Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin

We have used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F-alpha 2M for 30 min at 37 degrees C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F-alpha 2M for 15 min at 37 degrees C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F-alpha 2M microenvironment was 5.0 +/- 0.2. Using colloidal gold-alpha 2M for electron microscopic localizations we have verified that, under these conditions, alpha 2M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for 1/2 hr, we obtained a pH of 5.0 +/- 0.2 for the F-alpha 2M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 +/- 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.     

Increased binding of liposomes to cells by electric treatment.

The influence of electric field treatments on the interaction of large unilamellar vesicles (liposomes) with animal cells was monitored by the fluorescence assay based on the use of the liposomes loaded by a dye 1-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). It was shown that application of a short electric pulse (100 microseconds of 3-4 kV/cm) to the suspension of cells in presence of vesicles resulted in significant (more than 2 times) increase of the fluorescence associated with cells. The pH-sensitivity of the excitation spectrum of the dye and its interaction with the quencher were used to determine the nature of the phenomenon as the increase of the liposome binding onto the cell surface but not the consequence of a promotion of liposome uptake into the cells by endocytosis. The higher affinity for the liposome caused by the electric field has a lifetime of several minutes. The possible relation of the effect described to the electroporation of cell membranes and to macroscopic changes in membrane structure is discussed.     

Uptake of a natural surfactant and increased delivery of small organic anions into type II pneumocytes

The uptake of natural lung surfactant into differentiated type II cells may be used for the targeted delivery of other molecules. The fluorescent anion pyranine [hydroxypyren-1,3,6-trisulfonic acid, sodium salt (HPTS)] was incorporated into a bovine surfactant labeled with [3H]dipalmitoylphosphatidylcholine ([3H]DPPC). The uptake of [3H]DPPC and of HPTS increased with time of incubation and concentration, decreased with the size of the vesicles used, and was stimulated by 8-bromo-cAMP and partially inhibited by hypertonic sucrose. However, the amount of HPTS uptake was approximately 100 times smaller than that of [3H]DPPC. This large difference was due to a more rapid regurgitation of some of the HPTS from the cells but not to leakage from the surfactant before uptake. The acidification of the internalized surfactant increased linearly over 90 min to 7.13, and after 24 h, a pH of 6.83 was measured. In conclusion, after internalization of a double-labeled natural surfactant, the lipid moieties were accumulated in relation to the anions, which were targeted to a compartment not very acidic and in part rapidly expelled from the cells.     

Combined strategies for liposome characterization during in vitro digestion

Three types of pyranine (HPTS)-containing liposomes were prepared by high-pressure homogenization under optimized conditions. At 37°C, they were 1) fluid-state vesicles made from soybean phosphatidylcholine (SPC), 2) gel-state liposomes made from hydrogenated SPC (HSPC), and 3) solid-disordered membranes obtained from HSPC and cholesterol (HSPC-Chol). These liposome formulations were characterized before, during, and after in vitro digestion, which involved the presence of pH gradients, enzymes, and bile salts. Mean sizes and size distributions of the vesicles were determined by DLS; ³¹P-NMR (nuclear magnetic resonance) was used to quantify lyso-PC forms; internal pH was monitored throughout digestion with two different fluorescent pH probes; and changes in bilayer permeability and HPTS encapsulation were determined by size-exclusion chromatography and fluorimetry. Differential scanning calorimetry analysis was also performed in order to study the effect of digestion on HSPC vesicles. SPC liposomes were physically stable during digestion; they presented 8% lyso-forms and an HPTS encapsulation around 85% after in vitro digestion. However, they were extremely permeable to ions, so that the internal pH immediately equilibrated with the bulk pH. HSPC liposomes were the most affected by the digestive process. Even though they were chemically stable, as inferred from the low lyso-PC content, very important changes in their size distribution were observed. A final 50% HPTS leakage was quantified after in vitro digestion. Nevertheless, they were the least permeable to protons under pH gradients. HSPC-Chol vesicles presented intermediate permeability to protons, having their internal pH decreased from approximately 6.8 to 4.6 after 1 hour of incubation at pH 2. This was the most chemically stable formulation and showed the highest encapsulation, even after in vitro digestion. Therefore, HSPC-Chol liposomes would be the most adequate choice for the design of lipid products for oral administration.     

pH-Sensitive Liposomes

Abstract

pH sensitive liposomes are lipid compositions that can be destabilized when the external pH is changed; usually from a neutral or slightly alkaline pH to an acidic pH. They are designed to circumvent delivery of liposome contents to the lysosomes of cells following internalization of the vesicle via the endocytic pathway. In the majority of compositions, a lipid containing a pH titratable group is mixed with phosphatidylethanolamine containing unsaturated acyl chains in a molar ratio (pH sensitive component/PE) of 1/4 or greater. There are five major groups of phosphatidylethanolamine containing pH-senstive lipid compositions. These can be classified by their acid-titratable component: phospholipids, acylated amino acids, fatty acids, cholesterol derivatives and miscellaneous double chain amphiphiles. The biophysical mechanism of action involves a transition of the lipids from the lamellar phase to the hexagonal phase. In cell culture, pH sensitive vesicles can increase the delivery of fluorescent markers, proteins, cytotoxic compounds, RNA and DNA into the cytoplasm. The mechanism of delivery is suggested to involve the destabilization of the liposome in the endosome as the pH is reduced from 7.4 to 5.0 and subsequent destabilization of, or fusion with, the endosomal membrane; some of the liposome contents are introduced into the cytoplasm. In most cases, the extent of liposome contents delivery into the cytoplasm is less than 1% of the amount that becomes cell associated. However further studies, with more reliable assays to differentiate cytoplasmic from lysosomal delivery, are required to place an exact value on this efficiency. The efficiency of pH sensitive liposomes in vivo is limited by stability of certain of the liposome compositions in serum and targeting to the appropriate cell. Cholesterol hemisuccinate is a particularly attractive component for in vivo use since it stabilizes the liposome when in serum at pH 7.4. The use of pH sensitive liposomes in drug delivery should continue to expand due to the increasing number of macromolecular therapeutic agents with intracellular targets.     

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported¹. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases². Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells³. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis^3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.     

Visible light-induced destabilization of endocytosed liposomes

The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near infrared light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by associating selected amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), or the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer. The DiI sensitization of the polymerization of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes caused liposome destabilization with release of encapsulated aqueous markers. In separate experiments, similar photosensitive liposomes were endocytosed by cultured HeLa cells. Exposure of the cells and liposomes to 550 nm light caused a net movement of the liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) from low pH compartment(s) to higher pH compartment(s). This suggests that photolysis of DiI-labelled liposomes results in delivery of the contents of the endocytosed liposomes to the cytoplasm. The release of HPTS into the cytoplasm appears to require the photoactivated fusion of the labelled liposomes with the endosomal membrane. These studies aid in the design of visible light sensitive liposomes for the delivery of liposome-encapsulated reagents to the cytoplasm.     

pH-sensitive liposomes mediate cytoplasmic delivery of encapsulated macromolecules

Negatively charged liposomes are endocytosed by the coated vesicle system and accumulate in acidic intracellular vesicles. Liposomes that become unstable at acidic pH improve cytoplasmic delivery of membrane-impermeant macromolecules such as calcein (CAL) and FITC dextran (18 or 40 kDa). Oleic acid (OA): phosphatidylethanolamine (PE) (3:7 mole ratio) liposomes become permeable to CAL at pH less than 7.0. Control liposomes of phosphatidylserine:PE or OA:phosphatidylcholine are stable at pH 4-8. OA:PE liposomes promote cytoplasmic delivery of encapsulated CAL to CV-1 cells, as evidenced by the emergence of diffuse, cytoplasmic CAL fluorescence. Delivery requires metabolic energy and is partially inhibited by chloroquine or monensin, which raise the pH of intracellular vesicles.     

Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.     

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.     

The Effects of pH and Intraliposomal Buffer Strength on the Rate of Liposome Content Release and Intracellular Drug Delivery

Targeted liposomal drug formulations may enter cells by receptor-mediated endocytosis and then traffick by membrane flow into acidic intracellular compartments. In order to understand the impact of these intracellular pH changes on liposomal drug unloading, the effect of pH on the release from folate-targeted liposomes of three model compounds with distinct pH dependencies was examined. 5(6)-carboxyfluorescein, which titrates from its anionic to uncharged form following internalization by KB cells, displays strong endocytosis-dependent release, since only its uncharged (endosomal) form is membrane permeable. Endocytosis-triggered unloading of drugs of this sort is enhanced by encapsulating the drug in a weak buffer at neutral pH, so that acidification of the intraliposomal compartment following cellular uptake can occur rapidly. Sulforhodamine B, in contrast, retains both anionic and cationic charges at endosomal pH (~pH 5), and consequently, escapes the endosomes only very slowly. Doxorubicin, which is commonly loaded into liposomes in its membrane-impermeable (cationic) form using an acidic buffer, still displays endocytosis-triggered unloading, since sufficient uncharged doxorubicin remains at endosomal pHs to allow rapid re-equilibration of the drug according to the new proton gradient across the membrane. In this case, when the extraliposomal [H⁺] increases 250-fold from 4 × 10^–8 M (pH 7.4, outside the cell) to 10^–5 M (pH 5, inside the endosome), the ratio of doxorubicin inside to outside the liposome must decrease by a factor of 250. Therefore, the collapse of the transliposomal pH gradient indirectly drives an efflux of the drug molecule from the liposome. Since a change in intraliposomal pH is not required to unload drugs of this type, the intraliposomal compartment can be buffered strongly at acidic pH to prevent premature release of the drug outside the cell. In summary, pH triggered release of liposome-encapsulated drugs can be achieved both with drugs that increase as well as decrease their membrane permeabilities upon acidification, as long as the intraliposomal buffer strength and pH is rationally selected.     

Liposome immunoblotting assay using a substrate-forming precipitate inside immunoliposomes

A new immunoblotting assay which uses antibody-coupled liposomes containing horseradish peroxidase is proposed. A substrate 4-chloro-1-naphthol permeated through the phospholipid membrane of the antibody-coupled liposomes and formed a colored product precipitating inside the liposomes. The precipitates accumulated in the liposomes and could be detected at the positions where the liposomes coupled with a target in blotted samples. Combination of liposomes with average diameter of 350 nm and a PVDF membrane with a pore size of 450 nm, 0.02 ng of IgM was detected, while the conventional immunoblotting using antibody-HRP conjugates detected 2 ng of IgM. The sensitivity increased about two orders of magnitude by the liposome immunoblotting assay. This liposome immunoblotting assay gives a simple detection method of proteins with a high sensitivity, as well as a high sensitivity Western blotting assay.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

Fractionation of endocytic vesicles and glucose-transporter-containing vesicles in rat adipocytes.

We subfractionated intracellular vesicles from rat adipocytes in order to examine the subcellular distribution of endocytic vesicles or endosomes with respect to insulin-regulatable glucose-transporter (GT)-containing vesicles [James, Lederman & Pilch (1987) J. Biol. Chem. 262, 11817-11824]. Vesicles mediating fluid-phase endocytosis sedimented as a single major peak of greater density than the single distinct peak of GT-containing vesicles. This difference was also apparent during cellular insulin exposure and after insulin removal. Endocytosis of insulin and IGF (insulin-like growth factor) II was also examined. In sucrose gradients, IGF II-containing vesicles were less dense than those containing internalized insulin. Receptor-mediated endocytic vesicles were distinct from fluid-phase endocytic vesicles, but overlapped with the GT-containing vesicles. Vesicles containing internalized ligand were further fractionated by agarose-gel electrophoresis after various times of internalization. At least three different vesicle subpopulations containing the iodinated ligands were resolved after 5 min of internalization. Endocytic vesicles containing rapidly internalized insulin (1.5 min at 37 degrees C) consistently co-migrated with GT-containing vesicles. These data indicate that fluid-phase and receptor-mediated endocytosis occur via different pathways in adipocytes. Furthermore, whereas the intracellular GT-containing vesicles are distinct from fluid-phase vesicles, a rapidly labelled pool of insulin-containing vesicles consistently co-fractionated with GT-containing vesicles when separation techniques based on size, density and charge were used. This suggests that the insulin receptor may directly interact with the intracellular GT-containing vesicles after insulin-induced endocytosis.     

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblast insulin receptors. These cells bind and internalize insulin normally. Biochemical assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 degrees C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 degrees C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The total number of complexes reached a maximum by 5 min and decreased rapidly thereafter with a t 1/2 of approximately 10 min. There was a distinct delay in the appearance, rate of rise, and peak of intracellular free and degraded insulin. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored, based on the ability of dissociated insulin to rebind to receptor upon neutralization of acidic intracellular vesicles with monensin. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.     

Ligand/receptor internalization: a kinetic, flow cytometric analysis of the internalization of N-formyl peptides by human neutrophils

Fluorescence flow cytometry was used to measure the internalization of the fluorescent ligand N-formyl-nle-leu-phe-nle-tyr-lys-fluorescein by human neutrophils. The internalization process was monitored by the accessibility of the receptor-bound fluorescent ligand to quenching following a change in the pH of the extracellular medium from 7.4 to 3.0. In such a pH change, extracellular ligand or fluorescein are quenched immediately (excitation 488 nm). In contrast, intracellular fluorescein (derived from fluorescein diacetate) or intracellular ligand are quenched with half-times of approximately 20 or approximately 40 sec, respectively, at 37 degrees C. The fraction of internalized ligand is calculated by resolving the fast and slow components of the quenching process. Temporal resolution of the internalization process in this system depends upon two factors. We have previously shown that it is possible to examine essentially continuously the kinetics of ligand binding in the nM concentration range without removing the free ligand (Sklar LA, Finney DA, Cytometry 3:161, 1982). We have now modified a Becton Dickinson FACS IV sample head assembly to permit direct addition of reagents into the cell suspension while on-line. This enables us to change the suspension pH and evaluate internalization with a time resolution of a few seconds. We observe that internalized ligand can be detected within 1 min and that the rate is proportional to the number of receptors occupied. The rate is essentially linear over the first few minutes and approximately 60% of the receptor-bound ligand is internalized after 3 min.     

The role of pH in the intracellular acetylcholine receptor ligand processing

Previous studies have shown that the internalized AChRs are transported through many vesicular compartments: Golgi associated vesicles, coated vesicles, smooth vesicles, endosome-like structures and lysosomes. These compartments have an acidic pH ranging from 4.5 to 6.5. The pH differences between organelles suggests that these differences may influence the sorting and final expression of AChRs. To test this hypothesis, we measured the number of counts of 125I-alpha BTX or 125I-Mab35 dissociated from myotube membranes containing AChRs as a function of pH. Neither the 125I-alpha BTX nor 125I-Mab35 showed an enhanced dissociation in the pH range 4.0-7.0, whereas lowering the pH to 6.0 or below enhanced the dissociation of 125I-alpha 2-macroglobulin from myotubes. In other experiments using Torpedo membrane we showed that neither 125I-alpha BTX nor 125I-Mab35 appreciably dissociated from the AChR unless the pH was less than 4 or above 11. Double-label studies using a novel membrane permeable acidotropic molecule DAMP (3-(2,4 nitroanilino) 3'amino-N-methyl-dipropylamine), facilitated mapping the pH of the intracellular compartments containing internalized AChRs. This molecule accumulates inside acidic compartments in the cell and has a dinitrophenol (DNP) group recognized by DNP specific antibodies. Cells were treated with 30 micrograms DAMP for 30 min and allowed to internalize Mab35-gold (15 nm) for various periods (0-15 h). At each time point we fixed and washed the cells, and incubated with anti-DNP monoclonal antibodies followed by incubation with anti-mouse IgG and protein A colloidal gold (5 nm). Different sized gold particles allowed us to simultaneously identify the AChR compartments and estimate their pH. Sister cultures were exposed to acidotropic drugs to destroy pH gradients. Under those conditions, AChR delivery to lysosomes was blocked. Our studies show that AChRs are transported through acidic compartments ranging from pH 4.5 to 6.5 and in contrast to other ligands they do not dissociate from the intracellular membranes at low pH.     

Encapsulation of Living Leishmania Promastigotes in Artificial Lipid Vacuoles

After phagocytosis by mammalian macrophages, promastigote forms of Leishmania parasites settle inside intracellular parasitophorous vacuoles (PVs) in which they transform into amastigote forms and replicate. Here, using a variant of the ‘inverted emulsion’ method, we succeeded in encapsulating living L. amazonensis parasites in giant artificial liposomes that serve as model PVs. We were able to control the size of liposomes, the pH and the composition of their internal volume, and the number of internalized parasites per liposome. L. amazonensis promastigotes encapsulated in liposomes filled with RPMI-Dextran solution at pH 7.5 or 6.5 survived up to 96 h at 24°C. At 37°C and pH 5.5, parasites survived 48h. This method paves the way to identifying certain effectors secreted by the parasite and to unraveling specific mechanisms of fusion between the PV and intracellular vesicles of the host cell. This method will also facilitate the study of the temporal evolution of biophysical properties of the PV during its maturation.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.