Stimulation of the pathway of porphyrin synthesis in the liver of rats and mice by griseofulvin, 3,5-Diethoxycarbonyl-1,4-dihydrocollidine and related drugs: evidence for two basically different mechanisms.

Griseofulvin and isogriseofulvin cause, like 3,5-diethoxycarbonyl-1,4-dihydrocollidine, a fall in the activity of the hepatic enzyme porphyrin-metal chelatase and accumulation of protoporphyrin in the liver. Analogues of either griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine which do not decrease the chelatase activity are not porphyrogenic on their own, but can potentiate the porphyria caused by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. This suggests the existence of two basically different mechanisms by which drugs stimulate the pathway of porphyrin synthesis in the liver.     

Ferrochelatase and N-alkylated porphyrins

Summary The final step in heme synthesis is catalyzed by the mitochondrial enzyme, ferrochelatase. Characterization of this enzyme has been complicated by a number of factors including the dependence of enzyme activity on lipids. Purification of ferrochelatase from rat and bovine sources has been achieved only relatively recently using blue Sepharose CL-6B chromatography. When 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is given to animals, it produces a hepatic porphyria resembling human variegate porphyria thus providing an experimental system in which to study this disease. DDC has been found to cause the accumulation of a green pigment, identified as N-methyl protoporphyrin IX (N-MePP), which is a potent inhibitor of ferrochelatase. The source of the N-methyl substituent of N-MePP was found to be the 4-methyl group of DDC. Considerable evidence indicates that the protoporphyrin IX moiety of N-MePP originates from the heme moiety of cytochrome P-450 and that DDC is a suicide substrate for this hemoprotein. Some studies suggest that cytochrome P-450 isozymes differ in their susceptibility to destruction by DDC and its 4-alkyl analogues. Griseofulvin has also been reported to inhibit hepatic ferrochelatase in rodents but not in the 17-day old chick embryo nor in hepatocyte culture systems. Thus, the mechanism by which griseofulvin produces an experimental porphyria in chick embryo liver cell culture is different from that for rodents.     

Drug-induced protoporphyria in the olfactory mucosa of the hamster

Administration of 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (4-ethyl-DDC) to hamsters resulted in a marked loss of cytochrome P-450-dependent reactions (peroxidase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase) in both liver and olfactory epithelium within 2 hr. This inactivation of cytochrome P-450 was accompanied by inhibition of ferrochelatase (FK), stimulation of 5-aminolevulinate synthase (ALA-S), and accumulation of protoporphyrin both in the liver and to a lesser degree, in the olfactory epithelium. These results suggest that the mechanism of induction of protoporphyria in nasal tissues is similar to that occurring in the liver, namely, suicidal metabolism of 4-ethyl DDC by cytochrome P-450 resulting in formation of N-ethylprotoporphyrin, a potent inhibitor of FK. The consequent depletion of heme leads to stimulation of ALA-S and, thus, porphyrin accumulation. Investigation of the dose-response to 4-ethyl DDC demonstrated that, in liver, maximal inhibition of FK and accumulation of protoporphyrin occurred at a dose of 50 mg/kg while ALA-S activity continued to increase up to a dose of 100 mg/kg. This is compatible with an additional effect of the drug on ALA-S involving induction of cytochrome P-450 and, thus, further depletion of heme. In the olfactory epithelium, stimulation of ALA-S was significantly less marked, suggesting that this secondary effect does not operate in nasal tissue. This is consistent with reports that olfactory cytochrome P-450s are noninducible.     

Identification of N-methylprotoporphyrin IX in livers of untreated mice and mice treated with 3, 5-diethoxycarbonyl- 1, 4-dihydrocollidine: source of the methyl group

Administration of the porphyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to mice, leads to the accumulation of N-methylprotoporphyrin IX in liver. This porphyrin is a potent inhibitor of ferrochelatase activity and accounts for the porphyria produced after DDC administration. The N-methylprotoporphyrin IX extracted from DDC-treated mice is primarily of one isomeric form, as shown by nuclear magnetic resonance spectroscopy. The methyl group of N-methylprotoporphyrin IX isolated from DDC-treated mice is derived mostly from the 4-methyl group of DDC. The transfer of this methyl group and its subsequent covalent attachment to protoporphyrin IX may be mediated by a form of hepatic microsomal cytochrome P-450. N-Methylprotoporphyrin IX is also found in livers of untreated mice at levels that are low but significant.     

N-Alkylprotoporphyrin IX formation in 3,5-dicarbethoxy-1,4-dihydrocollidine-treated rats. Transfer of the alkyl group from the substrate to the porphyrin

Administration of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) to rats causes the accumulation of N-methylprotoporphyrin IX, a potent inhibitor of ferrochelatase. To clarify the origin of the porphyrin N-methyl group, we have synthesized and administered to rats N-ethyl-3,5-dicarbethoxy-1,4-dihydrocollidine (N-ethyl DDC) and 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), the DDC analogue with a 4-ethyl rather than 4-methyl group. Only N-methylprotoporphyrin IX is isolated from rats treated with the former agent, and only N-ethylprotoporphyrin IX from those treated with the latter. All four isomers of N-ethylprotoporphyrin IX are formed biologically. The structure of the isolated porphyrins has been confirmed by complete spectroscopic comparison with the four synthetic isomers of N-ethylprotoporphyrin IX. DDEP has been shown to cause NADPH- and time-dependent in vitro loss of hepatic microsomal cytochrome P-450. These results unequivocally establish that the 4-alkyl groups in DDC and dDEP are the source of the N-alkyl group in N-methyl- and N-ethylprotoporphyrin IX, respectively, and strongly suggest that the alkyl group is transferred to the prosthetic heme of cytochrome P-450 during catalytic processing of the substrate by the enzyme. The mechanism of the group transfer is discussed.     

Stimulation of liver 5-aminolaevulinate synthetase by drugs and its relevance to drug-induced accumulation of cytochrome P-450. Studies with phenylbutazone and 3,5-diethoxycarbonyl-1,4-dihydrocollidine

The relevance of the stimulation of 5-aminolaevulinate synthetase to the accumulation of cytochrome P-450 after administration of drugs was examined in rats treated with phenylbutazone and with 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine alone stimulated 5-aminolaevulinate synthetase without increasing the concentration of cytochrome P-450, whereas phenylbutazone alone increased the microsomal cytochrome P-450 without significantly affecting the activity of the enzyme. When the two drugs were given together both effects were found. It is concluded that if an increased amount of 5-aminolaevulinate and haem must be made to provide for the accumulation of cytochrome P-450, it need only be a small amount. It is also concluded from these findings that stimulation of the drug-metabolizing system on the one hand and marked enhancement of 5-aminolaevulinate synthetase activity and porphyria on the other are likely to result from different actions of the drugs. Evidence is presented suggesting that porphyrogenic drugs stimulate markedly the activity of 5-aminolaevulinate synthetase by lowering the concentration of haem in the liver, thereby decreasing the normal feedback control. With 3,5-diethoxycarbonyl-1,4-dihydrocollidine a rapid inhibition of mitochondrial ferrochelatase and of liver haem synthesis may be the primary mechanism involved.     

The effect of N-methylprotoporphyrin and succinyl-acetone on the regulation of heme biosynthesis in chicken hepatocytes in culture

The essential features of hepatic protoporphyria, namely inhibition of ferrochelatase, accumulation of protoporphyrin and stimulation of 5-aminolevulinic acid synthase (ALA-S) were all obtained by treating chicken hepatocytes in culture with small doses of N-methylprotoporphyrin. Both N-methylprotoporphyrin and succinyl-acetone, another inhibitor of heme biosynthesis, stimulated ALA-S when given on their own and also enhanced the stimulation of ALA-S caused by phenobarbital.     

Decreased liver activity of porphyrin–metal chelatase in hepatic porphyria caused by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Studies in rats and mice

1. A difference has been found between rats and mice in their sensitivity to the porphyrogenic effect of drugs. Mice are more sensitive than rats to 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but less sensitive than rats to 2-allyl-2-isopropylacetamide. 2. Use has been made of this difference in sensitivity to ascertain the importance of the decrease of liver porphyrin-metal chelatase activity in porphyria caused by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mice, which are more sensitive than rats to the stimulation of 5-aminolaevulinate caused by this drug, are also more sensitive with respect to the decrease of chelatase activity. 3. In both species, after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, the ratio between chelatase activity and 5-aminolaevulinate activity is linear with respect to the reciprocal of the liver porphyrin concentration. This suggests that under these conditions the degree of porphyrin accumulation depends on the balance between rate of porphyrin formation and rate of porphyrin utilization. 4. Compound SKF 525-A (2-diethylaminoethyl 3,3-diphenylpropylacetate) when given before 3,5-diethoxycarbonyl-1,4-dihydrocollidine prevents the appearance of porphyria in the rat and also largely prevents the decrease of chelatase activity. In the mouse it is much less effective in preventing porphyria and it is almost completely inactive in protecting the chelatase from a decrease in activity. 5. Cycloheximide, when given before 3,5-diethoxycarbonyl-1,4-dihydrocollidine also inhibits the induction of 5-aminolaevulinate synthetase and the appearance of porphyria in the rat, but does not prevent the decrease of chelatase activity. These results suggest that two successive stages can be distinguished in the induction process: a first stage leading to inhibition of haem synthesis and a second stage requiring synthesis of protein in the liver and leading to stimulation of 5-aminolaevulinate synthetase.     

The formation of N-alkylprotoporphyrin IX and destruction of cytochrome P-450 in the liver of rats after treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine and its 4-ethyl analog

The effects of two porphyrogenic agents, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), have been studied in rats. The administration of these compounds leads to the formation and accumulation in the liver of N-methylprotoporphyrin IX and N-ethylprotoporphyrin IX, respectively. In each case, the alkyl group of the porphyrin is derived from the 4-alkyl group of the porphyrogenic chemical. Each N-alkylporphyrin is a potent inhibitor of protoheme ferrolyase (EC 4.99.1.1) (ferrochelatase) activity. N-Methylprotoporphyrin IX is somewhat more potent than N-ethylprotoporphyrin IX as an inhibitor of ferrochelatase activity in vitro. However, more N-ethylprotoporphyrin IX accumulates in rat liver than does the N-methyl analog. Since alkylporphyrins are formed during the catabolism of heme (or hemoprotein), the effects of DDC and DDEP on hepatic microsomal cytochrome P-450 were also studied. Whereas DDC treatment led to only a slight decrease in cytochrome P-450 levels (25%), DDEP administration led to a marked decrease (75%) in the total cytochrome P-450 level. In phenobarbital- and 3-methylcholanthrene-treated rats, DDC administration did not alter the hepatic microsomal cytochrome P-450 content, while administration of DDEP to either phenobarbital-treated or 3-methylcholanthrene-treated rats led to marked reduction of levels in cytochrome P-450. Although the N-methylprotoporphyrin IX level was not increased following DDC administration to either phenobarbital- or 3-methylcholanthrene-treated rats, there was a marked increase in N-ethylprotoporphyrin IX accumulation in both phenobarbital- and 3-methylcholanthrene-treated rats after the administration of DDEP. These results suggest that DDC and DDEP react with different forms of rat hepatic microsomal cytochrome P-450.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tanger?s, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Effect of some antineoplastics on metabolic heme pathway

1. The porphyrinogenic ability of several antineoplastics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of delta-aminolaevulinate synthase (ALA-S), were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan, notwithstanding the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo.     

Ribosome function in livers of porphyric mice.

1. A porphyrinogenic drug, 3,5-diethoxycarbonyl-1,4-dihydrocollidine, caused a decrease in the proportion of single ("run off") ribosomes, and an increase in the number of polyribosomes, in the livers of treated animals. 2. No change could be detected in the distribution of amino acid incorporation among hepatic polyribosomes.     

A difference between two strains of rats in their liver non-haem iron content and in their response to the porphyrogenic effect of hexachlorobenzene.

Female Agus rats developed hepatic porphyria at a much faster rate than female Porton-Wistar rats when fed a diet containing 0.01% of hexachlorobenzene (HCB). They also showed a greater inhibition of liver uroporphyrinogen decarboxylase [EC 4.1.1.37] activity and a marked stimulation of 5-aminolaevulinate synthetase [EC 2.3.1.37]. The difference between the two strains could not be correlated with differences in the liver concentrations of HCB. However, control Agus rats were found to possess significantly higher levels of total non-haem iron in their livers than the Porton animals. This was particularly apparent after 24 h of starvation and is further evidence for the involvement of iron in the pathogenesis of HCB-induced porphyria. The posterior lobes of the livers from the Agus rats given HCB became porphyric more slowly than the remainder with less severe inhibition of uroporphyrinogen decarboxylase. In contrast to their increased susceptibility to HCB, the Agus rats were less susceptible to another prophyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine.     

Ursodesoxycholic acid and heme-arginate are unable to improve hematopoiesis and liver injury in an erythropoietic protoporphyria mouse model

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin overproduction which is responsible for painful skin photosensitivity. Chronic liver disease is the most severe complication of EPP, requiring liver transplantation in some patients. Data from a mouse model suggest that cytotoxic bile formation with high concentrations of bile salts and protoporphyrin may cause biliary fibrosis by damaging bile duct epithelium. In humans, cholestasis is a result of intracellular and canalicular precipitation of protoporphyrin. To limit liver damage two strategies may be considered: the first is to reduce protoporphyrin production and the second is to enhance protoporphyrin excretion. Bile salts are known to increase protoporphyrin excretion via the bile, while heme arginate is used to decrease the production of porphyrins in acute attacks of hepatic porphyrias. The Griseofulvin-induced protoporphyria mouse model has been used to study several aspects of human protoporphyria including the effects of bile salts. However, the best EPP animal model is an ethylnitrosourea-induced point mutation with fully recessive transmission, named ferrochelatase deficiency (Fech(m1Pas)). Here we investigate the effect of early ursodesoxycholic acid (UDCA) administration and heme-arginate injections on the ferrochelatase deficient EPP mouse model. In this model UDCA administration and heme-arginate injections do not improve the protoporphyric condition of Fech(m1Pas)/Fech(m1Pas) mice.     

Mallory-Denk-bodies: lessons from keratin-containing hepatic inclusion bodies

Inclusion bodies are characteristic morphological features of various neuronal, muscular and other human disorders. They share common molecular constituents such as p62, chaperones and proteasome subunits. The proteins within aggregates are misfolded with increased beta-sheet structure, they are heavily phosphorylated, ubiquitinylated and partially degraded. Furthermore, involvement of proteasomal system represents a common feature of virtually all inclusions. Multiple aggregates contain intermediate filament proteins as their major constituents. Among them, Mallory-Denk bodies (MDBs) are the best studied. MDBs represent hepatic inclusions observed in diverse chronic liver diseases such as alcoholic and non-alcoholic steatohepatitis, chronic cholestasis, metabolic disorders and hepatocellular neoplasms. MDBs are induced in mice fed griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine and resolve after discontinuation of toxin administration. The availability of a drug-induced model makes MDBs a unique tool for studying inclusion formation. Our review summarizes the recent advances gained from this model and shows how they relate to observations in other aggregates. The MDB formation-underlying mechanisms include protein misfolding, chaperone alterations, disproportional protein expression with keratin 8>keratin 18 levels and subsequent keratin 8 crosslinking via transglutaminase. p62 presence is crucial for MDB formation. Proteasome inhibitors precipitate MDB formation, whereas stimulation of autophagy with rapamycin attenuates their formation.     

Rb and p53 Liver Functions Are Essential for Xenobiotic Metabolism and Tumor Suppression

The tumor suppressors Retinoblastoma (Rb) and p53 are frequently inactivated in liver diseases, such as hepatocellular carcinomas (HCC) or infections with Hepatitis B or C viruses. Here, we discovered a novel role for Rb and p53 in xenobiotic metabolism, which represent a key function of the liver for metabolizing therapeutic drugs or toxins. We demonstrate that Rb and p53 cooperate to metabolize the xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). DDC is metabolized mainly by cytochrome P450 (Cyp)3a enzymes resulting in inhibition of heme synthesis and accumulation of protoporphyrin, an intermediate of heme pathway. Protoporphyrin accumulation causes bile injury and ductular reaction. We show that loss of Rb and p53 resulted in reduced Cyp3a expression decreased accumulation of protoporphyrin and consequently less ductular reaction in livers of mice fed with DDC for 3 weeks. These findings provide strong evidence that synergistic functions of Rb and p53 are essential for metabolism of DDC. Because Rb and p53 functions are frequently disabled in liver diseases, our results suggest that liver patients might have altered ability to remove toxins or properly metabolize therapeutic drugs. Strikingly the reduced biliary injury towards the oxidative stress inducer DCC was accompanied by enhanced hepatocellular injury and formation of HCCs in Rb and p53 deficient livers. The increase in hepatocellular injury might be related to reduce protoporphyrin accumulation, because protoporphrin is well known for its anti-oxidative activity. Furthermore our results indicate that Rb and p53 not only function as tumor suppressors in response to carcinogenic injury, but also in response to non-carcinogenic injury such as DDC.     

Intermediate filament cytoskeleton of the liver in health and disease

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising approximately 70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.     

Drug-induced accumulation of uroporphyrin in chicken hepatocyte cultures. Structural requirements for the effect and role of exogenous iron.

The ability of drugs to cause uroporphyria in hepatocytes from 17-day-old chick embryos has been investigated and the response of the cells in culture compared with that of the intact liver of the embryos in ovo. In this chick-embryo system, drugs that cause accumulation of uroporphyrin within 19-24 h can only do so in culture; in contrast, 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which stimulate production of protoporphyrin, are effective both in culture and in ovo. A role of exogenous iron in worsening drug-induced uroporphyria was demonstrated in cultures of hepatocytes; iron also caused preferential accumulation of uroporphyrin from added 5-aminolaevulinate in the absence of a porphyrogenic chemical. Uroporphyria was induced in cultures of hepatocytes by drugs of widely different structures, suggesting that the primary molecular target with which they interact may be relatively aspecific in its binding characteristics. These results are briefly discussed, and two alternative hypotheses for the drug-induced effect in uroporphyrinogen metabolism are considered.     

Hepatic cyclic GMP formation is regulated by similar factors that modulate activation of purified hepatic soluble guanylate cyclase

The same factors that regulate the activation of purified hepatic soluble guanylate cyclase by diverse agents possessing distinct requirements for enzyme activation were found to modulate cyclic GMP formation in intact viable hepatic cells. A comparison was made between activation of heme-deficient or heme-reconstituted guanylate cyclase and stimulation of cyclic GMP formation in mouse hepatic slices that were 95% viable and showed no active efflux of cyclic GMP. Heme-dependent activators of guanylate cyclase elicited a greater -fold increase in hepatic cyclic GMP levels in slices from phenobarbital-pretreated than control mice. Brilliant cresyl blue and KCN inhibited both enzyme activation and hepatic cyclic GMP accumulation caused by agents that generate nitric oxide. Hepatic slices from 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated mice, which are known to develop sharp increases in hepatic protoporphyrin IX/heme concentration ratios, showed elevated resting cyclic GMP levels whereas phenobarbital pretreatment produced decreased resting cyclic GMP levels compared to controls. Guanylate cyclase activation by azide required added catalase, and both enzyme activation and hepatic cyclic GMP formation were inhibited by aminotriazole. Enzyme activation by glyceryl trinitrate and NaNO2 required added thiols. Hepatic slices from acetaminophen-pretreated mice showed marked depletion of sulfhydryls and decreased cyclic GMP formation in response to these enzyme activators. Both effects were completely restored by treatment of thiol-depleted mice with N-acetylcysteine. These observations lend support to the general view that information gained from studies on the regulatory properties of purified soluble guanylate cyclase bears a close relationship to studies on regulatory mechanisms that modulate cyclic GMP formation in intact cells.     

Induction of liver apolipoprotein A-IV mRNA in porphyric mice.

We have isolated cDNA clones for mRNAs that are induced by porphyria from a mouse liver library. Of the three inducible clones isolated, we have identified one as being apolipoprotein A-IV (apo A-IV) by its extensive homology with a rat apolipoprotein A-IV cDNA sequence. The level of liver apo A-IV mRNA increases rapidly in response to either of two porphyrogenic drugs. When the ferrochelatase-inhibited drug, 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) is used, a 6 and 28 fold induction of liver apo A-IV mRNA is observed in male and female mice, respectively. If the heme-destroying porphyrogenic drug, allylisopropylacetamide (AIA) is the inducing agent, liver apo A-IV mRNA levels increase 2-3 fold in both males and females. The level of apo A-IV mRNA reaches a maximum within 6-10 hr. after drug administration. Intestine apo A-IV mRNA levels do not change during either of these drug-induced porphyrias. RNA from acute-phase responsive liver or liver from mice treated with bilirubin, porphobilinogen, or protoporphyrin IX show no increase in apo A-IV mRNA. These results indicate that apo A-IV induction is tied to a disruption in porphyrin-heme biosynthesis but is not directly affected by several heme intermediates nor by the major heme degradation product, bilirubin.