Richard D. Levere

Effect of heme arginate administration on blood pressure in spontaneously hypertensive rats.

Cytochrome P450 content and activities are increased in the kidneys of spontaneously hypertensive rats (SHR) as compared with those of normotensive, Wistar-Kyoto (WKY), control rats during the period of rapid elevation of blood pressure. We studied the effect of heme arginate, a potent inducer of heme oxygenase (EC 1.14.99.3), on microsomal cytochrome P450 levels and activities and blood pressure in SHR at 7 wk of age. Administration of heme arginate (15 mg/kg body weight for 4 d) resulted in a marked decrease in blood pressure from 156.3 +/- 4.7 to 129.8 +/- 4.5 mm Hg (P less than 0.001), whereas blood pressure in SHR receiving the vehicle control was not affected. The blood pressure of age-matched WKY was not affected by heme arginate. Heme oxygenase activity increased in both hepatic and renal microsomes of SHR and WKY by two- to four-fold after treatment with heme arginate. Maximal increase of heme oxygenase mRNA occurred 5-7 h after the last injection of heme arginate and returned to control levels after 24 h. The increase in heme oxygenase activity was associated with a parallel decrease in cytochrome P450 content and in the activity of cytochrome P450 omega/omega-1 arachidonate hydroxylases in kidneys of SHR. It is postulated that heme arginate treatment resulted in induction of heme oxygenase which consequently led to a diminution of cytochrome P450, especially the arachidonate omega/omega-1 hydroxylases leading to a marked decrease in 19-hydroxyeicosatetraenoic acid (HETE) and 20-HETE. The effect of heme arginate on blood pressure may be mediated via these biochemical events inasmuch as both 19-HETE and 20-HETE produced by the kidney may promote hypertension by causing vasoconstriction and sodium retention.

Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta.

Cytochrome P-450-dependent mixed function oxidase activity is present in vascular tissue; however, as far as we could determine, the distribution of monooxygenase activity across the blood vessel wall has not previously been assessed. The aryl-hydrocarbon hydroxylase activity was examined by metabolism of benzo[a]pyrene in microsomes prepared from intimal and smooth muscle cell scrapings of the hog thoracic aorta. Microsomes of intimal cells comprising 95% endothelial cells showed an approximately 2.5-fold increase in aryl-hydrocarbon hydroxylase activity compared with that in microsomes prepared from medial smooth muscle cells. Michaelis-Mentin kinetics for the intimal enzyme yielded an apparent Km value of 11.11 microM and an apparent Vmax of 3-OH benzo[a]pyrene of 40 pmol/mg protein/10 min. Aryl-hydrocarbon hydroxylase activity was dependent on nicotinamide adenine dinucleotide phosphate and was inhibited by 7,8 benzoflavone, SKF 525A, and carbon monoxide. The localization of cytochrome P-450-dependent mixed function oxidase primarily to the intimal surface of the aorta may indicate a role for this enzyme system in vasoregulation and the pathogenesis of atherosclerosis.

The role of haem biosynthetic and degradative enzymes in erythroid colony development: the effect of haemin

Summary. In vitro culture of murine bone marrow has proved to be a useful system for defining the haem biosynthetic and degradative enzymatic pathways during erythroid colony (CFU‐E) growth and development. Previous attempts to elucidate these pathways during erythropoiesis have been limited by the amount of biological material available as well as the sensitivity of specific enzyme assays. These conditions were overcome in the present study. In order to obtain an enrichment of CFU‐E, nonadherent mouse bone‐marrow cells were cultured in special culture plates using a modified methyl‐cellulose medium with erythropoietin. CFU‐E yields were increased approximately threefold. Utilizing the sensitive radiochemical assay, direct measurement of ALAS activity was determined in very early developing CFU‐E cultures as well as mature cultures. ALAS activity was found to reach a peak after 60 h of culture growth and then started to decline in activity. Cellular synthesis of haem was determined (with 14C ALA) and minor modifications of standard assays were also made in order to determine spectrophotometrically δ‐aminolaevulinic acid dehydratase (ALAD) and haem oxygenase activity in developing CFU‐E cultures. These assays were reproducible with as few as four or five culture plates. It was found that ALAD activity rose progressively after 36 h of culture growth and reached a plateau at about 60 h of growth. A continuous increase in 14C ALA incorporation into haem was seen at later hours of culture growth, suggesting that the induction of other haem enzymes beyond ALAS is still rate limiting in haem synthesis. Conversely, haem oxygenase activity declined up to 60 h of growth and was elevated at later culture periods without a subsequent increase in ALASA and ALAD. The observed increase in haem enzymes brought about by haemin was completely suppressed by addition of cycloheximide to the cultures. These results suggest that

Expression of heme oxygenase gene in rat and human liver

Developmental changes of microsomal heme oxygenase were studied. In human fetal liver, the enzyme activity was 8 times higher than that detected in the adult liver. On the other hand, adult livers contained 4 times more cytochrome P450 than fetal livers. Elevated heme oxygenase activity in fetal liver was not due to modulators present in the microsomes. Similar to human, rat fetal liver also contained high enzyme activity which appeared to be regulated at the transcriptional level. Hybridization analysis of rat liver RNAs with cDNA for rat heme oxygenase revealed that the level of mRNA for the enzyme was 3-fold higher in the fetus than in the adult. The low cytochrome P450 content may be due in part to the high heme oxygenase activity in fetal livers.

Identification of heme oxygenase and cytochrome P-450 in the rabbit heart.

The regulation of cardiac heme oxygenase and cytochrome P-450 mixed function oxidase was studied in the rabbit heart. Heme oxygenase activity is found in ventricular and atrial microsomal fractions. This activity is NADPH dependent, and is inhibited by tin and zinc protoporphyrin, but not by either SKF 525A or 7,8-benzoflavone. Immunologic studies of cardiac heme oxygenase demonstrate that antibodies prepared against human purified hepatic heme oxygenase recognize rabbit atrial heme oxygenase and inhibit the enzyme activity by 92%. In contrast, control immunoglobulin does not inhibit heme oxygenase activity. Further, the western blotting technique demonstrates that a similar band of protein with a molecular weight of 32,000 exists in cardiac microsomes and that no protein cross-reacts with purified hepatocyte heme oxygenase. Marked induction of atrial heme oxygenase is observed in microsomal fractions prepared from rabbits treated with cobalt chloride. Atrial microsomes possess 0.24 nmol of cytochrome P-450 as compared to 0.68 nmol/mg protein in microsomes from the liver. The levels of aryl hydrocarbon hydroxylase (AHH) activity, a cytochrome P-450-dependent enzyme, in ventricle and atrium are stimulated by a NADPH-generating system and are sensitive to 7,8-benzoflavone, and SKF 525A, known inhibitors of cytochrome P-450 mixed function oxidase. AHH activity in ventricular and atrial microsomes is 2-3% of that seen in liver microsomes whereas the P-450 content/mg protein is about 20% of that observed in the liver. AHH activity is mediated by a form of cytochrome P-450 that is inducible by 3-methylcholanthrene/beta-naphthoflavone. A possible new role of the heart cytochrome P-450 system in cardiac function is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

The toxic effects of heavy metals on rat bone marrow in vitro erythropoiesis: Protective role of hemin and zinc

The effects of gold (Au), lead (Pb), and cadmium (Cd) on rat bone marrow in vitro erythropoiesis (CFUE) were studied. Au was found to be significantly toxic to CFUE growth at concentrations as low as 10(-9) M whereas Pb and Cd displayed toxicity at 10(-7) M. Addition of Pb plus Cd in combination had a greater toxic effect on CFUE growth than when the metals were added singly, and the toxic effect of Cd was reduced when Zinc (Zn) was added in combination to the cultures. When hemin (10(-6) M) was added to cultures containing 10(-6) M Au, Pb, or Cd, CFUE numbers were obtained that were equivalent to control cultures without hemin. Thus, hemin exerted a protective effect on erythropoiesis in the presence of otherwise toxic amounts of Au, Pb, and Cd. It is concluded that Au, Pb, and Cd have toxic effects on in vitro erythropoiesis and that this toxicity may be overcome in part by Zn or hemin. The possible involvement of the heme biosynthetic and degradative pathways is discussed with respect to these results.

Inhibition of Human Adult and Fetal Heme Oxygenase by New Synthetic Heme Analogues

Heme oxygenase (HO) is the rate-limiting enzyme for heme degradation, and elevated levels of HO may be associated with a variety of pathologic disturbances. A limited number of HO inhibitors such as the metallo-porphyrins have been proposed as possible chemotherapeutic agents for the treatment of hyperbilirubinemia. We undertook the study of various natural newly synthesized heme analogues as possible inhibitors of HO in human adult and fetal liver microsomes. We investigated two compounds with substitutions at the 2 and 4 position of the porphyrin ring, iron deuteroporphyrin 2,4 disulfonic (1a) and iron deuteroporphyrin 2,4 bis glycol (1b), and two compounds with substitutions of aromatic groups on the methene bridges of the porphyrin molecule, meso-tetra-4-carboxyphenyl-porphine (2a) and meso-tetra-4-sulfonatophenyl-porphine (2b). When these heme analogues were incubated in the reaction media in the presence of heme, two of the analogues (1a) and (1b) inhibited the conversion of heme to bilirubin. This inhibition was 97% and 65% respectively for (1a) and (1b) when both were present in 30 μM concentrations. Both of these compounds exhibited competitive type inhibition. The kI for the more potent inhibitor, (1b), was determined to be 0.30 μM. Porphyrins with aromatic substitutions at the methene bridges (2a, 2b) did not inhibit the conversion of heme to bilirubin, even at relatively high concentrations. Furthermore, the specific activity of HO was significantly greater (5×) in fetal microsomes as contrasted with adult microsomes. Even though fetal microsomes had greater HO activity, 5 μM of compound (1b) caused a similar degree of inhibition in both adult and fetal preparations. These studies represent the discovery of a new class of inhibitors of HO, distinct in structure from known inhibitors, the metallo-porphyrins. These compounds thus represent potentially novel drugs in the treatment of hyperbilirubinemic states or research tools in understanding mechanisms of heme oxidation.

Properties of human kidney heme oxygenase: Inhibition by synthetic heme analogues and metalloporphyrins

Heme oxygenase activities in human kidney microsomes were found to be from 0.238 to 0.620 nmol of bilirubin/mg/hr (mean 0.375, SD 0.134), which represent approximately 30% of activities determined for human adult liver. There was interindividual variation in heme oxygenase activity of a 2-5-fold difference. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from human kidney was identified on Western blots by its reaction with the anti-heme oxygenase liver antibody similar to the purified enzyme protein. Thus, a homology exists between human hepatic and kidney heme oxygenase. The enzyme activity was sensitive to inhibition by metalloporphyrins, such as tin-protoporphyrin IX and, to a lesser degree, by zinc and cobalt protoporphyrin IX. In a study of different synthetic heme analogues for in vitro inhibition of heme oxygenase, we found that replacement of iron by zinc in deuteroporphyrin IX 2,4 bis glycol dramatically potentiated the inhibition of heme oxygenase activity. This finding demonstrated that zinc deuteroporphyrin IX 2,4 bis glycol is a most potent inhibitor of heme oxygenase activity.

Differential Effects of Partial Hepatectomy on Hepatic and Renal Heme and Cytochrome P450 Metabolism

Partial hepatectomy has been suggested to affect hepatic and renal cytochrome P450 content and the related drug metabolizing enzyme system. In addition, cytochrome P450 and its dependent activities have been shown to be regulated by the availability of cellular heme. We, therefore, studied cytochrome P450 in addition to the level of heme oxygenase, the rate-limiting enzyme of heme catabolism, and delta-aminolevulinic acid (ALA) synthase, the rate-limiting enzyme of heme synthesis, in the remnant liver and intact kidneys of rats after two-thirds hepatectomy. The level of hepatic heme oxygenase was elevated threefold in partially hepatectomized rats as compared to sham-operated rats, while ALA synthase was decreased by 40%. This was reflected in decreased hepatic cytochrome P450 content, ie, from 0.689 +/- 0.175 nmole/mg to 0.505 +/- 0.089 nmole/mg protein and associated decreased drug metabolizing enzymes: aryl hydrocarbon hydroxylase, 7-ethoxycoumarin-O-deethylase, and benzphetamine N-demethylase, by 40%, 40%, and 47%, respectively. In contrast, renal heme oxygenase was not changed after hepatectomy, whereas renal ALA synthase was increased by fourfold. Renal cytochrome P450, aryl hydrocarbon hydroxylase, 7-ethoxycoumarin-O-deethylase, and benzphetamine N-demethylase were increased after partial hepatectomy by 84%, 360%, 165% and 406%, respectively. These data indicate that partial hepatectomy decreases liver cytochrome P450 levels by inducing heme oxygenase and inhibiting ALA synthase activities. In this situation the kidney plays a substitutive role in metabolizing endogenous substrates oxygenated by cytochrome P450 isozymes.

Effect of age on rat liver heme and drug metabolism

Old (24-months) rats have lower activities of hepatic delta-aminolevulinic synthase and the microsomal cytochrome P-450 monooxygenase activities--aminopyrine N-demethylase and aniline hydroxylase--as compared to young (2-months) animals. In contrast, the activity of the heme degradative enzyme, heme oxygenase, is higher in the old rats. Cytochrome P-450 and microsomal heme contents were maintained in the old. When inducibility and inhibition of these enzymes were studied, the old rats responded to the same degree as the young. These results indicate that the ability of the heme synthetic and degradative enzymes to respond to decreasing cellular heme levels is not significantly altered with age. The observations that there is a lower baseline activity of ALA-synthase and good maintenance of microsomal heme and cytochrome P-450 content, in spite of elevated heme oxygenase activity in the old, suggest that, at least in the senescent rat, hepatic heme utilization and degradation are only loosely coupled to heme production. It appears, therefore, that alternate sources of heme for cytochrome P-450 are available in the old animals. Furthermore, it is suggested that the old rat has a baseline change in ALA-synthase, heme oxygenase, and cytochrome P-450 that may be overcome under the appropriate conditions.