Isoform Of Angiotensin-converting Enzyme Research Articles

URINARY SOMATIC ANGIOTENSIN CONVERTING ENZYME AND SOLUBLE N-DOMAIN ISOFORMS (90 AND 65KDA) EXPRESSION AND CORRELATION WITH DIFFERENT NUTRITIONAL STATUS/CARDIOVASCULAR RISK PROFILE

Abstract Objective: Angiotensin converting enzyme (ACE) has been involved in physiopathology of several diseases including hypertension, diabetes, obesity and metabolic syndrome. The somatic ACE weights from 130–190 KDa and presents N- and C-domains. Two soluble N-domain isoforms have been described in human urine, weighting 65 and 90KDa. Studies have supported that N-domain ACE with 90KDa is a biomarker for hypertension, pre-eclampsia and inflammation. We evaluated the expression of somatic ACE and soluble N-domain isoforms (90 and 65KDa) in urine of children and adolescents with different nutritional status and cardiovascular risk profile. Design and method: The participants aged from 6 to 19 years were classified into four groups according to their BMI percentile; underweight (n = 51), normal weight (n = 53), overweight (n = 53) and obese (n = 49). Waist-height-ratio (WHtR) was used to assess cardiovascular risk profile dividing the participants into normal risk (n = 105) and high risk (n = 101). The urines were concentrated 10-fold and dialyzed with Tris-HCl pH 8 and pure water. Then, we performed western blot analysis using 50 mg of lyophilized urinary protein. ACE expression was proved against the polyclonal antibody Y1. Protein detection was performed by chemiluminescent and analysis was done in Image Labâ (BioRad) software utilizing total protein stain for normalization. Results: ACE expression is augmented in obese children when compared with normal weight children (0.09 vs 0.53 arbitrary units, p = 0,04). The higher cardiovascular risk group also presented increased expression of ACE (0.27 vs 0.09 arbitrary units, p = 0.046). The 90KDa N-domain isoform is frequently found in the high cardiovascular risk children (p = 0.02). Additionally, according to Spearman correlation test, the expression of 90KDa N-domain isoform correlates positively with waist circumference (cm), WHtR, BMI percentile and Z-score of BMI. Conclusions: Increased ACE expression in obese children contributes to higher cardiovascular risk once this enzyme biosynthesizes Angiotensin II which promotes blood pressure increase, sympathetic nervous system activation and release of glucocorticoids from adrenal gland. ACE expression is also augmented in children with high cardiovascular risk. Presence of 90 KDa N-domain ACE in urine of children and adolescents is a biomarker of poor prognostic for cardiovascular disease in childhood obesity.

Abstract P394: Ace Activity and Its Expression as a Hypertension Biomarker in the Urine of Pediatric Renal Transplant Recipients

Pediatric hypertension is a risk factor for hypertension in adults. Primary hypertension results from genetic, environmental, and behavioral factors. Renin Angiotensin System is important to control cardiovascular and renal functions. Angiotensin Converting Enzyme (ACE) has isoforms: somatic (130-190 kDa) and testicular (90-110 kDa). Casarini et al. described the N-domain isoform of 90kDa, in urine of hypertensive humans as a possible hypertension biomarker. Objective: Analyze ACE isoform in urine of pediatric renal transplant recipients before and after transplantation and urine of donor, to verify the 90 kDa isoform transmission and correlating with hypertension. Methods: 30 pediatric recipients with living donors; ACE activity was performed before, 1, 3 and 6 months post transplantation using ZPhe and HHL as substrates and its ratio. Results: 69% had pre-transplant hypertension and 85.7% family history. After 6 months 25% remained. From the pre-transplant until the sixth month after, ACE activity was reduced. In the model of linear regression between ACE activity and variables, including pre-transplantation for the substrates ZPhe-HL remained significant, time and uropathy (0.023 more); with HHL, time and uropathy with higher activity of ACE (0.019 more) and for weight, reduction of ACE activity ( 0.00038 at 1 kg increase in weight). At post-transplant only time and weight remained significant with ZPhe-HL, and for HHL uropathy, proteinuria (0.010 more ACE activity) and weight. Recipients before transplantation had higher urinary ACE activity when compared to controls. Western blotting data showed that all recipients without ACE 90KDa when received a kidney from the donor that had this isoform, started to express it. Conclusions: Increased ACE activity in uropathies may be associated with renal injury or intra-renal production. Proteinuria, a marker of renal injury, can be associated with increase on ACE activity. With nutritional recovery and a healthy kidney after transplantation, patients can reverse the degree of renal damage and decrease the production of ACE.

Pericardial Parietal Mesothelial Cells: Source of the Angiotensin-Converting-Enzyme of the Bovine Pericardial Fluid.

BackgroundAngiotensin II (Ang II), the primary effector hormone of the renin-angiotensin system (RAS), acts systemically or locally, being produced by the action of angiotensin-converting-enzyme (ACE) on angiotensin I. Although several tissue RASs, such as cardiac RAS, have been described, little is known about the presence of an RAS in the pericardial fluid and its possible sources. Locally produced Ang II has paracrine and autocrine effects, inducing left ventricular hypertrophy, fibrosis, heart failure and cardiac dysfunction. Because of the difficulties inherent in human pericardial fluid collection, appropriate experimental models are useful to obtain data regarding the characteristics of the pericardial fluid and surrounding tissues.ObjectivesTo evidence the presence of constituents of the Ang II production paths in bovine pericardial fluid and parietal pericardium.MethodsAlbumin-free crude extracts of bovine pericardial fluid, immunoprecipitated with anti-ACE antibody, were submitted to electrophoresis (SDS-PAGE) and gels stained with coomassie blue. Duplicates of gels were probed with anti-ACE antibody. In the pericardial membranes, ACE was detected by use of immunofluorescence.ResultsImmunodetection on nitrocellulose membranes showed a 146-KDa ACE isoform in the bovine pericardial fluid. On the pericardial membrane sections, ACE was immunolocalized in the mesothelial layer.ConclusionsThe ACE isoform in the bovine pericardial fluid and parietal pericardium should account at least partially for the production of Ang II in the pericardial space, and should be considered when assessing the cardiac RAS.

An ACE Up Your Sleeve

Although the renin angiotensin system (RAS) is a well established regulator of blood pressure and intravascular volume,1 considerable evidence has emerged demonstrating the importance of the RAS directly on the cardiovascular system (please refer to the Figure for description of the enzymatic pathways involved in RAS regulation). Indeed, angiotensin converting enzyme (ACE) inhibitors, which prevent the conversion of angiotensin I into angiotensin II (Ang II), and Ang II receptor type 1 (AT1R) antagonists, which inhibit the receptor-mediated actions of Ang II, have demonstrated direct favorable effects on the heart. These include a reduction in cardiac fibrosis during hypertensive left ventricular (LV) hypertrophy, protection against LV enlargement following myocardial infarction, and reduced LV remodeling following coronary artery bypass graft surgery.2 Hence, ACE1 is generally thought of as a villain contributing to the development of cardiovascular disease due to its generation of Ang II. Fortunately, the RAS may also have a potential hero in ACE2, which can counter many of the negative consequences of ACE1-produced Ang II. In this issue of Circulation Research , Patel et al3 demonstrate an important role for the RAS in the development of diabetic cardiomyopathy. If diabetic Akita mice are deficient for ACE2 (Akita/ACE2KO), the enzyme responsible for converting Ang II into Ang 1–7, a progressive systolic dysfunction occurred that is associated with oxidative stress, excessive extracellular matrix degradation, vascular dysfunction, and impairment of the Akt-signaling pathway. Ang II activation of the AT1R was implicated in these actions, since a 1-month treatment with the AT1R antagonist, irbesartan, reversed this systolic dysfunction, oxidative stress, vascular dysfunction, and Akt signaling in the Akita/ACE2KO mice. Figure. Angiotensin converting enzyme (ACE)1 and 2 in the renin angiotensin system. The ACE isoforms 1 and 2 play a critical role in renin angiotensin system regulation. ACE1 …

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), each of which bears an active site exhibiting different biochemical properties. The ACE isoforms consisted of one domain were also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when crystal their structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control distinct physiological functions. The C-terminal catalytic domain, playing important role in regulation of blood pressure, catalyzes angiotensin I cleavage in vivo. The N-domain contributes to processing of other bioactive peptides for which it exhibits high affinity. Domain-selective inhibitors able to block selectively either the N- or C-domain of ACE have been developed.

CARDIOPROTECTIVE EFFECTS OF GRANULOCYTE COLONY‐STIMULATING FACTOR IN ANGIOTENSIN II‐INDUCED CARDIAC REMODELLING

1. Granulocyte colony stimulating factor (G-CSF) is reported to have a beneficial effect on cardiac dysfunction in postinfarction and doxorubicin-induced cardiomyopathy. Thus, the aim of the present study was to investigate the effects of G-CSF on cardiac remodelling in angiotensin (Ang) II-induced hypertrophy. 2. Four groups of mice were investigated. The first group served as a control group. The second group was injected with recombinant human G-CSF (10 microg/kg per day, s.c.) on the first 5 days of each week and treatment was continued for 4 weeks. An osmotic minipump was implanted subcutaneously into each mouse in the third group so that pressor doses of AngII (2.88 mg/kg per day) or saline could be administered over a period of 4 weeks. The fourth group was infused with AngII (2.88 mg/kg per day) and injected with G-CSF (10 microg/kg per day, s.c.) for 4 weeks. 3. Angiotensin II treatment significantly elevated blood pressure and caused cardiac hypertrophy and fibrosis in mice. Treatment of mice with G-CSF did not reduce the AngII-induced increase in blood pressure, but ameliorated the development of cardiac fibrosis and hypertrophy. Infusion of AngII induced upregulation of angiotensin-converting enzyme (ACE) expression and downregulation of ACE2 expression. Treatment with G-CSF reduced cardiac levels of ACE and increased ACE2 expression. In addition, G-CSF treatment reduced the expression of osteopontin (OPN) and phospho-p70S6 kinase, which were upregulated by AngII infusion. 4. These results suggest that G-CSF reduces AngII-induced hypertrophy. Modulation of the expression of the ACE isoforms contributes to regression of AngII-induced cardiac hypertrophy. The effect of G-CSF to prevent cardiac fibrosis and hypertrophy may be mediated, in part, via inhibition of OPN expression and p70S6 kinase phosphorylation.

Stunting growth: association of the blood pressure levels and ACE activity in early childhood

Angiotensin converting enzyme (ACE) converts angiotensin I to angiotensin II and inactive bradykinin. Several studies carried out in our laboratory have consistently identified three isoforms of ACE, at 65, 90 and 190 kDa, with the 90-kDa isoform being a possible genetic marker of hypertension. Based on these observations and the fact that nutritional stunting can be associated with hypertension, we have investigated the expression and activity of ACE in stunted children and its association with blood pressure (BP) levels and nutritional state. Sixty children aged 2-7 years were selected for this study. A urine sample was collected from each child. Angiotensin converting enzyme activity was evaluated using two different substrates, and ACE expression was detected by Western blotting. Our results show that nutritional stunting is associated with high ACE activity in childhood and that adjustment by gender does not modify the strength of this association. A greater percentage of stunted children had increased BP levels, and this clinical parameter was inversely correlated with anthropometric indicators. A greater urinary protein expression of the three ACE isoforms was observed in the group of children with growth stunting. Our findings suggest that the reported high risk of hypertension in stunted adolescents and adults are, at least partly, associated with abnormalities in the renin-angiotensin system.

Presence of angiotensin converting enzyme isoforms in larval lepidoptera ( Spodoptera littoralis)

In this research the presence of angiotensin converting enzyme (ACE) in larvae of the lepidopteran Spodoptera littoralis was evaluated. Making use of the substrate Abz-FRK-(Dnp)P-OH and the specific inhibitor captopril at 10 μM, ACE activity was determined in a fluorescence assay for intact larvae, hemolymph, head, midgut and dorsal tissue. In dorsal tissue and hemolymph, ACE activity was highest. These data are consistent with a possible role for ACE in contractions of the dorsal vessel and metabolism of circulating peptide hormones in the hemolymph. After the presence of ACE was confirmed, a sequential procedure of anion exchange and size exclusion chromatography was applied to purify ACE from whole wandering larvae (last stage). With this procedure, three different ACE pools were collected that cleaved the fluorogenic substrate Abz-FRK-(Dnp)P-OH. Activity could be inhibited by a final concentration of 2.5 μM captopril. In addition, two out of three samples eluted at different salt concentration and thus ACE 1, 2 and 3 represent at least two different ACE isoforms. These data reveal that ACE is present in S. littoralis and that at least two out of three isolated ACE forms are truly isoforms.

Relationship between spermatozoon movement velocity and expression of testicular isoform of angiotensin-converting enzyme on their surface

The expression of testicular isoform of angiotensin-converting enzyme on the surface of human spermatozoa was evaluated by flow cytometry with monoclonal antibodies to epitopes of C-terminal domain of human angiotensin-converting enzyme and the mean path, mean curvilinear and straight-line velocities were determined by computer analysis of spermatozoon movement. A positive correlation between the expression of testicular isoform of angiotensin-converting enzyme on the cell surface and spermatozoon movement velocity was revealed.

Novel mechanism of action of ACE and its inhibitors

angiotensin-converting enzyme (ACE) is a dipeptidyl peptidase transmembrane-bound enzyme (for review see Ref. [2][1]). A soluble form of ACE in plasma is derived from the plasma membrane-bound form by proteolytic cleavage of its COOH-terminal domain. There are two distinct isoforms of ACE: somatic

Tissue isoforms of angiotensin-converting enzyme

Tissue isoforms of angiotensin-converting enzyme

Expression and localization of N-domain ANG I-converting enzymes in mesangial cells in culture from spontaneously hypertensive rats

The angiotensin-converting enzyme (ACE) profile in urine of hypertensive patients and spontaneously hypertensive rats (SHR; 90- and 65-kDa N-domain ACEs) is different from that of healthy subjects and Wistar rats (190 and 65 kDa). In addition, four ACE isoforms were purified from mesangial cells (MC) of Wistar rats in the intracellular compartment (130 and 68 kDa) and as secreted forms (130 and 60 kDa). We decided to characterize ACE forms from SHR MC in culture. Analysis of the ACE gene showed that SHR MC are able to express ACE mRNA. The concentrated medium and cell homogenate were separately purified by gel filtration and then subjected to lisinopril-Sepharose chromatography. The molecular masses of purified enzymes, 90 kDa for ACEm1A and 65 kDa for ACEm2A (secreted enzymes) and 90 kDa for ACEInth1A and 65 kDa for ACEInth2A (intracellular), were different from those of Wistar MC. The purified enzymes are Cl- dependent, inhibited by enalaprilat and captopril, and able to hydrolyze AcSDKP. Immunofluorescence and cell fractionation followed by Western blotting showed predominant immunoreaction of the 9B9 antiserum for N-domain ACE in the nuclei. The N-domain ACE was localized in the glomerulus from Wistar rats and SHR. ANG II and ANG-(1-7) were localized in the cell cytoplasm and nuclei. The 90-kDa N-domain ACE, described recently as a possible genetic marker of hypertension, was found inside the cell nuclei of SHR MC colocalized with ANG II and ANG-(1-7). The presence of ANG II in the cell nuclei could suggest an important role for this peptide in the transcription of new genes.

Effects of spironolactone and eprosartan on cardiac remodeling and angiotensin-converting enzyme isoforms in rats with experimental heart failure

Angiotensin-converting enzyme (ACE)-2 is a newly described enzyme with antagonistic effects to those of the classical ACE (ACE-1). Both ANG II and aldosterone play an important role in the pathophysiology of congestive heart failure (CHF) and in the adverse cardiac remodeling during its development. In this study, we examined the effects of experimental CHF induced by an aortocaval fistula (ACF) and of its treatment with ANG II and aldosterone inhibitors on the relative levels of ACE-1 and ACE-2. We also compared the effects of spironolactone, an aldosterone antagonist, and eprosartan, an ANG II receptor antagonist, on heart hypertrophy and fibrosis in rats with ACF. Spironolactone (15 mg x kg(-1) x day(-1) ip, via minipump) or eprosartan (5 mg x kg(-1) x day(-1) ip, via minipump) was administered into rats with ACF for 14 and 28 days. Specific antibodies were used to determine the protein levels of myocardial ACE-1 and ACE-2. ACF increased the cardiac levels of ACE-1 and decreased those of ACE-2. Heart-to-body weight ratio significantly increased from 0.30 +/- 0.004% in sham-operated controls to 0.50 +/- 0.018% and 0.56 +/- 0.044% (P < 0.001) in rats with ACF, 2 and 4 wk after surgery, respectively, in association with increased plasma levels of aldosterone. The area occupied by collagen increased from 2.33 +/- 0.27% to 6.85 +/- 0.65% and 8.03 +/- 0.93% (P < 0.01), 2 and 4 wk after ACF, respectively. Both spironolactone and eprosartan decreased cardiac mass and collagen content and reversed the shift in ACE isoforms. ACF alters the ratio between ACE isoforms in a manner that increases local ANG II and aldosterone levels. Early treatment with both ANG II and aldosterone antagonists is effective in reducing this effect. Thus ACE isoform shift may represent an important component of the development of cardiac remodeling in response to hemodynamic overload, and its correction may contribute to the beneficial therapeutic effects of renin-angiotensin-aldosterone system inhibitors.

What’s new in the renin-angiotensin system?

Angiotensin-converting enzyme (ACE) is a zinc- and chloride-dependent metallopeptidase that plays a vital role in the metabolism of biologically active peptides. Until recently, much of the inhibitor design and mechanism of action of this ubiquitous enzyme was based on the structures of carboxypeptidase A and thermolysin. When compared to the recently solved structures of the testis isoform of ACE (tACE) and its Drosophila homologue (AnCE), carboxypeptidase A showed little structural homology outside of the active site, while thermolysin revealed significant but less marked overall similarity. The ellipsoid-shaped structure of tACE, which has a preponderance of alpha-helices, is characterised by a core channel that has a constriction approximately 10 A from its opening where the zinc-binding active site is located. Comparison of the native protein with the inhibitor-bound form (lisinopril-tACE) does not reveal any striking differences in the conformation of the inhibitor binding site, disfavouring an open and closed configuration. However, the inhibitor complex does provide insights into the network of hydrogen-bonding and ionic interactions in the active site as well as the mechanism of ACE substrate hydrolysis. The three-dimensional structure of ACE now paves the way for the rational design of a new generation of domain-selective ACE inhibitors.

N-Domain angiotensin I-Converting enzyme expression in renal artery of Wistar, Wistar kyoto, and spontaneously hypertensive rats

One of the most intriguing features in kidney transplantation is the finding that kidneys from hypertensive rats can transfer arterial hypertension on transplantation into normotensive rats. Some evidence also suggest that, in humans undergoing renal transplantation, the genotype of the donor kidney determines the blood pressure in the recipient. The renin-angiotensin-aldosterone system is the major etiological candidate in hypertension because it plays an important role in the control of cardiovascular homeostasis. Angiotensin-converting enzyme (ACE) cleaves the C-terminal from angiotensin I as well as from bradykinin. Thus, by generating the potent vasoconstrictor angiotensin II and by degrading the vasodepressor bradykinin, ACE is considered to play a role in blood pressure regulation. We have previously described the presence of N-domain ACE in urine of Wistar (W), Wistar Kyoto (WKY), and spontaneously hypertensive rats (SHR), all of which can hydrolyze the vasodilator peptide Angiotensin 1–7 and also the N-Acetyl-Ser-Asp-Lys-Pro, two peptides described as specific for N-domain ACE. These findings suggest that the 90 kd ACE isoform found in urine and in tissues of SHR is a possible genetic marker of hypertension. Based on the fact that the renal artery has an important role in the control of renal blood flow, we evaluated the presence of N-domain ACE in the renal artery of hypertensive and normotensive rats. Using Western blotting techniques on the renal arteries of W and WKY rats, we detected the 190-kd ACE (similar to somatic ACE) and also the 65-kd ACE previously described in urine and renal tissue as N-domain ACE. The 65-kd and 90-kd isoforms of ACE were also detected in renal arteries in SHR rats. Further studies are required to understand the role of 90-kd enzyme described as a possible local marker of hypertension, its contribution in angiotensin catabolism, and whether this abnormal form of the enzyme has any link with the development and transfer of hypertension after transplantation.

Species-specific splicing and expression of angiotensin converting enzyme

Angiotensin converting enzyme (ACE) is a critical determinant in the pathogenesis of various cardiovascular diseases and in the control of male fertility. Multiple isoforms of ACE protein are present in body fluids and tissues, but their formation and functions in vivo remain to be investigated. To determine whether alternative splicing contributes to the formation of ACE isoforms, this study was designed to clone all possible spliced transcripts in rat. We found that the splicing of intron 13 in testicular ACE was species-dependent. Compared with human and mouse testicular ACE, rat testicular ACE ( rtACE) retained intron 13 in its mature transcripts. The insertion of the intron 13 did not change or shift the reading frame. Cloning and characterization of the rtACE showed that, in addition to testicular tissue, it was wildly expressed in somatic tissues, such as lung, kidney, cardiac ventricle, and skeletal muscle from both genders. Furthermore, we demonstrated that the expression of rtACE was developmentally up-regulated in testicular tissue and increased during cardiac hypertrophy. Our data suggests that the inclusion of intron 13 produces a novel ACE isoform. This isoform likely participates in local angiotensin II formation in both somatic and germinal tissues, and associates with certain physiological or pathophysiological events.

The Germinal Isozyme of Angiotensin-converting Enzyme Can Substitute for the Somatic Isozyme in Maintaining Normal Renal Structure and Functions

The angiotensin-converting enzyme (ACE) gene encodes two structurally related isozymes, somatic ACE and germinal ACE, that are uniquely expressed in discrete locations in the body. The importance of ACE in these cell types was revealed by generating Ace -/- mice, which exhibit multiple abnormalities including renal structural defects and functions, hypotension, and male sterility. To test the hypothesis that specific physiological functions of ACE are mediated by isozyme-specific and tissue-specific expression patterns, we have used a transgenic approach to develop mouse strains that express just one ACE isoform in the target tissue of Ace -/- mice. The mice described in this report produce germinal ACE in sperm and serum. These mice were as healthy as wild type mice, and the males were fertile. Interestingly, they had normal kidney structure, fluid homeostasis, and partially restored urine concentration despite having low blood pressure. This result demonstrated that circulating germinal ACE is sufficient for maintaining normal kidney structure and fluid homeostasis but insufficient for restoring blood pressure to normal levels.

The unknown alternative splicing generates a novel isoform of human angiotensin-converting enzyme as a soluble form

Human angiotensin-converting enzyme (ACE) gene exists in the chromosome 17, and two kinds of molecules are produced from the same gene locus according to the alternative splicing as the somatic and testis form. It is said theACE activity in serum derives from proteolytic release of the somatic ACE located in endothelial cell surface. V~ report here, in human somaticACE mRNA, the unrecognized alternative splicing occurs in common, and as a result, a new isoform of ACE is generated. The samples were Jurkat cell (humanT cell leukemia line) and peripheral blood leukocytes obtained healthy subjects.The mRNA was extracted from these cells, and RT-PCR was executed with the primer set in the 15th exon and the 18th exon.The electroophoresis of the PCR product visualized two bands of DNA fragment; one was the expected band derived from somaticACE mRNA, and the other was the unexpected, 150bp-longer one. This longer DNA fragment was subcloned and sequenced. As a result, the structure of this fragment was revealed to be exon15-exon16-exon17-intronf7-exon18 of human ACE gene, and a novel alternative splicing was confirmed.This alternative splicing arose a new stop codon immediately after the 17th exon, and it was surmised that a novel isoform of ACE was £1enerated as a soluble form which deleted the transmembrane portion and C domain of two active centers of somaticACE. This isoform mRNAwas also detected in lung, kidney, aorta and pancreas, and not detected in brain, heart, liver, skeletal muscle, testis and placenta by screening the human cDNA library of each organ. In leukocytes and pancreas, the expression level was about 20 percent of somaticACE, and much lower in lung, kidney and aorta. The other research group have reported that the transgenic mouse of the equivalent artificial molecule had shown a similar phenotype of theACE-knockout mouse, therefore this spontaneously expressed molecule in human is also expected to have similar functions in vivo. Further, we have investigated the relationship between this alternative splicing and theACE Insertion/Deletion poiymorphism in the 16th intron. At this moment, any correlation have been found within both phenomena. The 2nd Annual Scientific Meeting • JHFS 67

Gene expression and tissue localization of the two isoforms of angiotensin I converting enzyme.

Angiotensin converting enzyme exists in two different isoforms, somatic and germinal, whose respective distributions and intracellular localizations have not been precisely determined. The differing biochemical and molecular characteristics of the two isozymes allowed the preparation of antibodies specific for each of the two angiotensin converting enzyme isoforms and of two nucleic acid probes, one of which was specific for the germinal isoform. Immunohistochemistry and in situ hybridization were used to determine the cell distribution of, respectively, the two isoforms and their corresponding messenger RNAs in the classically studied tissues of male adult humans and marmosets. Results provided by the two different methods were always concordant and were identical in the two species. The somatic angiotensin converting enzyme form was expressed uniquely in somatic tissues (vascular endothelial cells and at the brush border of renal proximal convoluted tubule, jejunal villus, and epididymal duct epithelia), and the germinal form was expressed uniquely in germinal cells with a precise stage-specific pattern, starting in round spermatids and finishing in spermatozoa. In situ hybridization documented the presence of somatic angiotensin converting enzyme messenger RNA in renal tubule epithelium, jejunal enterocytes, and epididymal epithelium and demonstrated that there was no direct correlation between the levels of angiotensin converting enzyme messenger RNA and the enzyme it encodes for, i.e., angiotensin converting enzyme, in a given epithelium. The significance of the ultraselective expression of germinal angiotensin converting enzyme and of its specific messenger RNA at a very precise stage of spermatogenesis remains uncertain.