Acetylcholine Synthesis by Adult Bovine Adrenal Chromaffin Cell Cultures

Adrenal chromaffin cells normally synthesize and release catecholamines. In the present study, [3H]acetylcholine synthesis and another characteristic of cholinergic neurons, [3H]choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells. Chromaffin cell cultures took up [3H]choline from the medium and acetylated the [3H]choline to form [3H]acetylcholine. The rate of [3H]acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture. [3H]Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+. The ability for K+ to stimulate synthesis of [3H]acetylcholine developed only after 28 days in culture. [3H]Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline. [3H]Choline uptake was enhanced by Na+ omission in day-14 cultures, but was at least partially Na+-dependent in day-29 cultures. Hemicholinium-3 (IC50 less than 10 muM) inhibited [3H]choline uptake into chromaffin cell cultures. It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.     

Ouabain-Sensitive Choline Transport System in Capillaries Isolated from Bovine Brain

In physiological conditions, there is a net transport of choline from brain to blood, despite the fact that the choline concentration is higher in plasma than in CSF. Because of the blood-brain barrier characteristics, such passage against the concentration gradient takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, [3H]choline uptake properties have been analyzed in capillaries isolated from bovine brain. [3H]Choline uptake was linear with time for up to 1 h. Nonlinear regression analysis of the uptake rates at different substrate concentrations gave the best fit to a system of two components, one of which was saturable (Km = 17.8 +/- 4.8 microM; Vmax = 11.3 +/- 3.4 pmol/min/mg of protein) and the other of which was nonsaturable at concentrations up to 200 microM. The [3H]choline transport was significantly reduced in the absence of sodium and after incubation with 10(-4) M ouabain for 30 min. Ouabain also inhibited choline uptake in purified cerebral endothelial cells, but not in the endothelium isolated from bovine aorta. Accordingly, cerebral endothelial cells were able to concentrate [3H]choline, with this effect being abolished by ouabain, whereas in aortic endothelial cells the [3H]choline intracellular concentration was never higher than that of the incubation medium. These results suggest that the blood-brain barrier endothelium is specifically provided with an energy-dependent choline transport system, which may explain the choline efflux from the brain and the maintenance of a low choline concentration in the cerebral extracellular space.     

Promotion of the release of 11-cis-retinal from cultured retinal pigment epithelium by interphotoreceptor retinoid-binding protein.

This study investigates whether the interphotoreceptor retinoid-binding protein (IRBP) is necessary for the release of 11-cis-retinaldehyde (RAL) or if the retinoid is constitutively released from the retinal pigment epithelium (RPE) following synthesis. The strategic location of IRBP in the interphotoreceptor matrix (IPM) and its retinoid-binding ability make it a candidate for a role in 11-cis-RAL release. Fetal bovine RPE cells were grown in permeable chambers, and their apical surfaces were incubated with medium containing either apo-IRBP, the apo form of cellular retinaldehyde-binding protein (CRALBP), the apo form of serum retinol-binding protein (RBP), or bovine serum albumin (BSA) or with medium devoid of binding proteins. [3H]-all-trans-Retinol (ROL) was delivered to the basal surface of the cells by RBP. High-performance liquid chromatography demonstrated that [3H]-11-cis-RAL was optimally released into the apical medium when apo-IRBP was present. The most surprising result was the diminished level of [3H]-11-cis-RAL when apo-CRALBP was in the apical medium. Circular dichroism demonstrated that CRALBP had not been denatured by the photobleaching required for endogenous ligand removal. Therefore, apo-CRALBP should have been able to bind [3H]-11-cis-RAL if it was constitutively released into the apical medium. In addition, when proteins other than apo-IRBP were present, or if the cells were incubated with medium alone, the observed decrease in apical [3H]-11-cis-RAL was concomitant with a buildup of intracellular [3H]-all-trans-retinyl palmitate and [3H]-all-trans-ROL in the basal culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate

Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (3H-test and 14C-extracellular marker) technique. [3H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible 3H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [3H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [3H]folate or [3H]methotrexate in either direction was not detectable within 5-6 min. At the brush-border side (maternal) uptake of [3H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells.     

Ligand-dependent modulation of membrane phospholipid metabolism in ConA-stimulated lymphocytes

Human peripheral lymphocytes were activated by ConA in serum-free culture medium, supplemented by BSA. Incorporation of [³H]thymidine into DNA, of [³H]uridine into RNA and of oleate or acetate into membrane phospholipids was investigated. DNA synthesis could be completely inhibited by αMM or by anti-ConA-IgG. Fab and F(ab)₂ fragments of the anti-ConA were equally active. When αMM or anti-ConA was added to cultures at different times after stimulation with ConA, incorporation of [³H]thymidine into DNA (measured after 72 h) could be prevented up to 6–8 h completely and up to 20–30 h partially. Incorporation of [³H]uridine into RNA could be arrested at any time of the culture up to 40 h at the level it had reached but did not reverse to the level of unstimulated cells for a long time. In contrast, incorporation of oleate into lecithin returned to the level of unstimulated cells within 2–3 h after removal of ConA. This suggests that the activation of the phospholipid turnover in stimulated cells is a direct consequence of the presence of the mitogen at the membrane and thus may be a critical initial event in lymphocyte activation.     

Sulfate transport in porcine thyroid cells. Effects of thyrotropin and iodide

In porcine thyroid cells, thyroglobulin sulfation is controlled by thyrotropin (TSH) and iodide, which contribute to regulating the intracellular sulfate concentration, as we previously established. Here, we studied the transport of sulfate and its regulation by these two effectors. Kinetic studies were performed after [(35)S]sulfate was added to either the basal or apical medium of cell monolayers cultured without any effectors, or with TSH with or without iodide. The basolateral uptake rates were about tenfold higher than the apical uptake rates. TSH increased the basolateral and apical uptake values (by 24 and 9%, respectively, compared with unstimulated cells), and iodide inhibited these effects of TSH. On the basis of results of the pulse-chase experiments, the basolateral and apical effluxes appeared to be well balanced in unstimulated cells and in cells stimulated by both TSH and iodide: approximately 40-50% of the intracellular radioactivity was released into each medium, whereas in the absence of iodide, 70% of the intracellular radioactivity was released on the basolateral side. The rates of transepithelial sulfate transport were increased by TSH compared with unstimulated cells, and these effects decreased in response to iodide. These results suggest that TSH and iodide may each control the sulfate transport process on two sides of the polarized cells, and that the absence of iodide in the TSH-stimulated cells probably results in an unbalanced state of sulfate transport.     

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 μmol/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. © 1993 Wiley-Liss, Inc.     

Retinoic acid treatment of fibroblasts causes a rapid decrease in [3H]inositol uptake

NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of [3H]inositol compared to solvent-treated controls. The onset of RA-induced inhibition of [3H]inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10(-8) to 10(-5) M and the effect was fully reversible within 48 h after RA removal. The Vmax and Kt for the controls were 10 nmol/2.5 x 10(6) cells/2 h and 51 microM; and for RA-treated cells the values were 4 nmol/2.5 x 10(6) cells/2 h and 52 microM. The decreased [3H]inositol uptake was not due to a change in the affinity (Kt) of the transporter for the inositol but to a decrease in the Vmax. The maximal effect on inositol uptake was dependent on RA treatment of the cells after they reached saturation density or if made quiescent by serum starvation. RA was the most active of the different retinoids examined in the order RA greater than 13-cis-RA = retinyl acetate greater than all-trans-retinol greater than 5,6-dihydroxyretinoic acid methyl ester greater than N-4-hydroxyphenyl retinamide. In contrast to this effect on inositol, the uptake of fucose, mannose, galactose, and glucose was either not affected or enhanced (for mannose and fucose) by RA treatment. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of [3H]inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for [3H]inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of [3H]inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner.     

Randomized controlled trial of exercise and blood immune function in postmenopausal breast cancer survivors.

The objective was to determine the effects of exercise training on changes in blood immune function in postmenopausal breast cancer survivors. Fifty-three postmenopausal breast cancer survivors were randomly assigned to an exercise (n=25) or control group (n=28). The exercise group trained on cycle ergometers three times per week for 15 wk. The control group did not train. The primary end point was change in natural killer cell cytotoxic activity in isolated peripheral blood mononuclear cells. Secondary end points were changes in standard hematological variables, whole blood neutrophil function, the phenotypes of isolated mononuclear cells, estimations of unstimulated and phytohemaglutinin-stimulated mononuclear cell function (rate of [3H]thymidine uptake), and the production of proinflammatory [interleukin (IL)-1alpha, tumor necrosis factor-alpha, IL-6] and anti-inflammatory cytokines (IL-4, IL-10, transforming growth factor-beta1). Statistical tests were two-sided (alpha <0.05). Fifty-two participants completed the trial. Intention-to-treat analyses, which included the baseline value as a covariate, showed significant differences between groups for change in percent specific lysis of a target natural killer cell at all five effector-to-target ratios (adjusted mean between-group change over all 5 effector-to-target ratios = +6.34%; P <0.05 for all comparisons), the lytic activity per cell (adjusted mean between-group change = -2.72 lytic units; P=0.035), and unstimulated [3H]thymidine uptake by peripheral blood lymphocytes (adjusted mean between-group change = +218 per dpm x 10(6) cells; P = 0.007). There were no significant differences between groups for change in any other end point. Exercise training increased natural killer cell cytotoxic activity and unstimulated [3H]thymidine uptake by peripheral blood lymphocytes in postmenopausal breast cancer survivors.     

Uptake of a natural surfactant and increased delivery of small organic anions into type II pneumocytes

The uptake of natural lung surfactant into differentiated type II cells may be used for the targeted delivery of other molecules. The fluorescent anion pyranine [hydroxypyren-1,3,6-trisulfonic acid, sodium salt (HPTS)] was incorporated into a bovine surfactant labeled with [3H]dipalmitoylphosphatidylcholine ([3H]DPPC). The uptake of [3H]DPPC and of HPTS increased with time of incubation and concentration, decreased with the size of the vesicles used, and was stimulated by 8-bromo-cAMP and partially inhibited by hypertonic sucrose. However, the amount of HPTS uptake was approximately 100 times smaller than that of [3H]DPPC. This large difference was due to a more rapid regurgitation of some of the HPTS from the cells but not to leakage from the surfactant before uptake. The acidification of the internalized surfactant increased linearly over 90 min to 7.13, and after 24 h, a pH of 6.83 was measured. In conclusion, after internalization of a double-labeled natural surfactant, the lipid moieties were accumulated in relation to the anions, which were targeted to a compartment not very acidic and in part rapidly expelled from the cells.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate

Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (³H-test and ¹⁴C-extracellular marker) technique. [³H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible ³H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [³H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [³H]folate or [³H]methotrexate in either direction was not detectable within 5–6 min. At the brush-border side (maternal) uptake of [³H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells.     

Receptor-mediated uptake of horseradish peroxidase in innervated and denervated skeletal muscle

The in vitro uptake of [3H]inulin and horseradish peroxidase (HRP) has been studied in innervated and 6 days denervated extensor digitorum longus muscle of the mouse. Both markers were taken up at a higher rate in denervated muscle. The increase in uptake after denervation was, however, larger for HRP than for [3H]inulin. After 2 h incubation at 37 degrees C, pH 7.3, in the presence of equimolar concentrations of HRP and [3H]inulin (approx. 2.1 microM), the uptake of HRP was approx. 8 times as great as the uptake of [3H]inulin in the same innervated muscles. In denervated muscle the HRP uptake was approx. 19 times as great as the [3H]inulin uptake in the same muscles. Various possible explanations of these differences in uptake have been considered and tested experimentally. [3H]Inulin uptake in skeletal muscle has previously been shown to obey bulk kinetics. The present investigation shows the HRP uptake to obey saturation kinetics. The HRP uptake shows dependency on divalent cations and is reduced if incubation is carried out at pH 6.4. The uptake of HRP, when used at a low, non-saturating concentration (10 micrograms/ml approx. 0.25 microM), is inhibited greater than or equal to 60% by yeast mannan (0.1 mg/ml), ribonuclease B (0.1 mg/ml, approx. 7.4 microM), mannose (30 mM), monodansylcadaverine (1 mM), chloroquine (100 microM), trifluoperazine (25 microM) or maleic acid (2 mM). It is concluded that HRP is taken up in innervated and denervated skeletal muscle by a process of receptor-mediated endocytosis and that this uptake is under neurotrophic control.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Flux of catecholamines through chromaffin vesicles in cultured bovine adrenal medullary cells

Primary cultures of bovine adrenal medullary chromaffin cells were pulse-labeled with [3H]dopamine or [3H]norepinephrine and examined for radioactive and total catecholamine contents by high performance liquid chromatography after additional incubations of 15 min to 10 days. [3H]Dopamine was rapidly taken up by chromaffin vesicles in situ and converted to norepinephrine with a half-time of approximately 6 h. [3H] Norepinephrine taken up by the cells was metabolized in three phases. 1) During its brief transit through the cytoplasm, 20 to 35% of this amine was converted to [3H]epinephrine. 2) Following vesicular accumulation, 65 to 70% of the remaining [3H]norepinephrine was methylated to form [3H]epinephrine with a half-time of approximately 30 h, corresponding to the rate of vesicular catecholamine loss from reserpine-treated cells. 3) The residual [3H]norepinephrine decreased with a half-time of 5 days, probably representing loss from norepinephrine-storing cells. [3H]Epinephrine formed endogenously had a half-life in the cultures of approximately 15 days. These data suggest that leakage of norepinephrine from chromaffin vesicles into the cytoplasm limits the rate of dopamine conversion to epinephrine in the adrenal medulla. The kinetic data indicate that approximately 18% of the endogenous norepinephrine and 73% of the endogenous dopamine are present in epinephrine cells.     

Induction of chromosomal aberrations in cultured Chinese hamster cells by short-term treatment with cadmium chloride

Inducibility of chromosomal aberrations and cytotoxicity in cultured Chinese hamster cells by cadmium chloride (CdCl2) was investigated under 3 different treatment conditions: (i) 2-h treatment in MEM medium supplemented with 10% fetal bovine serum (MEM + 10% FBS) or (ii) in HEPES-buffered Hanks' solution (HEPES-Hanks), and (iii) continuous treatment for 24 h in MEM + 10% FBS. Two-h treatment with CdCl2 in HEPES-Hanks or continuous treatment for 24 h in MEM + 10% FBS was respectively 2 or 3 times more cytotoxic than 2-h treatment with the metal in MEM + 10% FBS. Continuous treatment for 24 h with a CdCl2 concentration in excess of 5 X 10(-6) M was too toxic to the cells to allow chromosomal analysis, and moreover, only a slight increase in incidence of chromosomal aberrations was observed at a concentration of 5 X 10(-6) M CdCl2. In contrast, a marked and concentration-dependent increase in incidence of chromosomal aberrations was observed after post-treatment culture for 22 h follows 2-h treatment with 1 X 10(-6) M to 5 X 10(-5) M of CdCl2 in both MEM + 10% FBS and HEPES-Hanks. Two-h treatment with cadmium in HEPES-Hanks was approximately 3 times more potent for the induction of chromosomal aberrations than that in MEM + 10% FBS. Types of aberrations induced by CdCl2 mainly consisted of chromatid gaps and breaks, although a few exchanges, dicentrics and fragmentations were observed at high concentrations of cadmium. Increase in incidence of tetraploidy was also observed with a concentration dependency after 2-h treatment with CdCl2. Potency of CdCl2 to induce chromosomal aberrations after 2-h exposure was comparable to that of benzo[a]pyrene activated with S9 at equitoxic concentrations. Two-h treatment with cadmium markedly inhibited incorporation of [3H]thymidine, even at concentrations at which incorporation of [3H]uridine or [3H]leucine was less inhibited. However, the inhibition of [3H]thymidine incorporation by cadmium was reversible and the incorporation restored to the control level during 2-6 h of post-treatment incubation. These findings suggest that restoration of DNA synthesis after cadmium exposure is required for the efficient detection of chromosomal aberrations induced by the metal.     

Inhibition of hepatic proteolysis by insulin. Role of hormone-induced alterations of the cellular K+ balance

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

Putrescine uptake in saintpaulia petals

Putrescine uptake and the kinetics of this uptake were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments of [³H] or [¹⁴C] putrescine were done on single petals at room temperature at various pH values. The results show that putrescine uptake occurs against a concentration gradient at low external putrescine concentration (0.5-100 micromolar) and follows a concentration gradient at higher external putrescine concentrations (100 micromolar to 100 millimolar). 2,4-Dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone, two uncouplers, had no effect on putrescine uptake. Uptake rates were constant for 2 hours, reaching a maximum after 3 to 4 hours. Putrescine uptake depended markedly on the external pH and two maxima were observed: at low external concentrations of putrescine, the optimum was at pH 5 to 5.5; at higher concentrations the optimum was at pH 8.     

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors.