Effect of glucose on induction of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead

In the present work we have been able to demonstrate that phenobarbital and lead exert an inducing effect on the biosynthesis of delta-aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of delta-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. he present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.     

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of insulin

In the present work we demonstrate that insulin decreases the phenobarbital-induced activities of delta-aminolevulinic acid synthase and ferrochelatase in isolated hepatocytes from normal and experimental-diabetic rats. Insulin concentrations required to produce significant inhibition in diabetic hepatocytes were higher than in normal cells. Under similar experimental conditions, insulin decreased the basal activities of delta-aminolevulinic acid synthase and ferrochelatase in hepatocytes from normal rats; no inhibitory effect was observed on the basal activity of delta-aminolevulinic acid synthase in hepatocytes from diabetic rats. Cytochrome P-450 content of both normal and diabetic cells was not affected by insulin in absence or presence of phenobarbital. The inhibitory action of insulin was exerted even when effective concentrations of glucagon, dexamethasone, or 8-(p-chlorophenylthio)-cAMP were present.     

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of cAMP

In the present work we have been able to demonstrate the existence of some interrelationship between intracellular level of cAMP content and phenobarbital induction of delta-aminolevulinic acid synthase, ferrochelatase, and cytochrome P-450 biosynthesis in isolated rat hepatocytes. The increase of the level of intracellular cAMP produced by activators of adenylate cyclase, inhibitors of phosphodiesterase, or added cyclic nucleotides is reflected by an increase of the phenobarbital induction effect. The greater induction observed in hepatocytes of diabetic rats may be due to a higher level of the intracellular cAMP. The lack of potentiation of added cAMP in diabetic cells is mainly due to the fact that the maximum induction that could be attained is already achieved by the effect of the preexisting high level of the endogenous cAMP.     

Effect of glucose on induction of δ-aminolevulinic acid synthase,ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead

In the present work we have been able to demonstrate the phenobarbital and lead exert an inducing effect on the biosynthesis of δ-aminovulenic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the induction effect produced by phenobarbital in this in vitro system. Glucose inhibits the induction of δ-aminolevulinic acid synthase and ferrochelatase. This repression effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. No glucose effect was observed on the phenobarbital- and lead-mediated inductions of cytochrome P-450. The present results add more experimental evidence to support the concept that the last enzyme of the heme pathway is inducible, and as such may have a significant role in regulatory mechanisms of porphyrin and heme biosynthesis.     

Effect of glucose on the induction of δ-aminolevulinic acid synthase and ferrochelatase in isolated rat hepatocytes by allylisopropylacetamide

The present work shows that allylisopropylacetamide exerts an inducing effect on δ-aminolevulinic acid synthase and ferrochelatase activities in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the inducing effect produced in both enzymes. Glucose inhibits the induction of δ-aminolevulinic acid synthase and ferrochelatase in this in vitro system. A similar effect was observed with fructose and 2-deoxyglucose. No glucose effect was observed with galactose, mannose, glycerol, pyruvate and lactate. The glucose effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. The simple in vitro method used in the present work promises to be a very useful tool for studies of regulatory mechanisms of porphyrin and heme biosynthesis in hepatocytes under normal and pathological conditions (hepatic porphyrias).     

Increased rat testicular heme oxygenase activity associated with depressed microsomal heme and cytochrome P-450 levels after repeated administration of human chorionic gonadotropin

Repeated administration of human chorionic gonadotropin to rats results in a maximal depression of testicular microsomal heme and cytochrome P-450 levels at 24 h, followed by increases that plateau at pretreatment levels by day six. Associated with the depressed levels of microsomal heme and cytochrome P-450 is an increase of testicular microsomal heme oxygenase activity at 12-24 h. Testicular mitochondrial delta-aminolevulinic acid synthase activity was increased at 24 h, and remained elevated throughout the 9-day treatment period. Pretreatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, failed to prevent the depression of testicular microsomal heme or cytochrome P-450 or increased heme oxygenase activity caused by repeated administration of human chorionic gonadotropin, and administration of estradiol benzoate failed to alter testicular microsomal heme oxygenase activity suggesting that these parameters were not related to altered testicular estrogen content caused by increased aromatase activity. These results suggest that increased testicular heme oxygenase activity is associated with decreased microsomal heme and cytochrome P-450 content during human chorionic gonadotropin-induced desensitization.     

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.     

Coordinate elevations of liver delta-aminolevulinate synthase and cytochrome P-450 RNA by phenobarbital in chicken embryos: the effects of heme

1. The role of heme in the coordinate elevations of liver delta-aminolevulinate (ALA) synthase activity and microsomal cytochrome P-450 concentration induced by phenobarbital (PB) was investigated in the chicken embryo. 2. Eighteen day old chicken embryos were given PB, and the changes in liver content of PB-inducible cytochrome P-450 RNA and of ALA synthase RNA were determined at different times after exposure to the drug. 3. The concentrations of both types of RNA increased rapidly after PB administration, and by 9 hr the level of ALA synthase RNA was 55-fold higher than control and that of cytochrome P-450 RNA was 7-fold higher than normal. 4. While the rate of increase in ALA synthase activity paralleled closely that of the enzyme's RNA concentration, the rate of increase of spectrally active cytochrome P-450 concentration in microsomes lagged behind that of the apoprotein's RNA by several hours. 5. To test whether heme depletion was responsible for the coordinate inductions of the two enzymes, embryos were loaded with ALA 2 hr before exposure to PB. 6. The protocol led to a drop in the PB-inducible ALA synthase RNA concentration and to an increase in that of cytochrome P-450 RNA, measured 6 hr after drug administration. 7. In primary cultures of hepatocytes, hemin in the culture medium caused a modest drop in ALA synthase RNA concentration but had a variable effect on that of cytochrome P-450 RNA in cells incubated with PB for 9 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of 5-aminolaevulinate synthase, haem oxygenase and ligand formation in the mechanism of maintenance of cytochrome P-450 concentration in hepatocyte culture.

The present work shows that the ability of pyridines e.g. metyrapone, to maintain the cytochrome P-450 concentration in cultured hepatocytes is not due to their ability to alter the 5-aminolaevulinate synthase and haem oxygenase activities of the hepatocytes. Since ligands such as metyrapone will prevent the cobalt-mediated loss of hepatic cytochrome P-450 in rats, the hypothesis that ligand formation is the mechanism of maintenance of the cytochrome in hepatocyte culture was tested. The observation that non-pyridine ligands will maintain the cytochrome P-450 concentration supports this hypothesis.     

Regulation of phenobarbital-induced ferrochelatase mRNA activity by dibutyryl cAMP and glucose in normal and diabetic rat hepatocytes.

The induction of ferrochelatase activity by phenobarbital and its potentiation by dibutyryl cAMP assayed in normal rat hepatocytes are associated with increased activity of ferrochelatase mRNA. Glucose inhibits this stimulatory effect. This inhibition can be reversed with increasing concentrations of dibutyryl cAMP. The inducing effect exerted by phenobarbital on the activity of ferrochelatase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response in diabetic rat hepatocytes is neither potentiated by adding dibutyryl cAMP nor repressed by glucose. The absence of a glucose effect persists even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study are consistent with those reported in other published studies of ferrochelatase activity. This adds more experimental evidence to support the concept that ferrochelatase is inducible. The results obtained suggest that ferrochelatase is more susceptible to induction with phenobarbital in diabetic rat hepatocytes than in normal rat hepatocytes.     

Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes

Maintenance of microsomal cytochrome P-450 content by cultured rat hepatocytes has proven an elusive goal. It is reported here that exogenous heme maintains cytochrome P-450 content of cultured rat hepatocytes at high levels during the first 72 h of incubation. The maintenance studies have been expanded to demonstrate the in vitro induction of cytochrome P-450 by phenobarbital treatment. The induction of P-450 in vitro by phenobarbital required the trace element, selenium, in the presence of exogenous heme. The present findings suggest that selenium, and other trace elements, may have an essential role in the formation of holocytochrome P-450 in vitro.     

Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011 beta, P-450C21, and adrenodoxin by prostaglandins E2 and F2 alpha and cholera toxin

To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined. These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex. Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment. Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin. Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins. Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes.     

Role of cytochrome P-450IIE1 in N-nitroso-N-methylaniline induced hepatocyte cytotoxicity.

1. The cytotoxicity of N-nitrosomethylaniline (NMA) towards hepatocytes isolated from rats was prevented by acetone or ethanol (inhibitors for cytochrome P-450IIE1) but not by metyrapone or SKF525A (inhibitors for cytochrome P-450IIB1/2). Various alcohols, secondary ketones and isothiocyanates that induced cytochrome P-450IIE1 were also found to be protective. Various aromatic and chlorinated hydrocarbon solvents that are substrates or inducers of cytochrome P-450IIE1 also prevented NMA cytotoxicity. Nitrogen-containing heterocycles that induced cytochrome P-450IIE1 were less effective. Further evidence that cytochrome P-450IIE1 was responsible for the activation of NMA was the marked increase in hepatocyte susceptibility if hepatocytes from pyrazole-induced rats were used. 2. NMA was more cytotoxic to hepatocytes isolated from phenobarbital-pretreated rats than uninduced rats. However, metyrapone now prevented and SKF525A delayed the cytotoxicity whereas ethanol, acetone, allyl isocyanate, isoniazid or trichloroethylene had no effect on the susceptibility of phenobarbital-induced hepatocytes. Furthermore, microsomes isolated from phenobarbital-pretreated rats had higher NMA-N-demethylase activity which was more inhibited by metyrapone and SKF525A than that of uninduced microsomal activity. By contrast the N-demethylase activity of phenobarbital induced microsomes was more resistant to acetone, ethanol, hexanal, trichloroethylene and toluene than uninduced microsome. 3. The above results suggest that cytochrome P-450IIE1 catalyses the cytotoxic activation of NMA in normal or pyrazole-induced hepatocytes whereas cytochrome P-450IIB1/2 is responsible for cytotoxicity in phenobarbital-induced hepatocytes.     

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes.

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.     

Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum.

A solubilized preparation of cytochrome P-450, obtained by treatment of mitochondria from bovine corpora lutea with phospholipase A, contained all of the necessary components for the cholesterol side chain cleavage activity. The solubilized cytochrome -450 preparation could be isolated essentially free of endogenous cholesterol side chain cleavage activity by various fractionation techniques. A cholesterol side chain cleavage enzyme system was reconstituted using the isolated cytochrome P-450 preparation and purified adrenodoxin and adrenodoxin reductase (components of the enzyme system purified from the adrenal cortex). Protein kinase was partially purified from the cytosol fraction of bovine corpora lutea. It was purified 43-fold and the activity was highly dependent on cyclic adenosine 3:5-monophosphate (cyclic AMP). When ATP and this partially purified cyclic AMP-dependent protein kinase were added to the reconstituted cholesterol side chain cleavage enzyme assay in which cytochrome P-450 was limiting, a stimulation (20 to 74%) of the conversion of cholesterol into pregnenolone was observed. This stimulation was statistically significant with p value less than 0.001. The stimulatory effect of the protein kinase appeared to be dependent on ATP and was not mimicked by bovine serum albumin, indicating that the effect was specific for protein kinase. Protein kinase caused a phosphorylation of the cytochrome P-450 preparation when large amounts of this preparation were used in the assay. It is concluded from these results that the direct activation of the cytochrome P-450 component of the cholesterol side chain cleavage by protein kinase may be one of the ways by which cyclic AMP mediates the effect of luteinizine.     

Regulation of aromatase in estrogen-producing cells

Human adipose stromal cells in monolayer culture aromatize androstenedione to estrone. The rate of aromatization is stimulated 20- to 30-fold by glucocorticoids when fetal calf serum is present in the culture medium and by dibutyryl cyclic AMP in the absence of serum. The action of dibutyryl cyclic AMP to stimulate aromatase activity is potentiated markedly by phorbol esters and inhibited by growth factors, such as EGF. In order to investigate the mechanisms underlying this multifactorial regulation, we have prepared polyclonal and monoclonal antibodies specific for aromatase cytochrome P-450. By use of these antibodies it was demonstrated that the action of these various factors to regulate aromatase activity was caused by alterations in the rate of synthesis of aromatase cytochrome P-450, whereas the synthesis of the reductase component of the aromatase enzyme complex was relatively unaffected. The changes in the rate of synthesis of aromatase cytochrome P-450 were, in turn, reflective of changes in the levels of translatable mRNA specific for this protein. In order to analyze the levels of aromatase cytochrome P-450 mRNA directly, we have isolated a cloned cDNA insert complementary to the mRNA encoding aromatase cytochrome P-450, by screening a lambda gt 11 human placental cDNA library utilizing the polyclonal anti-aromatase P-450 IgG. Use of this cDNA probe in Northern analysis of RNA extracted from human adipose stromal cells revealed that the changes in translatable mRNA resulting from incubation of the cells with the various regulatory factors were due to changes in the absolute levels of mRNA encoding this protein.     

Mechanism of hexachlorobenzene-induced porphyria in rats. Effect of phenobarbitone pretreatment.

The effect of a pretreatment with phenobarbitone (PB) on the porphyrinogenic action exerted by hexachlorobenzene (HCB) was examined in female rats. Kinetic studies of enzyme function after HCB poisoning showed that porphyrinogen carboxy-lyase was the only enzyme of haem biosynthesis that markedly lowered its activity. Both stages of uroporphyrinogen (UPG) III decarboxylation were decreased. This enzyme, together with UPG I synthase (increased levels) were the first enzymes altered. Subsequently, an increase in delta-aminolaevulinate (AmLev) synthase and ferrochelatase was detected; AmLev dehydratase was the last to increase. On long-term exposure, PB alone did not modify the basal values of haem intermediates; only the content of cytochrome P-450 increased. All the enzyme activities studied showed no significant changes, except ferrochelatase, which increased. With both drugs the metabolic impairment promoted by HCB was accelerated and enhanced by prior PB treatment leading to the onset of an earlier and stronger porphyria. A more noticeable accumulation and excretion of higher carboxylated porphyrins and precursors was more promptly observed as a consequence of the early porphyrinogen carboxy-lyase blockade and the concomitant induction of AmLev synthase. Although the enzymic activities of both AmLev dehydratase and ferrochelatase were enhanced, this response differed in time. For UPG I synthase this pretreatment elicited lower values than those found in the HCB group. Cytochrome P-450 contents were immediately and slightly enhanced by all the drugs, but the values for the combined treatment were the lowest. Of the several hypotheses that could explain the action of HCB on the haem pathway, our results would suggest that the porphyrinogenic action of HCB is mediated by some of its metabolic products.     

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.