Yasuhiro Teranishi

Cardiac Angiotensin II Type 2 Receptor Activates the Kinin/NO System and Inhibits Fibrosis

Abstract—We have previously demonstrated that stimulation of the angiotensin (Ang) II type 2 receptor in vascular smooth muscle cells caused bradykinin production by activating kininogenase in transgenic mice. The aim of this study was to determine whether overexpression of AT2 receptors in cardiomyocytes attenuates Ang II–induced cardiomyocyte hypertrophy or interstitial fibrosis through a kinin/nitric oxide (NO)-dependent mechanism in mice. Ang II (1.4 mg/kg per day) or vehicle was subcutaneously infused into transgenic mice and wild-type mice for 14 days. The amount of cardiac AT2 receptor relative to AT1 receptor in transgenic mice was 22% to 37%. Ang II caused similar elevations in systolic blood pressure (by ≈45 mm Hg) in transgenic mice and wild-type mice. Myocyte hypertrophy assessed by an increase in myocyte cross-sectional area, left ventricular mass, and atrial natriuretic peptide mRNA levels were similar in transgenic and wild-type mice. Ang II induced prominent perivascular fibrosis of the intramuscular coronary arteries, the extent of which was significantly less in transgenic mice than in wild-type mice. Inhibition of perivascular fibrosis in transgenic mice was abolished by cotreatment with HOE140, a bradykinin B2 receptor antagonist, or L-NAME, an inhibitor of NO synthase. Cardiac kininogenase activity was markedly increased (≈2.6-fold, P <0.001) after Ang II infusion in transgenic mice but not in wild-type mice. Immunohistochemistry indicated that both bradykinin B2 receptors and endothelial NO synthase were expressed in the vascular endothelium, whereas only B2 receptors were present in fibroblasts. These results suggest that stimulation of AT2 receptors present in cardiomyocytes attenuates perivascular fibrosis by a kinin/NO-dependent mechanism. However, the effect on the development of cardiomyocyte hypertrophy was not detected in this experimental setting.

Cardioprotective Role of AT2 Receptor in Postinfarction Left Ventricular Remodeling

Abstract—The aim of this study was to determine the role of the AT2 receptor (AT2R) in left ventricular (LV) remodeling after myocardial infarction (MI). The left anterior descending arteries were ligated in AT2R gene knockout (Agtr2-) and wild-type (Agtr2+) mice. The LV remodeling was evaluated by echocardiography and histology over a period of 2 weeks after MI. The infarct sizes in hearts excised from Agtr2+ and Agtr2- mice on day 1 were similar. The mortality rate of Agtr2- mice (62.9%) on day 14 after MI was significantly (P <0.05) higher than that of Agtr2+ mice (39.7%). Accordingly, LV/body weight ratios (3.7±0.2 versus 3.0±0.1 on day 14) and LV end-diastolic (4.8±0.3 versus 3.9±0.4 mm on day 7) and end-systolic (4.4±0.3 versus 3.2±0.6 mm on day 7) dimensions evaluated by echocardiography were significantly greater in Agtr2- than in Agtr2+ mice. The rates of ventricular arrhythmia, rates of cardiac rupture, and blood pressures in the 2 strains were similar after MI. Myocyte cross-sectional areas were increased after MI, but the magnitudes were similar in Agtr2+ and Agtr2- mice, indicating the greater increases in LV dimensions and weight in Agtr2- mice are due to elongation of myocyte length and/or an increase in the interstitial weight (including vasculatures, infiltrated cells, and interstitial fluid). Interstitial fibrosis in remote myocardium was not evident in either strain. These results indicate AT2R plays a significant role in the protection against early development of LV dilation, thereby reducing the early mortality rate after MI.

Apoptosis Is Not Increased in Myocardium Overexpressing Type 2 Angiotensin II Receptor in Transgenic Mice

Abstract—To determine whether angiotensin type 2 (AT2) receptor stimulation induces apoptosis in cardiomyocytes in vivo, we developed transgenic mice overexpressing the AT2 receptor in a cardiac-specific manner, using the &agr;-myosin heavy-chain promoter. Ten- to 12-week-old male homozygous transgenic mice (n=44) and wild-type mice (n=44) were used. Both transgenic and wild-type mice were given either saline (control), a subpressor dose of angiotensin II (100 ng · kg−1 · min−1), a pressor dose of angiotensin II (1000 ng · kg−1 · min−1) for 14 days, a pressor dose of angiotensin II for 28 days to investigate the effects of stimulation on both angiotensin type 1 (AT1) and AT2 receptors, the AT1 antagonist L158809 alone, or a combination of angiotensin II (1000 ng · kg−1 · min−1) and L158809 for 14 days to investigate the effects of selective AT2 receptor stimulation. Apoptosis was analyzed in paraffin-embedded ventricular sections by the terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) technique. In both transgenic and wild-type mice, administration of a subpressor dose of angiotensin II, L158809, or a combination of angiotensin II and L158809 did not significantly affect the tail-cuff blood pressure or heart-to-body weight ratio, whereas administration of a pressor dose of angiotensin II for 14 or 28 days significantly increased blood pressure and the heart-to-body weight ratio. However, there was no statistical difference between the effects of angiotensin II in transgenic and wild-type mice. The number of TUNEL-positive nuclei was ≈0 to 10 per 100 000 cardiomyocytes, with no difference between transgenic and wild-type mice, regardless of saline infusion or any stimulation. In infarcted canine myocardial tissue sections for positive control, the number of TUNEL-positive nuclei was increased by 13.8 to 19.1 times compared with those in the noninfarcted myocardium. In conclusion, angiotensin II infusion for a period of 28 days failed to induce cardiomyocyte apoptosis regardless of the presence or absence of cardiac AT2 receptor overexpression. It is unlikely that in mice the AT2 receptor is a strong signal to induce cardiomyocyte apoptosis in vivo.

Disruption of the type 2 dopamine receptor gene causes a sodium-dependent increase in blood pressure in mice☆

BACKGROUND Dopamine D(2) receptors (D(2)Rs) are expressed in the kidney. It has not been determined whether D(2)Rs are involved in the mechanism of sodium handling and blood pressure (BP) control. METHODS The function of D(2)Rs was investigated in mice disrupted with D(2)R gene (D(2)KO mice). Six-week-old male D(2)KO mice and wild-type (WT) mice were fed high-salt (4% NaCl) or low-salt (0.01% NaCl) diets for 8 weeks. RESULTS Before starting the metabolic diet, there were no significant differences in body weight, food consumption, and 24-h urine excretions of creatinine, sodium and potassium. The high-salt diet caused a significant elevation in systolic BP in D(2)KO mice but not in WT mice. Calculation of sodium and potassium balances revealed a significantly high level of sodium retention in D(2)KO mice placed on the high-salt diet. Twenty-four-hour urine norepinephrine excretions and heart rates, indicators of sympathetic activity, were not different in D(2)KO and WT mice on the high-salt diet. Administration of nemonapride, a specific D(2)-like receptor antagonist, to WT mice given 0.9% NaCl in drinking water caused suppression of urinary sodium excretion but had no effect in mice without salt loading. CONCLUSIONS These results suggest that D(2) receptors promote sodium excretion during a period of high salt intake. A defect in this mechanism may result in sodium-dependent BP elevation.

Enhanced expression of mitochondrial genes in senescent endothelial cells and fibroblasts

It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.

Effect of BCL-2 down-regulation on cellular life span

Reactive oxygen species (ROS) are toxic for cells. BCL-2 is known as the anti-death protein and acts as an antioxidant. When theBCL-2 level of normal fibroblasts was suppressed by antisense bcl-2oligodeoxynucleotide or antisense bcl-2 RNA expression, the life span of the culture was shortened by about 11 population doublings(approx. 15% of the total life span) in comparison to the control culture. Since about twice as many cell deaths were observed in the antisense culture than in the vector culture, the life span shortening was probably caused by ROS-induced death. Acceleration of telomere shortening was not evident in the antisense culture. Other BCL-2 family proteins showed no significant change in expression. Cell death was suppressed by N-acetyl-L-cysteine, an antioxidant, suggesting that ROS were the major cause of cell death. In conclusion, reduction of BCL-2 makes cells more sensitive to death induced by ROS and leads to shortening of the cultures lifespan.

Life span shortening of normal fibroblasts by overexpression of BCL-2: a result of potent increase in cell death

It is well known that BCL-2 protects against cell death by both apoptosis and necrosis. The culture of bcl-2-transfected normal fibroblasts showed a shorter life span by about 12 population doubling levels compared to that of vector transfectants (64 vs 76 population doubling levels, respectively). An MTT assay revealed that BCL-2-overexpressing cells (HCA2/bcl-2) showed more severe growth suppression due to hydrogen peroxide or doxorubicin treatment than vector control cells (HCA2/vector). We observed a significant number of dead cells in the HCA2/bcl-2 culture, but not in the HCA2/vector culture. Other BCL-2 family proteins with both antiapoptotic and proapoptotic activity and other apoptosis-related factors were maintained at similar levels, indicating that overexpression of BCL-2 is the major reason that normal fibroblasts are sensitized to cell death. A broad caspase inhibitor (z-Val-Ala-Asp-fmk) and inhibitors of specific caspases (acetyl-Asp-Glu-Val-Asp-CHO, acetyl-Ile-Glu-Thr-Asp-CHO, and acetyl-Leu-Glu-His-Asp-CHO) suppressed cell death of HCA2/bcl-2 effectively, suggesting involvement of caspase 3-, 8-, and 9-dependent pathways in cell death and that the form of death is apoptosis. Unexpectedly, involvement of active MEK in cell death was shown by the use of its inhibitor, suggesting that crosstalk between BCL-2 and the MAP kinase cascade regulates death as well as life span.

Compensatory vasoconstrictor effects of sodium pentobarbital on the hindquarters of conscious normotensive control and lumbar-sympathectomized Wistar rats

The aim of this study was to compare the vasoconstrictor effect of sodium pentobarbital on the hindquarter resistance of intact control Wistar rats with the effect on lumbar-sympathectomized rats. For this purpose, mean arterial pressure (MAP) and hindquarter (supplied terminal aorta) flow (HQF) were simultaneously measured in these conscious rats with an arterial in dwelling cannula and electromagnetic flow probe implanted around the terminal aorta. Hindquarter resistance (HQR) was calculated as MAP divided by HQF. In the intact control conscious rats, subsequent pentobarbital anesthesia (30 mg/kg, i.v.) caused an increase in HQR (+43.5 +/- 7.4%, mean +/- S.E.M.) and a decrease in MAP (-17.0 +/- 3.2%). After pentobarbital anesthesia, subsequent ganglionic blockade with hexamethonium bromide (C6; 25 mg, i.v.) induced a significant decrease in HQR (-30.9 +/- 3.0%) with a further lowering of MAP (-20.9 +/- 1.6%). However, in rats not anesthetized with sodium pentobarbital, C6 alone induced almost no change in HQR (-3.4 +/- 5.3%), even when MAP was lowered (-24.2 +/- 2.5%). In the lumbar-sympathectomized rats, pentobarbital anesthesia produced almost no change in HQR (-11.7 +/- 4.4%), although MAP decreased significantly (-24.3 +/- 2.2%). These findings suggest that: (1) sodium pentobarbital anesthesia newly generates a compensatory vasoconstrictor tone in the hindquarters acting against the depressor effect, and (2) the vasocompensator tone is controlled by the efferent fibers, including those in the lumbar sympathetic nerves.

Antagonistic effects of antimuscarinic drugs on α1-adrenoceptors

Abstract. We previously observed that noradrenaline (NA)-induced contraction of the portal vein of rabbit was relaxed by the antimuscarinic drugs of atropine sulfate, but not scopolamine hydrobromide. In the present study we examined the possible effect of the antimuscarinic drugs of atropine sulfate, scopolamine hydrobromide, p-fluoro-hexa-hydro-sila-difenidol (p-F-HHSiD, the M3-receptor antagonist) and pirenzepine (the M1-receptor antagonist) on α1-adrenoceptor (AR).Atropine and p-F-HHSiD relaxed the α1-AR agonist methoxamine-induced contraction of the rabbit portal vein in a concentration-dependent manner; however, scopolamine and pirenzepine had no such inhibitory effect. Radioligand binding studies with the α1-AR ligand 2-[2-(4-hydroxy-3-[125I]iodo-phenyl)ethylaminomethyl]-α-tetralone ([125I]HEAT) in membrane preparations from mouse whole brain showed that atropine (pKi=5.33) and p-F-HHSiD (pKi=5.88) had higher affinities than scopolamine (pKi=3.17) and pirenzepine (pKi<2.70). Furthermore, atropine and p-F-HHSiD had higher affinities for all human cloned α1-ARs than scopolamine and pirenzepine. The results show that the antimuscarinic drugs atropine and p-F-HHSiD have a direct but weak antagonistic activity against α1-ARs.

Vasodilating effect of benidipine hydrochloride in the renal and hindquarter vascular regions (supplied by terminal aorta) of spontaneously hypertensive rats

One of two Ca antagonists, benidipine (3-30 microg/kg) or nifedipine (30-600 g/kg), was administered in a bolus injection through the jugular vein, and the changes in mean arterial pressure (MAP), renal flow (RF), and hindquarter flow (HQF) in conscious spontaneously hypertensive rats (SHRs) and normotensive control rats (NCRs). Renal vascular resistance (RR) and hindquarter resistance (HQR) were calculated as MAP divided by RF and HQF, respectively. When a high dose was administered to decrease the blood pressure by about 20%, the RR was significantly lower with benidipine than with nifedipine. The decrease in HQR was not significantly different between benidipine and nifedipine. When a low dose was administered to decrease the blood pressure by about 7%, the decrease in RR was not significantly different between benidipine and nifedipine, but the HQR was significantly lower with benidipine than with nifedipine. In the NCRs, no pharmacological properties were significantly different between these two Ca antagonists.