Uptake of dibucaine into large unilamellar vesicles in response to a membrane potential

Local amine anesthetics appear to exert their effects in the charged (protonated) form on the cytoplasmic side of excitable membranes. Two features of interest are the mechanism whereby these drugs move across the membrane to the inner monolayer and the actual membrane concentrations achieved. In this work, we have investigated the influence of a K+ diffusion potential, delta psi, on the transmembrane distribution and concentration of the local anesthetic dibucaine employing large unilamellar vesicle systems. It is demonstrated that egg phosphatidylcholine large unilamellar vesicles exhibiting a delta psi (interior negative) actively accumulate dibucaine to achieve high interior concentrations. 31P and 13C nuclear magnetic resonance studies show that the internalized drug is localized to the vesicle inner monolayer, and suggest that the protonated form of the anesthetic is the species that is actively transported. The inner monolayer anesthetic concentrations thus achieved can be an order of magnitude or more larger than predicted on the basis of anesthetic lipid-water partition coefficients. It is suggested that these effects may be related to the mechanisms whereby local anesthetics are localized and concentrated at their sites of action in nerve membranes.     

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Influence of ion gradients on the transbilayer distribution of dibucaine in large unilamellar vesicles

The uptake of dibucaine into large unilamellar vesicles in response to proton gradients (delta pH; inside acidic) or membrane potentials (delta psi; inside negative) has been investigated. Dibucaine uptake in response to delta pH proceeds rapidly in a manner consistent with permeation of the neutral (deprotonated) form of the drug, reaching a Henderson-Hasselbach equilibrium where [dibucaine]in/[dibucaine]out = [H+]in/[H+]out and where the absolute amount of drug accumulated is sensitive to the buffering capacity of the interior environment. Under appropriate conditions, high absolute interior concentrations of the drug can be achieved (approximately 120 mM) in combination with high trapping efficiencies (in excess of 90%). Dibucaine uptake in response to delta psi proceeds more than an order of magnitude more slowly and cannot be directly attributed to uptake in response to the delta pH induced by delta psi. This induced delta pH is too small (less than or equal to 1.5 pH units) to account for the transmembrane dibucaine concentration gradients achieved and does not come to electrochemical equilibrium with delta psi. Results supporting the possibility that the charged (protonated) form of dibucaine can be accumulated in response to delta psi were obtained by employing a permanently positively charged dibucaine analogue (N-methyldibucaine). Further, the results suggest that delta psi-dependent uptake may depend on formation of a precipitate of the drug in the vesicle interior. The uptake of dibucaine into vesicles in response to ion gradients is of direct utility in drug delivery and controlled release applications and is related to processes of drug sequestration by cells and organelles in vivo.     

pH-dependent binding of local anesthetics in single batrachotoxin-activated Na+ channels. Cocaine vs. quaternary compounds.

The effects of internal and external pH on the binding kinetics of local anesthetics (LAs) were studied in single batrachotoxin-activated Na+ channels incorporated into planar bilayers. With internal quaternary QX-314 and RAC421-II drugs, the binding interactions were little affected by either external or internal pH. With tertiary cocaine, the binding kinetics were drastically altered by pH. A decrease in the internal pH from 9.3 to 6.2 decreased the apparent equilibrium dissociation constant (Kd) of internal cocaine by more than 100-fold. This increase in the binding affinity was mostly accounted for by an increase in the apparent cocaine on-rate constant (kon) of approximately 80-fold. The cocaine off-rate constant (koff) was little changed (between 3-4 s-1). These results demonstrate quantitatively that the charged form of cocaine is the active form for BTX-activated Na+ channels. Surprisingly, the apparent pKa of cocaine near its binding site was estimated to be 1.4 units lower than that in bulk solution (7.1 vs. 8.5), indicating that the LA drug encounters a relatively hydrophobic environment. Opposite to the internal pH effect, a decrease of external pH from 8.4 to 6.2 increased the Kd value of internally and externally applied cocaine by approximately 8- and approximately 25-fold, respectively. External pH effect was primarily mediated by modulation of kon; koff was again relatively unaffected. Our findings support a model in which neutral cocaine can readily cross the membrane barrier, but needs to be protonated internally to bind to its binding site.     

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.     

Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances

With few exceptions, weakly basic compounds that are sufficiently lipophilic in their neutral forms and sufficiently hydrophilic in their protonated forms accumulate in lysosomes. When the concentration within the lysosomes becomes sufficiently high, osmotic swelling occurs. The cells than take on a vacuolated appearance. The concentrations at which different weak bases cause lysosomal vacuolation vary over almost three orders of magnitude. For any particular weak base, it is the concentration of the neutral form that determines the extent of uptake and the degree of vacuolation. Chloroquine is anomalous in that concentrations greater than approximately 30 microM cause less uptake and less vacuolation than do lower concentrations.     

The inhibition of calcium efflux from rat liver mitochondria by halogenated anesthetics

The halogenated anesthetics halothane, enflurane and isoflurane inhibit the calcium efflux induced by Ruthenium Red in isolated rat liver mitochondria. The extent of the inhibition is higher for enflurane (approximately 50%) than for either isoflurane (approximately 35%) or halothane (approximately 15%), and does not increase significantly between 0.1 and 0.6-1.0 mM anesthetic. Both the mitochondrial respiratory rate and transmembrane electrical potential are unaffected by the halogenated anesthetics concentrations capable to inhibit the efflux of calcium.     

Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae

Picrophilus oshimae is an extremely acidophilic, thermophilic archaeon that grows optimally at 60°C and at pH 0.7. It is an obligatory acidophile that does not grow at pH values above 4.0. The proton motive force in respiring cells is composed of a large transmembrane pH gradient, inside less acid, and a reversed transmembrane electrical potential, inside positive. Cells maintain an intracellular pH at around 4.6 at extracellular pH values ranging from 0.8 to 4.0. Above pH 4.0 cells lyse rapidly and lose their viability. Liposomes prepared from lipids derived from P. oshimae have an extremely low proton permeability at acidic pH. However, at neutral pH, the lipids are unable to assemble into regular liposomal structures. These observations suggest that the loss of viability and cell integrity above pH 4.0 is due to an impairment of the barrier function of the cytoplasmic membrane. Received: July 18, 1997 / Accepted: November 25, 1997     

The pH-dependent rate of action of local anesthetics on the node of Ranvier

Local anesthetic solutions were applied suddenly to the outside of single myelinated nerve fibers to measure the time course of development of block of sodium channels. Sodium currents were measured under voltage clamp with test pulses applied several times per second during the solution change. The rate of block was studied by using drugs of different lipid solubility and of different charge type, and the external pH was varied from pH 8.3 to pH 6 to change the degree of ionization of the amine compounds. At pH 8.3 the half-time of action of amine anesthetics such as lidocaine, procaine, tetracaine, and others was always less than 2 s and usually less than 1 s. Lowering the pH to 6.0 decreased the apparent potency and slowed the rate of action of these drugs. The rate of action of neutral benzocaine was fast (1 s) and pH independent. The rate of action of cationic quaternary QX-572 was slow (greater than 200 s) and also pH independent. Other quaternary anesthetic derivatives showed no action when applied outside. The result is that neutral drug forms act much more rapidly than charged ones, suggesting that externally applied local anesthetics must cross a hydrophobic barrier to reach their receptor. A model representing diffusion of drug into the nerve fiber gives reasonable time courses of action and reasonable membrane permeability coefficients on the assumption that the hydrophobic barrier is the nodal membrane. Arguments are given that there may be a need for reinterpretation of many published experiments on the location of the anesthetic receptor and on which charge form of the drug is active to take into account the effects of unstirred layers, high membrane permeability, and high lipid solubility.     

Bioenergetic properties and viability of alkalophilic Bacillus firmus RAB as a function of pH and Na+ contents of the incubation medium.

The bioenergetic properties and viability of obligately alkalophilic Bacillus firmus RAB have been examined upon incubation in alkaline and neutral buffers in the presence or absence of added Na+. At pH 10.5, cells incubated in the absence of Na+ exhibited an immediate rise in cytoplasmic pH from less than 9.5 to 10.5, and they lost viability very rapidly. Viability experiments in the presence or absence of an energy source further suggested that the Na+-dependent mechanism for pH homeostasis is an energy-requiring function. The Na+/H+ antiporter, which catalyzes the vital proton accumulation at alkaline pH, was only slightly operational at pH 7.0; both whole cells and vesicles exhibited net proton extrusion even in the presence of Na+. Moreover, cells incubated in buffer at pH 7.0 were actually more viable in the presence of Na+ than in its absence. Thus, the inability of B. firmus RAB to grow at neutral pH is not due to excessive acidification of the cytoplasm. Rather, the transmembrane electrical potential, delta psi, generated at pH 7.0 was found to be much lower than at alkaline pH. The very low delta psi compromised several cell functions, e.g., Na+/solute symport and motility, which in this and other alkalophiles specifically depend upon delta psi and Na+.     

Dopamine accumulation in large unilamellar vesicle systems induced by transmembrane ion gradients

Transmembrane movement of dopamine in response to K+ or H+ ion gradients has been investigated. It is shown that dopamine can accumulate rapidly into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine exhibiting either a K+ diffusion potential (delta psi; negative inside) or a pH gradient (inside acidic). This can result in entrapped dopamine concentrations of 30-40 mM and inside-outside concentration gradients of nearly 300-fold. The transmembrane dopamine gradients formed in LUV systems exhibiting delta pH (inside acidic) indicate that the transport process can be dictated by movement of the neutral form of dopamine which redistributes according to a simple Henderson-Hasselbach equilibrium. The mechanism of dopamine transport in response to a valinomycin-induced K+ potential is more complex. Although generation of a K+ diffusion potential results in acidification of the vesicle interior, the magnitude of the induced delta pH (approx. 1 pH unit) is insufficient to account for the dopamine concentration gradient achieved (greater than 200-fold). Further, data presented here suggest that higher uptake levels of dopamine can be achieved when certain anions (ATP and citrate) are entrapped within the LUV system. These anions may complex with the protonated form of dopamine creating a non-equilibrium trapping phenomena resulting in interior concentrations of dopamine in excess of that predicted by a simple Henderson-Hasselbach equilibrium.     

Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP-dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the K0.5 of inhibition of calcium uptake are the following: 23 microM (dibucaine), 0.44 mM (lidocaine), 1.5 mM (procaine), and 0.8 mM (tetracaine). There is a good correlation between these K0.5 values and the concentrations of the local anesthetics that inhibit the Ca2(+)-dependent Mg2(+)-ATPase of these membranes. In addition, except for procaine, these local anesthetics stimulate severalfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP-dependent Ca2+ uptake, e.g., 80-700 microM dibucaine, 2-10 mM lidocaine, and 1-3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations.     

Comparison of the binding of anthracycline derivatives to purified DNA and to cell nuclei

Fluorescence method was used to study the interactions of anthracyclines with purified DNA and with cell nuclei at 37 degrees C, at pH ranging from 6.8 to 8. Four anthracyclines were used; adriamycin (ADR), 4'-o-tetrahydropyranyladriamycin (THP-ADR), daunorubicin (DNR) and aclacinomycin (ACM). The values of pKa of deprotonation of these four drugs in the pH range 6.5-8.5 are 8.4, 7.7, 8.4 and 7.0 for ADR, THP-ADR, DNR and ACM, respectively. The overall binding constants K* of these four drugs to purified DNA was determined at various pH values. The binding constants K0 and K+ of the respectively neutral form and once protonated form of the drugs to DNA were calculated. Using cell nuclei from K562 cells, the amount of drug intercalated (CN) within the nuclei of K562 cells and the amount of free drug (CE) in the solution were determined at various pH values: measuring at the same pH values, a linear correlation occurred between K* and CN/CE.     

Evidence for a sodium influx pump in sunflower roots

Summary Transmembrane electrical potential differences in the cortical cells of the root of the sunflower (Helianthus annuus) have been measured using microelectrodes. The plants were grown in culture solution with a range of sodium concentrations. It was found that increasing the external sodium concentration had virtually no effect on the transmembrane potential. The vacuolar content of sodium did not change significantly with the age of the tissue indicating that sodium was in flux equilibrium in our experiments. This allowed the Nernst equation to be used to calculate the electrochemical potential gradient for sodium between the vacuole and the external solution. It was concluded that sodium was being transported into the vacuole against the electrochemical potential gradient. The location and role of the inwardly directed sodium pump implied by these results is discussed in relation to the efflux pumps for sodium reported for roots of other species. Potassium was also accumulated against the electrochemical potential gradient by these cells.Sodium was found to stimulate the growth of H. annuus when present in the culture solution at very low concentrations.     

The protonmotive force and beta-galactoside transport in Bacillus acidocaldarius.

The acidophilic and thermophilic bacterium, Bacillus acidocaldarius maintains a cytoplasmic pH between 5.85 and 6.31 over a range of external pH from 2.0 to 4.5. Consistently, the pH optimum of beta-galactosidase, as assayed in cell extracts, is between pH 6.0 and 6.5. An electrical potential (delta-psi), interior positive, is also maintained across the membrane. A delta-psi of approximately 34 mV was calculated from determinations of thiocyanate uptake by cells at pH 3.5. Addition of the proton conductor carbonyl cyanide m-chlorophenylhydrazone increased the delta-psi. Treatment of cells with valinomycin (in the absence of external potassium ions) or high concentrations of thiocyanate, to abolish the delta psi, resulted in collapse of the transmembrane proton gradient (delta pH). Active transport of methylthio-beta, D-galactoside occurred optimally at pH 3.5. Transport of the galactoside was inhibited by various compounds which could dissipate the transmembrane delta pH and by respiratory inhibitors. A decrease in the delta pH and an increase in the delta psi occurred upon addition of methylthio-beta, D-galactoside to cells of B. acidocaldarius. Thus the transport of this solute appears to involve an electrogenic symport with protons. The transport system is most active at 50 degrees C and shows little activity at 25 degrees C, although the delta pH is the same at the two temperatures. Gramicidin inhibits methylthio-beta, D-galactoside transport equally effectively at 50 degrees C and 25 degrees C, while nigericin inhibits only after a lag at 25 degrees C.     

Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH.

Methanolobus taylorii GS-16, a moderately halophilic and alkaliphilic methanogen, grows over a wide pH range, from 6.8 to 9.0. Cells suspended in medium with a pH above 8.2 reversed their transmembrane pH gradient (delta pH), making their cytosol more acidic than the medium. The decreased energy in the proton motive force due to the reversed delta pH was partly compensated by an increased electric membrane potential (delta psi). The cytosolic acidification by M. taylorii at alkaline pH values was accompanied by K+ extrusion. The cytosolic K+ concentration was 110 mM in cells suspended at pH 8.7, but it was 320 mM in cells suspended at neutral pH values. High external K+ concentrations (210 mM or higher) inhibited the growth of M. taylorii at alkaline pH values, perhaps by preventing K+ extrusion. Cells suspended at pH 8.5 and 300 mM external K+ failed to acidify their cytosol. The key observation indicative of the involvement of K+ transport in cytosolic acidification was that valinomycin (0.8 microM), a K+ uniporter, inhibited the growth of M. taylorii only at alkaline pH values. Experiments with resting cells indicated that at alkaline pH values valinomycin uncoupled catabolic reactions from ATP synthesis. Thus, K+/H+ antiport activity was proposed to account for the K+ extrusion and the uncoupling effect of valinomycin at alkaline pH values. Such antiport activity was demonstrated by the sharp drop in pH of the bulk medium of the cell suspension upon the addition of 0.1 M KCl. The antiporter appeared to be active only at alkaline pH values, which was in accordance with a possible role in pH homeostasis by M. taylorii growing at alkaline pH values.     

Uncoupling by Acetic Acid Limits Growth of and Acetogenesis by Clostridium thermoaceticum

When cells of the anaerobic thermophile Clostridium thermoaceticum grow in batch culture and homoferment glucose to acetic acid, the pH of the medium decreases until growth and then acid production cease, at about pH 5. We postulated that the end product of fermentation limits growth by acting as an uncoupling agent. Thus, when the pH of the medium is low, the cytoplasm of the cells becomes acidified below a tolerable pH. We have therefore measured the internal pH of growing cells and compared these values with those of nongrowing cells incubated in the absence of acetic acid. Growing cells maintained an interior about 0.6 pH units more alkaline than the exterior throughout most of batch growth (i.e., ΔpH = 0.6). We also measured the transmembrane electrical potential (ΔΨ), which decreased from 140 mV at pH 7 at the beginning of growth to 80 mV when the medium had reached pH 5. The proton motive force, therefore, was 155 mV at pH 7, decreasing to 120 mV at pH 5. When further fermentation acidified the medium below pH 5, both the ΔpH and the ΔΨ collapsed, indicating that these cells require an internal pH of at least 5.5 to 5.7. Cells harvested from stationary phase and suspended in citrate-phosphate buffer maintained a ΔpH of 1.5 at external pH 5.0. This ΔpH was dissipated by acetic acid (at the concentrations found in the growth medium) and other weak organic acids, as well as by ionophores and inhibitors of glycolysis and of the H⁺-ATPase. Nongrowing cells had a ΔΨ which ranged from about 116 mV at external pH 7 to about 55 mV at external pH 5 and which also was sensitive to ionophores. Since acetic acid, in its un-ionized form, diffuses passively across the cytoplasmic membrane, it effectively renders the membrane permeable to protons. It therefore seems unlikely that mutations at one or a few loci would result in C. thermoaceticum cells significantly more acetic acid tolerant than their parental type.     

Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes.

The generation of transmembrane ion gradients by Oxalobacter formigenes cells metabolizing oxalate was studied. The magnitudes of both the transmembrane electrical potential (delta psi) and the pH gradient (internal alkaline) decreased with increasing external pH; quantitatively, the delta psi was the most important component of the proton motive force. As the extracellular pH of metabolizing cells was increased, intracellular pH increased and remained alkaline relative to the external pH, indicating that O. formigenes possesses a limited capacity to regulate internal pH. The generation of a delta psi by concentrated suspensions of O. formigenes cells was inhibited by the K+ ionophore valinomycin and the protonophore carbonyl cyanide-m-chlorophenylhydrazone, but not by the Na+ ionophore monensin. The H+ ATPase inhibitor N,N'-dicyclohexyl-carbodiimide inhibited oxalate catabolism but did not dissipate the delta psi. The results support the concept that energy from oxalate metabolism by O. formigenes is conserved not as a sodium ion gradient but rather, at least partially, as a transmembrane hydrogen ion gradient produced during the electrogenic exchange of substrate (oxalate) and product (formate) and from internal proton consumption during oxalate decarboxylation.