Trichloroacetic Acid-soluble Radioactivity Research Articles

Deficiency of Phospholipase A2 Receptor Exacerbates Ovalbumin-Induced Lung Inflammation

Secretory phospholipase A2 (sPLA2) plays a critical role in the genesis of lung inflammation through proinflammatory eicosanoids. A previous in vitro experiment showed a possible role of cell surface receptor for sPLA2 (PLA2R) in the clearance of extracellular sPLA2. PLA2R and groups IB and X sPLA2 are expressed in the lung. This study examined a pathogenic role of PLA2R in airway inflammation using PLA2R-deficient (PLA2R(-/-)) mice. Airway inflammation was induced by immunosensitization with OVA. Compared with wild-type (PLA2R(+/+)) mice, PLA2R(-/-) mice had a significantly greater infiltration of inflammatory cells around the airways, higher levels of groups IB and X sPLA2, eicosanoids, and Th2 cytokines, and higher numbers of eosinophils and neutrophils in bronchoalveolar lavage fluid after OVA treatment. In PLA2R(-/-) mice, intratracheally instilled [(125)I]-labeled sPLA2-IB was cleared much more slowly from bronchoalveolar lavage fluid compared with PLA2R(+/+) mice. The degradation of the instilled [(125)I]-labeled sPLA2-IB, as assessed by trichloroacetic acid-soluble radioactivity in bronchoalveolar lavage fluid after instillation, was lower in PLA2R(-/-) mice than in PLA2R(+/+) mice. In conclusion, PLA2R deficiency increased sPLA2-IB and -X levels in the lung through their impaired clearance from the lung, leading to exaggeration of lung inflammation induced by OVA treatment in a murine model.

Microtubules Support Production of Starvation-induced Autophagosomes but Not Their Targeting and Fusion with Lysosomes

Autophagy is a major catabolic pathway in eukaryotic cells whereby the lack of amino acids induces the formation of autophagosomes, double-bilayer membrane vesicles that mediate delivery of cytosolic proteins and organelles for lysosomal degradation. The biogenesis and turnover of autophagosomes in mammalian cells as well as the molecular mechanisms underlying induction of autophagy and trafficking of these vesicles are poorly understood. Here we utilized different autophagic markers to determine the involvement of microtubules in the autophagic process. We show that autophagosomes associate with microtubules and concentrate near the microtubule-organizing center. Moreover, we demonstrate that autophagosomes, but not phagophores, move along these tracks en route for degradation. Disruption of microtubules leads to a significant reduction in the number of mature autophagosomes but does not affect their life span or their fusion with lysosomes. We propose that microtubules serve to deliver only mature autophagosomes for degradation, thus providing a spatial barrier between phagophores and lysosomes.

Regulation of Intracellular Accumulation of Mutant Huntingtin by Beclin 1

Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.

Insulin-like growth factor-I inhibits dexamethasone-induced proteolysis in cultured L6 myotubes through PI3K/Akt/GSK-3β and PI3K/Akt/mTOR-dependent mechanisms

We and others reported previously that IGF-I inhibits dexamethasone-induced proteolysis in cultured L6 myotubes. Recent evidence suggests that this effect of IGF-I at least in part reflects PI3K/Akt-mediated inhibition of Foxo transcription factors. The potential role of other mechanisms, downstream of PI3K/Akt, is not well understood. Here we tested the hypothesis that PI3K/Akt-mediated inactivation of GSK-3β and activation of mTOR contribute to the anabolic effects of IGF-I in dexamethasone-treated myotubes. Cultured L6 myotubes were treated with 1 μM dexamethasone in the absence or presence of 0.1 μg/ml of IGF-I and inhibitors of GSK-3β and mTOR. Protein degradation was measured by determining the release of trichloroacetic acid soluble radioactivity from myotubes that had been prelabeled with 3H-tyrosine for 48 h. IGF-I reduced basal protein breakdown rates and completely abolished the dexamethasone-induced increase in myotube proteolysis. These effects of IGF-I were associated with increased phosphorylation of Akt, GSK-3β, and the mTOR downstream targets p70 S6K and 4E-BP1. The PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin reversed the anabolic effect of IGF-I in dexamethasone-treated myotubes. In addition, the GSK-3β inhibitors LiCl and TDZD-8 reduced protein degradation in a similar fashion as IGF-I. Our results suggest that PI3K/Akt-mediated inactivation of GSK-3β and activation of mTOR contribute to the anabolic effects of IGF-I in dexamethasone-treated myotubes.

Insulin-Like Growth Factor-I Blocks Dexamethasone-Induced Protein Degradation in Cultured Myotubes by Inhibiting Multiple Proteolytic Pathways

In previous studies, insulin-like growth factor-I (IGF-I) inhibited glucocorticoid-induced muscle protein breakdown, but the intracellular mechanisms of this effect of IGF-I are not well understood. The purpose of the present study was to test the hypothesis that IGF-I inhibits multiple proteolytic pathways in dexamethasone-treated cultured L6 myotubes. Myotubes were treated with 1 microM dexamethasone for 6 hours in the absence or presence of 0.1 microg/ml of IGF-I. Protein degradation was determined by measuring the release of trichloroacetic acid-soluble radioactivity from proteins prelabeled with 3H-tyrosine. The contribution of lysosomal, proteasomal-dependent, and calpain-dependent proteolysis to the inhibitory effect of IGF-I on protein degradation was assessed by using inhibitors of the individual proteolytic pathways (methylamine, beta-lactone, and E64, respectively). In addition, the influence of IGF-I on cathepsin B, proteasome, and calpain activities was determined. Treatment of L6 myotubes with dexamethasone resulted in an approximately 20% increase in protein degradation. This effect of dexamethasone was completely blocked by IGF-I. When the different protease inhibitors were used, results showed that IGF-I inhibited lysosomal, proteasomal-dependent, and calpain-dependent proteolysis by 70, 44, and 41%, respectively. Additionally, IGF-I blocked the dexamethasone-induced increase in cathepsin B, proteasome, and calpain activities. The present results suggest that IGF-I inhibits glucocorticoid-induced muscle proteolysis by blocking multiple proteolytic pathways.

Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions

Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1 h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40–60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins.

Insulin and Analogue Effects on Protein Degradation in Different Cell Types: DISSOCIATION BETWEEN BINDING AND ACTIVITY

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activity in vitro and in vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (Lys(B28),Pro(B29)-insulin (LysPro), Asp(B10)-insulin (B10), and Glu(B4),Gln(B16),Phe(B17)-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [(3)H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC(50) of 4.6 x 10(-9) m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC(50) = 4.2 x 10(-11) and 1.6 x 10(-10) m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.

In Vivo Clearance of Ternary Complexes of Vitronectin-Thrombin-Antithrombin Is Mediated by Hepatic Heparan Sulfate Proteoglycans

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

LDL receptor family-dependent and -independent pathways for the internalization and digestion of lipoprotein lipase-associated beta-VLDL by rat vascular smooth muscle cells

Lipoprotein lipase (LPL) promotes the binding and internalization of beta-VLDL (very low density lipoprotein) by many cell types. We examined the function of receptors in the LDL receptor family (LRF) and heparan sulfate proteoglycans (HSPG) in the metabolism of LPL-associated beta-VLDLa by rat vascular smooth muscle cells (VSMCs) in culture. These cells express LDL receptor-related protein and the VLDL receptor, but not the LDL receptor. LPL greatly increased the binding of 125I-labeled beta-VLDL to VSMCs at 4 degrees C. Binding was almost entirely inhibited by heparin, but essentially unaffected by the potent LRF-antagonist, receptor-associated protein (RAP), indicating that LRFs do not contribute significantly to the VSMC binding capacity for LPL-associated beta-VLDL. At 37 degrees C, RAP inhibited the rapid internalization of LPL-associated 125I-labeled beta-VLDL and the digestion of the beta-VLDL into trichloroacetic acid soluble radioactivity; these processes still occurred, but at a decreased rate. RAP did not inhibit the ability of beta-VLDL-LPL complex to stimulate VSMC ACAT activity. Furthermore, in Oil red-O histochemistry experiments, which model foam cell transformation in vitro, RAP paradoxically increased cholesteryl ester storage in VSMCs treated with beta-VLDL and LPL under specific cell culture conditions. These results support a model in which the internalization of LPL-associated beta-VLDL by VSMCs is mediated by two pathways, one involving LRFs and a second that is independent of LRFs, probably involving direct uptake by HSPG. The LRF-dependent pathway leads to less cellular storage of cholesteryl ester and thus may be antiatherogenic under certain conditions.

Epidermal Growth Factor and Platelet-derived Growth Factor-BB Induce a Stable Increase in the Activity of Low Density Lipoprotein Receptor-related Protein in Vascular Smooth Muscle Cells by Altering Receptor Distribution and Recycling

Low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor, expressed by vascular smooth muscle cells (VSMCs) in normal arteries and in atherosclerotic lesions. In this investigation, we demonstrate a novel mechanism for the regulation of LRP activity in cultured rat aortic VSMCs. Cells that were treated with platelet-derived growth factor-BB (PDGF-BB) or epidermal growth factor (EGF) for 24 h bound increased amounts of the LRP ligand, activated alpha2-macroglobulin (alpha2M), at 4 degrees C. The Bmax for activated alpha2M was increased from 56 +/- 5 to 178 +/- 24 and 143 +/- 11 fmol/mg cell protein by PDGF-BB and EGF, respectively, while the KD was unchanged. Northern and Western blot analyses demonstrated that neither PDGF-BB nor EGF increase LRP mRNA or protein levels. Instead, LRP was redistributed to the cell surface and remained localized primarily in coated pits, as determined by surface protein biotinylation, affinity labeling, and immunoelectron microscopy studies. The increase in cell-surface LRP was partially explained by a 50% decrease in receptor endocytosis rate; however, at 37 degrees C, PDGF-BB- and EGF-treated VSMCs still bound/internalized increased amounts of activated alpha2M and subsequently released increased amounts of trichloroacetic acid-soluble radioactivity. The cytokine-induced shifts in LRP subcellular distribution were stable when VSMCs were challenged with a saturating concentration of ligand and then incubated, in the absence of cytokine, for 2.5 h at 37 degrees C. Regulation of LRP distribution and activity may be an important aspect of the VSMC response to the atherogenic cytokines, PDGF-BB and EGF.

Stability of Gastrin in the Gastrointestinal Lumen of Suckling, Weanling and Adult Pigs

It has been reported in the literature that a large quantity of gastrin is released into the gastric lumen in various species. This study was aimed to examine the stability of gastrin in the gastrointestinal (GI) lumen of pigs. Iodine-labelled little (G17) and big gastrin (G34) were incubated in vitro with the GI luminal fluids of suckling, weanling and adult pigs at 37 degrees C for 20 min, and the degradation of the peptide was measured by monitoring the generation of trichloroacetic acid soluble radioactivity. The degradation rate of G17 in the gastric fluids of all animals was less than 10%, while the degradation of G34 was less than 15% in the gastric fluids of suckling and adult pigs and about 25% in the gastric fluids of weanling pigs. The degradation rates of G17 and G34 in the small intestinal fluids of suckling pigs were between 18 and 30%, and were significantly lower than the corresponding rates in the intestinal fluids of weanling and adult pigs, the latter were between 35 and 67%. Addition of defatted porcine colostrum or its components, the casein or acid-soluble fraction, inhibited gastrin degradation in the intestinal fluids with the casein fraction having highest inhibition potency. These results indicate that gastrin is stable in the GI lumen of the suckling pigs and porcine colostrum protects gastrin from luminal hydrolysis in the small intestine, suggesting a potential physiological role of luminally released gastrin in suckling animals.

Stimulation of “Prohormone Thiol Protease” (PTP) and [Met]Enkephalin by Forskolin.: BLOCKADE OF ELEVATED [Met]ENKEPHALIN BY A CYSTEINE PROTEASE INHIBITOR OF PTP

Proenkephalin and other prohormones require proteolytic processing at paired basic and monobasic residues for the biosynthesis of active neuropeptides. The novel "prohormone thiol protease" (PTP) has been proposed as a candidate proenkephalin processing enzyme for the production of [Met]enkephalin in chromaffin granules (Krieger, T. J., and Hook, V. Y. H. (1991) J. Biol. Chem. 266, 88376-8383). In this study, PTP was examined during elevation of cellular [Met]enkephalin by forskolin, a direct activator of adenylate cyclase that produces cAMP. Treatment of chromaffin cells with forskolin for 72 h increased enkephalin precursor cleaving activity (measured by following the conversion of the model substrate [35S-Met]preproenkephalin to trichloroacetic acid-soluble radioactivity) in isolated chromaffin granules by 170-180% over controls (100%). The increased activity was associated with the membrane fraction, rather than the soluble fraction, of chromaffin granules. The elevated activity was inhibited by E-64c, which is a potent inhibitor of PTP and cysteine proteases; however, the activity was not inhibited by serine or aspartic protease inhibitors. The elevated activity was identified as PTP based on immunoprecipitation by anti-PTP immunoglobulins. Stimulation of PTP synthesis was involved in the forskolin-induced increase in PTP activity, as demonstrated by a 10-fold increase in [35S]PTP pulse labeling in forskolin-treated chromaffin cells. Forskolin elevation of PTP protein levels within chromaffin granules was also detected in Western blots. Importantly, the forskolin-mediated rise in cellular [Met]enkephalin levels was completely blocked when cells were preincubated with the cysteine protease inhibitor Ep453, which is known to be converted by intracellular esterases to the more effective inhibitor E-64c (Buttle, D. J., Saklatvala, J., Tamai, M., and Barrett, A. J. (1992) Biochem. J. 281, 175-177). Both E-64c and Ep453 inhibit PTP, with E-64c being more potent (Azaryan, A. V., and Hook, V. Y. H. (1994b) Arch. Biochem. Biophys. 314, 171-177). These results demonstrate a role for PTP in proenkephalin processing in chromaffin cells and indicate that [Met] enkephalin formation and PTP are both regulated by cAMP.

Metabolic Fate of Human Urinary Kallidinogenase(SK-827) (5): Metabolism in Rats.

The metabolism of SK-827 was s tudied by gel filtration of plasma, urine, bile and liver homogenate after intravenous injection of 125I-SK-827 in rats. 1. Gel filtration (Sephacryl S-300 superfine) chromatograms of radioactivity in plasma, obtained after intravenous injection of 125I-SK-827, demonstrated that the radioactivity in plasma was mainly in the form of 125I-SK-827 bound to an inhibitor and free 125I-SK-827. The elimination half-lives of 125I-SK-827-inhibitor complex and free 125I-SK-827 in the plasma were calculated to be 5.8 min and 39 min, respectively. 2. Gel filtration chromatograms of radioactivity in the liver demonstrated that the radioactivity in the liver was mainly in the form of 125I-SK-827 bound to an inhibitor and free 125I-SK-827. The half-lives of 125I-SK-827-inhibitor complex and free 125I-SK-827 in the liver were calculated to be 6.6 min and 10.2 min, respectively. 3. Appearance of trichloroacetic acid soluble (TCA-soluble) radioactivity during incubation at 37°C for 2 hr of the liver slices obtained at 5, 15 and 30 min after intravenous injection of 125I-SK-827 were 9%, 39% and 51% of the radioactivity present in liver slice before incubation, respectively. This result suggests that the degradation of injected 125I-SK-827 occurred in the liver. It was found that TCA-soluble degradation product in liver slices was mainly (90%) inorganic iodine. 4. Gel filtration chromatogram of radioactivity in urine within 1 hr after injection demonstrated that the radioactivity excreted to the urine was in the form of free 125I-SK-827 (16.8% of total radioactivity) and degraded product. No radioactivity related to the 125I-SK-827-inhibitor complex was observed. 5. Gel filtration chromatogram of radioactivity in bile at 1 hr after injection demonstrated that the radioactivity in bile was in the form of 125I-SK-827 bound to inhibitors (51.2%), free 125I-SK-827 (34.3%) and degraded product (14.6%). 6. From these results, it became clear that 125I-SK-827 administered intravenously was bound by an inhibitor, and complex was cleared from the plasma by the liver. The complex taken up by the liver was rapidly degraded into low molecular weight compounds. TCA-insoluble radioactivity detected in the liver at 24 hr after administration were considerded to be endogenous high molecular weight compounds into which TCA-soluble radioactivity produced by degradation of 125I-SK-827 was incorporated.

Inhibition of mouse embryonic, yolk sac, and limb-bud functions by the methyl isocyanate metabolite S-(N-methylcarbamoyl)glutathione.

We previously reported that S-(N-methylcarbamoyl)glutathione (SMG), a conjugate formed by the reversible reaction between methyl isocyanate and glutathione, inhibited the development of mouse embryos in culture. The present study was done to determine whether SMG produced embryotoxicity by inhibiting yolk sac functions. For this purpose we determined the effects of an embryotoxic concentration of SMG on mouse yolk sac uptake mechanisms and lysosomal proteolysis as well as on the incorporation of [3H]leucine in mouse embryonic and limb-bud proteins. After 5 h of culture, SMG inhibited the uptake of [14C]sucrose and 125I-labelled bovine serum albumin in isolated day 15 yolk sacs to 62 and 77% of control, respectively. Lysosomal proteolysis was not inhibited, as judged by the release of trichloroacetic acid soluble radioactivity into the culture media. Uptake and incorporation of [3H]leucine from free [3H]leucine or from [3H]leucine-labelled protein in SMG-treated day 9 embryos were inhibited, respectively, to 61 and 25% of the control uptake during a 16-h labelling period. SMG also inhibited the incorporation of free [3H]leucine into limb-bud proteins. SMG-induced inhibition of 125I-labelled bovine serum albumin uptake by yolk sacs was partially prevented by the thiol donors N-acetylcysteine and glutathione but not by acivicin (gamma-glutamyl transpeptidase inhibitor) and aminooxyacetic acid (cysteine conjugate beta-lyase inhibitor). These data suggest that SMG suppresses embryonic growth primarily by an inhibition of nutrient uptake by the yolk sac. We postulate that this inhibition is due to tissue carbamoylation by methyl isocyanate released from SMG.

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin‐like growth factor‐I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [3H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [3H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [3H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 +/- 0.03 nmol/l and 34.8 +/- 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 mumol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells.

Parathyroid hormone transport effects and hormonal processing in primary cultured rat proximal tubular cells.

The development of satisfactory cell culture models for the study of parathyroid hormone (PTH)-induced inhibition of Pi transport has proven difficult. Using subcellular fractionation techniques we investigated the response of primary cultures of rat proximal tubular cells to PTH-(1-34). Specific binding of 125I-bPTH-(1-34) occurred at 2 degrees C. After 5 min of rewarming, trypsin-releasable radioactivity decreased from 90 to 50%, indicating internalization of the ligand. Cell disruption, followed by density centrifugation with 17% Percoll either directly after binding at 2 degrees C or post-rewarming for 20 min, showed a shift of 125I label from the plasma membrane (5'-nucleotidase) to lysosomal fractions (beta-D-glucosaminidase), confirming the sequential occurrence of cell surface binding, internalization and transport to lysosomes of 125I-bPTH-(1-34). Reculture at 37 degrees C revealed steady accumulation of trichloroacetic acid-soluble radioactivity in the medium, indicating degradation of 125I-bPTH-(1-34). Phosphate transport in the absence of sodium was minimal. Incubation of the cells with bPTH-(1-34) resulted in up to 50% inhibition of sodium-dependent phosphate transport. Prior phosphate depletion abrogated the response to PTH.

Receptor-mediated endocytosis of vitronectin is regulated by its conformational state

Vitronectin is a structurally labile molecule with a native, non-heparin binding form and a conformationally altered, heparin binding form. To understand the physiological significance of the two conformers of vitronectin, we examined the metabolism of both conformers by cultured human skin fibroblasts. Both native and altered vitronectin bound to confluent fibroblast monolayers. Both conformers of vitronectin competed equally well for the binding of altered vitronectin to the cell layer, suggesting that both conformers bound to the same site in the cell layer. In contrast, 125I-altered vitronectin, but not 125I-native vitronectin, was degraded to trichloroacetic acid-soluble radioactivity by the fibroblast monolayer. Degradation of vitronectin was saturable, sensitive to chloroquine, and occurred intracellularly, suggesting that vitronectin was degraded through a lysosomal pathway. Heparin and thrombospondin inhibited the degradation of altered vitronectin. The degradation of native vitronectin was induced by addition of gamma-thrombin which exposes vitronectin's cryptic heparin-binding domain. These studies suggest that the heparin-binding domain in vitronectin is required for the clearance of vitronectin from the matrix. In addition, these data demonstrate that the conformation of vitronectin regulates its half-life in the matrix. These studies provide the first evidence for a distinct function for the conformers of vitronectin.

Characterization of a high affinity binding site for pancreatic-type phospholipase A2 in the rat. Its cellular and tissue distribution.

We showed in an earlier study (Arita, H., Hanasaki, K., Nakano, T., Oka, S., Teraoka, H., and Matsumoto, K. (1991) J. Biol. Chem. 266, 19139-19141) that there is a high affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) in Swiss 3T3 fibroblast cells. Analysis of the cellular distribution in rat using 125I-PLA2-I as a radioligand indicated the presence of this site in various cells, including vascular smooth muscle cells (VSMC), vascular endothelial cells, synovial cells, chondrocytes, and gastric mucosal cells. Scatchard analysis of rat VSMC revealed the existence of a single class of binding site with a Kd value of 1.60 nM and a Bmax value of 51.0 fmol/10(6) cells. The mammalian mature type of PLA2s-I derived from several animal species specifically recognized the same site in cells and stimulated DNA synthesis, whereas its inactive zymogen, mammalian group II PLA2s, and snake and bee venom PLA2s showed much lesser activities. 125I-PLA2-I bound to VSMC was rapidly internalized and subsequently released from the cells as trichloroacetic acid-soluble radioactivity. Down-regulation of the PLA2-I site was observed in the treatment of VSMC with cAMP-elevating agents, as well as glucocorticoids. Affinity labeling experiments indicated that PLA2-I binds to a single polypeptide with a mass of approximately 200,000 daltons. These results suggest a novel PLA2-I action on the cellular function via its specific binding site.

Intracellular catabolism of radiolabeled anti-CD3 antibodies by leukemic T cells

The endocytosis and intracellular metabolism of radiolabeled anti-CD3 MoAb 64.1 by the malignant human T cell line HPB-ALL were studied using biochemical, morphological, electrophoretic, and Chromatographic techniques. Biosynthetically labeled [ 3H]64.1 and externally radioiodinated 125I-64.1 were similarly internalized and degraded by tumor cells, with approximately 70% of the initially bound radioactivity being released to the culture supernatant as trichloroacetic acid-soluble radioactivity in the first 24 hr of culture. Radiolabeled 64.1 was routed from the cell membrane to endosomes where initial proteolysis began and finally to lysosomes where terminal catabolism to single amino acids occurred. SDS-PAGE demonstrated four major intracellular metabolite species (46, 25, 15, and <10 kDa). Thin-layer chromatography demonstrated that >95% of the trichloroacetic acid-soluble radioactivity in culture supernatants was 125I-monoiodotyrosine, indicating that proteases, not deiodinases, were of primary importance in catabolism of 125I-64.1. In the presence of inhibitors of lysosomal function (leupeptin, monensin, and ammonium chloride), 125I-64.1 degradation was impeded, causing prolonged retention of radioactivity in the lysosomal compartment of cells. However, although the pace of catabolism was markedly diminished by these agents, no major changes in the sizes of intermediate metabolites generated were observed. Our results suggest that judicious administration of lysosomal inhibitors (e.g., chloroquine, verapamil, monensin) may significantly enhance retention of radioimmunoconjugates by lymphoid malignancies, improving radioimmunoscintigraphic and radioimmunotherapeutic efforts.

Plasma clearance and tissue binding of rANP [99–126] and iso-rANP [1–45] in the rat

Plasma clearance and tissue binding of atrial natriuretic peptide (ANP) and iso-ANP were compared in Inactin-anaesthetized rats. It was found that the plasma half-life of iso-ANP was comparable to ANP. Appearance of trichloroacetic acid-soluble radioactivity of iso-ANP in the plasma was considerably slower than that of ANP, suggesting that the metabolic process of these two peptides may be different. Although the binding distribution of these two peptides was similar, the total binding of iso-ANP to organs other than the kidney was much lower. The kidney, lung, heart and adrenal gland appeared to be major target organs for iso-ANP. Autoradiography showed that iso-ANP bound specifically to the renal glomerulus and proximal part of the proximal tubule. This latter binding site in the kidney was not apparent with ANP, suggesting that iso-ANP may exerts its physiological action at different sites in this organ.