Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.     

Rapid and sensitive HPLC assay for simultaneous determination of procaine and para-aminobenzoic acid from human and rat liver tissue extracts

A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery C(18) column using 10mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 microM to 750 microM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 microM and the lower limit of quantitation was 10 microM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.     

Role of extracellular calcium in the hyperthermic killing of CHL V79 cells

Two major questions are addressed by this study: Can an influx of calcium ion sensitize CHL V79 cells to hyperthermia, and, if so, does this occur during heating and does it play a crucial role in cell death? V79 cells are sensitized to hyperthermia by the calcium ionophore A23187 which also induces an influx of calcium at both 37 and 43 degrees C. Sensitization is at least partially dependent on the presence of extracellular calcium. In the absence of A23187, survival is independent of calcium concentration (from 0 to 25 mM) during heating, which differs from the behavior of hepatocytes which are sensitized to hyperthermia by 15 mM CaCl2. Calcium influx, as assayed by uptake of 45Ca measured after washing in LaCl3, is detectable in 3 mM CaCl2 only after 30 min at 45 degrees C, an exposure which reduces reproductive survival to less than 0.1%. Calcium uptake reaches 6 nmol/10(6) cells after 180 min at 45 degrees C. This is not due to a general loss of membrane permeability since there is no trypan blue staining during this time. In 15 mM CaCl2, influx occurs earlier (15 min) but still succeeds the loss of reproductive survival which is less than 1% at this time. Uptake is much higher in 15 mM CaCl2, reaching 10 nmol/10(6) cells by 30 min and 25 nmol/10(6) cells at 180 min, but the temporal pattern of uptake does not correlate with loss of reproductive survival. Thus, although A23187 sensitizes V79 cells to hyperthermia, probably by increased influx of calcium ion, and increased influx occurs during exposure to 45 degrees C, influx is not a crucial early event in the killing of V79 cells. This does not eliminate the possibility of intracellular calcium redistribution during hyperthermia.     

Effect of cold shock and cooling rate on calcium uptake of ram spermatozoa

Rapid cooling (cold shock) of washed ejaculated ram sperm irreversibly reduced motility and respiration and greatly increased uptake of ⁴⁵Ca²⁺. The effect was greater as the temperature of cooling was reduced from 15°C to 0°C, and a substantial increase in sperm calcium levels was even observed after slow cooling to temperatures below 10°C. The rise in calcium uptake on freezing sperm to −79°C was not as great as that on cold shocking sperm to 0°C.Inactivation of sperm by mild heat (50°C) had no significant effect on calcium uptake but subsequent cold shock increased the sperm calcium. Reverse immobilization of sperm by low concentrations of formaldehyde significantly reduced calcium uptake on cold shock. Addition of detergents to sperm immediately reduced motility, respiration and calcium uptake of control and cold-shocked sperm to zero.     

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.     

Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes

2-Deoxy[1-¹⁴C]glucose uptake in rat adipocytes was measured as a function of time in the absence and presence of unlabelled glucose or 2-deoxyglucose. Uptake of tracer alone was linear from 2 s to 6 min. At 37°C the rate of uptake in insulin-stimulated cells decreased markedly after a few seconds in the presence of glucose (0.5–10 mM) and after 0.5–2 min in the presence of deoxyglucose (2–10 mM). Similar data were obtained at 22°C. With 10 mM glucose (37°C, 30 s) approx. 80% of the intracellular radioactivity was non-phosphorylated deoxyglucose and with 10 mM deoxyglucose approx. 40% was non-phosphorylated. The results show that deoxy[¹⁴C]glucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose and at least partially in the presence of deoxyglucose. The data suggest caution in using deoxyglucose uptake as a measure of transport, especially in complex kinetic studies.In addition, the initial velocity of tracer ¹$\text{3-O-}\text{methylglucose}$ was found to be approx. 2-fold higher than that of tracer deoxyglucose even though both sugars inhibited the initial velocity of labelled methylglucose half-maximally at a concentration of 5 mM. These data suggest a fundamental difference between deoxyglucose and methylglucose transport.     

Calcium and pancreatic β-cell function: 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools

β-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of ⁴⁵Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37°C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable ⁴⁵Ca. The stimulatory effect of phosphate was additive to that produced by glucose. ⁴⁵Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of ⁴⁵Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated ⁴⁵Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic β-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.     

Sulfate influx on band 3 protein of equine erythrocyte membrane (Equus caballus) using different experimental temperatures and buffer solutions

The aim of this study was to assess the anion transport in equine erythrocytes through the measurement of the sulfate uptake operating from band 3 using different experimental temperatures and buffer solutions. Blood samples of six clinically healthy horses were collected via jugular vein puncture, and an emochrome‐citometric examination was performed. The blood was divided into four aliquots and by centrifugation and aspiration the plasma and buffy coat were carefully discarded. The red blood cells were washed with an isosmotic medium and centrifuged. The obtained cell suspensions were incubated with two different experimental buffer solutions (buffer A: 115 mM Na2SO4, 10 mM NaCl, 20 mM ethylenediaminetetraacetic acid, 30 mM glucose; and buffer B: 115 mM Na2SO4, 10 mM NaCl, 20 mM ethylenediaminetetraacetic acid, 30 mM MgCl2) in a water bath for 1 h at 25 °C and 37 °C. Normal erythrocytes, suspended at 3% hematocrit, were used to measure the influx by absorption spectrophotometry at 425 nm wavelength. Unpaired Student's t‐test showed a statistically significant decrease (P < 0.01) of rate constants in equine erythrocytes at 25 °C versus 37 °C using both experimental buffer solutions. Comparing the buffer A with buffer B unpaired Student's t‐test showed statistically lower values (P < 0.0001) for A solution versus B solution both at 25 °C and at 37 °C. The greater inhibition of SO₄⁼ influx measured in equine erythrocytes indicates the increased formation of the sulfydryl bonds in band 3 and the modulation of the sulfydryl groups, culminating in the conformational changes in band 3. Copyright © 2012 John Wiley & Sons, Ltd.     

Calcium transport in isolated bone cells. II. Calcium transport studies

Calcium transport was studied in bone cells isolated from fetal rat calvaria. ⁴⁵Ca uptake experiments revealed an active component of calcium exchange. Calcium uptake was inhibited by iodoacetamide, DNP, CCCP and oligomycin and appeared to be dependent on medium phosphate concentration. Initial influx values exhibited saturation kinetics from 0.6 mM to 1.5 mM extracellular calcium. Efflux of ⁴⁵Ca from loaded cells increased in the presence of iodoacetamide, DNP and CCCP. Incubation of the cells af 4° C inhibited both influx and efflux of calcium. Parathyroid hormone had no consistent effect on calcium uptake although characteristic increases in cyclic AMP levels were seen with the hormone. Calcitonin appeared to cause a transient increase in calcium uptake.     

Calcium enhances the hemolytic action of bile salts

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.     

NMR investigation of the influence of procaine and its metabolites on the water exchange through human erythrocyte membranes

The effect of procaine hydrochloride and its metabolites on the diffusional water exchange through erythrocyte membranes was investigated at 37 degrees C and at concentrations ranging between 5 X 10(-5) M and 5 X 10(-1) M by using the NMR manganese doping method. Procaine hydrochloride and 2-diethylaminoethanol have a moderate stimulating effect on the water exchange, of up to 20% at concentrations ranging between 10(-3) and 10(-2) M, while an increasing inhibitory effect was found at higher concentrations. The p-aminobenzoic acid has no effect on the water exchange up to 10(-2) M and, at higher concentrations, and apparent decreasing inhibition was noticed which is thought to be an artefact due to the uptake of Mn2+ by the cells. The temperature dependence studies suggest that procaine HCl enhances the uptake of Mn2+ by the cells. An opposite effect was found for rigid erythrocytes. The p-aminobenzoic acid and 2-diethylaminoethanol appeared to be more effective than procaine hydrochloride in increasing the uptake of Mn2+.     

Glucose metabolism in the hypothermic perfused rat heart.

Although hypothermic whole organ perfusion is widely used in attempts to preserve organs for transplantation and to preserve the myocardium during cardiac surgery, little is known about substrate metabolism during hypothermia. A knowledge of metabolism utilization during hypothermic whole organ perfusion might allow optimal substrate choice for preservation of energy stores and functional capacity. Separate groups of hearts from fed rats were perfused 30 minutes with Krebs Henseleit bicarbonate buffer containing 5mM glucose-U-¹⁴C, at 37°, 25°, 20°, 15° and 10°C. From 37° to 15°C, heart rate decreased 90% and coronary flow decreased 25%. Glucose uptake decreased 5 fold from 37° to 10°C while ¹⁴CO₂ and lactate production decreased 50 fold and 28 fold, respectively. Myocardial glycogen was stable until 10°C at which point increased glycogenolysis occured. The incorporation of ¹⁴C in glycogen was stable at 37°, 30° and 25° but decreased progressively with lower temperatures. The percent recovery of glucose as ¹⁴CO₂, lactate and ¹⁴C in glycogen decreased from 73% at 37° at 10°C. Our studies indicate that metabolism of glucose is greatly reduced but significant above 15°C.     

Production of 3-hydroxypropionaldehyde using a two-step process with Lactobacillus reuteri

3-Hydroxypropionaldehyde (3-HPA) produced by Lactobacillus reuteri is a broad-spectrum antimicrobial substance of glycerol conversion. The aim of the present work was to optimize 3-HPA production by Lb. reuteri ATCC 53608 using a two-step process. The first step was the production of Lb. reuteri cells in optimal conditions. Cells were then harvested by centrifugation and suspended in glycerol solution, which the resting cells bioconverted to 3-HPA. The effect of biomass concentration, temperature, glycerol concentration, anaerobic/micro-aerophilic conditions, and incubation time was studied for high 3-HPA production. 3-HPA accumulation was limited by the death of cells in contact with high concentrations of 3-HPA. However, a very high 3-HPA concentration of 235±3 mM was obtained after 45 min of incubation at 30°C in 400 mM glycerol for an initial free-cell concentration of 1.6±0.3×10¹⁰ viable cells/ml. A high viability was maintained at low temperatures in the range 5–15°C, but with a slightly lower yield of 3-HPA at 5°C compared with higher temperatures, up to 37°C. Successive 1-h incubations of Lb. reuteri cells in 200 mM glycerol at 15°C to tentatively reuse the cells resulted in decreasing 3-HPA concentrations at the end of each cycle, with two successful production cycles yielding high 3-HPA concentrations of 147±1 mM and 128±2 mM.     

The release of cytosolic proteins from cells treated with concanavalin A during scraping from plastic culture dishes

When rat hepatoma cells (HTC and R117-21B), treated with concanavalin A (conA) at 37 °C, were scraped from plastic culture dishes with a silicone-rubber policeman, the cell membranes were broken and the cytoplasm was released. This phenomenon was also observed in cells treated with conA at 4 °C, even though it took a longer time to show the same effect. The effect of 10 μg/ml of conA on the release of the cellular proteins reached a plateau when the treatment was carried out at 37 °C. Ninety percent of this effect was abolished by 10 mM of α-methyl-d-mannoside. The effect was completely nullified by 100 mM. At 4 °C, however, even 100 mM of this sugar could not abolish this effect. The apparent decrease in the cellular proteins with conA after scraping was observed not only in the logarithmic phase, but also in the stationary phase of cell growth. The breakdown of plasma membranes with conA eventually caused decrease in tyrosine aminotransferase activity, even though the lectin induced the enzyme activity in cultured cells.     

pH-dependent effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor

Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37°C for 20 min, its ability to bind [³H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2–3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (>5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [³H]progesterone-receptor complexes; 70–80% of which remained after a 20 min incubation at 37°C in the presence of 2–3 mM tungstate at pH 7. At pH 8, presence of tungstate (1–10 mM) during the 37°C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4°C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.     

Adaptation of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45°C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. A 60-min preincubation of cells at 37°C or pretreatment with low concentrations of H₂O₂ (0.5 mM) or menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Loss of acrosin from bovine spermatozoa following cold shock: Protective effects of seminal plasma

Loss of the alkaline proteinase acrosin and other proteins from the acrosome of bovine spermatozoa was investigated following cold shock and/or incubation of the spermatozoa at either 5, 21, or 37 °C for 4 hr. As detected by electrophoretic analyses of the acrosomal material two bands of acrosin activity and 10 proteins were lost from the acrosome after cold shock and incubation for 4 hr at 5 or 21 °C, whereas one acrosin band and 10 protein bands were lost after cold shock and incubation at 37 °C. Only 45% of the total acrosin activity remained in the acrosome after both cold shock and 4-hr incubation at 37 °C. Egg yolk, present at levels above 15%, and seminal plasma prevented much of the loss of acrosin from the cells.     

Short‐term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short‐term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30–37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30‐min preincubation of isolated mitochondria at 25–40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short‐term high temperature events associated with global warming.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

The Cellular Regulation of Vesicle Exocytosis by Entamoeba histolytica1,2

ABSTRACT. We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed ¹²⁵iodine (¹²⁵I) labeled tyrosine conjugated dextran; ¹²⁵I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of ¹²⁵I-dextran released after 120 min at 37°C, 31°C, 25°C, and 4°C, respectively (P < 0.01 for each). Exocytosis at 37°C was inhibited by cytochalasin D (10 μg/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 μM) (P < 0.01 for each at ≥ 60 min). Calcium ionophore A23187 (1 μM) enhanced exocytosis at 5 and 15 min (P < 0.01). Elevation of vesicle pH with NH₄Cl (10 mM) had no effect on release of ¹²⁵I-dextran; phorbol myristate acetate (10^?6 M) increased exocytosis by 46% at 30 min (P < 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased ¹²⁵I-dextran release into the cell supernatant after 30 and 60 min at 37°C (by 40% and 42%, respectively, P < 0.01); release of ¹²⁵I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.