Induction of inducible nitric oxide synthase by ginsenosides in cultured porcine endothelial cells

Mechanism of Nitric oxide (NO) production by ginsenosides was investigated in cultured porcine endothelial cells. Beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) staining showed that the NO production was significantly enhanced by the presence of 40 microg/ml ginsenosides with 10 microM L-arginine after 12 h incubation. NO production was suppressed by addition of 0.5 microM Nomega-Nitro-L-arginine (L-NNA), an inhibitor of NO synthases (NOSs), to the incubation medium. In addition, the immunoreactive signals of inducible NOS (iNOS) were appeared in endothelial cells after 12-h incubation of ginsenosides, whereas the signals were not observed in non-treated cells. Our findings suggest that ginsenosides can enhance NO production by induction of iNOS in addition to its direct effect on endothelial cells by increasing intracellular Ca2+ concentration.     

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.     

Selective induction of peroxisomal enzymes by the hypolipidemic drug bezafibrate. Detection of modulations by automatic image analysis in conjunction with immunoelectron microscopy and immunoblotting

Quantitative immunoelectron microscopy in conjunction with quantitative analysis of immunoblots have been used to study the effects of bezafibrate (BF), a peroxisome-proliferating hypolipidemic drug, upon six different enzyme proteins in rat liver peroxisomes (Po). Antibodies against following peroxisomal enzymes: catalase, urate oxidase, alpha-hydroxy acid oxidase, acyl-CoA oxidase, bifunctional enzyme (hydratase-dehydrogenase) and thiolase, were raised in rabbits, and their monospecificities were confirmed by immunoblotting. Female Sprague-Dawley rats were treated for 7 days with 250 mg/kg/day bezafibrate and liver sections were incubated with the appropriate antibodies followed by the protein A-gold complex. The labeling density for each enzyme was estimated by automatic image analysis. In parallel experiments immunoblots prepared from highly purified peroxisome fractions of normal and BF-treated rats were incubated with the same antibodies. The antigens were visualized by an improved protein A-gold method including an anti-protein A step and silver amplification. The immunoblots were also quantitated by an image analyzer. The results revealed a selective induction of beta-oxidation enzymes by bezafibrate with thiolase showing the most increase followed by bifunctional protein and acyl-CoA oxidase. The labeling density for catalase and alpha-hydroxy acid oxidase was reduced, confirming fully the quantitative analysis of immunoblots which in addition revealed reduction of uricase. These observations demonstrate that hypolipidemic drugs induce selectively the beta-oxidation enzymes while other peroxisomal enzymes are reduced. The quantitative immunoelectron microscopy with automatic image analysis provides a versatile, highly sensitive and efficient method for rapid detection of modulations of individual proteins in peroxisomes.     

Proliferation of peroxisomes and induction of peroxisomal beta-oxidation enzymes in rat hepatoma H4IIEC3 by ciprofibrate

For the analysis of the molecular mechanism of the action of peroxisome proliferators, we attempted to establish the optimal conditions for obtaining the effects of the chemicals in vitro, employing an established cell line, Reuber rat hepatoma H4IIEC3. Histochemical analyses revealed a marked increase in the number, size, and catalase content of peroxisomes in the cells cultured on a medium containing 0.5 mM ciprofibrate, a peroxisome proliferator. The activity of acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was increased by more than 10-fold by the same treatment. Catalase was also induced significantly, whereas the activities of glutamate dehydrogenase and lactate dehydrogenase, mitochondrial and cytosolic marker enzymes, did not change upon the treatment. Immunoblotting and RNA-blotting analyses confirmed the increases in the amount of protein and mRNA for all the three enzymes of the peroxisomal beta-oxidation system. Cell fractionation experiments gave a partial separation of peroxisomes from other organelles for the induced culture. Thus, H4IIEC3 cells offer a good in vitro model system of the induction of peroxisomes and peroxisomal beta-oxidation enzymes by peroxisome proliferators.     

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells.

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.     

Induction of drug metabolism enzymes by dihalogenated biphenyls

The effects of pretreatment with symmetrically dihalogenated biphenyls (DXBs, X-F, Cl(C), Br(B) and I) on rat liver drug metabolism enzymes were investigated. 4,4'-DFB, -DCB, and -DBB as well as 2,2'-DFB appeared to be inducers of microsomal cytochrome P-450-linked monoxygenases (N-demethylases of aminopyrine and ethylmorphine). However, no structure-induction relationship was found. 4,4'-DXBs also induced a cytochrome P-448-linked mono-oxygenase (ethoxyresorufin O-deethylase), and their order of induction potential seemed to parallel the increase of the size of the halogen substituent. Therefore, 4,4'-DXB's may be categorized as mixed-type inducers, the cytochrome P-450 component being the more pronounced. Data on the cytochrome P-448 induction by dihalogenated biphenyls with only para substituents may be considered as a refinement of the previously described structure-activity relationship in this respect. All of the DXBs except 3,3'-DCB and 4,4'-DIB, enhanced, like phenobarbital, the activity of UDP-glucuronyltransferase toward 4-hydroxybiphenyl. Only 4,4'-DFB was able to induce the activity of glutathione S-transferase toward 1,2-epoxy-3-(p-nitrophenoxy)propane. Studies after 4,4'-DBB-treatment revealed, like phenobarbital, a preferential induction of ethylmorphine N-demethylase on rough endoplasmic reticulum-derived microsomes, whereas UDP-glucuronyltransferase activity toward 4-hydroxybiphenyl was induced to a larger extent on smooth endoplasmic reticulum microsomes, suggesting a dissimilar enzyme induction in microsomal subfractions.     

Induction of drug metabolizing enzymes by vitamin E

Vitamin E is an essential micronutrient involved in various processes relevant to human health and disease. Although it has long been considered just as an antioxidant, it has now become clear that vitamin E has functions far exceeding that as an antioxidant. These include regulation of cellular signaling processes and gene expression. Expression control of enzymes involved in drug metabolism was recognized during the investigation of vitamin E degradation. Vitamin E is metabolized by side chain degradation initiated by an omega-hydroxylation, catalyzed by a cytochrome P450 enzyme (CYP). This mechanism is identical for all forms of vitamin E. The degree to which they are degraded, however, varies dramatically, and may, in part, explain their different biological activities. CYPs degrade various endogenous and exogenous compounds and many of them are induced by their substrates. Also, gamma-tocotrienol, identified as substrate of CYPs, increased endogenous CYP3A4 in human HepG2 cells. In two studies with mice undertaken independently, alpha-tocopherol induced Cyp3a11, the murine homolog to human CYP3A4, whereas neither gamma-tocopherol nor gamma-tocotrienol, due to rapid degradation, showed any effect. CYPs are induced via the activation of the pregnane-X-receptor (PXR), a member of the family of nuclear receptors. They are activated by a large number of lipophilic xenobiotics. Also, vitamin E induced a reporter gene driven by PXR. The induction was highest with alpha- and gamma-tocotrienol and low but significant with alpha-tocopherol. This roughly correlates with the in vitro binding of vitamin E to PXR. These findings reveal that, in principle, vitamin E is able to directly influence gene activity. They also raise the question of whether vitamin E may interfere with drug metabolism in humans. Related research is urgently deeded.     

Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.     

Induction of heme oxygenase by delta 12-prostaglandin J2 in porcine aortic endothelial cells.

delta 12-Prostaglandin (PG)J2 stimulated the synthesis of a 31,000-dalton protein (termed p31) and the induction of cellular heme oxygenase activity in porcine aortic endothelial cells. A good correlation was observed between the time courses and dose dependencies of the induction of p31 synthesis and that of heme oxygenase activity by delta 12-PGJ2. Hemin, a known inducer of heme oxygenase, also induced p31 synthesis as well as heme oxygenase activity in the cells. On two-dimensional gel electrophoresis, p31 induced by delta 12-PGJ2 exhibited an isoelectric point of 5.4, which coincided exactly with that induced by hemin. These results indicate that the p31 induced by delta 12-PGJ2 in porcine aortic endothelial cells is heme oxygenase.     

Induction of hepatic drug metabolizing enzymes by DL-methionine in rats

A single intraperitoneal injection of DL-methionine (500 mg/kg body wt.) to adult male Wistar rats was shown to significantly induce all the components of the hepatic microsomal mixed function oxidase system such as NADPH cytochrome C reductase activity, cytochromes P-450 and b₅, as well as activities of drug metabolizing enzymes such as aminopyrine demethylase and uridine 5′ -diphosphate-glucuronosyltransferase. Combined administration of nicotinamide (250 mg/kg body wt.) and DL-methionine (500 mg/kg body wt.) was shown to bring about an additional increase (25-30%) in the activities of these enzymes as compared to their induction on independent administration of the two endobiotics. In rats bearing Yoshida sarcoma (ascites) tumour as well as in normal rats injected with serum from tumour bearing animals, the decreased activities of hepatic mixed function oxidases could be restored to their normal levels by administration of DL-methionine (500 mg/kg body wt.) to these rats. Whereas actinomycin D (1 mg/kg body wt.) had no effect on the increased incorporation of [¹⁴C] labelled leucine into microsomal proteins following administration of nicotinamide, the enhanced incorporation of the label following DL-methionine administration was completely inhibited by the same dose of actinomycin D. Administration of cycloheximide (0·5 mg/kg body wt.) to rats could completely inhibit the increased incorporation of [¹⁴C] leucine into hepatic microsomal proteins following independent administration of nicotinamide and DL-methionine. Similar inhibitory pattern with actinomycin D and cycloheximide was also demonstrated in case of induction of NADPH cytochromeC reductase activity by both these endobiotics.     

Induction of rat hepatic drug metabolizing enzymes by dimethylcyclosiloxanes

Low molecular weight dimethylcyclosiloxanes (DMCS) are important precursors in the synthesis of polydimethysiloxane polymers widely used in industry, and in medical and personal care products. The objective of this study was to characterize the ability of two DMCS, octamethylcyclosiloxane (D4) and decamethylcyclopentasiloxane (D5) to induce drug metabolizing enzymes in rats. Male and female Sprague-Dawley rats were administered 1, 5, 20, or 100 mg/kg D4 or D5 in corn oil daily by gavage for 4 days. Changes in the levels of activity and/or immunoreactivity of CYP1A1/2, CYP2B1/2, CYP3A1/2 and NADPH cytochrome P450 reductase in liver microsomes were examined. Significant increases were observed in the liver to body weight ratio in female rats administered either D4 or D5 at doses > or = 20 mg/kg. Increases in the liver to body weight ratio were observed in male rats treated with > or = 100 mg/kg D5 but not with D4. Relatively large increases in CYP2B1/2 enzymatic activity and immunoreactive protein were observed with increasing concentrations of both D4 and D5. Significant increases in 7-pentoxyresorufin O-depentylase (PROD) activity were also detected in male and female rats given D4 at doses > or = 5 mg/kg. D5 increased PROD activity in male rats at doses > or = 20 mg/kg and in female rats at doses > or = 5 mg/kg. 7-Ethoxyresorufin O-deethylase (EROD) activity was increased in both male and female rats receiving > or = 20 mg/kg D4 or > or = 5 mg/kg D5; however, no changes were detected in CYP1A1/2 immunoreactive protein in rats of either sex. D4 and D5 caused significant increases in CYP3A1/2 immunoreactive protein in only male rats treated with 100 mg/kg of either compound. However, significant increases were detected in CYP3A1/2 immunoreactive protein in female rats at D4 doses > or = 20 mg/kg and D5 doses > or = 5 mg/kg. Induction of NADPH cytochrome P-450 reductase immunoreactive protein was observed with D4 in female rats and in both male and female rats with D5. Induction of CYP2B/1/2, CYP3A1/2 and NADPH cytochrome P450 reductase was observed in rats treated with 50 mg/kg phenobarbital by intraperitoneal injection. Maximal CYP2B induction detected with D4 was approximately 50% of the increase observed with phenobarbital. In summary, D4 and D5 induced CYP2B1/2 in adult rat liver in a manner similar to that observed with phenobarbital; however, differences were observed between D4 and D5 in their ability to induce CYP3A1/2 and NADPH cytochrome P450 reductase. Female rats were more sensitive to the inductive properties of low doses of both DMCS than male rats whereas male rats were more responsive to phenobarbital induction.     

Growth-dependent subcellular redistribution of protein kinase C in cultured porcine aortic endothelial cells

We have previously observed major differences in the phosphorylation of membrane proteins in sparse, proliferating versus confluent, quiescent pig aortic endothelial cells (EC) (Kazlauskas and DiCorleto, 1987). In the present study we examined whether EC growth state can influence the activity of a specific phosphorylating enzyme, protein kinase C (PKC) in cytosolic and membrane fractions of pig aortic EC. Levels of PKC were measured using two methods: 1) Ca2+ and phospholipid-dependent phosphorylation of exogenous histones using gamma-labeled [32P]ATP, and 2) [3H]phorbol-12,13-dibutyrate (PDBu) binding activity. The total amount of PKC activity in the quiescent versus proliferating cells was similar but the percentage of PKC activity in the membrane fraction correlated with the proliferative index of the cells: confluent, quiescent cultures exhibited a majority of PKC activity in the cytosolic fraction (67%), whereas sparse, proliferating cultures contained principally membrane-bound PKC (70%). We also examined the role of PKC in the mitogenic response of pig aortic EC to fetal calf serum. Following serum stimulation of sparse, serum-deprived pig aortic EC, PKC activity redistributed from the cytosolic to the membrane fraction in a rapid process that correlated with subsequent DNA synthesis. A potent activator of PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced a minimal mitogenic response in pig aortic EC when added alone but acted synergistically with low concentrations of fetal calf serum to greatly stimulate DNA synthesis. Furthermore, pig aortic EC treated with TPA for 24 h to down-regulate PKC exhibited only 25% of the serum-stimulated mitogenic activity of control cultures. These results suggest a role for PKC activation and translocation in the proliferation of pig aortic EC.     

Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells.

Potato (Solanum tuberosum) lectin, which is a very highly glycosylated glycoprotein, has been completely deglycosylated by use of the trifluoromethanesulphonic acid reagent described by Edge, Faltnek, Hof, Reichert & Weber [(1981) Analyt. Biochem. 118, 131-137]. This shows that both hydroxyproline-arabinofuranoside and serine-galactopyranoside linkages are hydrolysed. The deglycosylated lectin is still functional and cross-reacts with one component of an anti-(potato lectin) antiserum.     

Endothelin immunoreactivity in medium from cultured porcine aortic endothelial cells correlates with the biological activity

Using the radioimmunoassay (RIA) of endothelin (ET), we measured immunoreactive ET (IR-ET) in culture medium of porcine aortic endothelial cells. The immunoreactivity in the medium was compared with the biological activity. The amount of IR-ET released into the medium was calculated at 250-350 pg/10(6) cells/hr. The amount of IR-ET released into the culture medium increased progressively with 3-24 hr of incubation and corresponded to the increase in medium-induced vasoconstriction of rat isolated aorta. When the vasoconstrictor activities in the culture medium were plotted against the IR-ET concentration determined by RIA, the concentration-response curve showed similarity to that obtained with synthetic porcine ET. This RIA system will be a useful for investigating mechanisms of ET secretion from endothelial cells.     

Expression of liver peroxisomal proteins as compared to other organelle marker enzymes in rats treated with hypolipidemic agents

Peroxisome proliferation induced by 2 hypolipidemic agents (clofibrate and ciprofibrate) was studied in rats by complementary approaches, ie cell fractionation, electron microscopy, marker enzyme activities, immunoblotting and nucleic acid hybridization techniques. Administration of clofibrates for 2 and 52 weeks in doses of 500 ppm and 50 ppm respectively, or ciprofibrate for 2,28 and 52 weeks in doses of 250, 25 and 25 ppm respectively, did not alter the behavior of the peroxisomes after induction as shown by ultracentrifugation profiles. The peroxisome mass was increased as shown by the purification procedure. Specific enzymes (catalase and mostly cyanide insensitive palmitoyl CoA oxidase) were induced. A mechanism of peroxisome biogenesis might have been initiated ie cytosolic factor, ligand-receptor interaction and/or post-translational modification of the import. Increase in marker enzyme activities showed that the peroxisomes are the most responsive organelles in comparison to lysosomes, mitochondria and smooth endoplasmic reticulum (except for cytochrome P-450 LA omega-hydroxylase). Peroxisomal integral membrane proteins appeared to be differently induced: some of them were virtually absent in untreated rat liver but were strongly expressed in treated liver. Induction was sustained for 52 weeks, indicating that there was no compensatory mechanism.     

Induction of apoptotic cell death in vascular endothelial cells cultured in three-dimensional collagen lattice

Endothelial cells derived from fetal bovine aorta (BAECs) undergo apoptosis in three-dimensional (3-D) type I collagen lattice in the absence of specific angiogenic factor. In the presence of angiogenic factor, BAECs survive and form a capillary-like tube structure in 3-D culture. In the present study we elucidate the mechanisms of BAECs apoptosis or survival and tube formation in 3-D culture. When BAECs embedded in collagen lattice were cultured with angiogenic factor (fibroblast growth factor-2 (FGF-2) or 4beta-phorbol 12-myristate 13-acetate (PMA)) in the presence of PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, BAECs did not form tube structures and underwent apoptosis in collagen lattice. Function-blocking antibody against alphavbeta3 integrin also inhibited tube formation and induced apoptosis in 3-D culture in the presence of angiogenic factors. Exposure of BAECs to FGF-2 and PMA had no effect on the alphavbeta3 integrin expression but induced the activation of alphavbeta3 integrin. PD98059 attenuated alphavbeta3 integrin activation in response to angiogenic factor. KB-R8301, a hydroxamic acid-based matrix metalloproteinase (MMP) inhibitor, prevented apoptotic cell death in the absence of angiogenic factor in 3-D culture and enhanced capillary-like tube formation in the presence of angiogenic factor, which was not inhibited by the anti-alphavbeta3 integrin antibody. The results suggest that angiogenic factor-induced alphavbeta3 integrin activation through the MEK-ERK pathway regulates the BAEC fate between apoptosis and angiogenesis in collagen lattice. MMP derived from BAECs seems to play a key role in the release of cryptic ligands for alphavbeta3 integrin from intact collagen.     

Impact of diabetes-associated lipoproteins on oxygen consumption and mitochondrial enzymes in porcine aortic endothelial cells

Impairments in mitochondrial function have been proposed to play an important role in the pathogenesis of diabetes. Atherosclerotic coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated low density lipoproteins (glyLDL) and oxidized LDL (oxLDL) were detected in patients with diabetes. Our previous studies demonstrated that oxLDL and glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). The present study examined the effects of glyLDL and oxLDL on mitochondrial respiration, membrane potential and the activities and proteins of key enzymes in mitochondrial electron transport chain (mETC) in cultured porcine aortic EC (PAEC). The results demonstrated that glyLDL or oxLDL significantly reduced oxygen consumption in Complex I, II/III and IV of mETC in PAEC compared to LDL or vehicle control using oxygraphy. Incubation with glyLDL or oxLDL significantly reduced mitochondrial membrane potential, the activities of mitochondrial ETC enzymes - NADH dehydrogenase (Complex I), succinate cytochrome c reductase (Complex II + III), ubiquinol cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV) in PAEC compared to LDL or control. Treatment with oxLDL or glyLDL reduced the abundance of subunits of Complex I, ND1 and ND6 in PAEC. However, the effects of oxLDL on mitochondrial activity and proteins were not significantly different from glyLDL. The findings suggest that the glyLDL or oxLDL impairs mitochondrial respiration, as a result from the reduction of the abundance of several key enzymes in mitochondria of vascular EC, which potentially may lead to oxidative stress in vascular EC, and the development of diabetic vascular complications.     

Induction of peroxisomal beta-oxidation enzymes by dehydroepiandrosterone and its sulfate in primary cultures of rat hepatocytes.

Treatment of cultured rat-hepatocytes with 50 microM dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) for up to 5 days resulted in a progressive increase in peroxisomal beta-oxidation and carnitine acetyltransferase activity. After 5 days, the increases in activity were 2.6- and 4.8-fold for peroxisomal beta-oxidation and 11.7- and 17.1-fold for carnitine acetyltransferase over the initial activity, in DHEA- and DHEAS-treated cells, respectively. The stimulation of the activity of these enzymes by the respective agents was dose-related; it was maximum with 50 to 100 microM DHEA and 50 to 250 microM DHEAS, although DHEAS was more effective for stimulation than DHEA. Western blot analyses revealed the induction of acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme and carnitine acetyltransferase in the treated cells. Moreover, induction of fatty acid omega-hydroxylase proteins (P-450IVAS) was also revealed. These results indicate that DHEA and DHEAS act directly on hepatocytes. The induction of hepatic peroxisomal beta-oxidation enzymes and several other enzymes in rats administered with DHEA could be accounted for, at least in part, by the direct action of DHEA and its sulfate-conjugate (DHEAS) on liver cells.     

Noninactivation of thyrotropin by cultured porcine thyroid cells.

Freshly isolated porcine thyroid cells were cultured in the presence of highly purified porcine thyrotropin. Cells associate into follicles between the second and tenth day of culture and later form a monolayer. The biological and immunological activity of thyrotropin was measured daily in the media. Thyrotropin concentration and biological activity remained unchanged from the onset of the culture up to day 14. Limiting factors influencing thyroglobulin biosynthesis do not appear before day 13. The loss of follicular organization at day 10 cannot be explained by thyrotropin degradation in the medium. Considering the number of receptors per cell and the half life of the thyrotropin . receptor complex in the two dissociation compartments previously demonstrated, it appears in terms of both biological activity and affinity for the receptors that the thyrotropin molecules released from the first compartment do not differ from native molecules. It can be calculated that at least 31% of the molecules released from the second compartment are not inactivated. Thus, it is probable that the catabolism of thyrotropin on the receptor, or near the receptor site, does not play an important role in the regulation of thyroid cell function in vitro.     

Receptor-mediated uptake of lipoproteins by cultured porcine granulosa cells

Receptor-mediated uptake of low density lipoprotein (LDL) has been shown to provide a major source of cholesterol for a variety of cell types, particularly steroidogenic cells. In this study, the functional significance of lipoproteins in porcine ovarian granulosa cells and their mechanism of uptake by the cell was examined. Porcine LDL and high density lipoprotein (HDL) were isolated using a KBr density gradient, and the purity of both lipoproteins was confirmed by single corresponding bands on agarose gel stained for lipid and protein. Purified LDL and HDL were radioiodinated and labelled with colloidal gold for binding and tracer studies respectively. Both lipoproteins bind to cell surface and are internalized within 30 min at 37 degrees C. The cultured granulosa cells possess more HDL binding sites than LDL binding sites and are more responsive in progesterone production when supplemented with HDL. These results suggest that granulosa cells may preferentially utilize HDL over LDL as a source of cholesterol for steroidogenesis.