Maternal perinatal undernutrition modifies lactose and serotranferrin in milk: relevance to the programming of metabolic diseases?

A close link between intrauterine growth restriction and development of chronic adult diseases such as obesity, diabetes, and hypertension has been established both in humans and animals. Modification of growth velocity during the early postnatal period (i.e., lactation) may also sensitize to the development of metabolic syndrome in adulthood. This suggests that milk composition may have long-lasting programming/deprogramming metabolic effects in the offspring. We therefore assess the effects of maternal perinatal denutrition on breast milk composition in a food-restricted 50% (FR50) rat model. Monosaccharides and fatty acids were characterized by gas chromatography, and proteins were profiled by surface-enhanced laser desorption/ionization-time-of-flight analysis in milk samples from FR50 and control rat dams. Milk analysis of FR50 rats demonstrated that maternal undernutrition decreases lactose concentration and modulates lipid profile at postnatal day 10 by increasing the unsaturated fatty acids/saturated fatty acids and diminishes serotransferrin levels at postnatal day 21. Our data indicate that maternal perinatal undernutrition modifies milk composition both quantitatively and qualitatively. These modifications by maternal nutrition open new perspectives to identify molecules that could be used in artificial milk to protect from the subsequent development of metabolic diseases.

Proteomic analysis in cardiovascular diseases.

1. Cardiovascular diseases are a major cause of morbidity and mortality in western countries. The molecular mechanisms responsible for heart dysfunction are still largely unknown, except in cases of genetic defects or alteration of genes and proteins. 2. The publication of genome sequences from humans and other species has demonstrated the complexity of biology, including the finding that one gene does not encode for only one protein but for several, due to mRNA splicing and post-translational modifications. 3. Proteomic analysis can provide an overall understanding of changes in the levels of protein expression. Differential proteomics is a powerful tool for improving our understanding of integrated biochemical responses. The main techniques used are two-dimensional electrophoresis (2D-gel) and Surface-Enhanced Laser Desorption/Ionization Time of Flight (SELDI-TOF) to separate proteins associated with mass spectrometry. Bioinformatic tools make it possible to compare protein profiles obtained from diverse biological samples. 4. The combination of these approaches has proved to be particularly interesting for studying cardiovascular diseases and thereby improving our understanding of the mechanisms involved and identifying new biochemical factors and biomarkers involved in these diseases.

Regulation of prorenin secretion in cultured human transfected juxtaglomerular cells.

The regulation of renin secretion was studied in continuous culture of human juxtaglomerular cells (JGC), which provided a permanent source of human renin production (Pinet, F., M. T. Corvol, F. Dench, J. Bourguignon, J. Feunteun, J. Ménard, and P. Corvol, 1985, Proc. Natl. Acad. Sci. USA, 82:8503-8507). 95% of the renin species secreted was prorenin, and therefore this study concerned primarily prorenin secretion. Renin production was stable, since the cells had been maintained in culture for more than two years. In culture, these human cells formed colonies of smooth musclelike cells, and electron microscopy showed the presence of cytoskeleton structures including myofibrils and attachment bodies. This human model was used to investigate the control of prorenin secretion in vitro at cellular level. Various pharmacological agents known to stimulate or inhibit renin secretion were tested in the cell cultures. The variations in prorenin secretion were measured in the supernatant. Forskolin, an independent receptor activator of adenylate cyclase, stimulated prorenin secretion in a dose-dependent manner and this stimulation was mediated by 3',5' cyclic-AMP (cAMP). Angiotensin II (AII) was found to inhibit prorenin secretion directly in a dose-dependent manner and atrial natriuretic factor (ANF), whose effects on human JGC were characterized for the first time, was also shown to exert such inhibition. When the effects of this inhibition by AII and ANF were tested on forskolin-mediated stimulation of prorenin secretion, the latter was inhibited and no change occurred in cAMP release. When JGC were treated with histamine, bradykinin, or one or two bradykinin analogues, the responses suggested that in these cells, H2-histamine receptors and kinin receptors are dependent on adenylate cyclase. One peptide, substance P, had an inhibitory effect on prorenin secretion but it was less important than AII and ANF. The present results demonstrate that the adenylate cyclase system of human JGC remains intact during culture and supports the hypothesis that cAMP is the second messenger and Cai2+, the final messenger involved in renin secretion. The cell system used here permits the evaluation of cellular responses and intracellular events in granulated cells in a human model.

[Proteomics in cardiology]. / La protéomique en cardiologie.

Cardiovascular diseases are among the major causes of morbidity and mortality in the developed world. The molecular mechanisms responsible for dysfunction of the heart in most cardiac pathologies are still largely unknown, except that the expression of certain genes/proteins is altered. Proteomic analysis is a technology which can provide an overall understanding of changes in the level of protein expression. Especially with differential analysis, it now represents a powerful tool for interpreting all biochemical responses and their regulation. The principal technique employed is two dimensional electrophoresis (2-D gel) to separate the proteins followed by mass spectometry in order to identify them. Recently SELDI-TOF analysis, which is a complementary 2-D electrophoresis technique based on the combination of two principles, chromatography by retention on protein chips and mass spectometry, has allowed the comparison of protein profiles obtained from diverse biological samples. The publication of genome sequences for humans as well as for other species has provided evidence for the biochemical complexity, and in particular the fact that a gene does not just code for a single protein but for several, due to various alternative splicing processes, post-translational modifications etc... The combination of these various approaches has proved to be particularly interesting in the study of cardiovascular diseases with the aim of understanding the molecular mechanisms involved, providing evidence for protein interactions and identifying new biochemical factors / markers involved in the different cardiovascular pathologies.

[The best of fundamental research in 2006]. / L'essentiel de 2006 en recherche fondamentale.

The Cardiovascular Research Study Group attempts to organise round tables during its annual meetings based around themes that will allow clinical and fundamental researchers, especially the younger ones, to present their work. For example, certain round table subjects at the Spring cardiology meeting, such as 'Genetics: monogenic and polygenic diseases', 'Cellular therapy', and 'Ionic homoeostasis and cardiac arrhythmias', allowed several French laboratories to publish in some of the best journals such as Nature Genetics, Nature Methods, Circulation, and Circulation Research. During the Toulouse congress, eight poster prizes were awarded to young nonstatutory researchers under the age of 32 years. For the American Heart Association congress, eight travel grants were allocated to students whose abstracts had been accepted, paving the way for future publications from young French researchers in the best journals.

[The best of fundamental research in 2005]. / L'essentiel de 2005 en recherche fondamentale.

The cardiovascular research and reflection group aims to hold round tables between the annual congresses on themes allowing clinicians, fundamental research workers, especially the younger generation, to present their work. For example, a number of subjects of these round tables, "Genomic Modification and Integrated Physiology", "Adhesion and Extracellular Matrix", or "Electrophysiological Features of Ventricular Arrhythmias" of the 2005 Congress led to many laboratories publishing their results in the most reputable journals, such as Nature Medicine, Circulation. During the Strasbourg Congress, six poster prizes were attributed to young, nonstatutory research workers, under 32 years of age. Six voyage grants for the American Heart Association Meeting were given to students who had their summaries accepted, in the expectation of upcoming publications by young French workers in the best journals.

The endothelin system and renin in human fetal membranes.

Term human fetal membranes express prorenin, a key enzyme within the renin-angiotensin system. High levels of another vasoactive peptide, endothelin-1 (ET-1), are found in human amniotic fluid. To address the question of the relationship between these two vasoactive systems, we analyzed the expression of the components of the ET-1 system in fetal membranes in which cell types had been identified using different markers. Immunohistochemistry was performed with antibodies raised against the human proteins of the ET system. Term fetal membranes displayed ubiquitous labeling of endothelin-converting enzyme-1 (ECE-1) and ET-1. ETA receptors were detected in the chorionic connective tissue and the attached decidua; ETB receptors were localized to chorionic trophoblast cells and decidua. The localization of the ET-1 receptor subtype was confirmed by in-situ receptor binding. Renin immunoreactivity was detected in the chorionic connective tissue and the decidua. These findings suggest that ET-1 is produced ubiquitously in human fetal membranes, and its targets may be, trophoblast cells following ETB receptor activation, vascular structures and fibroblasts in the connective tissue and decidua via ETA and ETB receptors. It appears possible that renin and ET may contribute to the pathophysiological changes associated with premature labor and preeclampsia.

[Contribution of proteomic analysis to study cardiovascular physiopathology]. / Apport de l'analyse protéomique pour étudier la physiopathologie cardiovasculaire.

Proteomic is an innovative approach for determining without any priori the coordinated changes in protein levels contained in a biological sample. This technique now constitutes a powerful tool for revealing, in particular by differential analysis, biological responses and regulation of proteins. This field is becoming essential in each domain for fundamental and clinical biology, mainly in human medicine. It leads to improve the understanding of complex physiopathological processes like those involved in human pathologies, but also for identifying new biomarkers in order to consider new diagnostic and prognostic approaches and original therapeutic targets. The different proteomic techniques, essentially bi-dimensional electrophoresis and mass spectrometry, which benefit from recent technological developments, are presented in this article. A literature review summarize data obtained from proteomic works performed to have better understanding of mechanisms involved in cardiovascular diseases, first cause of morbidity and mortality in the world. Proteomic analysis performed in this area of research are promising, because of the lack of knowledge in the molecular interactions and the complexity of mechanisms involved, and of the impact of environmental risk factors. This impact is being generally only detectable at protein level. These works are only at the beginning and these approaches applied in cardiovascular field will meet a large success in the immediate future and should be promising.

Characterization of precursor and secreted forms of rat angiotensinogen.

Angiotensinogen precursors synthesized by rabbit reticulocyte lysate primed with rat liver RNA were compared with angiotensinogen secreted by rat hepatoma cells and rat hepatocytes using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of glycosylation with tunicamycin permitted identification of the nonglycosylated form of secreted angiotensinogen. Whereas angiotensinogen secreted by hepatoma cells and hepatocytes showed electrophoretic heterogeneity (mol wt, 52-62 X 10(3], tunicamycin-treated cells secreted only a single angiotensinogen species [mol wt, 48.3 +/- 0.7 X 10(3) (mean +/- SD)], which could be cleaved by renin. Two putative angiotensinogen precursors were synthesized in the reticulocyte lysate: a major protein of 52.5 +/- 1.0 X 10(3) mol wt and a minor protein of 55.7 +/- 1.3 X 10(3) mol wt. Evidence that these proteins represent separate angiotensinogen precursors includes the following. 1) Both proteins were recognized by five different polyclonal antibodies and two monoclonal antibodies. 2) Both proteins increased in parallel in reticulocyte lysates primed with liver RNA from rats nephrectomized and given hormones that increase liver angiotensinogen production. 3) Both proteins were cleaved by renin to produce a single protein of 47.6 +/- 0.8 X 10(3) mol wt. 4) The des-angiotensin I-angiotensinogen generated by renin treatment of the lysate had an electrophoretic mobility identical to that of des-AI-angiotensinogen produced by renin treatment of nonglycosylated angiotensinogen secreted by tunicamycin-treated hepatoma cells and hepatocytes. These studies suggest that rat liver synthesizes two separate angiotensinogen precursors which may differ only in the size of their prepro sequence. The heterogeneity of secreted angiotensinogen can be fully accounted for by differences in N-glycosylation of asparagine residues of the molecule. Glycosylation of angiotensinogen is not essential for its synthesis, processing, and secretion or its hydrolysis by renin.

Isolation and characterization of renin-producing human chorionic cells in culture.

Chorionic tissue is one of the major extrarenal sites of renin production, and as such, cultured chorionic cells are a potential model for in vitro studies of renin biosynthesis and regulation. Human chorionic cells were isolated from four chorions and maintained in tissue culture for a total of eight subcultures. Total renin production was considerable in the primary cultures, but fell gradually with successive passages. The cells could be frozen and thawed without losing their ability to divide or produce renin. Both the primary cultures and the subcultures contained a single type of elongated cell containing abundant rough endoplasmic reticulum and myofibrils, but no renin granules, suggesting that the cells had smooth muscle-like features. Immunocytochemistry indicated that they contained both renin and prorenin. The renin produced by the chorionic cells was not stored within the cells, but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had biochemical and immunological properties similar to those of pure human renin. The cells contained a renin mRNA that had the same size as that for renal renin (1.6 kilobases), confirming the synthesis of renin by these cells. The cells were also examined for the presence of other components of the renin-angiotensin system. Angiotensinogen and angiotensin I were not detected, but angiotensin-converting enzyme was present in extracts of primary and secondary cultured cells. beta hCG and progesterone were also found in the medium of primary culture. However, the production of beta hCG and progesterone fell after the primary culture, and beta hCG and progesterone were indetectable in secondary and tertiary cultures, respectively. These experiments suggest that these two hormones do not influence renin synthesis or vice versa. Thus, these cultures of human chorionic cells synthesized considerable quantities of prorenin and can provide a permanent source of nonrenal prorenin-producing cells.

Expression of steroidogenic enzyme messenger ribonucleic acids and corticosteroid production in aldosterone-producing and "nonfunctioning" adrenal adenomas.

"Nonfunctioning" adrenal adenomas are often diagnosed in patients without recognizable clinical symptoms of adrenocortical hyperfunction. The objective of this study was to determine directly the steroidogenic activity of such adenomas (n = 12) and compare them histologically and functionally to normal human adrenals (n = 6) and aldosterone-producing adenomas (n = 15). The histological appearances of nonfunctioning and aldosterone-producing adenomas were surprisingly similar. Nonfunctioning adrenal adenomas expressed all mRNAs of P450scc, P450c17, P450c21, adrenodoxin, and adrenodoxin reductase with relative levels comparable to those found in normal adrenals. Consistent with their hormone-producing nature, these adenomas had cortisol and aldosterone contents as high as those in normal adrenal tissues, a significantly (P < 0.05) increased 17-hydroxyprogesterone content, and a disproportionally low expression of P450c21 mRNA compared to aldosterone-producing adenomas. Cells isolated from both aldosterone-producing and nonfunctioning adrenal adenomas exhibited highly ACTH-sensitive cortisol and aldosterone production, suggesting again the presence of both zona glomerulosa-like and zona fasciculata-like steroidogenesis in these adenoma tissues. These results indicate that so-called nonfunctioning adrenal adenomas are not without steroidogenic activity. Therefore, the assumption that adrenal adenomas are entirely nonfunctioning in the absence of recognizable hormonal hyperfunction may not be correct.

Coexpression of renin, angiotensinogen, and their messenger ribonucleic acids in adrenal tissues.

The presence of the two components of the renin-angiotensin system (RAS) has been systematically investigated in human normal and pathological adrenal tissues with two aims: 1) the detection of renin and especially angiotensinogen, which has not been reported before; and 2) to study possible differences in the coexpression of renin and angiotensinogen in tissue of cortical and medullary origin. The relative levels of renin and angiotensinogen mRNAs were determined by Northern blot analysis in normal (n = 5) and pathological adrenal tissues of cortical (n = 23) and medullary (n = 10) origin. Renin, prorenin, and angiotensinogen levels were also measured. Renin concentrations in normal and pathological adrenals were around 30-fold higher than those in the plasma of normal subjects, except for a Cushing's adenoma, which contains an extremely high renin content. Renin accounted for 56% of the total renin in normal adrenals and up to 87% in neoplastic tissues. This high proportion of renin indicates a likely conversion of prorenin to renin within these tissues. Renin mRNA was detected in each group of adrenal tissues. There was a significant correlation between the concentration of renin and its mRNA (r = 0.75; P less than 0.05). Angiotensinogen and its mRNA were detected in all normal and pathological adrenals. Compared to normal adrenal tissues, the relative amount of angiotensinogen mRNA was significantly higher in pheochromocytomas. However, the increased mRNA level in these tissues was not accompanied by a parallel increase in tissue angiotensinogen levels. Since the translational efficiency of angiotensinogen was verified by in vitro cell-free translation, the low level of angiotensinogen compared to the relatively high amount of its mRNA suggests a lack of storage of this protein in adrenal cells, as in liver cells. This study demonstrates that renin and angiotensinogen are coexpressed in normal and pathological tissues. Tissues of different cellular origin (zona glomerulosa, fasciculata, and medullary tissue), were able to express, store, and process renin and synthesize angiotensinogen. There was no obvious relationship between the expression of these proteins and the pathophysiology of the adrenal gland.

A live-cell assay for studying extracellular and intracellular endothelin-converting enzyme activity.

Endothelin-1 (ET-1) is formed from its precursor preproET-1 via the cleavage of the intermediate bigET-1 by endothelin-converting enzyme (ECE-1). However, the subcellular site at which this step occurs is not clear: It could occur intravesicularly along the secretory pathway or bigET-1 might be released and processed extracellularly. To address this point, we have developed an integrated autocrine system that uses a recombinant Chinese hamster ovary (CHO) luciferase reporter cell line that permanently expresses the human ET(A) receptor. Into these cells we transiently transfected human ECE-1a cDNA, either together with the human preproET-1 cDNA (as an endogenous source of bigET-1), or alone (in which case exogenous bigET-1 was added). Phosphoramidon inhibited the conversion of exogenous bigET-1 (IC50 = 5 to 30 micromol/L) much better than that of endogenous bigET-1 (IC50 > 1 mmol/L). Both conversions showed similar high yields (20% to 100%) that depended on the amount of ECE-1a expressed. Thus, ECE-1a has two equally relevant activities in this recombinant system for CHO cells: (1) an intracellular, probably intravesicular activity, corresponding to the ECE-1a-mediated step of ET-1 biosynthesis and (2) an extracellular activity at the plasma membrane. If this is also the case for endothelial cells, ECE-1a inhibitors would have to cross the plasma and vesicle membranes to be effective. The present system could be useful for screening such inhibitors.

Regulation of human renin secretion and gene transcription in Calu-6 cells.

Calu-6 cells were characterized for studying the transcriptional regulation of the human renin gene. Analysis of cis-acting elements of the renin promoter showed the highest activity within the first 582 bp in serum-free conditions and of the 892 bp in the presence of serum. cAMP activates renin mRNA synthesis parallel to renin production (20-fold increase) as well renin promoter activity (2-fold). cAMP response element and the (-77 to -67) element are both necessary for activation of the renin promoter but do not act independently. Functional analysis of Intron A revealed the presence of a silencer specific to renin-producing cells.

Construction, expression and characterization of a soluble form of human endothelin-converting-enzyme-1.

Endothelin-converting-enzyme-1 (ECE-1) belongs to the family of zinc metallopeptidases and is responsible for generating endothelin (ET) peptides from their inactive precursors the big endothelins (bigET). The enzyme is a type II integral membrane protein consisting of a short amino-terminal cytosolic domain of 56 amino acids, a single transmembrane domain and a large putative extracellular domain containing the catalytic site. Recombinant and native ECE-1 are expressed as a dimer. We have constructed a soluble form of ECE, named sECE*, by fusing the cleavable signal peptide of pro-opiomelanocortin in frame to the complete extracellular domain of human ECE-1. Stable expression of this construct in CHO cells resulted in the secretion of a fully active enzyme. In contrast to membrane-bound ECE, sECE* was expressed as a monomer, highly glycosylated, as assessed by gel filtration and Western blot. However, recombinant sECE* converted bigET-1 with similar specific activity as ECE-1a. This activity was completely inhibited by phosphoramidon, but not by thiorphan and captopril. sECE* was active in a broad range of pH, showing an optimum of 6.6-6.8 for bigET-1. Thus, the extracellular domain alone is sufficient for conferring full ECE-1 activity, inhibitors recognition and substrate specificity.

Renin gene expression in the aging kidney: effect of sodium restriction.

The activity of the renin-angiotensin system as well as the ability of the kidney to retain sodium following salt restriction are reduced with age. The relationship between these age-related changes in renal function and the renin gene expression was presently investigated. The concentrations of renin and its mRNA were measured in kidney of 10- and 30-month-old control female WAG/Rij rats and of animals which were salt restricted for 4 days. In the senescent rats, the kidney renin concentration, like the plasma concentration of angiotensin II, was half that in adult rats. The intrarenal content of renin mRNA did not differ between 10- and 30-month-old animals, suggesting that the transcriptional rate of the renin gene is unchanged with age. During the early phase of adaptation to sodium depletion, the systemic angiotensin II concentration was not modified in either age groups. Four-days salt restriction did not significantly change the renal storage of renin. In contrast, this short term salt restriction induced a 2.3-fold increase in the renin mRNA in adult kidney, and a 1.9-fold increase in the senescent kidney. These data suggest that the age-related decrease in renal concentration of renin is linked to a modification in the rate of translation of renin mRNA, or to an alteration in the protein maturation. The difference in adaptation to the early phase of salt restriction with age should not be linked to changes in renin gene transcription, but more likely to a change in the tissue response to the local renin-angiotensin system.

Co-expression of PC2 and proenkephalin in human tumoral adrenal medullary tissues.

Expression of PC2, a Kex2-related protease, and of one of its possible substrates, proenkephalin, was examined in normal adrenal glands (n = 7) and pheochromocytomas (n = 20). PC2 could only be detected in normal adrenal glands using the sensitive RT/PCR technique. By Northern blot, PC2 and proenkephalin were expressed in 85% and 90% of the 20 pheochromocytomas studied, respectively. Moreover, in situ hybridization and immunohistochemistry confirmed expression of PC2 in human tumoral adrenal medullary tissue. These results show for the first time expression of PC2 in human pheochromocytomas which may be involved in the processing of proenkephalin.

Dissection of silencer elements in first intron controlling the human renin gene.

OBJECTIVE: A silencer within the renin first intron (intron A) was identified using Calu-6 cells, a pulmonary carcinoma cell line which produced renin. In the present study, a dissection of the first intron was performed to determine precisely the cis-regulatory elements involved in the silencer transcriptional effects. MATERIALS AND METHODS: Intron A was completely sequenced to characterize potential binding sites for known transcription factors. Partial portions of intron A were subcloned upstream the 892 bp of the renin promoter and transfected in different models of renin-producing cells: primary culture of human chorionic cells, human Calu-6 cells and mouse As4.1 cells. RESULTS: There is significant DNA homology (67%) between the 3' and 5' ends of the human and rat renin first intron. Several transcription factor binding sites identified in human first intron, but not in rat intron, do not contribute to the reported silencer activity. Transfections of renin/ luciferase constructs containing partial portions of first intron inserted upstream of the 892 bp in both renin-producing cells do not allow the precise characterization of cis-elements involved in the silencer effect. CONCLUSIONS: The silencer located renin intron A is cell specific. The integrity of the human first intron seems necessary for its repressor activity on renin proximal promoter in renin-producing cells.

Renin-secreting tumors.

An analysis of the renin-secreting tumors published in the literature suggests the diagnosis of JGC tumor should be evoked systematically in a young patient with severe hypertension and hypokalemia in whom a renovascular lesion has been eliminated by arteriography. A very high PRA usually is observed and blood pressure drops during converting enzyme treatment. Under acute administration of captopril, plasma renin may or may not increase, showing the inconsistency of the secretory autonomy of the tumor. The most useful examination for the localization of the tumor is the CT scan. Excessive renin production may provoke vascular lesions, left ventricular hypertrophy, and impairment of renin function that all disappear after surgical treatment, at the time when blood pressure returns to normal. Primary reninism has great physiologic importance for the hypothesis that favors the essential role of the kidney in determining the level of blood pressure. It can be considered as a unique, purely renin-dependent form of hypertension. This syndrome has no experimental equivalent and is the most caricatural form of other renin-dependent hypertension, such as renovascular disease, and probably some other forms of essential hypertension. The discovery of a renin-secreting tumor therefore constitutes a life-saving diagnosis for the patient, a subject of reflection for the specialist, and a useful tool for studies of the general mechanisms involved in enzyme biosynthesis and tumoral endocrine cell function.

Cellular distribution of endothelin-converting enzyme-1 in human tissues.

Endothelin-converting enzyme-1 (ECE-1) is the key enzyme of endothelin biosynthesis, catalyzing the final processing step. As shown by the targeted disruption of the ECE-1 gene, mature endothelins must be produced at specific sites for normal embryonic development. Therefore, it is important to know the exact pattern of ECE-1 gene expression. In this study we investigated the cellular distribution of ECE-1 in a variety of human tissues by in situ hybridization and immunohistochemistry. Widespread expression of the ECE-1 gene was noted, with a similar distribution pattern for mRNA and protein in normal human tissues, suggesting a major biological role for ECE-1. ECE-1 levels were particularly high in the cardiovascular, reproductive, and endocrine systems. There was strong and consistent labeling for ECE-1 in the vascular endothelial cells of all organs examined and in various nonvascular cells, especially some glandular cells. A large amount of ECE-1 protein and mRNA was detected in the Leydig cells of the testis and in the granulosa and theca cells of the ovary. In the adrenal gland, ECE-1 was detected in the cortex and medulla, with the strongest labeling in the zona glomerulosa. Therefore, ECE-1 may be involved in other systems, such as the regulation of hormone secretion, rather than exclusively generating ET-1 from its precursor. These results point out the potential side effects of ECE-1 inhibitors that are currently under development for treatment of cardiovascular diseases. (J Histochem Cytochem 47:447-461, 1999)