Membrane potentials of BHK (baby hamster kidney) cell line: ionic and metabolic determinants

The transmembrane potential of cells from a continuous cell line (BHK-21) has been investigated by a combination of electrophysiological and flame photometric techniques. The ratio of sodium permeability to potassium permeability (P_Na/P_K) determined from membrane potentials recorded at varying external potassium concentrations was 0.082; from membrane potential measurements and the intracellular sodium and potassium concentrations of cells in 6.8 mM K⁺ media the value was 0.075. The P_Na/P_K ratio was not temperature dependent. Dinitrophenol (1 mM) did not significantly alter the membrane potential of cells incubated for one hour with the inhibitor. However, iodoacetate (1 mM) and sodium fluoride (30 mM) caused a significant depolarization during a one-hour incubation. Measurements of sodium and potassium concentrations during incubation at 4°C showed a decrease in internal potassium and an increase in internal sodium accompanied by a decreased membrane potential. Ion concentrations and membrane potentials were measured in cells recovering at 37°C following 24 hours at 4°C. Membrane potentials in excess of E_K during the first ten minutes of recovery may indicate electrogenic pumping.     

The use of hyperosmolar,intracellular-like solutions for the isolation of epithelial cells from guinea-pig small intestine

Isolated small intestinal epithelial cells were prepared by using either (a) hyperosmolar, low sodium, high potassium containing (intracellular-like) solutions, or (b) isoosmolar, high sodium, low potassium containing (extracellular-like) solutions. Both (a) and (b) cells show high viability as estimated by Trypan blue exclusion, oxygen consumption, cellular ATP content, lactate-dehydrogenase liberation, intracellular ion concentrations and significant Na⁺-dependent alanine and uridine uptakes. Although (a) and (b) cells show in the cold similar ion concentration, after reincubation at 37° C for 30 min (a) cells show intracellular ion concentrations of 31 mM Na, 129 mM K and 88 mM Cl, whilst (b) cells have 71 mM Na, 93 mM K and 102 mM Cl. Cells prepared with (a) concentrate much more alanine and uridine than cells prepared with (b), probably because the latter have a lower Na⁺ gradient across the plasma membrane. Cells prepared with intracellular-like solutions would be an ideal system to study Na⁺-dependent transport mechanisms and the regulatory systems of intracellular ion concentrations.     

Effects of neurotoxins on pancreatic islets: Elucidation of a possible role for sodium channels in the secretory response

The neurotoxins veratridine and Leiurus toxin were used to characterize the nature of the sodium channel in the pancreatic β-cell membrane in relation to metabolc and secretory events. Insulin release and glycolytic flux were measured on batch-incubated rat islets. Veratridine, 200 μM, but not 10 μM, elicited a secretory response in the presence of 5.6 mM (basal) glucose, but did not influence the response to 15.3 mM glucose. Leiurus toxin, 20 nM, together with basal glucose and 10 μM veratridine induced insulin release, although Leiurus toxin, alone, was not effective. The secretory responses to the neurotoxins, but not 15.3 mM glucose, were blocked by tetrodotoxin. Glucose utilization was enhanced by 200 μM veratridine in the presence of basal glucose. Leiurus toxin at 20 nM increased the glycolytic rate which was further enhanced by the addition of 10 μM veratridine. The increments in glycolytic flux were partially or completely blocked by tetrodotoxin. Ouabain, 1.0 mM, had no effect on the secretory response to veratridine, but completely blocked the veratridine-induced increase in glycolytic flux. These observations indicate that the sodium channels in the β-cell membrane are pharmacologically similar to those in neuronal plasma membranes. Furthermore, the secretory response elicited by neurotoxins may occur independently of an increase in glycolytic flux. The major role of glycolytic flux may be to provide energy for extrusion of sodium from the β-cell.     

Sodium and potassium content and membrane transport properties in red blood cells from newborn puppies

The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.     

Action potential of the Garfish non-myelinated olfactory nerve.

The interesting Garfish olfactory nerve has been described by D. Easton (6). His structural analysis of the preparation shows homogeneous, uniform diametered axons measuring about 0.24 microns. The present study is an attempt to characterize its electrical and ionic property. The duration of the extracellularly measured action potential is about 60 msec at 20°C and increases to about 500 msec at 5°C, a time dispersion of about 8X. The conduction velocity varies from 20 cm/sec at 20°C to 4 cm/sec at 0°C. Recordings from small isolated fiber bundles have been shown to be viable. Reduction of sodium concentration to 5 mM in the external medium annihilates the A.P. response. Increase in external potassium concentration to about 16 mM makes the nerve inexcitable.     

Regulation of ooplasmic sodium and potassium activities in developing locust eggs

Ooplasmic activities of potassium and sodium were measured with ion sensitive microelectrodes before and during the period of maximal water uptake which occurs 3–5 days after oviposition for eggs incubated at 37°C. Potassium activity increased from 84 mM in eggs before fertilization at 118 mM in eggs 1 day after fertilization (d1). Sodium activity increased from 8 mM to 29 mM over the same period. These changes exceeded those predicted from the decrease in water content (8%) during the first day after oviposition. Between d1 and d3, potassium and sodium activities decreased to values predicted on the basis of the 88% increase in egg water content. Although water content increased an additional 46% between d3 and d5, ooplasmic sodium activity remained constant at 11 mM and potassium activity increased from 64 mM to 74 mM during this time. Declines in concentrations of sodium and potassium measured in whole eggs by atomic absorption spectrometry mirrored the increase in egg water content. The results suggest that regulation of ooplasmic sodium and potassium activities is accomplished by release of these ions from internal stores, possibly the york spheres. © 1992 Wiley-Liss, Inc.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

Differential effects of veratridine and calcium on the release of [3H]noradrenaline and [14C]α-aminoisobutyrate from rat brain cortex slices

The efflux of [³H]noradrenaline (NA) and of the non transmitter, non metabolizable, amino acid [¹⁴C]α-aminoisobutyrate (AIB), was followed simultaneously from superfused rat brain cortex thin slices, that had been preloaded with those substances. Short (2 min) “pulses” of increasing veratridine concentrations were applied at 10 min intervals. When calcium in the superfusion fluid was 1 mM, [³H]NA efflux increased progressively with pulses of 1, 3, 10 and 30 μM veratridine, but further increase to 100 μM resulted in a decrease of the induced ³H-efflux. Veratridine-enhanced [³H]NA efflux decreased considerably in 0.1 mM calcium and was virtually suppressed when no calcium was added to the superfusion fluid. In 1 mM calcium, the efflux of [¹⁴C] AIB was increased progressively by pulses of 10, 30 and 100 μM veratridine, but no increase in efflux was seen with 1 or 3 μM drug. In 0.1 mM, or without added calcium, the induced efflux of [¹⁴C]AIB was markedly increased. Similar findings were seen when a long (10 min) pulse of 10 μM veratridine was given. After such long pulses there was a rapid return of AIB efflux to pre-veratridine levels if calcium was 1 mM, but in the absence of added calcium, the return to baseline levels of both [³H]NA and, especially, that of [¹⁴C]AIB efflux, was greatly impaired. The veratridine enhanced efflux of both NA and AIB was entirely blocked by 1 μM tetrodotoxin.     

Properties of single potassium channels in guinea pig hepatocytes

The patch-clamp technique of cell-attached and inside-out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K⁺ concentrations. A linear single-channel current-voltage relationship was obtained at the voltage range of -80 to -20 mV with slope conductance of 70 ± 6 pS (n = 10). Under symmetrical high K⁺ concentration of 148 mM in the cell-attached patch membrane, the I-V curve exhibited a mild inward rectification at potentials positive to +20 mV. The values of reversal potential was +5 ± 2 mV (n = 10). When the external potassium concentration ([K⁺]₀) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 ± 2 pS (n = 4) and 24 ± 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K⁺]₀, indicating that this channel was highly selective for K⁺. Open probabilities (P₀) of the channel were 73-93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K⁺ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34-36°C. P₀ in inside-out patches was not changed by application of 1 μM Ca²⁺ nor 1 mM Mg²⁺ to the internal side of patch membranes. It is concluded that a novel type of the K⁺ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca²⁺]_i, from those in other species. © 1994 Wiley-Liss, Inc.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

The influence of phosphate,fluoride and potassium ions on the activity of amp-deaminase from human skeletal muscle

• 1.1. Kinetic properties of the inhibitory effect of inorganic phosphate and fluoride and of the activatory effect of potassium ion on human skeletal muscle AMP-deaminase (E.C. 3.5.4.6) have been investigated
• 2.2. It has been shown that phosphate is a competitive inhibitor (K₁, ≈0.8 × 10⁻³M) and fluoride a noncompetitive inhibitor (K₁≈3.2 × 10⁻³ M) of human muscle AMP-deaminase.
• 3.3. The changes of potassium ion concentration between 20 and 200 mM did not influence the Michaelis constant which was about 0.9 x 10⁻³ M at 30°C. Also the change of substrate concentration in the range 40–300 μM did not influence the activation constant for potassium (K_a≈0.4 × 10⁻¹ M).
• 4.4. Higher concentraion of potassium (200mM) was found to diminish the “temperature sensitivity” of the enzyme activity.
• 5.5. The energy of activation (E) in the presence of 150 mM KC1 calculated from Arrhenius plot was about 4600 cal/mole of substrate. The heat of the enzyme-substrate complex formation obtained from the plot of log K_m vs T⁻¹ was shown to have positive value (+2200 cal/mole) at the temperatures lower than 23°C and negative value (—4100 cal/mole) at the temperatures higher than 23°C.

     

Influence of batrachotoxin, veratridine, grayanotoxin 1 and tetrodotoxin on uptake of Na-22 by rat brain membrane preparations

The actions of a number of sodium channel-specific neurotoxins on the uptake of Na-22 by osmotically sensitive membrane preparations from rat brain were studied using a glass-fiber filter assay. Under control conditions, there was Na-22 uptake that reached saturation within 5 min, and was insensitive to tetradotoxin (10 μM). Batrachotoxin (K_dapp = 0.2 μM), veratridine (K_dapp = 1 μM) and grayanotoxin I (K_dapp = 30 μM), which increase sodium conductance in electrogenic membranes, stimulated Na-22 uptake approximately 2-fold over control levels. This additional Na-22 uptake was markedly dependent on the ionic strength of the media, associated with subfractions of the preparation enriched in plasma membranes, and completely inhibited by tetrodotoxin (10 μM). It was highly labile, showing only a minor decrease in activity within the first 4–6 h after preparation of the membranes, but disappearing within 24 h at 4° C. It is suggested that the toxins-activated Na-22 uptake, which is tetrodotoxin-sensitive, results from the actions of these toxins on the macromolecular channel complex which controls resting and action potential sodium conductance.     

Restriction of calcium uptake in normal and sickle red cells by procaine hydrochloride and P-aminobenzoic acid.

A reduced capacity for calcium uptake by human red cells after procaine hydrochloride and p-aminobenzoic acid treatment has been measured by atomic absorption spectrophotometry and X-ray microprobe techniques. Cell-associated calcium was lower by 45% and 38% in deoxygenated sickle cells pre-exposed to 10 mM procaine HCl and PABA and incubated 2 hr. at 37°C in plasma, as compared with untreated cells. Prior exposure of washed red cells to 10 mM PABA reduced the intracellular calcium level by 39% after incubation for 45 min. at 37°C with 40 μM calcium ionophore A23187. Procaine HCl was more efficient than PABA in inhibiting calcium uptake at equimolar concentrations.     

Detection and automatic control of ammonium ion concentration in miceobial culture with an ammonium ion selective electrode

An ammonium ion selective electrode (AISE) had a membrane of polyvinyl chloride in which the antibodies nonactin and monacin were embedded. The detection range was 0.1–200 mM. The step response was 90% in 20 seconds. The output of the AISE increased 6% with a 1°C rise temperature. The output of the AISE was constant between pH 4–7. The selectively coefficient of potassium ion was 0.158 and hence its interferring effect must be considered. The selectivity coeficcients of other cations were small enough to be negligible. Throughout a batch culture of Escherichia coli, values calculated by subtrating (selectivity coefficient) × (potassium ion concentration) from the detected output of the AISE agreed with actual concentrations of ammonium ion. An automatic. constant-value, feebdack control system of ammonium ion concentration was attempted by on-off controlled supply of solution containing both ammonium and potassium ions, the proportion of whose concentration was made equal to the proportion of their average volumetric consumption rates by a microorganism in batch culture. By this control system, ammonium ion concentrations in culture supernatants of fed-batch cultures of Escherichia coli and Saccharomyces cerevisiae could be maintained vitrually at constant levels (5±0.8 mM for the cultivation of E. coli and 50±5 M for the cultivation of S. cerevisiae).     

Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid?

Cycloleucine accumulation by frog muscle was studied at o °C and 25 °C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 °C than at 25 °C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 °C.The steady state data are consistent with an absortion model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model.Diffusion-limited flux is ruled out on the basis of“influx profile analysis” of the time course of cycloleucine entry at both 0 °C and 25 °C.At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 °C. The rate constant of efflux does not vary with cellular concentration.These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflex only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 °C and 25 °C. Therefore, an absorption model for cycloleucine accumulation in frog muscle appears to be untenable.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

SODIUM AND CHLORIDE FLUXES IN SYNAPTOSOMES IN VITRO

Synaptosomes incubated in a physiological saline extrude sodium and take up potassium. As would be expected this process is completely blocked by metabolic inhibitors such as cyanide and iodoacetate. However, when metabolic inhibitors are replaced by ouabain (100 μM) there is an increase in the steady state intrasynaptosomal sodium and chloride content even though there is no change in the potassium content. The increases are prevented when synaptosomes are incubated with metabolic inhibitors in addition to ouabain. There is therefore a ouabain-insensitive process that transports sodium, chloride and concomitantly water into synaptosomes. It appears not to function when the supply of metabolic energy is inhibited. The diuretic furosemide (1 mM) in the presence of ouabain inhibits the entry of sodium and chloride without affecting the intrasynaptosomal potassium concentration. Ethacrynic acid (1 mM) has a somewhat similar effect but in addition appears to damage the synaptosome membrane. Kinetic measurements were made of the uptake of sodium, potassium and chloride under conditions of metabolic inhibition and the permeability constants of the membrane determined. Values of 0.068, 0.117 and 0.032 × 10^-6 (cm s^-1) were found for the permeability constants of the membrane to (respectively) sodium, potassium and chloride. Measurements of the rate of uptake in the presence of ouabain revealed an inwardly directed sodium and chloride flux of 5-20 pmol cm^-2 s^-1. Calculation of the fluxes from the steady state ion concentrations also reveals an inwardly directed sodium and chloride flux, though of lesser magnitude. The influx of water is less than would be expected to preserve osmotic equality suggesting that the translocation of sodium and chloride is the primary event. Although its function remains uncertain the flux has a considerable effect on the ion content of synaptosomes.     

Temperature dependency of calcium responses in mammary tumour cells

Changes of intracellular calcium concentration ([Ca²⁺]_i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24°C and 35°C. ATP (0·1–100 μM ) and bradykinin (0·1 nM –1 μM ) induced the increase of [Ca²⁺]_i at both temperatures and Ca²⁺-depletion did not affect these [Ca²⁺]_i responses. Both [Ca²⁺]_i responses became more sensitive at 35°C than at 24°C. A clear latency of [Ca²⁺]_i increased after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35°C. These results suggested the involvement of a regenerative or threshold process in the [Ca²⁺]_i responses in mammary tumour cells. © 1997 John Wiley & Sons, Ltd.     

In vitro stability of nitrate reductase from barley leaves

Barley seedling nitrate reductase was stabilized in vitro without the use of extraneous protein by optimizing the buffer components. The extraction buffer (NRT 8.5) consists of 0.25 M Tris-HCl, pH 8.5, 3 mM DTT, 5 μM FAD, 1 μ M sodium molybdate and 1 mM EDTA. This buffer stabilizes the extracted nitrate reductase at O° and 30°, whereas the addition of extraneous protein to standard extraction buffers stabilizes the enzyme only at 0°.     

Diacytosis of 125I-asialoorosomucoid by rat hepatocytesa. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation

Diacytosis of ¹²⁵I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37°C for 30 min or 18°C for 2 h, washing free of cell surface receptor-bound tracer at 4°C and then reincubating at 37°C. The cells preloaded at 37°C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 39}\text{min}$). When the preloaded cells were incubated in the presence of 100 μg/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 μM colchicine, 20 μM cytochalasin B, 20 μM chloroquine, 10 mM NH₄Cl, 10 μM monensin or 20 μM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoocrosomucoid were observed with cells preloaded at 18°C. These results indicate that diacytosis of ¹²⁵I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.