Abstracts

Abstract

Oral PresentationsEffect of Renin-Angiotensin-Aldosterone System Suppression During Lactation on Salt Sensitivity in SHRSP. L. Otani,1 T. Matsui, 2 and T. Murakami.1,3 1Division of Advanced Life Sciences, Graduate School of Agriculture; 2Division of Bioscience and Bioenvironmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka, Japan; 3Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University, Nara, Japan.Several studies have indicated that the intrauterine or lactation environments affect not only fetal growth, but also the onset of hypertension and cardiovascular disease in adult life. Treatment of SHR during very early life with agents targeting the renin-angiotensin-aldosterone system leads to persistent reductions in blood pressure. In the present research, we started antihypertensive therapy during the lactation period and studied the effect of SHRSP on blood pressure and salt sensitivity.Experiment 1: using male SHRSP at 2 weeks of age, the rats were divided into a control group and an experimental group (AT1 receptor antagonist; losartan) and were fed a purification diet (AIN-76). As drinking water, the control group was given tap water, and the experimental group was given water containing losartan (100 mg/L). The treatment with losartan was discontinued at 4 weeks of age. After being fed the commercial diet (Funahashi SP diet) from weaning, both groups of rats were divided into a tap water group and 1% saline solution group, each consisting of 10 rats at 10 weeks of age. Blood pressure was measured by the tail-cuff method. The blood samples were collected from the tail vein at 4 and 15 weeks of age. Plasma aldosterone was determined by an EIA kit.Experiment 2: male SHRSP at 2 weeks of age were given either water or water containing Val-Tyr (VY; 400 mg/L), which is derived from sardine muscle hydrolysate and which has shown ACE inhibition activity in vitro. The treatment was stopped at 4 weeks of age, when both groups of rats were fed a commercial diet and tap water ad libitum. The drinking water was switched to 1% saline after the rats reached 10 weeks of age.The body weights of SHRSP that ingested losartan during the lactation period were similar to those of the control rats at the end of the treatment. However, at 10 weeks of age, the body weights of losartan-treated rats were lower than those of the control rats. There were no significant differences in blood pressure between the control rats and the losartan-treated rats. After the salt-loading from 10 weeks of age, the blood pressure elevation in the losartan-treated rats was gentle as compared with that of the control rats (p< 0.05). After 4 weeks of salt-loading, the control rats showed weakness, and their body weights decreased markedly. The losartan-treated rats, however, did not exhibit these changes. The life span of the losartan-treated rats (104 ± 78 days) was significantly longer than that of the control rats (37 ± 17 days). The body weight and blood pressure were not affected by the VY treatment from 2 to 4 weeks of age. On the other hand, the life span of the VY-treated rats (43 ± 13 days) tended to be extended in comparison with the control rats (29 ± 13 days).We have demonstrated that suppression of the renin-angiotensin-aldosterone system between 2 and 4 weeks of age blunted salt sensitivity. Thus, we suggest that the lactational period is a crucial stage of the development of salt sensitivity in SHRSP. Moreoever, a similar result was recognized in VY, which was derived from sardine muscle hydrolysate. We showed that nutrition in the lactational stage plays an important role in the prevention of stroke and vascular disease.Effect of Aldosterone Treatment on Stroke Incidence in Young or Mature SHRSP. T. Abe,1 L. Otani,1 Y. Hamada,2 and T. Murakami.1,3 1Division of Advanced Life Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan; 2Department of Ophthalmology, Saiseikai Tondabayashi Hospital, Osaka, Japan; 3Departrment of Food Science and Nutrition, Faculty of Agriculture, Kinki University, Nara, Japan.The renin angiotensin system (RAS) in the early stage of life may play a key role in the establishment of elevated blood pressure and vascular injury in later life. It is well known that angiotensin II is a major factor in the vascular injury. Recently, it has been clarified that aldosterone contributes directly to the acceleration of the development of vascular injury and stroke in SHRSP. These findings have led us to hypothesize that aldosterone in the early stage plays a role in the development of stroke and end-organ damage in SHRSP. The present study was undertaken to investigate the effect of aldosterone treatment on stroke incidence in young or mature SHRSP.The SHRSP used were 8-week-old (180 g) and 12-week-old males (250 g). Rats were implanted subcutaneously with osmotic pumps (alzet) that infused 40 mg/kg per day aldosterone, and were fed a commercial diet (Funabashi SP) for 4 weeks. Sham-infused rats served as controls. The animals had free access to diet and drinking water (tap water). The systolic blood pressure (tail-cuff method) and body weight were measured once a week. The blood was sampled under ether anesthesia from the abdomen aorta. Creatinine concentration and thiobarbituric-acid reactive substances (TBARS) in serum were determined by Buege and Aust's methods. Urinary albumine excretion was measured using a commercially available kit.In rats treated with aldosterone from 8 weeks of age, blood pressure was significantly elevated after the aldosterone treatment. The body weight of the aldosterone group decreased markedly in the 2 weeks after the aldosterone treatment. Urinary albumine and the plasma creatinine of the aldosterone group were higher than those of the control group. The aldosterone group's TBARS values showed a marked increase but were not shown in the control group. The incidence of cerebral stroke lesion in the control group was not shown, while that in the aldosterone group was 100%. In rats treated with aldosterone from 12 weeks of age, however, body weight and blood pressure showed no significant difference between the aldosterone and control group. At 4 weeks after aldosterone administration, no significant difference was found in urinary albumine excretion, plasma creatinine concentration, and TBARS between the aldosterone group and the control group. The incidence of cerebral stroke lesion was 0% in both the control and aldosterone groups.These results have been interpreted to imply that the aldosterone triggers or sets in motion a mechanism that determines cerebral stroke and end-organ damage. In SHRSP, the early stage of life is a critical window for cerebral stroke induced by aldosterone. However, in the mature stage, rats did not appear to be vulnerable to stroke induced by aldosterone treatment.Effects of Angiotensin II on the Activation and Nitric Oxide Production in Leukocyte. K. Takemori,1 H. Ishida,1 K. Yamamoto,2 and H. Ito.1 1Department of Pathology, and 2Division of Basic Medical Science, Kinki University School of Medicine, Osaka, Japan.We have already reported that nitric oxide (NO)-related radical derived from leukocytes may play an important role in cerebral microvascular injury in SHRSP. However, the mechanisms of leukocyte activation and subsequent NO production in severe hypertension has not yet been clarified. In the present study, we investigated the role of angiotensin II (AII) in activation and enhanced NO production in leukocytes, because it is well known that AII is the most important humoral factor in hypertension.Ten-week-old male SHRSP were continuously infused with AII (0.75 g/kg/hr) using an osmotic mini pump for 1 week. Leukocytes were isolated from these rats and the expression of Mac-1(adhesion molecule of leukocyte) was examined by flow cytometric analysis. The AT1 receptor and inducible nitric oxide synthase (iNOS) expression were examined by RT-PCR. Furthermore, the plasma NO metabolites level was investigated by HPLC. Leukocyte-rich fractions were obtained from 20-week-old WKY and SHRSP. Cells (105 cell/ml) were incubated in PBS-containing DAF-2 DA (5 M), and fluorescence in cells was observed by confocal microscope. In addition, NO production in the culture medium was measured using DAF-2 (0.5 mM) after either stimulation by AII (10 M) or lipopolysaccharide (LPS, 100 ng/ml) or inhibition by AT1 receptor blocker (CV11974, 10 M) or iNOS inhibitor (s-methylisothiourea, 10 M). The fluorescence intensity of each medium was measured by plate reader.The Mac-1 expression in leukocytes was higher in SHRSP than in the WKY. In the WKY, the expression of Mac-1 was upregulated by the AII treatment, whereas no significant difference was found in the SHRSP. Both in the WKY and SHRSP, the AT1 receptor expression was upregulated by the AII stimulation. In the SHRSP treated with AII, the iNOS expression was remarkably higher than in the control, and plasma NO metabolite levels were high among the experimental groups. In the fluorescence image of the leukocyte-rich fractions, DAF-2 DA positive cells (NO-generating cells) were closely coincident to antigranulocyte antibody positive cells. The fluorescence intensity was much greater in the SHRSP than in the WKY. In culture medium of the leukocytes stimulated by AII or LPS, the fluorescence intensity was higher than in the control. However, increased fluorescence in culture medium of SHRSP leukocytes was suppressed by AT1 receptor blocker and iNOS inhibitor, being at similar levels in the control.These results clearly indicate that NO production in leukocytes is enhanced in severe hypertension and this may be induced by AII via AT1 receptors in leukocytes.Beneficial Effects of Voluntary Long-Term Exercise on Blood Pressure and Vascular Inflammatory Parameters in Stroke-Prone SHR. A. Niwa, K. Ooshima, M. Tabuchi, and H. Higashino. Department of Pharmacology, Kinki University School of Medicine, Osaka-Sayama, Japan.Epidemiological studies clearly have documented that regular physical exercise reduced the risk of attack in patients with established coronary heart disease. However, the mechanisms remain poorly understood. This study planned to test how a long-term and moderate physical exercise affected on the vascular dysfunction caused in aged SHRSP.Male SHRSP 6-weeks-old at prehypertensive stage were divided into 3 groups: treadmill exercise (TE), voluntary wheel-running (WR), and sedentary control (SED). TE rats ran on a treadmill 1 h/day at a speed of 10 m/min and a 10° angle. WR rats were allowed to run voluntarily in a wheel with a 100 cm around running way. The only rats running 2 to 3 km/day were selected as WR rats. SED rats were placed in the cage without running. At the end of experimental period after 8 weeks, rats were sacrificed under pentobarbital anesthesia and thoracic aortas were quickly removed and frozen in liquid N2 for the analyses of endothelial nitric oxide synthase (eNOS) expression, eNOS activity, and oxidative stress. Plasma and/or urine were collected before the sacrifice for the measurements by ELISA of sICAM-1, MCP-1, and 8-iso-PGF2α.Expression of Akt, eNOS, and their phosphorylated ones were determined by Western blotting, and NADPH oxidase mRNA was measured by RT-PCR. Activities of eNOS were determined by measuring [3H]1-citrulline production from [3H]1-arginine. Superoxide (O2−) production was measured by flow cytometer using dihydroethidium in blood samples collected every 4 weeks. Another experiment to observe the occurrence of apoplexy and death was performed similarly with the experiment above by keeping those to death.WR rats significantly decreased the level of systolic blood pressure after 5 weeks of exercise compared with SED or TE rats and reduced the incidences of stroke and mortality. In aortas of WR rats, phosphorylated Akt and eNOS protein levels, eNOS activity, and basal cGMP level were significantly elevated compared with those of SED by 30%, 14%, 106%, and 73%, respectively. On the other hand, TE significantly suppressed the phosphorylation of eNOS on Ser1177 (0.76/1 in TE/SED: arbitrary units). Nitrotyrosine contents of aortas were significantly decreased in WE compared with SED or TE. The level of 8-iso-PGF2α in plasma and urine were greater in TE than in SED. Level of mRNA of nox 1, a NADPH oxidase subunit, was lower in the aortas of WR than those of SED or TE, but other two components, p47phox and p67phox, were not different among the three groups.At the time-course study, the degree of O2− production was estimated in peripheral monocytes and neutrophils. Both of the numbers and O2− productions were significantly decreased in the peripheral blood cells of WR compared with SED. WR rats showed significantly lower serum levels of MCP-1 than those in SED or TE. Serum levels of sICAM-1 were not different among the three groups.From our results, the following conclusions were obtained. Voluntary long-term and moderate exercise has beneficial effects on vascular function, possibly via increased NO bioavailability. At the cellular level, this beneficial training effect is closely related to Akt-dependent increases in eNOS contents, the phosphorylation or Ser1177, and a decrease in oxidative stress. Therefore, the exercise training can be effective intervention to mitigate the inflammation associated with hypertension in the vessels and ischemic changes in the brain even in the genetic hypertensive animals with stroke risk.Enhanced Nitric Oxide Production in Aortas from SHR/NDmcr-cp Rats' An Animal Model of the Metabolic Syndrome. S. Kagota, M. Kunitomo, and K. Shinozuka. Department of Pharmacology, School of Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan.Metabolic syndrome, a cluster of metabolic abnormalities including hypertension, hyperlipidemia, obesity, insulin resistance, and diabetes, can lead to atherosclerotic vascular diseases. The vascular endothelium regulates the tonus of underlying smooth muscle cells by releasing vasorelaxing factors such as nitric oxide (NO). Impairment of NO production or bioactivity is known to promote the development of atherosclerosis. The obese spontaneous hypertensive SHR/NDmcr-cp (cp/cp) rat (SHR-cp) is a strain with obesity superimposed on a background of SHR. SHR-cp rats display simultaneous onset of obesity, hypertension, hyperlipidemia, hyperglycemia, and hyperinsulinemia. Using this animal model, we investigated changes in the vasorelaxation of aortas of SHR-cp in comparison with their lean littermates SHR/NDmcr-cp ( + / + ) rats (SHR).Male SHR-cp and SHR, established at the Disease Model Cooperative Research Association, were purchased from Japan SLC, Inc. at 8 and 18 weeks of age (n = 6, respectively). The thoracic aortas were removed, and aortic rings were prepared. The rings were fixed vertically under a resting tension of 1.0 g in a 10-ml organ bath filled with Krebs-Henseleit solution. Each preparation was preconstricted with phenylepherine (0.1–0.3 µM), and then acetylcholine (ACh) or sodium nitroprusside (SNP) was cumulatively added to the bath medium. The isometric tension change was measured with a force-displacement transducer coupled to a dual channel chart recorder. The protein levels of endothelial NO synthase (eNOS) and soluble guanylate cyclase (sGC) in the aortas were determined by Western blot analysis.Furthermore, the rings were preconstricted with phenylepherine (0.1 µM) for 5 min and then stimulated with ACh (0.03 µM) or SNP (0.01 µM) for 1 min or 3 min, respectively, and then the cyclic GMP content in each preparation was determined with an enzyme immunoassay kit. The plasma levels of lipid peroxide and NO2 plus NO3 were determined using the fluorometric method and the Griess method, respectively.SHR-cp displayed the onset of obesity, hypertension, hypercholesterolemia, hypertriglycemia, and hyperglycemia from 8 to 18 weeks of age. At 8 weeks of age, the relaxations in response to ACh and SNP did not change significantly in aortic rings from SHR-cp and SHR. On the other hand, at 18 weeks of age, the relaxations in response to ACh slightly but significantly increased in SHR-cp compared with SHR, whereas there was no difference in SNP-induced relaxations. The protein levels of eNOS significantly increased in SHR-cp compared with SHR, but the protein levels of sGC did not change significantly in the two strains. The cyclic GMP levels induced by Ach were significantly higher in SHR-cp than SHR, whereas the cyclic GMP levels induced by SNP did not change significantly. The plasma NO2 plus NO3 levels showed a tendency to increase. The plasma thiobarbituric-acid reactive substances levels markedly increased in SHR-cp compared with SHR at 18 weeks of age.These results indicate that in aortas of SHR-cp, NO production from the endothelium seems to increase with aging. This effect probably results from a variety of factors, including increased oxidative stress in the context of the metabolic syndrome.Effect of Anthocyanin on Activation and Expression of Endothelial Nitric Oxide Synthase. J.-W. Xu,1,2 K. Ikeda,1,2 and Y. Yamori.3 1Frontier Health Science, School of Human Environmental Science; and 2Research Center for Life-Style Related Diseases, Mukogawa Women's University, Nishinomiya, Japan; 3International Center for Research on Primary Prevention of Cardiovascular Diseases, Kyoto, Japan.Dietary anthocyanins have an endothelium-dependent vasorelaxation effect. However, very few studies have examined their molecular basis. Here, we report that cyanidin-3-glucoside (Cy3G), a typical anthocyanin pigment, regulates expression and activation of the endothelial nitric oxide synthase (eNOS) in vascular endothelial cells.The Western blot analysis, immunoprecipitation, and transfection of Src and Akt cDNA into BAECs were performed; intracellular cyclic GMP activation and chromatin immunoprecipitation (ChIP) assay were performed using assay kits according to the manufacturers' instructions.Experiment 1: Cy3G induced eNOS protein expression in time- and concentration-dependent manner and stimulated phosphorylation of Src kinase and ERK1/2 MAP kinase in time-dependent manner. Cy3G also significantly increased nitric oxide output 2-fold. Moreover, Src kinase inhibitor pp2 and MEK inhibitor PD98059 blocked eNOS expression and phosphorylation of Src kinase, ERK1/2MAP kinase, and SP1 caused by Cy3G. The dominant-negative Src cDNA transfection blocked the Cy3G-caused eNOS expression in BAECs. Cy3G enhanced the binding activity of the transcription factor Sp1 to the GC box in the proximal eNOS promoter of BAECs, as revealed by chromatin immunoprecipitation (ChIP) assay.Experiment 2: Cy3G induced phosphorylation at Serine-1179 and at the same time dephosphorylation at Serine-116 of eNOS in BAECs. The peak periods of phosphorylation and dephosphorylation were at 15 min. Src kinase inhibitor pp2, PI3 kinase inhibitor wortmannin, and MEK inhibitor PD98059 restrained the phosphorylation of eNOS at Serine-1179, and calcineurin inhibitor cyclosporine A blocked the dephosphorylation of eNOS at Serine-116. The dominant-negative Akt cDNA transfection blocked the Cy3G-caused eNOS phosphorylation. Cy3G also caused formation of the eNOS/sGC complex, increased production of cGMP, and phosphorylation at Ser239 of the vasodilator-stimulated phosphoprotein, a sensitive monitor of defective nitric oxide/cGMP signaling.Our data showed that Cy3G induces eNOS expression via an Src-ERK1/2-Sp1 signaling pathway in vascular endothelial cells, enhances vascular eNOS activity, and may help to improve vascular endothelial function.Bone Marrow-Derived Endothelial Progenitor Cells Senescence in Spontaneously Hypertensive Rats. K. Kobayashi, T. Imanishi, C. Moriwaki, T. Hano, and I. Nishio.Recent studies have revealed an association between coronary risk factors and both number and function of bone marrow-derived endothelial progenitor cells (BM-EPCs). The aim of the present study is to investigate an effect of hypertension on BM-EPCs senescence. BM-EPCs were obtained from the tibias and femurs of age-matched (5- and 10-week-old) spontaneously hypertensive rat (SHR/Izm) and Wister-Kyoto rat (WKY/Izm). After ex vivo cultivation, we counted the number of BM-EPCs assessed by both DiLDL uptake and lectin binding. BM-EPCs senescence was detected by senescence-associated β-galactosidase staining (SA-β-Gal staining).Although the blood pressure (BP) in 5-week SHR/Izm was almost the same compared with 5-week WKY/Izm, BP in 10-week SHR/Izm was significantly higher than that of WKY/Izm (116 ± 17 mmHg and 156 ± 28 mmHg, respectively). There were no significant differences in the number of attached, differentiated BM-EPCs between SHR/Izm and WKY/Izm. However, the occurrence of senescence, assessed by SA-β-Gal staining, of BM-EPCs from 5- and 10-week SHR/Izm were significantly increased compared with that from WKY/Izm (p< 0.01). We concluded that BM-EPCs senescence is age-dependently and BP-independently accelerated in SHR/Izm.The Significance of Bradykinin B1 Receptor in the Hypertensive Cardiac Hypertrophy. H. Sasaki, N. Moniwa, J. Agata, N. Ura, K. Higashiura, Y. Miyazaki, Y. Ito, D. Yoshida, Y. Shinshi, and K. Shimamoto. Second Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.The kallikrein-kinin system plays important roles in blood pressure regulation, renal water-sodium metabolism, and organ protection. It is well known that angiotensin-converting enzyme inhibitor (ACEI) suppresses cardiac remodeling, and bradykinin B2 receptor antagonist partially abolishes this effect of ACEI, especially cardiac fibrosis. However, kinins elicit their effects via selective activation of two different receptors: the bradykinin B1 receptor (B1R) and bradykinin B2 receptor (B2R). Bradykinin binds to B2R, and its metabolite, Des-Arg9-BK, binds to B1R. B2R is continuously expressed in the cardiovascular system and mediates most of the vascular actions of bradykinin. B1R is not expressed or is expressed at low levels in tissues under normal conditions. Although B1R is generally believed to be induced in the several pathological conditions including hypertension, the role of B1R is not well known.The aim of this study is to elucidate the expression level of B1R in the hypertensive hypertrophic heart in stroke-prone spontaneously hypertensive rats (SHRSP), and to determine the functional role of cardiac B1R.WKY(n = 5) and SHRSP(n = 5) aged 16 weeks were used, and the cardiac B1R mRNA expression in each group was examined by reverse transcription-polymerase chain reaction (RT-PCR) and compared in both groups. Next, WKY and SHRSP and 12 weeks were employed and treated with B1R antagonist or vehicle for 4 weeks. They were acclimated to handling before randomization, then were divided into 3 groups: WKY group (n = 6), SHRSP group (n = 6), and SHRSP with antagonist group (n = 6). SHRSP with antagonist group were treated with 500 g/kg/day of Des-[Arg]9-[Leu]8-BK, B1 receptor antagonist using osmotic pump for 4 weeks. After 4 weeks, rats were anesthetized intraperitoneally with sodium pentobarbital (50 mg/kg), catheterized into right carotid artery, and blood pressure and heart rate were measured by direct method. We evaluated the phospholylation of various types of mitogen-activated protein kinase (MAPK) by Western immunoblotting, and quantitatively analyzed the cardiac fibrosis and the size of the myocardial cell by immunostaining.The B1R mRNA expression in heart was significantly higher in SHRSP than WKY. Blood pressure and left ventricular weight of SHRSP were significantly higher and were even higher by chronic infusion of B1R antagonist. The phospholylation of various types of MAPK were significantly increased in SHRSP with B1R antagonist compared with WKY and SHRSP. In the cardiac tissue, cardiac fibrosis and the size of the myocardial cell were increased in SHRSP more than in WKY and were more increased by chronic infusion of B1R antagonist in SHRSP. These results suggest that the B1R mRNA expression in heart was enhanced in SHRSP, a genetic severe hypertension model, and the B1R may suppress cardiac remodeling and hypertrophy through the inhibition of MAPK.Endothelin Antagonism Reverses Upregulated Renin-Angiotensin System in the Hypertrophied Heart of SHRSP Independently of Blood Pressure. S. Jesmin,1 H. Togashi,2 I. Sakuma,1 M. Yoshioka,2 and T. Miyauchi.3 1Department of Cardiovascular Medicine and 2Department Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 3Department of Cardiovascular Medicine, University Tsukuba School of Medicine Tsukuba, Japan.It is well recognized that one of the most potent stimuli for endothelin-1 (ET-1) release from cultured myocardial cells is angiotensin II (AII). ET-1 produced by cardiomyocytes is a mediator for AII-induced hypertrophy of cardiomyocytes via an autocrine/paracrine mechanism. Moreover, the renin-angiotensin system (RAS) contributes to activation of circulating endothelin in congestive heart failure. Thus, ET-1 is regulated by AII. But it is unknown whether endothelin (ET)-1 affects RAS in the heart. We hypothesized that ET-1 may act on the upstream of RAS in the hypertrophied heart, and ET antagonism may have an important role on RAS.In the present study, we investigated the effects of the ET blockade on the expression of the various components of RAS including angiotensin II (AII) in the heart of stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were treated for 12 weeks with SB209670 (ET-A/-B dual receptor antagonist) or saline (vehicle) starting from the early stage of hypertension (8 weeks old). Blood pressure was measured every month. Long-term treatment with SB209670 significantly decreased heart weight and left ventricular weight in SHRSP, while it did not alter blood pressure. The present study was carried out using immunoblotting, ELISA, immunohistochemistry, real-time PCR, in situ hybridization, and morphometric evaluation.When compared with age-matched control Wistar-Kyoto (WKY) rats, peptide level of AII was 15-fold higher in vehicle-treated SHRSP hearts. AII was measured in both plasma and left ventricular tissues by ELISA. Plasma AII level also was high in vehicle-treated SHRSP. Serum renin activity also was significantly higher in vehicle-treated SHRSP rats.Both the peptide and mRNA levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and AII type 1 receptor (AT1R) were significantly upregulated in the heart of vehicle-treated SHRSP. Left ventricular levels of angiotensin I were increased in vehicle-treated SHRSP. All these upregulated components of RAS were remarkably reversed by long-term ET blockade starting from the early stage of hypertension. AII type 2 receptor (AT2R) was about 40% downregulated in vehicle-treated SHRSP and was reversed by SB209670 treatment.Thus, AII receptors were differentially distributed in vehicle-treated SHRSP and these differential alterations were ameliorated by endothelin dual receptors antagonism. Moreover, the higher plasma activity of renin and ACE, and plasma level of AII in SHRSP were reversed by ET antagonism. The Chymase system, the alternate pathway for AII production, was 60% upregulated in vehicle-treated SHRSP and was reversed by ET antagonism. Significant cardiac hypertrophy and myocardial fibrosis at histological levels in SHRSP was ameliorated by ET antagonism, and left ventricular hypertrophy demonstrated by echocardiography also was suppressed by ET blockade. Moreover, isovolumetric relaxation time (IRT) corrected by R-R interval was altered in SHRSP hearts significantly and was ameliorated by ET antagonism. Left atrial diameter (LAD) was higher in vehicle-treated SHRSP and normalized by endothelin antagonism.It should be noted that a similar treatment protocol in spontaneously hypertensive rats could not inhibit cardiac hypertrophic changes.Thus, these findings indicate that ET antagonism suppressed the different components of upregulated RAS in the SHRSP hearts independently of blood pressure. Furthermore, we suggest that ET system may act on the upstream of RAS in the heart and that ET has some regulatory roles on cardiac RAS in SHRSP. It is interesting to note that ET-1 blockade also reversed upregulated plasma and left ventricular ET-1 levels in SHRSP. In addition, both the upregulated ET-1 receptors in SHRSP hearts were reversed by ET antagonism. Now we are trying to investigate whether the observations we have from this present study are specific to SHRSP strain or not. Moreover, specific ET-1 receptor blocker would be used in future studies to gain more insights regarding the effect of ET-1 on cardiac RAS.This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by Health Sciences Research grants for comprehensive research on aging and health from the Ministry of Health, Welfare, and Labor of Japan.The Order of Ligation and Development of Cataract in Unilateral Delayed-Bilateral Carotid Artery Ligation in SHR. T. Katsube,1 R. Horie,2 M. Okuma,3 A. Ohira,4 and T. Harada.2 1Eye Clinic, Hirata Municipal Hospital, Hirata, Japan; 2Department of Pathology; 3Okuma Eye Clinic, Kyoto, Japan; and 4Opthalmology, Faculty of Medicine, Shimane University, Izumo, Japan.We previously reported that ligation first on the left side in unilateral delayed-bilateral carotid artery ligation (UD-BCL) at a 1-week interval in SHR/Izm resulted in development of contralateral cataract in the right eye. In this study, we ligated the right carotid artery first in UD-BCL and evaluated the possible development of contralateral cataract.The animals used were 6 male and 6 female spontaneously hypertensive rats (SHR/Izm, B2 strain) aged 6 months that had been maintained at the 2nd Department of Pathology, Faculty of Medicine, Shimane University, with a strain preservation grant from the Ministry of Education, Culture, Sports, Science and Technology. After intraperitoneal pentobarbital anesthesia (30 mg/kg), double ligation of the right carotid artery with no. 3 silk was performed. At a 1-week interval, the left carotid artery was ligated. Body weight and blood pressure were serially measured before and after ligation, and the possible development of cataract was observed using a portable slit lamp (Kowa) after mydriatics instillation without anesthesia. Then 5 months after UD-BCL (11 months after birth), all the rats were sacrificed, and for pathological examinations autopsy mainly of the brain, heart, kidneys, and aortic arch was performed. The eyeballs were removed and fixed with 4% glutaraldehyde fixative, and the ciliary body was observed by electron microscopy.Of the 6 female rats, 1 developed cataract in the left eye after previous ligation of the right carotid artery. In this female rat with cataract, blood pressure was the highest among the 6 female rats both before (199 mmHg) and after ligation (186 mmHg).After ligation of the right carotid artery in UD-BCL, contralateral cataract developed in the left eye in this experiment. This contralateral development of cataract was consistent with the result of our prevous study, supporting our previously proposed hypothesis that ischemic changes initially occur in the ciliary tissue contralateral to the carotid artery ligated via ciliary arterioles branching from the ocular artery. In this model experimental system for acute cataract, it is possible that oxidation stress of the lens due to ischemia is an important factor for the development of cataract.In our prevous study, electron microscopy of the ciliary body showed more marked intracellular vacuolar degeneration on the right side than the left side after previous ligation of the left carotid artery. No development of cataract in the same experiment in WKY/Izm and significantly slighter expression of thioredoxin in SHR/Izm than in WKY/Izm on the vascular wall in response to oxidation stress have been reported (Yodoi and Horie 2002). SHR/Izm may be more sensitive to this ischemia in ciliary arterioles and develop cataract.Paradoxical Inhibitory Effects of Mild Stress on Atherogenesis in the Cerebrospinal Arterioles of Atherogenesis-Prone SHR. Y. Horie,1 K. Ishitobi,2 W. Saito,3 and R. Horie.4 1Totori University School of Medicine, Yonago, Japan; 2Department of Endocrinology; 3Department of Psychiatry and 4Neuropathology, Faculty of Medicine, Shimane University, Izumo, Japan.It has been reconfirmed that A1 and A3 strains of stroke-prone spontaneously hypertensive rats (SHRSP) (Okamoto et al. 1974) develop atherogenesis in the mesenteric arterioles. In addition when mild stress is present during the initial stage of atherogenesis, its paradoxical inhibitory effects on atherogenesis appear (Saito and Horie, 2003).In this study, we investigated attenuation of atherogenesis in the central nervous system by stress in rats. Animals were 30 male atherogenesis-prone SHR (SHRAP; Horie 1990). They were divided into three groups, stress group (group S) with (n = 10) or without (n = 10) uppercervical sympathectomy before restraint stress, and a control group as (group C, n = 10) receiving neither stress nor sympathectomy.They were all fed a high fat cholesterol (HFC) diet including 20% seut, 5% cholesterol, and 2% cholic acid (Funahashi Chiba, Japan) at the age of 2 months after birth for 2 weeks ad libitum. Water containing 1% NaCl was given ad libitum. In group S, restraint stress was given in the supine position 5 min/day, 5 days/week for 2 weeks.In Group C, animals were fasted without restraint during the restriction time established in group S. Half the animals in group S underwent bilateral uppercervical sympathectomy prior to restraint stress.During the experiment period, we measured blood pressure (BP), body weight (BW), food and water intakes, blood lipid levels (TG, Cho, HDL, LDL), 24-hr urine volume, and urinary catecholamine excretion (NA, AD; ECD method). After the end of the experiment, all the animals were sacrificed under anesthesia. Pathological investigation of the main organs was performed. The amount of fat deposition in the cerebral and spinal arterioles was semiquantitatively calculated using the BCSS method (Horie 1975).In the control group, atherogenesis developed in over 80% of the animals. In contrast, stressed animals showed significantly less atherogenesis, especially when they were sympathectomized as shown in the following table.ControlStressed without sympathectomyStress with sympathectomyFat deposition in the cerebral artery10/10 (100%)3/10 (30%)*0/10 (0%)*Fat deposition in the anterior spinal artery9/10 (90%)2/10 (20%)*2/10 (20%)*Number of sudanophilic rings in the arteries8.0 ± 0.55.9 ± 0.7*6.1 ± 0.3*Of control nervous system (M ± SE) blood pressure (mmHg, M ± SE)184 ± 5192 ± 5198 ± 7Gain in body weight (g, M ± SE)43 ± 645 ± 541 ± 7Serum cholesterol (mg/dL, M ± SE)150 ± 12147 ± 18153 ± 8*Significant difference from control (p< 0.05).These results suggest that mild stress attenuates atherogenesis through the endocrine and autonomic nervous systems.Cerebral Blood Flow Improvement and Antihypertensive Effect of Chinese Herbal Medicine on Stroke-Prone Spontaneously Hypertensive Rats. M. Gao,1 T. Murakami,2 J. Y. Han,3 X.P. Lin,3 K. Ikeda,2 Z. Yamamoto,2 and Y Yamori.2 1Department of Acupuncture Medicine, Oriental Medicine, Suzuka University of Medical Science, Suzuka, Japan; 2International Center for Research on Primary Prevention of Cardiovascular Diseases, Kyoto, Japan; 3Liaoning College of Traditional Chinese Medicine, Shenyang, China.The preventive effect of yoketuseinoukaryu (YS), a Chinese herbal medicine, on hypertension and stroke was evaluated using stroke-prone spontaneously hypertensive rats (SHRSP).Five-week-old animals were divided into 2 groups randomly: YS group (4% YS diet), and the control group (plain laboratory diet), 10 rats in each group. After oral administration of the two kinds of diets, blood pressure, body weight, incidence of stroke and survival rate were evaluated. On the 28th day of experiment, cerebral blood flow was measured in additional BYS group and B control group, 5 rats in each group, by laser Doppler perfusion imager (PIM II).Systolic blood pressures in YS group was significantly decreased, compared with that in the control group (on the 21st and 28th day, YS group versus control group p< 0.01). The mean cerebral blood flow value in BYS group was significantly higher than that in the B control group. The incidence of stroke in the YS group was decreased, whereas its survival rate was increased significantly by the 100th day of experiment.These results indicate that ingestion of Chinese herbal medicine YS attenuated the elevation of blood pressure and the development of cerebral ischemia, suggesting that YS treatments might be beneficial for the prevention of hypertension and the development of stroke.Expression and Regulation of a Novel Membrane-Bound Fatty Acid Receptor, GPR40, in a Genetically Obese-Diabetic Rat Model. T. Tomita,1 H. Masuzaki,1 H. Iwakura,1 J. Fujikura,1 T. Tanaka,1 M. Fujimoto,1 H. Chusho,1 K. Ebihara,1 J. Hiraoka-Yamamoto,2 K. Ikeda,2 T. Hayashi,1 K. Hosoda,1 Y. Yamori,3 S. Hinuma,4 and K. Nakao.1 1Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2Frontier Health Science Forum, Mukogawa Women's University, Hyogo, Japan; 3International Center for Research on Primary Prevention of Cardiovascular Diseases, Kyoto, Japan; 4Discovery Research Laboratories, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Ibaraki, Japan.GPR40, previously known as an orphan G-protein coupled receptor, was shown to be a novel membrane-bound fatty acid (FA) receptor. GPR40 is activated by medium- to long-chain FAs and augments glucose-stimulated insulin secretion (GSIS) from pancreatic β cells. However, little is known about the physiological and pathophysiological roles of GPR40. To explore the possible physiological and pathophysiological link between GPR40 and insulin secretion from pancreatic β cells, we measured GPR40 gene expression in isolated islets of a genetically diabetic-obese rat model, Koletsky rat (fak/fak). This model is a substrain of spontaneous hypersensitive rat (SHR) and manifests marked hyperlipidemia and hyperglycemia at 14 weeks of age. At this age, tissue triglyceride concentration in isolated islets from fak/fak was markedly increased as compared with the lean littermate (SHR).Here we show for the first time that mRNA level of GPR40 was substantially decreased in islets from 14-week-old male fak/fak as compared with SHR. A 24-hr culture of isolated islets from 8-week-old Wistar rats with long-chain FAs such as linoleic and oleic acids significantly decreased GPR40 mRNA level ( ~ 30–38% versus nontreated, p< 0.01), suggesting long-chain FAs play a possible role in the decrease in GPR40 mRNA.Of note, augmentation of GSIS by linoleic and oleic acids was markedly attenuated in islets from 14-week-old male fak/fak ( ~ 35% of + / + , p< 0.05). Our results suggest a possible link between decrease in GPR40 mRNA and β cell dysfunction in lipotoxicity, highlighting the potential importance of GPR40 in the mechanisms of fatty acid action on β cells.Metabolic Profiling of Spontaneously Hypertensive Rats by Nuclear Magnetic Resonance-Based Metabonomics. K. Akira, M. Imachi, and T. Hashimoto. School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.Spontaneously hypertensive rat (SHR) provide a simple and accessible model for studying essential hypertension that is a polygenic, heterogeneous, and multifactorial disease. SHR genetic and metabolic features are of great interest because they may provide insight into the pathophysiological processes underlying essential hypertension. Recently, many studies have been reported about genes responsible for hypertension of SHR, whereas metabolic changes of SHR have not been fully studied because of lack of suitable techniques. Recently, a metabonomic approach using 1H nuclear magnetic resonance (1H NMR) and chemometric techniques has been developed to study metabolic differences associated with gene function and pathophysiological and toxicological stimuli. Although metabonomics has been used successfully to study toxicity of various compounds, its application to understanding pathophysiological processes has been extremely limited. In this study, we have applied 1H NMR-based metabonomics to characterize the urinary metabolite profiles of SHR, stroke-prone SHR (SHRSP), and their normotensive control Wistar Kyoto rats (WKY) and to explore metabolic differences between the strains.Rats were individually placed in metabolism cages for urine collection and allowed free access to water, but no food was given during urine collection. Blood pressures were measured using a tail-cuff method. Urine samples were collected in the frozen state using dry ice, from 4 male SHR/Izm and WKY/Izm aged 22 and 29 weeks (experiment 1, 18-hr urine), and 5 or 6 male SHRSP/Izm and WKY/Izm aged 12 and 26 weeks (experiment 2, 24-hr urine). Urine samples (0.1–0.6 ml) were mixed with deuterium oxide for field-frequency lock so that the total volume amounts to 0.7 ~ 0.8 ml, and their 1H NMR spectra were acquired using 5-mm o.d.NMR tubes on a Bruker DRX500 spectrometer under the conditions of water suppression.The following treatments of NMR data and principle components analysis (PCA) were performed using Bruker AMIX software: The NMR spectra (δ 0.5–9.5) were automatically reduced to 225 integrated segments of equal width (0.04 ppm). The region between δ 4.5 and δ 6.5 was removed prior to PCA to remove the effects of water suppression and urea. Each spectral data set was normalized to the total sum of the integrals to compensate for variation in urine volumes. PCA was applied to the NMR data, and scores plots of the principle components (PCs) were constructed to visualize separation of the urine samples between the groups. The NMR spectral regions and metabolities that contribute to the separation were identified from the PC loadings that are coefficients of correlation of individual variable to the PC scores.The urine samples of SHR and SHRSP were clearly differentiated from those of WKY at any age in the score plots of PC1 and PC2. The separation between the samples from the hypertensive and normotensive strains were observed on the PC1 axis, the former having higher scores. The loadings plot showed that taurine (δ 3.22–3.26, 3.26–3.30, 3.38–3.42, 3.42–3.46), creatine(δ 3.90–3.94), and unidentified metabolites resonating at ≈ δ 2.46, 3.10, and 3.59 predominantly contributed to the separation in both experiments. The urinary levels of taurine and creatine were higher and those of the unknown components much lower in the hypertensive strains than in WKY. These differences were confirmed by visual inspection of the NMR spectra. Although the pathophysiological significance of these components remains to be elucidated, this study indicated that SHR and SHRSP can be distinguished from WKY in terms of urinary metabolic profiles, especially the above metabolic changes.Genetic Analysis of Metabolic Syndrome in SHR Strains. Takehiro Watanabe,1 H. Inomata,1 Y.-Q. Liang,1 T. Nabika,2 M. Mizunuma,1 L. Jin,1 M. Isobe,3 K. Yanai,1 and N. Kato.1 1Department of Gene Diagnosis and Therapeutics, Research Institute, International Medical Center of Japan, Tokyo, Japan; 2Shimane University, Shimane, Japan; 3Tokyo Medical and Dental University, Tokyo, Japan.Cardiovascular risk factors including hypertension tend to cluster in certain individuals and this is known as the metabolic syndrome, to which insulin resistance has been considered to play an important role. However, it remains unknown whether some “common” genetic impairments underlie the clustering of cardiovascular risk traits. To clarify the genetic basis of the metabolic syndrome, we systemically searched for quantitative trait loci (QTLs) affecting insulin resistance phenotypes in SHR and stroke prone SHR (SHRSP), which are widely used as unique model organisms of insulin resistance.First, we undertook genome-wide screens to investigate QTLs for metabolic traits in two F2 crosses involving SHR, SHRSP, and Wistar-Kyoto (WKY) rats. Second, to validate and narrow down the QTLs, we developed congenic rat strains derived from SHR and SHRSP, where principal QTLs for the metabolic traits were identified. Third, to explore genes differentially expressed between the progenitor strains, we performed expression studies on positional candidate genes by real-time PCR.Genome screens showed significant linkages to multiple metabolic traits on 6 rat chromosomes in SHR and 8 rat chromosomes in SHRSP, and congenic strains were constructed for these 14 rat chromosomes. Significant linkage appeared to be partially overlapped on rat chromosomes 1, 3, and 15 between two F2 crosses. Subsequent expression studies on several positional candidate genes of these chromosomal regions revealed significant changes between the progenitor strains. A panel of 14 congenic rat strains derived from SHR and SHRSP will be useful for studies on the clustering of cardiovascular risk factors.Further Analysis of the Blood Pressure QTL on Chromosome 1. T. Nabika,1 Z.-H. Cui,1 Y. Harada,1 N. Kato,2 and K. Ikeda.3 1School of Medicine, Shimane University, Shimane, Japan; 2International Medical Center of Japan, Tokyo, Japan; 3Mukogawa Women's University, Nishinomiya, Japan.We identified a potent QTL for blood pressure on the rat chromosome 1 through genetic studies on SHRSP. In this study, we constructed subcongenic strains for this region as well as performed genetic analysis on substrains of SHR to do further dissection of the QTL.A series of subcongenic strains were constructed through backcross of the original congenic strain (SHRSPwch1.0) to SHRSP. We established 9 subcongenic strains that have small genomic fragments of Wistar-Kyoto rats (WKY) on SHRSP background. Blood pressure was monitored at 12 weeks old with the tail-cuff method. In the analysis of substrains, we selected more than 80 simple sequence repeat (SSR) markers in the region and genotyped 4 substrains of SHRSP, 5 of SHR, and 1 WKY.Sequential cut-down of the QTL region by constructing subcongenic strains resulted in ups and downs of blood pressure, suggesting more than one gene regulating blood pressure in this region. Through genotyping of the 10 substrains, SSR markers of which 1 allele was shared with all substrains of SHR and SHRSP were found in narrow genome regions. As WKY did not have the same allele there, such regions were thought to be “promising” to harbor “hypertension genes.” Combined studies on subcongenic strains and substrains would provide an efficient way to narrow down the QTL region.Comparison of Arix Gene Structure and Expression Between SHRSP/Izm and WKY/Izm. Y. Harada,1 K. Nemoto,2 T. Mashimo,3 Z.-H. Cui,1 and T. Nabika.1 1Department of Functional Pathology, Shimane University Faculty of Medicine, Izumo, Japan; 2School of Pharmaceutical Science, University of Shizuoka, Japan; 3Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan.In the study of congenic rats targeting a quantitative trait locus (QTL) for blood pressure (BP) on chromosome (Chr) 1, we found enhanced BP response to several stresses. Further, the electrophysiological activities of neurons in the rostal ventrolateral medulla (RVLM) of the congenic rats were greater than those of Wistar-Kyoto rats (WKY). These observations indicated that genes regulating activities of the sympathetic neurons in the central nervous system might contribute to the pathogenesis of hypertension. Arix is a transcription factor regulating the differentiation of noradrenergic neurons as well as the expression of the tyrosine hydroxylase. Recently, this gene was reported to exist in the QTL region on rat Chr 1. To evaluate the role of the Arix gene in the genetic hypertension in rats, we studied the gene structure, the promoter activity, and the expression of the Arix gene in WKY and SHRSP.We determined the full length sequence of the Arix gene (8 kbp) including about 3 kbp of the promoter sequence on a genomic clone obtained through the screening of a cosmid library (the sequence datum was submitted to DDBJ under accession number AB186362). Sequences of the promoter, the three exons, and the exon-intron junctions were compared between WKY and SHRSP. The promoter activity was evaluated by the luciferase assay using constructs containing 3 kbp of promoter sequences from both strains of rats. The Arix gene expression in the kidney and the brain stem was analyzed by the real time (RT)-PCR.Several synonymous changes in exons were found between WKY and SHRSP. In spite of several nucleotide substitutions in the promoter region, the promoter activity was not significantly different between WKY and SHRSP. Consistent with these results, the Arix gene expression did not differ significantly between the two strains of rats. These results did not support a significant role of Arix in the pathogenesis of hypertension in SHRSP. Further analyses in utero may be necessary.Comprehensive Genome Searches of Quantitative Trait Loci for Blood Pressure and Associated Traits in the Spontaneous Hypertensive Rat Strains. H .Inomata, T. Watanabe, Y.-Q. Liang, M. Isobe, K. Yanai, and N. Kato. 1Department of Gene Diagnostics and Therapeutics, Research Institute, International Medical Center of Japan, Tokyo, Japan; 2Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan.The spontaneous hypertensive rat (SHR) and stroke-prone SHR (SHRSP) are considered as unique model organisms for hypertension and its cardiovascular complications. Genetic analysis of these strains is expected to elucidate pathophysiological mechanisms of the disorders. The identification of susceptibility gene for multifactorial diseases is not feasible even in animal models. In this study, we attempted to develop efficient strategies for producing congenic rat strains and positional cloning.First, we performed genome-wide searches of quantitative trait loci (QTLs) controlling blood pressure, serum total cholesterol and triglyceride levels, and fasting plasma glucose levels in two F2 intercrosses derived from SHR or SHRSP and Wistar-Kyoto rats (WKY). In 326 F2 (SHR × WKY) and 578 F2(SHRSP × WKY) rats, 6 and 8 significant QTLs were observed, respectively. Then, we produced congenic rat strains by using a speed congenic strategy for a total of 14 QTLs, where we had developed and applied a high-throughput typing marker-set for genome screens at each backcrossing stage. Thereby, we could produce congenic rat strains with four to five backcrossing, which took ≈ 2 years.We have succeeded in the rapid and parallel development of multiple congenic rat strains. These congenic strains seem to constitute valuable molecular tools for genetic analysis of cardiovascular risk factors and the findings should be useful in humans.Rat Phenome Project: The Untapped Potential of Existing Rat Strains. T. Mashimo, B. Voigt, K. Naoi, Y. Nakane, K.-I. Yamazaki, S. Nakanishi, T. Tsurumi, T. Kuramoto, and T. Serikawa. Institiute of Laboratory Animals, Kyoto University Graduate School of Medicine, Kyoto, Japan.The National Bio Resource Project for the Rat in Japan (NBRP-Rat) collects, preserves, and distributes rat strains. More than 250 inbred strains have been deposited thus far into the NBRP-Rat and are maintained as specific pathogen-free (SPF) rats or cryopreserved embryos. We are now comprehensively characterizing deposited strains as part of the Rat Phenome Project to reevaluate their value as models of human diseases. Phenotypic data are being collected for 7 categories, 109 parameters: functional observational battery (FOB, neurobehavior), behavior studies, blood pressure, biochemical blood tests, hematology, urology, and anatomy. Furthermore, genotypes are being determined for 370 SSLP markers distributed through the whole rat genome.Here we report these large-scale, high-throughput screening data already collected for 54 rat strains. This comprehensive, original phenotypic data can be systematically viewed by “Strain ranking” for each parameter. This allows investigators to explore the relationship between several rat strains, to identify new rat models, and to select the most suitable strains for specific experiments. The discovery of several potential models for human diseases, such as hypertension, hypotension, renal diseases, hyperlipemia, hematological disorders, and neurological disorders, illustrates the potential of many existing rat strains. All deposited strains and obtained data are freely available for any interested researcher worldwide at www.anim.med.kyoto-u.ac.jp/nbr.Caffeic Acid Inhibits Cell Proliferation Induced by Angiotensin II in Vascular Smooth Muscle Cells from SHRSP. P.-G. Li,1,2 J.-W. Xu,1,3 K. Ikeda,1,3 A. Kobayakawa,4 Y. Kayano,4 T. Mitani,4 T. Ikami,4 and Y. Yamori.5 1Frontier Health Science, School of Human Environmental Sciences, Mukogawa Women's University, Nishinomiya, Japan; 2Department of Nutrition and Food Hygiene, Capital University of Medical Sciences, Beijing, P.R. China; 3Research Center for Life-Style Related Diseases, Mukogawa Women's University, Nishinomiya, Japan; 4Research and Development Institute, Miki Corporation, Nishinomiya, Japan; 5International Center for Research on Primary Prevention of Cardiovascular Diseases, Kyoto, Japan.Caffeic acid, a phenolic acid that is abundant in normal diet, has been shown to be beneficial to cardiovascular diseases and have a hypotensive effect in spontaneously hypertensive rats. However, the mechanisms implicated are far from being clarified. The purpose of the present study is to investigate whether caffeic acid can inhibit the proliferative cellular events induced by angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) derived from stroke-prone spontaneously hypertensive rats (SHRSP).In this study, we exposed VSMCs to Ang II and caffeic acid found that JAK2, STAT1, as well as ERK1/2, were rapidly phosphorylated with Ang II stimulation (reaching maximal of 7.4 ± 0.6, 1.8 ± 0.2, and 99.2 ± 10.2-fold at 2 min, respectively). However, phosphorylation of these three kinases were significantly abolished by simultaneous caffeic acid treatment (decreased to 2.4 ± 0.6, 0.5 ± 0.1, and 49.8 ± 10.9-fold at 2 min, respectively, n = 3, p< 0.05). But when cells were incubated with caffeic acid and Ang II simultaneously for the same period, cell number did not increase and was significantly lower than that of the Ang II group (5.3 ± 0.5 × 104 cells/well, n = 6, p< 0.05). Moreover, we observed that caffeic acid abolished the increase of intracellular superoxide anion level (decreased from 127.3 ± 6.3% to 100.3 ± 6.6% of the control cells, n = 5, p< 0.05) and the increase of HSP90 protein expression (decreased from 1.99 ± 0.23 to 1.05 ± 0.15 fold, n = 3, p< 0.05) induced by Ang II.In conclusion, considering that reactive oxygen species (ROS) were thought to be upstream to JAK2, HSP90, and ERK1/2, our findings suggest that caffeic acid inhibits the proliferation of VSMCs induced by Ang II through inhibiting the generation of ROS and then abolishing the JAK/STAT pathway and the Ras/Raf-1/ERK1/2 pathway. Our findings contribute to the better definition of the effect of antioxidant caffeic acid on Ang II-mediated mitogenic signaling and therefore may be beneficial for the prevention and alleviation of hypertension and other vascular diseases.Studies for Oxidation Stress of Osteonecrosis of Femoral Head in Stroke-Prone Spontaneously Hypertensive Rats. K. Kumagai and M. Niwa. Departments of Orthopedic Surgery and Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.Spontaneously hypertensive rats (SHR) are thought to be an animal model of osteonecrosis of the femoral head (ONFH). Studies for human ONFH have shown a close parallelism between the steroid hormone and oxidation stress. The object of these experiments is to observe the causal relationship between the steroid hormone and the ostenecrosis of the femoral head, especially about oxidation stress in the pathologic condition analysis of avascular necrosis in the SHRSP femoral head.A total of 142 male SHRSP/Ngsk, maintained and bred at Biomedical Research Center, Division of Comparative Medicine Center for Frontier Life Sciences, Nagasaki University, WKY/Izm from Disease Model Cooperative Research Association, were used. At 15 weeks of age, each strain rat was grouped to 2 at random. The first group was control without drug. The second group was injected with steroid hormone at 17 weeks of age. These rats were decapitated at 19 weeks. As a steroid hormone, 5 mg methylpredonisolon was injected into the dorsal muscular layer at 17 weeks of age.The histopathological criteria of the osteonecrosis were adapted that from Jacques Arlet et al. with hematoxylin-eosin staining. The type 1 of their criteria, which were the minimal degeneration changes in fatty cells and myelocytes, was diagnosed to be necrosis as no heating procedure in this experiment.GroupONFHIncidence (%)SHRSP/Ngsk numbersWKY/Izm numbersONFHincidence (%)Control404252.52012.5Steroid hormone315995.11000The above findings indicated that steroid hormone was very effective in osteonecrosis in SHRSP/Ngsk but not in WKY/Izm.Immunohistological observation was applied on the intervention of oxidation stress. As primary antibodies, anti-8-hydroxy-deoxyguanosine, anti-4-hydroxy-2-nonenal, antiAGE (advanced glycation endoproduct), and anti-Rage (receptor for advanced glycation endoproducts) were used. The evaluation of immunostaining was done in the area of epiphysis, diaphysis, and lateral epiphysial vessels. Each group was compared there.The staining of antibodies was negative in the control group but was slightly positive in steroid hormone group in WKY. In comparison with WKY, the oxidation stress was observed more powerfully in SHRSP. In SHRSP, AGE was stained in both groups despite the existence of osteonecrosis. The other antibody did not show any tendency about osteonecrosis and steroid hormone administration.Furthermore, the staining of oxidation stress in lateral epiphysical vessels was not always stronger than that in the femoral head. An incidence of necrosis rose to only SHRSP/Ngsk by the steroid hormone administration. The oxidation stress increased by the steroid hormone in WKY, but more oxidation stress was observed in SHRSP.Some of these experiments were supported by a grant of the scientific research foundation in Japan.The Effects of the Residual Substance Powder after Sesame Supercriticality Extraction and Sesamin to Cerebral Blood Flow in Stroke-Prone Spontaneously Hypertensive Rats. T. Noguchi,1 K. Ikeda,2 and Y. Yamori.3 1Human Nutrition, Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan; 2Frontier Health Science, Mukogawa Women's University, Nishinomiya, Japan; 3International Center for Research on Primary Prevention of Cardiovascular Diseases, Kyoto, Japan.The research was carried out as a substance in which sesamin, a kind of lignan from sesame oil, has powerful antioxidant power, and we showed in recent years that sesamin and vitamin E prevent hypertension and cerebral thrombus formation in stroke-prone spontaneously hypertensive rats (SHRSP). To investigate the possibility of effective use of the further sesame, the hypertension and the cerebral circulation improvement effect by the residual substance (sesame powder) that carried out supercriticality extraction of the sesame and removed a part for oil and fats, were investigated.Four-week-old male SHRSP/Izm were divided into three groups: control group (c group); sesamin group (l group), sesamin 1.0 g/food kg; sesame powder group (G group), sesame powder 5.0 g/food kg. The supercritical fluid sampling process was used for sesamin, and it condensed and dissociated. At the age of 5 weeks, rats were fed specially prescribed food for 5 weeks, and food intake and the amount of drinking water were measured. Weight, blood pressure, and the number of heart rates were measured once every week.Rats bred in the metabolism cage once at 2 weeks, and the moisture content in biochemistry analysis and excrement was measured. The amount of cerebral blood flow was measured under Nembutal anesthesia (60 mg/(kg)) after the breeding period using two-dimensional scanning method laser Doppler blood-flow equipment(PIM II, Lisca).The upward tendency weight was seen in L group. As compared with C group (10-week age: 265.2 ± 13.3 mmHg), the systolic blood pressure was intentionally controlled by L group (10-week age; 230.3 ± 10.3 mmHg) and G group (10-week age; 226.2 ± 15.5 mmHg) (p< 0.05). The amount of cerebral blood flow was increased intentionally as compared with C group (4.2 ± 0.8) by L group (7.5 ± 1.6) and G group (7.8 ± 1.5) (p< 0.05).The residual substance after extracting sesame oil has an improvement effect on cerebral blood flow of the same grade as sesamin from this research. We want to extend the possibility of effective use of sesame.Optimal Method of Increasing Potassium Intake to Prevent Hypertensive Complications. R. Horie. Scientific Committee, International Medical Academy for Life Harmony Science, Department of Pathology, and Faculty of Medicine, Shimane University, Izumo, Japan.It has been reported that high blood pressure (BP) in stroke-prone SHR rats (SHRSP; Okamoto et al. 1974) is attenuated when the animals are given 2% KC1, and inversely aggravated when 1% NaCl is given, respectively, in drinking water during a 4-week period(Yamori, 1981). Further, our epidemiological studies in Daiwa village of Izumo district in Japan showed a linear correlation between BP levels and sodium-to-potassium ratios in 24-hr urine in elderly inhabitants aged 60 to 69 years (Yamori et al. 1981).Researchers demonstrated that 1% KC1 and 1% NaCl loading in drinking, respectively, for a sufficient duration resulted in shortening the life span of SHRSP. This was due to stroke caused by an aggravation of high BP, especially around 100 days after birth, compared with the life span of controls given tap water to drink (Ikeda et al. 1989). In the present study, the effect of a potassium-rich diet on the development of stroke in SHRSP was investigated in comparison to animals receiving 1% NaCl and 1% KCl loading, respectively, in drinking water.A total of 60 3-month-old male SHRSP were divided into 4 groups. Animals in group I (n = 15) were fed a standard laboratory chow diet (SP diet containing 193 mg of sodium and 197 mg of potassium per 2.7 g ash/100 g chow diet, Na to K ratio; Ca. 0.5) with untreated tap water to drink as controls. Those in groups II and III also were fed SP diet, with 1% NaCl and 1% KCl, respectively, in drinking water. Animals in group IV were fed a special SP diet including 20% dry powdered plum (containing 1 mg of sodium and 740 mg of potassium/2.4 g ash/100 g diet, Na to K ratio: Ca 0.001) with 1% NaCl in drinking water. BP (UR5000, Ueda, Japan) and body weight were frequently checked. Autopsy was performed at the time of natural death for pathological examinations.None of animals fed the special SP diet with 1% NaCl in the drinking water (group IV) developed cerebral stroke, whereas over 85% of animals in groups I, II, and III developed stroke as shown in the following table.GroupDrinking waterFoodStroke incidence %Life span (days, M ± SE)Maximal BP (mmHg, M ± SE)ITap waterSP13/15 (85%)254 ± 10247 ± 7II1%NaClSP15/15 (100%)174 ± 7253 ± 5III1%KClSP15/15 (100%)185 ± 11254 ± 8IV1%NaClSP with plum0/15 (0%)472 ± 19203 ± 10These findings suggest that a potassium-rich SP diet and dry powdered plums prevents stroke despite 1% NaCl loading in drinking water. Such stroke prevention might have resulted not only from the added potassium, but also from the other nutrients that are abundantly found in plum fruits, such as calcium, fiber, and so on. It should be emphasized that an excellent natural balance of nutrients prevents stroke, and such balance could have been promoted by the addition of dry powdered plum to the diet.Role of the Brain Renin-Angiotensin System in the Hypertension of Rats with Chronic Renal Failure. M. Nishimura1 and H. Takahashi.2 1Cardiovascular Division, Toujinkai Hospital, Kyoto, Japan; 2Department of Clinical Sciences and Laboratory Medicine, Kansai Medical University, Moriguchi, Japan.Increased peripheral sympathetic activity is one of the major mechanisms of hypertension in patients with end-stage renal disease. In the present study, we determined whether the brain renin-angiotensin system is involved in the pressor mechanism of chronic renal failure caused by subtotal nephrectomy in rats.A total of 50 8-week-old male spontaneously hypertensive rats (SHR: Charles River Japan) were used for this study. Under anesthesia of sodium pentobarbital, 26 SHR underwent subtotal nephrectomy performed by right subcapsular nephrectomy and infarction of approximately two-thirds of the left kidney by selective ligation of two of three to four extrarenal branches of the left renal artery. The other 24 SHR underwent sham surgery consisting of laparotomy and manipulation of both kidneys before wound closure. Rats were housed in a temperature (22°C) controlled room with ad libitum access to commercial standard rat chow(MF, Oriental Yeast Co, Kyoto) and tap water. Systolic arterial pressure was measured using tail-cuff method, and diurnal urinary excretion of unconjugated norepinephrine was measured once a week for 12 weeks.At the end of the experiment, expression of the renin-angiotensin system mRNAs in the hypothalamus and lower brain stem was measured using competitive reverse transcription polymerase chain reaction method in sham-operated SHR (n = 6) and SHR with subtotal nephrectomy (n = 8). In addition, effects of a 6-day intracerebroventricular infusion of CV-11974 (10 µg/kg/d or 50 µg/kg/d), a blocker of angiotensin II type-1 receptor, or vehicle (artificial cerebrospinal fluid) on systolic arterial pressure, heart rate, and urinary excretion of norepinephrine were investigated in sham-operated SHR (n = 18) and SHR with subtotal nephrectomy (n = 18).Compared with sham-operated SHR, SHR with subtotal nephrectomy showed higher plasma creatinine concentration (0.51 ± 0.03 mg/dl vs. 2.16 ± 0.06 mg/dl, p< 0.0001) and greater excretion of urinary protein (29.1 ± 2.1 mg/d vs. 279.9 ± 12.1 mg/d, p< 0.0001). Plasma renin activity was lower in SHR with subtotal nephrectomy than in sham-operated SHR(2.85 ± 0.29 ng/ml/h vs. 14.12 ± 0.64 ng/ml/h, p< 0.0001). Systolic arterial pressure was higher in SHR with subtotal neprectomy than in sham-operated SHR (221 ± 23 mm HG vs. 170 ± 14 mm Hg, p< 0.001), and urinary excretion of norepinephrine was greater in SHR with subtotal neprectomy than in sham-operated SHR(2.712 ± 0.572 ng/g BW/d vs.1.696 ± 0.244 ng/g BW/d, p< 0.001).Expression of renin, angiotensin-converting enzyme, and AT1 receptor mRNAs were greater in SHR with subtotal nephrectomy than in sham-operated SHR in the hypothalamus or lower brain stem. Continuous intracerebroventricular infusion of CV-11974, a nonpeptide AT1 receptor blocker, attenuated increases in systolic blood pressure (vehicle, 226 ± 13 mm Hg: 10 µg/kg/d of CV-11974, 180 ± 12 mm Hg, p< 0.001 vs. vehicle: 50 µg/kg/d of CV-11974, 161 ± 12 mm Hg, p< 0.001 vs. vehicle) and urinary excretion of norepinephrine in SHR with subtotal nephrectomy.The brain renin-angiotensin system is likely to be activated in SHR with chronic renal failure caused by subtotal nephrectomy and may be involved in the hypertensive mechanism of this chronic renal failure model.Role of Reactive Oxygen Species in the Cardiovascular Center of Stroke-Prone Spontaneously Hypertensive Rats in Neural Mechanisms of Hypertension. Y. Hirooka, T. Kishi, Y. Kimura, Y. Sagara, A. Takeshita, and K. Sunagawa. Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.An increase in reactive oxygen species (ROS) contributes to the pathogenesis and the development of hypertensive vascular disease. However, little is known regarding the role of ROS in the rostral ventrolateral medulla (RVLM) which determines basal sympathetic nerve activity within the brain stem in hypertension. The aim of the present study was to determine whether ROS in the brain, particularly in the RVLM, are increased in the stroke-prone spontaneously hypertensive rats (SHRSP), and if so, whether this contributes to hypertensive mechanisms of SHRSP. Thiobarbituric-acid reactive substances (TBARS) were measured in the brain of SHRSP and normotensive Wistar-Kyoto rats (WKY). Electron spin resonance (ESR) spectroscopy analysis also was performed to quantify ROS generation more directly. Furthermore, adenovirus vectors encoding Mn-superoxide dismutase (AdMnSOD) were transfected into the RVLM.Blood pressure and heart rate were measured using the radiotelemetry system in a conscious state. TBARS levels were significantly increased in the brain including the RVLM of SHRSP compared with WKY. Intensity of signal decay of ESR was significantly greater in the RVLM than in WKY, and it was attenuated by dimethylthiourea, a scavenger of hydroxyl radical. Blood pressure and heart rate were significantly decreased in SHRSP after AdMnSOD transfection. This time course was consistent with expression of transfected AdMnSOD. Urinary norepinephrine excretion was significantly higher in SHRSP than in WKY. After transfection of AdMnSOD, it decreased to the level of WKY. In addition, TBARS levels in the RVLM were decreased after AdMnSOD transfection in SHRSP.These results indicate that ROS generation from superoxide to hydroxyl radical in the RVLM is increased in SHRSP. This might be involved in neural mechanisms of hypertension.Increase participates in the disease state and the formation/development of hypertensive vessel change by hypertension. However, it is not clear about a role of active oxygen in cephalad medulla oblongata retroperitoneum outside area(RVLM) which there is it in a role in a brain, brainstem part in particular, and prescribe sympathetic nerve action.It was to clarify whether you decreased by a depression treatment more whether the active oxygen which increased participated in hypertonic mechanism through sympathetic nervous system in SHRSP whether active oxygen in RVLM in a brain of a cerebral apoplexy spontaneously hypertension rat (SHRSP) in particular increased as for the purpose of this study.[a method] I used the Thiobarbituric acid-reactive substances (TBARS) measurement and electron paramagnetic resonance (ESR) law with SHRSP and a normal blood pressure rat (WKY) and did determination of intracerebral active oxygen.Furthermore, I observed alteration of blood pressure/cardiac rate/sympathetic nerve action when I let a MnSOD gene appear as vector with an adenovirus under non-anesthesia awakening with telemetry method to RVLM focus and erased active oxygen.I evaluated a change of intracerebral active oxygen in front and back that did a treatment by various antihypertensive agent on 30th by TBARS determination last.[a result] the TBARS levels in RVLM of SHRSP increased in comparison with WKY.I compared ESR signal attenuation speed with WKY in SHRSP, and this was restrained in dimethylthiourea which was hydroxyl radical scavenger greatly.I agree in time progress of gene expression, and depression/cardiac rate degradation/sympathetic nerve action decrease was accepted by a MnSOD gene.Furthermore, a brainstem region TBARS level fell by amlodipine and a depression treatment by a Terumi sultan with quantity to produce hypotensive effect of the degree in SHRSP, and urinary norepinephrine defluxion dosage fell.The TBARS level did not change in spite of depression of the degree in hydralazine.[a conclusion] hydroxyl radicals production from superoxide increases in RVLM of SHRSP and center as hypertonic mechanism-related adaptation is aberrant and suggests what can decrease the intracerebral active oxygen which increased by participating, an antihypertensive agent treatment of a certain modality.Long-Term Endothelin Antagonism Reverses the Hypertension-Induced Cerebrovascular Remodeling in SHRSP. S. Jesmin,1 H. Togashi,2 I. Sakuma,1 C. N. Mowa,3 M. Yoshioka,2 and T. Miyauchi.4 1Department of Cardiovascular Medicine, and 2Department of Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 3Department of Neurobiology and Pharmacology, NE Ohio University College of Medicine, Rootstown, Ohio, USA; 4Department of Cardiovascular Medicine, University of Tsukuba, School of Medicine, Tsukuba, Japan.Stroke-prone spontaneously hypertensive rats (SHRSP) progressing to the malignant hypertensive stages suffer spontaneous stroke in part due to abnormal cerebrovascular development. Several reports have described the involvement of endothelin (ET)-1 in the pathogenesis of stroke in different ways. ET-specific antibodies have been shown to slightly reduce blood pressure in SHRSP. Evidence that the binding of ET, a vasoconstrictor with vasoproliferative activity, is upregulated in the salt-loaded SHRSP.Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, is known to play physiological and pathological roles in the development of cerebral capillaries. We have reported that ET-1, its type A receptor (ETAR), VEGF, and its receptors were upregulated in the frontal cortex of SHRSP at the typical hypertensive stage, whereas ET type B receptor (ETBR), endothelial nitric oxide synthase (eNOS), and Akt, which act downstream of VEGF, were downregulated as compared with its genetic normotensive control Wistar-Kyoto rats (WKY).The upregulated VEGF/KDR system is believed to be a hypertension-induced compensatory adaptation in frontal cortex of SHRSP brain. Immunohistochemistry, immunoblotting and real-time PCR techniques were applied. The expression patterns of ET-1, ETAR, ETBR, VEGF and its receptors, Akt, and eNOS were different between SHRSP and SHR.The current study investigated whether long-term treatment with the ET-A/-B dual receptor antagonist SB209670 (1 mg/g/day, s.c for 12 weeks) starting from the early stage of hypertension (8 weeks old) can reverse these alterations in the SHRSP brain. Blood pressure was measured every month. Systolic blood pressure was slightly decreased after endolethin antagonism but diastolic pressure was unchanged in SHRSP. ET antagonism by long-term treatment with the ET receptor antagonist SB209670 suppressed serum and cerebral ET-1 levels and ETAR expression in the frontal cortex of SHRSP. Serum and cerebral ET-1 levels were measured by ELISA. Upregulated expression of VEGF and its receptors (KDR and Flt-1) and downregulated Akt and eNOS, the downstream components of VEGF signaling, observed in the vehicle-treated SHRSP brain were remarkably reversed by ET antagonism.Serum and frontocortical nitric oxide (NO) levels were recovered by endothelin antagonism in SHRSP. NO was measured as nitrite by colorimetric method. The 35% decrease in ETBR expression in the frontal cortex of vehicle-treated SHRSP also was ameliorated by SB209670 treatment. Thus, hypertension-induced alterations in the SHRSP brain were normalized by long-term ET antagonism. Usually ETBR activation induces endothelium-dependent relaxation through the release of NO and prostacyclin. In the present study, ETBR and eNOS were altered in parallel in the SHRSP brain. Cerebral blood flow of SHRSP was halved at the malignant hypertension stage associated with upregulated ET-1 and ETAR. Cerebral capillary density also was decreased in SHRSP with the development of malignant hypertension.Although VEGF was upregulated in SHRSP at typical hypertensive stage, its angiogenic downstream molecules were downregulated and as a result there was no increase of cerebral blood flow rather cerebral flow tended to decrease at typical hypertensive stage of SHRSP rats. Therefore, we suggest that ET antagonism from the early stage of hypertension prevents the progression of hypertension-induced neurovascular remodeling in the SHRSP frontal cortex, although the question remains what will happen if ET antagonism treatment would start when SHRSP rats have already established hypertension.Further studies are required to rule out completely whether the reversal effect by ET antagonism on the VEGF signaling and ET receptors was due to the reduction in systolic blood pressure or independent of its effects on blood pressure. Also specific endothelin receptor blocker should be used to clarify the differential roles of ET receptors in stroke-prone hypertension-induced cerebrovascular remodeling. Finally, the effects of ET antagonism on regional cerebral blood flow in SHRSP would provide the functional significance of ET-1 receptors blockade treatment in stroke-prone hypertensive models.This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by Health Sciences Research grants for comprehensive research on aging and health from the Ministry of Health, Welfare, and Labor of Japan.Sympathoexcitatory by the Caudal Pressor Area is Mediated by the AT1 receptor in the Rostral Ventrolateral Medulla of Spontaneously Hypertensive Rats. Y. Yajima, S. Ito, S. Tomioka, Y. Masubuchi, M. Hirastuka, T. Suzuki, H. Kawato, N. Katsunuma, T. Kawabe, K. Komatsu, K. Tsukamoto, and K. Matsumoto. Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan.The caudal pressor area (CPA), located in the caudal ventrolateral medulla, tonically activates sympathoexcitatory outflow through rostral ventrolateral medulla (RVLM) vasomotor neurons and participates in blood pressure regulation. Sympathoexcitation by the CPA is independent of excitatory amino acid (EAA)-sensitive RVLMs neurons in Sprague-Dawley (SD) rats. Therefore, other types of receptors must mediate this effect. It has been reported that in spontaneously hypertensive rats (SHR), one of reason for maintaining high blood pressure is overexpression of angiotensin II type1(AT1) receptor in the RVLM. We have reported that sympathoexcitatory input from the CPA is enhanced in SHR. Thus, we hypothesized that the sympathoexcitatory influence of the CPA in the RVLM is mediated by AT1 receptors in SHR.In chloralose-anesthetized male SHR, injection of glutamate into the CPA elicited a pressor response (27 ± 2 mmHg, n = 6) that was substantially greater than in Wistar-Kyoto rats (13 ± 2 mm Hg, n = 8). In SHR, preinjection of the AT1 receptor antagonist valsartan bilaterally into the RVLM significantly attenuated the pressor response (7 ± 4 mm Hg, n = 6). In contrast, preinjection of artificial cerebrospinal fluid had no effect.These results indicate that enhanced sympathoexcitation evoked from CPA in SHR is mediated by AT1 receptors in the RVLM vasomotor neurons. From these results, overexpression or increased activation of AT1 receptors in the RVLM may play a key role in the induction and/or maintenance of high blood pressure in animal models of essential hypertension.