate specific binding diminished to 14.2 pmol/mg protein with a Kd-value of 550 nM. Under both of the above conditions, similar Kd were obtained in membranes isolated from cultures and from the newborn brain. However, Bmax-values were significantly lower in culture membranes than in intact cells or newborn membranes. Displacement studies showed that NMDA was the most potent compound to inhibit [³H]glutamate binding in membranes obtained from cultured neurons as well as from the newborn brain, whereas quisqualate, AMPA, kainate andtrans-ACPD were equally effective. According to these data and to the ionic dependence of glutamate binding, it was concluded that cultured neurons from the rat embryo forebrain express various glutamate receptor subtypes, mainly L-AP4 and NMDA receptors, with characteristics close to those in the newborn brain, and which display functional properties since a transient cell exposure to glutamate led to a 70% inhibition of [³H]TPP⁺ uptake.     

Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells

Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na⁺ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg²⁺ and can be increased by 40-fold by loading cells with Mg²⁺ to 10 mm. The purpose of this study was to verify the connection of AS Na⁺ influx with Na-dependent, amiloride-sensitive Mg²⁺ efflux and to utilize AS Na⁺ influx to explore that pathway.Determination of unidirectional influx of Na⁺ and net loss of Mg²⁺ in parallel sets of cells showed that activation by extracellular [Na⁺] follows a simple Michaelis-Menten relationship for both processes with a K _m of 105–107 mm and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg²⁺] with a [Mg²⁺]_0.5 of 2.1–2.3 mm and a Hill coefficient of 1.8. Comparison of V_max for both sets of experiments indicated a stoichiometry of 2 Na: l Mg. Amiloride inhibits Na⁺ influx and Mg²⁺ extrusion in parallel (K _i = 0.3 mm). Like Mg²⁺ extrusion, amiloride-sensitive Na⁺ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na⁺ influx in hamster red cells appears to be through the Na-Mg exchange pathway.There was no amiloride-sensitive Na⁺ efflux in hamster red cells loaded with Na⁺ and incubated with high [Mg²⁺] in the medium with or without external Na⁺, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier.     

Temperature-dependent modification of divalent cation flux in the rat parotid gland basolateral membrane

Divalent cation (Mn²⁺, Ca²⁺) entry into rat parotid acinar cells is stimulated by the release of Ca²⁺ from the internal agonist-sensitive Ca²⁺ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn²⁺ influx into internal Ca²⁺ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca²⁺-free medium for 10 min) and passive ⁴⁵Ca²⁺ influx in basolateral membrane vesicles (BLMV). Mn²⁺ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn²⁺ entry appeared to be inactivated since it was not increased by raising extracellular [Mn²⁺] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn²⁺ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q₁₀=1.7) at 21–37°C to 25 kcal/mol (Q₁₀=3.0) at 21-8°C. Under the same conditions, Mn²⁺ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E _a of 7.8 kcal/mol (Q₁₀=1.5) and 6.2 kcal/mol (Q₁₀ < 1.5), respectively. These data suggest that depletion-activated Mn²⁺ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn²⁺ entry into unstimulated acini.As in intact acini, Ca²⁺ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca²⁺ influx in BLMV at 37°C demonstrated the presence of two Ca²⁺ influx components: a saturable component, with K _Ca =279 ± 43 m, V_max = 3.38 ± 0.4 nmol Ca²⁺/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but V_max decreased by 61% to 1.3 ± 0.4 nmol Ca²⁺/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca²⁺ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca²⁺ uptake at 37°C there was no significant loss of Ca²⁺ influx. These data suggest that the temperature sensitive high affinity Ca²⁺ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca²⁺ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work.     

Effects of membrane potential on sodium-dependent calcium uptake by sarcolemma-enriched preparations from canine ventricle

Summary The effect of membrane potential on sodium-dependent calcium uptake by vesicles in an isolated cardiac sarcolemma preparation was examined. Initial time course studies showed that the reaction deviated from initial velocity conditions within minutes. This appeared to be due, in part, to loss of the sodium gradient. Assays carried out to 10 sec revealed a linear component of uptake (2 to 10 sec) and a faster component (complete by 2 sec). The latter was eliminated by loading the preparation with ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid (EGTA). This maneuver did not affect the slow component, and subsequent studies used preparations containing EGTA. Potassium Nernst potentials (E _K), established by potassium gradients in the presence of valinomycin, were varied from –100 to +30 mV by changing [K⁺] _o from 1.18 to 153.7mM ([K⁺] _i =50mM). The initial velocity of sodium-dependent calcium uptake was stimulated twofold by changingE _K from –100 to 0 mV and another twofold by raisingE _K from 0 to +30 mV. For the total range ofE _K and [K⁺] _o , 32 to 36% of the increase appeared to reflect stimulation by extravesicular potassium. The remainder appeared to be due to membrane potential. The profile of sodium-dependent calcium uptake versusE _K suggested that calcium influx through electrogenic sodium/calcium exchange may be much more affected by the positive region of the cardiac action potential than by the negative region.