Angiotensin-Converting Enzyme Activity Is Reduced in Brain Microvessels of Spontaneously Hypertensive Rats

Abstract: Angiotensin-converting enzyme (ACE) activity in brain microvessels of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls was measured. Cerebral microvessels, prepared from the cerebral cortices by the albumin flotation and glass bead filtration technique, were free of neuronal and glial elements. ACE activity in brain microvessels of SHR was lower than that of WKY. A Woolf-Augustinsson-Hofstee plot showed that the reduction of the enzyme activity in SHR was due to a 30% decrease in V_max without any change in K_m for substrate. The decrease of ACE activity in brain micro-vessels of SHR may indicate an impairment of the central renin-angiotensin system and may be related to cerebral microvascular dysfunctions occurring in hypertension.     

Angiotensin-converting enzyme activity in isolated brain microvessels.

The localization of angiotensin-converting enzyme (kininase II; ACE) in bovine cerebral cortex was studied by mechanically isolating microvessels from surrounding brain parenchyma. ACE specific activity, as assayed by generation of L-histidyl-L-leucine from the synthetic substrate hippuryl-L-histidyl-L-leucine, was enriched approximately 30 times in microvessels compared to homogenates of intact cerebral cortical gray matter. The nonapeptide 9a, SQ20,881), the orally active anti-hypertensive drug, 2-D-methyl-3-mercaptopropanoyl-L-proline (SQ14,225), and the vasoactive peptides bradykinin and angiotensin II inhibited this activity in a dose-dependent fashion. Brain microvessel ACE required chloride for optimal activity, was potentiated by cobalt nitrate, and was inhibited by the chelating agents EDTA and o-phenanthroline. Enzymatic generation of histidyl-leucine also was observed with the naturally occurring decapeptide substrate angiotensin I. In addition, microvessels obtained from bovine cerebellar cortex, hippocampus and corpus striatum, as well as from the cerebral cortex of Sprague-Dawley rats, were enriched in ACE activity. The presence of angiotensin-converting enzyme in brain microvessels suggests that cellular components of the blood-brain barrier may participate in the metabolism of peptide hormones such as angiotensin I and bradykinin within the central nervous system.     

Nutritional Prevention on Hypertension, Cerebral Hemodynamics and Thrombosis in Stroke-Prone Spontaneously Hypertensive Rats

1. Stroke-prone spontaneously hypertensive rats (SHRSP/Izm), which become severely hypertensive and exhibit a very high incidence of stroke (cerebral hemorrhage and/or infarction), are used widely for the study of the hypertension and stroke. In the previous study, we indicated that high thrombotic tendency of cerebral microvessels in SHRSP/Izm compared with stroke-resistant SHR (SHR/Izm) and normotensive Wistar Kyoto rats (WKY/Izm) at aged period. 2. L-arginine, a substrate of nitric oxide (NO), and voluntary exercise reduced blood pressure and thrombotic tendency in cerebral microvessels caused by highly production of NO in vivo. Furthermore, antioxidants show that the effects of antihypertensive and antithrombosis in SHRSP/Izm. 3. Although SHRSP/Izm become genetically hypertensive and exhibit stroke, a number of nutritional factors, particularly antioxydative nutrient, have preventive effects on hypertension, cerebral blood flow dysfunction, thrombus formation, and neuronal cell death in SHRSP/Izm. Our results indicate that those treatments are beneficial in the prevention of hypertension and stroke and that the nutritional science is very important for "prediction and prevention medicine."     

Angiotensin-converting enzyme (kinase II) in pituitary gland of spontaneously hypertensive rats

Angiotensin-converting enzyme (ACE) (kinase II; dipeptidyl carboxypeptidase, EC 3.4.15.1) activity was measured in pituitary gland of young (4-week-old) and adult (18-week-old) male, spontaneously hypertensive rats (SHR) and in age-matched normotensive male Wistar-Kyoto (WKY) control rats. In the three lobes of the pituitary gland ACE activity was significantly higher in young than in adult animals, in both SH and WKY rats. In the anterior lobe, ACE activity was lower in SHR when compared to age-matched Wistar-Kyoto controls. In contrast, ACE activity in the intermediate lobe of the pituitary gland was higher in SHR, and in particular in young animals. The observed differences between young WKY and SH rats in both the intermediate and anterior lobes did not appear to be due to a modified affinity of ACE for the substrate hippuryl-His-Leu, but to alterations in ACE maximal velocity or number of available molecules. No differences in ACE activity were detected between SHR and WKY rats in the posterior lobe. Total protein content was higher in the intermediate lobe and lower in the posterior lobe of young SHR when compared to normotensive controls. The present results suggest the possibility for a role of pituitary ACE in spontaneous (genetic) hypertension in rats.     

Plasma angiotensin-converting enzyme activity and blood pressure during the first year of life in normotensive and spontaneously hypertensive rat.

Plasma angiotensin-converting enzyme (ACE) activity and systolic blood pressure were studied every consecutive month during the first years of life in male spontaneously hypertensive (SHR) and in normotensive rats (NWR). During the first month after birth neither ACE activity nor systolic blood pressure showed significant difference between SHR and NWR. ACE activity in SHR was significantly reduced from 2nd till 12th month of age in comparison with age-matched NWR. In the 2nd month of age the systolic blood pressure was significantly higher in SHR than in NWR, it increased further until the 5th month and was maintained at this high level till the 12th month. No correlation between changes in the systolic blood pressure and the ACE activity was found in SHR after the 2nd month of age. During the investigation period no age-related dynamics of ACE activity was observed in SHR. The observed difference of ACE activity was not due to an increase of plasma his-leu hydrolyzing activity in SHR and was not abolished after a 24-hour dialysis of plasma. This difference could not be caused by the altered effect of chloride ion on the enzyme since similar pattern of Cl-dependent activation of plasma ACE in 4-month-old SHR and NWR was observed. Lineweaver-Burke plot analysis revealed that this difference appears to be due to a decrease of the enzyme maximal velocity in SHR but to a change of the Km value of ACE for the substrate hippuryl-1-his-1-leu. Our data provide evidence for a lower concentration of the available active enzyme molecules in SHR plasma in respect to NWR after the 1st month of life. Whether the reduced ACE activity in SHR is a consequence of the increased blood pressure remains to be elucidated.     

Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats

Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.     

Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel, Mytilus edulis

The angiotensin I converting enzyme (ACE) inhibitory activity of fermented blue mussel sauce (FBMS) was investigated. Blue mussels were fermented with 25% NaCl (w/w) at 20 degrees C for 6 months and the resultant mixture was passed through a 40-mesh sieve, desalted using an electrodialyzer and then lyophilized. The IC(50) value of FBMS for ACE activity was 1.01 mg/ml. An ACE inhibitory peptide was purified from FBMS using Sephadex G-75 gel chromatography, SP-Sephadex C-25 ion exchange chromatography and reversed-phase high-performance liquid chromatography on a C(18) column. The IC(50) value of purified ACE inhibitory peptide was 19.34 microg/ml, and 10 amino acid residues of the N-terminal sequence was EVMAGNLYPG. The purified peptide was evaluated for antihypertensive effect in spontaneously hypertensive rats (SHR) following oral administration. Blood pressure significantly decreased after peptide ingestion. This result suggested that FBMS may have beneficial effects on hypertension.     

Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks

Oligopeptides of 1 KDa or less were obtained by hydrolysis of chicken egg yolks with a crude enzyme, and by dialysis with a semipermeable membrane filter. Since the extracted peptides had an inhibitory action on the activity of angiotensin I-converting enzyme (ACE) in vitro, they were orally administered at 20, 100 and 500 mg/kg body weight to spontaneously hypertensive rats (SHR) for 12 weeks to analyze the physiological role on cardiovascular functions. The administered oligopeptides suppressed the development of hypertension at all dosages. After 12 weeks at 500 mg/kg body weight, the values for systolic, mean, and diastolic blood pressure were approximately 10% less in SHRs administered than controls. Furthermore, serum ACE activity of the peptide-administered groups was significantly lower than that of the control group in a dose-related manner. Our results imply that oligopeptides extracted from hen's egg yolks could potentially suppress the development of hypertension in SHR, and this effect might be induced by the inhibition of ACE activity.     

Chronobiologic Evaluation of Angiotensin-Converting Enzyme Activity in Serum and Lung Tissue from Normotensive and Spontaneously Hypertensive Rats

Angiotensin-converting enzyme (ACE) activity in serum and lung tissue from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was determined at six different circadian times. In WKY rats serum ACE varied significantly within 24 h, mainly due to reduced enzyme activity at 12:00 h. In SHR the 24-h profile of serum ACE did not exhibit time-dependent differences. Mean serum ACE activity over 24 h was significantly higher in WKY than in SHR. In lung tissue ACE activity did not depend on the circadian time in either strain. Mean enzyme activity in lung tissue was not different between WKY and SHR. We conclude that circadian changes in the activity of serum and tissue ACE are unlikely to play an important role in the regulation of the circadian blood pressure profile in both normotensive and spontaneously hypertensive rats.     

Chronobiologic Evaluation of Angiotensin-Converting Enzyme Activity in Serum and Lung Tissue from Normotensive and Spontaneously Hypertensive Rats

Angiotensin-converting enzyme (ACE) activity in serum and lung tissue from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) was determined at six different circadian times. In WKY rats serum ACE varied significantly within 24 h, mainly due to reduced enzyme activity at 12:00 h. In SHR the 24-h profile of serum ACE did not exhibit time-dependent differences. Mean serum ACE activity over 24 h was significantly higher in WKY than in SHR. In lung tissue ACE activity did not depend on the circadian time in either strain. Mean enzyme activity in lung tissue was not different between WKY and SHR. We conclude that circadian changes in the activity of serum and tissue ACE are unlikely to play an important role in the regulation of the circadian blood pressure profile in both normotensive and spontaneously hypertensive rats.     

Pathways of bradykinin degradation in blood and plasma of normotensive and hypertensive rats

Kinins are vasoactive peptide hormones that can confer protection against the development of hypertension. Because their efficacy is greatly influenced by the rate of enzymatic degradation, the activities of various kininases in plasma and blood of spontaneously hypertensive rats (SHR) were compared with those in normotensive Wistar-Kyoto rats (WKY) to identify pathogenic alterations. Either plasma or whole blood was incubated with bradykinin (10 microM). Bradykinin and kinin metabolites were measured by high-performance liquid chromatography. Kininase activities were determined by cumulative inhibition of angiotensin I-converting enzyme (ACE), carboxypeptidase N (CPN), and aminopeptidase P (APP), using selective inhibitors. Plasma of WKY rats degraded bradykinin at a rate of 13.3 +/- 0.94 micromol x min(-1) x l(-1). The enzymes ACE, APP, and CPN represented 92% of this kininase activity, with relative contributions of 52, 25, and 16%, respectively. Inclusion of blood cells at physiological concentrations did not extend the activities of these plasma kininases further. No differences of kinin degradation were found between WKY and SHR. The identical conditions of kinin degradation in WKY and SHR suggest no pathogenic role of kininases in the SHR model of genetic hypertension.     

Antihypertensive effect of angiotensin I-converting enzyme inhibitory peptides from a sesame protein hydrolysate in spontaneously hypertensive rats

Sesame peptide powder (SPP) exhibited angiotensin I-converting enzyme (ACE) inhibitory activity, and significantly and temporarily decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) by a single administration (1 and 10 mg/kg). Six peptide ACE inhibitors were isolated and identified from SPP. The representative peptides, Leu-Val-Tyr, Leu-Gln-Pro and Leu-Lys-Tyr, could competitively inhibit ACE activity at respective Ki values of 0.92 microM, 0.50 microM, and 0.48 microM. A reconstituted sesame peptide mixture of Leu-Ser-Ala, Leu-Gln-Pro, Leu-Lys-Tyr, Ile-Val-Tyr, Val-Ile-Tyr, Leu-Val-Tyr, and Met-Leu-Pro-Ala-Tyr according to their content ratio in SPP showed a strong antihypertensive effect on SHR at doses of 3.63 and 36.3 microg/kg, which accounted for more than 70% of the corresponding dosage for the SPP-induced hypotensive effect. Repeated oral administration of SPP also lowered both SBP and the aortic ACE activity in SHR. These results demonstrate that SPP would be a beneficial ingredient for preventing and providing therapy against hypertension and its related diseases.     

Effect of acute oral administration of captopril and MK-421 on vascular angiotensin converting enzyme activity in the spontaneously hypertensive rat

Vascular angiotensin converting enzyme (ACE) may be important as a potential site of action for the antihypertensive effect of ACE inhibitors. ACE activity, estimated by hydrolysis of [³H] HipGlyGly, was similar in the aorta, mesenteric and carotid arteries of SHR. ACE activity in veins was not as consistent and was significantly lower in the jugular veins and vena cava of SHR than in the arteries. Nevertheless, ACE activity in all the blood vessels examined, although less than lung ACE activity, was higher than ACE activity found in other tissues such as brain, heart and kidney. Equieffective antihypertensive doses of captopril (10 mg/kg p.o.) and MK-421 (1.0 mg/kg p.o.) dramatically inhibited ACE activity in all the arteries and veins examined. Maximal ACE inhibition occured within 15 minutes after the oral administration of captopril. In contrast, maximal ACE inhibition was slower in onset and of longer duration after MK-421 than after captopril for all the vessels. Thus, relatively high ACE activity can be measured in both arteries and veins from the spontaneously hypertensive rat (SHR) and ACE was dramatically inhibited after antihypertensive oral doses of captopril or MK-421. Furthermore, vascular ACE inhibition can be used to compare the onset and duration of activity of ACE inhibitors; MK-421 has a longer onset and duration in SHR than captopril based on inhibition of ACE in blood vessels.     

Antihypertensive Effect of Angiotensin I-Converting Enzyme Inhibitory Peptides from a Sesame Protein Hydrolysate in Spontaneously Hypertensive Rats

Sesame peptide powder (SPP) exhibited angiotensin I-converting enzyme (ACE) inhibitory activity, and significantly and temporarily decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) by a single administration (1 and 10 mg/kg). Six peptide ACE inhibitors were isolated and identified from SPP. The representative peptides, Leu-Val-Tyr, Leu-Gln-Pro and Leu-Lys-Tyr, could competitively inhibit ACE activity at respective Ki values of 0.92 μM, 0.50 μM, and 0.48 μM. A reconstituted sesame peptide mixture of Leu-Ser-Ala, Leu-Gln-Pro, Leu-Lys-Tyr, Ile-Val-Tyr, Val-Ile-Tyr, Leu-Val-Tyr, and Met-Leu-Pro-Ala-Tyr according to their content ratio in SPP showed a strong antihypertensive effect on SHR at doses of 3.63 and 36.3 μg/kg, which accounted for more than 70% of the corresponding dosage for the SPP-induced hypotensive effect. Repeated oral administration of SPP also lowered both SBP and the aortic ACE activity in SHR. These results demonstrate that SPP would be a beneficial ingredient for preventing and providing therapy against hypertension and its related diseases.     

Renal Effects and Underlying Molecular Mechanisms of Long-Term Salt Content Diets in Spontaneously Hypertensive Rats

Several evidences have shown that salt excess is an important determinant of cardiovascular and renal derangement in hypertension. The present study aimed to investigate the renal effects of chronic high or low salt intake in the context of hypertension and to elucidate the molecular mechanisms underlying such effects. To this end, newly weaned male SHR were fed with diets only differing in NaCl content: normal salt (NS: 0.3%), low salt (LS: 0.03%), and high salt diet (HS: 3%) until 7 months of age. Analysis of renal function, morphology, and evaluation of the expression of the main molecular components involved in the renal handling of albumin, including podocyte slit-diaphragm proteins and proximal tubule endocytic receptors were performed. The relationship between diets and the balance of the renal angiotensin-converting enzyme (ACE) and ACE2 enzymes was also examined. HS produced glomerular hypertrophy and decreased ACE2 and nephrin expressions, loss of morphological integrity of the podocyte processes, and increased proteinuria, characterized by loss of albumin and high molecular weight proteins. Conversely, severe hypertension was attenuated and renal dysfunction was prevented by LS since proteinuria was much lower than in the NS SHRs. This was associated with a decrease in kidney ACE/ACE2 protein and activity ratio and increased cubilin renal expression. Taken together, these results suggest that LS attenuates hypertension progression in SHRs and preserves renal function. The mechanisms partially explaining these findings include modulation of the intrarenal ACE/ACE2 balance and the increased cubilin expression. Importantly, HS worsens hypertensive kidney injury and decreases the expression nephrin, a key component of the slit diaphragm.     

Antihypertensive effect of spent brewer yeast peptide

Numerous studies have investigated dietary approaches to prevent chronic lifestyle-related diseases, including hypertension. Spent brewer’s yeast is the second largest byproduct originated by the brewing industry and it deserves considerable attention because of its high nutritional value, ca. 40% of its dry mass is rich in protein which can be hydrolyzed into biologically active peptides. To upgrade this byproduct, the aim of this study was initially in vitro assessment of biological properties, e.g. ACE inhibition and antioxidant activity, and then, the in vivo effect in short-term oral antihypertensive effect of hydrolyzed yeast fractions on a well characterized model to study hypertension - Spontaneously Hypertensive Rats (SHR). Here, it was demonstrated that the fraction with molecular weight below 3 kDa containing tri and tetra- peptides with hydrophobic amino acid residues – SPQW, PWW and RYW, causes the most noticeable decrease in systolic, diastolic and mean blood pressure of SHR and shows highest antioxidant effect. These properties highlight the potential use of yeast extract as nutraceutical or functional food ingredient for the management and treatment of hypertension with antioxidant effect.     

Reduced adenosine deaminase activity in the CNS of spontaneously-hypertensive rats

The activity of adenosine deaminase (ADA) has been measured in the hypothalamus, pons medulla and cerebral cortex from 30-day-old and 100-day-old spontaneously-hypertensive rats (SHR) and age-matched WKY controls. At 100 days there was a significant reduction in ADA activity in the hypothalamus (18.0%), pons medulla (20.6%) and cerebral cortex (14.7%). In 30-day-old SHR animals (prior to the development of significant hypertension) no significant changes were seen in the cerebral cortex or pons medulla but there was a small but significant reduction in ADA activity in the hypothalamus (9.2%). There was no significant reduction in the ADA activity in heart or kidney. Extracts of 100-day-old pons medulla which had been briefly heated to destroy endogenous ADA activity did not differentially affect the activity of exogenous purified ADA.     

Pressor responsiveness to angiotensin in soy-fed spontaneously hypertensive rats

Dietary soy may attenuate the development of arterial hypertension. In addition, some soy-containing foods exhibit angiotensin-converting enzyme (ACE) inhibitory properties. Accordingly, we tested the hypothesis that ACE inhibition contributes to the antihypertensive effect of dietary soy. Mean arterial blood pressure (MAP) was recorded from conscious spontaneously hypertensive rats (SHR) at least 24 h after the implantation of catheters. Cumulative dose-response curves to intravenous angiotensin I (AI) (5-100 ng x kg(-1) x min(-1)) and angiotensin II (AII) (1-20 ng x kg(-1) x min(-1)) were constructed for male, sham-operated female, and ovariectomized female (OVX) SHR that were maintained on either casein or soy diets. The soy diet was associated with a significant reduction in baseline MAP in the OVX SHR (approximately 20 mmHg, 1 mmHg = 133.322 Pa). AI and AII infusions caused graded increases in MAP in all groups. However, there was no significant attenuation of the pressor responses to AI in the soy-fed SHR. Conversely, we observed a significant rightward displacement of the AII dose-response curves in the soy-fed sham-operated and OVX SHR. We conclude that ACE inhibition does not account for the antihypertensive effect of dietary soy in mature SHR.     

Angiotensins in plasma of hypertensive rats and human

The plasma levels of des-aspartate-angiotensin I (DAA-I) in three models of hypertensive rats and hypertensive subjects were determined and compared with their normotensive controls. The rationale for the study was based on our earlier findings showing that DAA-I is a physiological angiotensin peptide that is involved in the pathophysiology of the cardiovascular system. The determination was carried out by the technique of capillary electrophoresis. Plasma level of angiotensin I, angiotensin II, and angiotensin III was also determined as a measurement of the status of the renin-angiotensin system in the different models of hypertension. DAA-I was found to be significantly lower in the spontaneously hypertensive rats (SHR) (46.6 +/- 2.5 pmol/l compared to 66.1 +/- 3.4 pmol/l for the normotensive control Wistar Kyoto rats), renal hypertensive rats (54.2 +/- 5.1 pmol/l compared to 72 +/- 2.5 pmol/l for the normotensive control Sprague-Dawley rats), and essential human hypertensive subjects (15.2 +/- 0.9 pmol/l compared to 19.5 +/- 2.5 pmol/l for the normotensive adult), whilst plasma concentration of angiotensin I and angiotensin II is reflective of the state of the renin-angiotensin system in the particular model of hypertension. When the SHR and human hypertensive subjects were treated with an angiotensin converting enzyme (ACE) inhibitor, the plasma level of DAA-I increased significantly. These findings suggest that the low plasma level of DAA-I could be a characteristic defect of the renin-angiotensin system in the two genetic models of hypertension (SHR and human essential hypertensive subjects). The increase of the nonapeptide following ACE inhibitor treatment could be an important hitherto unrecorded contributory factor to the effectiveness of ACE inhibitors in combating heart pathology.     

Serotonin metabolism in thrombocytes and microvascular hemostasis in spontaneous arterial hypertension

Serotonin content and accumulation in platelets and its release from them, as well as changes in thrombus formation in mesenteric arterioles and venules of the small intestine have been investigated in control rats and rats with spontaneous hypertension (SHR). Serotonin accumulation in platelets was determined upon its incubation with platelets. Disodium ADP salt was used as an inductor of release. Laser-induced thrombosis was caused by microvessels exposure to impulse laser irradiation. The control animals revealed a significant difference between the initial serotonin platelet level and serotonin level upon incubation and release; in values, the values of basic thrombus-forming parameters were higher than in arterioles. In SHR there is a decrease in biogenic amine content in platelets, a depression in its accumulation and release, an increase in the time of thrombus growth, its size up to the separation of the first embolus and its length along the vascular wall. It is concluded that spontaneous hypertension is characterized by decreased functional activity of platelets and depressed resistance of arterioles and venules to thrombus formation.