On the mechanism of transbilayer transport of phosphatidylglycerol in response to transmembrane pH gradients

Previous work [Hope et al. (1989) Biochemistry 28, 4181-4187] has shown that asymmetric transmembrane distributions of phosphatidylglycerol (PG) in PG-phosphatidylcholine (PC) large unilamellar vesicles can be induced in response to transbilayer pH gradients (delta pH). Here the mechanism of PG transport has been investigated. It is shown that PG movement in response to delta pH is consistent with permeation of the uncharged (protonated) form and that the half-time for transbilayer movement of the uncharged form can be on the order of seconds at 45 degrees C. This can result in rapid pH-dependent transmembrane redistributions of PG. The rate constant for transbilayer movement exhibits a large activation energy (31 kcal/mol) consistent with transport of neutral dehydrated PG where dehydration of the (protonated) phosphate presents the largest barrier to transmembrane diffusion. It is shown that acyl chain saturation, chain length, and the presence of cholesterol modulate the rate constants for PG transport in a manner similar to that observed for small nonelectrolytes.     

Uptake of basic amino acids and peptides into liposomes in response to transmembrane pH gradients.

The uptake of derivatives of lysine and a pentapeptide (ala-met-leu-trp-ala) into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been examined. In these derivatives, the C-terminal carboxyl functions have been converted to methyl esters or amides. It is shown that the presence of a pH gradient (interior acidic) results in the rapid and efficient accumulation of these weak base amino acid and peptide derivatives into LUVs in a manner consistent with permeation of the neutral (deprotonated) form. It is suggested that this property may have general implications for mechanisms of transbilayer translocation of peptides, such as signal sequences, which exhibit weak base characteristics.     

Putting the pH into phosphatidic acid signaling

Background

Camouflage patterns that hinder detection and/or recognition by antagonists are widely studied in both human and animal contexts. Patterns of contrasting stripes that purportedly degrade an observer's ability to judge the speed and direction of moving prey ('motion dazzle') are, however, rarely investigated. This is despite motion dazzle having been fundamental to the appearance of warships in both world wars and often postulated as the selective agent leading to repeated patterns on many animals (such as zebra and many fish, snake, and invertebrate species). Such patterns often appear conspicuous, suggesting that protection while moving by motion dazzle might impair camouflage when stationary. However, the relationship between motion dazzle and camouflage is unclear because disruptive camouflage relies on high-contrast markings. In this study, we used a computer game with human subjects detecting and capturing either moving or stationary targets with different patterns, in order to provide the first empirical exploration of the interaction of these two protective coloration mechanisms.

Results

Moving targets with stripes were caught significantly less often and missed more often than targets with camouflage patterns. However, when stationary, targets with camouflage markings were captured less often and caused more false detections than those with striped patterns, which were readily detected.

Conclusions

Our study provides the clearest evidence to date that some patterns inhibit the capture of moving targets, but that camouflage and motion dazzle are not complementary strategies. Therefore, the specific coloration that evolves in animals will depend on how the life history and ontogeny of each species influence the trade-off between the costs and benefits of motion dazzle and camouflage.     

Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane- permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3- (5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]     

Lipid asymmetry induced by transmembrane pH gradients in large unilamellar vesicles

We have investigated the influence of transmembrane pH gradients across large unilamellar vesicle membranes on the transbilayer distributions of simple lipids with weak base and weak acid characteristics. Trinitrobenzenesulfonic acid labeling results consistent with a rapid and complete migration of stearylamine and sphingosine to the inner monolayer of the large unilamellar vesicles are observed when the large unilamellar vesicles' interior is acidic. Alternatively, when the vesicle interior is basic, oleic and stearic acid cannot be removed by external bovine serum albumin, indicating a localization in the inner monolayer. Moreover, effects corresponding to the decrease in external surface charge predicted upon the migration of stearylamine or stearic acid to the inner monolayer are readily detected employing ion exchange chromatography. These results are consistent with transbilayer distributions of these agents dictated by a Henderson-Hasselbach equilibrium. The possible implications for metabolic regulation by pH gradients, as well as factors giving rise to phospholipid transbilayer asymmetry, are discussed.     

Measurement of transmembrane pH gradients in human erythrocytes using 19F NMR

A new high-sensitivity method has been described for measuring transmembrane pH gradients in vesicular systems using ¹⁹F NMR. The ¹⁹F resonance of trifluoroethylamine has been shown to have a large pH-dependent chemical shift and the position of the resonance was measured with high precision and sensitivity. In suspensions of human erythrocytes, trifluoroethylamine distributed itself across the membrane and separate ¹⁹F resonances were obtained from the trifluoroethylamine inside and outside of the cells. The pH in each compartment was calculated from the resonance positions.     

Intervesicular exchange of lipids with weak acid and weak base characteristics: influence of transmembrane pH gradients

Transmembrane pH gradients have previously been shown to induce an asymmetric transmembrane distribution of simple lipids that exhibit weak acid or basic characteristics (Hope, M.J. and Cullis, P.R. (1987) J. Biol. Chem. 262, 4360-4366). In the present study we have examined the influence of proton gradients on the inter-vesicular exchange of stearylamine and oleic acid. We show that vesicles containing stearylamine immediately aggregate with vesicles containing phosphatidylserine and that disaggregation occurs subsequently as stearylamine equilibrates between the two vesicle populations. Despite visible flocculation during the aggregation phase, vesicle integrity is maintained. Stearylamine is the only lipid to exchange, fusion does not occur and vesicles are able to maintain a proton gradient. When stearylamine is sequestered to the inner monolayer in response to a transmembrane pH gradient (inside acidic) aggregation is not observed and diffusion of stearylamine to acceptor vesicles is greatly reduced. The ability of delta pH-dependent lipid asymmetry to modulate lipid exchange is also demonstrated for fatty acids. Oleic acid can be induced to transfer from one population of vesicles to another by maintaining a basic interior pH in the acceptor vesicles. Moreover, it is shown that the same acceptor vesicles are capable of depleting serum albumin of bound fatty acid. These results are discussed with respect to the mechanism and modulation of lipid flow between membranes both in vitro and in vivo.     

Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients

Studies from this laboratory (Mayer et al. (1986) Biochim. Biophys. Acta 857, 123-126) have shown that doxorubicin can be accumulated into liposomal systems in response to transmembrane pH gradients (inside acidic). Here, detailed characterizations of the drug uptake and retention properties of these systems are performed. It is shown that for egg phosphatidylcholine (EPC) vesicles (mean diameter of 170 nm) exhibiting transmembrane pH gradients (inside acidic) doxorubicin can be sequestered into the interior aqueous compartment to achieve drug trapping efficiencies in excess of 98% and drug-to-lipid ratios of 0.36:1 (mol/mol). Drug-to-lipid ratios as high as 1.7:1 (mol/mol) can be obtained under appropriate conditions. Lower drug-to-lipid ratios are required to achieve trapping efficiencies in excess of 98% for smaller (less than or equal to 100 nm) systems. Doxorubicin trapping efficiencies and uptake capacities are related ito maintenance of the transmembrane pH gradient during encapsulation as well as the interaction between doxorubicin and entrapped citrate. This citrate-doxorubicin interaction increases drug uptake levels above those predicted by the Henderson-Hasselbach relationship. Increased drug-to-lipid ratios and trapping efficiencies are observed for higher interior buffering capacities. Retention of a large transmembrane pH gradient (greater than 2 units) after entrapment reduces the rate of drug leakage from the liposomes. For example, EPC/cholesterol (55:45, mol/mol) liposomal doxorubicin systems can be achieved which released less than 5% of encapsulated doxorubicin (drug-to-lipid molar ratio = 0.33:1) over 24 h at 37 degrees C. This pH gradient-dependent encapsulation technique is extremely versatile, and well characterized liposomal doxorubicin preparations can be generated to exhibit a wide range of properties such as vesicle size, lipid composition, drug-to-lipid ratio and drug release kinetics. This entrapment procedure therefore appears well suited for use in therapeutic applications. Finally, a rapid colorimetric test for determining the amount of unencapsulated doxorubicin in liposomal systems is described.     

Estimation of transmembrane pH gradients from phase equilibria of spin-labeled amines.

Spin-labeled secondary amines have been used to measure transmembrane proton gradients in sonicated liposomes. The electron paramagnetic resonance spectra of these probes show changes in the ratio of membrane associated to free aqueous probe as a function of transmembrane pH gradient. As the pH gradient is increased, inside acidic, the amount of membrane associated probe increases. The results are accounted for by a simple thermodynamic theory.     

Osmotic and pH transmembrane gradients control the lytic power of melittin.

Transmembrane osmotic gradients applied on large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles were used to modulate the potency of melittin to induce leakage. Melittin, an amphipathic peptide, changes the permeability of vesicles, as studied using the release of entrapped calcein, a fluorescent marker. A promotion of the ability of melittin to induce leakage was observed when a hyposomotic gradient (i.e., internal salt concentration higher than the external one) was imposed on the vesicles. It is proposed that structural perturbations caused by the osmotic pressure loosen the compactness of the outer leaflet, which facilitates the melittin-induced change in membrane permeability. Additionally, we have shown that this phenomenon is not due to enhanced binding of melittin to the vesicles using intrinsic fluorescence of the melittin tryptophan. Furthermore, we investigated the possibility of using a transmembrane pH gradient to control the lytic activity of melittin. The potency of melittin in inducing release is known to be inhibited by increased negative surface charge density. A transmembrane pH gradient causing an asymmetric distribution of unprotonated palmitic acid in the bilayer is shown to be an efficient way to modulate the lytic activity of melittin, without changing the overall lipid composition of the membrane. We demonstrate that the protective effect of negatively charged lipids is preserved for asymmetric membranes.     

Use of the fluorescent weak acid dansylglycine to measure transmembrane proton concentration gradients

The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient.     

Influence of pH on phosphatidic acid multilayers a rippled structure at high pH values

The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5–14 (2.6 M K⁺), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.     

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential gradient, and that the maximal rate of accumulation may exist when both components of the protonmotive force are present.     

Influence of pH on phosphatidic acid multilayers. A rippled structure at high pH values.

The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5--14 (2.6 M K+), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.     

Proton flux in large unilamellar vesicles in response to membrane potentials and pH gradients.

The transport of protons across liposomes composed of phosphatidylcholine in response to electrical potentials or pH gradients has been investigated. The results support three major conclusions. The first of these concerns the need for reliable measurements of electrical potentials and pH gradients. It is shown that the potential probe tetraphenylphosphonium and the pH probe methylamine provide accurate and self consistent measures of electrical potentials and pH gradients respectively in these systems. Second, it is shown by two independent techniques that the pH gradients induced in response to valinomycin and potassium dependent electrical potentials are significantly smaller than would be expected for electrochemical equilibrium. The pH gradients observed are stable over an 8 h time course and are sensitive to the ionic composition of the buffers employed, where the presence of external sodium results in the smallest induced pH gradients. These results are discussed in terms of current models of proton conductance across membranes. In a final area of investigation, it is shown that valinomycin and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) can transport sodium ions in a synergistic manner.