pH Of Vesicles Research Articles

A Role for Presenilins in Autophagy Revisited: Normal Acidification of Lysosomes in Cells Lacking PSEN1 and PSEN2

Presenilins 1 and 2 (PS1 and PS2) are the catalytic subunits of the γ-secretase complex, and genes encoding mutant PS1 and PS2 variants cause familial forms of Alzheimer's disease. Lee et al. (2010) recently reported that loss of PS1 activity lead to impairments in autophagosomal function as a consequence of lysosomal alkalinization, caused by failed maturation of the proton translocating V0a1 subunit of the vacuolar (H+)-ATPase and targeting to the lysosome. We have reexamined these issues in mammalian cells and in brains of mice lacking PS (PScdko) and have been unable to find evidence that the turnover of autophagic substrates, vesicle pH, V0a1 maturation, or lysosome function is altered compared with wild-type counterparts. Collectively, our studies fail to document a role for presenilins in regulating cellular autophagosomal function. On the other hand, our transcriptome studies of PScdko mouse brains reveal, for the first time, a role for PS in regulating lysosomal biogenesis.

B/Ca in coccoliths and relationship to calcification vesicle pH and dissolved inorganic carbon concentrations

Coccolithophorid algae are microscopic but prolific calcifiers in modern and ancient oceans. When the pH of seawater is modified, as may occur in the future due to ocean acidification, different species and strains of coccolithophorids have exhibited diverse calcification responses in laboratory culture. Since their biomineralization is a completely intracellular process, it is unclear why their response should be affected by extracellular seawater pH. Variations in the B/Ca in coccoliths are potential indicators of pH shifts in the intracellular coccolith vesicle where calcification occurs, because B/Ca in abiogenic calcites increases at higher pH due to the greater abundance of borate ions, the only B species incorporated into calcite. We used a SIMS ion probe to measure B/Ca of coccoliths from three different strains of Emiliania huxleyi and one strain of Coccolithus braarudii braarudii cultured under different seawater pH conditions to ascertain if the B/Ca can be used to elucidate how coccolithophorids respond to changing ocean pH. These data are interpreted with the aid of a conceptual model of cellular boron acquisition by coccolithophorids. Based on uptake in other plants, we infer that boron uptake by coccolithophorid cells is dominated by passive uptake of boric acid across the lipid bilayer. Subsequently, in the alkaline coccolith vesicle (C.V.), boron speciates according to the C.V. pH, and borate is incorporated into the coccolith. At increasing seawater pH, the relative abundance of the neutral boric acid in seawater decreases, lowering the potential B flux into the cell. Homeostasis or constant pH of the coccolith vesicle results in a decrease of the B/Ca in the coccolith with increasing seawater pH. In contrast, if coccolith vesicle pH increases with increasing seawater pH, then the B/Ca will increase as the fraction of borate in the coccolith vesicle increases. The coccolith B/Ca is also expected to depend inversely on the dissolved inorganic carbon (DIC) concentration in the coccolith vesicle. The B/Ca in cultured coccoliths is much lower than that of foraminifera or corals and limits precision in the analysis. Modest variations in DIC or pH of the coccolith vesicle can account for the observed trends in B/Ca in cultured coccoliths. The model shows that paired measurements of B/Ca and B isotopic composition of the calcite could distinguish between regulation of pH or DIC in the coccolith vesicle.

Real‐time gene delivery vector tracking in the endo‐lysosomal pathway of live cells

Using live-cell confocal microscopy and particle tracking technology, the simultaneous transport of intracellular vesicles of the endo-lysosomal pathway and nonviral polyethylenimine (PEI)/DNA nanocomplexes was investigated. Due to potential problems associated with the use of acid-sensitive probes in combination with a gene vector that is hypothesized to buffer the pH of intracellular vesicles, the biological location of PEI/DNA gene vectors was revealed by probing their trafficking in cells expressing fluorescent versions of either early endosome antigen 1, a protein that localizes to early endosomes, or Niemann Pick C1, a protein that localizes to late endosomes and lysosomes. Studies directly show that PEI/DNA nanoparticles are actively transported within both early and late endosomes, and display similar overall transport rates in each. Additionally, gene vector transfer between endosomes is observed. Over time post-transfection, gene vectors accumulate in late endosomes/lysosomes; however, real-time escape of vectors from membrane-bound vesicles is not observed.

Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles

The self-assembly of cationic and anionic amphiphile mixtures into vesicles in aqueous media was studied using two different systems: (i) decanoic acid and trimethyldecylammonium bromide and (ii) hexadecanedioic acid (a simple bola-amphiphile) and trimethyldecylammonium bromide. The resulting vesicles with varying amphiphile ratios were characterized using parameters such as the critical vesicle concentration, pH sensitivity, and encapsulation efficiency. We also produced and observed giant vesicles from these mixtures using the electroformation method and confocal microscopy. The mixed catanionic vesicles were shown to be more stable than those formed by pure fatty acids. Those containing bola-amphiphile even showed the encapsulation of a small hydrophilic solute (8-hydroxypyrene-1,3,6-trisulfonic-acid), suggesting a denser packing of the amphiphiles. Compression and kinetics analysis of monolayers composed of these amphiphiles mixtures at the air/water interface suggests that the stabilization of the structures can be attributed to two main interactions between headgroups, predominantly the formation of hydrogen bonds between protonated and deprotonated acids and the additional electrostatic interactions between ammonium and acid headgroups.

Spectroscopic Microscopy Analysis of the Interior pH of Individual Phospholipid Vesicles

The use of phospholipid vesicles as reaction containers, as vehicles for pharmaceutical drug delivery, and as model systems for cells has prompted the development of new methods for analyzing the structure of vesicles and their contents. The pH of the interior of vesicles is of particular interest when analytes are encapsulated and concentrated with the use of a pH gradient. While the interior pH is generally measured for large populations of vesicles, we report the measurement of the interior pH of individual vesicles as their buffer contents are titrated by transfer of N-methylbutylamine (NMBA) into the vesicle by a pH gradient. The initially acidic buffer within the vesicles is titrated along with a small concentration of an encapsulated pH sensitive dye, 5,6-carboxy SNARF-1-dextran. Images of the indicator fluorescence from each vesicle and its dispersed fluorescence spectrum are recorded using epi-illumination spectral fluorescence microscopy. From a fit of the spectra to the respective acid and base forms of the fluorescent indicator, the interior pH of individual vesicles as a function of the concentration of the NMBA titrant in the external solution could be determined.

Measurements of the Acidification Kinetics of Single SynaptopHluorin Vesicles

Uptake of neurotransmitters into synaptic vesicles is driven by the proton gradient established across the vesicle membrane. The acidification of synaptic vesicles, therefore, is a crucial component of vesicle function. Here we present measurements of acidification rate constants from isolated, single synaptic vesicles. Vesicles were purified from mice expressing a fusion protein termed SynaptopHluorin created by the fusion of VAMP/synaptobrevin to the pH-sensitive super-ecliptic green fluorescent protein. We calibrated SynaptopHluorin fluorescence to determine the relationship between fluorescence intensity and internal vesicle pH, and used these values to measure the rate constant of vesicle acidification. We also measured the effects of ATP, glutamate, and chloride on acidification. We report acidification time constants of 500 ms to 1 s. The rate of acidification increased with increasing extravesicular concentrations of ATP and glutamate. These data provide an upper and a lower bound for vesicle acidification and indicate that vesicle readiness can be regulated by changes in energy and transmitter availability.

Potential cross-talk between (pro)renin receptors and Wnt/frizzled receptors in cardiovascular and renal disorders

The renin-angiotensin system and Wnt/frizzled receptor signaling pathways are important in the development of essential organs, and their aberrant activation results in cardiovascular and renal pathologies. The (pro)renin receptor ((P)RR)-bound (pro)renin is enzymatically active generating angiotensin-II and activating mitogen-activated protein kinases, leading to cell proliferation and to upregulation of profibrotic genes expression, resulting in end-organ damage. The (P)RR does more than bind to (pro)renin, because it is functionally linked to the vacuolar-H(+)-ATPase (v-H(+)-ATPase) that regulates pH of cellular and intracellular vesicles, and to Wnt signaling. This signaling pathway is essential for cell survival, embryonic development and has had a role in various disease states as evidenced by mutation or genetic ablation of the (P)RR gene. This suggests two types of functions of (P)RRs, first one as a receptor for (pro)renin and second one as an accessory subunit of the v-H(+)-ATPase and a cofactor of the Wnt/Fz receptor complex. This review will discuss both of these functions of (P)RRs thereby giving new perspectives as to the roles of (P)RRs in cardiovascular and renal pathologies.

Herpesviruses and Intermediate Filaments: Close Encounters with the Third Type

Intermediate filaments (IF) are essential to maintain cellular and nuclear integrity and shape, to manage organelle distribution and motility, to control the trafficking and pH of intracellular vesicles, to prevent stress-induced cell death, and to support the correct distribution of specific proteins. Because of this, IF are likely to be targeted by a variety of pathogens, and may act in favor or against infection progress. As many IF functions remain to be identified, however, little is currently known about these interactions. Herpesviruses can infect a wide variety of cell types, and are thus bound to encounter the different types of IF expressed in each tissue. The analysis of these interrelationships can yield precious insights into how IF proteins work, and into how viruses have evolved to exploit these functions. These interactions, either known or potential, will be the focus of this review.

Concurrent imaging of synaptic vesicle recycling and calcium dynamics

Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

Intravesicular factors controlling exocytosis in chromaffin cells.

Chromaffin granules are similar organelles to the large dense core vesicles (LDCV) present in many secretory cell types including neurons. LDCV accumulate solutes at high concentrations (catecholamines, 0.5-1 M; ATP, 120-300 mM; or Ca(2+), 40 mM (Bulenda and Gratzl Biochemistry 24:7760-7765, 1985). Solutes seem to aggregate to a condensed matrix to elude osmotic lysis. The affinity of solutes for LDCV matrix is responsible for the delayed release of catecholamines during exocytosis. The aggregation of solutes occurs due to a specific H(+) pump denominated V-ATPase that maintains an inner acidic media (pH ≈5.5). This pH gradient against cytosol is also responsible for the vesicular accumulation of amines and Ca(2+). When this gradient is reduced by modulation of the V-ATPase activity, catecholamines and Ca(2+) are moved toward the cytosol. In addition, some drugs largely accumulate inside LDCV and not only impair the accumulation of natural solutes, but also act as false neurotransmitters when they are co-released with catecholamines. There is much experimental evidence to conclude that the physiological modulation of vesicle pH and the manipulation of intravesicular media with drugs affect the LDCV cargo and change the kinetics of exocytosis. Here, we will present some experimental data demonstrating the participation of drugs in the kinetics of exocytosis through changes in the composition of vesicular media. We also offer a model to explain the regulation of exocytosis by the intravesicular media that conciliate the experimentally obtained data.

Development of pH-responsive fluorescent false neurotransmitters.

We introduce pH-responsive fluorescent false neurotransmitters (pH-responsive FFNs) as novel probes that act as vesicular monoamine transporter (VMAT) substrates and ratiometric fluorescent pH sensors. The development of these agents was achieved by systematic molecular design that integrated several structural elements, including the aminoethyl group (VMAT recognition), halogenated hydroxy-coumarin core (ratiometric optical pH sensing in the desired pH range), and N- or C-alkylation (modulation of lipophilicity). Of 14 compounds that were synthesized, the probe Mini202 was selected based on the highest uptake in VMAT2-transfected HEK cells and desirable optical properties. Using Mini202, we measured the pH of catecholamine secretory vesicles in PC-12 cells (pH approximately 5.9) via two-photon fluorescence microscopy. Incubation with methamphetamine led to an increase in vesicular pH (pH approximately 6.4), consistent with a proposed mechanism of action of this psychostimulant, and eventually to redistribution of vesicular content (including Mini202) from vesicles to cytoplasm. Mini202 is sufficiently bright, photostable, and suitable for two-photon microscopy. This probe will enable fundamental neuroscience and neuroendocrine research as well as drug screening efforts.

Real-Time Measurement of Endosomal Acidification by a Novel Genetically Encoded Biosensor

Genetically encoded fluorescent proteins are optimal reporters when used to monitor cellular processes as they can be targeted to any subcellular region by fusion to a protein of interest. Here, we present the pH-sensitive fluorescent protein E1GFP which is ideally suited to monitor pH changes in dynamic intracellular structures in real time with high spatio temporal resolution. E1GFP is a ratiometric pH indicator by emission with a pK close to 6.0. We describe an application of this novel pH reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E1GFP to the HIV-Tat protein which is endowed with cell-penetrating properties, we were able to monitor multi-step endocytosis. It is well established that Tat protein enters T-cells using clathrin-coated pits and progressively translocates into well-characterized low-pH endosomes. By using E1GFP conjugated to Tat we monitored the changes in vesicle pH, starting from the initial cell-surface value through the intracellular endocytic network, in real time. We demonstrate a progressive acidification within Tat-loaded vesicles that is crucial to the intracellular fate of Tat. The low endosomal pH exploited by Tat-E1GFP after prolonged incubation in live cells, leads to protein degradation in the lysosomal compartment. We also show that treatment with chloroquine raises the vacuolar pH, demonstrating the ability of E1GFP to respond effectively to environmental pH changes. We believe that this implementation illustrates the suitability of E1GFP for use in the surveillance of intracellular pH during dynamic cellular events with high spatio-temporal resolution.

Simultaneous Optical-Electrical Measurement of the Delay between formation of the Fusion Pore and Proton Equilibration in Exocytosis of Single Vesicles

Optical detetction of neutralization of pH in granules or vesicles is often used to define exocytotic events. However, combined measurents of ensemble capacitance and pH-dependent vesicular fluorescence changes have suggested that the movement of protons only becomes possible after fusion pore expansion with a mean delay of > 300 ms (1). To enhance the temporal resolution of such measurements, we have combined capacitance recordings of single vesicle fusion in RBL cells transfected with synapthpHluorin as a reporter of vesicular pH. To monitor cell capacitance steps due to exocytosis of single granules in whole cell patch-clamp mode, we used the piecewise linear technique. Internal solution contained 10 μM free Ca2+ and 300 μM GTPγS.View Large Image | View Hi-Res Image | Download PowerPoint Slide Before establishing whole cell recordings, punctate fluorescence signals could be detected with excitation at 460 nm, while during perfusion with internal solution and excitation at 480 nm, punctate fluorescence signals gradually appeared at corresponding sites. Fluorescence increases clearly lagged capacitance steps by several 100 ms- seconds, supporting the idea that pH equilibration through the fusion pore is delayed.(1) Barg et al.: Neuron, 33, 287-299, 2002.

Forward transport of the receptor P63 is necessary for secretagogue‐enhanced surfactant protein‐A (SP‐A) binding to type II pneumocytes

SP‐A plays an important role in surfactant lipid turnover in the lung. P63 (CKAP4) located on both the plasma membrane (PM) and ER of type II pneumocytes has been identified as an SP‐A receptor. Interference with specific calcium (Ca++)‐dependent binding of SP‐A to type II cells occurs in competition studies using anti‐P63 pAb and upon treatment with siRNA to reduce P63 protein levels. A 2.5‐fold enhancement of 125I‐SP‐A Ca++‐dependent binding to type II cells plated on microporous membranes was achieved by exposure of the cells to the secretagogue cAMP (0.1mM), without effect on non‐specific (Ca++‐independent) SP‐A cell association. Anti‐P63 Ab blocked the cAMP‐augmented specific binding of SP‐A, evidence for the importance of P63 in this process. Inhibitors of receptor recycling were utilized to evaluate whether the forward transport of the ER‐resident P63 to the PM was necessary for receptor function. Monensin, a carboxylic ionophore, increases the pH of intracellular vesicles and brefeldin A (BFA) blocks formation of transport vesicles. In the presence of monensin (2uM) or BFA (10ug/ml), cAMP‐augmentation of the specific binding of SP‐A was abolished. Thus, secretagogue stimulation of SP‐A binding required the movement of P63 protein from the ER to the PM. Our results provide further evidence for the role of P63 as a SP‐A receptor protein on lung type II cells. [HL 19737]

On the role of intravesicular calcium in the motion and exocytosis of secretory organelles

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient towards the cytosol (5.5 vs. 7.2) promoted by the V-ATPase activity. This gradient of pH is mainly responsible for the accumulation of amines. The secretory vesicles contain large amounts of total Ca2+, but the free intragranular [Ca2+], the mechanisms for Ca2+ uptake and release from the granules and their physiological relevance regarding exocytosis are still matters of debate.We have recently shown that disruption of the pH gradient of secretory vesicles slowed down exocytosis. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that the V-ATPase inhibitor bafilomycin A1 directly released Ca2+ from freshly isolated vesicles. Accordingly, vesicle alkalinization released Ca2+ from the granules to the cytosol, measured with fura-2 in intact chromaffin cells. Using TIRFM in cells over-expressing the EGFP-labeled synaptobrevin (VAMP2-EGFP) protein, we have then shown that the Ca2+ released from the vesicles to the cytosol in the presence of bafilomycin, dramatically increased the granule motion of chromaffin- or PC12-derived granules, and triggered exocytosis (measured by amperometry).We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of the local cytosolic Ca2+ around the vesicles and in two of the major functions of secretory cells, vesicle motion and exocytosis.1

Low-pH Triggering of Human Metapneumovirus Fusion: Essential Residues and Importance in Entry

Human metapneumovirus (HMPV) is a significant respiratory pathogen classified in the Pneumovirinae subfamily of the paramyxovirus family. Recently, we demonstrated that HMPV F protein-promoted cell-cell fusion is stimulated by exposure to low pH, in contrast to what is observed for other paramyxovirus F proteins. In the present study, we examined the potential role of histidine protonation in HMPV F fusion and investigated the role of low pH in HMPV viral entry. Mutagenesis of the three ectodomain histidine residues of the HMPV F protein demonstrated that the mutation of a histidine in the heptad repeat B linker domain (H435) ablated fusion activity without altering cell surface expression or proteolytic processing significantly. Modeling of the HMPV F protein revealed several basic residues surrounding this histidine residue, and the mutation of these residues also reduced fusion activity. These results suggest that electrostatic repulsion in the heptad repeat B linker region may contribute to the triggering of HMPV F. In addition, we examined the effect of inhibitors of endosomal acidification or endocytosis on the entry of a recombinant green fluorescent protein-expressing HMPV. Interestingly, chemicals that raise the pH of endocytic vesicles resulted in a 30 to 50% decrease in HMPV infection, while the inhibitors of endocytosis reduced infection by as much as 90%. These data suggest that HMPV utilizes an endocytic entry mechanism, in contrast to what has been hypothesized for most paramyxoviruses. In addition, our results indicate that HMPV uses the low pH of the endocytic pathway to enhance infectivity, though the role of low pH likely differs from classically described mechanisms.

Secretory vesicle rebound hyperacidification and increased quantal size resulting from prolonged methamphetamine exposure

Acute exposure to amphetamines (AMPHs) collapses secretory vesicle pH gradients, which increases cytosolic catecholamine levels while decreasing the quantal size of catecholamine release during fusion events. AMPH and methamphetamine (METH), however, are retained in tissues over long durations. We used optical and electron microscopic probes to measure the effects of long-term METH exposure on secretory vesicle pH, and amperometry and intracellular patch electrochemistry to observe the effects on neurosecretion and cytosolic catecholamines in cultured rat chromaffin cells. In contrast to acute METH effects, exposure to the drug for 6-48 h at 10 microM and higher concentrations produced a concentration-dependent rebound hyperacidification of secretory vesicles. At 5-10 microM levels, prolonged METH increased the quantal size and reinstated exocytotic catecholamine release, although very high (> 100 microM) levels of the drug, while continuing to produce rebound hyperacidification, did not increase quantal size. Secretory vesicle rebound hyperacidification was temperature dependent with optimal response at approximately 37 degrees C, was not blocked by the transcription inhibitor, puromycin, and appears to be a general compensatory response to prolonged exposure with membranophilic weak bases, including AMPHs, methylphenidate, cocaine, and ammonia. Thus, under some conditions of prolonged exposure, AMPHs and other weak bases can enhance, rather than deplete, the vesicular release of catecholamines via a compensatory response resulting in vesicle acidification.

A comparison between the responses of neutral red and acridine orange: Acridine orange should be preferential and alternative to neutral red as a dye for the monitoring of contaminants by means of biological sensors

The acridine orange (AO) and neutral red (NR) dyes, commonly used as probes to measure the internal pH in acidic vesicles, are compared in this article. The comparison between the two dyes (arising from calculations taking into account their analytical constants) illustrated that the use of AO is preferential to that of NR because the AO response is sensitive over the whole pH range between 4.0 and 7.4, whereas the NR response is effective only between pHs 4.0 and 6.0. In addition, it became evident from the mitochondrial respiration response that NR, unlike AO, is a protonophore. When taken into consideration, these two properties suggest that AO is more suitable than NR as an indicator of toxicity measurements in water samples because the environmental toxic compounds induce pH changes in the acidic vesicles of biological structures that are used as environmental biosensors.

Intravesicular Calcium Release Mediates the Motion and Exocytosis of Secretory Organelles: A STUDY WITH ADRENAL CHROMAFFIN CELLS

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.

Mechanisms by which amphetamine redistributes dopamine out of vesicles: A computational study

Computer simulations of dopamine (DA) and amphetamine interactions associated with dopaminergic storage vesicles were developed in order to better explain how amphetamine causes redistribution of DA out of the vesicles. In the model, DA can be transported into vesicles via the vesicular monoamine transporter. Amphetamine competitively inhibits DA uptake either as a substrate for the transporter or by interference with DA binding to the transporter. Both of the amines can passively diffuse across the membrane in both directions, but only the neutral species can cross the membrane in this manner. The abundance of neutral and positive moieties of the amines is governed by the Henderson-Hasselbalch equation. The model reproduces experimentally observed steady-state DA levels in vesicles, vesicles emptying faster after a change of pH inside the vesicle than after a change in access of DA for the vesicular monoamine transporter, and the impact of amphetamine on DA uptake and efflux in a variety of experimental paradigms. The simulations indicate that a small percentage of DA is constantly diffusing out of vesicles and is replaced by DA entering the vesicle by the vesicular monoamine transporter. Low doses of amphetamine cause DA redistribution out of vesicles primarily by inhibiting DA storage while an enhancement of efflux rates as a result of a change in vesicle pH is added at higher concentrations of amphetamine.