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PubMed1986

The mechanism of the hormonal activation of respiration in isolated hepatocytes and its importance in the regulation of gluconeogenesis.

The effects of hormones on the cytochrome spectra of isolated hepatocytes were recorded under conditions of active gluconeogenesis from L-lactate. Glucagon, phenylephrine, vasopressin and valinomycin, at concentrations that caused stimulation of gluconeogenesis, increased the reduction of the components of the cytochrome bc1 complex, just as has been observed in liver mitochondria isolated from glucagon-treated rats [Halestrap (1982) Biochem. J. 204, 37-47]. The effects of glucagon and phenylephrine were additive. The time courses of the increased reduction of cytochrome c/c1 and NAD(P)H/NAD(P)+ caused by hormones, valinomycin, A23187 and ethanol were measured by dual-beam spectrophotometry and fluorescence respectively. Ethanol (14 mM) produced a substantial rise in NAD(P)H fluorescence, beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, no change in cytochrome c/c1 reduction, a 10% decrease in O2 consumption and a 60% decrease in gluconeogenesis. Glucagon, phenylephrine and vasopressin caused a substantial and transient rise in NAD(P)H fluorescence, but a sustained increase in cytochrome c/c1 reduction and the rates of O2 consumption and gluconeogenesis. The transience of the fluorescence response was greater in the absence of Ca2+, when the cytochrome c/c1 response also became transient. The fluorescence response was smaller and less transient, but the cytochrome c/c1 response was greater, in the presence of fatty acids. Both responses were greatly decreased by the presence of 1 mM-pent-4-enoate. Valinomycin (2.5 nM) caused a decrease in NAD(P)H fluorescence coincident with an increase in cytochrome c/c1 reduction and the rate of gluconeogenesis and O2 consumption. A23187 (7.5 mM) caused increases in both NAD(P)H fluorescence and cytochrome c/c1 reduction. The effects of hormones and valinomycin on the time courses of NAD(P)H fluorescence, cytochrome c/c1 reduction and light-scattering by hepatocytes were compared with those of 0.5 microM-Ca2+ or 1 nM-valinomycin on the same parameters of isolated liver mitochondria. It is concluded that hormones increase respiration by hepatocytes in a biphasic manner. An initial Ca2+-dependent activation of mitochondrial dehydrogenases rapidly increases the mitochondrial [NADH], which is followed by a volume-mediated stimulation of fatty acid oxidation and electron flow between NADH and cytochrome c. 10. Amytal (0.5 mM) was able to reverse the effects of hormones on the reduction of cytochromes c/c1 and the rates of gluconeogenesis and O2 consumption without significantly lowering tissue [ATP].(ABSTRACT TRUNCATED AT 400 WORDS)

Amobarbital↗
PubMed1987

Diverse effects of insulin-induced hyperpolarization on 3-O-methyl-D-glucose (3-O-MG) transport in frog skeletal muscles.

It has been suggested that the insulin-induced hyperpolarization might be a mediator of the stimulatory action of insulin on glucose transport. The purpose of the present study was to investigate the relationship between the insulin-induced hyperpolarization and the stimulatory action of insulin on glucose transport in skeletal muscle. Satorius muscles dissected from bullfrogs (Rana catesbeiana) were used. Insulin induced a hyperpolarization of the membrane and an increase in the 3-O-Methyl-D-glucose (3-O-MG) uptake and extrusion. In the presence of valinomycin, insulin had no significant effect on the membrane potential. Insulin still had the stimulatory action on both the 3-O-MG uptake and extrusion even in the presence of valinomycin, under whose condition insulin had no significant effect on the membrane potential. The magnitude of the stimulatory action of insulin on the 3-O-MG uptake in the presence of valinomycin was smaller than that in the absence of valinomycin. The magnitude of the stimulatory action of insulin on the 3-O-MG extrusion was, on the contrary, larger than that in the absence of valinomycin. The abolishment of the insulin-induced hyperpolarization decreased the 3-O-MG uptake and increased the 3-O-MG extrusion. The observation in the present study concludes that insulin has two different actions on glucose transport. One of them is developed through the insulin-induced hyperpolarization, which increases the 3-O-MG uptake and decreases the 3-O-MG extrusion. The other action is irrelevant of the insulin-induced hyperpolarization and stimulates both the 3-O-MG uptake and extrusion.

3-O-Methylglucose↗
PubMed1982

Effects of ionophores and dicyclohexylcarbodiimide on Mycoplasma gallisepticum adherence to erythrocytes.

To test the influence of the electrochemical ion gradient across mycoplasma membranes on the capacity of organisms to adhere to host cells, Mycoplasma gallisepticum cells were treated with valinomycin, carbonylcyanide m-chlorophenylhydrazone, and N,N'-dicyclohexylcarbodiimide (DCCD) singly or in combination. Uptake of [3H]tetraphenylphosphonium by the treated cells was employed as a measure of the effects of the ionophores on membrane potential. In the absence of K+, valinomycin increased, whereas carbonylcyanide m-chlorophenylhydrazone, and DCCD decreased [3H]tetraphenylphosphonium uptake. However, with a high level of K+ or with DCCD, uptake of [3H]tetraphenylphosphonium in the presence of valinomycin decreased below control levels, indicating that, generally, the ionophores affected membrane potential in the expected manner. The treated organisms were tested for their capacity to attach to glutaraldehyde-fixed human erythrocytes. DCCD was the best inhibitor of mycoplasma attachment, and in combination with valinomycin attachment, capacity decreased by about 40%. The combination of valinomycin plus carbonylcyanide m-chlorophenylhydrazone was less effective; it decreased attachment by about 15 to 25%. It was concluded that the dissipation of ion gradients across cell membranes decreases only partially mycoplasma adherence, in line with previous findings that isolated mycoplasma membranes retain the major part of the attachment capacity of intact cells.

Adhesiveness↗
PubMed1978

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.

Bacillus subtilis↗
PubMed1981

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.

Chemotaxis↗
PubMed1989

Gastric H+-K+-ATPase in situ: evidence for compartmentalization.

Gastric glands, isolated from rabbit, were permeabilized with digitonin to permit measurement of H+-K+-adenosinetriphosphatase (ATPase) activity and proton transport in situ. Measurement of proton gradient formation using acridine orange fluorescence showed two phases of ATP-driven proton accumulation; one phase occurs spontaneously in KCl medium and one phase requires the K+ ionophore valinomycin. Valinomycin was found to increase H+-K+-ATPase activity, indicating that the second phase is because of increased proton transport rather than a decrease in proton leak rate. The acid-activated, irreversible inhibitor, omeprazole, was used to selectively eliminate the H+-K+-ATPase molecules associated with the spontaneous component of proton transport. After omeprazole treatment a residual, valinomycin-dependent component of proton transport could be demonstrated. These results are interpreted as evidence for two compartments of H+-K+-ATPase, separated by a barrier that prevents K+ diffusion and pH equilibration. The two compartments may be separated also on the basis of anion selectivity. The spontaneously active compartment was found to be functional with various anions, including sulfate and isethionate, whereas the valinomycin-dependent component is highly selective for chloride. The proportion of H+-K+-ATPase that exists in each compartment was quantitated by measuring the fraction of total ATPase activity that could be inhibited by omeprazole in the absence and presence of valinomycin. For glands that were preconditioned with cimetidine, approximately 30% of the inhibitable enzyme was found associated with the spontaneous compartment, and this fraction increased to approximately 70% with histamine preconditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1987

K-Cl transport systems in rabbit renal basolateral membrane vesicles.

The transport pathways for chloride in basolateral membrane vesicles from the rabbit renal cortex were investigated. 36Cl uptake was stimulated by the presence of potassium in the uptake media compared with sodium or N-methyl-D-glucamine. In addition, potassium (86Rb) uptake was stimulated more by chloride than by nitrate or gluconate. Neither of these processes was further stimulated by potassium gradients plus valinomycin, suggesting the presence of an electrically neutral K-Cl cotransport system. A magnesium-induced chloride conductance was also found in the basolateral membrane vesicles. In the absence of magnesium, the chloride conductance was low; valinomycin and an inwardly directed potassium gradient did not stimulate 36Cl uptake, anthracene-9-carboxylic acid did not inhibit 36Cl uptake, and valinomycin did not stimulate chloride-dependent 86Rb uptake. However, in the presence of 1 mM magnesium, opposite results were obtained; valinomycin and an inwardly directed potassium gradient stimulated 36Cl uptake, anthracene-9-carboxylic acid inhibited 36Cl uptake, and valinomycin stimulated chloride-dependent 86Rb uptake. Therefore, an electrically neutral K-Cl cotransport and magnesium-induced chloride conductance were found in renal cortical basolateral membrane vesicles prepared from the rabbit renal cortex.

Animals↗
PubMed1999

Effects of ionophores on the phospholipid flippase activity of gastric vesicles.

Recently, a gastric Mg(2+)-ATP-dependent phospholipid flippase was found. Here, the effects of ionophores and monovalent cations on the gastric flippase were examined. We found that translocation of the fluorescent analogue of phosphatidylcholine was inhibited by valinomycin in the presence of K(+). The inhibition depended on both the concentrations of valinomycin and K(+). Valinomycin did not inhibit translocation in the absence of K(+). Protonophores, carbonylcyanide-m-chlorophenylhydrazone (CCCP) and carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP), accelerated translocation by 190-270%. These increases were completely abolished by 2-methyl-8-(phenylmethoxy)imidazo-[1, 2-a]pyridine-3-acetonitrile (SCH 28080), a gastric flippase inhibitor. Since these protonophores did not affect the Mg(2+)-dependent ATPase activity that is responsible for phospholipid translocation by the flippase, the coupling ratio of the amount of transported phospholipids/the amount of hydrolyzed ATP was variable and seemed to depend on the state of the membrane bilayer, for example fluidity. Inhibition by the valinomycin-K(+) complex was abolished in the presence of CCCP or FCCP, indicating the valinomycin-K(+)-CCCP(FCCF) ternary complex did not inhibit the flippase.

4-Chloro-7-nitrobenzofurazan↗
PubMed1989

Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient.

Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH.

Animals↗
PubMed1987

Calcium ionophore (A23187)- and arachidonic acid-stimulated prostaglandin release from microvascular endothelial cells: effects of calcium antagonists and calmodulin inhibitors.

Calcium ionophore (A23187)-stimulated prostaglandin (PG) E2 and I2 (measured as 6-keto PGF1 alpha) release from cultured rabbit coronary microvessel endothelial (RCME) cells in a time- and dose-dependent manner. A23187-stimulated PG release was reduced by the calcium channel blockers nifedipine, verapamil and diltiazem and by the intracellular calcium blocker, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate. A23187-stimulated PG release was significantly reduced by lowering the calcium concentration in the buffer to concentrations of 0.2 mM or less. A23187-stimulated 45Ca uptake was not inhibited by nifedipine (0.5 microM), diltiazem (10 micron) or verapamil (50 microM) although these same concentrations of calcium channel blockers significantly inhibited A23187-stimulated PG release. However, these concentrations of calcium blockers did inhibit K+ (10 mM)-valinomycin (5 microM)-stimulated 45Ca uptake, indicating that, although RCME cells probably have voltage-dependent calcium channels and although calcium influx via these channels is blocked by the calcium channel blockers, voltage-dependent calcium influx plays little or no role in A23187-stimulated 45Ca influx and PG release. KCl-valinomycin significantly stimulated PG release, but this increase was not significantly affected by either nifedipine (0.5 microM) or diltiazem (10 microM) despite complete inhibition of KCl-valinomycin-stimulated 45Ca influx. Verapamil (50 microM) exhibited a small but significant suppression of KCl-valinomycin-stimulated PG release. These observations most likely indicate that calcium influx by voltage-dependent calcium channels plays little or no role in the events leading to either A23187- or KCl-valinomycin-stimulated PG release. The calmodulin antagonists, trifluoperazine and calmidazolium, also reduced A23187-stimulated PG release. In vitro studies of porcine pancreatic phospholipase (PL) A2 activity suggested that the inhibitory actions of trifluoperazine, but not the calcium antagonists, may be mediated by direct inhibitory actions on PLA2. Studies with [3H]arachidonic acid (AA)- and [14C]stearic acid-prelabeled RCME cells suggested that A23187 stimulated both PLA2 and PLC activity, leading to the release of AA. Studies with exogenous AA indicated that reducing calcium availability by reducing buffer calcium concentrations resulted in an enhanced conversion of exogenous AA to PGs. RCME cells incubated in nominally calcium-free buffer exhibited a decreased rate of AA incorporation. The observed increase in AA conversion to PGs in low calcium buffer suggests that calcium may stimulate AA uptake and acylation as well as AA release.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

The lysosomal proton pump is electrogenic.

Lysosomes were purified approximately 40-fold from rat kidney cortex by differential and Percoll density gradient centrifugation. In a sucrose medium, the lysosomes quenched the fluorescence of the potential sensitive dye diS-C3-(5) (3,3'-dipropylthiocarbo-cyanine iodide) in a time-dependent manner, indicating that the dye accumulates within the lysosomal interior. After treatment of the lysosomes with valinomycin, the dye fluorescence displayed a logarithmic dependence upon the external K+ concentration; thus, the fluorescence signal provides a semiquantitative measure of the lysosomal membrane potential (delta psi). In the absence of valinomycin, lysosomal quenching of diS-C3-(5) fluorescence was partially reversed by agents which collapse the lysosomal pH gradient (ammonium sulfate, chloroquine, and K nigericin), suggesting that the proton gradient across the lysosomal membrane contributes to delta psi. A rapid increase in diS-C3-(5) fluorescence, indicative of an increase in delta psi, was observed upon the addition of Mg-ATP to the lysosomes. The ATP-dependent fluorescence change was inhibited by protonophores, K valinomycin, permeable anions, and N-ethylmaleimide, but was unaffected by ammonium sulfate, K nigericin, or sodium vanadate. Oligomycin had no effect at concentrations below 2 micrograms/ml; at higher concentrations, oligomycin partially inhibited the fluorescence response to Mg-ATP, but it also inhibited the fluorescence response to K valinomycin, suggesting that it had modified the permeability of the lysosomal membrane. Dicylohexylcarbodiimide behaved similarly to oligomycin. Mg-ATP also altered the lysosomal distribution of 86Rb+ (in the presence of valinomycin) and S[14C]CN-, consistent with an increase in the potential of the lysosomal interior of 40-50 mV. The results demonstrate that the lysosomal proton pump is electrogenic.

Adenosine Triphosphatases↗
PubMed1978

Proton electrochemical gradient and phosphate potential in mitochondria.

The paper reports an analysis of the relationship between deltamuH the proton electrochemical potential difference, and deltaGp, the phosphate potential. Depression of deltamuH and deltaGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b) nigericin (+ valinomycin); (c) KCl (+ valinomycin); and (d) rotenone. The uncoupler depresses deltamuH more than nigericin (+ valinomycin), KCl (+ valinomycin) and rotenone at equivalent deltaGp. The deltaGp/deltamuH ratio is about 3 at high values of deltamuH. When deltaGp and deltamuH are depressed by nigericin (4 valinomycin) the deltaGp/deltamuH ratio remains constant. When deltaGp and deltamuH are depressed by uncouplers, the deltaGp/deltamuH ratio increases hyperbolically tending to infinity while deltamuH tends to zero. The absence of constant proportionality between deltaGp and deltamuH indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.

Adenosine Triphosphate↗
PubMed1978

Selective ion binding and membrane activity of synthetic cyclopeptides.

Four cyclic peptides related to the membrane-active complexones PV, cyclo-(L-Pro-Lval-D-Pro-D-Val)3, and valinomycin were synthesized: (1) cyclo-(L-Pro-L-Ala-D-Val)3 or PVPA, (2) cyclo-(L-Ala-L-Val-D-Pro-D-Val)3 or PVAV, (3) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2-L-Pro-D-Val or PV-10, (4) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2 or PV-8. In a two-phase extraction assay the affinity of PV and PVPA for alkali picrates was about three orders or magnitude greater than that of valinomycin. It was about equal to valinomycin for PVAV and much lower for PV-10 and PV-8. PV, PVPA and PVAV showed a selectivity sequence similar to that of valinomycin, namely K+ approximately Rb+ greater than Cs+ greater than Na+ greater than Li+. In the series PV, PV-10, PV-, the preference for K+ over Na+ was 700, 5 and less than 1, respectively. Thus, it was possible to reverse the selectivity of PV for K+ over Na+ by reducing the ring size from 12 to 8 amino acid residues. In sheep red cell lipid bilayer membranes PVPA increased the membrane conductance significantly in the presence of either KCl or NaCl but it was less potent than PV. PV-10, PV-8 and PVAV on the other hand were ineffective in this assay. The inactivity of PVAV as a potassium carrier in membrane was in contrast to its high affinity for potassium picrate in two-phase assays. Such a behaviour may be observed of a compound that has too low an aqueous cation binding constant to use the solution-complexation mechanism of PV (Davis et al. (1976) Biochemistry 15, 768--774 and Pinkerton et al. (1969) Biochem. Biophys. Res. Commun. 35, 512--518) and too slow binding and release kinetics to use the interfacial-complexation mechanism of valinomycin.

Cations, Monovalent↗
PubMed1997

Diffusion membrane potential in liposomes: setting by ion gradients, absolute calibration and monitoring of fast changes by spectral shifts of diS-C3(3) fluorescence maximum.

A novel fluorescent technique for direct assessment of membrane potential was tested on suspensions of large unilamellar vesicles (LUV). The method is based on monitoring shifts in the fluorescence maximum, lambda(max), of the redistribution dye diS-C3(3) caused by dye binding to the LUV membrane. A theory describing the behavior of this dye in LUV suspensions was elaborated and tested. The diffusion potentials across the LUV membrane were adjusted by ion gradients in the absence of valinomycin. When using KCl and choline chloride without valinomycin the potential can be set as high as -70 mV. These potentials exhibited long-term stability and the theory allowed to determine the upper limits of membrane permeabilities for Cl-, choline cations, protons and hydroxyls relative to the K+ permeability. The absolute values of membrane potential set by ion gradients were calibrated using valinomycin. The monitoring of the lambda(max) shift permitted us to show real-time changes in membrane potential, since addition of valinomycin to the LUV was followed by an immediate depolarization. The setting of the potential and the dye re-equilibration after valinomycin addition took place within a second.

Carbocyanines↗
PubMed1996

Cell-mediated mineralization in culture at low temperature associated with subtle thermogenic response.

In both the growth plate and in marrow stromal cell cultures cell-mediated mineralization is preceded by characteristics of anaerobic and low efficiency energy metabolism. Reagents that increase mineralization like malonate and dexamethasone (DEX) also increase the mitochondrial membrane potential (MtMP) especially 1 week after DEX stimulation. Contrarily, levamisole, which decreases mineralization, also decreases MtMP. Modulation of MtMP and energy metabolism could be linked to regulation of mineralization by the uncoupling of oxidative phosphorylation. This uncoupling should be associated with thermogenesis in cells that induce mineralization. We examined whether cold temperature affects mineralization, and whether cellular thermogenesis takes place at cold temperature in parallel to changes in MtMP. Osteoprogenitor cells (OPC) induced, in DEX stimulated rat marrow stroma, higher mineralization at 33 degrees C than at 37 degrees C. Increased mineralization by cold temperature required long incubation since incubation in the cold during short intervals, 3-4 days, did not increase mineralization relative to (37 degrees C) controls. Marrow stromal cells in the presence of valinomycin responded to incubation at 33 degrees C by retaining all the vital dye after 4 h, unlike the cells at 37 degrees C; however, after 24 h the level of dye retention at 33 degrees C was the same as at 37 degrees C. The delayed response of the temperature-dependent (> 37 degrees C) K+ ionophor to incubation in the cold indicated that certain cells may respond to low temperature by local intracellular heating, and by heat conduction to the plasma membrane. DEX-stimulated stromal cells, unlike unstimulated cells, showed increased mitochondrial rhodamine 123 retention in the presence of valinomycin after 24 h in the cold, which corresponds to day 4 of OPC induction. This is consistent with the concept that valinomycin-induced cell damage is mediated by (cold-induced) local heating. The mechanism of this cell damage should selectively prefer nonthermogenic (rhodamine retaining) over thermogenic (rhodamine leaking) cells such as OPC. At cold temperature DEX-stimulated stromal cells showed the best anti-OPC selection under exposure to valinomycine between days 3-7, concurrent with the period of rhodamine leakage from the mitochondria. These results indicate that thermogenesis is enhanced during the period of low MtMP in mineralizing cells, and prolonged exposure to cold increases mineralization also due to induction of subtle thermogenesis.

Animals↗
PubMed1997

NMR studies of inorganic phosphate compartmentation in the isolated rat liver during acidic perfusion.

Mitochondrial inorganic phosphate has been shown to be undetectable by 31P NMR in the isolated rat liver perfused under physiological conditions. Cold perfusion (4 degrees C) with valinomycin (K+ ionophore) induced the appearance of an additional resonance assigned to P(i) from mitochondrial compartment (P(i,mito)) (Thiaudière et al., 1993, FEBS Lett. 330, 232-235). Here we have demonstrated that P(i,mito) can be detected by NMR under normothermic conditions (37 degrees C) in acidic (pH 6.5, bicarbonate-free) perfused liver using 50 nM valinomycin or 10 microM N,N'-dicyclohexylcarbodiimide (DCCD, a mitochondrial H+-ATP synthase inhibitor). These conditions resulted in a significant increase in mitochondrial P(i) content. In the presence of valimomycin, pH values of 7.00+/-0.07 and 6.60+/-0.10 (n = 7) for mitochondria and cytosol, respectively, were determined from the chemical shift values of P(i) resonances. Electron microscopy demonstrated a large matrix swelling under valinomycin perfusion, explaining the increased level of mitochondrial P(i). The amount of mitochondrial P(i) measured by NMR increased linearly with the cellular ATP depletion, suggesting a mitochondrial influx of P(i) from the cytosolic compartment with valinomycin perfusion. Moreover, the level of matrix P(i) was dependent on the cytosolic pH value, the resonance being not detectable at physiological cytosolic pH. During mitochondrial swelling, P(i) influx was likely to be associated with proton influx, owing to the stability of transmembrane pH gradient and matrix proton concentration.

Adenosine Triphosphate↗
PubMed1975

Transmembrane electrophoresis of 8-anilino-1-naphthalenesulfonate through egg lecithin liposome membranes.

Valinomycin has been shown to increase the amount of 8-anilino-1-naphthalenesulfonate (ANS) bound to egg lecithin liposomes and also to increase the maximum fluorescence value, as derived from double reciprocal plots. The assay conditions were such that addition of valinomycin would not produce a transmembrane potential. The formation of a valinomycin potassium ANS complex in the micelle membrane is proposed. This could account for the increase in the maximum fluorescence value and, by acting as an ANS transporter, could also account for the increase in ANS bound. Tributylamine was also shown to increase the binding and maximum fluorescence of ANS. In assay conditions where the addition of valinomycin would produce a transmembrane potential negative inside, the tributylamine-induced fluorescence was reversed. The fluorescense decrease is interpreted as transmembrane electrophoresis of ANS in response to a transmembrane potential.

Anilino Naphthalenesulfonates↗
PubMed1977

Use of 1-anilino-8-naphthalene-sulfonate as a probe of gastric vesicle transport.

The interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with a KD of 7.5 micronM and those slowly accessible, but rapidly accessed in the presence of valinomycin with a KD of 2.5 micronM. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number of KD of those sites. The time course of this increase correlates with H+ uptake and Rb+ extrusion by those vesicles and H+ carries such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC14N and valinomycin there is transient uptake of SCN-. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H+ gradient.

Anilino Naphthalenesulfonates↗