Bicarbonate- and calcium-dependent induction of rapid guinea pig sperm acrosome reactions by monovalent ionophores

The monovalent cationic ionophores monensin and nigericin stimulated rapid guinea pig sperm acrosome reactions in the presence of extracellular Na+, Ca2+ and bicarbonate (HCO3-/CO2). Extracellular K+ (mM concentrations), in contrast, was not required for the stimulatory effect of the ionophores. The effect of HCO3-/CO2 is concentration, pH and temperature dependent, with maximal responses obtained with 50 microM monensin or 25 microM nigericin at a concentration of 30 mM HCO3-, 2.5% CO2 and pH 7.8 at 25 degrees C. At a constant HCO3- concentration (30 mM), monensin stimulated acrosome reactions within the pH range 7.5-7.8, whereas a higher or lower pH did not support acrosome reactions at 25 degrees C. At constant extracellular pH (7.8), monensin stimulated acrosome reactions in the presence of 30 mM HCO3-, whereas higher and lower concentrations did not support acrosome reactions at 25 degrees C. The permeant anions pyruvate and lactate were essential to maintain sperm motility when treated with monensin under these conditions. NH4Cl, sodium acetate and 4,41-diisothiocyano-2, 21-disulfonic acid stibene (DIDS; 25 microM), an anion transport inhibitor, blocked the ability of monensin to stimulate acrosome reactions. Verapamil (100 microM), a putative Ca2+ transport antagonist, in contrast, did not prevent the monensin-induced acrosome reactions. Physiological concentrations of Na+ were needed for monensin to stimulate acrosome reactions, but high concentrations of Mg2+ prevented the monensin stimulation. The Ca2+ ionophore A23187 (75 nM) also required physiological concentrations of Na+ for the rapid induction of maximal acrosome reactions at an elevated pH (8.3) but did not require the presence of extracellular HCO3-. These studies suggest that a monovalent ionophore-induced rise in sperm intracellular Na+ concentrations is a pre-Ca2+ entry event, that stimulates an endogenous Ca2+/Na+ exchange that allows a Ca2+ influx which in turn induces the acrosome reaction. The possible regulatory role of the sperm intracellular pH and Na+, K+-ATPase during the capacitation process under physiological conditions is discussed.     

ATP-dependent and DCCD-insensitive Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions

ATP-dependent Cl- uptake by membrane vesicles from the rat brain plasma membrane fractions was not affected by the addition of 40 mM of K+, Na+ or HCO3- to the assay medium. Na+ and K+ did not alter the uptake even in the presence of a K+ ionophore, valinomycin (10 microM), or a H+/K+ exchanger, nigericin (10 microM), whereas in the presence of both of these ionophores, K+, but not Na+, reduced the Cl- uptake. Inhibitors of proton pump activity, N,N'-dicyclohexylcarbodiimide (1 mM) and 5-(N,N-hexamethylene)amiloride (40 microM), however, did not affect the Cl- uptake. These findings suggest the presence of a primary Cl- transport system probably associated with passive H+ flux in the brain plasma membranes.     

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

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

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles.

The Mg2+-dependent, K+-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H+ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth. Valinomycin stimulated both dye uptake and K+-ATPase (valinomycin-stimulated K+-ATPase); stimulation by valinomycin was due to increased K+ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K+-ATPase and H+ accumulation as an index, the sequence for anion permeation was NO-3 greater than Br- greater than Cl- greater than I- greater than acetate approximately isethionate. When permeability to both K+ and H+ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K+-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an 'uncoupling' of ATP hydrolysis from H+ accumulation. Thiocyanate interacts with valinomycin inhibiting the typical action of the K+ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN- to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K+-ATPase, it appears that SCN- is a very permeant anion which can be placed before NO-3 in the sequence of permeation. Valinomycin-stimulated ATPase and H+ uptake showed similar dependent correlations, including: dependence on [ATP] and [K+], pH optima, temperature activation, and selective inhibition by SH- or NH2-group reagents. These results are consistent with a pump-leak model for the gastric microsomal K+-ATPase which was simulated using Nernst-Planck conditions for passive pathways and simple kinetics for the pump. The pump is a K+/H+ exchange pump requiring K+ at an internal site. Rate of K+ entry would depend on permeability to K+ as well as the counterion, either (1) the anion to accompany K+ or (2) the H+ efflux path as an exchange ion. The former leads to net accumulation of H+ and anion, while the latter results in non-productive stimulation of ATP hydrolysis.     

Proton-coupled calcium transport by intact cells of Azotobacter vinelandii.

Addition of ionophores to resting aerobic cultures of Azotobacter vinelandii OP resulted in 45Ca2+ uptake (Km = 60 microM Ca2+; Vmax 1.1 nmol/min per mg of cell protein) which was inhibited by cations (La3+ greater than Mn2+ greater than Sr2+ greater Ba2+). The rate of Ca2+ entry correlated with the magnitude of a transmembrane proton gradient (inside acid) which developed in the respective order: valinomycin less than tetrachlorosalicylanilide less than nigericin less than gramicidin D less than tetrachlorosalicylanilide plus valinomycin. A process of calcium-proton exchange (antiport) is responsible for calcium accumulation under these conditions.     

Ion permeability of isolated chromaffin granules.

The passive ion permeability, regulation of volume, and internal pH of isolated bovine chromaffin granules were studied by radiochemical, potentiometric, gravimetric, and spectrophotometric techniques. Chromaffin granules behave as perfect osmometers between 340 and 1,000 mosM in choline chloride, NaCl, and KCl as measured by changes in absorbance at 430 nm or from intragranular water measurements using 3H2O and [14C]polydextran. By suspending chromaffin granules in iso-osmotic media of various metal ions and selectively increasing the permeability to either the cation or the anion by intrinsically permeable ions or specific ionophores, it was possible to determine by turbidity and potentiometric measurements the permeability to the counterion. These measurements indicate that the chromaffin granule is impermeable to the cations tested (Na+, K+, and H+). Limited H+ permeability across the chromaffin granule membrane was also shown by means of the time course of pH re-equilibration after pulsed pH changes in the surrounding media. The measurement of [14C]methylamine distribution indicates that a significant deltapH exists across the membrane, inside acidic, which at an external value of 6.85 has a value of 1.16. The deltapH is relatively insensitive to changes in the composition of the external media and can be enhanced or collapsed by the addition of ionophores and uncouplers. Measurement at various values of external pH indicates an internal pH of 5.5. Use of the ionophore A23187 indicates that Ca++ and Mg++ can be accumulated against an apparent concentration gradient with calcium uptake exceeding 50 nmol/mg of protein at saturation. These measurements also show that Ca++ and Mg++ are impermeable. Measurement of catecholamine release under conditions where intravesicular calcium accumulation is maximal indicates that catecholamine release does not occur. The physiological significance of the high impermeability to ions and the existence of a large deltapH are discussed in terms of regulation of uptake, storage, and release of catecholamines in chromaffin granules.     

The Limulus sperm motility-initiating peptide initiates acrosome reactions in sea water lacking potassium

During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.     

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.     

Role of proton motive force in genetic transformation of Bacillus subtilis

This study explored the role of the proton motive force in the processes of DNA binding and DNA transport of genetic transformation of Bacillus subtilis 168 strain 8G-5 (trpC2). Transformation was severely inhibited by the ionophores valinomycin, nigericin, and 3,5-di-tert-4-hydroxybenzylidenemalononitrite (SF-6847) and by tetraphenylphosphonium. The ionophores valinomycin and nigericin also severely inhibited binding of transforming DNA to the cell envelope, whereas SF-6847 and carbonylcyanide-p-trifluoromethoxyphenylhydrazone hardly affected binding. The proton motive force, therefore, does not contribute to the process of DNA binding, and valinomycin and nigericin interact directly with the DNA binding sites at the cell envelope. The effects of ionophores, weak acids, and tetraphenylphosphonium on the components of the proton motive force and on the entry of transforming DNA after binding to the cell envelope was investigated. DNA entry, as measured by the amount of DNase I-resistant cell-associated [3H]DNA and by the formation of DNA breakdown products, was severely inhibited under conditions of a small proton motive force and also under conditions of a small delta pH and a high electrical potential. These results suggest that the proton motive force and especially the delta pH component functions as a driving force for DNA uptake in transformation.     

Evidence for proton countertransport by the sarcoplasmic reticulum Ca2(+)-ATPase during calcium transport in reconstituted proteoliposomes with low ionic permeability

To ascertain the coupling between Ca2+ and H+ fluxes during Ca2+ transport by the Ca2(+)-pumping ATPase of the sarcoplasmic reticulum, we used well characterized reconstituted proteoliposomes. The method for the functional reconstitution of the Ca2(+)-ATPase was an extension of our recently published procedure (Rigaud, J. L., Paternostre, M. T., and Bluzat, A. (1988) Biochemistry, 27, 2677-2688). The reconstituted vesicles which sustained high Ca2+ transport activities in the absence of Ca2+ precipitating anions exhibited low ionic passive permeability. Proton fluxes generated by external acid pulses have been monitored by using the fluorescence of the pH-sensitive probe pyranine trapped inside proteliposomes. When K+ was the only permeant ion, low proton-hydroxyl passive permeability was found (permeability coefficient congruent to 5 x 10(-5) cm s-1). In the presence of Cl-1 ions, a higher proton permeability was observed, presumably due to diffusion of HCl molecules. It was further demonstrated that systematic characterization of the passive permeability is essential for understanding and controlling the ATP-dependent Ca2+ accumulation in the reconstituted liposomes. The first line of evidence for Ca2(+)-H+ countertransport during operation of the Ca2(+)-ATPase came from Ca2+ uptake measurements. The ATP-dependent Ca2+ accumulation into proteoliposomes was shown to be critically dependent upon the ionic composition of the medium and the presence of ionophores. In K2SO4 medium a very low Ca2+ uptake was obtained which was only slightly affected by the presence of valinomycin. On the contrary, Ca2+ accumulation was increased 3-4-fold in the presence of the protonophore carbonyl-cyanide-p-trifluoromethoxy phenylhydrazone, indicating that a transmembrane pH gradient was built up during Ca2+ uptake that inhibited the transport activity of the pump. Accordingly, we found that Ca2+ loading capacity increased with internal buffer capacity. Finally in KCl medium, high Ca2+ accumulation was observed even in the absence of protonophore in agreement with a rapid dissipation of the pH gradient in the presence of chloride ions. Additional evidence that the Ca2+ pump of sarcoplasmic reticulum operated as a Ca2(+)-H+ countertransport was provided by measurements of ATP-dependent intraliposomal alkalinization using entrapped 8-hydroxyl-1,3,6-pyrene trisulfonate (pyranine) and accumulation of the weak acid acetate. In K2SO4 medium, transmembrane pH gradients of about 1 pH unit were generated with kinetics parallel to those of the Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that ATP-dependent Ca2+ transport in rat parotid microsomal membranes requires charge compensation.

ATP-dependent Ca2+ transport was investigated in a rat parotid microsomal-membrane preparation enriched in endoplasmic reticulum. Ca2+ uptake, in KCl medium, was rapid, linear with time up to 20 s, and unaffected by the mitochondrial inhibitors NaN3 and oligomycin. This Ca2+ uptake followed Michaelis-Menten kinetics, and was of high affinity (Km approximately 38 nM) and high capacity (approximately 30 nmol/min per mg of protein). In the presence of oxalate, Ca2+ uptake continued to increase for at least 5 min, reaching an intravesicular accumulation approx. 10 times higher than without oxalate. Ca2+ uptake was dependent on univalent cations in the order K+ = Na+ greater than trimethylammonium+ greater than mannitol and univalent anions in the order Cl- greater than acetate- greater than Br- = gluconate- = NO3- greater than SCN-. Ca2+ uptake was not elevated if membranes were incubated in the presence of a lipophilic anion (NO3-) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Ca2+ transport was altered by changes in the K+-diffusion potential of the membranes. A relatively negative K+-diffusion potential increased the initial rate of Ca2+ accumulation, whereas a relatively positive potential decreased Ca2+ accumulation. In the presence of an outwardly directed K+ gradient, nigericin had no effect on Ca2+ uptake. In aggregate, these studies suggest that the ATP-dependent Ca2+-transport mechanism present in rat parotid microsomal membranes exhibits an electrogenic Ca2+ flux which requires the movement of other ions for charge compensation.     

Ion transport and respiratory control in vesicles formed from reduced nicotinamide adenine dinucleotide coenzyme Q reductase and phospholipids.

NADH-coenzyme Q reductase from bovine heart mitochondria (complex I) was incorporated into phospholipid vesicles by the cholate dialysis procedure. Mixtures of purified phosphatidylcholine and phosphatidylethanolamine were required. Oxidation of NADH by coenzyme Q1 catalyzed by the reconstituted vesicles was coupled to proton translocation, directed inward, with an H+/2e ratio greater than 1.4. Similar experiments measuring proton translocation in submitochondrial particles gave an H+/2e ratio of 1.8. The proton translocation in both systems was not seen in the presence of uncoupling agents and was in addition to the net proton uptake from the reduction of coenzyme Q1 by NADH. Electron transfer in the reconstituted vesicles also caused the uptake of the permeant anion tetraphenylboron. The rate of electron transfer by the reconstituted vesicles was stimulated about 3-fold by uncouplers or by valinomycin plus nigericin and K+ ions. The results indicate that energy coupling can be observed with isolated NADH-coenzyme Q reductase if the enzyme complex is properly incorporated into a phospholipid vesicle.     

The proteoliposomal steady state. Effect of size, capacitance and membrane permeability on cytochrome-oxidase-induced ion gradients.

1. The flux pathways for H+ and K+ movements into and out of proteoliposomes incorporating cytochrome c oxidase have been investigated as a function of the electrical and geometrical properties of the vesicles. 2. The respiration-induced pH gradient (delta pH) and membrane potential (delta psi) are mutually dependent and individually sensitive to the permeability properties of the membrane. A lowering or abolition of delta psi by the addition of valinomycin increased the steady-state level of delta pH. Conversely, removal of delta pH by the addition of nigericin resulted in a higher steady-state delta psi. 3. Vesicles prepared by sonication followed by centrifugation maintained similar pH gradients at steady state to those in vesicles prepared by dialysis, although the time taken to reach steady state was longer. Higher pH gradients can be induced in non-centrifuged sonicated preparations. 4. No significant differences were found in H+ and K+ permeability between proteoliposomes prepared by dialysis or by sonication. The permeability coefficient of the vesicle bilayers for H+ was 6.1 x 10(-4) cm.s-1 and that for K+ was 7.5 x 10(-10) cm.s-1. An initial fast change in internal pH was seen on the addition of external acid or alkali, followed by a slower, ionophore-sensitive, change. The initial fast phase can be increased by the lipid-soluble base dibucaine and the weak acid oleate. In the absence of ionophores, increasing concentrations of oleate increased the rate of H+ translocation to a level similar to that seen in the presence of nigericin. Internal alkalinization could also be induced by oleate upon the addition of potassium sulphate. 5. The initial, pre-steady-state and steady-state delta pH and delta psi changes can be simulated using a model in which the enzyme responds to both delta pH and delta psi components of the protonmotive force. At steady state, the electrogenic entry of K+ is countered by electroneutral exit via a K+/H+ exchange. 6. The permeability coefficient, PH, calculated from H+ flux under steady-state turnover conditions, was approx. 100 times higher than the corresponding 'passive' measurements of PH. Under conditions of oxidase turnover, the vesicles appear to be intrinsically more permeable to protons.     

Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.     

Bile salt-induced calcium fluxes in artificial phospholipid vesicles

The ionic permeability of selected biological membranes is increased by bile salts. To examine changes in calcium permeability during the exposure of artificial membranes to bile salts, we investigated calcium uptake by unilamellar and multilamellar phospholipid vesicles. In the presence of 750 microM taurodeoxycholate, uptake of radiolabelled calcium by unilamellar vesicles increased 2.5-fold over control values. Calcium uptake by multilamellar vesicles as measured with a free calcium indicator, arsenazo III, increased 2.2- or 21-fold in the presence of 60 microM lithocholate or 3 beta-hydroxy-5-cholenoate, respectively. Results were directly influenced by experimental variables such as bile salt hydrophobicity, external calcium concentration, and the bile salt/lipid molar ratio. Observed membrane solubilization was minimal despite increased calcium permeability. Comparison of radiolabelled calcium uptake with radiolabelled sodium or radiolabelled rubidium uptake indicated that bile salt-dependent calcium uptake was 60-140-times greater than bile salt-dependent uptake of either monovalent cation. In an effort to delineate forces affecting calcium translocation, vesicles were exposed either to valinomycin, which induced an electrochemical gradient across the membrane, or to nigericin, which induced a proton gradient. Exposure to valinomycin minimally influenced bile salt-induced calcium uptake while exposure to nigericin significantly promoted uptake by 40-70%. The results suggest that bile salts promote calcium uptake by a mechanism which may be similar to those of other carboxylic ionophores.     

The role of sodium ions in methanogenesis. Formaldehyde oxidation to CO2 and 2H2 in methanogenic bacteria is coupled with primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2

Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (delta G0' = -27 kJ/mol CO2), when methanogenesis was inhibited by 2-bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings. Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2-3 mol Na+/mol formaldehyde oxidized were extruded. Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores of Na+/H+ antiport inhibitors. Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores. Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+-translocating-ATP-synthase inhibitor, dicyclohexylcarbodiimide. With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.     

Cholesterol Stimulation of Penetration of Unilamellar Liposomes by Hydrophobic Compounds.

The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of negericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution. Cholesterol similarly increases the ability of A-23187, 1799, or NH4SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb+ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca45 trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb+ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

Effect of ionophore A23187 on the membrane permeability in mouse fibroblasts.

Addition of the divalent cation ionophore A23187 to transformed mouse fibroblasts (3T6) resulted in an increase in the cell membrane permeability to normally impermeant solutes (e.g., nucleotides). The membrane permeability was assessed by following the efflux of prelabeled adenine nucleotides, the influx of p-nitrophenyl phosphate in cells attached to plastic dishes and reconstitution of intracellular protein synthesis in the presence of exogenously added normally impermeant factors required for macromolecular synthesis. The permeability change of 3T6 cells was found to be dependent on the specific presence of external calcium ion. The permeabilization was found to occur preferably in alkaline pH and specific to certain transformed cells. It is preceded by rapid efflux of K+, influx of Na+ and partial hydrolysis of cellular nucleotides in 3T6 cells. Similar ion fluxes were previously found to precede cell permeabilization by electrogenic ionophores for monovalent ions and by exogenous ATP. Our data suggest that a calcium dependent process caused the K+ release and excess Na+ entry, causing dissipation of the membrane potential and subsequent formation of aqueous channels.     

Anion/calcium ion ratios and proton production in some mitochondrial calcium ion uptakes.

The uptake of Ca2+ by liver mitochondria, when phosphate movement is inhibited, occurs when Co2 is present and not in its absence. Uptake of Ca2+ to form CaCO3 yields 2H+/Ca2+. Heart mitochondria, when phosphate movement is inhibited, will take up Ca2+ with the exact equivalent of hydroxybutyrate, lactate or acetate. By providing a carrier for Cl- with tributyltin, a stoicheiometric uptake of Cl- with the Ca2+ takes place. The uptakes appear to occur without significant pH change; there appears to be no CO2-dependent uptake into heart mitochondria. Oxygenation of anaerobic heart mitochondria, in the presence of an inhibitor of phosphate movement and of generation of phosphate from internal ATP, does not yield significant change of external acidity in relation to the amount of O2 added. Use of Bromothymol Blue as an indicator of the distribution of a weak acid anion confirms that the transient nature of the response of the dye distribution to Ca2+ is connected with movement of endogenous phosphate. Bromothymol Blue accumulated in response to Ca2+ is discharged when entry of the Ca2+ (in the presence of mersalyl) is mediated with nigericin. It is concluded that Ca2+ uptakes will occur alternatively with the equivalent of anions or in exchange for endogenous K+ and that proton production is connected with the changes of ionization of phosphate (unless phosphate movement is inhibited) and in liver mitochondria with the hydration of CO2.     

The use of ionophores of rapid loading of human red cells with radioactive cations for cation-pump studies.

Techniques are described for the rapid loading of intact human red cells with radioactive isotopes of alkali cations or Ca2+ by using ionophorous compounds (nigericin, gramicidin D and A 23187). Loading was rapid and efficient if the membrane potential of the cells was rendered more negative inside. After cation loading the ionophores could be bound to albumin and removed by repeated washings. The ATP and 2,3-DPG contents of the cells were practically unaltered by this treatment. Passive membrane permeability to Na+ and Ca2+ returned to normal. Loaded erythrocytes pumped out Na+ in a ouabain-sensitive and Ca2+ in a lanthanum-sensitive way. Ca2+ -loaded red cells were microspherocytes and exhibited a rapid K+ -efflux. Parallel with the extrusion of Ca2+ cells regained their biconcave shape and normal passive permeability to K+.