Spontaneously Hypertensive Rats Cells Research Articles

The antihypertensive effect of remote ischemic conditioning in spontaneously hypertensive rats.

Limb remote ischemic conditioning (LRIC) may be an effective method to control hypertension. This study investigated whether LRIC decreases blood pressure by regulating the hypertensive inflammatory response in spontaneously hypertensive rats (SHR). The SHR and aged-matched Wistar rats with different ages were randomly assigned to the SHR group, SHR+LRIC group, Wistar group, and Wistar + LRIC group. LRIC was conducted by tightening a tourniquet around the upper thigh and releasing it for three cycles daily (10 mins x3 cycles). Blood pressure, the percentage of monocytes and T lymphocytes, and the concentration of pro-inflammatory cytokines in the blood were analyzed. The blood pressure of SHR was significantly higher than that of age-matched Wistar rats. LRIC decreased blood pressure in SHR at different ages (4, 8, and 16 weeks old), but had no effect on the blood pressure in Wistar rats. Flow cytometry analysis showed that blood monocytes and CD8 T cells of SHR were higher than those of Wistar rats. LRIC significantly decreased the percentage of monocytes and CD8 T cells in SHR. Consistent with the changes of immune cells, the levels of plasma IL-6 and TNF-α in SHR were also higher. And LRIC attenuated the plasma IL-6 and TNF-α levels in SHR. LRIC may decreased the blood pressure via modulation of the inflammatory response in SHR.

Loss of SR‐PM Contact Sites May Contribute to The Development of Hypertension in Spontaneously Hypertensive Rats

Sarcoplasmic reticulum (SR)‐plasma membrane (PM) contact sites in vascular smooth muscle (VSM) cells bring the SR Ca2+ release channels into proximity of plasma membrane large‐conductance Ca2+‐activated K+ (BKca) channels and regulate intracellular Ca2+ through BKca‐mediated membrane hyperpolarization. Loss of BKca channel activity leads to increased intracellular Ca2+ concentration, vascular hypercontractility, and activation of hypertrophic genes. We hypothesized that the loss of close contact between the SR and the PM contributes to the development of vascular hypercontractility and hypertrophy in the Spontaneously Hypertensive Rat (SHR). To evaluate this hypothesis, we compared BKca channel activity in response to the activation of inositol 1,4,5‐trisphosphate receptor (IP3R), an SR Ca2+ channel between SHR and normotensive Sprague Dawley (SD) rats. We found that in endothelium‐denuded pressurized mesenteric arteries, paxilline (1μM), a selective BKca channel blocker, significantly increased norepinephrine (NE, 0.5μM)‐induced vasoconstriction in SD arteries (n=5) by ~15% (% initial diameter from 78.58±3.93 to 66.77±3.07). However, paxilline failed to increase NE‐induced vasoconstriction in SHR arteries (Figure 1). Paxilline treatment significantly augmented the hypertrophic effect of angiotensin II (ANG II) in SD, where the total cell area of paxilline treated ANG II group was ~11% (from 3471.48±121.35 to 3854.86±129.99μm2) greater compared to the non‐paxilline‐treated ANG II group. In contrast, the paxilline‐induced increase in hypertrophic response was absent in SHR. Similarly, in WST‐1 cell proliferation assay, paxilline treatment significantly increased ANG II‐induced proliferation in SD by ~15% (Absorbance(A440nm‐A630nm) from 0.15±0.01 to 0.17±0.01) while having no effect on cell proliferation in SHR. Next, we compared the effect of 5‐HT on BKca channel activity between SHR and SD rat mesenteric VSM cells using patch‐clamp whole‐cell mode. 5‐HT activates IP3Rs by stimulating the production of IP3 in VSM cells. At +60mV, 5‐HT (0.3μM) significantly increased BKca current density at +60mV in SD VSM cells from 50.78±2.19 to 69.40±5.50 pA/pF (n=6 cells, P<0.05) but the increase in current density was non‐significant in SHR (from 36.77±3.11 to 47.45±8.83 pA/pF, n=6 cells, Figure 2). We also examined the Ca2+‐ and voltage‐sensitivity of BKca channels using inside‐out patch‐clamp. BKca channels in VSM cells isolated from mesenteric arteries of SHR and SD rats displayed identical Ca2+‐ and voltage‐sensitivity. In conclusion, our data suggest a lack of BKca activation in response to IP3R activation in VSM cells ofSHR, which may be due to the loss of close contact between the SR and the PM.

Sex differences in TLR4 expression in SHR do not contribute to sex differences in blood pressure or the renal T cell profile.

Hypertension is a primary risk factor for the development of cardiovascular disease. Mechanisms controlling blood pressure (BP) in men and women are still being investigated; however, there is increasing evidence supporting a role for the innate immune system. Specifically, Toll-like receptors (TLRs), and TLR4 in particular, have been implicated in the development of hypertension in male spontaneously hypertensive rats (SHR). Despite established sex differences in BP control and inflammatory markers in hypertensive males and females, little is known regarding the role of TLR4 in hypertension in females. Our hypotheses were that male SHR have greater TLR4 expression compared with females, and that sex differences in TLR4 contribute to sex differences in BP and the T cell profile. To test these hypotheses, initial studies measured renal TLR4 protein expression in 13-wk-old male and female SHR. Additional SHR were implanted with telemetry devices and randomized to treatment with either IgG or TLR4 neutralizing antibodies. Untreated control male SHR have greater TLR4 protein expression in the kidney compared with females. However, treatment with TLR4 neutralizing antibody for 2 wk did not significantly alter BP in either male or female SHR. Interestingly, neutralization of TLR4 increased renal CD3+ T cells in female SHR, with no alteration in CD4+ T cells or CD8+ T cells in either sex. Taken together, our data indicate that although male SHR have greater renal TLR4 expression than females, TLR4 does not contribute to the higher BP and more proinflammatory renal T cell profile in males versus females.

NAP-22 and MARCKS mRNA expression levels in spontaneously hypertensive rat kidneys: the effect of drinking water with different calcium contents

Objective. The aim of study was to assess the effect of the level of calcium intake with drinking water on NAP-22 and MARCKS mRNA expression in cortical and medullar kidney layers of spontaneously hypertensive rats.Design and methods. The study involved 90-day-old SHR (n = 8) and WKY (n = 8) strain rats of both sexes. We assessed tissue samples from cortical and medullar kidney layers. NAP-22 and MARCKS mRNA expression levels were determined by RT-PCR.Results. Sufficient drinking water calcium intake was associated with similar the expression of NAP-22 and MARCKS mRNA in kidneys of spontaneously hypertensive and normotensive rats. Consumption of drinking water with insufficient calcium content it decreases in both rat strains, being more evident in spontaneously hypertensive rats, especially in the medullar layer.Conclusions. Our results show that genetically determined impairments of calcium metabolism in cells of spontaneously-hypertensive rats (SHR line) and their effect on intracellular signaling processes are more evident with the reduced intake of exogenous calcium.

NAP-22 and MARCKS mRNA expression levels in spontaneously hypertensive rat kidneys: the effect of drinking water with different calcium contents

Objective. The aim of study was to assess the effect of the level of calcium intake with drinking water on NAP-22 and MARCKS mRNA expression in cortical and medullar kidney layers of spontaneously hypertensive rats.Design and methods. The study involved 90-day-old SHR (n = 8) and WKY (n = 8) strain rats of both sexes. We assessed tissue samples from cortical and medullar kidney layers. NAP-22 and MARCKS mRNA expression levels were determined by RT-PCR.Results. Sufficient drinking water calcium intake was associated with similar the expression of NAP-22 and MARCKS mRNA in kidneys of spontaneously hypertensive and normotensive rats. Consumption of drinking water with insufficient calcium content it decreases in both rat strains, being more evident in spontaneously hypertensive rats, especially in the medullar layer.Conclusions. Our results show that genetically determined impairments of calcium metabolism in cells of spontaneously-hypertensive rats (SHR line) and their effect on intracellular signaling processes are more evident with the reduced intake of exogenous calcium.

The abnormalities of adrenomedullary hormonal system in genetic hypertension: Their contribution to altered regulation of blood pressure.

It is widely accepted that sympathetic nervous system plays a crucial role in the development of hypertension. On the other hand, the role of adrenal medulla (the adrenomedullary component of the sympathoadrenal system) in the development and maintenance of high blood pressure in man as well as in experimental models of hypertension is still controversial. Spontaneously hypertensive rats (SHR) are the most widely used animal model of human essential hypertension characterized by sympathetic hyperactivity. However, the persistence of moderately elevated blood pressure in SHR subjected to sympathectomy neonatally as well as the resistance of adult SHR to the treatment by sympatholytic drugs suggests that other factors (including enhanced activity of the adrenomedullary hormonal system) are involved in the pathogenesis of hypertension of SHR. This review describes abnormalities in adrenomedullary hormonal system of SHR rats starting with the hyperactivity of brain centers regulating sympathetic outflow, through the exaggerated activation of sympathoadrenal preganglionic neurons, to the local changes in chromaffin cells of adrenal medulla. All the above alterations might contribute to the enhanced release of epinephrine and/or norepinephrine from adrenal medulla. Special attention is paid to the alterations in the expression of genes involved in catecholamine biosynthesis, storage, release, reuptake, degradation and adrenergic receptors in chromaffin cells of SHR. The contribution of the adrenomedullary hormonal system to the development and maintenance of hypertension as well as its importance during stressful conditions is also discussed.

The antihypertension effect of hydrogen sulfide (H2S) is induced by activating VEGFR2 signaling pathway

AimsPrevious studies demonstrated that H2S has an antihypertension effect on hypertension, but the mechanism involved is unclear until now. The aim of the study is to elucidate the effect of H2S on PH and the mechanism involved. Main methodsIn this study, GYY4137 (a H2S donor) were administered to spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) by intraperitoneally injection daily for consecutive 14 days. Systolic blood pressure (SBP), endothelial-dependent relaxation (EDR), plasma malondialdehyde (MDA), superoxide dismutase (SOD), and H2S levels were measured. Human umbilical vein endothelial cells (HUVECs) were also used to elucidate the mechanism involved in the protect effect of H2S on the injured vessels. Key findingsOur results showed that GYY4137 normalized the SBP (P < 0.0001), increased EDR (P < 0.01), reduced oxidative stress (increased the content of SOD and reduced the content of MDA) of SHR. Meanwhile, GYY4137 could promote the proliferation (P < 0.01) and migration (P < 0.01) of HUVECs, increase the expression of endothelial NO synthase (eNOS) and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) both in SHR and HUVECs treated with GYY4137. In addition to the above results, the PIP3/Akt signaling pathway was activated and the expression of caspase 3 was increased by GYY4137. However, all the above effects of GYY4137 were blocked by ZD6474 (a VEGFR2 inhibitor). SignificanceGYY4137 had a hypotensive and vascular protect effect on PH. This effect might be mediated through upregulating the expression of VEGFR2, which subsequently alleviating oxidant-provoked vascular endothelial dysfunction, and promoting the proliferation and migration of endothelial cells in SHR.

Adenosine A1 and A2a receptors modulate the nitrergic system in cell culture from dorsomedial medulla oblongata.

Adenosine and nitric oxide act on the fine-tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). Although the interaction between adenosine and NO is well known in the periphery, the mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, related to neural control of circulation, are not completely understood and might be relevant for individuals predisposed to hypertension. In this study we evaluate the interaction between adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Using quantification of nitrite levels, RT-PCR analysis and RNA interference we demonstrate that adenosine A1 (A1R) and A2a receptor (A2aR) agonists induce a concentration-dependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells from WKY and SHR, respectively. These effects in nitrite levels are attenuated by the administration of A1R and A2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A1R and A2aR show an increase and decrease of nNOS mRNA levels, respectively. Pretreatment with the nonselective inhibitor of NOS, L-NAME, abolishes nitrite-increased levels triggered by CGS 21680 in WKY and SHR cells. Finally, it is shown that the cAMP-PKA pathway is involved in A1R and A2aR-mediated decrease and increase in nitrite levels in SHR and WKY cells. Our results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of neonate WKY and SHR rats. In part, the modulatory profile is different in the SHR strain.

Cannabinoid type 2 receptor agonist JWH133 decreases blood pressure of spontaneously hypertensive rats through relieving inflammation in the rostral ventrolateral medulla of the brain.

Neuroinflammation in the rostral ventrolateral medulla (RVLM) has been reported to be associated with hypertension. The upregulation and activation of the cannabinoid type 2 (CB2) receptor may be part of the active process of limiting or downregulating the inflammatory process. This study was designed to determine the role of the CB2 receptor in blood pressure (BP) through relieving neuroinflammation in the RVLM in spontaneously hypertensive rats (SHRs). The long-term effects of intracerebroventricular injection of JWH133, a selective CB2 receptor agonist, on BP, heart rate (HR) and renal sympathetic nerve activity (RSNA) in SHR and Wistar-Kyoto (WKY) rats were determined. ELISA was used to measure the levels of proinflammatory cytokines, and western blotting was employed to detect protein expression of the CB2 receptor. Immunofluorescence staining was used to localize the CB2 receptor. Gene silencing of the CB2 receptor was realized by injecting adeno-associated virus (AAV) expressing CB2-specific shRNA (AAV2-r-CB2shRNA) into the RVLM. We found that SHRs exhibited higher levels of basal BP, HR, RSNA and proinflammatory cytokines (TNFα, IL-6 and IL-1β) than those in WKY rats. The protein level of the CB2 receptor in the RVLM was robustly increased in SHRs. In addition, the CB2 receptor was mainly expressed on microglia cells of SHRs but not in WKY rats. No expression of the CB2 receptor was found on neurons of either WKY rats or SHRs. Furthermore, intracerebroventricular injection of JWH133 (1 mmol/l, 10 μl) for 28 days decreased the BP, HR, RSNA and proinflammatory cytokines significantly in SHRs, but it had no such effects in WKY rats. These effects were abolished by microinjection of 300 nl AAV2-r-CB2shRNA into the RVLM to knock down the CB2 receptor. Taken together, our results suggest that exciting the CB2 receptor relieves proinflammatory cytokine levels in the RVLM to decrease the BP, HR and RSNA in SHRs.

Progressive Myocardial Apoptosis and Cardiac Dysfunction in Spontaneously Hypertensive Rats During Aging

Abstract Objective To investigate myocardial apoptosis and the expression of apoptosis-related proteins [B-cell lymphoma-2 (Bcl-2) and Bax], cardiac hypertrophy, and function in spontaneously hypertensive rats (SHR) during aging. Methods Male SHR (n = 36) and Wistar-Kyoto rats (WKY, n = 30) were monitored for their cardiac function from 14 to 102 weeks of age using echocardiography. Myocardial apoptosis in the subendocardium and subepicardium was analyzed using TUNEL staining. The apoptosis-related proteins (Bcl-2 and Bax) were assessed by Western blot analysis. Results Left ventricular mass index (LVMI) in SHR increased progressively with age. Left ventricular ejection fraction (LVEF) showed no significant difference from 14 to 66 weeks of age, but decreased significantly in SHR from 84 to 102 weeks of age. The apoptotic index (APOI) of myocardial cells in SHR increased with age, in which subendocardial APOI (APOI-endo) increased at 66 weeks and was significantly higher than the age-matched WKY (9.83 ± 3.97 vs. 4.03 ± 2.06, P &amp;lt; 0.05). Additionally, the subepicardial APOI (APOI-epi) increased significantly at 84 weeks of age (P &amp;lt; 0.05). Bax/Bcl-2 ratio was higher in SHR than WKY at 66 weeks (3.98 ± 0.80 vs. 0.29 ± 0.06, P &amp;lt; 0.05), and enhanced further at 102 weeks of age (10.54 ± 1.60 vs. 0.70 ± 0.13, P &amp;lt; 0.05). Myocardial APOI was positively correlated with LVMI (r = 0.83, P &amp;lt; 0.01), negatively with LVEF in 66–102 weeks (r = −0.81, P &amp;lt; 0.01), and positively with Bax/Bcl-2 ratio (r = 0.79, P &amp;lt; 0.01). Conclusion Myocardial apoptosis increases with aging and is associated with progressive left ventricular remodeling and cardiac dysfunction in SHR.

Role of complement 3 in the pathogenesis of hypertension.

The complement system has recently been reported to contribute to the development and pathogenesis of hypertension, several cardiovascular and renal diseases, and cardiometabolic disorders accompanied by inflammation and tissue remodeling. We have demonstrated that complement 3 (C3) is highly expressed in mesenchymal tissues in spontaneously hypertensive rats (SHRs) and induces the synthetic phenotype and exaggerated growth of mesenchymal cells by maintenance effect on dedifferentiated cells. To verify the role of C3 in the pathogenesis of hypertension, we targeted the C3 gene from SHRs by zinc-finger nuclease gene-editing technology and demonstrated that the increased expression of C3 induces salt-sensitive hypertension with activation of the renal renin-angiotensin system in SHRs. We recently found that increased expression of C3 is associated with the suppression of miR145 and induces Krüppel-like factor 5 and the synthetic phenotype of mesenchymal cells in SHRs. We also demonstrated that C3 is involved in the epithelial-to-mesenchymal transition and dedifferentiation of epithelial cells in kidneys subjected to unilateral ureteral obstruction with elevation of blood pressure. Thus, C3 is an essential factor in the pathogenesis of hypertension due to its maintenance effect on undifferentiated mesenchymal cells.

P6376The role of circulating lymphocytes on hypertensive heart disease

Abstract Aims and background Hypertension is a disease accompanied by moderate inflammation. Physical activity and pharmacological intervention protect the organisms against hypertension-dependent end-organ damage. They should therefore interfere with the mobilization of lymphocytes. Different subsets of lymphocytes should specifically interfere with vascular and cardiac remodeling. In this study we correlated the amount of circulating lymphocytes with the expression of vascular and cardiac specific genes in the aorta and left ventricle to identify the tissue-specific effects of these cells. Methods 50 female spontaneously hypertensive rats (SHR) and 6 female normotensive Wistar rats (Wis) were analyzed. They were grouped in 7 subgroups exposed to different treatment regimes (modification of activity, type of activity, aldosterone blockade, and a combination thereof. Indicator genes of mal-adaptive vascular and cardiac remodeling were quantified by qRT-PCR. Results SHRs had an increased number of circulating NK cells and monocytes, but lower numbers of T cells. T cells inversely correlated with the vascular expression of p22phox, a member of the NADPH oxidase complex. Thus, lower number of T-cells favors the vascular expression of p22phox and thereby oxidative stress. In the left ventricle, T cells are inversely correlated with the expression of arginase-1 and UCP-2 but positively correlated with Glut-4 expression. The lower number of T cells in SHR therefore favors the expression of proteins often associated with oxidative stress and glucose metabolism. Monocytes are positively correlated with key markers of left ventricular hypertrophy (such as ANP, collagen-1, and β-MHC). Thus, the increased number of monocytes in SHRs supports cardiac hypertrophy. NK cells are inversely correlated with the expression of anti-inflammatory cytokines in the aorta (IL-10, IL-15). Thus, high number of circulating NK cells seems to favor a pro-inflammatory phenotype in the vasculature. Conclusion In a standard model of essential hypertension the profile of circulating lymphocytes differs from that in normotensive rats. The different subsets of lymphocytes specifically interfere with specific processes in the ventricle and vessel. Collectively, the difference between hypertensive and normotensive rats (less T cells, more NK cells and monocytes) supports a mal-adaptive role under hypertensive conditions.

Apoptosis contributes to the pro‐inflammatory T cell profile in blood of male and female spontaneously hypertensive rats, but not the control of blood pressure

It is well established that cell death is a hallmark of hypertension. Apoptosis is a controlled physiologic form of cell death in which dying cells are cleared via phagocytosis limiting further tissue inflammation and damage. We have previously shown that spontaneously hypertensive rats (SHR) have greater apoptosis compared to age matched normotensive Wistar Kyoto rats. We also showed that there is a sex difference in renal cell death in SHR with females having greater renal apoptosis compared to age matched male SHR. The contribution of apoptosis to the control of blood pressure (BP) in established hypertension is unknown, however, 13 week old female SHR have a lower BP and a less pro‐inflammatory T cell profile compared to males. The goal of the current study was to test the hypothesis that greater apoptosis in female SHR contributes to the lower BP and anti‐inflammatory T cell profile vs. male SHR. Male (M) and female (F) SHR were randomized to receive vehicle (Veh) or ZVAD‐FMK, a cell permeable, non‐selective apoptosis inhibitor, from 13–15 weeks of age (1 mg/kg via IP injection 5 days/week; n=4–8) and BP was measured via telemetry. Following 2 weeks of treatment, kidney and blood were collected to measure apoptosis and the T cell profile by flow cytometry. Treatment with ZVAD for 2 weeks reduced renal apoptosis in both male and female SHR and abolished the sex difference in renal apoptosis (expressed as % total kidney cells‐ M Veh: 2.2±0.3 vs M ZVAD: 1.2±0.4; F Veh: 4.5±0.9 vs F ZVAD: 0.7±0.2; Effect of ZVAD: P&lt;0.0001, Effect of sex: P=0.082, Interaction: P=0.0075). Inhibition of apoptosis did not alter BP in either male or female SHR (BP in mmHg in M ZVAD: 143±5; M Veh: 145±4 F ZVAD: 139±2; F Veh: 140±3 mmHg). However, inhibition of apoptosis decreased the numbers of circulating CD3+ T cells in both male (expressed as % total blood cells‐ M Veh: 12.2±1% vs M ZVAD: 10.3±1.1%) and female SHR (F Veh: 14.7±1.3% vs F ZVAD: 11.8±1.3%; Effect of ZVAD: P=0.07, Effect of sex: P=0.096, Interaction: P= 0.75). Inhibition of apoptosis also increased Tregs in female SHR (expressed as %CD3+CD4+FoxP3+ cells‐ F Veh: 6.3±0.3 vs F ZVAD: 8.1±0.1) with no effect in males (M Veh: 7.5±0.4% vs M ZVAD: 7.6±0.6%; Effect of ZVAD: P=0.025, Effect of sex: P=0.48, Interaction: P=0.041). The renal T cell profile was not altered by inhibition of apoptosis. Taken together, our data suggest that while apoptosis does not contribute to the control of BP in established hypertension in either male or female SHR, it does contribute to the pro‐inflammatory T cell profile in the blood of both sexes. Further studies will determine the contribution of apoptosis to the maturation induced increases in BP in SHR prior to the development of established hypertension.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A8234 Role of GRK4 in the regulation of renal ETB receptor in hypertension

Objectives: To study the role of G protein-coupled receptor kinase 4 (GRK4) in the regulation of endothelin receptor B (ETBR) in hypertension. Methods: Male anaesthetic spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were used to test ETBR-mediated natriuresis and diuresis. The ETBR agonist, BQ-3020 (0.1, 0.5, 1.0Ymg/kg/min) were infused via supra-renal artery at a rate of 0.04Yml/min for 40Ymin. The same experiments were conducted in GRK4 A142 V transgenic mice to confirm the relationship between GRK4 and ETBR hyper-phosphorylation. Co-immunoprecipitation and confocal microscopy were conducted to test interaction between GRK4 and ETBR. Results: Activation of ETBR induced natriuresis and diuresis in WKY rats, which was impaired in SHRs. ETBR phosphorylation was higher in SHRs as compared with WKY rats, although ETBR expression had no difference in kidneys from WKY and SHRs. Activation of ETBR induced natriuresis in wild-type mice, however, in GRK4 transgenic mice, the ETBR-mediated natriuresis and diuresis was lost, accompanied with higher ETBR hyper-phosphorylation. There were co-localization and co-immunoprecipitation between ETBR and GRK4 in RPT cells both from WKY and SHRs, the ETBR/GRK4 linkage was higher in SHR cells than in WKY cells. Down-regulation of GRK4 with siRNA recovered ETBR function, which is pathophysiological significance, because down-regulation of renal GRK4 expression via ultrasound-targeted microbubble destruction restored ETBR-mediated natriuresis and diuresis in SHRs, accompanied with lowered ETBR phosphorylation. Conclusion: This study provides a mechanism by which GRK4, via regulation of renal ETBR function, participates in the pathogenesis of hypertension.

IcarisideII improves left ventricular remodeling in spontaneously hypertensive rats by inhibiting the ASK1-JNK/p38 signaling pathway

Inhibition or removal of excess reactive oxygen species can effectively protect cellular function or reduce cell death because oxidative stress is the main cause of cellular damage in many diseases. The flavonoid compound IcarisideII having a slight inhibitory effect on PDE5, is the main active components of epimedium in vivo and has a wide range of pharmacological effects on oxidation and apoptosis. However, whether IcarisideII has the same protective effect on ventricular remodeling in spontaneously hypertensive rats (SHR) is unknown. We found that compared with WKY rats, SHRs exhibited noticeable arterial hypertension. Additionally, echocardiography showed that the diameter of the left ventricle was enlarged, wall thickness was increased, and ejection fraction and short axis shortening rate were reduced. H&E staining demonstrated that SHR cells were disordered and noticeably hypertrophic. Masson trichrome staining revealed significant myocardial fibrosis in the myocardium. Tunel staining indicated that 4.39 times the percent of apoptotic cells were present in SHRs compared to WKY rats. In our study, intra-gastric administration of IcarisideII decreased blood pressure, promoted heart function recovery and improved ventricular remodeling in SHRs. Additionally, it reduced myocardial fibrosis, inhibited myocardial apoptosis, decreased the generation of reactive oxygen species and improved SOD activity. IcarisideII down-regulated the activation of the oxidative stress associated proteins ASK1, p38 and JNK; inhibited the expression of p53, Bax and cleaved-caspase3 in the mitochondrial apoptosis pathway; and up-regulated the expression of Bcl-2. In conclusion, this study indicates that IcarisideII can inhibit myocardial apoptosis and improve left ventricular remodeling in SHRs. It can be inferred that this mechanism may be related to the inhibition of the ASK1-JNK/p38 signaling pathway.

Short hairpin RNA interference targeting interleukin 1 receptor type I in the paraventricular nucleus attenuates hypertension in rats.

Blood pressure is controlled by tonic sympathetic activities, excessive activation of which contributes to the pathogenesis and progression of hypertension. Interleukin (IL)-1β in the paraventricular nucleus (PVN) is involved in sympathetic overdrive and hypertension. Here, we investigated the therapeutic effects of IL-1 receptor type I (IL-1R1) gene silencing in the PVN on hypertension. Recombinant lentivirus vectors expressing a short hairpin RNA (shRNA) targeting IL-1R1 (Lv-shR-IL-1R1) or a control shRNA were microinjected into PVN of spontaneously hypertensive rats (SHRs) and normotensive WKY rats. The fluorescence of green fluorescent protein-labelled vectors appeared at 2weeks after injection and persisted for at least 8weeks. IL-1R1 protein expression in the PVN was reduced 4weeks after Lv-shR-IL-1R1 injection in SHRs. IL-1R1 interference also reduced basal sympathetic activity, cardiac sympathetic afferent reflex in SHRs. Depressor effects were observed from week 2 to 10 after Lv-shR-IL-1R1 treatment in SHRs, with the most prominent effects seen at the end of week 4. Furthermore, Lv-shR-IL-1R1 treatment decreased the ratio of left ventricular weight to body weight and cross-sectional areas of myocardial cells in SHRs. Additionally, Lv-shR-IL-1R1 treatment prevented an increase in superoxide anion and pro-inflammatory cytokines (PICs, TNF-α and IL-1β) in the PVN of SHR, and upregulated anti-inflammatory cytokine (AIC, IL-10) expression. These results indicate that shRNA interference targeting IL-1R1 in the PVN decreases arterial blood pressure, attenuates excessive sympathetic activity and cardiac sympathetic afferent reflex, and improves myocardial remodelling in SHRs by restoring the balance between PICs and AICs to attenuate oxidative stress.

Moringa oleifera-rich diet and T cell calcium signaling in spontaneously hypertensive rats.

Moringa oleifera is a plant whose fruits, roots and leaves have been advocated for traditional medicinal uses. The physicochemical analysis shows that Moringa oleifera contains more dietary polyunsaturated fatty acids (PUFA) than saturated fatty acids (SFA). The consumption of an experimental diet enriched with Moringa oleifera extracts lowered blood pressure in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto (WKY) rats as compared to rats fed an unsupplemented control diet. Anti-CD3-stimulated T cell proliferation was diminished in both strains of rats fed the Moringa oleifera. The experimental diet lowered secretion of interleukin-2 in SHR, but not in WKY rats compared with rats fed the control diet. Studies of platelets from patients with primary hypertension and from SHR support the notion that the concentration of intracellular free calcium [Ca(2+)](i) is modified in both clinical and experimental hypertension. We observed that the basal, [Ca(2+)](i) was lower in T cells of SHR than in those of WKY rats fed the control diet. Feeding the diet with Moringa oleifera extracts to WKY rats did not alter basal [Ca(2+)](i) in T cells but increased basal [Ca(2+)](i) in SHR. Our study clearly demonstrated that Moringa oleifera exerts antihypertensive effects by inhibiting the secretion of IL-2 and modulates T cell calcium signaling in hypertensive rats.

Abstract P217: Nox Compartmentalization, Protein Oxidation and ER Stress in Vascular Smooth Muscle Cells in Hypertension

In hypertension, activation of NADPH oxidases (Noxs) is associated with oxidative stress and vascular dysfunction. The exact role of each isoform in hypertension-associated vascular injury is still unclear. We investigated the compartmentalization of Noxs in VSMC from resistance arteries of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Expression of Nox1 and Nox4 was increased in SHR cells (96.6±28.7% and 48.2±21.2% vs WKY, p&lt;0.05), as well as basal ROS levels measured by chemiluminescence (110.2±26.4% vs WKY, p&lt;0.05) and amplex red (105.2±33.2% vs WKY, p&lt;0.05). Phosphorylation of unfolded protein response activators, PERK and IRE1α, and expression of ER chaperone BiP were elevated in SHR cells (p&lt;0.05 vs WKY), indicating activation of ER stress response. Immunoblotting after organelle fractionation demonstrated that Noxs are expressed in an organelle-specific manner, with Nox1, 2 and 4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHR cells, NoxA1ds (Nox1 inhibitor, 10μM) and GKT136901 (Nox1/4 inhibitor, 10μM) decreased AngII-induced ROS levels (p&lt;0.001 vs Ctl). Additionally, mito-tempol (mitochondrial-targeted antioxidant, 50nM) and 4-PBA (ER stress inhibitor, 1mM) decreased basal ROS levels in SHR cells (p&lt;0.05 vs Ctl). Furthermore, oxidation of the antioxidant enzymes Peroxiredoxins (Prx) was increased in SHRSP compared to WKY (2.51±0.14 vs 0.56±0.07, p&lt;0.001). One-dimensional isoelectric focusing revealed that cytosolic Prx2 and mitochondrial Prx3 were more oxidized in SHRSP than WKY cells. Using a biotin-tagged dimedone-based probe (DCP-Bio) we identified oxidation of ER stress proteins BiP and IRE1. To investigate the effect of protein oxidation in vascular function, vascular reactivity was evaluated in isolated mesenteric arteries. Inhibition of general oxidation (DTT 1mM; Emax: 111.7±33.1) and peroxiredoxin (Conoidin A 10nM; Emax: 116.0±7.3) reduces vascular contraction in response to noradrenalin in WKY rats (Emax: 166.6±30.2; p&lt;0.05). These findings suggest an important role for Nox1/4 in redox-dependent organelle dysfunction and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.

Effects of RMF on BKCa and Kv channels in basilar arterial smooth‑muscle cells of SHR.

The current study observed the effects and investigated the mechanism of remifentanil (RMF) on the isolated cerebral basilar arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. A pressure myograph system was used to observe and compare the effects of different concentrations of RMF (10−10-10−5 mol/l) on the diameter changes of freshly isolated cerebral basilar arteries, which have been pre-shrunk by phenylephrine (PE), an endothelium-independent vasoconstrictor. Vascular smooth-muscle cells of the cerebral basilar artery (BASMCs) were freshly obtained via enzymolysis. BKCa (large-conductance calcium-activated potassium channels) current (IBKCa) and Kv (voltage-gated potassium channels) current (IKv) were recorded using a whole-cell patch-clamp technique. The changes in IBKCa and IKv produced by different concentrations of RMF (10−10 to 10−5 mol/l) on the two types of rats with the holding potential of −40 mV were observed and compared. The cerebral basilar arteries of the SHR and WKY rats were relaxed by RMF in a concentration-dependent manner (P<0.05; n=5). At the same concentration, the diastolic effect of RMF on SHR was weaker than that observed in WKY rats (P<0.05, n=5). When the rats were pre-perfused with 10−3 mol/l of the BKCa channel blocker tetraethylammonium (TEA), the diastolic amplitudes of RMF in SHR and WKY rats were decreased, and the fitting curves shifted down (P<0.05; n=7 and 6, respectively). However, no statistically significant difference was observed with 10−3 mol/l of the Kv channel blocker 4-aminopyridine (4-AP; n=6 and 9, respectively; P>0.05). Outward currents were increased by RMF in both BASMCs of SHR and WKY rats in a voltage- and dose-dependent manner (P<0.05; n=6). At the same concentration, the effect of RMF on the outward currents in BASMCs of WKY rats was stronger than that on SHR (P<0.05; n=6). The enhancing effect of RMF can be partially blocked by either 10−3 mol/l TEA (P<0.05; n=6) or 10−3 mol/l 4-AP (P<0.05 or 0.01; n=6 and 9, respectively) however can be totally blocked by the mixture of TEA and 4-AP (P<0.05, n=7). RMF served a diastolic role in the cerebral basilar arteries of rats in a dose-dependent manner, likely by activating the BKCa and Kv channels. However, SHR demonstrated a less pronounced diastolic reaction to RMF than that observed in WKY rats.

Effect of D3 dopamine receptor on dopamine D4 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto rats and spontaneously hypertensive rats.

Dopamine receptors induce natriuresis in kidney. Previous studies have shown interactions between different subtypes of dopamine receptors in renal proximal tubule (RPT) cells. We hypothesize that D3 receptors have an interaction with D4 receptors in RPT cells from normotensive rats (Wistar-Kyoto, WKY) and spontaneously hypertensive rats (SHRs). Immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine the expression of D3 and D4 receptors. Na-K-ATPase activity was used to measure the function of receptors. The distribution and colocalization of D3 and D4 receptors were detected by confocal microscopy and co-immunoprecipitation. D3 receptor agonist PD128907 increased the mRNA and protein expression of D4 receptors in RPT cells from WKY rats, but decreased that from SHRs. In the presence of PLC blocker (U73122, 10-mol/l) or PKC inhibitor 19 -31 (10-mol/l), the up-regulation of D3 receptor on D4 receptor was lost in WKY cells. Moreover, stimulation with PD128907 for 30 minutes decreased D4 receptor degradation in WKY cells, not in SHR cells. D3 and D4 receptors colocalized and co-immunoprecipitated in RPT cells. PD128907 increased co-immunoprecipitation of D3 and D4 receptors in WKY RPT cells, but not in SHR RPT cells. Pre-treatment with D3 receptor agonist also increases D4 receptor mediated inhibitory effect on Na-K-ATPase activity in WKY cells, but not in SHR cells. Renal D3 receptor regulates the expression and function of D4 receptor in RPT cells via PLC /PKC signaling pathway, the loss of this interaction might be involved in the pathogenesis of hypertension.