Driving forces in hepatocellular uptake of phalloidin and cholate

Active uptake of phalloidin and cholate in isolated rat liver cells depends upon both Na⁺ gradient and membrane potential. Omission of Na⁺ or inhibition of the (Na⁺ + K⁺)-ATPase diminished both phalloidin and cholate uptake. Dissipation of the sodium, potassium or proton gradient by monensin, nigericin, gramicidin and valinomycin blocked phalloidin uptake and also caused reduction of cholate transport. Chelation of Ca²⁺ and Mg²⁺ by EGTA or incubation of liver cells with NH₄Cl neither influenced phalloidin nor cholate uptake. Hyperpolarization of liver cells by the lipophilic anions NO₃⁻ or SCN⁻ enhanced phalloidin but reduced cholate uptake. Depolarization induced by a reversed K⁺ gradient reduced both kinds of transport. The results indicate that sodium ions and the membrane potential are driving forces for phalloidin and cholate uptake in hepatocytes.     

The Relation between Membrane Potential and the Transport Activity of Systems a and L in Plasma Membrane Vesicles of the Ehrlich Cell

Plasma membrane vesicles isolated from Ehrlich ascites tumor cells have been used to investigate the role of the transmembrane potential in the energetics of Systems A and L. As expected, Na⁺-dependent System A was responsive to changes in membrane potential. System L activity, as measured by transport of 2-aminonorbornane-2-carboxylic acid (BCH), was shown to be Na⁺-independent and was not altered by changes in the membrane potential. The combination of valinomycin and nigericin decreased accumulation of MeAIB but not that of BCH. The presence of nigericin alone caused a significant decrease in uptake by System A and a decrease in uptake by System L to a lesser degree. The inhibitory action of nigericin might reflect its ability to dissipate the Na⁺ gradient rather than an effect on K⁺ or H⁺ flows. The results indicate that modes of energization not produced through the transmembrane potential must account for any uphill operation of System L.     

The use of phospholipid-impregnated millipore filters for recording nonelectrogenic cation flows in the presence of Me/nH exchangers

It has been shown that with a cation (K⁺, Na⁺, Ca²⁺) concentration gradient on a Millipore filter impregnated with a decane solution of phospholipid, in the presence of a Meⁿ⁺/nH⁺ exchanger (nigericin, monensin, A23187), addition of a protonophore induces the formation of an electric potential positively charged on the side where the concentration of the cation is lower. The formation of the potential is induced by the hydrogen ion concentration gradient in the filter and in the unstirred layers as a result of the Meⁿ⁺/nH⁺ exchange. In such a system, with a pH gradient on the filter in the presence of monensin and valinomycin, a potential is generated with the plus on the side of the lower concentration of hydrogen. The effect is the result of the formation of a potassium ion concentration gradient in the unstirred layers in the course of the K⁺/H⁺ exchange. It is concluded that phospholipid-impregnated filters can be used for search and identification of electroneutral membrane ionophores of the Meⁿ⁺/nH⁺ exchanger type.     

Incorporation de la vitellogénine tritiée dans les ovocytes du Crustacé Amphipode Orchestia gammarellus (Pallas) / Incorporation of tritiated vitellogenin into the oocytes of the crustacean amphipod Orchestia gammarellus (Pallus)

Summary

During the secondary vitellogenesis the oocytes of Orchestia gammarellus accumulate yolk spheres and lipid droplets. We studied the uptake of tritiated vitellogenin by the oocyte and its accumulation in the yolk spheres.     

Cation transport and electrogenesis byStreptococcus faecalis

Summary Uptake of the lipid-soluble cations dibenzyldimethylammonium (DDA⁺) and triphenylmethylphosphonium (TPMP⁺) byStreptococcus faecalis is biphasic. The initial phase is a rapid binding of the ions which does not require a source of metabolic energy and apparently consists of cation exchange at the cell surface. Upon addition of glucose further uptake of the cations occurs, by exchange for Na⁺ and H⁺. Evidence is presented suggesting that this metabolic uptake of DDA⁺ and TPMP⁺ is not due to active transport. It rather appears that uptake results from the generation of an electrical potential, interior negative, by the extrusion of H⁺ and, indirectly, of Na⁺. Accumulated DDA⁺ and TPMP⁺ are discharged by proton-conducting uncouplers. The cationconducting antibiotics valinomycin, monactin, nigericin and monensin do not inhibit uptake. Potassium and, under certain conditions, H⁺ displace DDA⁺ and TPMP⁺. Generation of an electrical difference across the membrane was verified by the accumulation of K⁺ in the presence of valinomycin. The concentration ratios achieved correspond to potentials of the order of –150 to –200 mV.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Na+ transport by human placental brush border membranes: Are there several mechanisms?

Na⁺ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na⁺ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na⁺ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na⁺ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na⁺ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na⁺ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na⁺ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na⁺ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na⁺. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na⁺ uptake, whereas benzamil, the Na⁺ channel blocker, had no effect, it was concluded that the amiloride sensitive Na⁺ uptake was predominantly or exclusively due to a Na⁺-H⁺ exchanger activity. K⁺ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na⁺ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10⁻⁷ M insulin, 10⁻¹⁰ M angiotensin II, or 10⁻⁸ M human parathyroid hormone (PTH) did not influence Na⁺ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na⁺-H⁺ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co-substrates such as phosphate and aminoacids, the Na⁺-H⁺ exchange is probably the unique mechanism of Na⁺ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. © 1996 Wiley-Liss, Inc.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

Membrane Potential Mediates H+-ATPase Dependence of ``Degradative Pathway' Endosomal Fusion

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as ``degradative endocytosis.' The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for ``degradative endocytosis.' These endosomal pathways also have a unique distribution of their H⁺-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H⁺-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H⁺-ATPase dependent in baby hamster kidney cells, and H⁺-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H⁺-ATPase dependent, we inhibited v-type H⁺-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H⁺-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H⁺-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H⁺-ATPase in mammalian homotypic endosomal fusion. Received: 29 October 1996/Revised: 8 December 1997     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Sucrose transport by the alkaliphilic, thermophilic Bacillus sp. strain TA2.A1 is dependent on a sodium gradient

An alkaliphilic Bacillus designated strain TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C. Sodium chloride (>5 mM) was an obligate requirement for the growth of strain TA2.A1 on sucrose, and growth on sucrose was inhibited by monensin, an ionophore that collapses the sodium gradient (ΔpNa⁺) across the cell membrane. Sucrose transport by strain TA2.A1 was sodium dependent and was inhibited by monensin. The K_t for sucrose tran-sport was 33 μM and the Eadie–Hofstee plot was linear, suggesting one high-affinity uptake system for sucrose. The affinity for sodium was low (0.5 mM), and the Hill plot had a slope of 1.6, suggesting that sodium binding was noncooperative and that the sucrose transporter had more than one binding site for sodium. Based on these results, Bacillus strain TA2.A1 uses a sodium gradient for sucrose uptake, in addition to the sodium-dependent glutamate uptake system reported previously. Received: March 15, 2000 / Accepted: July 17, 2000     

Endothelial cell oxidant generation during K+-induced membrane depolarization

We tested the hypothesis that membrane depolarization may initiate oxidant generation in the endothelial cell. Depolarization was produced in bovine pulmonary arterial endothelial cells (BPAEC) in monolayer culture with varying external K⁺, or with glyburide (10 μM), tetraethylammonium (TEA, 10 mM), gramicidin (1 μM), or nigericin (2 μM). Evaluation of bisoxonol fluorescence of BPAEC indicated concentration-dependent depolarization by high K⁺ (2% change in fluorescence/mV change in membrane potential in the 5.9–48 mM range of K⁺) and essentially complete depolarization with glyburide. Generation of oxidants was assessed with o-phenylenediamine dihydrochloride (o-PD) oxidation in the presence of horseradish peroxidase (HRP). There was a time-dependent increase in o-PD oxidation with 24 mM K⁺, nigericin, and gramicidin over 2 hours compared with control. In 1 hour o-PD oxidation increased 2.8-fold for 24 mM and 3.7-fold for 48 mM K⁺ compared with control. Catalase reduced 24 mM K⁺-induced o-PD oxidation by 50%, while Cu/Zn-superoxide dismutase (SOD) abolished the increase. Oxidation of o-PD was reduced by 57% in the absence of HRP in the system. With K⁺ channel blockade, o-PD oxidation increased 3.8-fold with glyburide and 4.6-fold with TEA compared with control. These data indicate formation of H₂O₂ and possibly other oxidants with depolarization and suggest involvement of K⁺-channels in this process. © 1996 Wiley-Liss, Inc.     

l-glutamate transport in Lactobacillus helveticus

An energy source (glucose or lactose) was required for the transport of l-glutamic acid by Lactobacillus helveticus. Mg²⁺, K⁺ and Li⁺ increased its accumulation while Ca²⁺ and Na⁺ decreased it. It was inhibited by NaF, indicating that ATP may be involved in uptake. Optimum transport was at pH 7.3 and 45°C. l-Glutamic acid transport showed a high degree of stereospecificity, as neither d-glutamate nor d-aspartate were active. Proton-conducting uncouplers, like carbonyl cyanide-m-chlorophenylhydrazone, and ionophores (nigericin, monensin and gramicidin) were strongly inhibitory. These results indicate that a proton motive force may be involved in the transport of l-glutamic acid.The authors are with the Centro de Referencia para Lactobacilos, Chacabuco 145 4000 S.M. de Tucumán, Argentina and the Cátedra de Microbiologia Superior, Universidad Nacional de Tucumán, Argentina.     

Glutamate,aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain

To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K_m of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K⁺ and Rb⁺ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na⁺; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na⁺ and K⁺ gradients. Monensin inhibits uptake by selectively dissipating the Na⁺ gradient. For both glutamate and GABA transport, the Na⁺ and K⁺ gradients are synergistic and not additive.     

Analisi ultrastrutturale degli effetti della monensina in Euglena gracilis,con particolare riguardo all'apparato di Golgi

Abstract

Ultrastructural analysis of the effects of monensin in Euglena gracilis, with special reference to the Golgi apparatus. - The monovalent ionophore, monensin, is known to affect the mature (trans) half of the dictyosomes of several organisms, including Euglena gracilis. We demonstrated that the exposure to high concentrations of this compound (2.5 × 10^?5 to 10^?4M) provoked remarkable swelling also in the forming (cis) half of Euglena cisternae. Additional dilations affected the thylakoids of both mature chloroplasts and proplastids of greening cells in which the organelle development was slower than in the control group. No osmotic swelling was observed for the mitochondria. Since monnesin exchanges one proton for each monovalent cation (Na⁺ or K⁺) transported, it follows that an energy driven influx of H⁺ is necessary to accumulate sufficient osmotically active ions in a membrane compartment. Thus it is possible that H⁺-ATPases are present on both forming and mature half of Euglena dictyosomes.     

Studies on isolated subcellular components of cat pancreas

Summary Transport of alanine was studied in isolated plasma membrane vesicles from cat pancreas using a rapid filtration technique. The uptake is osmotically sensitive and the kinetics ofl-alanine transport are biphasic showing a saturable and a nonsaturable component. The saturable component is seen only when a sodium gradient directed from the medium to the vesicular space is present. Under this condition an overshooting uptake ofl-but not ofd-alanine occurs. The Na⁺ gradient stimulated uptake ofl-alanine is inhibited byl-serine andl-leucine and stimulated when the membrane vesicles had been preloaded withl-alanine,l-serine orl-leucine.The ionophore monensin inhibits stimulation of uptake caused by a sodium gradient. In the presence of valinomycin or carbonyl cyanidep-trifluoromethoxyphenylhydrazone (CFCCP), the sodium-dependent transport is augmented in vesicles preloaded with K₂SO₄ or H⁺ ions (intravesicular pH 5.5), respectively. In the presence of different anions, the Na⁺-dependent transport is stimulated according to increasing anionic penetration through membranes (lipid solubility). We conclude that a sodium dependent electrogenic amino acid transport system is present in pancreatic plasma membranes.     

The effects of alterations of transmembrane Na+ and K+ gradients by ionophores (nigericin,monensin) on serotonin transport in human blood platelets

Ionophores (monensin, nigericin) capable of transporting both Na⁺ and K⁺ across cell membranes down their concentration gradients reduce the rate and total magnitude of serotonin uptake by platelets. The effect of the ionophores was time dependent, so that inhibition increased progressively until eventually uptake ceased entirely. Nigericin and monensin produced loss of platelet K⁺ and an equivalent molar uptake of Na⁺ thereby abolishing the normal transmembrane Na⁺ and K⁺ gradients. The time course of these ionophore-induced cation shifts at 37° C corresponded to the rate at which inhibition of serotonin transport developed. The ionophores did not affect total ATP concentration of platelets nor the metabolic pool of ATP formed from [¹⁴C] adenine. Nigericin and monensin released about 80% of platelet ¹⁴C and endogenous serotonin over a 30 min period, without release of platelet adenine nucleotides, calcium or β-glucuronidase. The ionophores did not elicit platelet aggregation nor did they prevent maximal aggregation brought about by ADP, collagen or A23187. Replacement of Na⁺ in the medium by K⁺ abolished serotonin uptake but only 10–20% of endogenous serotonin was released. In KCl medium the Na⁺ gradient was initially reversed, but quickly dissipated as Na⁺ reequilibrated with the extracellular fluid. At 37° C the ionophores did not affect either the rate of Na⁺ reequilibration or the efflux of [¹⁴C] serotonin. Na⁺ reequilibration was slower at 20° C and the ionophores significantly increased platelet Na⁺ loss and strongly inhibited the efflux of [¹⁴C] serotonin. The data support a mechanism of serotonin transport due to a Na⁺-dependent carrier-mediated process which need not be directly dependent on metabolic energy, but which does require metabolic energy to maintain normal $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$ gradients.