Charles A. Frolik

Transforming growth factor-β controls receptor levels for epidermal growth factor in NRK fibroblasts

NRK fibroblasts exposed to transforming growth factor-beta (TGF-beta) show increased binding of radiolabeled epidermal growth factor (EGF) relative to untreated cells. The binding of another growth factor, rat insulin-like growth factor-II, is unaffected. The increase in EGF binding induced by TGF-beta is not due to inhibition of EGF processing nor to an alteration in the affinity of plasma membrane EGF receptors. However, treatment of the cells with TGF-beta does cause a rapid increase in the number of plasma membrane receptors for EGF. TGF-beta has little effect on the rate of overall protein synthesis, but the increase it induces in EGF binding can be completely inhibited by cycloheximide and tunicamycin. Thus a selective synthetic mechanism underlies TGF-beta action. Cells incubated with TGF-beta also show altered down regulation of their EGF receptors in response to the ligand; concentrations of EGF that can induce strong biological responses no longer decrease the plasma membrane receptor level below the basal state. These results agree well with the known specificity and synergism of the interaction between TGF-beta and EGF. Moreover, they describe a mechanism of growth control in which bioactive peptides act coordinately through a regulatory effect on the number of cell-surface receptors.

Metabolism of Retinoids

Publisher Summary This chapter discusses metabolism of retinoids. Dietary retinol is quickly esterified in the intestinal mucosa with long-chain fatty acids and is incorporated into chylomicra and transported through the lymph to the liver. In the liver, the retinyl esters are hydrolyzed and then re-esterified for storage. Retinyl palmitate is the major ester in both the lymph and in the liver, with saturated esters comprising two-thirds to three-fourths of the retinyl esters. The enzyme responsible for retinol esterification in the liver is found in the microsomal fraction. When spontaneously transformed mouse fibroblasts are grown in the presence of certain retinoids, the cells display an increased adhesion and a change in their morphology to that more characteristic of a normal phenotype. The enzymatic activity required for the synthesis of 4-hydroxyretinoic acid from retinoic acid is located in the microsomal fraction of the hamster liver.

High-pressure liquid chromatographic determination of 13-cis-retinoic acid and all-trans-retinoic acid in human plasma.

Abstract A simple and selective method is described for the determination of 13- cis -retinoic acid and all- trans -retinoic acid in plasma. The assay involves lyophilization of the sample, extraction of the residue with methanol, and separation of the retinoids by reverse-phase high-pressure liquid chromatography. The procedure can detect as little as 0.05 μg of either compound. It is capable of being readily modified to determine the amount of retinoic acid in other tissues as well as tissue concentrations of other retinoids. Using this assay, the absorption and disappearance from human plasma of an oral dose of 13- cis -retinoic acid have been followed.

Dose-dependent kinetics of all-trams-retinoic acid in rats: Plasma levels and excretion into bile, urine, and faeces

Abstract Plasma concentration-time curves for all-trans-retinoic acid (RA) after 0.015.0.25 or 5 mg/kg, i.V., deviated from first-order kinetics in the rat. Within 10 min after the i.v. infusion, a rapid, dose-dependent decrease in RA concentration was observed (slope steepest at the lowest dose). During a secondary phase of slower decline, the times required to halve the RA concentration after 0.015, 0.25 and 5 mg/kg were 40, 65 and 120 min respectively. At later times, the concentration-time curves for all three dose levels assumed a fast rate of decline (half-life about 19 min at the lower dose). The dose-dependent kinetics of RA in plasma were not due to enterohepatic recirculation of RA, since RA levels in plasma were not lower in rats with biliary fistulas given comparable doses. In contrast, circulating levels of RA metabolites remained elevated for several hours and were significantly diminished by interruption of the enterohepatic circulation. After a dose of [10-3H]RA, the rate of biliary excretion of radiolabeled material was initially slower after 5 mg/kg RA than after 0.015 mg/kg RA. Within the first 24 hr, however, approximately the same proportion of both doses appeared in bile. All-trans-retinoyl-β-glucuronide is only a minor biliary metabolite of RA. Glucuronidation of RA was dose-dependent, since the percentage of total biliary metabolites represented by all-trans-retinoyl-β-glucuronide increased with increasing dose. Renal excretion of RA and its metabolites was significantly decreased by interruption of the enterohepatic circulation. The percentage of dose excreted in the urine decreased with increasing dose.

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

Abstract The metabolism of 13-cis-[11-3H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.

Metabolism of all-trans-retinyl acetate to retinoic acid in hamster tracheal organ culture

All-trans-[3H]retinyl acetate has been shown to be metabolized to all-trans-[3H]retinoic acid in a target tissue of vitamin A action, the hamster trachea in organ culture. That the compound produced is indeed all-trans-retinoic acid is demonstrated by chromatography of the biosynthetically produced retinoic acid with synthetic all-trans-retinoic acid in two different high-pressure liquid chromatographic systems, either as the free acids in a reverse-phase system or as the methyl esters in a normal-phase system. The all-trans-[3H]retinoic acid was also found in the tracheal epithelium and cartilage as well as in the medium. In addition the tracheal tissue also contained retinyl palmitate and other esters. Finally, further in vitro metabolism of [3H]retinyl acetate paralleled the metabolism of [14C]retinoic acid suggesting that these two compounds are being metabolized through similar pathways.

Inhibition of transforming growth factor-induced cell growth in soft agar by oxidized polyamines

An inhibitor of the transforming growth factor-induced growth in soft agar of normally anchorage-dependent rat kidney fibroblasts has been detected in the acid-ethanol extracts of human placenta, bovine lung and kidney, and human rhabdomyosarcoma cells (A673). The inhibitor has been purified from human placenta by gel-filtration and cation-exchange chromatography followed by acetylation and HPLC. Acetylation destroys inhibitory activity and deacetylation, by treatment with 6 N HCl at 110 degrees C for 16 h, restores full activity. The purified compound has been identified as spermine by mass spectral and NMR analyses and by cochromatography on HPLC of the acetylated material with acetylated spermine. Both the compound isolated from the placenta and spermine and spermidine show approximately equal activity in inhibiting the transforming growth factor-induced growth of cells in soft agar with an ED50 of 0.7-1.1 microM, while putrescine displays no inhibitory activity. Evidence suggests that the polyamines must first be oxidized by serum polyamine oxidase before inhibition will occur. Acrolein, a product of polyamine oxidation, will also inhibit cell growth in soft agar with an ED50 of 6.8 microM. It is concluded that an oxidation product of spermine is responsible for the previously reported inhibition of colony growth in soft agar following treatment of normal fibroblasts with transforming growth factors.

Variables in the high-pressure cation-exchange chromatography of proteins☆

The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and p I range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.