Akio Kinoshita

Cellular localization and regional distribution of an angiotensin II-forming chymase in the heart.

The human heart is a target organ for the octapeptide hormone, angiotensin II (Ang II). Recent studies suggest that the human heart contains a dual pathway of Ang II formation in which the major Ang II-forming enzymes are angiotensin I-converting enzyme (ACE) and chymase. Human heart chymase has recently been purified and its cDNA and gene cloned. This cardiac serine proteinase is the most efficient and specific Ang II-forming enzyme described. To obtain insights into the cardiac sites of chymase-dependent Ang II formation, we examined the cellular localization and regional distribution of chymase in the human heart. Electron microscope immunocytochemistry using an anti-human chymase antibody showed the presence of chymase-like immunoreactivity in the cardiac interstitium and in cytosolic granules of mast cells, endothelial cells, and some mesenchymal interstitial cells. In the cardiac interstitium, chymase-like immunoreactivity is associated with the extracellular matrix. In situ hybridization studies further indicated that chymase mRNA is expressed in endothelial cells and in interstitial cells, including mast cells. Tissue chymase levels were determined by activity assays and by Western blot analyses. Chymase levels were approximately twofold higher in ventricles than in atria. There were no significant differences in chymase levels in ventricular tissues obtained from non-failing donor hearts, failing ischemic hearts, or hearts from patients with ischemic cardiomyopathy. These findings suggest that a major site of chymase-dependent Ang II formation in the heart is the interstitium and that cardiac mast cells, mesenchymal interstitial cells, and endothelial cells are the cellular sites of synthesis and storage of chymase. In the human heart, because ACE levels are highest in the atria and chymase levels are highest in ventricles, it is likely that the relative contribution of ACE and chymase to cardiac Ang II formation varies with the cardiac chamber. Such differences may lead to differential suppression of cardiac Ang II levels during chronic ACE inhibitor therapy in patients with congestive heart failure.

Increased Chymase-Dependent Angiotensin II Formation in Human Atherosclerotic Aorta

Locally formed angiotensin II (Ang II) and mast cells may participate in the development of atherosclerosis. Chymase, which originates from mast cells, is the major Ang II-forming enzyme in the human heart and aorta in vitro. The aim of the present study was to investigate aortic Ang II-forming activity (AIIFA) and the histochemical localization of each Ang II-forming enzyme in the atheromatous human aorta. Specimens of normal (n=9), atherosclerotic (n=8), and aneurysmal (n=6) human aortas were obtained at autopsy or cardiovascular surgery from 23 subjects (16 men, 7 women). The total, angiotensin-converting enzyme (ACE)-dependent, and chymase-dependent AIIFAs in aortic specimens were determined. The histologic and cellular localization of chymase and ACE were determined by immunocytochemistry. Total AIIFA was significantly higher in atherosclerotic and aneurysmal lesions than in normal aortas. Most of AIIFA in the human aorta in vitro was chymase-dependent in both normal (82%) and atherosclerotic aortas (90%). Immunocytochemical staining of the corresponding aortic sections with antichymase, antitryptase or anti-ACE antibodies showed that chymase-positive mast cells were located in the tunica adventitia of normal and atheromatous aortas, whereas ACE-positive cells were localized in endothelial cells of normal aorta and in macrophages of atheromatous neointima. The density of chymase- and tryptase-positive mast cells in the atherosclerotic lesions was slightly but not significantly higher than that in the normal aortas, and the number of activated mast cells in the aneurysmal lesions (18%) was significantly higher than in atherosclerotic (5%) and normal (1%) aortas. Our results suggest that local Ang II formation is increased in atherosclerotic lesions and that chymase is primarily responsible for this increase. The histologic localization and potential roles of chymase in the development of atherosclerotic lesions appear to be different from those of ACE.

Differences in Tissue Angiotensin II–Forming Pathways by Species and Organs In Vitro

Angiotensin (Ang) II plays an important role in cardiovascular homeostasis, not only in the systemic circulation but also at the tissue level, and is involved in the remodeling of the heart and vasculature under pathological conditions. Although alternative Ang II-forming pathways are known to exist in various tissues, the details of such pathways remain unclear. The aim of this study was to examine tissue Ang II-forming activities and to identify the responsible enzyme in several organs (lung, heart, and aorta) in various species (human, hamster, rat, rabbit, dog, pig, and marmoset). Among the organs examined, the lung contained the highest Ang II-forming activity. The responsible enzyme for pulmonary Ang II formation was angiotensin I-converting enzyme (ACE) in all of the species except the human lung, in which a chymaselike enzyme was dominant. In the heart, the highest total Ang II-forming activity was observed in humans, and a chymaselike enzyme was dominant in all of the species except rabbit and pig. Aorta exhibited a relatively high total Ang II-forming activity, with a predominance of chymaselike activity in all of the species except rabbit and pig, in which ACE was dominant. Our results indicate that there were remarkable differences in Ang II-forming pathways among the species and organs we examined. To study the pathophysiological roles of ACE-independent Ang II formation, one should choose species and/or organs that have Ang II-forming pathways similar to those in humans.

Effect of acute exercise on plasma immunoreactive — atrial natriuretic factor☆

The effect of acute exercise on plasma immunoreactive-atrial natriuretic factor (IR-ANF) was studied in 5 healthy young males subjected to graded exercise on a bicycle ergometer at four different work intensities (approx. 30, 50, 70, and 90% VO2max respectively). Except for the final exercise period, which was continued until exhaustion, all the others were of 30 min duration. Venous blood samples were obtained in the last 2 min of each exercise period. The plasma IR-ANF response to exercise was similar to that of heart rate and systolic blood pressure in that mild exercise in the first stage (corresponding to approx. 30% VO2 max) caused a striking increase of plasma IR-ANF concentrations with a further augmentation in the next stages and a levelling off at exhaustion. Plasma arginine-vasopressin (AVP) and aldosterone (ALDO) rose significantly only in the last two stages, and the highest concentrations were observed at exhaustion. In conclusion, acute exercise stimulates ANF secretion in proportion to the intensity of exercise, while concomitant increases in plasma AVP and ALDO occur only when the work load exceeds the blood lactate (BLA) threshold.

Effects of angiotensin II generated by an angiotensin converting enzyme-independent pathway on left ventricular performance in the conscious baboon.

Human chymase is a serine proteinase that converts angiotensin (Ang) I to Ang II independent of angiotensin converting enzyme (ACE) in vitro. The effects of chymase on systemic hemodynamics and left ventricular function in vivo were studied in nine conscious baboons instrumented with a LV micromanometer and LV minor axis and wall thickness sonomicrometer crystal pairs. Measurements were made at baseline and after [Pro11DAla12] Ang I, a specific substrate for human chymase, was given in consecutive fashion as a 0.1 mg bolus, an hour-long intravenous infusion of 5 mg, a 3 mg bolus, and after 5 mg of an Ang II receptor antagonist. [Pro11DAla12]Ang I significantly increased LV systolic and diastolic pressure, LV end-diastolic and end systolic dimensions and the time constant of LV relaxation and significantly decreased LV fractional shortening and wall thickening. Administration of a specific Ang II receptor antagonist reversed all the hemodynamic changes. In separate studies, similar results were obtained in six of the baboons with ACE blockade (20 mg, intravenous captopril). Post-mortem studies indicated that chymase-like activity was widely distributed in multiple tissues. Thus, in primates, Ang I is converted into Ang II by an enzyme with chymase-like activity. This study provides the first in vivo evidence of an ACE-independent pathway for Ang II production.

Genetic analysis of the epithelial sodium channel in Liddle's syndrome.

Background Liddles syndrome is an autosomal inheritable disorder that causes hypertension due to excess function of sodium channel. Objective To analyze the DNA sequence of the amiloride-sensitive epithelial sodium channel (ENaC) in three patients who had low-renin hypertension with hypokalemia. The patients included a 24-year-old woman and her 20-year-old brother whose mother was hypertensive. The third patient was a 15-year-old girl with no family history of hypertension. Methods The DNA sequence of the ENaC was analyzed as follows. Venous blood samples were collected from the patients and total genomic DNA was prepared by standard methods. Specific primers were used for direct polymerase chain reaction; one set of primers for amplifying the C terminus (codon 523–638) of the b subunit of ENaC, and two sets of primers for amplifying the C terminus (codons 525–587 and 568–650) of the γ subunit of ENaC. Polymerase chain reaction products were purified and subjected to direct DNA sequence analysis. Results Direct sequence analysis demonstrated the presence of a single-base substitution in one segment of the b subunit of ENaC, a C→T transition that changed the encoded Pro (CCC) at codon 616 to Ser (TCC) in the siblings (cases 1 and 2). In case 3, we found a missense mutation of Pro (CCC) to Leu (CTC) at codon 616. Case 3 is considered to be sporadic, since DNA sequencing of the PY motif of her parents gave normal results. Conclusions The DNA sequences of the ENaC in three patients with Liddles syndrome were analyzed. In one family case, we found a new missense mutation of Pro (CCC) to Ser (TCC) at codon 616 in the β subunit of ENaC. A genetic analysis of the amiloride-sensitive epithelial sodium channel is recommended in assessing patients with low-renin, salt-sensitive hypertension whose blood pressure is not responsive to spironolactone treatment.

What types of hypertensives respond better to mild exercise therapy

Twenty-one patients with essential hypertension were treated with aerobic (lactate threshold) exercise (n = 21) and the change in blood pressure was compared with that in a non-exercising hypertensive control group (n = 10). After 10 weeks of exercise therapy blood pressure in the exercise group was significantly reduced, from 154 ± 3/100 ± 2 to 141 ± 3/93 ± 3 mmHg, and there was a significant reduction in plasma volume, cardiac index and plasma noradrenaline whereas there were no changes in the non-exercise group. Responders (10 mmHg or more fall in mean blood pressure) in the exercise group had a significantly higher cardiac index, serum Na: K ratio and lower total peripheral resistance in pretraining studies than non-responders. In conclusion, exercise at lactate threshold is effective in lowering blood pressure in those mild hypertensives with higher cardiac index and serum Na: K ratio.

Mild Exercise Therapy Increases Serum High Density Lipoprotein2 Cholesterol Levels in Patients with Essential Hypertension

In ten patients with essential hypertension who were on a prescribed regimen of supervised mild exercise, we examined serum lipids, HDL cholesterol subfractions (HDL2 and HDL3), and apolipoproteins. The findings were compared with data on age- and sex-matched hypertensives not on this regimen of exercise. Blood samples were obtained from the auricle during the multistage exercise test, and changes in levels of lactate were measured. The exercise was done for 30 minutes three times weekly for 10 weeks. A significant reduction of both systolic and diastolic blood pressure was evident at 10 weeks. Serum concentrations of HDL2 cholesterol increased significantly at 10 weeks, but there were no changes in total and HDL3 cholesterol. No significant changes were observed in serum concentrations of total cholesterol, triglyceride, and apolipoproteins (apo) A-I, apo A-II, apo B, apo C-II, apo C-III and apo E following 10 weeks of exercise therapy. In the hypertensive controls who were not on exercise therapy, blood pressure as well as all parameters related to lipoproteins remained unchanged. Thus, mild exercise lowers blood pressure and improves the lipoprotein profile.

Mild exercise activates renal dopamine system in mild hypertensives

Objective: The role of renal dopamine in the early depressor effect of exercise was evaluated in hypertensives. Methods: After a general clinical observation period of 4 weeks, 29 essential hypertensives were divided into two groups. The exercise group (n = 16) underwent blood lactate threshold exercise using a cycle ergometer for 60 min three times a week for 4 weeks. Results: In the non-exercise group (n = 13), blood pressure (BP) and humoral variables did not change significantly (from 150 ± 3/93 ± 2 to 145 ± 2/94 ± 1 mm Hg). In the exercise group (n = 16), resting BP was significantly reduced from 158 ± 2/92 ± 2 at week 0 to 145 ± 3/85 ± 3 mm Hg at week 4. The increase in urinary free dopamine excretion (from 248 ± 14 to 276 ± 24 ng/mg Cr) at week 4 was significantly higher than that in the non-exercise group (from 220 ± 31 to 196 ± 27 ng/mg Cr). In the exercise group, urinary kallikrein activity also increased significantly from 173.0 ± 35.4 at week 0 to 320.3 ± 63.3 ng bradykinin/min/mg Cr at week 4. These changes in urinary free dopamine excretion and urinary kallikrein activity were negatively correlated with the change in BP. The change in urinary sodium excretion was also negatively correlated with the change in plasma volume index. Moreover, the change in urinary free dopamine excretion was positively correlated with the changes in urinary kallikrein activity and urinary sodium excretion. The change in renal decarboxylation rate of DOPA (3,4-dihydroxyphenylalanine) positively correlated with the changes in urinary free dopamine excretion and urinary sodium excretion, and was negatively correlated with the change in systolic BP. Conclusion: These results suggest that exercise triggered renal dopamine generation and activation of renal kallikrein-kinin system, resulting in natriuresis and BP reduction in the early phase (4 weeks) of mild exercise.

Urinary kallikrein activity is increased during the first few weeks of exercise training in essential hypertension

Objective To determine whether the renal kallikrein–kinin and dopamine systems participate in lowering blood pressure during mild exercise in hypertensives. Design After a general clinical observation period of 4 weeks, 27 essential hypertensives were divided into two groups. The exercise group underwent blood lactate threshold exercise, using a cycle ergometer for 60 min three times a week for 10 weeks. The non-exercise group was observed at the outpatient clinic. Blood pressure and humoral parameters were measured at weeks 0, 1, 2, 4 and 10 in both groups. Methods Blood pressure was measured indirectly with an automatic blood pressure recorder. Twenty-four-hour urinary kallikrein activity (by kininogenase assay), total or free dopamine and total noradrenaline (by high-performance liquid chromatography) were also measured. Results In the non-exercise group blood pressure and humoral parameters did not change. In the exercise group the change in resting blood pressure between weeks 0 and 10 was statistically significant. The change in 24-h urinary kallikrein activity of the exercise group was significantly greater than that of the non-exercise group between weeks 0 and 1 and weeks 0 and 2. Moreover, the change in systolic blood pressure (SBP) between weeks 0 and 2 was negatively correlated with the change in urinary kallikrein activity between weeks 0 and 2, the change in total dopamine between weeks 0 and 2 was negatively correlated with the change in diastolic blood pressure in the same period, and the change in SBP between weeks 0 and 10 was positively correlated with the change in total noradrenaline in the same period in the exercise group. Subjects with a relatively high baseline urinary kallikrein activity had a significantly greater change in SBP between weeks 0 and 10 than subjects with a relatively low baseline activity. Conclusions The renal kallikrein—kinin and dopamine systems may participate in lowering blood pressure during the first few weeks of exercise training. The subsequent reduction of sympathetic activity may be involved in maintaining the lowered blood pressure. Mild exercise is more effective in reducing blood pressure in hypertensives who have a relatively high basal renal kallikrein-kinin system activity.