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The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)

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In standard culture conditions, three human hepatoma cell lines, Hep3B, PLC/PRF/5 and HepG2, were characterised by a predominant transcription of only two (H2 and L) among the four genes involved in the synthesis of inter-alpha-trypsin inhibitor (ITI)-related proteins. Pulse-chase experiments followed by immuno-precipitation with specific anti-L and anti-H ITI antisera showed that the proteins synthesised displayed a restricted L and/or H2 antigenic reactivity. Furthermore, while Hep3B and PLC/PRF/5 lines only synthesised ITI precursors (mainly the L-form), HepG2 cells were able to secrete an ITI-like protein. Immunocytochemical analyses substantiated these results with uneven distribution of heavy and light-chain polypeptide reactivity among the cells. The use of hepatoma cell models for the study of protein synthesis and assembly must therefore be considered cautiously.

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The effects of interleukin 6 (IL-6), the major inducer of the acute-phase reaction, on the expression of inter-alpha-trypsin inhibitor (ITI) genes were examined using human HepG2 hepatoma cells. The three ITI heavy-chain genes H1, H2 and H3 were transcriptionally regulated by IL-6 in a dose- and time-dependent manner. The treatment of HepG2 cells with IL-6 resulted in an increase of H1 and H3 mRNA levels and a decrease of H2 and L mRNA levels. Actinomycin D blocked the action of IL-6, suggesting that IL-6 regulated the H1, H2, H3 gene expression. Moreover, the kinetics of the ITI mRNA degradation in untreated and IL-6-treated cells confirmed these data. The nuclear run-on assay supports the regulatory effect of IL-6 at the transcription level of the L and H2 genes. Primer extension experiments showed that the effect of IL-6 on L, H2 and H3 mRNA synthesis was not related to the transcription starting point. Although H1, H2, H3 and L gene products are supposedly present in similar amounts in the ITI and pre-alpha-trypsin inhibitor molecules, the present work shows that these genes are regulated in a different manner, at least under the influence of IL-6.

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In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion. The enzyme system involved in the ester linkage between H and L chains is localized in the trans-Golgi network since no ITI-like protein could be obtained in the presence of monensin; instead free heavy-chain protein forms and bikunin were secreted in culture supernatants. The ITI-like protein synthesized by HepG2 cells is therefore composed of two heavy chains HC2 linked to two bikunin chains by chondroitin sulphate bridges, although the GAG linkage between HC2 chains is presumably different. Further, a different maturation route leading to restricted heavy-chain forms, Hm and Hd, could be shown.

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The Hep G2 hepatoma cell line synthesizes the inter-alpha-trypsin inhibitor (ITI). This protease inhibitor and the other proteins of this family include four polypeptides chains: three heavy chains (HC1, HC2, HC3) and one light chain (bikunin). In the present study, we have demonstrated by immunofluorescence that ITI is detected mainly in perinuclear cytoplasmic zones comparable to those of albumin or alpha-1-antitrypsin. The presence of the mRNAs of the four polypeptide chains in all Hep G2 cells of a non-synchronized culture have been demonstrated by in situ hybridization. An evaluation of the transcription of the four ITI genes through an analysis of markings brings to the fore a clearly much higher rate of mRNAs from the light chain than from the heavy chains. The mRNAs corresponding to the HC2 chains are more heavily represented than are those corresponding to the HC1 and HC3 chains. In Hep G2 cells in culture, a quantification of mRNAs based on the in situ hybridization technique shows that their relative quantities, in decreasing order, are those of L, HC2, HC3 and HC1.

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The role of the renin angiotensin system (RAS) during pregnancy is not fully understood but numerous studies point to its importance in the homoeostasis of the fetal blood pressure and in the physiology of the fetal kidney near term. The aim of this study was to investigate the tissue distribution of angiotensin-converting enzyme (ACE) in the pregnant rabbit and its fetus and to assess the effect of Perindopril (PIL) a new ACE inhibitor on maternal and fetal tissue ACE activity. On day 28 of gestation, animals of the experimental groups were gavage-fed with 1 mg PIL or 10 mg PIL. ACE activity was assayed in tissue homogenates and serum with a radio-enzymatic method using (Gly-1-14C)-hippuryl-L-histidyl-leucine as specific substrate. In the kidney of control pregnant rabbit, decreasing values of ACE were found with a concentration gradient from the cortex to the inner papilla. ACE values in lung were comparable to those seen in kidney cortex. A significant effect of PIL was found with a percentage of inhibition of ACE activity in the renal cortex above 74% after 1 mg PIL and 88% after 10 mg PIL. In the control group, ACE activity was predominant in lung of fetuses. After maternal administration of 1 mg PIL, ACE activity fell significantly in fetal serum, placenta and fetal lung, but not in fetal kidney. Ten mg PIL produced a further significant decrease in ACE activity in fetal organs and serum. Plasma renin activity (PRA) was significantly stimulated after PIL administration in both mothers and fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

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Perindopril, a new specific and potent inhibitor of angiotensin-I-converting enzyme was used to evaluate the possible participation of the inhibition of the renin-angiotensin system in the development of aminoglycoside-induced renal failure. Kidney function, morphology and biochemistry were evaluated at regular intervals throughout the study. Perindopril was given orally to rats at a daily dose of 2 mg/kg for 15 days prior to and during 15 day gentamicin treatment given intraperitoneally at a daily dose of 50 mg/kg. Perindopril treatment alone induced no modification in renal function or structure. Gentamicin treatment alone induced typical renal lesions which were scored as moderate and a slight but significant decrease in ACE blood levels. Concurrent treatment with perindopril and gentamicin induced a greater drop in ACE blood levels than after the administration of perindopril alone and produced more marked renal impairment than after the administration of gentamicin alone. These observations suggest that the integrity of the renin-angiotensin system may play an important role in limiting kidney injury during aminoglycoside induced nephrotoxicity.

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Kidney is the main source of the production of renin and angiotensin, while also being one of their main target organs. This study was designed to determine the regional distribution of angiotensin-I-converting enzyme (ACE) in the kidney using a biochemical approach. Interspecies variations were analyzed in human, monkey, rabbit, dog and rat kidneys. Kidney ACE content differed among species with decreasing contents as follows: rabbit greater than human greater than monkey greater than dog greater than rat. In rabbit, human, monkey and dog kidneys, we observed predominant cortical distribution of ACE compared with the medulla or papilla; median cortex/papilla ACE activity ratio was 19, 14, 9 and 7 for the rabbit, human, dog and monkey, respectively. In rat kidney, ACE predominantly distributes in the outer medulla, while cortex ACE content appears to be low. The difference in ACE distribution in the rat kidney and to a lesser extent in the dog kidney when compared to rabbit, monkey or man should be taken into account when extrapolating to the human renal hemodynamic studies, which are frequently performed in rats or dogs.

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The tissue distribution of Perindopril, a new potent inhibitor of the angiotensin-converting enzyme (ACE), was studied after in vivo intravenous injection into rabbits of tracer amounts of the tritiated drug either alone (no modification of the renin angiotensin system) or along with a pharmacologic dose of 10 mg/kg unlabelled Perindopril. Lung and kidneys were the most densely labelled tissues. Kidney distribution of tritiated Perindopril was studied either by histo-autoradiography or by measurement of radioactivity in the homogenates of dissected kidney zones. A close parallelism was found between the distribution patterns of ACE and radioactivity throughout the kidney. Tritiated-Perindopril binding was inhibited by concurrent treatment of the animals with the unlabelled drug or pretreatment with Captopril. Autoradiographic study of kidney slices after the administration of tracer amounts of Perindopril showed an intense labelling of the glomerular mesangium and of endothelial structures of blood vessels, and a lack of labelling of tubular epithelial cells. The possible occurrence of two pools of "ACE-like" activity in the kidney is discussed, namely i) one pool which is labelled by tritiated Perindopril, located in glomerular mesangium and endothelial structures which may be involved in the pharmacological action of ACE inhibitors; and ii) a second pool located in the proximal-tubule cell brush-border, remaining unlabelled by Perindopril, for which the high amount of neutral endopeptidase present at this site may be responsible.

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Perindopril, a new specific and potent inhibitor of angiotensin-I-converting enzyme, was used to evaluate the possible participation of inhibition of the renin-angiotensin system in the development of aminoglycoside-induced renal failure. Kidney function, morphology and biochemistry were evaluated at regular intervals throughout the study. Perindopril was given orally to rats at a daily dose of 2 mg/kg for 15 days prior to and during 15-day gentamicin treatment given intraperitoneally at a daily dose of 50 mg/kg. Perindopril treatment alone induced no modification in renal function or structure. Gentamicin treatment alone induced typical renal lesions which were scored as moderate and a slight but significant decrease in ACE blood levels. Concurrent treatment with perindopril and gentamicin induced a greater drop in ACE blood levels than after the administration of perindopril alone and produced more marked renal impairment than after the administration of gentamicin alone. These observations suggest that the integrity of the renin-angiotensin system may play an important role in limiting kidney injury during aminoglycoside-induced nephrotoxicity.

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Angiotensin I-converting enzyme (ACE) activity was measured with hippurylhistidylleucine as a substrate in isolated human glomeruli. The mean level was 2.2 +/- 0.47 mIU/mg glomerular protein. S9780, a newly designed competitive inhibitor of ACE, inhibited this activity by 85% at 0.3 microM. [3H]S9780 specifically bound to isolated human glomeruli. The Kd value and the number of sites were 23 nM and 83 fmol/mg, respectively. The prodrug, S9490, and Captopril were less potent than S9780 in displacing [3H]S9780 from its binding sites. Angiotensin I had no effect. Binding of [3H]S9780 was inhibited after preincubation of the glomeruli with a specific polyclonal anti-human ACE antibody. These results demonstrate that ACE is present in human adult glomeruli.

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