Effects of temperature, metabolic inhibitors and some other factors on fluid-phase and adsorptive pinocytosis by rat peritoneal macrophages.

Low temperature, NaF and 2,4-dinitrophenol could each abolish the pinocytic uptake of 125I-labelled poly(vinylpyrrolidone) or colloidal [198Au]gold by rat peritoneal macrophages cultured in vitro. Cytochalasin B caused only partial inhibition, even at 10 microgram/ml, and colchicine (10 or 25 microgram/ml) inhibited uptake of colloidal [198Au]gold much more than that of 125I-labelled poly(vinylpyrrolidone). Dibutyryl cyclic AMP and ouabain were without effect on uptake of 125I-labelled poly(vinylpyrrolidone), and slight stimulation was seen with ATP and theophylline. Uptake of 125I-labelled poly(vinylpyrrolidone) was abolished by EGTA (5mM), but restored by adding CaCl2 (5mM). The results appear not to support the conventional criteria for the division of pinocytic phenomena into macropinocytosis, requiring a metabolic energy supply and cytoskeletal components, and micropinocytosis, requiring neither.     

A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages.

A method for the culture of rat peritoneal macrophages in vitro is described, in which pinocytic uptake of colloidal [198 Au]gold, 125I--labelled poly(vinylpyrrolidone) and [14C]sucrose proceeds at contant and fairly reproducible rates for several hours. The rat of uptake of colloidal [198 Au]gold, which wxhibited some inter-batch variation, was approx. 100 times that of the other two substrates. Colloidal gold did not affect the rate of uptake of 125I-labelled poly(vinylpyrrolidone) and therefore its own high rate of uptake could not be attributed to a stimulation of the formation of pinocytic vesicles. It conclude that uptake of collodial gold is highly dependent on adsorption on binding sites on the plasma membrane. Uptake of formaldehyde-treated 125I-labelled bovine serum albumin was followed by the release of [125I]iodo-L-tyrosine into the culture medium and took place at a rate intermediate between those of collodial [198Au]gold and the other two non-digestible substrates, 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose.     

The pinocytosis of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by rat yolk sac cultured in vitro.

The rates of uptake of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by 17.5-day rat yolk sac cultured in vitro were studied. Over a 6.5h period each substrate was accumulated at a constant and reproducible rate of approx. 2microliter/h per mg of protein. After accumulation in vitro, the three substances were released from the tissue into substrate-free medium at low rates. Sucrose present in the medium at concentrations up to 10 mg/ml was without effect on the accumulation of either [14C]sucrose or 125I-labelled poly(vinylpyrrolidone), but at higher concentrations inhibited the uptake of both substrates. Some batches of colloidal [198Au]gold had a significantly higher Endocytic Index (up to 5 microliter/h per mg of protein). The Endocytic Index of such a batch decreased with increasing substrate concentration, but colloidal gold did not decrease the Endocytic Index of 125I-labelled poly(vinylpyrrolidone). It is concluded that the three substrates enter the yolk sac by pinocytosis in the liquid phase. Those batches of colloidal [198Au]gold with higher Endocytic Indices are considered to enter also by adsorption on membrane binding-sites.     

Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

Different glycosides were grafted on the surface of liposomes containing ¹²⁵I-labelled γ-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of $\text{p-}\text{aminophenyl-}\text{d}\text{-glycosides}$ to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ¹²⁵I-labelled γ-globulin was determined in mouse tissues from 5–60 min after a single injection of these liposomes. The liver uptake of encapsulated ¹²⁵I-labelled γ-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ¹²⁵I-labelled γ-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of ¹²⁵I-labelled γ-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of ¹²⁵I-labelled γ-globulin from liposomes containing asialogangliosides depends upon the phospholipid/ glycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.     

Uptake by rat peritoneal macrophages of125I-labelled polyvinylpyrrolidone entrapped within liposomes

Rat peritoneal macrophages in vitro capture¹²⁵I-labelled polyvinylpyrrolidone entrapped within either negatively or positively charged liposomes more rapidly than they do the free macromolecule. The uptake of negatively charged liposomes was linear with time over l0 h, whilst the uptake of positively charged ones, although more rapid, was more transient. Neither type of liposome was taken up in the presence of 2,4-dinitrophenol (100 g/ml), and 5 mM calcium chloride increased the uptake of negatively charged liposomes. The enhanced uptake of 125I-labelled polyvinylpyrrolidone when presented in liposomes must have been a consequence of entrapment rather than of a simple interaction between lipid and polyvinylpyrrolidone, since the presence of the lipids employed or of empty liposomes had no effect on the uptake of unentrapped¹²⁵I-labelled polyvinylpyrrolidone.     

Adsorptive pinocytosis of polycationic copolymers of vinylpyrrolidone with vinylamine by rat yolk sac and rat peritoneal macrophage

Polycationic copolymers of vinylpyrrolidone and vinylamine (10:0.77) were prepared, and ¹²⁵I-labelled with either Bolton-Hunter reagent or methyl 3,5-di-[¹²⁵I]iodohydroxybenzimidate. The rate of pinocytic capture of the copolymer was compared with that of ¹²⁵I-labelled polyvinylpyrrolidone, using rat visceral yolk sacs and rat macrophages cultured in vitro as test systems. Whereas polyvinylpyrrolidone was captured entirely by non-adsorptive pinocytosis, the cationic derivative was captured more efficiently, probably because it adsorbs to the cell surface. Copolymer of M_r 120 000 was internalized by macrophages somewhat more rapidly than copolymer of M_r 46 000, but was excluded from the yolk sac.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, ^99mTc, and an aqueous marker, ¹²⁵I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped ¹²⁵I-labelled poly(vinyl pyrrolidone) were labelled with ^99mTc by the SnCl₂ method [2]. ^99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, ^99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than ¹²⁵I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60–70% of ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of ^99mTc-radioactivity was co-eluted with liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all ^99mTc-radioactivity does not represent the 60–70% of intact liposomes present in lymph nodes. Using the aqueous marker ¹²⁵I-labelled poly(vinyl pyrrolidone), the lymphnode localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that ¹²⁵I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier [2], where the authors used ^99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became ‘leaky’ to low-molecular-weight drugs, for example, methotrexate (M_r 454) to a much greater extent than with a large-molecular-weight substance, ¹²⁵I-labelled poly(vinyl pyrrolidone) (M_r 30 000–40 000), when incubated with rat lymph at 37°C. Using the two markers ^99mTc and ¹²⁵I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining λ-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with ^99mTc rather than using the SnCl₂ technique [2], or using other radionuclides as markers for γ-scan imaging.     

Pinocytic uptake of divinyl ether-maleic anhydride (pyran copolymer) and its failure to stimulate pinocytosis

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of ¹²⁵I-labelled PVP and colloidal ¹⁹⁸Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [¹⁴C]DIVEMA and ¹²⁵I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.     

Effect of molecular size of 125I-labelled poly(vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and rat peritoneal macrophages.

Rates of pinocytosis of different molecular-weight distributions of 125I-labelled poly(vinylpyrrolidone) by rat visceral yolk sacs and rat peritoneal macrophages were measured in vitro. Four preparations of mean molecular weights 50 000, 84 000, 700 000 and 7 000 000, were used. Macrophages captured the highest-molecular-weight preparation more rapidly than the other preparations. In contrast, rate of capture by the yolk sac decreased with increasing molecular weight. Incubations with a very-high-molecular-weight fraction derived from the 7 000 000-average-mol. wt. preparation clearly demonstrated that very large polymer molecules are not accumulated by the yolk sac, but are preferentially captured by macrophages. Analysis of the 125I-labelled poly(vinylpyrrolidone) internalized by the two cell types confirmed that low-molecular-weight material is preferred by the yolk sac, whereas the macrophage is less discriminating.     

Studies on the mechanism of transferrin iron uptake by rat reticulocytes

Mechanism of transferrin iron uptake by rat reticulocytes was studied using ⁵⁹Fe- and ¹²⁵I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound ⁵⁹Fe was located in the cytoplasmic fraction, only 25–30% of ¹²⁵I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic ¹²⁵I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound ¹²⁵I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering ⁵⁹Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.     

Pinocytosis in the rat visceral yolk sac effects of temperature, metabolic inhibitors and some other modifiers

Low temperature, 2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone, ¹²⁵I-labelled bovine serum albumin or colloidal ¹⁹⁸Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the oresence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.     

Insulin regulates protein metabolism in mouse blastocysts

Mouse blastocysts, in vitro, endocytosed 100 μg/ml ¹²⁵I-labelled bovine serum albumin (BSA) at a rate equivalent to 192 ± 27 μl/hr/mg embryonic protein over the first 20 min. Insulin stimulated this initial uptake by 30% (P < 0.05). After this time, accumulation of ¹²⁵I-labelled BSA began to plateau as the endocytosed ¹²⁵I-labelled BSA was catabolized and ¹²⁵I was released from the cells. Insulin caused an ≈?72% (P < 0.05) increase in the amount of uncatabolized ¹²⁵I-labelled BSA remaining in insulin-treated blastocysts after 2 hr as compared to control blastocysts. Insulin partially inhibited catabolism of endocytosed ¹²⁵I-labelled BSA during the first 2 hr following transfer to nonradioactive medium. After this time, degradation ceased in both control and insulin-treated blastocysts, leaving a small, uncatabolized protein pool remaining in the embryos; however, as a result of insulin's inhibitory effects on the initial catabolic rate, the uncatabolized protein pool was 30% (P < 0.05) larger in insulin-treated blastocysts after the 4 hr chase. Insulin inhibited endogenous protein degradation in blastocysts by 37% (P < 0.05). Combined with previous studies showing a 90% increase in endogenous protein synthesis in blastocysts following short-term stimulation with insulin (Harvey and Kaye, 1988), these results suggest that insulin acts to increase the endogenous protein-reserves in the embryo. Dose-response studies indicated an EC₅₀ of 0.5 pM for insulin's stimulation of ¹²⁵I-labelled BSA accumulation, consistent with action via its own receptor. Insulin-like growth factor-1 (IGF-1) also stimulated protein accumulation at concentrations similar to those observed with insulin, suggesting that IGF-1 may act via its own receptor rather than the insulin receptor to exert its effects on endocytosis. © 1993 Wiley-Liss, Inc.     

The streptokinase–human plasminogen activator complex. Composition and identity of a subcomponent with activator activity

Reactions between purified plasminogen and streptokinase, labelled with ¹³¹I and ¹²⁵I respectively, were investigated by polyacrylamide-gel discontinuous electrophoresis. A molecular complex consisting of both ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase migrated between plasminogen and streptokinase. This complex contained bovine plasminogen activator activity. The relative quantities of ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase in this complex were markedly affected by reaction conditions. A fragment that retained 50% or more of the parent activator activity was released from the complex after exposure to mercaptoethanol. This subcomponent had an estimated molecular weight of 70000, and contained both ¹³¹I-labelled plasminogen and ¹²⁵I-labelled streptokinase.     

Fluid endocytosis by rat liver and spleen. Experiments with 125I-labelled poly(vinylpyrrolidone) in vivo.

1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.     

Iron and transferrin distribution in reticulocytes incubated with heme synthesis inhibitors

A high level of non-heme iron (either labelled or unlabelled) in mitochondria, ferritin and low-molecular-weight pool of reticulocytes was induced by preincubation with isonicotinic acid hydrazide or penicillamine together with either ⁵⁹Fe- or ⁵⁶Fe-labelled transferrin. Addition of apotransferrin during reincubation of ⁵⁹Fe-labelled reticulocytes was accompanied by the transfer of ⁵⁹Fe from low-molecular-weight pool to transferrin, which was found in the reticulocyte cytosol both free and bound to a carrier. Similarly, when cells were reincubated with ¹²⁵I-labelled transferrin, more ¹²⁵I-labelled radioactivity was found, in both free and carrier-bound transferrin peaks, in reticulocytes with a high level of low-molecular-weight cold iron than in control ones. These results suggest that transferrin enters reticulocytes takes up iron from low-molecular-weight pool.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of ¹²⁵I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on ¹²⁵I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of ¹²⁵I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

A comparative study of the effects of polyamino acids and dextran derivatives on pinocytosis in the rat yolk sac and the rat peritoneal macrophage

Poly-l-lysine, poly-l-α-ornithine, poly-l-glutamic acid, dextran, DEAE-dextran and dextran sulphate all fail to affect greatly the rate of pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone by 17.5-day rat visceral yolk sac or rat peritoneal macrophages cultured in vitro. It is concluded that these agents do not much affect the rate of pinocytic ingestion of liquid. Polycations accelerate the accumulation of colloidal ¹⁹⁸Au in both systems, and this is ascribed to the formation of substrate · modifier complexes which either adsorb to plasma membrane, and thus gain rapid entry, or initiate another mode of endocytosis. Pinocytic uptake of formaldehyde-denatured ¹²⁵I-labelled bovine serum albumin was accelerated by poly-l-lysine and poly-l-ga-ornithine in the macrophage, buth inhibited in the yolk sac.     

Studies on the inhibitory effect of bacitracin on 125I-labelled insulin internalization in the rat hepatocyte

Previous studies have suggested that transglutaminase has a role in the internalization of some polypeptide hormones and is inhibited by the antibiotic, bacitracin. Bacitracin has been used in insulin-receptor studies to inhibit extracellular degradation of ¹²⁵I-labelled insulin. The aim of this study was to investigate bacitracin's effect on ¹²⁵I-labelled insulin-receptor interactions in isolated rat hepatocytes. 1 g/l bacitracin increased cell-associated ¹²⁵I-labelled insulin at 20, 30 and 37°C (P < 0.001, 0.0005 and 0.0005, respectively). At 5 and 15°C (internalization does not occur), bacitracin did not affect cell-associated ¹²⁵I-labelled insulin. The bacitracin effect was concentration dependent, increasing to 2 g/l. Scatchard analysis showed that bacitracin did not alter insulin receptor affinity or number. 1 g/l bacitracin abolished the effect of chloroquine. The increased cell-associated radioactivity with bacitracin was surface-bound in nature. 0.5 g/l bacitracin decreased ¹²⁵I-labelled insulin degradation in hepatocyte suspensions (P < 0.001) and in buffer previously incubated with hepatocytes (P < 0.0005). More ¹²⁵I-labelled insulin remained associated with cells during dissociation studies at 37°C when the buffer contained 1 g/l bacitracin. Label that appeared in the buffer after 60 min was significantly more intact in the presence of bacitracin (P < 0.025). These results suggest that bacitracin retards the internalization of ¹²⁵I-labelled insulin in isolated rat hepatocytes.     

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of ¹²⁵I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of ¹²⁵I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10⁸ cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of ¹²⁵I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.