Distribution and disappearance rate of submaxillary renin.

Highly purified submaxillary renin (SR) labeled with 125I was injected intravascularly into adult male mice following removal of submaxillary glands and kidneys, and the disappearance of this labeled SR from the circulating vascular volume was studied on the basis of a two compartment system. There was a fast and a slow component to the disappearance curves. Mean half-times of the fast and slow component were 12.4 +/- 0.4 min and 86 +/- 3 min in sialoadenectomized mice, while in mice whose submaxillary glands and kidneys were removed the half-times were 14.7 +/- 0.4 min and 108 +/- 7 min, respectively. The uptake of radioactivity by various organs of the mouse was also measured. Accumulation of radioactivity occurred in the kidneys and liver. Only trace amounts of radioactivity were found in the other organs. The findings suggest that the fast component of the disappearance curve was probably due to equilibration of the injected labeled SR in the circulation. However, the fast component may be related to some extent to the rapid uptake of labeled SR by the kidneys. The half-time of the slow component may represent the true halflife of SR in mice, since a significant reciprocal relationship between the half-times of the slow component and metabolic rate constant k10 was observed both in sialoadenectomized mice and in nephrectomized-sialoadenectomized mice.     

THE IN VIVO REUTILIZATION OF LYMPHOCYTIC AND SARCOMA DNA BY CELLS GROWING IN THE PERITONEAL CAVITY

An ascites tumor, Sarcoma I, was transplanted to isologous and homologous mice which had been labeled with tritiated thymidine from 1 to 24 hours previously. Radioautographic preparations revealed labeled host lymphocytes emerging to mingle with the transplanted tumor and the subsequent appearance of nuclear radioactivity in the sarcoma. Sarcoma cells cultured subcutaneously or in Millipore diffusion chambers in previously labeled mice did not demonstrate significant radioactivity. Transplantation of washed, H³-thymidine-labeled lymphocytes to non-radioactive, sarcoma-bearing mice was followed by the gradual appearance of nuclear radioactivity in the sarcoma. The label in the sarcoma was entirely removed by deoxyribonuclease but not by ribonuclease treatment prior to radioautography. Intraperitoneal injections of purified, H³-thymidine-labeled sarcoma or lymphoid DNA in normal or tumor-bearing mice were followed by radioactivity appearing in sarcoma or normal peritoneal mononuclear cells. It was concluded that reutilization of DNA and its metabolites may occur in vivo, and the conditions under which reutilization may be detected are discussed.     

Effects of prolonged moderate body deuteration on proliferative activity in major cell renewal systems in mice

To evaluate the effects of prolonged moderate body deuteration on incorporation of tritiated thymidine (³HTdR) into the DNA of major cell renewal systems, young adult mice were given drinking fluid containing 30 % heavy water for 7, 14, 21, 42 and 70 days. Control mice drank tap water. Three hours prior to sacrifice, 925 kBq of ³HTdR were injected intravenously. Following extraction of the bulk of the soluble activity with an aqueous formalin solution, the residual ³H-activity of the organs was assayed by liquid scintillation counting and by autoradiography. The total thymic ³H-activity and the thymic weight, particularly of the cortex, were significantly reduced in deuterated mice early in the course of the experiment. The fraction of labeled thymocytes diminished to less than one half of the control values of day 70. The ³H-activity of the bone marrow in deuterated mice was reduced to about $\text{3}\text{4}$ of control values. In contrast, the total ³H-activity of the small intestine, as well as mean labeling index and mitotic index of small intestinal epithelia, were not markedly altered in deuterated versus control mice. Drinking water containing 30 % of heavy water did thus not result in generalized, profound and progressive disturbance of ³HTdR incorporation in all the major cell renewal systems in the mouse. The thymus and, to a lesser extent, the bone marrow, were unquestionably affected, but the depression of ³HTdR incorporation did not increase markedly in the course of 10 weeks. In these terms, the toxicity of 30 % heavy water in the drinking fluid appears minor. This is of particular interest since exposure to similar concentrations is known to elicit immunodepressive and/or antineoplastic effects.     

Turnover of inositol phosphates in brain during ischemia-induced breakdown of polyphosphoinositides

Using either [³²]ATP or [³H]inositol as precursors which were injected intraventricularly into rat brain, decapitative ischemic treatment resulted in a more rapid loss of labeled phosphatidylinositol 4,5-biphosphates than phosphatidylinositol 4-phosphates in the initial 30 s-1 min. When polyphosphoinositides were labeled with [³H]inositol, the breakdown of these compounds was accompanied by a time-dependent appearance of labeled inositol phosphates. Although the level of radioactivity of inositol trisphosphate was low, a peak labeling activity was shown at 30 s. The radioactivity of inositol bisphosphate showed an increase after a delay of 30 s, and reached a peak at 1 min before declining to the baseline level at 5 min. There was also a lag period of 30 s for the appearance of labeled inositol monophosphate, after which the radioactivity continued to increase in a biphasic manner for the entire 5 min period. Results indicate that decapitative ischemic treatment to rats can serve as an experimental model for assessing in vivo stimulation of the receptor-mediated signal transduction mechanism related to polyphosphoinositide breakdown and subsequent turnover of inositol phosphates in brain.     

Disturbance of lymphocyte circulation byPseudomonas aeruginosa infection in oxazolone-sensitized mice

Pseudomonas aeruginosa infection depresses contact sensitivity to oxazolone in mice. To test whether an altered lymphocyte circulation plays a role in this depression⁵¹Cr-labeled lymphocytes fromP. aeruginosa-infected and oxazolone-sensitized donors were injected intravenously into infected and sensitized recipients, and the radioactivity uptake of several organs was determine. The controls consisted of normal mice receiving labeled lymphocytes from normal donors. While the radioactivity recovered from the liver, spleen, and mesenteric lymph nodes was similar in the test and the control group, significantly more radioactivity was recovered from the draining lymph nodes of infected and sensitized recipients. The concentration of labeled lymphocytes from sensitized donors in the draining lymph nodes of sensitized recipients was 18% greater than that of the controls but 31% lower than that of infected and sensitized animals receiving cells from infected and sensitized donors.P. aeruginosa infection enhances lymphocyte entrapment within the draining lymph nodes of oxazolone-sensitized mice.     

Degradation of horseradish peroxidase after microinjection into mammalian cells

Horseradish peroxidase (HRP) has been microinjected into mammalian cells in tissue culture by the erythrocyte ghost-mediated technique. This protein was selected because it can be localized and quantified after injection by cytochemical and spectrophotometric methods. HRP labeled by reductive methylation retained full catalytic activity, was efficiently loaded into erythrocyte ghosts, and did not associate to a significant degree with ghost membranes. A combination of cytochemical staining and autoradiography established that HRP injected into rat L6 myoblasts, HE(39)L human diploid fibroblasts, or HeLa cells was intracellular and uniformly distributed throughout the cell, while cell lysis techniques showed that the catalytically active HRP was not membrane bound. Inactivation of labeled HRP after injection paralleled the disappearance of the 40-kDa polypeptide, and was always more rapid than its overall degradation. This difference was associated with a pool of water-insoluble radioactivity in the injected cells. This material was of smaller molecular size than the native protein: many labeled peptides were detected in the range of 10 to 38 kDa. By the use of inhibitors of autophagic proteolysis or lysosomal function it was established that HRP degradation was not subjected quantitatively to the same regulatory processes as the average endogenous protein labeled in the same cultures.     

Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells

We have studied the hepatic uptake of retinol bound to the circulating retinol binding protein-transthyretin complex. Labeled complex was obtained from the plasma of donor rats that were fed radioactive retinol. When labeled retinol-retinol binding protein-transthyretin complex was injected intravenously into control rats, about 45% of the administered dose was recovered in liver after 56 h. Parenchymal liver cells were responsible for an initial rapid uptake. Perisinusoidal stellate cells initially accumulated radioactivity more slowly than did the parenchymal cells, but after 16 h, these cells contained more radioactivity than the parenchymal cells. After 56 h, about 70% of the radioactivity recovered in liver was present in stellate cells. For the first 2 h after injection, most of the radioactivity in parenchymal cells was recovered as unesterified retinol. The radioactivity in the retinyl ester fraction increased after a lag period of about 2 h, and after 5 h more than 60% of the radioactivity was recovered as retinyl esters. In stellate cells, radioactivity was mostly present as retinyl esters at all time points examined. Uptake of retinol in both parenchymal cells and stellate cells was reduced considerably in vitamin A-deficient rats. Less than 5% of the injected dose of radioactivity was found in liver after 5-6 h (as compared to 25% in control rats), and the radioactivity recovered in liver from these animals was mostly in the unesterified retinol fraction. Studies with separated cells in vitro suggested that both parenchymal and stellate cells isolated from control rats were able to take up retinol from the retinol-retinol binding protein-transthyretin complex. This uptake was temperature dependent.     

Autoradiographic studies with 3H 1,25 dihydroxyvitamin D3 in thyroid and associated tissues of the neck region

Rats and mice fed a vitamin D-deficient or vitamin D-complete diet were injected with 3H 1,25 (OH)2 vitamin D3. Autoradiograms prepared from cross sections through the neck region revealed nuclear concentration of radioactivity strongest in parathyroid chief cells, occasionally in thyroid follicular epithelial and interfollicular cells, in the epithelium of tubular remnants of the ultimobranchial body, in epithelium of the esophagus, in chondrocytes of tracheal cartilage, and in myoepithelial cells of tracheal glands. In the thyroid, most of the follicle epithelial cells did not show nuclear concentration of radioactivity which occurred only occasionally and predominantly in follicles located in marginal positions. Thyroglobulin in lumina of thyroid follicles contained varying amounts of radioactivity that correspond to the diameter of the follicles, with relatively high amounts in large follicles and little or no radioactivity in small follicles. Competition with excess of unlabeled 1,25 (OH)2 vitamin D3 abolished nuclear radioactivity, but not the radioactivity in the colloid, while 25 (OH) vitamin D3 did not affect either. When a combination of autoradiography and immunohistochemistry was applied, follicular and parafollicular C-cells positive for calcitonin antibodies, did not show nuclear concentration of radioactivity. Tubular remnants of ultimobranchial bodies, however, showed distinct nuclear labeling, but did not stain, or only weakly stain, with antibodies to calcitonin. When 3H 25 (OH) vitamin D3 was injected, no nuclear concentration of radioactivity was noted in any of the tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of exogenous gangliosides GM1 and chemically modified GM1 in mice

Ganglioside GM1(NeuAc), labeled at the C-3 position of sphingosine with tritium, was injected into C3H/He, C57BL/10, B10.AQR mice intraperitoneally. The incorporation and the distribution of the radioactivity in various organs were examined. The injected [3H]GM1(NeuAc) was mainly incorporated in the liver and hydrolyzed sequentially. Sialic acid of ganglioside GM1(NeuAc) and metabolites was converted to N-glycolyl type from N-acetyl type. An appreciable amount of the sphingosine moiety in the administered GM1(NeuAc), moreover, was reutilized, being converted to sphingomyelin, and incorporated into alkyl chain of the ether lipid in phosphatidylethanolamine. The distributions of radioactivity in the metabolites of GM1(NeuAc) administered to the three strains of mice were different from each other. In other organs, GM1(NeuAc) was incorporated and metabolized only slightly. The N-methylamide, at the carboxyl group of the sialic acid, of the labeled ganglioside GM1(GM1(NeuAc)-NMe) was injected into C3H/He mice. Most of the administered [3H]GM1(NeuAc)-NMe was incorporated in the liver, and was metabolized to GM3(NeuAc)-NMe, via GM2(NeuAc)-NMe, within 24 h. GM3(NeuAc)-NMe was the only radioactive compound in the subsequent 10 weeks, but disappeared from the liver gradually. N-Methylamide-modified gangliosides were resistant to hydrolysis by mouse hepatic sialidase, to elongation by glycosyltransferase and to N-glycolylation at N-acetylneuraminic acid by monooxygenase.     

Nepsilon-(3-[*I]Iodobenzoyl)-Lys5-Nalpha-maleimido-Gly1-GEEEK ([*I]IB-Mal-D-GEEEK): a radioiodinated prosthetic group containing negatively charged D-glutamates for labeling internalizing monoclonal antibodies

Novel methods are needed for the radiohalogenation of cell-internalizing proteins and peptides because rapid loss of label occurs after lysosomal processing when these molecules are labeled using conventional radioiodination methodologies. We have developed a radiolabeled prosthetic group that contains multiple negatively charged D-amino acids to facilitate trapping of the radioactivity in the cell after proteolysis of the labeled protein. N(epsilon)-(3-[(125)I]iodobenzoyl)-Lys(5)-N(alpha)-maleimido-Gly(1)-GEEEK ([(125)I]IB-Mal-D-GEEEK) was synthesized via iododestannylation in 90.3 +/- 3.9% radiochemical yields. This radioiodinated agent was conjugated to iminothiolane-treated L8A4, an anti-epidermal growth factor receptor variant III (EGFRvIII) specific monoclonal antibody (mAb) in 54.3 +/- 17.7% conjugation yields. In vitro assays with the EGFRvIII-expressing U87MGDeltaEGFR glioma cell line demonstrated that the internalized radioactivity for the [(125)I]IB-Mal-D-GEEEK-L8A4 conjugate increased from 14.1% at 1 h to 44.7% at 24 h and was about 15-fold higher than that of directly radioiodinated L8A4 at 24 h. A commensurately increased tumor uptake in vivo in athymic mice bearing subcutaneous U87MGDeltaEGFR xenografts (52.6 +/- 14.3% injected dose per gram versus 17.4 +/- 3.5% ID/g at 72 h) also was observed. These results suggest that [(125)I]IB-Mal-d-GEEEK is a promising reagent for the radioiodination of internalizing mAbs.     

Non-random tissue distribution of human naïve umbilical cord matrix stem cells

AIM: To determine the tissue and temporal distribution of human umbilical cord matrix stem (hUCMS) cells in severe combined immunodeficiency (SCID) mice. METHODS: For studying the localization of hUCMS cells, tritiated thymidine-labeled hUCMS cells were injected in SCID mice and tissue distribution was quantitatively determined using a liquid scintillation counter at days 1, 3, 7 and 14. Furthermore, an immunofluorescence detection technique was employed in which anti-human mitochondrial antibody was used to identify hUCMS cells in mouse tissues. In order to visualize the distribution of transplanted hUCMS cells in H&E stained tissue sections, India Black ink 4415 was used to label the hUCMS cells. RESULTS: When tritiated thymidine-labeled hUCMS cells were injected systemically (iv) in female SCID mice, the lung was the major site of accumulation at 24 h after transplantation. With time, the cells migrated to other tissues, and on day three, the spleen, stomach, and small and large intestines were the major accumulation sites. On day seven, a relatively large amount of radioactivity was detected in the adrenal gland, uterus, spleen, lung, and digestive tract. In addition, labeled cells had crossed the blood brain barrier by day 1. CONCLUSION: These results indicate that peripherally injected hUCMS cells distribute quantitatively in a tissue-specific manner throughout the body.     

Hepatic processing and biliary secretion of the cholesteryl esters from beta very-low-density lipoproteins in the rat

beta-Migrating very-low-density lipoproteins (beta-VLDL) are cholesteryl-ester-enriched lipoproteins which accumulate in the serum of cholesterol-fed animals or patients with type III hyperlipoproteinemia. In the rat, beta-VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, beta-VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after beta-VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded. It is concluded that beta-VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from beta-VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from beta-VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low-density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.     

Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands

It has been postulated that the stem cells of somatic tissues protect themselves from mutation and cancer risk by selective segregation of their template DNA strands. Self-renewing mammary epithelial stem cells that were originated during allometric growth of the mammary ducts in pubertal females were labeled using [3H]-thymidine (3HTdR). After a prolonged chase during which much of the branching duct morphogenesis was completed, 3HTdR-label retaining epithelial cells (LREC) were detected among the epithelium of the maturing glands. Labeling newly synthesized DNA in these glands with a different marker, 5-bromodeoxyuridine (5BrdU), resulted in the appearance of doubly labeled nuclei in a large percentage of the LREC. By contrast, label-retaining cells within the stroma did not incorporate 5BrdU during the pulse, indicating that they were not traversing the cell cycle. Upon chase, the second label (5BrdU) was distributed from the double-labeled LREC to unlabeled mammary cells while 3HTdR was retained. These results demonstrate that mammary LREC selectively retain their 3HTdR-labeled template DNA strands and pass newly synthesized 5BrdU-labeled DNA to their progeny during asymmetric divisions. Similar results were obtained in mammary transplants containing self-renewing, lacZ-positive epithelial cells suggesting that cells capable of expansive self-renewal may repopulate new mammary stem cell niches during the allometric growth of new mammary ducts.     

The sites of catabolism of murine monomeric IgA

The tissue sites of monomeric IgA (mIgA) catabolism were determined in a BALB/c mouse model. Mouse mIgA myeloma proteins were labeled either by direct iodination or by coupling the residualizing label, dilactitol-125I-tyramine (125I-DLT) to the proteins; catabolites from protein labeled with 125I-DLT accumulate at the site of protein degradation, allowing identification of the tissue and cellular sites involved in catabolism of the protein. The circulating half-lives of 125I- and 125I-DLT-mIgA were the same. The distribution of radioactivity in tissues was measured at 1, 3, 24, and 96 h after iv. injection of 125I-DLT-labeled mIgA, dimeric IgA (dIgA), IgG, or mouse serum albumin. The greatest uptake of 125I-DLT-mIgA was attributable to the liver. This organ accounted for more internal catabolism of mIgA than all other tissues combined. In contrast, 125I-DLT-IgG was catabolized equally in skin, muscle, and liver. These data indicate that, in mice, the liver is the major site of mIgA catabolism. To determine the cell types involved, collagenase digestion was used to isolate parenchymal and non-parenchymal cells from perfused liver of animals injected with 125-DLT-mIgA. Most of the radioactivity was associated with the hepatocyte fraction, even though both cell types showed uptake of 125I-DLT-mIgA. Inhibition studies, with asialofetuin and mouse IgA demonstrated that the uptake of mIgA by liver cells was mediated primarily by the asialoglycoprotein receptor.     

Hepatic uptake of phospholipid-depleted chylomicrons in vivo. Comparison with the uptake of chylomicron remnants.

1. Rats pretreated with Triton WR-1339 to prevent the formation of remnants were injected with [3H]cholesterol-labelled remnants, intact chylomicrons or chylomicrons depleted of most of their surface phospholipids by treatment with phospholipase A2. Within 5 min about 80% of the injected label of remnants and phospholipid-depleted chylomicrons was incorporated into the livers compared with less than 10% of the injected radioactivity of intact chylomicrons. A similar rapid hepatic uptake of radioactivity occurred when rats not pretreated with Triton were injected with [3H]cholesterol-labelled phospholipid-depleted chylomicrons. This rapid hepatic uptake of phospholipid-depleted chylomicrons occurred apparently without any alteration in the apoprotein composition of the particles. 2. The participation of hepatocytes in the uptake of remnants and phospholipid-depleted chylomicrons was examined. Both types of particles were taken up by the hepatocytes. However, small chylomicrons (Sf less than 400) were taken up more efficiently than were large chylomicrons (Sf greater than 400), but neither was taken up as efficiently as the remnants. 3. The results of this study lend support to the hypothesis that phospholipid-depleted chylomicrons and chylomicron remnants are taken up by the liver by a similar mechanism, which depends on the loss of surface phospholipids.     

Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets

The addition of thrombin to human platelets prelabeled with 32Pi led to significant loss of radioactivity in phosphatidylinositol 4,5-bisphosphate within 5 s, followed by recovery or even increase by 2 min. Loss of label from phosphatidylinositol phosphate was much less marked. Stimulated loss of label from phosphatidylinositol was not seen, while labeled phosphatidate increased severalfold. The principal labeled water-soluble phosphates observed, in addition to 32Pi and [32P] ATP, co-migrated with inositol diphosphate and inositol triphosphate. This suggests that a pool of polyphosphoinositides is constantly undergoing phosphodiesteratic cleavage and resynthesis. Thrombin addition led to rapid increase in radioactivity in inositol triphosphate, but not in inositol diphosphate. We conclude that this early consequence of the thrombin-platelet interaction is the result of an increase in the phosphodiesteratic cleavage of phosphatidylinositol bisphosphate.     

Murine allogeneic in vivo stem cell homing(,)

Stem cell homing has been studied in syngeneic models and appears to be rapid (<1 h) and dependent on cellular adhesion and migration factors. We utilized a full H2-mismatched transplantation model to determine the basics of allogeneic homing. C57BL/6J Lin-Sca-1+ cells were labeled with CFSE and injected in non-myeloablated BALB/c mice. Fluorescent cell detection was via high-speed FACS analysis. Alternatively, B6.SJL whole bone marrow cells were injected in lethally irradiated BALB/c mice (10 Gy). One, 3, 6, and 24 h after transplant, marrow was harvested and cells were either plated for high proliferative potential colony-forming cell (HPP-CFC) assay or secondarily injected into myeloablated (8 Gy) C57BL/6J mice using 10% competing C57BL/6J marrow. Chimerism was evaluated at 8 weeks. CFSE+ cells were detected in the bone marrow 1, 3, and 6 h after injection. The numbers were moderately lower when compared to syngeneic homing possibly due to strain effect. Conversely, utilizing a surrogate or secondary assay, we observed a decline of secondary engraftment of harvested cells over time, but not of HPP-CFC. Combining experiments and normalizing the 1-h time point to 100% (to allow comparison), we observed a mean relative engraftment of 87 +/- 29%, 72 +/- 21%, 84 +/- 35% of the 1 h level at 3, 6, and 24 h respectively. HPP-CFC assay showed no significant variation as a homing surrogate over 1-6 h. These data indicate a rapid homing into allogeneic recipients with a plateau at 1 h. The decline of secondary engraftability over time may indicate a phenotype alteration of homed cells.     

Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice

The knob domains from the fiber proteins of adenovirus serotypes 2 and 12 were labeled with radioiodine and then injected into the bloodstreams of mice. Knob proteins with functional binding sites for the coxsackie and adenovirus receptor (CAR) were cleared rapidly from the circulation, with radioactivity appearing predominantly in the stomach, while knob mutants unable to bind to CAR remained in the blood circulation for a prolonged period. The clearance of radiolabeled wild-type knob from the blood was slowed by coinjecting an excess of unlabeled wild-type knob protein. An earlier study showed that (99m)Tc-labeled knob protein with intact CAR-binding activity also cleared rapidly from the blood circulation of mice, with radioactivity accumulating predominantly in the liver (K. R. Zinn et al., Gene Ther. 5:798-808, 1998). Together these results suggest that rapid clearance of knob protein from the blood results from specific binding to CAR in the liver and that the bound knob then enters a degradative pathway. The elevated levels of radioiodine in the stomach observed in our experiments are consistent with deiodination of labeled knob by dehalogenases in hepatocyte microsomes and uptake of the resultant free radioiodine by Na/I symporters in the gastric mucosa. Although CAR has been shown to localize in tight junctions of polarized epithelial cells, where it functions in intercellular adhesion, the results of our study suggest that a subset of CAR molecules in the liver is highly accessible to ligands in the blood and able to rapidly deliver bound ligand to an intracellular degradative compartment.     

Secretagogue-responsive and -unresponsive pools of phosphatidylinositol in pancreatic islets

The effect of glucose on phosphatidylinositol turnover was studied. Phosphatidylinositol of rat pancreatic islets was labeled with myo[2-3H]inositol in the presence of various secretagogues (16.7 mM D-glucose, 22 mM D-mannose, 20 mM D-glyceraldehyde) and nonsecretagogues (3.3 mM D-glucose, 20 mM pyruvate, 16.7 mM D-galactose, 16.7 mM L-glucose). Upon subsequent stimulation with 16.7 mM D-glucose, only the islets that were labeled in the presence of secretagogues showed a loss of radioactivity from phosphatidylinositol. No loss of radioactivity from phosphatidylinositol occurred in the presence of 3.3 mM D-glucose even after labeling in the presence of secretagogues. A comparison of the subcellular distribution of labeled phosphatidylinositol in islets before and after stimulation with insulinotropic glucose revealed a loss of radioactivity from the plasma membrane fraction as judged by subcellular fractionation with a sucrose gradient. These results support a hypothesis advanced previously that pancreatic islets contain a unique pool of phosphatidylinositol that undergoes rapid turnover only in the presence of insulinotropic concentrations of D-glucose or other secretagogues [R. S. Rana, R. J. Mertz, A. Kowlura, J. F. Dixon, L. E. Hokin, and M. J. MacDonald (1985) J. Biol. Chem. 260, 7861-7867]. On the basis of the subcellular fractionation studies reported here, the secretagogue-responsive phosphatidylinositol pool appears to be located primarily in the plasma membrane of pancreatic islets.     

Serum half-life, distribution, hepatic uptake and biliary excretion of alpha-tocopherol in rats

The serum clearance of alpha-[3H]tocopherol has been studied after intravenous injection of intestinal lymph labeled in vivo with radioactive alpha-tocopherol. The half-life of the injected alpha-[3H]tocopherol was approx. 12 min. Fractionation of plasma by ultracentrifugation 10 min after injection of lymph showed that 91% of the radioactive alpha-tocopherol remaining in plasma was located in chylomicrons (d less than 1.006 g/ml) and 7.8% in high-density lipoproteins (HDL, 1.05 less than d less than 1.21 g/ml). 2 h after administration of alpha-tocopherol, about 35% of the radioactivity recovered in plasma was associated with chylomicrons and approx. 51% with HDLs. alpha-[3H]Tocopherol was initially taken up by the liver, which contained more than 50% of the injected radioactivity after 45-60 min. Separation of parenchymal and nonparenchymal cells demonstrated a preferential uptake of alpha-[3H]tocopherol by the parenchymal liver cells. After 24 h about 11% of the injected dose was recovered in the liver. Considering whole organs the liver, adipose tissue and skeletal muscle had the highest content of radioactivity after 24 h. Furthermore, about 14% of the administered dose was recovered in bile during 24 h draining.