David B. Donner

Identification and characterization of receptors for tumor necrosis factor-α in the brain

Specific receptors for murine TNF have been identified in homogenates of rodent brain. These receptors are saturable and bind TNF with sufficient affinity to ensure occupancy by cytokine elaborated during infection. 125I-mTNF was detected in four specific complexes of Mr 130,000, 90,000, 66,000 and 60,000 after affinity labeling. Solubilization of brain membranes into detergent increased binding capacity 4-fold which indicates the presence of latent receptors for mTNF in the brain. Specific binding was greatest in the brainstem, least in the cerebellum and was also detected in the cortex, thalamus and basal ganglia.

The effects of bioregulators upon amino acid transport and protein synthesis in isolated rat hepatocytes

Isolated rat hepatocytes prepared by an enzyme perfusion technique possess a functional amino acid transport system and retain the capacity to synthesize protein. Amino acid transport was studied using the non-metabolizable amino acid analog alpha-aminoisobutyric acid. The transport process was time, temperature and concentration dependent. Similarly, leucine incorporation into protein was time and temperature dependent being optimal at 3m degrees C. Amino acid, fetal calf serum, growth hormone and glucose all produced small, reproducible increases in protein synthesis rates. Bovine serum albumin diminished the uptake of alpha-aminoisobutyric acid and leucine incorporation into protein. The amino acid content on either side of the cell membrane was found to affect transport into or out of the cellular compartment (transconcentration effects). High cell concentrations decreased transport and protein synthesis as a result of isotopic dilution of labelled amino acids with those released by the hepatocytes. This was consistent with the capacity of naturally occurring amino aicds to compete with alpha-aminoisobutyric acid for uptake into the hepatocyte. In order to define more precisely the effects of bioregulators on transport and protein synthesis it will be necessary to define and subfractionate cellular compartments and proteins which are the specific targets of cellular regulation.

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH3 cells were characterized. Uptake of 125I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37 degrees C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with 125I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for 125I-labeled insulin binding sites. 125I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. 125I-labeled insulin was degraded at both 4 and 37 degrees C by GH3 cells, but not by medium conditioned by these cells. After a 5 min incubation at 37 degrees C, products of 125I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of 125I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of 125I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of 125I-labeled insulin, as well as intact hormone, were recovered from GH3 cells. After 30 min incubation at 37 degrees C, 80% of the cell-bound radioactivity was not extractable from GH3, cells with acetic acid.

Consequences of 125I-labeled insulin degradation by hepatocytes on the interpretation of receptor binding studies

The association of 125I-labeled insulin with hepatocytes was assayed by filtration or microcentrifugation. Assay by centrifugation resulted in a greater amount of retained radioactive label throughout the course of association of 125I-labeled insulin with hepatocytes. Similarly, saturation experiments assayed by microcentrifugation suggested greater binding than filtration. During dissociation, cells isolated by centrifugation release a greater amount of rapid-dissociating radioactive label. Control experiments of [3H]-inulin exclusion with cell pellets, which were isolated during microcentrifugation, demonstrated that the difference between the methods was not due to extracellular trapping of radioactivity. Therefore, the data suggested that there was more low-affinity retention when binding was assayed by centrifugation than filtration. The integrity of the 125I-labeled insulin extracted from hepatocytes was determined by column chromatography. A substantially greater proportion of the extracted radioactivity was fragments of 125I-labeled insulin in cells isolated by centrifugation. It is suggested that the extensive washing of the cells during filtration removes more fragments than does centrifugation. During dissociation, the low-affinity component of radioactivity, which was observed in the centrifugal assay, resulted from the transient retention of insulin fragments. The extensive degradation of insulin, which was assayed by either method, and the differences observed between these methods, should be considered in the interpretation of binding experiments with cells.

Acute metabolic effects of human recombinant tumor necrosis factor β in the rat

AbstractBackground: Cancer cachexia is associated with several alterations in host metabolism, including hypoaminoacidemia and an increase in gluconeogenesis (GLC) and lipolysis. Tumor necrosis factor β (TNFβ), a lymphokine released by mitogen-activated T lymphocytes and several cancer cell lines, causes an increase in lipolysis in 3T3L1 adipocytes. Since little is known about the metabolic effects of TNFβ in vivo, we examined its acute effects in the rat.nMethods: Twenty-eight male Fischer rats were injected intraperitoneally with TNFβ (250 µg/kg) or saline (CTL), and after 4 h, isolated hepatocytes were obtained (by in situ collagenase liver perfusion [n=12]) or aortic blood was collected (n=16). Hepatocytes were incubated with 10 mM alanine (ALA) or 10 mM lactate (LAC), and glucose production was measured. Rates of GLC (nmol glucose/106 cells/min) were determined by linear regression. Plasma lactate, glucose, insulin, and amino acids (AA) (nmol/ml) were measured, and values were expressed as means ± SEM. Comparisons between groups were made by unpairedt test or Mann-WhitneyU test, and significance was defined as p<0.05.nResults: TNFβ caused a 130% increase in gluconeogenesis from alanine (2.7±0.5 vs 1.2±0.2 nmol glucose/106 cells/min, TNF vs CTL), and a 60% increase from lactate (7.5±1.0 vs 4.6±0.5 nmol glucose/106 cells/min, TNF vs CTL). Plasma insulin levels in TNF treated rats were 1.2±0.2 ng/ml compared to 1.1±0.2 ng/ml in CTL. Total amino acid levels in TNF treated rats were 3,175±111 nmol/ml compared to 3,190±103 nmol/ml in CTL.nConclusion: In vivo TNFβ causes an increase in hepatic gluconeogenesis from alanine and lactate with no change in plasma insulin or amino acids.

Early Events in the Interaction of IFN-α with IFN-Sensitive and IFN-Resistant Daudi Cells

Daudi cells, and IFN-sensitive and -resistant Daudi subclones contain 2,300 (Kd=20 pM), 3,700 (Kd=52 pM), and 3,000 (Kd=45 pM) homogeneous IFN-α binding sites per cell, respectively. Thus, IFN-sensitive and — resistant Daudi cells have similar numbers of high affinity IFN-α receptors. The dissociation of IFN from its receptor on each Daudi cell line was resolved into fast and slow components. IFN-α dissociated from IFN-sensitive Daudi cells more slowly than from resistant cells. Also, a greater amount of the IFN-receptor complex becomes associated with the Triton-insoluble cytoskeletal matrix in the sensitive than in the resistant cells. Lectins, which increase the formation of Triton-insoluble IFN-receptor complexes in sensitive and resistant cells, decrease the dissociation of IFN-α from its receptor. Finally, IFN-α inhibits the anti-Ig-induced redistribution of surface Ig in IFN-sensitive but not in IFN-resistant cells. Our results suggest that association of IFN-receptor complexes with the Triton-insoluble matrix may be of functional significance. Such interaction may slow the dissociation of IFN-α from its receptor, impair mobility of cell surface receptors, and perhaps play an important role in transmembrane signalling through the IFN receptor.