Trichloroacetic Acid-precipitable Radioactivity Research Articles

Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.

Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion

The mechanism of thyroid hormone (TH) secretion from the thyroid gland into blood is unknown. Humans and mice deficient in monocarboxylate transporter 8 (MCT8) have low serum thyroxine (T4) levels that cannot be fully explained by increased deiodination. Here, we have shown that Mct8 is localized at the basolateral membrane of thyrocytes and that the serum TH concentration is reduced in Mct8-KO mice early after being taken off a treatment that almost completely depleted the thyroid gland of TH. Thyroid glands in Mct8-KO mice contained more non-thyroglobulin-associated T4 and triiodothyronine than did those in wild-type mice, independent of deiodination. In addition, depletion of thyroidal TH content was slower during iodine deficiency. After administration of 125I, the rate of both its secretion from the thyroid gland and its appearance in the serum as trichloroacetic acid-precipitable radioactivity was greatly reduced in Mct8-KO mice. Similarly, the secretion of T4 induced by injection of thyrotropin was reduced in Mct8-KO in which endogenous TSH and T4 were suppressed by administration of triiodothyronine. To our knowledge, this study is the first to demonstrate that Mct8 is involved in the secretion of TH from the thyroid gland and contributes, in part, to the low serum T4 level observed in MCT8-deficient patients.

Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Alzheimer disease beta-amyloid (Abeta) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and gamma-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Abeta production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Abeta. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

Modifications of H3 and H4 during Chromatin Replication, Nucleosome Assembly, and Histone Exchange

Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the "histone code" associated with chromatin replication and dynamic histone exchange in human cells.

Functionally Independent AU-rich Sequence Motifs Regulate KC (CXCL1) mRNA

Certain pro-inflammatory chemokine mRNAs containing adenine/uridine-rich sequence elements (AREs) in their 3' untranslated regions (3'-UTRs) are known to exhibit constitutive instability and sensitivity to proinflammatory stimuli resulting in the stabilization of the message. Using tetR-regulated transcription we now show that the 3'-UTR of the mouse CXCL1 (KC) mRNA contains at least two ARE motifs that are structurally and functionally distinct. A fragment of 77 nucleotides containing 4 clustered AUUUA pentamers located at the 5'-end of the KC 3'-UTR is only modestly unstable yet promotes markedly enhanced, post-transcriptional protein production in response to either interleukin-1alpha (IL-1alpha) or lipopolysaccharide (LPS), suggesting translational regulation. In contrast, a fragment containing 3 isolated AUUUA pentamers corresponding to the residual 3' 400 nucleotides of the KC 3'-UTR confers both instability and is stabilized in response to IL-1alpha. Although the clustered AUUUA pentamers in the upstream region are required for stimulus sensitivity, mutation of all three pentamers in the downstream region has little or no effect on either instability or stimulus sensitivity. The upstream region is comparably stabilized in response to either IL-1alpha or LPS, whereas the AUUUA-independent downstream determinant is differentially more sensitive to IL-1alpha. Finally, using UV-induced RNA cross-linking, these functionally independent sequences exhibit different patterns of interaction with RNA-binding proteins. Collectively, these findings document the presence of multiple independent determinants of KC mRNA function and demonstrate that these operate via distinct mechanisms.

Production, in Vitro Characterisation, in Vivo Clearance, and Tissue Localisation of Recombinant Barramundi (Lates calcarifer) Insulin-like Growth Factor II

Recombinant barramundi insulin-like growth-factor-II (bIGF-II) has been produced in Escherichia coli after modification of an expression plasmid that coded for a chicken IGF-II fusion protein. The bIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, and refolded. The protein was then released from the fusion protein by cleavage with subtilisin BPN′. Finally the protein was purified to homogeneity with a number of HPLC steps. In vitro analysis of recombinant bIGF-II demonstrated decreased potency in stimulating protein synthesis when compared to human and barramundi IGF-I (bIGF-I). The in vivo distribution of radiolabeled bIGF-II and bIGF-I in the circulation and tissue uptake of radiolabeled bIGF-II was also compared in juvenile barramundi (Lates calcarifer). Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that bIGF-II was degraded faster than bIGF-I. Moreover, neutral gel chromatography of these samples suggested this difference may be due to reduced affinity of bIGF-II, compared to bIGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi circulation. Based on these results, it would appear that elements important in the function of IGFs have been well conserved during vertebrate evolution. However, to clearly define the IGF system in fish it will be necessary to characterise the IGFBPs present and to determine how they influence the biological actions of native IGFs.

Ionizing-Radiation Resistance in the Desiccation-Tolerant Cyanobacterium Chroococcidiopsis

The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10 Chroococcidiopsis strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of Chroococcidiopsis strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration.

Comparison of Recombinant Barramundi and Human Insulin-like Growth Factor (IGF)-I in Juvenile Barramundi (Lates calcarifer): In Vivo Metabolic Effects, Association with Circulating IGF-Binding Proteins, and Tissue Localisation

The in vivo actions of human and fish insulin-like growth factor (IGF)-I have been compared to extend the understanding of the metabolism of IGFs in fish and to identify potential differences in their actions. The effects of acute administration of these proteins on the incorporation of glucose into muscle glycogen and leucine into liver protein in juvenile barramundi were investigated. In these in vivo metabolic assays, both baramundi IGF-I (bIGF-I) and human IGF-I (hIGF-I) increase the incorporation of d-[14C]glucose into muscle glycogen and [14C]leucine into liver protein. The distribution of radiolabeled human and barramundi IGF-I in the circulation and their uptake by tissue was also compared in juvenile barramundi (Lates calcarifer). Analysis of trichloroacetic acid-precipitable radioactivity in sequential samples following bolus injection of radiolabeled IGFs revealed that hIGF-I was degraded faster than bIGF-I. Neutral gel chromatography of these samples suggested that this difference is due to reduced affinity of hIGF-I, compared to bIGF-I, for the IGF-binding proteins (IGFBPs) present in the barramundi. Tissue uptake of [125I]-labeled hIGF-I and bIGF-I was similar except that [125I]bIGF-I uptake by the kidney exceeded that of hIGF-I. It is suggested that while some of the in vivo actions of IGFs in fish are conserved, functional differences between mammalian and teleostean IGFs exist, particularly with respect to their interactions with fish IGFBPs.

Measurement of muscle protein synthesis by positron emission tomography with L-[methyl-11C]methionine: effects of transamination and transmethylation.

Positron emission tomography with L-[methyl-11C]methionine provides a measure of regional protein synthesis rate (PSR) in skeletal muscle. However, the validity of the method depends on incorporation of methionine into protein with minimal transamination, transmethylation, or both. To test directly these assumptions, uptake of L-[methyl-14C]methionine in skeletal muscle was measured in control and cycloheximide-treated rats. Normal and cycloheximide-treated rats (n = 8/group) were injected with 50 microCi of L-[methyl-14C]methionine and arterial blood sampled over 90 minutes. After killing, thigh muscle was homogenized, centrifuged, and treated with trichloroacetic acid. PSR from circulating methionine was estimated from trichloroacetic acid-precipitable radioactivity, arterial time-activity curves, and plasma methionine concentrations. In normal rats, approximately 70% of the tissue radioactivity was precipitated with trichloroacetic acid. In normal animals, PSR was 0.22 nmoles x min(-1) x g(-1), in excellent agreement with previous results. In the cycloheximide-treated group, PSR was 0.0032 nmoles x min(-1) x g(-1); approximately 98% reduction compared with controls. These studies support the hypothesis that L-[methyl-11(14C]methionine accumulates in skeletal muscle as 11(14)C-labeled protein.

Influence of insulin-like growth factor-binding protein-2 on plasma clearance and transfer of insulin-like growth factors-I and -II from plasma into mammary-derived lymph and milk of goats

Plasma clearance of insulin-like growth factors-I and -II (IGF-I and -II) and insulin-like growth factor-binding protein-2 (IGFBP-2) from lactating goats (n = 4) was determined following a single intravenous injection of the corresponding 125I-labelled human protein. Transfer of these proteins out of the vascular space was monitored by their subsequent appearance in mammary-derived lymph and milk. Clearance of 125I-IGFBP-2 from circulation was 0.37 +/- 0.06 ml/min/kg, which is markedly greater than that of 125I-IGF-I or -II (0.11 +/- 0.01 and 0.12 +/- 0.01 ml/min/kg respectively). This was also reflected in longer elimination half-lives for IGF-I (353 +/- 6 min) and -II (254 +/- 8 min) compared with IGFBP-2 (110 +/- 9 min). Three hours after injection of the 125I-labelled protein, the plasma:lymph ratio of trichloroacetic acid-precipitable radioactivity was 1.54 +/- 0.04, 3.3 +/- 0.6 and 4.1 +/- 0.4 for IGFBP-2, IGF-I and -II respectively. The form of 125I-IGFBP-2 in lymph was not different from that of plasma. Elevation of plasma concentrations of IGFBP-2 by its intravenous infusion significantly decreased plasma half-life of both IGF-I and -II (251 +/- 8 and 198 +/- 7 min respectively). Although the amount and rate of transfer of IGF into mammary-derived lymph was decreased slightly by IGFBP-2, concentrations eventually obtained were not different from control. However, secretion of IGFs into milk was significantly reduced by IGFBP-2, particularly in the case of IGF-I. These results are consistent with the ability of all three compounds to cross the vascular endothelium intact and of IGFBP-2 to decrease the uptake of IGF by mammary epithelium and subsequent secretion into milk. IGFBP-2 may well have acted to target plasma IGF towards non-mammary tissues, thus explaining the more rapid plasma clearance of IGFs in the presence of elevated IGFBP-2.

Patellar tendon and anterior cruciate ligament have different mitogenic responses to platelet-derived growth factor and transforming growth factor beta.

The healing responses of the anterior cruciate ligament and the patellar tendon differ markedly. The anterior cruciate ligament fails to heal, whereas the patellar tendon heals slowly. The basis of these differences is unknown. Since cellular proliferation is a critical element of healing, we investigated the response to explants of anterior cruciate ligament and patellar tendon from sheep knees to platelet-derived growth factor-AB and transforming growth factor beta 1 as a function of time and dose. Explants cultured for 48, 72, and 96 hours with transforming growth factor beta 1 (0-100 ng/ml) or platelet-derived growth factor-AB (0-200 ng/ml) were radiolabeled for the final 24 hours with [3H]thymidine, and DNA synthesis was quantified as trichloroacetic acid-precipitable radioactivity normalized to dry tissue weight. Statistical analyses (analysis of variance) showed that transforming growth factor beta 1 induced a significant proliferative response in the anterior cruciate ligament at 96 hours with equivalent responses at 10, 50, and 100 ng/ml, whereas the patellar tendon only responded to one condition, 10 ng/ml at 96 hours. Conversely, the patellar tendon had a significant dose-dependent response to platelet-derived growth factor-AB at 72 and 96 hours, whereas the anterior cruciate ligament showed no proliferative response to platelet-derived growth factor-AB. The minimal response of anterior cruciate ligament to platelet-derived growth factor-AB could explain, at least in part, the poor repair capacity of this tissue. The response of the anterior cruciate ligament to transforming growth factor beta suggests that exogenous transforming growth factor beta may promote initial healing. Although growth factors have the potential to modulate soft-tissue repair, tissue responses in tendons and ligaments may vary at different anatomic sites.

The pharmacokinetics, tissue localization, and metabolic processing of recombinant human hepatocyte growth factor after intravenous administration in rats.

Hepatocyte growth factor (HGF) is a pluripotent mitogen thought to be involved in liver regeneration. It is synthesized as a single chain promitogen and requires proteolytic processing to a two-chain heterodimeric form for biological activity. The pharmacokinetics and tissue distribution of radioiodinated single chain recombinant human HGF ([125I]rhuHGF) were studied in male Sprague-Dawley rats after an iv bolus dose. Pharmacokinetic parameters were determined from trichloroacetic acid-precipitable radioactivity in serum samples. There was a rapid distribution phase (t1/2 alpha = 3.1 min) and a slower elimination phase (t1/2 beta = 114 min). Tissue distribution was assessed by whole body autoradiography 5, 60, and 1440 min after an iv bolus dose. rhuHGF rapidly distributed to the liver, kidney, adrenal gland, and spleen. The importance of the liver in the rapid clearance and subsequent conversion of single chain pro-rhuHGF to the mitogenically active two-chain form was demonstrated using an isolated rat liver perfusion system. TCA-precipitable radioactivity excreted into the bile (1.0 +/- 0.1%) and released into the venous effluent (38.9 +/- 6.1%) was monitored for 60 min after a portal vein injection. The appearance of radioactivity in both the bile and venous effluent was maximal between 20-35 min. Further characterization of the reduced samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the two-chain form of [125I]rhuHGF was the predominant form after hepatic perfusion. These studies suggest that the liver plays a major role in the rapid clearance and subsequent activation of pro-rhuHGF in vivo.

Clearance of surfactant-like particle proteins from circulation in rats

Surfactant-like particles (SLP) have been isolated from the apical surface of rat enterocytes but appear to arise within the enterocyte and to be secreted into neighboring lymphatics (K. DeSchryver-Kecskemeti, R. Eliakim, S. Carroll, W. F. Stenson, M. A. Moxley, and D. H. Alpers. J. Clin. Invest. 84: 1355-1361, 1989). The present study was undertaken to analyze the clearance and distribution of these particle-associated proteins in vivo in the rat. Labeling of particles isolated from apical scrapings of rat enterocytes with 125I enabled identification of proteins of 116, 92, 68, 58, 48, 31, and 25 kDa. After the intravenous administration of 125I-SLP, the trichloroacetic acid-precipitable radioactivity in the plasma disappeared in a biphasic pattern. The average half-life of the early phase was 7.5 min and the second phase was 75 min. The half-life of a subset of the proteins (116, 92, and 48 kDa) was shorter, measuring 4.5 min for the first phase and ranging from 20 to 52 min for the second phase. The amounts of labeled particle distributed to the liver, kidney, and spleen were much greater than could be accounted for by distribution in the extracellular space alone. The liver accounted for removal of approximately 50% of label in the first phase, with the kidney and spleen contributing 10 and 1%, respectively. Only 0.5 and 2.5% of labeled protein appeared in the bile and urine, respectively. The majority of labeled protein remaining in the serum after the initial phase of tissue uptake migrated at a density of 1.07-1.08, characteristic of intact particles, but slowly shifted to higher densities over 24 h. The maximum binding capacity was 56 nmol/mg membrane protein (Michaelis constant = 51 pM) using hepatic basolateral membranes. The particle was taken up into hepatocytes, as demonstrated by autoradiography. These data demonstrate the importance of the liver in clearing SLP from the blood in rats. The slow clearance of a portion of the particle protein provides support for the continued presence of these proteins in the serum of rats.

Effect of calcium depletion on the secretion of newly synthesised human chorionic gonadotropin by first trimester human placenta

Depletion of calcium in the extracellular medium used to incubate first trimester human placental minces resulted in a significant decrease in the quantity of immuno-reactive hCG in the medium and a corresponding increase in the tissue. In contrast, when secretion of newly synthesised hCG was monitored in the absence of calcium by using a radioactive amino acid precursor, a significant increase in the secretion of newly synthesised hCG in the medium was noticed. This was true of secretion of other proteins also as evidenced by the increase in the trichloroacetic acid precipitable radioactivity in the medium in the absence of calcium. These results suggest that newly synthesised hCG is preferentially released over stored hormone in the absence of calcium.

Distribution of the Recombinant Coagulation Factor 125I-rFVIIa in Rats

Recombinant human factor VIIa (rFVIIa; NovoSeven) is a two-chain activated clotting factor that is used in the treatment of haemophilia. The distribution of radioactivity in male and pregnant and non-pregnant female rats has been examined by whole-body autoradiography (WBA) after single intravenous doses of 125I-radiolabelled rFVIIa at a dosage level of ca. 0.1 mg/kg. Concentrations of radioactivity were highest in the blood and the highly perfused major thoracic and visceral organs and gonads. This distribution of radioactivity was generally similar in pregnant and non-pregnant females, and although radioactivity was concentrated in the foetal thyroid, it was present in other foetal tissues only at trace levels. Radioactivity in thyroid, urinary bladder and gastrointestinal tract of all rats was apparently associated with detached 125I-iodide. At early sacrifice times (up to 2 h), radioactivity was present in the bone marrow, but at later times (6-24 h) it was apparently associated with the mineralised bone structures. The quantitative distribution of total and trichloroacetic acid precipitable radioactivity in the tissues of rats also was studied after single intravenous doses of 125I-rFVIIa and 125I-rFVII, the non-activated single chain precursor of FVIIa, which is normally present in the circulation. These studies confirmed the WBA findings and showed that the tissue distribution of 125I-rFVII and 125I-rFVIIa was similar, indicating that the distribution of rFVIIa during therapy would be similar to that produced from endogenous FVII as a physiological response to vascular injury.

Disposition of rhIGF-I. (3). Plasma level, distribution and excretion of 125I-rhIGF-I after repeated subcutaneous administration to rats.

125I-rhIGF-I was administered subcutaneously to rats at the dose of 1 mg/kg for 7 consecutive days. After seventh subcut aneous administration of 125I-rhIGF-I, the plasma levels of total and trichloroacetic acid (TCA)precipitable radioactivity declined more slowly than those after a single administration. However, it might be caused by an endogenous proteins which had incorporated a free 125I and low molecular weight fragments of 125I-rhIGF-I and the plasma levels of rhIGF-I itself were thought not to be changed after repeated administration. Except the thyroid, the permeability of rhIGF-I to tissues did not change, and no accumulation of 125I-rhIGF-I in any particular tissue was observed after repeated administration. During repeat ed administration, the excretion of radioactivity in urine and feces was almost similar to that after a single administration, and 80% and 9 % of the administered dose was excreted within 168 hours after the last dosing to urine and feces, respectively. Most of radioactivity in t he urine consisted of free 125I which indicates that 125I-rhIGF-I was well metabolized. Thus, it appears that 125I-rhIGF-I does not accumulate in tissue.

Metabolic fate of KRN5702: Plasma level, distribution, metabolism and excretion of 125I-KRN5702 after repeated subcutaneous administration to rats.

The plasma levels, tissue distribution and excretion of 125I-KRN5702 after repeated subcutaneous administration were investigated in male rats. After seventh subcutaneous administration of 125I-KRN5702 at a dose of 200IU/kg, no difference in the plasma level profiles of total and trichloroacetic acid (TCA) precipitable radioactivity was observed, whereas immunoreactive radioactivity showed relatively low level when compared to that after a single administration. Both the area under the plasma concentration vs time curve (AUC) and the maximum level (Cmax) of immunoreactive radioactivity after repeated administration were 17 and 16% lower than that after single administration, respectively. We consider that the increase in utilization of this material by target organs might be responsible for increased plasma clearance. In the majority of tissues, the levels of radioactivity at 8 hours after each dose during repeated administration were same or slightly higher than those after initial administration. High levels of radioactivity were observed in the plasma, kidney and at the site of administration, independently of the numbers of injection. Whereas those in the spleen, bone marrow and thyroids were relatively higher as compared with those of the initial administration. After final administration, the most of tissues showed a maximum level of radioactivity at 8 hours after administration and then decreased in parallel with plasma level in any time interval. Beyond 48 hours after final administration, the radioactivity in the site of injection decreased to below 2 % of that at 2 hours after administration, and no accumulation in any particular organs or tissues was observed in repeated administration of 125I-KRN5702. Excretion of radioactivity in urine and feces was almost constant, i. e. 80 ?? 90% and 4 ?? 60% of each administered dose, respectively, during repeated administration. Finally, within 7th days after final administration, radioactivity excreted in urine and feces were 96.2% and 8.0% of total dosing, respectively.

Importance of the liver in plasma clearance of hepatocyte growth factors in rats.

After intravenous administration of 125I-labeled hepatocyte growth factor (HGF), trichloroacetic acid-precipitable radioactivity in the plasma disappeared rapidly with an early phase half-life of 4 min. The amounts of 125I-HGF distributed to the liver, adrenal, spleen, kidney, and lung tissues were much greater than those that could be accounted for by distribution to the extracellular space alone. The first-pass removal of 125I-HGF by the liver was approximately 26%; the liver accounted for approximately 70% of early-phase removal. The hepatic handling was also analyzed using a single-pass perfused liver system. The steady-state extraction ratio of tracer 125I-HGF was 0.48 but dropped to 0.23 in the presence of excess HGF (135 pM), demonstrating hepatic removal saturation of HGF. In the presence of excess HGF, the heparin-washable 125I-HGF, the heparin-resistant and acid-washable 125I-HGF, and the internalized 125I-HGF dropped to 54, 31, and 32% of the control values. The presence of at least two binding sites for HGF on the liver cell surfaces was made clear: the heparin-washable site and the heparin-resistant and acid-washable binding site, considered to have higher affinity for HGF. The internalization of 125I-HGF was observed to some extent even in the presence of excess HGF and phenylarsine oxide, known to be an inhibitor of polypeptides receptor-mediated endocytosis, suggesting the contribution of a relatively nonspecific internalization mechanism as well as receptor-mediated endocytosis.

Determination of the pharmacokinetic profiles of insulin-like growth factor binding proteins-1 and -2 in rats

The insulin-like growth factor binding proteins (IGFBPs) are present in serum and alter the half-life of IGF-I and II in the vascular compartment. Because both IGFBP-1 and 2 have been proposed as regulators of IGF transport, we directly determined their distribution and elimination characteristics in rats. 125I-IGFBP-1 and 2 were injected into anesthetized normal rats. Multiple blood samples were drawn over time and the trichloroacetic acid precipitable radioactivity used to determine their pharmacokinetic profiles. The mean residence time for IGFBP-1 was 129 +/- 31 min and for IGFBP-2 116 +/- 24 min. The volume of distribution at steady state was 300 +/- 87 ml/kg for IGFBP-1 and 242 +/- 32 ml/kg for IGFBP-2. The elimination half-life was 120 +/- 26 min for IGFBP-1 and 144 +/- 32 for IGFBP-2. The results show that two separate methods of analysis give equivalent results for estimation of half-lives of these forms of IGFBPs and that their half-lives are substantially greater than that reported for free IGF-I, but less than that reported for the 150-kilodalton IGF complex. The findings suggest that these proteins equilibrate with extravascular compartments and may have a role in modulating transvascular transport of IGF-I and II.