Renin-like Activity Research Articles

Lack of vitamin D receptor causes stress-induced premature senescence in vascular smooth muscle cells through enhanced local angiotensin-II signals

ObjectivesThe inhibition of the renal renin-angiotensin system by the active form of vitamin D contributes to the cardiovascular health benefits of a normal vitamin D status. Local production of angiotensin-II in the vascular wall is a potent mediator of oxidative stress, prompting premature senescence. Herein, our objective was to examine the impact of defective vitamin D signalling on local angiotensin-II levels and arterial health. MethodsPrimary cultures of aortic vascular smooth muscle cells (VSMC) from wild-type and vitamin D receptor-knockout (VDRKO) mice were used for the assessment of cell growth, angiotensin-II and superoxide anion production and expression levels of cathepsin D, angiotensin-II type 1 receptor and p57Kip2. The in vitro findings were confirmed histologically in aortas from wild-type and VDRKO mice. ResultsVSMC from VDRKO mice produced more angiotensin-II in culture, and elicited higher levels of cathepsin D, an enzyme with renin-like activity, and angiotensin-II type 1 receptor, than wild-type mice. Accordingly, VDRKO VSMC showed higher intracellular superoxide anion production, which could be suppressed by cathepsin D, angiotensin-II type 1 receptor or NADPH oxidase antagonists. VDRKO cells presented higher levels of p57Kip2, impaired proliferation and premature senescence, all of them blunted upon inhibition of angiotensin-II signalling. In vivo studies confirmed higher levels of cathepsin D, angiotensin-II type 1 receptor and p57Kip2 in aortas from VDRKO mice. ConclusionThe beneficial effects of active vitamin D in vascular health could be a result of the attenuation of local production of angiotensin-II and downstream free radicals, thus preventing the premature senescence of VSMC.

311. REGULATION OF THE RENIN ANGIOTENSIN SYSTEM (RAS) IN A TROPHOBLAST CELL LINE BY CYCLIC ADENOSINE MONOPHOSPHATE (cAMP) AND 5'-AZA-2'-DEOXYCYTIDINE (AZA)

Renin and renin-like activities have been detected in early gestation placentae1,2. To explore what regulates RAS expression in trophoblasts we studied the effects of cAMP and AZA (DNA demethylating agent) on the expression of mRNAs for prorenin receptor (ATP6AP2), prorenin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), ACE2, angiotensin II type 1 receptor (AGTR1), and a downstream target of the prorenin receptor, cyclooxygenase-2 (PTGS2) in the early human trophoblast cell line HTR-8/SVneo. Cells were cultured for 24 or 48 h with vehicle, 0.5 mM cAMP or 15 mM AZA. Messenger RNA abundances were measured relative to Alien RNA using real-time qRT-PCR. All mRNAs were detected except for ACE and ACE2. REN mRNA abundance was increased by cAMP and AZA (P < 0.0001). However, cAMP was more effective than AZA at both 24 and 48 h (P < 0.0001; P < 0.05) at the concentrations employed. AZA significantly increased AGT mRNA levels at 24 h (P = 0.001) and AGTR1 mRNA levels at 48 h (P < 0.0001), while cAMP had no effect. P TGS2 mRNA expression was increased by cAMP at 24 h (P < 0.0001) and by AZA at 48 h (P < 0.0001). cAMP and AZA had no effect on ATP6AP2 abundance. Thus, prorenin may function directly through the prorenin receptor, since cAMP upregulated both REN and its downstream target PTGS2 in HTR-8/SVneo cells, especially considering that ACE and ACE2 were not expressed. Our findings also suggest that expression of the RAS in trophoblast is regulated by DNA methylation as there was sustained over expression of REN, AGT, AGTR1 and PTGS2 with AZA. The ATP6AP2 promoter possesses CpG islands; however, its expression was not affected by AZA. This may suggest that either DNA methylation is not involved in the regulation of this gene, or that the over expressed prorenin binding to its receptor is causing suppression of ATP6AP2 abundance via a previously identified negative feedback pathway. (1) Pringle et al., Perinatal Society of Australia & New Zealand (PSANZ) 2010, Wellington, New Zealand (Abstract).(2) Itskovitz et al., Journal of Clinical Endocrinology and Metabolism, 1992, 75: 906–10.

Separation of Renin-Like Activity from Acid Protease Activity in Different Parts of Rat Brain

Separation of Renin-Like Activity from Acid Protease Activity in Different Parts of Rat Brain

Escherichia coli lipopolysaccharide inhibits renin activity in human mesangial cells

Hyperactivation of systemic renin-angiotensin system (RAS) during sepsis is well documented. However, the behavior of intrarenal RAS in the context of endotoxemia is yet to be defined. The present study evaluates the direct effect of Escherichia coli lipopolysaccharide (LPS) on immortalized human mesangial cell (HMC) RAS. Quiescent HMC were incubated with vehicle or LPS (1-100 microg/ml), and levels of angiotensin I and II (Ang I and II) and their metabolites were analyzed by high-performance liquid chromatography. In addition, angiotensin-converting enzyme (ACE) and renin activity were also investigated. Cell lysate and extracellular medium levels of Ang II were rapidly reduced (1 h) in a time- and concentration-dependent manner, reaching a significant -9 fold-change (P<0.001) after 3 h of LPS incubation. Similar results were obtained for Ang I levels (-3 fold-change, P<0.001). We ruled out Ang I and II degradation, as levels of their metabolic fragments were also significantly decreased by LPS. ACE activity was slightly increased following LPS incubation. On the other hand, renin activity was significantly inhibited, as Ang I concentration elevation following exogenous angiotensinogen administration was blunted by LPS (-60% vs vehicle, P<0.001). Renin and angiotensinogen protein levels were not affected by LPS according to Western blot analysis. Taken together, these data demonstrate for the first time that LPS significantly downregulates HMC RAS through inhibition of renin or renin-like activity. These findings are potentially related to the development of and/or recovery from acute renal failure in the context of sepsis.

EFFECTS OF PLASMA FREE HEMOGLOBIN (Hb) ON THE RENIN ANGIOTENSIN SYSTEM (RAS)

A growing body of evidence suggests that the presence of plasma free Hb, even in extremely low concentrations, may be associated with adverse clinical signs and symptoms. Hb is linked to vasoconstrictive, pro-oxidative and pro-inflammatory events. While Hb’s oxidative and inflammatory effects are well established, the complete pathomechanism of Hb-induced vasoconstriction has not yet been fully elucidated. The hemodynamic disturbances mediated by Hb are of rapid onset, occurring within minutes, and sustained, lasting hours. Recently, we discovered that Hb, in its ferryl form, possesses angiotensin converting enzyme (ACE)-like activity, being able to convert, within minutes, angiotensin (Ang)-I to Ang-II and other active Ang fragments. This present study evaluated further the effect of Hb on RAS, focusing on the impact of ferrous- and ferryl-Hb on angiotensinogen (AGT) and ACE. The possible renin-like activity of Hb toward AGT was evaluated by reversed phase C-18 HPLC. The effect of Hb on ACE was investigated by monitoring changes in activity of purified and human lung microvascular endothelial ACE. Results indicate that both forms of Hb had no impact on AGT. However, incubation of purified ACE with ferryl-Hb increased its enzymatic activity. Hb also activated lung microvascular ACE. These events occurred within hours and could contribute to the sustained vasoconstrictive episodes associated with the presence of free Hb. Based on this study, it can be concluded that Hb has an impact on various elements of RAS, which could be an important factor in the not yet fully understood Hb-mediated vasoconstriction.

Ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome

Myocardial enzymatic activity of renin and cathepsin D before and after bilateral nephrectomy.

A local renin-angiotensin system is present within the myocardium and can play a role in the initiation and maintenance of cardiac hypertrophy. The source of myocardial renin maybe direct cardiac renin gene expression, or plasma renin of renal origin. A primary indication that myocardial renin is derived from plasma renin of renal origin was from work showing that cardiac renin activity was no longer detected 30 hours after bilateral nephrectomy (BNX). However, more recent studies have been able to detect myocardial renin after BNX. We measured normal rat cardiac renin before and after 48-hour BNX using a myocardial renin assay with improved sensitivity. The myocardial renin assay was also used to assess normal rat cardiac myocyte renin levels. Since cardiac tissue contains cathepsin D, a lysosomal enzyme capable of renin-like activity, a rat cathepsin D assay was also developed to assess cathepsin D contribution to renin-like activity. Several artifacts were shown to contribute to myocardial renin-like enzymatic activity levels after BNX, including initial plasma renin stimulation during BNX surgery, assay pH, and cardiac cathepsin D activity. Myocardial renin concentration after 48-BNX was found to be only approximately 1% of normal control levels, and renin concentration in normal cardiac myocytes was only 2-fold greater than assay blanks. Both results were probably overestimated due to cathepsin D contamination. In conclusion, no evidence was found for myocardial renin synthesis in the normal adult rat heart, and myocardial renin decays to near zero levels after 48-hour BNX.

Response of serum total renin to ramipril and metoprolol in hypertensive patients

Renin-angiotensin system has long been thought to be a classic endocrine negative feedback system in the pathophysiology of hypertension. Furthermore, angiotensin II formation was believed to be regulated by renin secreted from the kidneys. In contrast to these considerations is the identification of local angiotensin II production in other tissues than pulmonary vasculature. Prorenin, the molecular precursor of renin, has been assumed to be involved in local angiotensin II production because of its renin-like activity. Prorenin has also been found to be secreted from extrarenal sources, although a major part of it is derived from the kidneys. Increased concentration of total renin in serum has been proposed to be useful in identifying patients with active proliferative retinopathy in insulin-dependent diabetic patients. Renin-angiotensin system is strongly affected by angiotensin-converting enzyme (ACE) inhibitors and therefore the interfering effect of ACE inhibitor medication on total renin concentration should be known in order to interpret serum total renin concentrations. Nine hypertensive outpatients, all men, treated at the department of internal medicine in Turku University Central Hospital, received randomly 5 mg of ramipril or 95 mg of metoprolol once a day for 4 weeks. Ramipril significantly increased the mean value of total renin (191.9 ng/l vs 312.0 ng/l, p<0.01), but the metoprolol-induced increase in the concentration of serum total renin was insignificant. We conclude that the negative feedback mechanism in regulating renin and prorenin secretion was inhibited by ACE inhibitor ramipril but beta1-selective adrenoceptor antagonist metoprolol did not significantly change total renin concentration in serum.

Paraneoplastic hypokalemia in acute myeloid leukemia: a case of renin activity in AML blast cells

Hypokalemia due to renal potassium loss has frequently been observed in patients with acute myeloid leukemia (AML). The pathogenic mechanism for this hyperkaluresis is unclear. In this report we describe a patient with AML FAB M4, in whom the clinical course, the electrolyte disturbances, the serum aldosterone levels, and the diffuse hyperplasia of the adrenal cortex documented a typical case of marked secondary hyperaldosteronism. On analysis of the leukemic cells of this patient compared with normal bone marrow cells, a significant increase of renin-like activity in the cytosol of the blast cells was noted. Activation of the renin-angiotensin-aldosterone system by paraneoplastic production of renin-like activity in AML blast cells might contribute to the hypokalemia often observed in patients with acute myeloid leukemia.

The Kallikrein–Kinin and Renin–Angiotensin Systems in the Kidney of an African Lungfish,Protopterus annectens

Renin-like activity (RLA), angiotensin I converting enzyme-like (ACELA), and kallikrein-like activity (KLA), activities of the key enzymes of renin–angiotensin and kallikrein–kinin systems, were sought in the kidney of the African lungfishProtopterus annectensduring the aquatic phase. RLA, examined by RIA (using porcine angiotensinogen as substrate), was 0.38 ± 0.05 ng angiotensin I/mg protein/hr. ACELA and KLA were investigated in assays spectrophotometrically. ACELA, measured at 37 and at 20°, was, respectively, 1.55 ± 0.55 and 0.61 ± 0.23 nmol hippurate/min/mg protein. KLA was 7.34 ± 0.93 mU/mg protein in the crude kidney extract and 31.05 ± 7.50 mU/mg protein after electrophoretic purification. Renal kininogenase activity was inhibited by 100% byD-Phe-Phe-Arg-chloromethyl ketone (10 μM), 98% by phenylmethylsulfonyl fluoride (2 nM), and 91% by aprotinin (1000 kIU). The apparent molecular weight of the renal kininogenase on SDS–PAGE was 27,000 Da. Both the renal enzyme and the purified glandular kallikrein, used as a control, have the same mobility on polyacrylamide gel electrophoresis. Immunoreactivities toward angiotensin II and bradykinin were localized by double immunostaining in the same cells of the proximal tubules. Putative angiotensin II receptors were demonstrated immunohistochemically, in the supranuclear region of proximal tubular cells, using an antibody to the sequence between amino acids 225 and 237 of the mammalian AT1receptor.

Glucose, insulin and renin activity after sodium loading and depletion in Vipera aspis

Sodium, potassium, chloride, glucose, insulin and renin activity were investigated in fasted Vipera aspis subjected for 3 days to administration of 3% NaCl 5 ml, or injection of a diuretic and water loading to produce sodium depletion. After sodium loading, plasma sodium and glucose were significantly elevated if compared with those of controls, while plasma renin-like activity and plasma insulin were depressed. The insulin and somatostatin producing cells (B- and D-cells) showed only a weak immunoreactivity, while in the glucagon producing cells (A-cells) the immunoreactivity was stronger if compared with the handled controls. After sodium depletion, plasma sodium and glucose were significantly depressed and plasma renin-like activity and plasma insulin were significantly elevated. A strong immunoreactivity was present in B- and D-cells and only a weak immunoreactivity was detectable in the A-cells. These data suggest that the secretory activity of the endocrine pancreas and kidney may be affected, in vipers, by sodium and/or volume status.

A local renin-angiotensin-like system in the heart of an African lungfish (Protopterus annectens)

Abstract The presence of components of a renin-angiotensin system was investigated in the heart of an African lungfish Protopterus annectens . A renin-like activity is present in heart homogenates. The activity reaches its maximum after 90 min of incubation at 20°C. By a spectrophotometric assay, an angiotensin converting enzyme-like activity was detected. By immunohistochemistry Ile 5 -Angiotensin II immunoreactive cardiocytes were demonstrated in the atrium and bulbus. Our data indicate that probably a local renin-angiotensin-like system is present in the heart of Protopterus annectens .

Angiotensin II immunoreactivity is elevated in ascites during severe ovarian hyperstimulation syndrome: implications for pathophysiology and clinical management

To investigate the ovarian renin-angiotensin system (RAS) during severe ovarian hyperstimulation syndrome (OHSS). Simultaneous sampling of blood and ascitic or peritoneal fluid (PF) during therapeutic paracentesis or laparoscopy. University Hospital. Twelve patients were investigated: three patients presenting severe OHSS, three patients with a spontaneous first trimester pregnancy, three normally cycling women during the early luteal phase, and three patients with ascites of nonovarian origin. Renin-like activity and angiotensin II (ANG II) immunoreactivity were measured simultaneously in the plasma and the ascites or PF. Angiotensin II immunoreactivity was much higher in the ascites or PF than in corresponding plasma during severe OHSS, first trimester pregnancy, and in the early luteal phase, while it was lower in ascites of nonovarian origin. Renin-like activity and ANG II immunoreactivity were the highest in the ascites of severe OHSS and in the PF from part of the patients with a spontaneous first trimester pregnancy. The present findings argue for the ovarian origin of the elevated renin-like activity and ANG II immunoreactivity in the ascites of severe OHSS and suggest a stimulatory role of hCG on the ovarian RAS whether during severe OHSS or first trimester spontaneous pregnancy. The vasoactive peptide ANG II may contribute to the maintenance of the ascites in severe OHSS but is probably not responsible for the formation of the ascites. The efficiency of paracentesis during severe OHSS could be explained at least partially by the removing of great amounts of ANG II from the peritoneal cavity.

Renin-like Activity, Angiotensin I-Converting Enzyme-like Activity, and Osmoregulatory Peptides in the Dogfish Rectal Gland

Renin-like activity (RLA) and angiotensin I-converting enzyme-like activity (ACELA), two key enzymes of the renin-angiotensin cascade (RAS), were sought in the dogfish rectal gland. RLA was 1.1 ± 0.2 ng Ang I/mg protein/hr after incubation with porcine angiotensinogen and 0.8 ± 0.1 ng Ang I/mg protein/hr after incubation with homologous plasma. ACELA was 7.22 ± 1.08 and 8.87 ± 1.9 nmol hippurate generated/min/mg protein respectively, at 0 and 37°. The presence of these enzymes may indicate the presence of an endogenous RAS-like system in the rectal gland. Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) binding sites were demonstrated autoradiographically in the subcapsular region of the gland, suggesting a possible interaction of the two hormones in the blind outer ends of the rectal gland tubules. Immunoreactivities toward Ang II, ANP, bombesin, vasoactive intestinal polypeptide (VIP), glucagon, and somatostatin were differentially localized in the rectal gland within three concentric zones with potentially different functional activities. In the capsule, there was a strong positive ir-glucagon reaction and a slightly weaker reaction for ir-somatostatin and VIP. In the blind outer ends of the tubeles (in the subcapsular zone), strong immunoreactivity was present toward all the tested peptides except glucagon and somatostatin. In the inner zone and in the central canal, only a weak immunoreactivity toward Ang II and glucagon was observed.

Gonadotropin stimulates ovarian renin-angiotensin system in the rabbit.

The present study was undertaken to assess the role of ovarian renin-angiotensin system (RAS) in the preovulatory cascade induced by gonadotropin exposure. In the in vitro perfused rabbit ovaries, exposure to human chorionic gonadotropin (hCG) enhanced the secretion rate of angiotensin II (Ang II) within 1 h. The secretion rate reached maximal levels at 6 h and then declined thereafter. The intrafollicular Ang II content and renin-like activity were also significantly increased at 2 and 4 h after exposure to hCG, compared with control ovaries perfused with medium alone. The level of intrafollicular Ang II after hCG exposure significantly exceeded the concentration of Ang II in an equivalent volume of plasma. The addition of 1 microM captopril to the perfusate significantly inhibited the secretion rate of Ang II stimulated by hCG; however, captopril affected neither the ovulatory efficiency nor prostaglandin production in ovaries treated with hCG. Captopril significantly inhibited the resumption of meiosis in the ovulated ova and follicular oocytes stimulated by hCG. The administration of 100 micrograms Ang II at 2-h intervals to the perfusate reversed the inhibitory effects of captopril on hCG-induced oocyte maturation. In conclusion, these data indicate that gonadotropin stimulates renin-like activity and Ang II production in the rabbit ovary. Ovarian renin-angiotensin system may play an important role in the process of oocyte maturation after exposure to gonadotropin.

Renin is not synthesized by cardiac and extrarenal vascular tissues. A review of experimental evidence.

A comprehensive review of physiological and molecular biological evidence refutes claims for synthesis of renin by cardiac and vascular tissues. Cardiovascular tissue renin completely disappears after binephrectomy. Residual putative reninlike activity, where investigated, has had the characteristics of lysosomal acid proteases. Occasional reports of renin or renin mRNA in vascular and cardiac tissues can be ascribed to failure to remove the kidneys 24 hours beforehand, overloading of detection systems, problems with stringency in identification, and illegitimate transcripts after more than 25 cycles of polymerase chain reaction. Others, using more stringent criteria, have failed to detect cardiac and vascular renin mRNA. Accordingly, a growing number of investigators have concluded that the kidneys are the only source of cardiovascular tissue renin. Although prorenin is secreted from extrarenal tissues as well as from the kidneys, there is no evidence that it is ever converted to renin in the circulation. The kidney is the only tissue with known capacity to convert prorenin to renin and to secrete active renin into the circulation. Accordingly, renin of renal origin determines plasma and hence, extracellular fluid renin levels. In these loci, angiotensin (Ang) I, formed by renin cleavage of circulating and interstitial fluid angiotensinogen, is in turn cleaved by angiotensin converting enzyme, located in plasma and extracellular fluids and on the luminal surface of pulmonary and systemic vascular endothelial cells, to Ang II, which perfuses and bathes the heart and vasculature. Consistent with this model, plasma renin and angiotensin and the antihypertensive action of renin inhibitors, converting enzyme inhibitor, or Ang II antagonists all disappear after binephrectomy. Thus, the plasma renin level, via Ang II formation, determines renin system vasoconstrictor activity, the antihypertensive potential of anti-renin system drugs, and the risk of heart attack in hypertensive patients. This analysis redirects renin research to renal mechanisms that create the plasma renin level, to renal prorenin biosynthesis and its processing to renin, and to their regulated secretion, extracellular distribution, and possible binding to by target tissues. In this context, it is still possible that changes in circulating and interstitial renin substrate or available converting enzyme might exert subtle modulating influences on Ang II formation. However, this analysis redefines the importance of plasma renin measurements to assess clinical situations, because plasma renin is the only known initiator driving the cardiovascular renin-angiotensin system, and its strength can be measured.

Renin and renin inhibition in anephric man.

Renin activity appears to be present in low concentrations in the plasma of anephric humans but could be artifactual secondary to inadvertent activation of prorenin during specimen collection and handling or from a renin-like enzyme. We studied the effects of specimen collection, storage, different assay conditions, trypsin activation, and the renin inhibitor EMD 56133 (E Merck, Darmstadt) on plasma renin activity (PRA) in anephric man. PRA was detectable in all seven bilaterally nephrectomized (BNX) patients (0.2 +/- 0.1 ng AI/ml/hr, range 0.1-0.7) but was significantly lower than normals (2.4 +/- 0.3 ng AI/ml/hr, range 1.5-3.1, p = 0.001). PRA was not different in BNX whether blood samples were collected on ice or at room temperature and assayed immediately or whether samples were frozen and assayed several days later. Prolonged cold storage of samples and five freeze-thaw cycles over six to seven months did not significantly increase PRA in normals or anephrics. However, deliberate repeated freezing and thawing over the period of a single day increased PRA 4.1-fold in BNX and 1.6-fold in normals. Renin-like activity was also detected in BNX individuals using renin concentration determinations with either excess human or sheep angiotensinogen. The inhibition of renin activity (IC-50% = 3.16 x 10(-9) molar) by EMD 56133 was not different between BNX and normals. Thus, active renin is present in the plasma of anephric humans and does not result from the inadvertent activation of prorenin due to sample handling. Although the source of PRA in BNX is unknown, the enzyme appears functionally normal as evidenced by the dose-response to a single renin inhibitor.

KRI-1314: An Orally Effective Inhibitor of Human Renin

Biochemical and pharmacological properties of KRI-1314, a newly synthesized, low molecular weight (M.W.: 690) renin inhibitor, were investigated in vitro and in vivo. The novel amino acid norstatine, which is shorter in chain length than the well-known statine, was incorporated into KRI-1314 as a tetrahedral transition-state analogue for the Leu10-Val11 scissile peptide in the renin substrate. KRI-1314 more strongly inhibited plasma renins from primates than those from dogs, rabbits, guinea pigs and rats. KRI-1314 competitively inhibited highly-purified human renin with a Ki value of 9.9 x 10(-10) M. KRI-1314 strongly inhibited the tissue renin-like activities of various organs from Japanese monkeys, with IC50 values on the order of 10(-8) M. KRI-1314 was also very stable in various tissue homogenates from Japanese monkeys. Both intravenous (from 0.25 to 3 mg/kg) and oral (10 and 30 mg/kg) administration of KRI-1314 to anesthetized and conscious sodium-depleted Japanese monkeys, respectively, significantly lowered the blood pressure and plasma renin activity without affecting the heart rate. In Japanese monkeys, KRI-1314 was continuously detected in the plasma up to at least 7 hr after oral administration of 10 and 30 mg/kg. These results demonstrate that KRI-1314 is a highly potent, primate-selective and long-lasting oral renin inhibitor with a blood pressure lowering effect.

Renin and angiotensin converting enzyme in elasmobranchs

Renin-like activity (RLA) and angiotensin I converting enzyme-like activity (ACELA), the two key enzymes of the renin-angiotensin system (RAS), were sought in the elasmobranch Scyliorhinus canicula. Renal extracts were desalted in a G-25 and eluted in a G-100 Sephadex column (calibration 15,000–70,000). The fractions were concentrated in a vacuum device. A 48,000-MW fraction incubated with synthetic and porcine angiotensinogen generated angiotensin I estimated by RIA. This same fraction was vasopressor in rats and dogfish. ACELA was sought in gill, heart, liver, spleen, pancreas, intestine, kidney, gonads, brain, skin, and muscle of dogfish using a spectrophotometric assay. The highest level of ACELA was found in the gills followed by spleen, kidney, and brain (33.79 ± 2.3, 29.56 ± 1.0, 14.62 ± 1.0, and 13.80 ± 2.3 nmol hippurate/min/mg protein, respectively). Intestine, gonads, skin and muscle contained no measurable amounts of ACELA. Captopril inhibited enzymatic activity from all ACELA containing tissues.

Studies on the renin-angiotensin system in primary monolayer cell cultures of the rat epididymis.

Monolayer cell cultures formed from the rat cauda epididymidis exhibited renin-like and angiotensin-converting enzyme (ACE) activities and contained immunoreactive angiotensin I (AI) and angiotensin II (AII). Renin-like activity, determined indirectly by radioimmunoassay of generated AI at a near-neutral pH of 6.0, was demonstrated in the cell lysate but was almost undetectable in the serum-free cell culture medium, suggesting that renin expression in epididymal cells is an intracellular phenomenon. In contrast, both AI and AII were detected in the cell lysate and cell culture medium. The level of AI was enhanced by pretreating the cells with the ACE inhibitor captopril (100 nmol/l). Incubating the cell monolayers with thoroughly washed sperm cells obtained from the intact cauda epididymides of rats increase (P less than 0.01) the AII content of the cell culture medium, with a parallel decline (P less than 0.01) in the AI concentration. However, adrenaline (0.23 mumol/l), which was found to stimulate electrogenic anion secretion by cell monolayers grown on previous supports, was without effect on the renin-like activity or concentration of angiotensins. The ACE activity in cells was confirmed by its strong dependence on chloride ion and its susceptibility to inhibition by captopril (100 nmol/l). Enzyme activity was significantly (P less than 0.005) higher in the culture medium than in the cell lysate and cell membrane fragments. Angiotensinogen, which is obligatory for an intrinsic renin-angiotensin system, is present in epididymal cells. Presumably, it is synthesized and processed in the cell cytosol by intracellular renin.(ABSTRACT TRUNCATED AT 250 WORDS)