TCA-precipitable Radioactivity Research Articles

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.

Labelling of interleukin-2 (IL-2) with 123-iodine with retention of its capacity to bind to activated lymphocytes.

Human recombinant interleukin-2 (IL-2) was labelled with Iodine-123 using modified Bolton and Hunter method. Separation from free iodine was performed by gel filtration chromatography using a Sephadex G10 column. HPLC analysis of labelled IL-2 showed that 98% of TCA precipitable radioactivity eluted in a single peak. The immunoreactivity of 123I-labelled IL-2 was determined by divert binding using the Fluorescence Activated Cell Sorter (FACS) and by receptor binding assay of IL-2 to activated lymphocytes. To demonstrate in vivo binding to activated lymphocytes, 123I-labelled IL-2 was injected intravenously into a newly diagnosed diabetic BB/Wistar rat. Higher radioactivity was detected in the pancreas and in the lymph nodes of the BB/W rat compared to a normal rat. These preliminary data show that 123I-labelled IL-2 retains its immunoreactivity and capacity to bind to activated lymphocytes both in vitro and in vivo and may be used for in vivo localization of lymphocytic infiltration in Type 1 diabetes.

HDLs in apoA-I transgenic Abca1 knockout mice are remodeled normally in plasma but are hypercatabolized by the kidney

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P < 0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.

ARL1 and membrane traffic in Saccharomyces cerevisiae.

To examine the functions of the Arf-like protein, Arl1p, in Saccharomyces cerevisiae, a null allele, arl1delta::HIS3, was constructed in two strains. In one background only, loss of ARL1 resulted in temperature-sensitive (ts) growth (suppressed on high-osmolarity media). Allelic variation at the SSD1 locus accounted for differences between strains. Strains lacking ARL1 exhibited several defects in membrane traffic. First, arl1delta strains secreted less protein as measured by TCA-precipitable radioactivity found in the media of [(35)S]-labelled cells. A portion of newly synthesized carboxypeptidase Y (CPY) was secreted rather than correctly targeted to the vacuole. Uptake of the fluid-phase marker, lucifer yellow, was reduced. All these phenotypes were exacerbated in an ssd1 background. The ts phenotype of the arl1deltassd1 strain was suppressed by YPT1, the yeast Rab1a homologue, suggesting that ARL1 and YPT1 have partially overlapping functions. These findings demonstrate that ARL1 encodes a regulator of membrane traffic.

Actions of an IGF-I-enhancing antibody on IGF-I pharmacokinetics and tissue distribution: increased IGF-I bioavailability.

We have previously demonstrated that a specific anti-IGF-I antibody will enhance the growth-promoting activity of IGF-I in vivo (Stewart et al. 1993). Since the antibody had a modest affinity for IGF-I we suggested that the antiserum might protect IGF-I from degradation whilst maintaining it in a bioavailable form. The aim of this investigation was to test that hypothesis by determining the plasma clearance and tissue distribution of tracer IGF-I in the presence of the enhancing anti-IGF-I immunoglobulin (anti-IGF-I Ig) or non-immune immunoglobulin (NI Ig). Dwarf rats were treated with saline, NI Ig or anti-IGF-I Ig for 4 days. On day 4, 125I-IGF-I (1.6 x 10(7) c.p.m.) was injected into the jugular vein and blood sampled over the next 330 min when the rats were killed: samples of liver, kidney and skeletal muscle were quickly dissected out. Mean plasma trichloroacetic acid (TCA)-precipitable 125I-IGF-I was always significantly greater (P < 0.001 for each time point) from anti-IGF-I Ig rats versus the NI Ig or saline groups (which exhibited practically identical decay curves), implying increased binding capacity for IGF-I in the anti-IGF-I Ig rats. Pharmacokinetic parameters were calculated by resolution of the decay curves using a two-phase model. The total clearance rate of 125I-IGF-I was significantly decreased (P < 0.001) by almost twofold in the anti-IGF-I versus the two control groups, consistent with the increased binding capacity in the anti-IGF Ig rats. The half-lives of the faster-decaying phase were not significantly different between treatment groups but, surprisingly, that for the slower-decaying phase was significantly decreased (P < 0.001) in the anti-IGF-I Ig rats versus the two control groups; this may reflect the low affinity of the anti-IGF-I Ig for IGF-I and its enhancing properties. The degradation of 125I-IGF-I was significantly decreased in animals receiving the anti-IGF-I Ig. In support of this, kidney TCA-precipitable radioactivity (c.p.m.) was seven-fold less (P < 0.001) in the anti-IGF-I Ig groups versus the controls, indicative of reduced excretion. Liver TCA-precipitable radioactivity was increased (P < 0.001) in the anti-IGF-I Ig rats, probably due to reticuloendothelial clearance of non-self antibodies: skeletal muscle TCA-precipitable radioactivity tended to increase in the anti-IGF-I Ig group versus the controls which might indicate increased targeting of IGF-I to muscle. Size exclusion chromatography of plasma 15 and 120 min after administration of 125I-IGF-I demonstrated a broad peak of radioactivity with a molecular mass of 150-300 kDa in the anti-IGF-I Ig-treated rats, which was responsible for more than 90% of the eluted radioactivity. This suggests that: (1) 125I-IGF-I was bound to the anti-IGF-I Ig and might also be able to associate with IGFBPs or (2) the polyclonal antibody might recognise more than one antigenic site on IGF-I. These data indicate that the anti-IGF-I Ig was protecting IGF-I from degradation, leading to a large plasma pool of IGF-I but that IGF-I could be transferred readily from the plasma pool to tissues. We suggest that administration of IGF-I in conjunction with a binding molecule similar to the antibody described here could provide the basis for effective IGF-I treatment strategy.

Effects of iodide on thyroglobulin biosynthesis in FRTL-5 cells.

Iodide inhibits several thyroid parameters through an organic intermediate, and this process has been related to thyroid autoregulation. The aim of this study was to determine the effect of iodine on thyroglobulin (Tg) synthesis in the rat thyroid cell line FRTL-5. TSH stimulated amino acid incorporation into the cells by 400% and iodine had no effect on this parameter. No effect of TSH or iodide on [35S] methionine incorporation into protein was found under our experimental conditions (approximately 80% of total [35S]methionine incorporated was found in TCA-precipitable material). TSH caused an increase in Tg synthesis, after 1 h, while iodide partially blocked the effect of TSH (control 6.4% of TCA precipitable radioactivity; TSH 10.7%; iodide 8.4%). After 24 h, the protein released into the medium was measured. TSH stimulated total protein liberation and iodide inhibited this parameter. TSH stimulated total RNA content, and iodide caused an inhibition. Northern analysis did not show inhibition by iodide of TSH-stimulated Tg mRNA levels. The present results show an inhibitory effect of excess iodide on TSH-stimulated thyroglobulin biosynthesis in FRTL-5 cells.

Thyroid hormone induces a 52 kDa soluble protein in goat testis Leydig cell which stimulates androgen release

Incubation of goat testicular Leydig cells with 3,5,3′-triiodothyronine(T 3) induces the generation of a proteinaceous factor (factors) which was located in the soluble supernatant fraction (100 000 × g supernatant, 100k sup) of sonicated Leydig cells. Addition of this factor to Leydig cell incubation greatly stimulated androgen release. This factor(s) was purified based on its biological properties, i.e., its addition to Leydig cell incubation augmented the release of androgen. This was designated as TIP (T 3-induced protein) activity. 100 k sup prepared from Leydig cells incubated in the absence (control) or presence of T 3 was gel filtrated through Sephadex G-100. 100 k sup from T 3 incubate gave two protein peaks, P-I and P-II, control 100 k sup had similar nature of P-I, but P-II was not well marked. Incubation in the presence of [ 14C]leucine clearly showed TCA precipitable radioactivity only in the P-II region of T 3 incubate. 5 μg of P-II protein stimulated androgen release from Leydig cells (1 · 10 6 cells/well) to more than 5-fold as compared to control. P-II protein was further purifies by FPLC Mono-Q column chromatography where one unadsorbed (MQ-I) and two adsorbed(MQ-II and MQ-III) protein peaks could be detected. MQ-II, which was eluted with 0.20 M NaCl gradient, demonstrated strong TIP activity (2 μg protein released 4.8-fold more androgen as compared to control). MQ-II was passed through FPLC Superose-6 column where it gave two peaks and Peak-I(SP-I) showed strong TIP activity (2 μg protein stimulated a 6-fold increase in androgen release as compared to control). Polyacrylamide gel electrophoresis (PAGE) indicated SP-I to be a homogeneous protein and SDS-PAGE demonstrated it to be a 52 kDa monomer protein. Results show that T 3 induces the synthesis of a 52 kDa protein in testicular Leydig cells which in turn causes stimulation of androgen release suggesting this protein to be a novel mediator of T 3 function in Leydig cells.

Studies on the Metabolic Fate of Modified Recombinant Tissu-Type Plasminogen Activator (E6010) (2): Blood or Plasma Concentration, Distribution, Metabolism and Excretion in Rats after Repeated Intravenous Administration of 125I-E6010.

Blood or plasma concentration, distribution, metabolism and excretion of E6010 were studied in male rats during and after repeated daily intravenous administration of 0.4 mg/kg of 125I-E6010 for 10 days. 1. At 5 min after the repeated intravenous administration of 125I-E6010 for 10 days, blood or plasma levels of total and TCA-precipitable radioactivity, immunoreactive E6010 and the unchanged E6010 and its metabolites were the same as those at 5 min after a single dosing. After the repeated intravenous administration of 125I-E6010 for 10 days, the levels of total and TCA-precipitable radioactivity in the blood or plasma decreased slower than those after a single administration, but the levels of immunoreactive E6010 and unchanged E6010 were the same as those after a single administration. 2. TCA-precipitable radioactivity level at 24 hr afte r the last dosing in most tissues did not exceed the levels after a single dose by 3 times, except in spleen (3.7 times), bone marrow (3.7 times) and thyroid (3.2 times). After repeated intravenous administration of 125I-E6010, the levels of total and TCA-precipitable radioactivity in each tissue decreased slower than those after a single dosing, but the levels of the immunoreactive E6010 in the liver and kidney were the same as those after a single dosing. 3. The excretion of radioactivity in the urine and feces was nearly the same th roughout the period of repeated intravenous administration of 125I-E6010. Within 7 days after the last dosing, 92.42% and 7.74% of the administered radioactivity was excreted into the urine and feces, respectively. Radioactivity excreted in the urine after the last dosing consisted almost of small molecular weight metabolites or free 125I, similar to that after a single administration.

Studies on the Metabolic Fate of Modified Recombinant Tissu-Type Plasminogen Activator (E6010) (3): Feto-Placental Transfer and Excretion into Milk in Rats after a Single Intravenous Administration of l25I-E6010.

The feto-placental transfer of 125I-E6010 and the excretion into milk were studied in pregnant and lactating rats after a single intravenous administration of 0.4 mg/kg of 125I-E6010. 1. At 15 min after a single intravenous administration of 125I-E6010 to rats on day 13 and 19 of gestation, the radioactivity level in the fetus was 245 ?? 348 times lower than that present in maternal plasma, but most of that was not TCA-precipitable. At later time on day 19 of gestation, the level of radioactivity in the fetal thyroid tissue was higher than that in the other fetal and maternal tissues, however, it was suggested that radioactivity in the thyroid results from the accumulation of free 125I. These results suggested that 125I-E6010 administered to pregnant rats was not transfered into the fetus as an intact E6010. 2. After a single intravenous administration of 125I-E6010 to lactating rats, the TCA-precipitable radioactivity was found in the milk, and its level increased and reached maximum at 24 hr, but the immunoreactive E6010 was not detected at any time. Then, most of the radioactivity in the milk was considered to be an endogeneous high molecular weight compounds into which a free iodine, produced by degradation of 125I-E6010, was incorporated.

Metabolic Fate of YM866, a Novel Fibrinolytic Agent (I). Plasma Concentration, Distribution, Metabolism and Excretion in Rats after Single Intravenous Administration.

We investigated the plasma concentration profile, distribution, metabolism and excretion of radioactivity after a single intravenous administration of 125I-YM866 in male rats. 1. After a single intravenous administration of 125I-YM866, more than 50% of this drug rapidly formed two high molecular complexes with plasma proteins. The ratio of trichloroacetic acid (TCA)-precipitable radioactivity to total radioactivity in plasma decreased with time, suggesting that this drug is metabolized into low molecular weight metabolite or free 125I. Immunoreactive YM866 concentrations decreased more rapidly than total or TCA-precipitable radioactivity. 2. After a single intravenous adminis tration of 125I-YM866, radioactivity levels were highest in the plasma, followed by whole blood, liver, kidney, adrenal gland, spleen, lung, pituitary gland and thyroid. Radioactivity levels in these tissues were one-quarter or half of that in the plasma, and the level in the brain was much lower than in the plasma. Except for the uptake of the released 125I by some specific tissues, the radioactivity in most tissues decreased rapidly. Although most of the radioactivity in tissues were precipitable with TCA at an early stage after administration, the ratio of TCA-precipitate to total radioacivity decreased rapidly with time. 3. After a single intr avenous administration of 125I-YM866, 91.2% and 3.9% of the dosed radioactivity was excreted in urine and feces within 144 hr, respectively. Most of the radioactivity in urine was not precipitable with TCA and eluted in the gel filtration chromatographic (GFC) analysis at a low molecular region, suggesting that the radioactivity was excreted as low molecular weight materials 4. The concentration of 125I-YM866 in plasma was higher than that of 125I-tissue plasminogen activator (125I-t-PA), probably because 125I-YM866 was distributed less extensively to the liver than 125I-t-PA.

Studies on the Metabolic Fate of Modified Recombinant Tissue-Type Plasminogen Activator (E6010) (1): Blood or Plasma Concentration, Distribution, Metabolism and Excretion in Rats after a Single Intravenous Administration of l25I-E6010 in Comparison with l25I-Recombinant Tissue-Type Plasminogen Activator (rt-PA).

Blood or plasma concentration, distribution, metabolism and excretion of E6010 were studied in male rats after a single intravenous administration of 125I-E6010 in comparison with 125I-rt-PA. 1. After a single intravenous administration of 125I-E6010, plasma clearances of TCA-precipitable radioactivity, immunoreactivity, fibrinolytic activity and the unchanged E6010 were 10.3, 11.2, 3.1 and 5.5 times lower than those after dosing of 125I-rt-PA, respectively. In the plasma, the complexes of 125I-E6010 with α2-macroglobulin and with α2-plasmin inhibitor were observed after dosing of 125I-E6010, as well as after dosing of 125I-rt-PA. 2. After a single intravenous administration of 125I-E6010, liver levels of TCA-precipitable radioactivity reached maximum level at 15 min after administration accounting for about 19% of administered radioactivity. In the case of 125I-rt-PA, it reached maximum level at 5 min accounting for about 54% of administered dose. These results suggest that the differences in the distribution to the liver between E6010 and rt-PA resulted in the difference of plasma clearances. 3. Within 7days after a single int ravenous administration of 125I-E6010, 96.05% and 5.32% of the administered radioactivity were excreted into urine and feces, respectively. Biliary excretion of radioactivity was 17.01% of the dose within 48hr after a single intravenous administration of 125I-E6010. Both radioactivity excreted in urine and in bile consisted almost of small molecular weight metabolites or free 125I.

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.

Pharmacokinetics and tissue distribution of recombinant human transforming growth factor beta 1 after topical and intravenous administration in male rats.

Recombinant human transforming growth factor beta (rhTGF-beta 1) enhances the healing process after topical application to various animal wound models. A detailed pharmacokinetic and tissue distribution study was performed to support the clinical development of rhTGF-beta 1 for wound healing indications. Rats received radioiodinated or unlabeled rhTGF-beta 1 as an intravenous (iv) bolus or as a topical formulation applied to a full thickness wound. Plasma concentrations of TGF-beta 1 were estimated from TCA-precipitable radioactivity or were measured by ELISA. Following iv administration, the initial half-life was rapid (< 11 min), regardless of whether radiolabeled or unlabeled rhTGF-beta 1 was used. The terminal half-life was long (163 min) when the test material was radioiodinated and administered as a trace dose and relatively short (< or = 61 min) when given at high doses and assayed by ELISA. Analysis of plasma radioactivity by SDS-PAGE revealed a time-dependent clearance of the 25-kDa parent molecule without a significant appearance of lower molecular weight radiolabeled metabolites. The majority of the radioactivity was associated with highly perfused organs, known iodide elimination pathways, and the thyroid at 1 and 8 hr after iv injection. After topical administration of a high dose (0.8 mg/kg), no immunoreactive TGF-beta 1 was detectable in plasma samples taken over a 48-hr period. However, trace amounts (< or = 0.05 ng/mL) of acid-precipitable radioactivity were detected in plasma after topical application of [125I]rhTGF-beta 1 (1 microgram/kg, 126 microCi/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of radioiodinated recombinant human TGF-beta 1 binding to bone matrix within rabbit skull defects.

Bone healing is regulated in part by the local production of TGF-beta 1 and other growth factors produced by cells at the site of injury. The single application of recombinant human TGF-beta 1 (rhTGF-beta 1) to calvarial defects in rabbits induces an accelerated recruitment and proliferation of osteoblasts within 3 days. This ultimately results in the formation of new bone and the complete closure of the defect within 28 days. The persistence and localization of [125I]rhTGF-beta 1 within an osseous defect was investigated after applying a single dose of [125I]rhTGF-beta 1 formulated in a 3% methylcellulose vehicle. Normal bone encompassing the defect site, the periosteum, and the gel film covering the dura were harvested at 0, 4, 8, and 24 h and 3, 7, and 16 days after [125I]rhTGF-beta 1 application. The defect site-associated radioactivity was quantitated, visualized by autoradiography, and characterized by TCA precipitation and SDS-PAGE. Radioactivity was observed in autoradiographs of gross specimens, histologic sections of the bone matrix, and periosteal tissue surrounding the defect. There was a time-dependent decrease in TCA-precipitable radioactivity; however, radioactivity was still associated with the bone matrix 16 days after application of [125I]rhTGF-beta 1. SDS-PAGE and autoradiography of the radioactivity in homogenized bone and periosteal samples revealed a 25 kD band, suggesting that the radioactivity remaining at the defect site represented intact [125I]rhTGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of Recombinant Factor VIIa in the Rat – A Comparison of Bio-, Immuno- and Isotope Assays

Recombinant human factor VIIa (rFVIIa) is an activated coagulation factor for intravenous use as a haemostatic agent in haemophiliacs who generate antibodies against factor VIII or IX. Plasma kinetic studies are important for the understanding of the action of rFVIIa which is exerted in the vascular compartment of the body, more specifically on the vessel walls at the site of injury. In the present study, rats were dosed 100 or 500 micrograms/kg 125I-rFVIIa i.v., without any side effects being observed, and the plasma profile of rFVIIa was studied by 3 different assays that were shown to correlate well at early times post-dose: trichloroacetic acid (TCA)-precipitable drug-related radioactivity, rFVIIa antigen determination by ELISA technique, and the assay of clot activity which is the only clinically applicable assay. The plasma concentration curve could be resolved into 1-3 exponentials, depending on the FVIIa detection principle that was employed. Initially, there was a short (ca. 10 min) phase of increasing concentrations before the attainment of Cmax. This was followed by a plasma recovery (Cmax x plasma volume/dose) in the vicinity of one half of the administered dose. The initial volume of distribution (V1) corresponded to the vascular compartment whereas the volume of distribution at steady state (Vss) was somewhat larger. Whole body clearance (CL-B) of rFVIIa was approx. 1 ml/min per kg, and mean residence time (MRT) and the half-life assumed to be associated with the loss of biological activity was approx. 1 h and 20-45 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutral metalloendopeptidase inhibitors as ANF potentiators: sites and mechanisms of action

Inhibition of the enzyme neutral metalloendopeptidase potentiates responses to atrial natriuretic factor and elicits reductions of blood pressure in desoxycorticosterone acetate sodium hypertensive rats. The present study evaluated the role of atrial natriuretic factor and bradykinin in the antihypertensive response to neutral metalloendopeptidase inhibition through the use of antibodies and antagonists, respectively. In addition, the pharmacokinetic mechanism by which neutral metalloendopeptidase inhibition interferes with atrial natriuretic factor metabolism was explored. The antihypertensive response to the neutral metalloendopeptidase inhibitor SCH 34826 was abruptly reversed by i.v. injection of a polyclonal antiserum to atrial natriuretic factor. In contrast, the antihypertensive response to SCH 34826 was unaffected by injection of the bradykinin antagonist Thi5,8-D-Phe7 bradykinin. The renal response to atrial natriuretic factor, SCH 34826, and phosphoramidon was inhibited by the bradykinin antagonist. The NEP inhibitor SCH 39370 significantly delayed the disappearance of TCA precipitable radioactivity from plasma following i.v. bolus dosing with 125I-labelled ANF 99-126. The effects were enhanced in the presence of the C receptor ligand. The results indicate that atrial natriuretic factor, but not bradykinin, plays an important role in the antihypertensive response to SCH 34826. Bradykinin plays a permissive role in the diuretic responses to atrial natriuretic factor and inhibitors of neutral metalloendopeptidase. Lastly, neutral metalloendopeptidase inhibition significantly alters the clearance and metabolism of tracer quantities of atrial natriuretic factor.

Effects of cyclic adenosine 3′,5′-monophosphate on amino acid transport and incorporation into protein in the rat aorta

To assess the effects of cyclic AMP on amino acid transport and incorporation into aortic tissue protein, rat aortic rings were incubated with the cyclic AMP analog, N6-monobutyryl cyclic AMP (MBcAMP), the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX), and radiolabeled amino acids. Subsequently, the aortic rings were homogenized in 5% trichloroacetic acid (TCA) and processed for liquid scintillation counting. Radioactivity present in the TCA supernatant following centrifugation was used to estimate amino acid transport. TCA-precipitable radioactivity was used as a measure of amino acid incorporation into protein. MBcAMP induced an increase in the uptake of [3H]alpha-aminoisobutyric acid into aortic rings and an increase in the incorporation of radiolabeled proline and leucine into TCA-precipitable protein. Similar effects were observed with low concentrations of MIX (0.025-0.25 mM); however, at higher concentrations of MIX, there was an attenuation of the effect or frank inhibition. Maximum stimulation of transport was observed within 90-120 min of the addition of MIX or MBcAMP to the incubation medium, whereas the effect on amino acid incorporation was not detectable until after 12 h of exposure to MIX or MBcAMP. The effects of cyclic AMP on transport were observed in both the tunica media and the tunica adventitia, whereas the effects on amino acid incorporation into protein were observed only in the tunica media. These data are consistent with a possible role for cyclic AMP in promoting changes in the tunica media that could lead to the development of vascular hypertrophy.

Studies on the metabolic fate of recombinant human erythropoietin(SNB-5001). II: Plasma level profiles, distribution, metabolism, excretion, accumulation in rats after intravenous and subcutaneous injection of 125I-SNB-5001.

Pharmacokinetics and accumulation of recombinant human erythropoietin (SNB-5001) were studied in rats after the intravenous(i.v.) and subcutaneous injection(s.c.) of 125I-SNB-5001. Plasma level declined biexponentially with a rapid phase (t1/2α : 0.4 ?? 0.9hr) and a slower phase (t1/2β :3.0 ?? 5.2hr) in male and female rats after a single i.v. (50 ?? 1250U/kg). Dosedependent Ccnax was observed at 6 ?? 9 hr after s.c. Plasma level after s.c. declined slower than after i.v. AUC0→∞ in rats after both injections increased in dose-dependent manner. Bioavailabilities after s.c. were 65 ?? 75%. Maximal levels of radioactivity in most tissues were observed at 10 min ?? 1hr after i.v. and at 6 ?? 12 hr after s.c. Higher radioactivity was found in the plasma and bone marrow following by kidney, liver and spleen. It was difficult to find the radioactivity in the brain and fat tissue. The decrease of tissue levels was proportional to that in the plasma. The levels in plasma and bone marrow at 24 hr after s.c. were two-fold higher than those after i.v. Urinary excretion of radioactivity was about 80% of injected dose in male and female rats until 96hr after i.v. Fecal excretions until 96hr accounted for 6% of administered dose, similar results were observed in rats after s.c. Biliary excretion was 7 % in male rats until 48 hr after i.v. A small amount of high molecular weight radioactive compound was observed in the urine and bile just after the injection, and other consisted of a low molecular weight radioactive compounds as revealed by HPLC analysis. During the repeated injection (50U/kg), the plasma levels at 1 hr after each i.v. and at 6 hr after each s.c. remained almost constant since the first day. There were no pronounced differences in pharmacokinetics between single and repeated injection (for 7 days) as measured the immunoreactive radioactivity. Plasma levels of total and TCA-precipitable radioactivity declined slower than those after the single injection. The tissue levels at 24hr after repeated injection increased than those after single injection but the ratios of tissue to plasma level after repeated injection scarcely changed. The excretion rates in urine and feces were nearly constant during the period of repeated injection, and 80% of injected radioactivity was recovered in urine and 8% in feces until 96hr after the last injection. Free 125I and high molecular weight metabolites were observed in plasma and tissues as determined by HPLC analysis. Free 125I gradually increased as the levels of 125I-SNB-5001 decreased. High molecular weight metabolites, such as M-3 in the plasma and M-1 in tissues, had a slight immunoreactivity. The immunoreactive metabolites were not detected in thyroid. These metabolites present in plasma, tissues and urine after repeated injection were almost same as those after a single injection. It was suggested that there was no specific tissue accumulating SNB-5001.

Inactive of atrial natriuretic factor in mice in vivo: crucial role of enkephalinase (EC 3.4.24.11)

Atrial natriuretic factor (ANF) is a hormone whose potent hemodynamic and renal actions might be beneficial in several cardiovascular disorders, but whose poor oral absorption and extremely rapid inactivation in vivo have so far prevented its therapeutic use. We have developed simple tests to study the peptides responsible for the hydrolysis of ANF in mice in vivo and to assess the effects of peptidase inhibitors. In mice injected with 125I-ANF in low amounts the radioactivity present in kidney, a major target organ for the hormone, was analysed by HPLC, precipitation with trichloracetic acid (TCA) and in a membrane binding assay. All three parameters indicated a rapid inactivation of the hormone: 20 s after injection of 125I-ANF the intact hormone represented less than 20% of the total kidney radioactivity. Oral pretreatment with acetorphan, a potent enkephalinase inhibitor resulted in a marked increase in the amount of intact 125I-ANF (6-fold), TCA-precipitated (5-fold) and membrane bound radioactivity (4-fold) in the kidney; the total kidney radioactivity was enhanced by ∼2-fold. A similar protective effect was observed with other enkephalinase inhibitors, i.e. thiorphan and kelatrophan; the latter was effective at a 10-fold higher dosage. In contrast, a large variety of inhibitors of metallo-, cysteine, serine and aspartic proteinases had no or only marginal effects. Instead, captopril, an angiotensin-converting enzyme inhibitor, reduced the total and TCA-precipitable radioactivity in the kidneys. Aminopeptidase inhibitors, used either alone or in conjunction with acetorphan, displayed significant but limited protective effects. The crucial role of enkephalinase in ANF inactivation in vivo suggests that inhibitors of this peptidase could be used in a novel therapeutic approach to cardiovascular or renal diseases by protecting endogenous ANF.

Differences between Brahman and Holstein cows in heat-shock induced alterations of protein synthesis and secretion by oviducts and uterine endometrium.

Our objectives were to test differences in protein synthesis and secretion by cultured oviducts and endometrium from Brahman and Holstein cows and the response of those tissues to in vitro heat shock. Explants of oviductal tissue obtained at estrus from Holstein (n = 5) and Brahman (n = 6) cows were cultured at a homeothermic (39 degrees C) or heat shock (43 degrees C) temperature. At 6 h, cultures were pulse-chase labeled (2 h, L[4,5-3H]leucine; 2 h, L-leucine). Endometrial explants were cultured similarly except that pulse labeling was performed for the first 0 to 15, 0 to 30, 30 to 60 and 60 to 90 min following onset of heat shock. A temperature of 43 degrees C increased secretion of nondialyzable 3H-labeled macromolecules by both oviducts of Brahmans but depressed secretion by the oviduct ipsilateral to the side of ovulation of Holsteins. For both breeds, 43 degrees C decreased incorporation of [3H]leucine into trichloroacetic acid (TCA)-precipitable radioactivity in oviducts from the ipsilateral side. Secretion of 3H-labeled macromolecules by pulse-labeled endometrial explants increased at 43 degrees C. Heat shock caused an immediate increase in TCA-precipitable radioactivity in tissue during pulse labeling for Holstein tissues. Incorporation was decreased at 43 degrees C in tissue from Brahmans in the first 30 min and increased thereafter. Incorporation of [3H]thymidine by endometrial explants from Brahmans was increased at 43 degrees C, whereas it was suppressed at 43 degrees C in explants from Holstein cows. Heat shock proteins of 72,000 and 90,000 molecular weight were present in endometrial tissues. A major secretory product of endometrium had a molecular weight of 57,500 for Brahmans and a lower molecular weight (55,600) for Holsteins.