Porphyria-induced hepatic porphyrinogen carboxy-lyase inhibitor and its interaction with the active site(s) of the enzyme.

Porphyrinogen carboxy-lyase is an enzyme that sequentially decarboxylates uroporphyrinogen III (8-COOH) to yield coproporphyrinogen III (4-COOH). In mammals this enzyme activity is impaired by hexachlorobenzene treatment, through generation of an enzyme inhibitor. The interaction of porphyrinogen carboxy-lyase inhibitor, extracted from the liver of hexachlorobenzene-treated rats, with substrate decarboxylation sites on the enzyme, was studied using four different carboxylated substrates belonging to the isomeric III series of naturally-formed porphyrinogens containing 8-,7-,6- and 5-COOH. Similar inhibitor effects were elicited against all the substrates assayed, with the exception of pentacarboxyporphyrinogen III in which decarboxylation was not inhibited to same extent. Enzyme protection assays in the presence of the different substrates, indicated that each porphyrinogen protects its own decarboxylation from inhibitor action. Preincubation of the inhibitor with normal enzyme increased its inhibitory effect. On the other hand, preincubation of both enzyme and inhibitor with superoxide dismutase or mannitol, did not alter inhibitory activity. Preincubation of the inhibitor with a number of amino acids showed that only arginine and its derivative N alpha-Benzoyl-L-Arginine ethyl ester interact with the inhibitor, noticeably reducing its ability to inhibit porphyrinogen carboxy-lyase. Albumin, histidine, serine, cysteine and imidazol, were unable to quench inhibitor activity. The present results indicate that the inhibitor acts at the binding site of each porphyrinogen. Taking into account that arginine is related to enzyme activity, and that histidine is found at the binding site of the substrates, the results suggest that the inhibitor could bind to arginine residues, blocking the access of substrates to histidine and altering the adequate orientation for decarboxylation by masking the positively charged active site necessary for porphyrinogen binding to the enzyme. In addition an indirect effect of the inhibitor mediated through free radicals could be discarded.

A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.

A rapid, reproducible method for the isolation of murine endothelial cells (ECs) has been developed. Murine ECs were highly enriched by collagenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (i.e., PECAM-1) monoclonal antibody-coated magnetic beads (Dynabeads). Pure EC populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendothelial cell contamination. Of the 300 cells plated, 29 clones (approximately 10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells from sponge implants (designated as SIECs) were then subjected to subsequent culture in vitro for 40 and 30 passages (up to 5 months), respectively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like "tubes" on Matrigel. However, 1G11 cells exhibited a "cobblestone" morphology, whereas SIECs had a fibroblast-like appearance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-alpha, the cells became positive for E-selectin (at 4 hours poststimulation) and the expression of ICAM-1, VCAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible, allowing the inclusion of further positive or negative selection steps.

Anti-tumor activity of cytokines against opportunistic vascular tumors in mice.

Polyoma middle T (PmT)-transformed endothelial cells may represent a unique murine model for human opportunistic vascular tumors. The present study was designed to evaluate the anti-tumor potential of a panel of 13 cytokines against murine PmT-transformed endothelial cells. Interferon gamma (IFNgamma) and transforming growth factor beta 1 (TGFbeta1) substantially decreased in a dose-dependent manner the proliferation of a panel of 6 PmT-transformed cell lines. IFNalpha and tumor necrosis factor alpha(TNFalpha) had marginal anti-proliferative activity, whereas other molecules (interleukins-1, -2, -4, -6 and -13, IFNbeta, leukemia inhibitory factor, oncostatin M, granulocyte-macrophage colony-stimulating factor) caused no growth inhibition. IFNgamma and TGFbeta1 were therefore selected for further analysis of their mechanism of action and in vivo relevance. IFNgamma and TGFbeta1 reduced the activity of phosphatidylinositol-3-kinase and the production of phosphatidylinositol 3,4-biphosphate, without modifying the tyrosine kinase(s) activity associated with PmT. IFNgamma and TGFbeta1 were also tested for their ability to modify the in vivo growth of the PmT-transformed endothelial cells H5V in syngeneic C57B1/6 mice. Treatment with IFNnu and TGFbeta1 significantly delayed tumor growth and increased survival time. In contrast, treatment with IFNalpha and TNFalpha failed to prolong survival. In nude mice, IFNgamma and TGFbeta1 had a transient effect on tumor growth but no effect on survival, suggesting a contribution of T cells to the in vivo anti-tumor activity of these cytokines.

Erythrocyte uroporphyrinogen I synthase in rats treated with the hepatic carcinogen diethylnitrosamine.

Uroporphyrinogen I Synthase (URO-S) activity was measured in erythrocytes of female and male rats which had received diethylnitrosamine (DENA) as an inducer of hepatic tumors. Twenty-two weeks after the last dose of the carcinogen, the rats showed statistically significant increases in the URO-S activity. Differences in the body weight, erythrocyte porphyrin content or the hematocrit between treated and control rats were not found. Fifty percent of female rats and thirty percent of male rats treated with DENA were found to have hepatic tumors but there was no correlation between blood URO-S activity and tumoral development in spite of the increase in URO-S activity observed in DENA treated rats. This was observed both in male and female rats.

Erythrocyte uroporphyrinogen I synthase in rats treated with the hepatic carcinogen diethylnitrosamine.

Uroporphyrinogen I Synthase (URO-S) activity was measured in erythrocytes of female and male rats which had received diethylnitrosamine (DENA) as an inducer of hepatic tumors. Twenty-two weeks after the last dose of the carcinogen, the rats showed statistically significant increases in the URO-S activity. Differences in the body weight, erythrocyte porphyrin content or the hematocrit between treated and control rats were not found. Fifty percent of female rats and thirty percent of male rats treated with DENA were found to have hepatic tumors but there was no correlation between blood URO-S activity and tumoral development in spite of the increase in URO-S activity observed in DENA treated rats. This was observed both in male and female rats.

Porphyrins and porphyrinogen carboxy-lase in hexachlorobenzene-induced porphyria.

1. Qualitative and quantitative studies of the porphyrins and the porphyrinogen carboxylyase of the liver, spleen, kidney, harderian gland and erythrocytes from normal rats and from those hexachlorobenzene-induced porphyria were carried out. 2. Hexachlorobenzene has no effect on erythrocyte porphyrin content, but produces a decrease in that of Harderian gland and an increase in the porphyrin content of the kidney and spleen, and a marked increase in the liver (1 mumol/g of tissue). Octacarboxylic (isomer III) and heptacarboxylic porphyrins accumulated in kidney, spleen and liver, the former porphyrin being predominant. 3. Hexachlorobenzene has no effect on the activity of porphyrinogen carboxy-lase in erythrocytes; there is a slight decrease in enzyme activity in the Harderian gland, and a marked decrease in the liver and kidney enzyme activities. In the liver the removal of each carboxyl group from uroporphyrinogen III appears to be affected by this treatment. 4. The liver is the principal site of action of hexachlorobenzene, with the kidney next in decreasing order of effect, and erythropoietic tissue is unaffected. The marked decrease in porphyrinogen carboxy-lyase activities observed in liver and kidney could explain the high accumulation of octacarboxylic and heptacarboxylic porphyrins found in these tissues. 5. The results are discussed in relation to changes promoted by hexachlorobenzene in other enzymes of the haem pathway.

Studies on porphyrin biosynthesis in lead-intoxicated rabbits.

The present report deals with studies on porphyrins and porphyrinogen carboxy-lyase of red cells and urinary porphyrins from lead-intoxicated rabbits. It was shown that the free erythrocyte porphyrins are mixture of protoporphyrin 9, the main component, and minor proportions of Coproporphyrin, Uroporphyrin III and Phyriaporphyrin. Analysis of the urinary porphyrins deomonstrates the presence of Coproporphyrins III as the major component, together with 15-20% of other porphyrins: 10-14% 5-COOH, 1-2% 6-COOH, 2-3% 7-COOH porphyrin and 1-2% Uroporphyrin III. We have not been able to detect an increase of Uroporphyrin I. Assays of porphyrinogen carboxy-lyase activity in hemolysate supernatant using Uroporphyrinogen III and Phyriaporphyrinogen (Phyria'gen) III as substrates, showed the existence of a slight decrease of both decarboxylase activities, being more affected during the second stage, the Phyria'gen decarboxylation. A possible regulation mechanism responsible for the porphyrin picture is discussed.