Blockade of AT1 receptors by losartan did not affect renin gene expression in kidney medulla.

This study was designed to determine particular changes in the renin gene expression and activity in renal cortex and medulla after AT(1) receptor blockade. It was found that two-week-treatment with AT(1) blocker losartan induced an increase in tissue renin activity in both parts of kidney causing subsequent elevation of plasma renin activity. Renin mRNA in losartan-treated rats was increased only in cortex, suggesting cortex origin of elevated renin activity in medulla. Medullary renin mRNA indicated local synthesis of renin within the whole kidney and supported the idea of the presence of tissue renin-angiotensin system. Our results show that gene expression of renin in kidney medulla is insensitive to AT(1) receptor blockade and this points out that the regulation of kidney renin-angiotensin system probably differs from that in cortex.

[Renal tissue angiotensins during converting enzyme inhibition of angiotensin I in spontaneously hypertensive rat]. / Angiotensines tissulaires rénales au cours de l'inhibition de l'enzyme de conversion de l'angiotensine I chez le rat spontanément hypertendu.

To compare the effects of an angiotensin-converting enzyme inhibitor on circulating and tissue renin-angiotensin system, we measured different renin-angiotensin system parameters during the first day of treatment (Day 1) as well as after two weeks of treatment (Day 14). Ramipril was given orally once daily to adult male spontaneously hypertensive rats. Renin activity, angiotensin-converting enzyme activity and levels of angiotensin I and angiotensin II in the plasma, renal cortex and renal medullar were assessed at Day 1 and Day 14 of the treatment. In the plasma, both renin activity and angiotensin I increased 10 to 15 fold one to four hours after acute as well as at Day 14 of ramipril treatment and then returned to basal values within 24 hours. Plasma angiotensin II levels were not significantly decreased at Day 1 or Day 14. The decrease in the angiotensin II/angiotensin I ratio suggested a sustained inhibition of plasma angiotensin-converting enzyme at Day 14. In the renal cortex and medulla, a clearly different pattern was observed: in ramipril treated rats, renin activity in the renal cortex and medulla did not change at Day 1 but at Day 14 we observed a slight and sustained increase in renin activity. Despite very high basal levels of renin activity, angiotensin I levels in the renal cortex were comparable to those in the plasma. The angiotensin I level increased only one-fold one hour after ramipril intake at Day 1 and Day 14. This suggests that angiotensinogen may have a limiting role in the synthesis of angiotensin I in the kidney. Angiotensin II levels were slightly higher in the renal cortex and medulla than in the plasma suggesting local synthesis of the peptide. In the kidney, angiotensin II levels decreased one and four hours after the acute or prolonged ramipril treatment and the angiotensin II/angiotensin I ratio was reduced at the same time. Our results show that the responses of the plasma and kidney components of the renin-angiotensin system to angiotensin-converting enzyme inhibition are different in the plasma and the kidney suggesting that the circulating and tissue renin-angiotensin system are at least in part independent.

[Effect of a non-antihypertensive dose of ramipril on the plasma and tissue renin-angiotensin system in 27 TGR (mRen2) rats]. / Effets d'une dose non antihypertensive de ramipril sur les systèmes rénine-angiotensine plasmatique et tissulaire chez le rat TGR (mRen-2) 27.

It is admitted that low dose of angiotensin converting enzyme (ACE) inhibitors allows the regression of left ventricular hypertrophy (HVG) in experimental models where plasma renin activity (PRA) is high. The use of low dose of ramipril, an ACE inhibitor, make it possible to explore the place of cardiac renin-angiotensin system (RAS) in the regression of HVG independently of blood pressure (BP). Twenty rats TGR (mRen2) 27, heterozygous male, 10 weeks old were treated by daily oral gavage during 6 weeks by 10 micrograms/kg/jour ramipril or distilled water and compared to 10 normotensive Sprague Dawley (SD) rats. BP was measured. After the period of treatment, plasma, left kidney and the ventricles were removed. On each tissue samples and plasma, angiotensinogen (Aogen), the renin activity, angiotensins I (Ang I) and II (Ang II) were determined by radioimmuno assay and the activity of ACE was measured by fluorimetry. BP does not differ between treated and untreated groups during 6 weeks of treatment but is significantly higher compared to SD rats. PRA of untreated rats is high (36 +/- 5 ng Ang I/mL/h). However, treatment did not make it possible to decrease HVG. In plasma and kidney treatment's effect on SRA is confirmed by the increase in renin activity (plasma: 63 +/- 9 vs 36 +/- 5 ng Ang I/mL/h; kidney: 127 +/- 11 vs 92 +/- 7 micrograms Ang I/g/h) which is accompanied by an increase of Ang I rates (plasma: 297 +/- 31 vs 15 +/- 10 fmol/mL; kidney: 241 +/- 37 vs 160 +/- 12 fmol/g) and of the reduction in Aogen. An inhibition of ACE is perceptible with low dose of ramipril in heart (left ventricle: 1.7 +/- 0.1 vs 2.5 +/- 0.3 nmol HisLeu/min/mg protein), but it does not appear significant modifications of the other elements of the RAS in this tissue. The Ang II cardiac rates are probably not solely defined by cardiac ACE activity, other ways of synthesis being described. The absence of regression of the HVG in TGR (mRen2) 27 rat with low dose of ramipril could be related to the absence of effect on cardiac Ang II rates. In addition, the relation between high PRA rates and the effectiveness of low dose of ACE inhibitor in the HVG are not confirmed.

[Effects of hydroxyl radicals on purified angiotensin I converting enzyme]. / Modulation de l'activité de l'enzyme de conversion de l'angiotensine I sous l'effet d'un générateur de radicaux hydroxylés.

Somatic angiotensin-converting enzyme (ACE) is a protein which contains two similar domains (N and C), each possessing a functional active site. The relationship between ACE, its natural substrates and oxygen free radicals is starting to be explored. On one hand, superoxide anions production is induced by angiotensin II and on the other hand, activated polynuclear neutrophils, through free radicals generation, alter endothelial ACE activity. In this study, we examined the impact of hydroxyl radicals (.OH) on purified ACE. .OH were produced using a generator: 2,2'-azo-bis 2-amidinopropane (GRH) provided by Lara-Spiral (Fr). GRH (3 mM), in a time-dependent fashion, inhibited ACE activity. When ACE was co-incubated for 4 h with GRH, its activity decreased by 70%. Addition of dimethylthiourea (DMTU: 0.03 to 1 mM) or mannitol + methionine (20/10 mM), two sets of .OH scavengers, produced a dose-dependent protection on ACE activity. To examine whether oxidation of thiol groups in the ACE molecule could be involved in the action of GRH, the effects of thiol reducing agents: mercaptoethanol and dithiotreitol (DTT) were investigated. These compounds produced a dose-dependent and significant protection; with 100% protection at 0.2 and 0.3 mM for mercaptoethanol and at 0.1 mM for DTT. The hydrolysis of two natural and domain-specific substrates were also explored. The hydrolysis of angiotensin I preferentially cleaved by the C domain was significantly (p < 0.01) inhibited by 57, 58 and 69% in contact with 0.3, 1 and 3 mM GRH [in nmol angio II formed/min/nmol of ACE, n = 4; 35.9 +/- 0.6 (control), 15.5 +/- 2.8 (GRH : 0.3 mM), 15.1 +/- 0.5 (1), 10.9 +/- 0.6 (3)]. The hydrolysis of the hemoregulatory peptide (hp), preferential substrate for the N domain was not affected by GRH at 0.3 mM and inhibited by 28% (not significant) by 1 mM GRH [in nmol ph hydrolized/min/nmol ACE, n = 4; 12.6 +/- 1.9 (control), 14.9 (GRH : 0.3 mM), 8.3 +/- 4.0 (1). These results demonstrated that .OH affect ACE activity and could suggest a privileged impact of GRH on the C domain. The precise sites of action of .OH remain unknown. The Cys residues near the active centers, by forming disulphide bridges during the oxidation could be of critical importance. Further studies will be needed to determine whether oxidative stress again ACE can be involved in the genesis of inflammatory vascular pathologies.

Renal tissue angiotensins during converting enzyme inhibition in the spontaneously hypertensive rat.

To compare the effects of an angiotensin-converting enzyme inhibitor on circulating and tissue renin-angiotensin system (RAS), we measured different RAS parameters during the first day of treatment (Day1) as well as after two weeks of treatment (Day14). Ramipril was given orally once daily to adult male spontaneously hypertensive rats (SHR). Renin activity (RA), angiotensin converting enzyme (ACE) activity and levels of angiotensin I (ang I) and angiotensin II (ang II) in the plasma, renal cortex and renal medulla were assessed at Day1 and Day14 of the treatment. In the plasma, both RA and ang I increased 10 to 15 fold one to four hours after acute as well as at Day14 of ramipril treatment and then returned to basal values within 24 hours. Plasma ang II levels were not significantly decreased at Day1 or Day14. The decrease in the ang II/ang I ratio suggested a sustained inhibition of plasma ACE at Day14. In the renal cortex and medulla, a clearly different pattern was observed: in ramipril treated rats, RA in the renal cortex and medulla did not change at Day1 but at Day14 we observed a slight and sustained increase in RA. Despite very high basal levels of RA, ang I levels in the renal cortex were comparable to those in the plasma. The ang I level increased only one-fold one hour after ramipril intake at Day1 and Day14. This suggests that angiotensinogen may have a limiting role in the synthesis of ang I in the kidney. Ang II levels were slightly higher in the renal cortex and medulla than in the plasma suggesting local synthesis of the peptide. In the kidney, ang II levels decreased one and four hours after the acute or prolonged ramipril treatment and the ang II/ang I ratio was reduced at the same time. Our results show that the responses of the plasma and kidney components of the RAS to ACE inhibition are different in the plasma and the kidney suggesting that the circulating and tissue RAS are at least in part independent.

Inhibitory effect of reactive oxygen species on angiotensin I-converting enzyme (kininase II).

1. Somatic angiotensin I-converting enzyme (ACE) is a protein that contains two similar domains (N- and C-terminal), each possessing an active site. We have examined the effects of a generator of hydroxyl radicals (g*OH: 2,2'-azo-bis(2-amidinopropane)) and hydrogen peroxide (H2O2) on ACE using an in vitro approach. 2. The generator of hydroxyl radicals inactivated ACE in a time (2-6 h)- and concentration (0.3-3 mmol/L)-dependent manner at 37 degrees C. When ACE was coincubated for 4 h with g*OH (3 mmol/L), its activity decreased by 70%. Addition of dimethylthiourea or mannitol + methionine, two *OH scavengers, resulted in a significant protection of ACE activity. Mercaptoethanol and dithiotreitol, two thiol-reducing agents, also efficiently protected ACE activity. 3. The hydrolysis of two natural and domain-specific substrates was explored. The hydrolysis of angiotensin I, preferentially cleaved by the C-domain, was significantly inhibited (57-58%) after 4 h exposure to g*OH (0.3-1 mmol/L). Under the same conditions of exposure, the hydrolysis of N-acetyl-Ser-Asp-Lys-Pro, a specific substrate for the N-domain, was only slightly inhibited by 1 mmol/L g*OH. 4. Hydrogen peroxide, another source of *OH, was used. After exposure to H2O2 (3 mmol/L; 4 h), an 89% decrease in ACE activity was observed. Pretreatment with the iron chelator deferoxamine (1 mmol/L) attenuated H2O2-mediated ACE inactivation, demonstrating that the effect of H2O2 was partly due to its conversion into *OH (Fenton reaction). 5. In summary, our findings demonstrate that g*OH and H2O2 inhibit ACE activity and suggest a preferential action of g*OH on the C-domain of the enzyme.