Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature

Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.     

Isolation and characterization of Mycoplasma mycoides subsp. mycoides mutants deficient in nucleoside monophosphate transport.

To select mutants lacking dAMP uptake, log-phase cells of Mycoplasma mycoides subsp. mycoides Y were incubated with high-specific-activity [32P]dAMP and then stored several weeks at -20 degrees C to allow 32P decay before plating out. Mutants were screened for lack of labeling by [32P]dAMP. Two mutants were studied further by uptake and growth experiments with other nucleotides.     

Cleavage of adenosine 5''-monophosphate during uptake by Streptomyces griseus.

Unlabeled adenine brought about a (delayed) decrease in radioactivity that had been taken up by phosphate-limited resting cells of Streptomyces griseus from [14C]adenine-labeled adenosine 5'-monophosphate (AMP). Inorganic phosphate, on the other hand, stimulated adenine uptake from AMP, presumably by activating an energy-dependent active transport mechanism. Unlabeled phosphate rapidly diluted the uptake of radioactivity from [32P]AMP. Adenine inhibited uptake of [32P]AMP but not that of [32P]orthophosphate; adenine is thought to act by inhibiting the cleavage of AMP. The uptake of 32P and 14C from double-labeled AMP showed marked differences; 32P was taken up much faster into both cells and nucleic acids. These data indicate that uptake of AMP components takes place after extracellular dephosphorylation of the nucleotide.     

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.     

The effect in vitro of ionizing irradiation and small rises in temperature on the uptake and release of labelled lipids by the human erythrocyte membrane

1. The effect of X-irradiation (50 000 rad) and an increase in temperature from 37 to 42 degrees C on the synthesis, uptake and release of labelled lipids by erythrocytes was studied in plasma incubations in vitro. 2. Both irradiation and a rise in temperature resulted in an inhanced synthesis of [32P]phosphatidic acid in the erythrocytes. 3. The uptake by the erythrocytes of 14C- and 3H-labelled cholesterol, [14C, 32P]phosphatidylethanolamine and [14C, 32P]phosphatidylcholine from plasma lipoproteins was increased by a rise in temperature but not by irradiation. These labelled lipids were apparently taken up in the ratio in which they were found in plasma. They were not released from the erythrocytes in the same manner.     

Temperature Dependence of Drug Interaction with the Platelet 5-Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

Although [3H]imipramine is a selective radioligand for the 5-hydroxytryptamine (5-HT) transporter in human platelets, its affinity for binding to the 5-HT transporter complex at 0 degrees C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5-HT uptake at the physiological temperature of 37 degrees C (Ki = 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5-HT transporter at assay temperatures between 0 degrees C and 37 degrees C, using as radioligands [3H]imipramine (0 degrees C and 20 degrees C) and [3H]paroxetine (20 degrees C and 37 degrees C), a newly available probe for the 5-HT transporter. At 20 degrees C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p less than 0.001), validating the use of these two radioligands to study the 5-HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5-HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5-HT transporter at 0 degrees C. At 37 degrees C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5-HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5-HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5-HT transporter similar to that of imipramine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of phospholipid vesicles with cultured mammalian cells. II. Studies of mechanism

The mechanism of interaction of artificially generated lipid vesicles (approximately 500 A diameter) with Chinese hamster V79 cells bathed in a simple balanced salt solution was investigated. The major pathways of exogenous lipid incorporation in vesicle-treated cells are vesicle-cell fusion and vesicle-cell lipid exchange. At 37 degrees C, the fusion process is dominant, while at 2 degrees C or with energy depleted cells, exchange of lipids between vesicles and cells is important. The fusion mechanism was demonstrated using vesicles of [14C]lecithin containing trapped [13H]inulin. Consistent with a fusion hypothesis, both components became cell associated at 37 degrees C in nearly the same proportions as they were present in the applied vesicles. Additional arguments in favor of vesicle-cell fusion and against phagocytosis or adsorption of intact vesicles are presented. At 2 degrees C or with inhibitor-treated cells, the [3H]inulin uptake was largely suppressed, while the lipid uptake was reduced to a lesser extent. Evidence for vesicle-cell lipid exchange was obtained using V79 cells grown on 3H precursors for cellular lipids. [14C]lecithin vesicles, incubated with such cells, showed no change in their elution properties when subjected to molecular sieve chromatography on Sepharose 4B. However, radioactivity and thin-layer chromatographic analyses revealed that a variety of cell lipiids had been exchanged into the uniamellar vesicles. Further evidence for the fusion and exchange processes was obtained using vesicles prepared from mixtures of [3H]lecithin and [14C]cholesterol. A two-step fusion mechanism consistent with the present findings is proposed as a working model for other fusion studies.     

Chylomicron-remnant uptake by freshly isolated hepatocytes. Effect of heparin and of hepatic triacylglycerol lipase.

Chylomicron remnants labelled biologically with [3H]cholesterol were efficiently taken up by freshly isolated hepatocytes during a 3 h incubation in Krebs bicarbonate medium. Their [3H]cholesteryl ester was hydrolysed (74% net hydrolysis), and 0.1 mM-chloroquine could partially inhibit this hydrolysis, provided that hepatocytes were first preincubated for 2 h 30 min at 37 degrees C. This hydrolysis was also measured in preincubated cells with remnants double-labelled (3H and 14C) on their free cholesterol moiety; [3H]cholesterol arising from [3H]cholesteryl ester hydrolysis was recovered in the free [3H]cholesterol pool. A dose-response study showed saturation of remnant uptake at 180 micrograms of remnant protein/10(7) cells. Heparin (10 units/ml) increased remnant uptake by 63% (P less than 0.01), [3H]cholesteryl ester accumulation in the cell pellet by 110% (P less than 0.025) and hepatic lipase activity secreted in the medium by 2.4-fold (P less than 0.01) and by 3.3-fold (P less than 0.01) at the end of the preincubation and incubation periods respectively. Addition of 100 munits of semi-purified hepatic lipase preparation/flask stimulated remnant uptake by 44-69%, and [3H]cholesteryl ester accumulation in the presence of chloroquine by 2.1-fold (P less than 0.025). When hepatic lipase was incubated solely with the remnants, it decreased their triacylglycerol and phospholipid contents by 24% and 26% respectively. Thus freshly isolated hepatocytes may be used to study chylomicron-remnant uptake. Hepatic lipase, which seems to underly the stimulating effect of heparin, facilitates remnant uptake in vitro, and this could be mediated by at least one (or both) of its hydrolytic properties.     

In vivo fate and scavenger receptor recognition of oxidized lipoprotein[a] isoforms in rats.

High levels of Lp[a] in blood form an independent risk factor for atherosclerosis. Oxidative modification of Lp[a] may be involved in the suggested atherogenic action of Lp[a]. After Cu(2+)-mediated oxidative modification of the 440 kDa and 610 kDa apo[a] isoforms of lipoprotein[a] (Ox-Lp[a]), the in vivo fate was investigated in rats. Ox-Lp[a], when injected into rats, was rapidly removed from the blood circulation by the liver, in which the intrahepatic fate is dependent on the degree of oxidation of the isoforms. Upon oxidation to a slightly increased negative charge of Lp[a], the high molecular weight form of Lp[a] is recognized more efficiently by the Kupffer cells than by the endothelial cells. When the liver uptake of Ox-Lp[a] is blocked by preinjection of polyinosinic acid (poly I), the association of Ox-Lp[a] with the rat heart is increased 20-fold. In vitro studies show that the association and degradation of 125I-labeled Ox-Lp[a] with liver endothelial and Kupffer cells was inhibited by oxidized LDL (Ox-LDL), poly I, or Ox-Lp[a] itself by 60-90%, while only a partial competition was found with acetylated-LDL (up to 25%). In conclusion, after oxidative modification of Lp[a], there is recognition of Ox-Lp[a] by specific oxidized-lipoprotein receptors on liver endothelial and Kupffer cells; the relative importance at low degrees of oxidation of Lp[a] is dependent on the molecular weight of the apo[a] isoforms. Under conditions in which liver uptake is not adequate, the deposition of Ox-Lp[a] in the heart may be of potential pathological importance.     

Hypoxanthine-guanine exchange by intact human erythrocytes

The uptake and release of [14C]hypoxanthine by human erythrocytes, suspended in a tris(hydroxymethyl)aminomethane (Tris)-glucose-NaCl isotonic medium (pH 7.4), have been studied at 37 degrees C. The uptake of hypoxanthine, mediated by its incorporation into inosine 5'-monophosphate (IMP), was markedly stimulated by preincubating the cells in phosphate-buffered saline. After a lag time, [14C]IMP-enriched erythrocytes released [14C]hypoxanthine in the medium. Formycin B, at concentrations known to inhibit purine nucleoside phosphorylase in intact erythrocytes, affected hypoxanthine uptake and release and led to an increase in the intracellular concentration of inosine, suggesting that the main catabolic path of IMP is the sequential degradation of the nucleotide to inosine and hypoxanthine. The addition of guanine to a suspension of [14C]IMP-enriched erythrocytes led to an increase in the rate of [14C]hypoxanthine release, which was unaffected by the presence of formycin B. During the guanine-induced hypoxanthine release, guanine was taken up by the cells as GMP. These results suggest that the presence of guanine in the incubation medium activates a catabolic path in human erythrocytes leading to IMP degradation without formation of inosine.     

Stimulus-aggregation coupling in platelets activated with PAF-acether

Contrary to most agonists, platelet-activating factor (PAF-acether) induces a more pronounced aggregation at 22 degrees C than at 37 degrees C. A possible explanation was sought in the mechanism that couples the PAF-acether-receptor complex with exposure and occupation of fibrinogen binding sites. Comparison of studies performed at 37 degrees C with those at 22 degrees C revealed: a faster binding of [3H]PAF-acether to its receptors; more accumulation of 32P-labelled phosphatidylinositol 4-monophosphate and a slower but more abundant formation of phosphatidic acid that lasted for 5 min; a 1.4-fold increase in phosphorylation of the Mr 47,000 protein and a 2-fold increase in phosphorylation of the myosin light chain. In contrast, less secretion occurred and less [32P]phosphatidylinositol accumulated at 22 degrees C than at 37 degrees C, and also the increase in cytosolic Ca2+ content and the formation of thromboxane B2 were considerably lower. No differences were found in [32P]phosphatidylinositol 4,5-bisphosphate formation and arachidonate metabolism. Fibrinogen binding studies revealed two types of binding at both temperatures, a high-affinity and a low-affinity binding. There were 6-fold more low-affinity binding sites at 22 degrees C than at 37 degrees C, whereas high-affinity binding did not change. These data suggest that the better aggregation found at 22 degrees C is the result of exposure of an increased number of fibrinogen binding sites. The increased protein phosphorylation and phosphatidic acid accumulation and the faster binding of PAF-acether to its receptors which accompany the better aggregation responses at 22 degrees C suggest that these processes are involved in the regulation of exposure of fibrinogen binding sites.     

Phosphate turnover of phosphatidylinositol in resting and thrombin-stimulated platelets

Human platelets were pulse-labelled with [32P]Pi and extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. Immediately after pulse-labelling, the specific 32P radioactivity of phosphatidylinositol (PI) was only 3.4% of that of the gamma-phosphoryl of ATP. Upon incubation of the platelets at 37 degrees C, the specific 32P radioactivity of ATP (beta- and gamma-phosphoryls) remained constant. However, specific 32P radioactivity in PI increased continuously to 17% of specific [gamma-32P]ATP at 90 min of incubation. Stimulation with 0.5 U/ml of thrombin induced a 35% decrease in mass of PI which was unaffected by the time after the pulse-labelling. In contrast, the thrombin-induced changes in [32P]PI differed markedly at the various times after the [32P]Pi-pulse. Immediately after pulse-labelling, [32P]PI initially decreased but increased thereafter to 260% of control values after 180 s. With increasing specific 32P-radioactivity in PI before stimulation, the thrombin-induced increase in [32P]PI gradually disappeared. After 90 min of incubation, thrombin induced a continuous decrease in [32P]PI that almost parallelled mass. The data are explained by an initial breakdown of PI to diacylglycerol through the PI cycle or the polyphosphoinositide cycle, followed by resynthesis of PI through phosphatidic acid. In contrast to pre-existing PI, the resynthesized PI is in full isotopic equilibrium with ATP. This allowed us to estimate that 14% of the PI that is consumed between 30 and 180 s of stimulation, is recycles. From our data we calculate that the rate of PI resynthesis increased from 2.4 to 20 nmol/min per 10(11) cells upon thrombin stimulation of platelets.     

Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation

The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.     

Secretion-coupled protein degradation: studies on mammary casein

Mammary explants from midpregnant rabbits were cultured for 18 h at 37 degrees C with insulin, prolactin and cortisol. Subsequently, explants were labelled for 2 h with inorganic [32P]phosphate, L-[5-3H]proline or L-[4,5-3H]leucine, washed and chased for up to 3 h. The radiolabelling profile of [32P]casein or [3H]casein during the chase period, obtained by isoelectric focussing or immunoprecipitation indicates extensive destruction of neosynthesized casein. The extent of casein destruction in mammary explants in culture (measured after radiolabelling with L-[5-3H]proline), is inversely related to casein secretion. Least casein degradation is observed in explants after 48 h in culture when casein secretion is maximal (observed histochemically). Subsequently, when the extracellular alveolar lumen is filled with secretion products (72 h), rapid intracellular casein destruction is again observed. When the chase was carried out in the presence of drugs which inhibit degradation and/or secretion, the results indicate that secretion-coupled casein degradation is dependent on an intact functional microfilamentous-microtubular network, casein is not degraded by an autophagosome requiring process, degradation is inhibited by leupeptin, amino-acid analogue containing casein does not undergo secretion-coupled degradation and inhibition of N-glycosylation of intracellular vesicular membrane proteins prevents secretion-coupled degradation. Secretion-coupled protein destruction is discussed in relation to the post-translational regulation of the net production of secretory proteins in eukaryotic cells.     

Temperature effect on kinetics of uptake, transfer, and clearance of [14C]noviflumuron in eastern subterranean termites (Isoptera: Rhinotermitidae)

[14C]Noviflumuron uptake, clearance, rate of excretion, and transfer from treated to untreated termite workers were evaluated at 15,19, 23, and 27 degrees C. Feeding units were constructed from plastic containers provisioned with washed sand, distilled water, [14C]noviflumuron-treated feeding discs (0.05 or 0.5% [AI]), and Reticulitermes flavipes (Kollar) workers. Feeding units were held in environmental growth chambers preset at 15, 19, 23, and 27 degrees C. The amount of [14C]noviflumuron present within R. flavipes was measured by scintillation counting and subsequently quantified. Uptake of noviflumuron by R. flavipes workers at 15 degrees C was approximately 2.8 times less than at 19 or 23 degrees C and approximately 4.4 times less than at 27 degrees ighest uptake of [14C]noviflumuron occurred at 27 degrees C and 144 h. Most transfer of [14C]noviflumuron from treated to untreated termite workers occurred between 19 and 27 degrees C. [14C]Noviflumuron had a half-life in R. flavipes workers of approximately 31-45 d, dependent on temperature. A higher amount of [14C]noviflumuron was lost through excretion at > or = 19 degrees C (approximately 15-22%) compared with 15 degrees C (0.27%). Results indicated that increased uptake, transfer, and clearance of noviflumuron by R. flavipes occurred at warmer temperatures (19-27 degrees C), and all of these processes were significantly lower at 15 degrees C.     

Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.     

Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR-31 and MonR-32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR-31 and MonR-32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR-31 or MonR-32 cells required more insulin than CHO cells. Specific binding of [125I]insulin was observed in these cell lines. The two MonR clones bound about one-half or less the [125I]insulin bound by CHO cells. Scatchard analysis for [125I]insulin binding at 4 degrees C and 37 degrees C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down-regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 microgram/ml insulin. A 50-60% decrease in levels of insulin surface binding capacities was observed in CHO after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2-[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.     

The mechanism of [3H]dexamethasone uptake into prolactin producing rat pituitary cells (GH3 cells) in culture

Glucocorticosteroids stimulate growth hormone (GH) synthesis and inhibit prolactin (PRL) synthesis and cell growth in cultured GH3 cells, a clonal cell strain derived from a rat pituitary tumour. This model system was used to study the mechanism by which glucocorticosteroids enter target cells. The cellular uptake of [3H]dexamethasone was temperature dependent and was further inhibited by addition of an excess amount of cold dexamethasone. Half maximal uptake was obtained after about 5 min at 37 degrees C. The initial rates of [3H]dexamethasone uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]dexamethasone in intact cells studied at different temperatures resulted in linear Arrhenius plots, with a calculated energy of activation of 91.0 kJ x mole-1 x degree-1. Scatchard analysis of specifically cell bound [3H]dexamethasone at equilibrium (0 degrees C) showed a straight line with a calculated dissociation constant (Kd) of 1.6 x 10(-9) M and a maximal uptake of 180 x 10(-15) mole/mg cell protein. Specific binding of [3H]dexamethasone to cytosol proteins could only be demonstrated at 0 degrees C. These results indicate that [3H]dexamethasone diffuses passively into the cell, and binds to specific receptors in an energy dependent way.     

Insulin processing and signal transduction in rat adipocytes

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.