Uptake and intracellular transport in rat liver of formaldehyde-treated bovine serum albumin labelled with 125I-tyramine-cellobiose

1. Endocytosis of formaldehyde-treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I-tyramine cellobiose (125I-TCfBSA). 125I-TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5-10 min after injection and is maintained for up to at least 180 min. During the first 5 min most of radioactivity remains acid-precipitable. After which, labelled acid-soluble components are produced at a constant rate for up to 30-40 min. 2. Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions. 3. Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma-membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid-precipitable and acid-soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long time (greater than 30 min) of injection a marked shift of radioactivity distribution towards higher densities occurs. 4. A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid-soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid-precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acid-soluble distribution without having a significant effect on the acid-precipitable distribution up to 30 min after 125I-TCfBSA injection. 5. Glycyl-1-phenylalanine-2-naphthylamide largely releases acid-soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I-TCfBSA injection. On the other hand the proportion of acid-precipitable radioactivity present in the fraction that can be released is almost zero at 10 min after injection, and it later increases. 6. The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I-TCfBSA is quickly delivered to the endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Renal uptake and degradation of trapped-label insulin

In order to study the kinetics of insulin degradation in the kidneys and liver, insulin was labelled by a trapped-label procedure and injected into rats. In contrast to conventional 125I-insulin, the trapped-label preparation allows quantitative measurements of the extent of degradation in vivo because the final degradation products do not leave the cells. One hour after injection, the amount of radioactivity in the kidneys from a trace dose of trapped-label insulin was 10 times higher that from conventionally labelled insulin; over 80% of the increase was due to low molecular weight degradation products which were retained in the kidneys. The amount of acid-precipitable radioactivity in the blood was the same for both labelled preparations, indicating that their rates of clearance were similar. In the kidney, we detected no degradation products of molecular weight intermediate between intact insulin and the end products of proteolysis. After 2 h, 33% of the injected dose remained in the kidneys and only 13% in the liver. Over 80% of the renal radioactivity was sedimentable in an isotonic density gradient, indicating that intact insulin, as well as degradation products in the cells, were enclosed within membrane-bound vesicles.     

Uptake and intracellular fate of gelonin,a ribosome-inactivating protein,in rat liver

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.     

Plasma clearance and liver metabolism of native and asialo-human transcortin in the rat.

Plasma kinetics and liver metabolism of iodinated human corticosteroid-binding protein have been studied in ovariectomized female rats. 125I-labeled human corticosteroid-binding globulin prepared by a modified chloramine T reaction was shown to be physically intact and biologically active. Intravenously injected 125I-labeled human corticosteroid-binding globulin was shown to give a complex clearance pattern from the plasma, with half-lives of 7.5 and 51 min. Estrogen injections had no effect on plasma clearance rate. Direct involvement of liver plasma membrane receptors for asialoglycoproteins in 125I-labeled human corticosteroid-binding globulin metabolism was demonstrated in vivo and in vitro using asialofetuin as a competitive inhibitor. 125I labeled human asialo-corticosteroid-binding globulin was cleared from the plasma with a half-life of less than 1 min, while the simultaneous injection of 5 mg asialofetuin maintained the circulating plasma lebels. Asialofetuin also slowed the clearance of intact 125I-labeled human corticosteroid-binding globulin from the plasma (t1/2 = 90 min). Binding of 125I-labeled human asialo-corticosteroid-binding globulin to rat liver plasma membranes in vitro was inhibited in a dose-dependent manner by asialofetuin, but not by intact human corticosteroid-binding globulin or fetuin. 125I-labeled human corticosteroid-binding globulin did not bind significantly to the membranes. It is concluded that human corticosteroid-binding globulin clearance from rat plasma is rapid and that the carbohydrate moiety of human corticosteroid-binding globulin is involved in its clearance and catabolism by the liver.     

Characterization of insulin degradation by rat-liver low-density vesicles

When incubated in vitro, isolated rat liver low-density vesicles degrade endocytosed insulin intraluminally. The rate of intravesicular degradation suggests that this pathway contributes significantly to insulin degradation in vivo. The vesicles can be selectively disrupted with digitonin at concentrations that abolish the latency of NADH pyrophosphatase, with minimal effect on the cisternal Golgi marker, galactosyl transferase. The results suggest that latent NADH pyrophosphatase may act as a marker enzyme for the vesicles within which insulin is degraded. The possible role of insulin-glucagon protease, a candidate enzyme for insulin degradation by the liver, was investigated. The activity of latent insulin-glucagon protease associated with low-density vesicles is sufficient to account for the rate of intravesicular proteolysis. However, the rate of intravesicular proteolysis is insensitive to membrane-permeant thiol reagents under conditions which strongly inhibit insulin-glucagon protease. This shows that insulin-glucagon protease is not rate-limiting for insulin degradation by these vesicles, and is unlikely to be involved in the regulation of degradation. After disruption with Brij, internalized insulin remains associated with the membrane. Degradation is not inhibited by addition of excess unlabelled insulin to the medium, and occurs more rapidly than the degradation of an equal activity of iodo-insulin added to the disrupted membranes. This implies that degradation of endocytosed insulin occurs while it is still bound to the inner surface of the vesicles. When bacitracin is coinjected with iodo-insulin, it inhibits degradation of internalized insulin both by intact and Brij-disrupted vesicles, but not the degradation of added exogenous insulin, confirming that degradation is membrane-associated, and that it does not require the release of insulin into free solution.     

Intracellular transport and degradation of 125I-tyramine cellobiose-labelled low-density lipoprotein endocytosed in vivo in rat liver cells studied by means of subcellular fractionation

The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand.     

In vivo fate of large unilamellar sphingomyelin-cholesterol liposomes after intraperitoneal and intravenous injection into rats

We investigated the fate of intraperitoneally and intravenously injected reverse phase evaporation vesicles of fairly uniform size (100–200 m) with respect to blood celarance, tissue distribution and integrity in vivo. The vesicles are composed of sphingomyelin and cholesterol in a molar ratio 3 : 2 and contain ¹²⁵I-labeled poly(vinyl pyrrolidone) in the aqueous compartment. It is shown that following an intrapersoneal injection the vesicles are transported intact, and not associated with cells, from the peritoneal activity to the blood and are subsequently taken up mainly by liver and spleen, where, particularly in liver, the phospholipid is partially metabolized. After an intraperitoneal injection the rate of vesicle-uptake by liver and spleen is reduced by a factor of 2–3 compared to the rate of vesicle-uptake by liver and spleen following an intravenous injection. The peritoneal cavity functions as a reservior of vesicles for some hours. The rates of blood clearance and uptake of the vesicles by liver and spleen appear to be slower than that found for vesicles of different lipid composition.     

Gadolinium chloride-induced Kupffer cell blockade increases uptake of oxidized low-density lipoproteins by rat heart and aorta.

Oxidized low-density lipoproteins (LDL) play a key role in the formation of atherosclerotic lesions of arteries. We analyzed the effect of hepatic resident macrophage (Kupffer cell) blockade on oxidized [125I]LDL accumulation in different organs and tissues of the rat. Kupffer cell blockade was induced by gadolinium chloride (GdCl3) which was injected intravenously 24 h prior to injection of oxidized [125I]LDL into the rats. Ten minutes after administration to intact animals, oxidized [125I]LDL was accumulated in the liver (86.8% of the dose administered), muscles (4.7%), spleen (2.1%), lungs (0.8%), kidney (0.6%), adrenal glands (0.2%), heart (0.15%), and thymus (0.04%). Kupffer cell blockade significantly decreased the clearance rate of oxidized [125I]LDL from the blood. Specific radioactivity (per g tissue) decreased in the liver (1.3-fold compared to control), but increased in the aorta (2.5-fold), heart (2-fold), lungs (1.6-fold), and kidney (1.3-fold). The results indicate that the accumulation of oxidized LDL in heart and aorta significantly depends on the functional state of the mononuclear phagocyte system in the liver.     

Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver.

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 X 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.     

Plasma clearance and endocytosis of mitochondrial malate dehydrogenase in the rat.

1. Pig mitochondrial malate dehydrogenase was labelled with 125I and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of only 7 min. 2. Radioactivity accumulated in liver, spleen, bone (marrow) and kidneys, reaching maxima of 3 1, 4, 6 and 9% of the injected dose respectively, at 10 min after injection. 3. Our data allow us to calculate that in the long run 59, 5, 11 and 13% of the injected dose is taken up and subsequently broken down by liver, spleen, bone and kidneys respectively. 4. Differential fractionation of liver showed that the acid-precipitable radioactivity was mainly present in the lysosomal and microsomal fractions, suggesting that the endocytosed protein is transported via endosomes to lysosomes, where it is degraded. 5. Radioautography of liver and spleen suggested that the labelled protein was taken up by macrophages of the reticuloendothelial system. 6. Mitochondrial malate dehydrogenase is probably internalized in liver, spleen and bone marrow by adsorptive endocytosis, since uptake of the enzyme of these tissues is saturable.     

Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1. Influx and efflux of l-tri-[(125)I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23 degrees C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3-5pm) influx into cells was saturable; an apparent K(t) value of 8.6+/-1.6pm was obtained. 3. At 5pm-l-tri-[(125)I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23 degrees C) uptake of l-tri-[(125)I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a K(d) value of 6.1+/-1.3pm. 4. Efflux of l-tri-[(125)I]iodothyronine from cells equilibrated with the hormone (5-123pm) was constant up to 21 s; the amount that flowed out was 17.7+/-3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[(125)I]iodothyronine (556-1226pm) 66.8+/-5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[(125)I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18-22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[(125)I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[(125)I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[(125)I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.     

Transplacental transfer of a growth hormone-releasing hormone peptide from mother to fetus in the rat

Previous studies showed that when growth hormone-releasing hormone (GHRH) was administered to either pregnant rats or pigs as a plasmid-mediated therapy, pituitary weight, somatotroph and lactotroph numbers, and postnatal growth rate of the offspring increased. To determine if these responses resulted from direct effects of GHRH on the fetus or were secondary to effects incurred in the mother, we studied in the rat the transplacental transfer of a GHRH analog (HV-GHRH) to the fetus from the maternal circulation. For the in vivo study, HV-GHRH was labeled with 125I and purified by reverse-phase high-performance liquid chromatography (HPLC). At 18 days of gestation, pregnant dams were administered a priming intravenous dose followed by a constant infusion of the labeled peptide. Approximately 2 days later, intact [125I]-HV-GHRH was isolated from the fetal liver, stomach contents, and brain. The amounts of tracer were positively correlated with those present in the corresponding dam's plasma. These data suggest that a GHRH analog of nonplacental origin, even at physiologic concentrations, can cross the placenta and, therefore, has the potential to influence fetal pituitary development directly.     

Characterization of Na+-dependent phosphate uptake in cultured kidney cells (JTC-12) from monkey.

O-(4-Diazo-3-[125I]iodobenzoyl)sucrose ([125I]DIBS), a novel labelling compound specifically designed to study the catabolic sites of serum proteins [De Jong, Bouma, & Gruber (1981) Biochem. J. 198, 45-51], was applied to study the tissue sites of degradation of serum lipoproteins. [125I]DIBS-labelled apolipoproteins (apo) E and A-I, added in tracer amounts to rat serum, associate with high-density lipoproteins (HDL) just like conventionally iodinated apo E and A-I. No difference is observed between the serum decays of chromatographically isolated [125I]DIBS-labelled and conventionally iodinated HDL labelled specifically in either apo E or apo A-I. When these specifically labelled HDLs are injected into fasted rats, a substantial [125I]DIBS-dependent 125I accumulation occurs in the kidneys and in the liver. No [125I]DIBS-dependent accumulation is observed in the kidneys after injection of labelled asialofetuin or human low-density lipoprotein. It is concluded that the kidneys and the liver are important sites of catabolism of rat HDL apo E and A-I.     

Coated vesicles participate in the receptor-mediated endocytosis of insulin

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.     

Effect of 4-aminopyrazolo[3,4-d]pyrimidine on the catabolism of high-density lipoprotein apolipoprotein A-I in the rat

The in vivo turnover and sites of catabolism of O-(4-diazo-3-[125I]iodobenzoyl)sucrose-labelled rat high-density lipoprotein (HDL) apolipoprotein A-I were studied in rats treated for 3 days with 4-aminopyrazolo-[3,4-d]pyrimidine (4APP). It was found that 4APP treatment decreases the serum cholesterol concentration to 6 mg/dl and stimulates the serum decay of labelled HDL. The latter effect could be attributed to an increased catabolism of radioactive HDL apolipoprotein A-I by the liver. When the serum cholesterol concentration was raised to physiological levels by a bolus injection of unlabelled rat HDL, at the time of administration of the labelled HDL, the serum decays and the tissue uptakes of apolipoprotein A-I labelled HDL were identical in 4APP-treated rats and control animals. When a bolus injection of unlabelled human low-density lipoprotein (LDL) was administered to 4APP-treated rats, the serum decay and tissue uptake of apolipoprotein A-I labelled HDL remained rapid. The recovery of radioactivity in the adrenal glands was increased almost 10 fold by 4APP treatment, a phenomenon which was reversed by a bolus injection of unlabelled HDL, but not by injection of unlabelled LDL. It is concluded that treatment of rats with 4APP does not affect the rate of catabolism of rat HDL apolipoprotein A-I, when the serum HDL concentration in the treated animals is restored to physiological levels.     

Effect of ketoconazole on the activity of key enzymes of biosynthesis of cholesterol and its esters in the liver of rats maintained on a cholesterol-enriched diet]

Ketoconazole in vivo has been studied for its effect on the activity of key enzymes of the cholesterol and its esters' biosynthesis in the liver and on the cholesterol concentration in certain fractions of blood lipoproteins in normal and cholesterol-fed rats. It is established that ketoconazole decreases cholesterol concentration in low-density lipoproteins and in very low-density lipoproteins as well as decrease the acyl-CoA-cholesterol acyl-transferase activity and increases the 3-hydroxy-3-methyl-glutaryl-CoA-reductase activity in the liver microsomes of intact and test animals. It is supposed that the possible cause of the observed changes can be a disturbance in regulation of basic links of cholesterol metabolism in the liver.     

Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment.

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.     

Drug delivery using vesicles targeted to the hepatic asialoglycoprotein receptor

We assessed the utility of liver-targeted vesicles as a drug delivery system for the treatment of liver diseases. Small, unilamellar vesicles (mean diameter, 60–80 nm) composed of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and digalactosyldiacylglycerol (mol ratios, 40:40:5:15) are rapidly cleared from the blood in rats after intravenous injection. In vivo organ distribution shows that the liver is the major site of vesicles accumulation, with roughly 60–80% of the vesicles contents delivered to the liver. Isolated, perfused rat liver experiments show that the uptake is due to the hepatic asialoglycoprotein receptor, and the uptake process occurs with minimal vesicle leakage. At low doses of the vesicles, the single pass extraction by the liver is around 50%, which means that this vesicle formulation operates close to optimal efficiency as a drug delivery system to the liver. Binding of vesicles to the liver was determined to saturate at 6.5 mg total lipid/kg body weight, with a maximum steady-state turnover rate of vesicles at 37° C of 79 μg lipid/min per kg body weight. This gives a receptor recycling time of around 80 min. We have incorporated this information into a pharmacokinetic model of vesicle distribution which quantitatively predicts the kinetics and dose dependence of vesicle uptake by the liver in vivo. This information can be used to optimize vesicle-mediated drug delivery to the liver.     

Fate of intraduodenally administered somatostatin in rats in vivo

Intraduodenally administered somatostatin-14 (a milk-borne peptide) has been shown to influence the pancreatic secretions in rats and dogs. To delineate the mechanism involved in the intraduodenal somatostatin-14, the fate of intraduodenal somatostatin-14 was investigated by administering [¹²⁵I][Tyr¹¹]somatostatin-14 ([¹²⁵I][Tyr¹¹]SS14) into the lumen of isolated duodenum in vivo of suckling and adult rats. At 2, 5, 10, and 30 min after administration, the radioactivity in the duodenal contents, duodenal wall, blood, liver, and kidney was measured, and the extracted radioactivity was analyzed for intact [¹²⁵I][Tyr¹¹]SS14 by reversed-phase high performance liquid chromatography. Radioactivity disappeared rapidly from the duodenal lumen with 50% of loss occurring at 2 min. No trace of intact [¹²⁵I][Tyr¹¹]SS14 was detected in any of the samples, except duodenal contents of suckling rats at 2 min, suggesting a rapid metabolism of SS14 in the duodenal lumen and a lack of duodenal absorption of intact SS14 in the rat.     

Drug delivery using vesicles targeted to the hepatic asialoglycoprotein receptor

We assessed the utility of liver-targeted vesicles as a drug delivery system for the treatment of liver diseases. Small, unilamellar vesicles (mean diameter, 60-80 nm) composed of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol and digalactosyldiacylglycerol (mol ratios, 40:40:5:15) are rapidly cleared from the blood in rats after intravenous injection. In vivo organ distribution shows that the liver is the major site of vesicle accumulation, with roughly 60-80% of the vesicle contents delivered to the liver. Isolated, perfused rat liver experiments show that the uptake is due to the hepatic asialoglycoprotein receptor, and the uptake process occurs with minimal vesicle leakage. At low doses of the vesicles, the single pass extraction by the liver is around 50%, which means that this vesicle formulation operates close to optimal efficiency as a drug delivery system to the liver. Binding of vesicles to the liver was determined to saturate at 6.5 mg total lipid/kg body weight, with a maximum steady-state turnover rate of vesicles at 37 degrees C of 79 micrograms lipid/min per kg body weight. This gives a receptor recycling time of around 80 min. We have incorporated this information into a pharmacokinetic model of vesicle distribution which quantitatively predicts the kinetics and dose dependence of vesicle uptake by the liver in vivo. This information can be used to optimize vesicle-mediated drug delivery to the liver.