Development Of Hypertension In SHRSP Research Articles

Contribution of afferent renal nerve signals to acute and chronic blood pressure regulation in stroke-prone spontaneously hypertensive rats.

The activation of sympathetic nervous system plays a critical role in the development of hypertension. The input from afferent renal nerves may affect central sympathetic outflow; however, its contribution to the development of hypertension remains unclear. We investigated the role of afferent renal nerves in acute and chronic blood pressure regulation using normotensive Wistar-Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Acute chemical stimulation of afferent renal nerves elicited larger increases in blood pressure and renal sympathetic nerve activity in young 9-week-old SHRSP compared to WKY. Selective afferent renal denervation (ARDN) and conventional total renal denervation (TRDN) ablating both afferent and efferent nerves in young SHRSP revealed that only TRDN, but not ARDN, chronically attenuated blood pressure elevation. ARDN did not affect plasma renin activity or plasma angiotensin II levels, whereas TRDN decreased both. Neither TRDN nor ARDN affected central sympathetic outflow and systemic sympathetic activity determined by neuronal activity in the parvocellular region of hypothalamic paraventricular nucleus and rostral ventrolateral medulla and by plasma and urinary norepinephrine levels, respectively. Renal injury was not apparent in young SHRSP compared with WKY, suggesting that renal afferent input might not be activated in young SHRSP. In conclusion, the chronic input from afferent renal nerves does not contribute to the development of hypertension in SHRSP despite the increased blood pressure response to the acute stimulation of afferent renal nerves. Efferent renal nerves may be involved in the development of hypertension via activation of the renin-angiotensin system in SHRSP.

Abstract 13: Cholic Acid Supplementation Attenuates Hypertension In Spontaneously Hypertensive Stroke Prone Rats

Recent studies have demonstrated a causal role of gut dysbiosis in the development of hypertension in several animal models. However, our understanding of the mechanisms linking gut dysbiosis to blood pressure (BP) regulation of the host is still lacking. One key mechanism by which the microbiota influences the host is through the generation/modification of metabolites, such as bile acids (BAs). BA signaling has been shown to influence many pathways involved in BP regulation, including systemic inflammation and vascular function. We previously observed that spontaneously hypertensive stroke prone rat (SHRSP) exhibited dysbiotic cecal microbiome, which included a significant increase in the genus Lactobacillus , known to sequester BAs within its cytosol and reduce BAs availability, when compared to WKY. Thus, we hypothesized that gut dysbiosis contributes to the development of hypertension by reducing bile acid signaling. We observed a significant reduction in 9 of 18 plasma BAs in SHRSPs, as compared to WKY. This included a 72% reduction in cholic acid (CA), a primary BA (n=7-8, p<0.05). We next examined the effects of CA supplementation (0.5% CA diet for 16 weeks) on systolic BP (SBP) in WKY and SHRSP. Within the 9 BAs that were reduced in SHRSP, CA and hyocholic acid were restored by CA treatment in SHRSP plasma to similar levels of that observed in WKY control plasma. Furthermore, CA treatment decreased SBP by 18 ±7mmHg at 20 weeks in SHRSP (n=7-8, p<0.05), but had no effect on SBP in WKY rats. Acetylcholine-induced vasodilation of the aorta was significantly impaired in SHRSP control by 40% (10 -6 μM ACh, 59.5% vs. 99.3%) as compared to WKY control (n=3-4, p<0.01). CA treatment significantly improved endothelium-dependent vasodilation in the aorta of SHRSP rats similar to that in WKY rats (n=3-4, p<0.05). CA treatment also altered a number of bacteria in the gut including restoration of relative abundance of Lactobacillus in SHRSP to the level of WKY controls. We conclude that reduced BA signaling contributes to the development of hypertension in SHRSP, and that CA treatment may be a potential therapeutic approach to attenuate vascular endothelial dysfunction and associated hypertension.

Cholic Acid Attenuates Systemic Hypertension in Spontaneously Hypertensive Rats

Gut dysbiosis, defined as a pathological imbalance of the gut microbiota, is associated with hypertension in humans and animal models. Recent studies have demonstrated a cause and effect relationship between gut dysbiosis and hypertension in several animal models. However, our understanding of the mechanisms linking gut dysbiosis to blood pressure (BP) regulation of the host is still lacking. One key mechanism by which the microbiota influences the host is through the generation/modification of metabolites, such as bile acids (BAs). BAs, which serve to emulsify fats, are derived from the liver, released into the gut, and further modified by bacteria into a variety of primary and secondary BAs species. These primary and secondary BAs diffuse into the systemic circulation, disperse throughout the body, and act as hormones by stimulating BA receptors. BA signaling has been shown to influence many pathways involved in BP regulation, including systemic inflammation and vascular function. Thus, we hypothesized that gut dysbiosis contributes to the development of hypertension by reducing bile acid signaling. By comparing 16s rRNA sequencing data from normotensive WKY and spontaneously hypertensive stroke prone rat (SHRSP) cecal content, we found that SHRSP had a significant increase in the genus Lactobacillus, known to sequester BAs within its cytosol and reduce systemic BAs availability, when compared to WKY (p<.05). Additionally, we observed a significant reduction in 9 of 18 plasma BAs in SHRSPs, as compared to WKY. This included a 77% reduction in cholic acid (CA), a primary BA. We next examined the effects of CA supplementation on systolic BP (SBP) in WKY and SHRSP. Starting at 6 weeks old, WKY and SHRSP were fed control or control + 0.5% CA diet for 16 weeks. SBP was measured weekly. At 22 wks, rats were euthanized, and the thoracic aorta was isolated to examine endothelial function by aortic ring assay. Cecal content was collected for 16s rRNA sequencing and BA measurements. CA treatment restored CA and hyocholic acid in SHRSP plasma to similar levels of that observed in WKY control plasma. Furthermore, CA treatment decreased SBP by 18 ±7mmHg at 20 weeks in SHRSP (p<.05) but had no effect on SBP in WKY rats. Acetylcholine‐induced vasodilation of the aorta was significantly impaired in SHRSP control as compared to WKY control. Interestingly, CA supplementation significantly improved endothelium‐dependent vasodilation in the aorta of SHRSP rats similar to that in WKY rats (p<.0005). Beta‐diversity analysis of the cecal microbiota showed that CA treatment significantly altered the community makeup in WKY and SHRSPs. Of interest, CA treatment restored relative abundance of Lactobacillus in SHRSP to the level of WKY controls. We conclude that reduced BA signaling contribute to the development of hypertension in SHRSP, and that CA treatment may be a potential therapeutic approach to attenuate vascular endothelial dysfunction and associated hypertension.Support or Funding InformationNational Heart, Lung, and Blood In stitute R01HL134838 (D.J. Durgan), and by National Institute of Neurological Disorders and Stroke R01NS080531 (R.M. Bryan)

Afferent renal nerve input is involved in blood pressure elevation through activation of angiotensin receptor type 1 in hypothalamic paraventricular nucleus in stroke‐prone spontaneously hypertensive rats

BackgroundRenal nerves contain both sympathetic efferent fibers and sensory afferent fibers. Activation of efferent renal nerves increases renin release and renal sodium reabsorption, causing hypertension. However, previous studies suggested that the contribution of afferent renal nerves to blood pressure (BP) regulation may differ among the animal models of hypertension. Activation of brain renin‐angiotensin system, specifically angiotensin receptor type 1 (AT1R), in hypothalamic paraventricular nucleus (PVN), which receives afferent renal input, is crucially involved in BP elevation. Therefore, the aim of this study was to examine whether the afferent renal input contributes to BP elevation through AT1R activation in the PVN in stroke‐prone spontaneously hypertensive rats (SHRSP).MethodsIn chronic experiment, total renal denervation (TRDN), selective afferent renal denervation (ARDN), or sham surgery was performed in male SHRSP at 10 weeks of age, which is the developing phase of hypertension. ARDN was produced by wrapping renal artery and vein with capsaicin‐soaked gauze. Systolic BP was measured by tail‐cuff method at baseline and 2 and 6 days after surgery. In acute experiment, we evaluated the effect of AT1R blockade with losartan in the PVN on pressor response to afferent renal nerve stimulation by intrapelvic infusion of 10 μM capsaicin under anesthesia in male Wistar‐Kyoto rats (WKY) and SHRSP at 10 weeks of age.ResultsSystolic BP was sequentially increased at day 2 and day 6 in sham‐operated SHRSP ([n=4], baseline, 188.3±9.2 mmHg; day 2, 192.1±8.1 mmHg; day 6, 201.1±3.9 mmHg). In both ARDN‐SHRSP and TRDN‐SHRSP, systolic BP was decreased from baseline at day 2 after surgery (ARDN [n=4], baseline, 187.3±10.3 mmHg; day 2, 170.5±5.2 mmHg; p=0.085; TRDN [n=4], baseline, 186.4±5.9 mmHg; day2 145.1±4.2 mmHg; p=0.003), and the decrease in systolic BP was smaller in ARDN‐SHRSP than in TRDN‐SHRSP (ARDN, −16.7±13.2 mmHg; TRDN, ‐ 41.4±10.0 mmHg; p=0.03). Although systolic BP was increased at day 6 compared to day 2 in ARDN‐SHRSP and TRDN‐SHRSP (ARDN, 197.8±14.9 mmHg; TRDN, 185.8±19.3 mmHg), the BP change from baseline to day 6 was smaller in ARDN‐SHRSP and TRDN‐SHRSP compared with sham‐operated SHRSP. We validated TRDN and ARDN by immunohistochemistry and western blotting: in the renal pelvis, both CGRP and tyrosine hydroxylase (a marker for afferent and efferent renal nerves, respectively) were abolished in TRDN‐SHRSP, and CGRP was selectively abolished in ARDN‐SHRSP. In acute experiment, BP elevation induced by afferent renal nerve stimulation was greater in SHRSP than in WKY (Δmean BP, SHRSP [n=4],12.2±4.1 mmHg; WKY [n=4], 6.6±5.4 mmHg; p=0.07). Prior microinjection of losartan into the PVN attenuated the pressor response to afferent renal nerve stimulation in SHRSP (Δmean BP, 1.1±4.4 mmHg, p=0.04 vs before losartan), but not in WKY (Δmean BP, 3.2±6.7 mmHg, p>0.10 vs before losartan).ConclusionNot only efferent renal nerve activation but afferent renal nerve input contributes to development of hypertension in SHRSP. The AT1R activation in the PVN may be involved in its mechanism.

Decreased proportion of Foxp3+ CD4+ regulatory T cells contributes to the development of hypertension in genetically hypertensive rats.

Dysregulation of the sympathetic nervous system and the immune system has been highlighted in the pathogenesis of hypertension. Foxp3 regulatory T (Treg) cells, which maintain immune homeostasis, have potent antihypertensive properties. We systematically explored whether Treg cells proportions are altered in stroke-prone spontaneously hypertensive rats (SHRSP) with increased sympathetic activity. Then, we focused on Treg cells in the spleen and determined whether Treg cells proportion in the spleen could be affected by splenic sympathetic input associated with the progression of hypertension. We also investigated effects of Treg cell induction on hypertension development and cardiac hypertrophy in SHRSP. We evaluated Treg cells in SHRSP. Compared with normotensive Wistar-Kyoto (WKY) rats, SHRSP exhibited decreased proportions of CD4CD25Foxp3 cells (Treg cells) in the spleen before the onset of hypertension. Splenic sympathetic denervation in prehypertensive SHRSP attenuated the reduction of Treg cells at 4 weeks after denervation (P < 0.05) and delayed the development of hypertension in SHRSP. Interleukin (IL)-2 and anti-IL-2 mAb complex selectively induced Treg cells in vivo (P < 0.01), delayed the development of hypertension and decreased the heart weight/body weight ratio (4.64 ± 0.06 vs. 4.25 ± 0.05 mg/g, P < 0.01) and the cross-sectional area of cardiac myocytes in SHRSP compared with vehicle-treated SHRSP (580 ± 3.91 vs. 438 ± 4.43 μm, P < 0.0001). Our findings indicate that a decrease in Treg cell proportion is crucial for the development of hypertension and cardiac hypertrophy in SHRSP, which is involved in sympathetic neural input to the spleen.

Is antioxidant therapy effective for advanced hypertension and renal injury?

nappropriate augmentation of reactive oxygen species(ROS) production reduces the bioavailability of anti-oxidant defenses [1,2] and plays a pivotal role in thepathophysiology of hypertension and renal injury [3–5].Experimental studies have indicated that enhanced ROSproduction is involved in vascular remodeling and endo-thelium dysfunction during the development of hyperten-sion [6]. Earlier clinical studies have also suggested a strongassociation between lower antioxidant levels and cardio-vascular risks [7].To date, much effort has been directed toward thebenefits of antioxidant therapy. Clinical studies, includingthe Cambridge Heart Antioxidant Study [8] and the Anti-oxidant Supplementation in Atherosclerosis PreventionStudy [9], have shown the beneficial effects of antioxidanttherapy for cardiovascular events. Large epidemiologicalstudies have also reported that dietary intake of anti-oxidants inverselycorrelateswithhypertension[9,10].Simi-larly, treatment with ascorbic acid significantly improvedSBP and DBP in mild-to-moderate hypertensive patients[11]. In experimental models of hypertension, vitamin Calone or in combination with vitamin E increased thesynthesis of nitric oxide and reduced blood pressure[12,13]. Furthermore, an antioxidant rich diet was shownto relieve hypertension and reduces renal immune cellinfiltration [14]. By contrast, many clinical trials failed todemonstrate any beneficial effects of antioxidant therapyon blood pressure [15–17] and cardiovascular events[18–20]. For example, the Heart Protection Study Collabo-rative Group studied high-risk individuals and showed thatalthoughantioxidantvitaminsupplementationsubstantiallyincreased blood vitamin concentrations, it did not produceany significant reductions in the incidence of any type ofcardiovascular disease [19].In this issue of Journal of Hypertension, the article‘Superoxide dismutase mimetic, tempol, aggravates renalinjury in advanced-stage stroke-prone spontaneouslyhypertensive rats’ by Sugama et al. [21] shows that anti-oxidanttherapywithasuperoxidemimetic,tempol,didnotreduce blood pressure in salt-loaded old stroke pronespontaneously hypertensive rats (SHRSP). The authors alsoshowed that although tempol effectively reduced oxidativestress in the kidney, it actually exacerbated the progressionof proteinuria and histological damage, such as glomerulo-sclerosis and interstitial fibrosis. These data suggestthat alternative pathways that are ROS-independent areinvolved in the pathogenesis of the advanced stage ofhypertension and renal injury in SHRSP. It is also possiblethat oxidative stress is merely a consequence of the acti-vation of these alternative pathways. However, Park et al.[22] had performed similar experiments in SHRSP andshown that long-term treatment with tempol from earlystages of hypertension did attenuate the progression ofhypertension through the prevention of vascular remodel-ing, and that this was associated with decreased vascularsuperoxide production and increased plasma antioxidantlevels.Thus, thequestion arisesastowhyantioxidanttherapiesshow such conflicting results in clinical and experimentalstudies [23]. Importantly, it is clear from the clinical studiesthatdosedifferencesdonotseemtoberesponsibleforsuchdiscrepant results [23]. One possible explanation for thediscrepancy in these studies is the different stage of hyper-tension and renal injury. In both clinical and experimentalstudies,beneficialeffectsofantioxidantsweremostlyfoundwhen interventions were started at an early stage. Forexample, Park et al. [22] administered tempol from an earlyage and showed an attenuation in the development ofhypertension in SHRSP, whereas Sugama et al. [21] failedto observe a blood pressure-lowering effect of tempolif treatment started at an advanced hypertensive stage.Inatomi et al. [24] have also shown that in patients withmild cardioembolic stroke, treatment with an antioxidant,edaravone, resulted in early functional improvement, butthese effects were not observed in late stages.Thefindingthatantioxidanttherapieshavenobeneficialeffects on advanced hypertension, as well as cardiovas-cular and renal diseases, suggest a potentialinvolvement of

Mineralocorticoid receptors/epithelial Na+ channels in the choroid plexus are involved in hypertensive mechanisms in stroke-prone spontaneously hypertensive rats

Increase in cerebrospinal fluid (CSF) Na(+) concentration ([Na(+)]) precedes hypertension and is a key step in the development of salt-induced hypertension. In the choroid plexus (CP), epithelial Na(+) channels (ENaCs) have an important role in Na(+) transport from the blood into the CSF. However, it remains unknown whether the mineralocorticoid receptors (MR)/ENaCs pathway in the CP of stroke-prone spontaneously hypertensive rats (SHRSP) is involved in neural mechanisms of hypertension. Therefore, we examined the role of the MR/ENaCs pathway in the CP in the development of hypertension in SHRSP associated with an increase in CSF [Na(+)]. As a marker of MR activation, serum/glucocorticoid-inducible kinase 1 (Sgk1) expression levels in the CP were measured and found to be greater in SHRSP than in Wistar-Kyoto (WKY) rats. CSF [Na(+)] levels were also higher in SHRSP than in WKY rats. In SHRSP, high-salt intake (8%) increased blood pressure and urinary norepinephrine excretion compared with those in animals fed a regular salt diet (0.5%) for 2 weeks. Furthermore, the expression levels of MR, Sgk1 and ENaCs in the CP and the increase in CSF [Na(+)] were greater in SHRSP fed a high-salt diet than in those fed a regular salt diet. These alterations were attenuated by intracerebroventricular infusion of eplerenone (10 μg kg(-1) per day), except for α-ENaC and β-ENaC. We conclude that activation of the MR/ENaCs pathway in the CP contributes to hypertension via an increase in CSF [Na(+)], thereby exaggerating salt-induced hypertension with sympathetic hyperactivation in SHRSP.

Gender-related differences in advanced glycation endproducts, oxidative stress markers and nitric oxide synthases in rats

An age- and blood pressure-associated increase in methylglyoxal (MG) and MG-induced advanced glycation endproducts (AGEs), including N(epsilon)-carboxyethyl-lysine (CEL) and N(epsilon)-carboxymethyl-lysine (CML), in the kidney of spontaneously hypertensive rats (SHR) has been shown. In the present study, gender-related changes in AGEs and nitric oxide synthase were investigated in Sprague-Dawley (SD) and stroke-prone SHR (SHRsp) rats. Immunohistochemical analyses were conducted on kidneys from 24-week-old male and female SD rats as well as SHRsp. The systolic blood pressure of SHRsp was significantly higher than that of SD rats. Male SD rats had more intense kidney staining for CEL than female SD rats. Both male and female SHRsp had more marked CEL and CML staining localized to kidney tubules, as opposed to SD rats. Female rats showed more staining in glomerular vessels than male rats in both SD and SHRsp. Nuclei containing nuclear factor-kappaB (NF-kappaB) p65 and activated macrophages were seen in the kidney from SHRsp, not so much in SD rats, localized to renal tubules in male and glomerular vessels in female SHRsp. A higher protein level of NF-kappaB p65 was found in SHRsp than in SD rats. SD rats had more intense kidney neuronal nitric oxide synthase staining than SHRsp. The intensity of inducible nitric oxide synthase staining was significantly higher in SHRsp than in SD rats, with no gender differences in either strain. SHRsp and male rats exhibited higher AGEs and oxidative stress than SD and female rats, respectively. These differences might partly account for the development of hypertension in SHRsp and the higher vulnerability of male animals to renal pathology.

Protective effect of docosahexaenoic acid against development of hypertension in young stroke-prone spontaneously hypertensive rats. Focus on the sympathoadrenal system and lipid metabolism

The present study was undertaken to elucidate the effects of acute and subacute administration of docosahexaenoic acid (DHA) on blood pressure in SHRSP between the ages of 6 and 11 weeks. The continuous administration of DHA for 5 weeks in SHRSP significantly inhibited blood pressure rise as compared with levels in non-treated SHRSP. DHA-treated SHRSP showed significantly decreased urinary epinephrine contents as compared with those in non-treated SHRSP. DHA-treated SHRSP produced a significant decrease in adrenal epinephrine concentration as compared with those in non-treated SHRSP at the age of 11 weeks. DHA-treated SHRSP also demonstrated significant decreases in serum triglycerides, LDL and lipid peroxide levels as compared with levels in non-treated SHRSP. However, a single intraperitoneal administration of DHA did not exert any influence on blood pressure in SHRSP at the age of 11 weeks. These findings suggest that the anti-hypertensive effect induced by a 5-week, continuous administration of DHA appears to be associated with the amelioration of increased sympathoadrenal activity and serum lipid derangement during the developmental stage of hypertension in young SHRSP.The present study clarified that a 5-week, continuous administration of DHA had a protective effect against the development of hypertension in SHRSP.

Transforming growth factor-beta expression in cardiovascular organs in stroke-prone spontaneously hypertensive rats with the development of hypertension.

Transforming growth factor (TGF)-beta activity is involved in several cardiovascular diseases owing to its effects on the growth of vascular smooth muscle cells and induction of extracellular matrix formation. We evaluated expression of TGF-beta in cardiovascular organs from stroke-prone spontaneously hypertensive rats (SHR-SP) which show severe cardiovascular damages with the development of hypertension. Twelve-week-old Wistar-Kyoto (WKY)/Izm rats and SHR-SP/Izm were loaded with 1% salt for 4 weeks. Aorta, heart and kidney were removed and evaluated histologically by hematoxylin-eosin staining. Expression of TGF-beta1 mRNA was evaluated by reverse transcription and polymerase chain reaction analysis in mRNA extracted with oligo dT-cellulose. Expression of TGF-beta1 protein was evaluated by Western blot analysis and immunohistochemical study in renal cortex. Whereas expression of TGF-beta1 mRNA was detected only in the heart of SHR-SP before salt loading, it was detected in the aorta, left ventricle of heart and renal cortex from both rat strains, and it was stronger in the renal cortex of SHR-SP than in the renal cortex of WKY rats. Expression of TGF-beta1 protein was markedly higher in the renal cortex of SHR-SP than in the renal cortex of WKY rats after salt loading. TGF-beta was localized at glomeruli and capillary arteries in the renal cortex, and immunostaining was stronger in SHR-SP than in WKY rats. Expression of TGF-beta1 was increased in glomeruli and capillaries of the renal cortex with the development of hypertension in SHR-SP. These results implicate TGF-beta in the renal damage observed in hypertension.

Attenuated Development of Hypertension by Antioxidant Vitamins in Stroke-Prone Shr Is Associated with Increased Vascular Superoxide Dismutase Activity and Decreased Oxidative Stress.

P34 Oxidative stress has been implicated in the pathophysiology of cardiovascular diseases. We investigated effects of antioxidant vitamins and tempol, superoxide dismutase (SOD) mimetic, on vascular superoxide anion (×O 2 - ) concentration, NADPH oxidase activity, SOD activity, plasma antioxidant levels and blood pressure in stroke-prone SHR (SHRSP). 16 week-old SHRSP on a high salt diet (4%NaCl) were divided into 4 groups: control (C), Vit C (1000mg/day), Vit E (1000 U/day) and tempol (1 mmol/d). Systolic blood pressure (SBP) and antioxidant status were measured weekly for 6 weeks. Aortic ×O 2 - concentration and NADPH oxidase activity were measured by luminometry using lucigenin (5 μmol/L) and aortic SOD activity and total plasma antioxidant status were measured using a spectrophotometric assay system. SBP and plasma antioxidant levels were similar in all groups prior to treatment. Table demonstrates results after 6 weeks treatment. Data are means ± SEM. Data from this study demonstrate that antioxidant vitamins and tempol attenuate the development of hypertension in SHRSP. This is associated with decreased vascular ×O2- production, increased activity of SOD and increased plasma antioxidant concentrations. We demonstrate the novel findings that Vits E and C induce their antioxidant effects, in part, by stimulating SOD activity. Thus increased oxidative stress is an important factor underlying vascular changes associated with severe hypertension in SHRSP.

Altered gene expression of uncoupling protein-2 and -3 in stroke-prone spontaneously hypertensive rats.

Uncoupling proteins (UCPs) are the inner mitochondrial membrane-associated proteins, which dissipate the proton gradient and generate heat instead of ATP. The involvement of UCPs in energy expenditure and glucose metabolism has been suggested. Recently, we succeeded in cloning of rat UCP2 and UCP3. The aim of this study was to elucidate the pathophysiological role of UCP2 and UCP3 in hypertension associated with hyperglycemia in stroke-prone spontaneously hypertensive rats (SHR-SP). UCP2 and UCP3 mRNA levels of cardiac and gastrocnemius muscles in SHR-SP and Wistar-Kyoto (WKY) rats were determined at 6 weeks (prehypertensive stage) and at 15 weeks (hypertensive stage). UCP2 and UCP3 mRNA levels in the heart of SHR-SP at 6 weeks were significantly higher than those of WKY rats (1.6-fold, 3.6-fold, respectively). These tendencies did not change in the heart at 15 weeks. UCP2 and UCP3 mRNA levels in the skeletal muscle of SHR-SP at 6 weeks were significantly higher than those of WKY rats (1.4-fold, 2.4-fold, respectively). In contrast, at 15 weeks, UCP2 and UCP3 mRNA levels in the skeletal muscle of SHR-SP were significantly lower than those of WKY rats (70 and 36% of WKY rats, respectively). Therefore, the decrease of UCP2 and UCP3 in the skeletal muscle was observed with the concomitant development of hypertension in SHR-SP. UCP2 mRNA levels in the epididymal fat of SHR-SP at 15 weeks were similar to that of WKY rats. Altered gene expression of UCP2 and UCP3 might be related to some pathophysiological aspects in hypertension and glucose metabolism in SHR-SP.

Effect of docosahexaenoic acid on smooth muscle cell functions

There is increasing evidence that fish oil-enriched diets attenuate the progression of several types of human and experimental renal, intestinal and cardiovascular disorders, including hypertension. Docosahexaenoic acid (DHA), may be one of the active biological component. We previously reported that dietary DHA suppressed the progression of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). The purpose of this study is to clarify the in vitro effect of DHA on cultured smooth muscle cell functions such as cell growth, hypertrophy, NO release, and intracellular Ca +2 metabolism, which are involved in the regulatory mechanisms of vascular tone. Addition of DHA to the culture medium of aortic smooth muscle cells isolated from SHRSP and normotensive Wistar Kyoto rats (WKY) had no significant effects on cell growth or on cell hypertrophy induced by angiotensin II as measured by flow cytometry. DHA had no stimulatory effect on interleukin-1β (10 mg ml )-induced nitric oxide release from smooth muscle cells of SHRSP, but rather slightly inhibited it. However, the treatment of smooth muscle cells with DHA (30 μM) for 2 days significantly suppressed the increase in intracellular Ca 2+ concentration induced by angiotensin II, but not by thapsigargin. This was due to the suppression of Ca 2+ influx, as determined by Mn 2+ influx experiment. These results indicate that DHA specifically suppresses Ca 2+ mobilization into smooth muscle cells. This may be one of the mechanisms by which dietary DHA prevents the development of hypertension in SHRSP.

Specific modulation of vascular smooth muscle cell functions by docosahexaenoic acid

There are increasing evidences that fish oil-enriched diets attenuate the progression of several types of human and experimental renal, intestinal and cardiovascular disorders including hypertension. Docosahexaenoic acid (DHA) may be one of the active biological component. We previously reported that dietary DHA suppressed the progression of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). The purpose of this study was to clarify the in vitro effect of DHA on vascular smooth muscle cell functions such as cell growth, hypertrophy, NO release, and intracellular Ca+2 dynamics which involves in the regulatory mechanisms of vascular tone. Addition of DHA to the culture medium of aortic smooth muscle cells isolated from SHRSP and normotensive Wistar Kyoto rats (WKY) had no significant effects on the cell growth, and cell hypertrophy induced by angiotensin II as measured by flow cytometer. DHA did not have a significant effect on interleukin-1 beta (10 ng/ml)-induced nitric oxide release from smooth muscle cells of SHRSP. However, the treatment of smooth muscle cells with DHA (30 microM) for 2 days significantly suppressed the increase in the intracellular Ca2+ concentration induced by 5-hydroxytryptamine, angiotensin II, depolarizing concentration of KCl, but not by thapsigargin. This suppression seems to be due to the suppression of Ca2+ influx, as determined by Mn2+ influx experiment. These results suggest that DHA specifically suppresses receptor-mediated Ca2+ influx in smooth muscle cells. This may be one of the mechanisms by which dietary DHA prevents the development of hypertension in SHRSP.

Dietary docosahexaenoic acid (22: 6n-3) prevents the development of hypertension in SHRSP.

1. We previously reported that hypertension in stroke-prone spontaneously hypertensive rats (SHRSP) caused renal membrane phospholipid degradation. Renal phospholipase A2 activity increased and membranous phospholipids decreased along with age in SHRSP. Membranous abnormalities induced by membrane fluidity and calcium permeability changes may contribute to the elevation of blood pressure in SHRSP. DHA, a major component of fish oil, constitutes a part of membrane phospholipid acylchains. 2. The purpose of this study was to clarify the effect of DHA on the relationship between the renal function and the development of hypertension in SHRSP. 3. Six week old male SHRSP were fed a semi-purified diet supplemented with DHA (0, 1 and 5%) for 14 weeks. 4. The systolic blood pressure of control SHRSP (DHA 0%) significantly increased from 120.2 mmHg to 202.9 mmHg. This increase in systolic blood pressure was significantly inhibited in a dose-dependent manner by 1 and 5% DHA diet to 167.8 to 149.8 mmHg, respectively. 5. Serum creatinine concentration and blood urea nitrogen (BUN) were significantly lower in DHA (5%)-treated SHRSP than in the control SHRSP. 6. These results indicate that DHA prevents the development of hypertension in SHRSP, which is associated with changes in renal function.

Genetic linkage of the human gene for phenylethanolamine N-methyltransferase (PNMT), the adrenaline-synthesizing enzyme, to DNA markers on chromosome 17q21-q22.

We have determined the genetic location of the human gene encoding phenylethanolamine N-methyltransferase (PNMT), the terminal enzyme of the catecholamine pathway catalyzing the synthesis of epinephrine (adrenaline) from norepinephrine. This gene is linked to DNA markers on the long arm of chromosome 17, q21-q22, most closely to the DNA markers MFD15 (D17S250) (Zmax = 15.0, theta = 0.065) and fLB17.1 (Zmax = 14.6, theta = 0.045). Multipoint linkage analysis placed the PNMT locus in the interval fLB17.1-CMM86 (D17S74), at 4 centiMorgans (cM) distal to fLB17.1, and at 17 cM proximal to CMM86. Mapping of the PNMT gene will provide the basis for genetic linkage studies in families with disease which might pathogenetically involve this enzyme. The human chromosomal region 17q21-22 identified here to harbour the PNMT gene may be syntenic to the chromosomal region in the stroke-prone spontaneously hypertensive rat (SHR-SP) recently linked to blood-pressure regulation. As an increase of PNMT activity has been associated with the development of hypertension in SHR-SP, it will be of interest to perform comparative mapping of the PNMT gene.

CHLORIDE ION INGESTED WITH SODIUM AFFECTS THE DEVELOPMENT OF CEREBRAL LESIONS IN STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

1. To determine the effect of chloride ion on the development of hypertension and the incidence of cerebral lesions in stroke-prone spontaneously hypertensive rats (SHRSP), groups of 10 rats were administered chronically with either 171 mmol/L sodium chloride or equimolar sodium provided as sodium citrate in the drinking water from the age of 12 weeks. 2. The life span was significantly extended in SHRSP given sodium citrate (336 +/- 28 vs 246 +/- 26 days, mean +/- s.e.m., P less than 0.05) but their development of hypertension was not different from SHRSP given sodium chloride. 3. In order to determine the role of calcium homeostasis, calcium in urine was collected. Urinary calcium in SHRSP given sodium citrate was significantly decreased (1.0 +/- 0.12 vs 1.8 +/- 0.18 mg/24 h urine, P less than 0.05). 4. If the normal life span is 320 +/- 35 days, this suggests that chloride ion ingested with sodium accelerates the development of cerebrovascular diseases, and that increased urinary calcium excretion may be related to this adverse chloride effect on the development of hypertension in SHRSP.

脳卒中易発症ラット（ＳＨＲＳＰ）の血圧および高血圧性臓器病変に対するＢｅｔａｘｏｌｏｌの効果

Effects of betaxolol, a cardioselective beta-adrenoceptor antagonist, on blood pressure and hypertensive complications in stroke-prone spontaneously hypertensive rats (SHRSP) were investigated. Betaxolol was provided in a dose of 33 +/- 1.8 mg/kg/day, orally in drinking water, throughout the experimental period. The chronic treatment with betaxolol inhibited the development of hypertension in SHRSP and reduced values of blood urea nitrogen, creatinine, total cholesterol, free cholesterol, triglyceride, phospholipid and HDL-cholesterol in serum. Treatment with betaxolol apparently inhibited the incidence of hypertensive lesions such as cardiac fibrosis, mesenteric vasculitis, proliferative and/or necrotic vasculitis and glomeruli showing collapse or vasculitis in the kidneys. To shorten the time before the onset of hypertension and the subsequent stroke, SHRSP were kept on a SP diet containing 0.39% Na instead of the F-2 diet. When the SHRSP were kept on the SP diet, all of the control SHRSP had cerebral apoplexy and severe hypertensive lesions in the heart and kidney. When betaxolol was chronically administered to SHRSP, cerebral apoplexy and hypertensive lesions in the heart and kidney were inhibited, but the effect on blood pressure was slight. Treatment with betaxolol reduced serum creatinine levels. Our observations show that betaxolol reduces blood pressure and potently inhibits hypertensive complications in SHRSP.

Effect of Green Tea on the Survival and Development of Hypertension in SHRSP

Effect of Green Tea on the Survival and Development of Hypertension in SHRSP

Effects of chronic alcohol consumption and alcohol withdrawal on blood pressure in stroke-prone spontaneously hypertensive rats

The development of blood pressure was monitored by the tail-cuff method in normotensive (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) receiving ethanol (alcohol) in drinking water from weaning (approximately 1 month of age). Alcohol administration over a 3-month period attenuated the development of hypertension in SHRSP and also caused a small reduction of the initial blood pressure rise in WKY. This was accompanied by a reduction of fluid intake and an increase of circulating antidiuretic hormone (arginine vasopressin; AVP). Circulatory volume remained constant. Direct measurement of arterial blood pressure in conscious rats before and after autonomic blockade confirmed the antihypertensive effect of alcohol in SHRSP and indicated that it is at least partly dependent on altered activity of neural mechanisms. Sudden withdrawal of alcohol caused an immediate increase of fluid intake followed by a rise of blood pressure lasting several days in both WKY and SHRSP. This withdrawal hypertension could not be attributed to changes in plasma catecholamines or AVP.