Net Proton Flux Research Articles

Comparative functional analysis reveals differential nucleotide sensitivity between human and mouse UCP1.

Mitochondrial uncoupling protein 1 (UCP1) is a unique protein of brown adipose tissue. Upon activation by free fatty acids, UCP1 facilitates a thermogenic net proton flux across the mitochondrial inner membrane. Non-complexed purine nucleotides inhibit this fatty acid-induced activity of UCP1. The most available data have been generated from rodent model systems. In light of its role as a putative pharmacological target for treating metabolic disease, in-depth analyses of human UCP1 activity, regulation, and structural features are essential. In the present study, we established a doxycycline-regulated cell model with inducible human or murine UCP1 expression and conducted functional studies using respirometry comparing wild-type and mutant variants of human UCP1. We demonstrate that human and mouse UCP1 exhibit similar specific fatty acid-induced activity but a different inhibitory potential of purine nucleotides. Mutagenesis of non-conserved residues in human UCP1 revealed structural components in α-helix 56 and α-helix 6 crucial for uncoupling function. Comparative studies of human UCP1 with other orthologs can provide new insights into the structure-function relationship for this mitochondrial carrier and will be instrumental in searching for new activators.

Evidence for the feasibility of transmembrane proton gradient regulating oxytetracycline extracellular biodegradation mediated by biosynthesized palladium nanoparticles

Extracellular biodegradation is a promising technology for removing antibiotics and repressing the spread of resistance genes, but the strategy is limited by the low extracellular electron transfer (EET) efficiency of microorganisms. In this work, biogenic Pd0 nanoparticles (bio-Pd0) were introduced in cells in situ to enhance oxytetracycline (OTC) extracellular degradation and the effects of transmembrane proton gradient (TPG) on EET and energy metabolism mediated by bio-Pd0 were investigated. The results indicated that the intracellular OTC concentration gradually decreased with increase in pH due to the simultaneous decreases of OTC adsorption and TPG-dependent OTC uptake. On the contrary, the efficiency of OTC biodegradation mediated by bio-Pd0@B. megaterium showed a pH-dependent increase. The negligible intracellular OTC degradation, the high dependence of OTC biodegradation on respiration chain and the results on enzyme activity and respiratory chain inhibition experiments showed that NADH-dependent (rather than FADH2-dependent) EET process mediated by substrate-level phosphorylation modulated OTC biodegradation due to high energy storage and proton translocation capacity. Moreover, the results showed that altering TPG is an efficient approach to improve EET efficiency, which can be attributed to the increased NADH generation by the TCA cycle, enhanced transmembrane electron output efficiency (as evidenced by increased intracellular electron transfer system (IETS) activity, the negative shift of onset potential, and enhanced one-electron transfer through bound flavin) and stimulation of substrate-level phosphorylation energy metabolism catalyzed by succinic thiokinase (STH) under low TPG conditions. The results of structural equation model that OTC biodegradation was directly and positively modulated by the net outward proton flux as well as STH activity, and indirectly regulated by TPG through NADH level and IETS activity confirmed the previous findings. This study provides a new perspective for engineering microbial EET and application of bioelectrochemistry processes in bioremediation.

The ebb and flow of protons: A novel approach for the assessment of estuarine and coastal acidification

The acidification of estuarine and coastal waters is a consequence of both natural (e.g., aerobic respiration) and anthropogenic (e.g., combustion of fossil fuels, eutrophication) processes and can negatively impact the surrounding ecosystems. Until recently it was difficult to accurately measure pH, and thus total proton concentrations ([HT+]), when salinities vary significantly and rapidly as a consequence of tidal mixing. Proton production and transport are ultimately responsible for acidification in nearshore environments, and the uncertainty surrounding proton concentrations measured at high frequency has hindered our understanding of the net impact of global and local processes on estuarine and coastal acidification. Here, we quantify the rate of proton exchange between an estuary and bay to assess the extent of acidification by using the novel combination of high frequency pHT (total hydrogen ion concentration scale) data from an autonomous SeapHOx™ sensor and continuous tidal discharge measurements made between the eutrophic Murderkill Estuary and Delaware Bay. Proton fluxes reverse with each tide. However, the net non-tidal proton fluxes are directed upstream and display seasonal changes in magnitude. Our results indicate that Delaware Bay contributes to the acidification of the Murderkill Estuary, yet the degree of acidification is reduced in the summer. Using proton concentrations measured at high temporal resolution to calculate proton fluxes provides a new and relatively simple approach for quantifying the acidification of dynamic nearshore environments.

V-ATPase expression during development ofArtemia franciscanaembryos: potential role for proton gradients in anoxia signaling

Under anoxia, Artemia franciscana embryos downregulate metabolic processes and approach an ametabolic state. Entrance into this quiescent state is accompanied by a profound acidification of the intracellular space, and more than two decades of research now clearly demonstrates that this acidification is critical to metabolic downregulation in anoxic embryos. However, the proximal mechanisms responsible for the pH shift remain largely unidentified. Here, we report evidence demonstrating expression of the V-ATPase in encysted embryos and present an argument for its involvement in the intracellular acidification induced by anoxia. We identified a single B-subunit cDNA sharing the greatest degree of sequence similarity with ;generalist-type' homologues from mammals (brain-type) and invertebrates. Quantitative analysis of B-subunit mRNA demonstrates differential expression throughout early development, and western blot analyses confirm the expression of at least six V-ATPase subunits in both heavy membranes and microsomal vesicles. The critical need for proton pumping during the anoxia-tolerant stage of development is demonstrated by incubation with the V-ATPase inhibitor bafilomycin A1, which halts embryonic development. Importantly, net proton flux from V-ATPase-acidified compartments to the surrounding cytoplasm is likely under anoxia and may significantly contribute to the enigmatic acidification critical to quiescence.

On the generation of Alfvén waves by solar energetic particles

A simple analytical theory of Alfven waves amplified by streaming solar energetic particles (SEPs) is studied. It is pointed out that a finite time-integrated net flux of energetic protons has to pass each point in space before we can expect Alfven waves to be significantly modified by the streaming instability. The time-integrated net proton flux needed for the time-integrated wave growth rate (or wave growth, for short) to exceed unity is evaluated. Assuming that protons stream much faster than the waves, we evaluate the wave growth as a function of position and wavenumber for a specified proton injection energy spectrum, dN/dE. The wave growth is found to be proportional to vp dN/dE, where v and p are the particle speed and momentum, and to the local Alfven speed V A . Thus, maximum wave growth is achieved at the location of maximum V A (at a few solar radii), and the minimum value of dN/dE required for the wave growth to exceed unity there is a few times 10 3 2 /vp protons per unit solid angle (in coordinate space) at the solar surface. If dN/dE is below this value, test-particle theory is a valid description of particle transport and acceleration. The value is not exceeded (above 1 MeV energies) in small gradual SEP events having peak 1-MeV proton intensities below ∼10 protons (cm 2 sr s MeV) - 1 at 1 AU. The spatial and momentum dependence of the wave growth can also be used to estimate the maximum emission strength of a moving proton source in the interplanetary medium. For a strong source moving through the solar wind at constant super-Alfvenic speed. the number of escaping particles per unit time and flux-tube cross section is approximately constant in time, predicting a plateau-type time-intensity profile observed ahead of the source. The model reproduces observations of streaming-limited intensities at energies around 10 MeV and explains the double peaked injection profiles observed in large SEP events.

On the mechanism of sodium-proton exchange in crayfish

In salt depleted crayfish net sodium and proton fluxes were coupled 1:1 as required by the frog skin–turtle bladder model. In addition, three proton pump inhibitors produced equal reductions of both fluxes. It is concluded that the model operates in these animals. Net Na + and H + fluxes were very small in tap water adapted animals, but regression analysis clearly showed that they were coupled, though perhaps not 1:1. Proton pump inhibitors, at concentrations that suppressed ++++fluxes in salt-depleted crayfish, had no measureable effect on proton movement in tap water-adapted animals. Two of them (dicyclocarbodiimide and N-ethyl maleimide), caused a small reduction in Na + influx without affecting proton efflux. These experiments provide no support for operation of the frog–turtle system in adult crayfish adapted to tap water. A 2Na + — H + exchanger is considered from an energetic point of view. Such a system might be able to couple Na + and H + fluxes in dilute, near neutral solutions ([Na +]∼1–2 mM; pH 7).

Oscillations in proton transport revealed from simultaneous measurements of net current and net proton fluxes from isolated root protoplasts: MIFE meets patch-clamp

Proton fluxes were measured non-invasively on patch-clamped protoplasts isolated from wheat roots using an external H + electrode to measure the electrochemical gradient in the external solution. Under voltage clamp in the whole-cell configuration, the H + fluxes across the plasma membrane could be measured as a function of voltage and time and correlated with the simultaneous measurements of membrane current. Protoplasts could exist in three states based on the current–voltage (I–V) curves and the flux–V curves. In the pump-state where the membrane voltage (Vm) was more negative than the electrochemical equilibrium potential for potassium (E K ), a net efflux of H + occurred that was voltage-dependent such that the efflux increased as Vm was clamped more positive. In the K-state, where Vm was close to E K , similar flux–V curves were observed. In the depolarised state where Vm was greater than E K the proton flux was characterised by a net influx of H + (H + -influx state) that reversed direction at more positive values of Vm. The inhibitory effect of DCCD and stimulatory effect of fusicoccin were used to correlate current and H + flux through the H + -ATPase for which there was reasonably good agreement within the limits of the flux measurements. Some protoplasts were kept in the whole-cell configuration for up to 3 h revealing slow sustained oscillations (period about 40 min) in H + flux that were in phase with oscillations in free-running Vm. These oscillations were also observed under voltage clamp, with membrane current in phase with H + flux, but which became damped out after a few cycles. The oscillations encompassed the pump-state, K + -state and H + -influx-state. The H +- flux–V curves and I–V curves were used to model the electrical characteristics of the plasma membrane with H + -ATPase, inward and outward K + rectifiers, a linear conductance, and a passive H + influx possibly through gated proton channels.

Calcification and measurements of net proton and oxygen flux reveal subcellular domains in Acetabularia acetabulum.

Vegetative adults of Acetabularia acetabulum (L.) Silva were studied as a model system for subcellular patterning in plants, and a description of several phenotypic and physiological characteristics that reveal patterns of subcellular differentiation in this unicellular macroalga was undertaken. Initially, calcification patterns were studied. Under favorable conditions, the rhizoid and most of the stalk calcified. Only the apical 10-20% of the stalk and a small region adjacent to the rhizoid remained uncalcified. Calcification in algae has been reported to result from a biologically mediated local increase in alkalinity. To test this model extracellular pH and extracellular hydrogen ion gradients were examined with ion-selective, self-referencing, electrodes. In the light, A. acetabulum displayed a general pattern of extracellular alkalinity around the entire alga, although in some individuals the region near the rhizoid and the rhizoid itself displayed extracellular acidity. Acetabularia acetabulum also displayed net hydrogen ion influx at the rhizoid and the apical half of the stalk, variable flux in the lower part of the stalk, and net hydrogen ion efflux at the base of the stalk next to the rhizoid. The lack of complete correlation between external pH patterns and calcification suggests that other factors contribute to the control of calcification in this alga. To examine whether net hydrogen ion flux patterns correlated with photosynthetic or respiration patterns, oxygen flux was measured along the stalk using self-referencing O2 electrodes. Photosynthetic oxygen evolution occurred at comparable levels throughout the stalk, with less evolution in the rhizoid. Respiration mainly occurred near and in the rhizoid, with less O2 consumption occurring more apically along the stalk. Our studies of calcification patterns, net hydrogen ion flux and O2 flux revealed several overlapping patterns of subcellular differentiation in A. acetabulum.

A sensitive computer‐controlled pH‐stat system allows the study of net H+ fluxes related to nitrogen uptake of intact plants in situ

ABSTRACTProton fluxes related to the acid–base balance of intact plants were investigated in detail. A multi‐channel titration system was developed in order to regulate the pH in two different sets of nutrient solutions. This system also allowed computation of the dynamics of proton fluxes associated with nutrient uptake in situ.The pH‐stat system presented here has proved to be very reliable and sensitive. By additions of acid or base to the nutrient solutions, the set pH could typically be maintained within 0·01 pH units. Experiments investigating net proton fluxes correlated with nitrogen uptake are described. The results show a rapid response of proton fluxes to changes in the form of nitrogen supplied, indicating that alterations in net proton fluxes are directly induced by the nature of the nitrogen source. The stoichiometry of proton fluxes connected to nitrogen uptake could be followed online, and the results are discussed in relation to the charge and acid–base balances of the whole plant.

Control of proximal tubule acidification by the endothelium of the peritubular capillaries

Guanosine 3',5'-cyclic monophosphate (cGMP), a nitric oxide mediator, stimulates Na+/H+ exchange in brush-border vesicles of the renal cortex. The aim of the present work was to test whether the endothelium of the peritubular capillaries modulated the rate of proximal luminal acidification through the release of endothelium-derived nitric oxide (EDNO). Perfusion of the tubule lumen with dibutyryl cGMP increased net proton flux (J(H)). Two agents that elicit EDNO production, bradykinin (BK) and carbamylcholine (Cch), increased J(H) when added to the peritubular capillary perfusate. Bradykinin did not affect J(H) when the peritubular capillaries and the lumen were perfused with Na-free solution. Methylene blue (MB) and N(G)-nitro-L-arginine methyl ester (L-NAME) blocked the elevation in J(H) by Cch and also decreased basal J(H). Bradykinin increased cGMP content of isolated proximal convoluted tubules, but only if they were coincubated with endothelial cells. This effect of BK was blocked by L-NAME. The results suggest that the endothelium of the peritubular capillaries affects proximal tubule acidification through changes of cGMP in proximal tubule cells, probably via stimulation of Na+/H+ exchanger.

Quinine, intracellular pH and modulation of hemi-gap junctions in catfish horizontal cells

Quinine increases the conductance of hemi-gap junctions in horizontal cells. We investigated the mechanisms of alkalinization and the hypothesis that quinine-induced alkalinization produced these conductance increases. We found that quinine-induced alkalinizations were not blocked by cobalt, amiloridie, or DIDS. Therefore, this suggests that the alkalinization is not likely due to net proton flux through opened hemi-gap channels nor is it likely due to an action on Cl −/HC0 3 − exchanger or Na +/H + exchanger, both of which are known to regulate pH i in the horizontal cells. Quinine increased hemi-gap conductance even when cells were recorded with patch pipets containing up to 80 mM HEPES. We conclude that quinine-induced alkalinization cannot account solely for the hemi-gap junctional conductance increases.

Proton flux measurements from tissues in buffered solution

ABSTRACTProton movement across plant cell membranes is part of many important physiological processes. The net proton flux to or from tissues can be determined non‐invasively by measuring the proton electrochemical potential gradient in the adjacent solution. In buffered solution, some of the protons crossing the tissue boundary diffuse as proto‐nated buffer whose flux is not included in the flux calculated from the proton (hydrogen ion) electrochemical gradient. In this theoretical paper, it is shown how experimenters can calculate the protonated buffer flux from the measured proton flux in solution. The ratio of these two components of total proton flux depends on the pH of the solution and on the concentration and pK of the buffer. For a given concentration of a buffer which has a single pK, the flux ratio rises with pH when the solution pH is lower than the buffer pK. The slope is about 2 on a log10 scale. As the pH increases above the pK, the flux ratio levels off to approach its maximum. With mixed buffers, or one having two or more pK values, the flux ratios are additive: each buffer acts independently based on its concentration and its pK value. Unbuffered solutions always have the buffering effects of water itself and also of carbonates due to carbon dioxide dissolved from the atmosphere. In unbuffered solutions at pH 6, the flux carried by water and carbonate is about 1 % of the measured proton flux. This validates measurements of proton flux from tissues, made by a number of workers, in unbuffered solutions below pH 6.

Kinetic modelling of geochemical processes at the Aitik mining waste rock site in northern Sweden

A steady-state geochemical model has been developed to study water-rock interactions controlling metal release from waste rock heaps at the Aitik Cu mine in northern Sweden. The Cu release in drainage waters from the site is of environmental concern. The waste rock heaps are treated as single completely mixed flow-through reactors. The geochemical model includes kinetices of sulphide and primary silicate mineral weathering, heterogeneous equilibrium with secondary mineral phases and speciation equilibrium. Field monitoring of drainage water composition provides a basis for evaluation of model performance. The relative rate of oxidative weathering of sulphides and dissolution of primary silicate minerals, using published kinetic data, are consistent with net proton and base cation fluxes at the site. The overall rate of Fe 2+ oxidation within the heap is three orders of magnitude faster than that which could be explained by surface-catalysed reaction kinetics. This suggests significant activity of iron-oxidizing bacteria. The absolute weathering rates of sulphides and silicate minerals, normalized to a measured BET surface area, are approximately two orders of magnitude lower at field scale than published rates from laboratory experiments. Because of the relative absence of carbonate minerals, the weathering of biotite and plagioclase feldspar are important sources of alkalinity.

The Na +/H + exchanger in transitional cells of the inner ear

The cytosolic pH (pH i) of transitional cells from the ampulla of the gerbil was measured micro-fluorometrically with the pH-sensitive dye 2′,7′-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) to assess the possible contribution of a Na +/H + exchanger to the regulation of H i. All experiments were conducted in virtually HCO 3 −free solutions. Under control conditions, pH i was 7.19 and addition of 10 −5 M ethylisopropylamiloride (EIPA), a blocker of Na +/H + exchange, caused a small but significant acidification of pH i. A transient exposure to 21.4 mM NH 4Cl caused a rapid cytosolic alkalinization followed by a brisk acidification and prompt recovery of pH i to its control value. The cytosolic buffer capacity (B i) was determined in the absence of Na + from changes in pH i which were elicited by [NH + 4] steps. B i was 4.7 mM/pH at pH i 7.19 and varied with pH i. The initial net proton flux J H, representative of Na +/H + exchange activity, was calculated from the product of the initial rate of alkalinization after an NH + 4-prepulse and B i at the corresponding pH i. J H was dependent on the extracellular Na + with an apparent K m of 64 mM, sensitive to the cytosolic [H +] with an apparent K m of 2.7 ∗ 10 −7 M (i.e. pH 6.6), and inhibited by EIPA with an IC 50 of 5 ∗ 10 −7 M. These data suggest that transitional cells contain in the basolateral membrane a Na +/H + exchanger of the amiloride-sensitive subtype.

Compartmental distribution and redistribution of abscisic acid in intact leaves

Using a computer model written for whole leaves (Slovik et al. 1992, Planta 187, 14-25) we present in this paper calculations of abscisic acid (ABA) redistribution among different leaf tissues and their compartments in relation to stomatal regulation under drought stress. The model calculations are based on experimental data and biophysical laws. They yield the following results and postulates: (i) Under stress, compartmental pH-shifts come about as a consequence of the inhibition of the pH component of proton-motive forces at the plasmalemma. There is a decrease of net proton fluxes by about 8.6 nmol · s(-1) · m(-2). (ii) Using stress-induced pH-shifts we demonstrate how 'stress intensities' can be quantified on a molecular basis. (iii) As the weak acid ABA is the only phytohormone which behaves in vivo and in vitro ideally according to the Henderson-Hasselbalch equation, pH-shifts induce a complicated redistribution amongst compartments in the model leaf. (iv) The final accumulation of ABA in guard-cell walls is intensive: up to 16.1-fold compared with only up to 3.4-fold in the guard-cell cytosol. We propose that the binding site of the guard-cell ABA receptor faces the apoplasm. (v) A twoto three-fold ABA accumulation in guard-cell walls is sufficient to induce closure of stomata. (vi) The minimum time lag until stomata start to close is 1-5 min; it depends on the stress intensity and on the guard-cell sensitivity to ABA: the more moderate the stress is, the later stomata start to close or they do not close at all. (vii) In the short term, there is almost no influence of the velocity of pH-shifts on the velocity of the ABA redistribution, (viii) Six hours after the termination of stress there is still an ABA concentration 1.4-fold the initial level in the guard-cell cytosol (delay of ABA relaxation, 'aftereffect'), (ix) The observed 'induction' of net ABA synthesis after onset of stress may be explained by a decrease in cytosolic ABA degradation. About 1 h after onset of stress the model leaf would start to synthesise ABA (and its conjugates) automatically, (x) This ABA net synthesis serves to 'inform roots' via an increased ABA concentration in the phloem sap. The stress-induced ABA redistribution is per se not sufficient to feed the ploem sap with ABA. (xi) The primary target membrane of 'stress' is the plasmalemma, not thylakoids. (xii) The effective 'stress sensor', which induces the proposed signal chain finally leading to stomatal closure, is located in epidermal cells. Mesophyll cells are not capable of creating a significant ABA signal to guard cells if the epidermal plasmalemma conductance to undissociated molecular species of ABA (HABA) is indeed higher than the plasmalemma conductance of the mesophyll (plasmodesmata open), (xiii) All model conclusions which can be compared with independent experimental data quantitatively fit to them. We conclude that the basic experimental data of the model are consistent. A stress-induced ABA redistribution in the leaf lamina elicits stomatal closure.

Evidence for Cotransport of Nitrate and Protons in Maize Roots

We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO(3) (-) and H(+) within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO(3) (-) uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H(+)-ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO(3) (-) (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO(3) (-) absorption. These results strongly indicate that the NO(3) (-) electrical response is due to the operation of a NO(3) (-) transport system in the plasmalemma of maize root cells. Furthermore, the results from the H(+)-ATPase inhibitor studies indicate that the NO(3) (-) transport system is linked to the H(+)-ATPase, presumably as a NO(3) (-)/H(+) symport. This is further supported by the pH response of the NO(3) (-) transport system (inhibition at alkaline pH values) and the change in net H(+) flux from a moderate efflux in the absence of NO(3) (-), to zero net H(+) flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO(3) (-)/H(+) flux data is a NO(3) (-)/H(+) symport with a NO(3) (-):H(+) flux stoichiometry >1, whose operation results in the stimulation of the H(+)-ATPase due to the influx of protons through the cotransport system.

Membrane electrogenesis in plasmodia of Physarum polycephalum: a dominant role for the photon pump

An electrogenic ion pump, previously identified in caffeine-induced plasmodial droplets of Physarum, is here shown to be the major source of the testing membrane potential of intact Physarum plasmodia, and to be an H +-ATPase. The plasma membrane of plasmodia is rapidly depolarized by the respiratory inhibitors, cyanide and azide, which cause an almost parallel depletion of intracellular ATP. It is also depolarized by the ATPase inhibitors, diethylstilbestrol and orthovanadate. Diethylstilbestrol, however, produces only a slight depletion of ATP. Plots of membrane potential versus intracellular ATP concentration, during the onset of cyanide inhibition, yield a saturation curve which can be fitted by the Michaelis equation (describing the pump) plus a constant term (describing ion diffusion), with maximal voltage of the pump ≊ −120mV, K m ≊ 1.1mM, and the diffusion term = − 4 mV. Intracellularly injected ATP hyperpolarizes plasmodial membranes to the expected level. Direct measurements of proton flux show net efflux of 14 pmol · s −1 · mg −1 (dry weight) in uninhibited plasmodia, which converts to 28 pmol · s −1 · mg −1 net infux during 80 s of cyanide inhibition. This efflux minimum coincides in time with the membrane potential minimum. Bidirectionality of net proton flux suggests a significant role of H + for passive diffusion.

On the Inhibition of the Mitochondrial Inner Membrane Anion Uniporter by Cationic Amphiphiles and Other Drugs

Depleting the mitochondrial matrix of divalent cations with the ionophore A23187 activates a pH-sensitive, anion uniport pathway which can transport many anions normally regarded as impermeant (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). Addition of valinomycin to respiring mitochondria can also induce the uptake of a wide variety of anions; however, the mechanism of anion transport during this "respiration-induced" swelling is less certain. In this paper, I demonstrate that both of these processes are inhibited by a variety of cationic amphiphiles including propranolol, quinine, amiodarone, imipramine and amitriptyline, and the benzodiazepine R05-4864. Although the IC50 values for the two processes are not equal, the ratio of IC50 values for the two processes appears to be the same for all drugs. Measurements of net transmembrane proton fluxes that occur during the assays reveal that respiration-induced swelling is associated with extensive proton ejection, the peak of which coincides with the maximum rate of anion transport. Moreover, from measurements of matrix buffering power, it is estimated that the matrix pH is 3 units more alkaline during respiration-induced swelling than during A23187-induced swelling. It is also shown that the IC50 for A23187-induced transport is pH-dependent in a manner consistent with modulation of drug binding by protonation of two sites. These findings allow the difference in IC50 values for the two types of assay to be explained by the pH dependence of the binding constant for the drug. Furthermore, the pH gradient generated during respiration-induced swelling is so large that the electrical component of the proton-motive force will be negligible. Thus, despite the fact that the mitochondria are "energized," rapid electrophoretic anion influx is possible. These data provide evidence that the transport of anions in these two types of assay occurs via the same pathway.

Net proton influx into bone during metabolic, but not respiratory, acidosis.

During acute metabolic acidosis there is a net influx of protons into bone, decreasing the elevated proton concentration. Whether there is an influx of protons into bone during acute respiratory acidosis is not known. To determine the effect of respiratory acidosis on net proton flux (JH) relative to bone, we compared JH from neonatal mouse calvariae incubated for 3 h in medium acidified by an increase in PCO2 (respiratory acidosis) with that from calvariae incubated in medium acidified to the same extent by a decrease in bicarbonate concentration (metabolic acidosis). The initial medium pH with respiratory acidosis was not different from that with metabolic acidosis (7.108 +/- 0.005 vs. 7.091 +/- 0.007, respectively, P = NS). During respiratory acidosis there was no JH from bone relative to the medium (JH = 236 +/- 93 neq.bone-1.3h-1, P = NS vs. 0); however, during metabolic acidosis there was net proton influx from the medium into bone (JH = -703 +/- 108, P less than 0.05 vs. 0, P less than 0.001 vs. respiratory acidosis). There was less calcium efflux from bone during respiratory than during metabolic acidosis (JCa = 68 +/- 6 nmol.bone-1.3 h-1 vs. 100 +/- 9, respectively, P less than 0.001). There is a net influx of protons into bone in vitro during acute metabolic, but not during acute respiratory, acidosis. The smaller calcium efflux during respiratory acidosis may indicate less net bone mineral dissolution and thus less buffer release into the medium.

Effects of parathyroid hormone on net proton flux from neonatal mouse calvariae.

Bone mineral buffers protons during acute metabolic acidosis; whether parathyroid hormone (PTH) augments proton buffering is controversial. To determine whether PTH augments proton buffering by bone, we cultured neonatal mouse calvariae with or without PTH (10(-8) M) for 3 h in medium that was physiologically acid (pH approximately 7.20), neutral (pH approximately 7.40), or alkaline (pH approximately 7.60). Over the entire pH range studied there was less influx of protons into calvariae treated with PTH than into control calvariae, indicating that PTH does not augment but instead inhibits proton buffering by bone. To determine whether chronic exposure to PTH is necessary to augment proton buffering, calvariae were incubated with PTH for 24 h before a 3-h culture. Calcium efflux from calvariae exposed to PTH (10(-8) M) for 24 h exceeded that of controls. When these same calvariae were recultured for 3 h in fresh medium, PTH-treated and control calvariae behaved similarly, with net efflux of protons into acid, neutral, and alkaline media. Regardless of whether PTH is added at the time of exposure to acid medium or 24 h before calvariae cultured with PTH do not buffer protons to a greater extent than controls.