Deoxycorticosterone Acetate Rats Research Articles

The Temporal Effects of DOCA‐Salt on the Renal T Cell Profile is Sex‐Specific

There is growing evidence for a role for T cells in the pathogenesis of hypertension. Previous studies by our laboratory show that hypertensive males have more pro‐inflammatory Th17 cells while females have more anti‐inflammatory T regulatory cells (Tregs); we propose that greater Tregs in females contribute to their lower blood pressure (BP) compared to males. The goal of the current study was to test the hypothesis that Tregs preferentially increase over time in female rats with increases in BP, while Th17 cells will increase to a greater extent in males. Male (M) and female (F) uni‐nephrectomized Sprague Dawley rats (10 weeks of age) were subcutaneously implanted with a deoxycorticosterone acetate (DOCA) pellet (200 mg/rat, 60‐day time release) and given 0.9% NaCl to drink. Subsets of rats were killed following 1, 2, and 3 weeks (wks) of DOCA treatment and kidneys were processed for flow cytometric analysis of T cells; n=6/group per time point. BP was measured in separate DOCA rats via telemetry (n=5–6). DOCA increased BP in both M (basal: 115±4, wk1: 146±3, wk2: 187±2, wk3: 190±2 mmHg; p<0.01) and F rats (basal: 105±3, wk1: 134±5, wk2: 165±8, wk3: 175±8 mmHg; p<0.01). BP was comparable between the sexes at baseline (p=0.2), wk 1 (p=0.07) and wk 3 (p=0.1); M had a higher BP than F at wk 2 (p=0.03). At all time points, females had more Tregs than males, and males had more Th17 cells (effect of sex: p<0.05 for all comparisons). Neither Tregs nor Th17 cells were altered vs. control following 1 wk of DOCA treatment (effect of treatment: p=0.9 for both). After 2 wks of DOCA treatment, Tregs decreased in treated males vs. control but were maintained in females (interaction: p=0.06) and Th17 cells increased in both sexes, although the increase was greater in males (effect of DOCA: p<0.001; interaction: p=0.02). Finally, at wk 3 of treatment, DOCA increased Tregs (effect of DOCA: p<0.01) and Th17 cells (effect of DOCA: p=0.03) in both sexes. We propose that the sex‐specific effects of DOCA on T cells at wk 2 contribute to sex differences in BP. The ability of females to maintain Tregs and thereby limit increases in effector Th17 cells at this time point may be a critical pathway by which they are able to maintain a lower BP relative to males.Support or Funding InformationR01HL127091‐02 Male Control Male DOCA Female Control Female DOCA Tregs: Wk 1 4±0.5 4±0.6 5±0.6 5±0.5 Tregs: Wk 2 5±0.8 3±0.5* 6±0.9 7±0.7 Tregs: Wk 3 2±0.3 4±0.3* 4±0.5 7±0.6* Th17 cells: Wk 1 16±2 17±1 11±1 11±1 Th17 cells: Wk 2 6±0.6 27±3* 4±0.5 17±1* Th17 cells: Wk 3 17±1 21±3* 11±2 17±3* All data are expressed as percent of CD3+CD4+ cells; indicates p<0.05 vs. same sex control.

Diuretics prevent Rho-kinase activation and expression of profibrotic/oxidative genes in the hypertensive aortic wall.

Diuretics are current antihypertensive drugs since they reduce blood pressure and cardiovascular risk. Increased vascular tone is modulated in a relevant way by the RhoA/Rho-kinase (ROCK) pathway, by acting on vascular smooth muscle cell contraction. This pathway has also proremodeling vascular effects. There are few data on the role of diuretics on both vascular ROCK activation and on proremodeling effects. We assessed the effects of hydrochlorothiazide (HCTZ) and spironolactone (spiro) alone and in combination with the ROCK inhibitor fasudil (FAS) on ROCK activation, gene expression of proremodeling markers and on hypertrophy in the aortic wall of hypertensive rats. Deoxycorticosterone acetate (DOCA)-salt hypertensive rats (male, Sprague-Dawley) were randomized to the specific ROCK inhibitor FAS, HCTZ, spiro or the combinations of FAS/HCTZ or FAS/spiro for 3 weeks. At the end of the study, ROCK activation (by western blot), gene expression of proremodeling markers (by reverse transcription polymerase chain reaction, RT-PCR) and vascular hypertrophy (by morphometry) were determined in the aortic wall. All treatments significantly reduced blood pressure. In the DOCA rats the p-myosin phosphatase target protein-1 (MYPT1)/t-MYPT1 ratio, index of ROCK activation was higher by 2.8 fold (p < 0.05) compared with control rats. All treatments reduced ROCK activation in the aortic wall to control levels (p < 0.05). Besides, significantly increased protein levels of transforming growth factor β1 (TGF-β1), gene expression of TGF-β1, connective tissue growth factor (CTGF), p22 phox and gp91 phox subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as well as increased media thickness and aortic media area/lumen area (AM/LA) in the untreated hypertensive rats were significantly reduced (p < 0.05) to control levels by all treatments. Similar effects were observed using both diuretics alone or in combination with FAS. In the aortic wall, both HCTZ and spiro in antihypertensive doses reduce ROCK activation, subsequent expression of genes that promote vascular remodeling and hypertrophy in this experimental model of hypertension. These effects could explain some of their clinical benefits in hypertensive patients.

CS-3150, a Novel Nonsteroidal Mineralocorticoid Receptor Antagonist, Shows Preventive and Therapeutic Effects On Renal Injury in Deoxycorticosterone Acetate/Salt-Induced Hypertensive Rats.

The present study was designed to assess both preventive and therapeutic effects of (S)-1-(2-Hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl) phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide (CS-3150), a novel nonsteroidal mineralocorticoid receptor antagonist, on renal injury in deoxycorticosterone acetate (DOCA)/salt-induced hypertensive rats (DOCA rats). From 7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt diet. In experiment 1, CS-3150 (0.3-3 mg/kg) was orally administered once a day for 4 weeks coincident with DOCA administration. In experiment 2, after establishment of renal injury by 4 weeks of DOCA/salt loading, CS-3150 (3 mg/kg) was orally administered once a day for 4 weeks with or without continuous DOCA administration. In experiment 1, DOCA/salt loading significantly increased systolic blood pressure (SBP), which was prevented by CS-3150 in a dose-dependent manner. Development of renal injury (proteinuria, renal hypertrophy, and histopathological changes in glomeruli and tubule) was also suppressed by CS-3150 with inhibition of mRNA expression of fibrosis, inflammation, and oxidative stress markers. In experiment 2, under continuous DOCA treatment, CS-3150 clearly ameliorated existing renal injury without lowering SBP, indicating that CS-3150 regressed renal injury independent of its antihypertensive action. Moreover, CS-3150 treatment in combination with withdrawal of DOCA showed further therapeutic effect on renal injury accompanied by reduction in SBP. These results demonstrate that CS-3150 not only prevents but also ameliorates hypertension and renal injury in DOCA rats. Therefore, CS-3150 could be a promising agent for the treatment of hypertension and renal disorders, and may have potential to promote regression of renal injury.

Remodeling and impaired dilation in middle cerebral arteries from Deoxycorticosterone acetate (DOCA)‐salt rats

Genetic hypertension causes inward remodeling and impaired dilation in the middle cerebral artery (MCA). DOCA‐salt rats are widely used model of hypertension, yet few studies have been performed on their cerebral arteries. We hypothesized that MCAs from DOCA‐salt hypertensive rats will have a reduced lumen diameter and impaired endothelium dependent dilation. Male Sprague‐Dawley rats had uninephrectomy, and received either DOCA (150mg/kg) and salt water (1% NaCl and 0.2% KCl) or tap water. MCAs were studied by pressure myography. Data are mean±SEM, SHAM vs DOCA. DOCA showed impaired dilation to 10 μM ADP (change from baseline: 41.2±5.2 vs 24.7±4.7%, p&lt; 0.05, ANOVA). Lumen diameter was reduced (295±18 vs 343±13 μm at 80 mmHg, p &lt;0.05, ANOVA) in the DOCA rats. Interestingly the wall/lumen ratio (0.06±0.02 vs 0.07±0.01 at 80 mmHg, p&gt;;0.05, ANOVA) did not change. These data suggest the MCAs from DOCA rats may exhibit inward hypotrophic remodeling. These data are contrary to the inward hypertrophic remodeling observed in MCAs from genetically hypertensive rats and imply that the mechanism of remodeling in DOCA‐salt rats may be different.

Abstract 324: In Vivo Cross-talk Between Mineralocorticoid Receptor and Ang-(1-7) Enhances Ca2+ Signaling in Ventricular Myocytes

Mineralocorticoids have been implicated in the pathogenesis of cardiac diseases. In contraposition, Angiotensin (Ang)-(1-7) has been shown to attenuate cardiac dysfunction induced by excessive mineralocorticoid receptor (MR) activation in vivo , suggesting a possible interaction between these two signaling pathways in the heart. Here, we investigated whether there is a cross-talk between MR signaling and Ang-(1-7) and its consequences for cardiomyocyte function. For this, we used a transgenic rat that overexpress an Ang-(1-7)-producing fusion protein, TGR(A1-7)3292 (TG), which induces an increase in circulating levels of this peptide, treated or not with deoxycorticosterone acetate (DOCA). After six weeks of DOCA-salt, rats presented increased blood pressure and cardiac ejection fraction when compared to SD control rats. Marked cardiac hypertrophy was also observed at six weeks of DOCA-salt treatment. In contrast, TG DOCA rats did not develop cardiac hypertrophy or changes in ventricular function. qPCR analysis of ventricular myocytes from SD DOCA-salt rats revealed a six fold increase in MR and a three fold increase in ß-myosin heavy chain mRNA transcripts. These changes were significantly attenuated in cardiomyocytes from TG DOCA rats. We also examined intracellular Ca 2+ levels in freshly isolated Fluo-4-loaded cells. The amplitude of the [Ca 2+ ] i transient was reduced in SD DOCA cardiomyocytes (F/F 0 =5.12+ 0.1 n=56 in SD versus F/F 0 =3.75+0.1 n=65 in SD DOCA myocytes, p&lt;0.05). These changes were at least partly explained by reduced SERCA2 protein and altered PLN phosphorylation in these cells. In contrast, TG DOCA cardiomyocytes presented enhanced Ca 2+ transient (F/F 0 =8.02+ 0.2 n=68 myocytes) and increased SERCA2 expression. Cardiomyocytes from TG rats presented Ca 2+ transient comparable to SD cells. SD DOCA rats also showed increased PP1 and PKC alpha expression in comparison to SD and TG DOCA cells. These results suggest that Ang-(1-7) and MR signaling pathways cross-talk to enhance Ca 2+ transient in cardiomyocytes preventing the Ca 2+ signaling dysfunction characteristic of excessive MR activation. These data provide a better understanding of the cardioprotective effects of this putative therapeutic peptide.

Splanchnic sympathetic nerves in the development of mild DOCA-salt hypertension

We previously reported that mild deoxycorticosterone acetate (DOCA)-salt hypertension develops in the absence of generalized sympathoexcitation. However, sympathetic nervous system activity (SNA) is regionally heterogeneous, so we began to investigate the role of sympathetic nerves to specific regions. Our first study on that possibility revealed no contribution of renal nerves to hypertension development. The splanchnic sympathetic nerves are implicated in blood pressure (BP) regulation because splanchnic denervation effectively lowers BP in human hypertension. Here we tested the hypothesis that splanchnic SNA contributes to the development of mild DOCA-salt hypertension. Splanchnic denervation was achieved by celiac ganglionectomy (CGX) in one group of rats while another group underwent sham surgery (SHAM-GX). After DOCA treatment (50 mg/kg) in rats with both kidneys intact, CGX rats exhibited a significantly attenuated increase in BP compared with SHAM-GX rats (15.6 ± 2.2 vs. 25.6 ± 2.2 mmHg, day 28 after DOCA treatment). In other rats, whole body norepinephrine (NE) spillover, measured to determine if CGX attenuated hypertension development by reducing global SNA, was not found to be different between SHAM-GX and CGX rats. In a third group, nonhepatic splanchnic NE spillover was measured as an index of splanchnic SNA, but this was not different between SHAM (non-DOCA-treated) and DOCA rats during hypertension development. In a final group, CGX effectively abolished nonhepatic splanchnic NE spillover. These data suggest that an intact splanchnic innervation is necessary for mild DOCA-salt hypertension development but not increased splanchnic SNA or NE release. Increased splanchnic vascular reactivity to NE during DOCA-salt treatment is one possible explanation.

Rho kinase inhibition activates the homologous angiotensin-converting enzyme-angiotensin-(1–9) axis in experimental hypertension

Angiotensin II (Ang II) levels depend on renin, angiotensin-converting enzyme (ACE), and on the homologous angiotensin-converting enzyme (ACE2). Increased ACE and Ang II levels are associated with higher Rho kinase activity. However, the relationship between Rho kinase activation and ACE2 in hypertension is unknown. The role of the Rho kinase signaling pathway in both enzymatic activity and aortic gene expression of ACE2 in deoxycorticosterone acetate (DOCA) hypertensive rats was assessed in the present study. Compared with sham animals, Rho kinase activity was higher by 400% (P<0.05) in the aortic wall of the DOCA hypertensive rats. In addition to blood pressure reduction, the specific Rho kinase inhibitor fasudil reduced aortic Rho kinase activity to levels observed in the sham control group and increased ACE2 enzymatic activity (by 83% in plasma and by 52% in the aortic wall, P<0.05), ACE2, and endothelial nitric oxide synthase (eNOS) aortic mRNA levels (by 340 and 40%, respectively, P<0.05) with respect to the untreated hypertensive DOCA rats. Fasudil also increased significantly plasma levels of Ang-(1-9) in normotensive and in the hypertensive rats. Aortic mRNA and protein levels of transforming growth factor-β1 (TGF-β1), plasminogen activator inhibitor 1 (PAI-1), and monocyte chemoattractant protein 1 (MCP-1) were significantly (P<0.05) higher in the untreated DOCA rats and were normalized by fasudil administration. In experimental hypertension, Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition reduces blood pressure and increases ACE2 levels and activity. At the same time, ROCK inhibition reduces angiotensin II and increases Ang-(1-9) plasma levels. Fasudil also increases vascular eNOS mRNA levels and reduces aortic overexpression of the remodeling promotion proteins TGF-β1, PAI-1, and MCP-1. This effect might additionally contribute to the antihypertensive and antiremodeling effects of ROCK inhibition in hypertension.

Concomitant Angiotensin AT1 Receptor Antagonism and Neprilysin Inhibition Produces Omapatrilat-like Antihypertensive Effects Without Promoting Tracheal Plasma Extravasation in the Rat

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy but cause high incidence of angioedema. We examined whether dual inhibition of angiotensin AT1 receptor (ARB) and NEP (ARB-NEPI, valsartan-candoxatril) provides similar efficacy to omapatrilat without the risk of angioedema. Activity of test compounds at the targets was assayed using fluorescence-based enzyme assays (ACE, NEP, aminopeptidase P) or competition binding assays (AT1). Target engagement in vivo (ACE, AT1, and NEP) was quantified by measuring inhibition of angiotensin-pressor responses and potentiation of atrial natriuretic peptide-induced urinary cyclic guanosine monophosphate (cGMP) output in rats. Tracheal plasma extravasation (TPE) was used as a surrogate to assess propensity of compounds to promote upper airway angioedema. Antihypertensive efficacy in renin-dependent and -independent states was measured in spontaneously hypertensive rats and deoxycorticosterone acetate salt hypertensive rats, respectively. Administration of omapatrilat and coadministration of valsartan and candoxatril blocked angiotensin induced vasopressor responses and potentiated atrial natriuretic peptide-induced increase in urinary cGMP output. In spontaneously hypertensive rats, valsartan, omapatrilat, and valsartan-candoxatril combination all produced reduction in blood pressure to a similar extent, whereas candoxatril was ineffective. In deoxycorticosterone acetate rats, omapatrilat, candoxatril, and valsartan-candoxatril combination but not valsartan produced reduction in blood pressure. Antihypertensive doses of omapatrilat produced robust increases in TPE; by contrast, valsartan, candoxatril, or their combination did not increase TPE. Pretreatment with icatibant, a bradykinin B2 antagonist, abolished omapatrilat-induced TPE but not its antihypertensive effects. On the background of NEP inhibition, suppression of the renin-angiotensin system through ARB and ACE inhibition shows a similar antihypertensive efficacy but exerts differential effects on bradykinin metabolism and TPE indicative of reduced risk of angioedema. Thus, dual AT1 receptor blockade and NEP inhibition is potentially an attractive approach to retain the excellent antihypertensive effects of omapatrilat but with a superior safety profile.

Mild DOCA-salt hypertension: sympathetic system and role of renal nerves

Excess sympathetic nervous system activity (SNA) is linked to human essential and experimental hypertension. To test whether sympathetic activation is associated with a model of deoxycorticosterone acetate (DOCA)-salt hypertension featuring two kidneys and a moderate elevation of blood pressure, we measured whole body norepinephrine (NE) spillover as an index of global SNA. Studies were conducted in chronically catheterized male Sprague-Dawley rats drinking water containing 1% NaCl and 0.2% KCl. After a 7-day surgical recovery and a 3-day control period, a DOCA pellet (50 mg/kg) was implanted subcutaneously in one group of rats (DOCA), while the other group underwent sham implantation (Sham). NE spillover was measured on control day 2 and days 7 and 14 after DOCA administration or sham implantation. During the control period, mean arterial pressure (MAP) was similar in Sham and DOCA rats. MAP was significantly increased in the DOCA group compared with the Sham group after DOCA administration (day 14: Sham = 109 ± 5.3, DOCA = 128 ± 3.6 mmHg). However, plasma NE concentration, clearance, and spillover were not different in the two groups at any time. To determine whether selective sympathetic activation to the kidneys contributes to hypertension development, additional studies were performed in renal denervated (RDX) and sham-denervated (Sham-DX) rats. MAP, measured by radiotelemetry, was similar in both groups during the control and DOCA treatment periods. In conclusion, global SNA is not increased during the development of mild DOCA-salt hypertension, and fully intact renal nerves are not essential for hypertension development in this model.

Uric Acid Does Not Affect the Acetylcholine-Induced Relaxation of Aorta from Normotensive and Deoxycorticosterone Acetate-Salt Hypertensive Rats

Uric acid (UA) results from xanthine oxidase (XO) catabolism of xanthine and is the final product of purine catabolism in humans. In this species, hyperuricemia is associated with gout, nephropathy, and increased cardiovascular disease risk. Although the effects of hyperuricemia in vascular biology are overall controversial, UA has been described as an antioxidant and as potentially improving endothelial function. Hypertension is associated with endothelial dysfunction. We hypothesized that UA improves the endothelial function of aorta from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. UA (100 microM) in the presence of the uricase inhibitor oxonic acid (10 microM) did not modify relaxation to acetylcholine (ACh) (1 nM-10 microM) in the aorta from nontreated, sham normotensive, and DOCA-salt hypertensive rats [response to 10 microM ACh for UA versus vehicle, respectively: nontreated = 37 +/- 7 versus 48 +/- 7%, sham = 53 +/- 15 versus 57 +/- 20%, DOCA = 81 +/- 4 versus 85 +/- 2% from 20 microM prostaglandin 2alpha (PGF(2alpha))-induced contraction]. Allopurinol (100 microM), a XO inhibitor, did not significantly alter the ACh-induced relaxation of sham and DOCA aortic rings (response to 10 microM ACh for allopurinol versus vehicle, respectively: sham = 61 +/- 5 versus 68 +/- 9%, DOCA = 87 +/- 6 versus 88 +/- 3% from 20 microM PGF(2alpha)-induced contraction). Uricemia, ranging from unmeasurable to 547 microM in sham and to 506 microM in DOCA rats, was not significantly different between these two groups. The expression and activity of XO, as well as the expression of uricase, were not different between sham and DOCA rat aorta. We conclude that, at least in vitro, UA does not affect the ACh-induced relaxation of normotensive and DOCA-salt hypertensive rats.

DOCA‐salt hypertensive rats display decreased vascular reactivity to urotensin‐II

IntroductionUrotensin‐II (U‐II) is a vasoconstrictor peptide and its potency can be up to 100 fold greater than that of endothelin‐1 (ET‐1). ET‐1 plays a major role in the vascular changes associated with salt‐sensitive hypertension, but the effects of U‐II in arteries from these animals have not yet been defined.ObjectiveWe hypothesized that DOCA‐salt rats have increased vascular reactivity to U‐II.Methods and ResultsWistar rats were unilaterally nephrectomyzed and deoxycorticosterone‐acetate (DOCA) (200mg/Kg) pellets were implanted. DOCA rats received water containing 1% NaCl/0.2% KCl, for 5 weeks. Aortic rings were mounted in myograph chambers and functional studies were performed. DOCA rats displayed increased systolic pressure (Control: 117±1 vs DOCA: 179±3; mmHg). Maximal contractions (mN) to U‐II were decreased in endothelium‐intact (Control: 16.6±1 vs DOCA: 8.8±1) and endothelium‐denuded (Control: 21.3±1.1 vs DOCA: 10.4±0.9) aortas from DOCA rats. This difference was abolished in endothelium‐intact vessels incubated with 10−5M urantide (U‐II antagonist) (Control: 13.5±0.9 vs DOCA: 12.7±0.9).ConclusionOur results suggest that U‐II production/release is augmented in mineralocorticoid hypertension and that decreased vascular reactivity to the peptide may represent counter‐regulatory mechanisms that limit additional vascular damage to this potent vasoconstrictor.

Blood pressure versus direct mineralocorticoid effects on kidney inflammation and fibrosis in DOCA-salt hypertension

We examined the contribution of high blood pressure versus direct mineralocorticoid effects to the progression of kidney inflammation and fibrosis in established experimental deoxycorticosterone-acetate (DOCA)-salt hypertension. Male Sprague-Dawley rats underwent unilateral nephrectomy and received subcutaneous DOCA pellets as well as 1% NaCl for drinking. After 4 weeks of DOCA-salt hypertension, rats were either killed (n = 6), or treated with a non-hypotensive dose of spironolactone (n = 7) or triple therapy (hydrochlorothiazide, reserpine and hydralazine, n = 8) to normalize blood pressure or with vehicle (n = 19) for two further weeks. Mean arterial pressure (MAP) was measured intra-arterially. Glomerulosclerosis, interstitial fibrosis, macrophage infiltration and complement deposition were evaluated on kidney sections. Expression of collagens, chemokines and cytokines was measured by real-time PCR. MAP was elevated in DOCA rats, not affected by spironolactone and normalized by triple therapy. Glomerulosclerosis and interstitial fibrosis of DOCA rats were alleviated by spironolactone and triple therapy. Macrophage infiltration, complement C3 deposition and nitrotyrosine staining in the kidney were significantly reduced by spironolactone as well as triple therapy. The expression of collagens, chemokines, adhesion molecules and profibrotic cytokines in the kidney was elevated in hypertension and decreased by triple therapy but not significantly affected by spironolactone. Direct mineralocorticoid effects as well as high blood pressure per se contribute to inflammation and fibrosis of the kidney. Oxidative stress may mediate the direct mineralocorticoid effects on kidney inflammation.

5-Hydroxytryptamine Lowers Blood Pressure in Normotensive and Hypertensive Rats

Arterial hyper-responsiveness to 5-hydroxytryptamine (5-HT) is a hallmark of hypertension, and plasma levels of free 5-HT are elevated in hypertension. We hypothesized that chronic administration of 5-HT would cause blood pressure to 1) rise in normotensive rats and 2) rise significantly more in hypertensive rats. The deoxycorticosterone acetate (DOCA)-salt hypertensive and sham normotensive rat were used. Animals were implanted with minipumps that delivered 5-HT (or vehicle) at a rate of 25 microg/kg/min for 7 days. Free plasma 5-HT was elevated significantly by this protocol. Within 48 h, mean arterial blood pressure measured telemetrically decreased in sham (106 +/- 2 to 83 +/- 2 mm Hg) and in DOCA-salt hypertensive (166 +/- 9 to 112 +/- 3 mm Hg) rats; vehicle did not change blood pressure in either group. Ganglionic blockade (hexamethonium) reduced blood pressure to a greater magnitude in DOCA vehicle-administered rats (peak fall arterial pressure, 91 +/- 14 mm Hg) compared with DOCA 5-HT-administered rats (40 +/- 6 mm Hg). Maximal acetylcholine-induced (NO-dependent) relaxation in phenylephrine-contracted aortic strips was greater in 5-HT-administered (69.2 +/- 9.1% relaxation) versus vehicle-administered (39.7 +/- 14.2%) DOCA rats; aortic endothelial cell nitric oxide synthase expression was higher in the 5-HT- versus vehicle-administered DOCA-salt rats. In normotensive and DOCA-salt hypertensive rats, the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine (0.5 g/l in water) prevented the fall in blood pressure to 5-HT. We conclude that chronic exogenous 5-HT reduces blood pressure in normotensive and hypertensive rats through mechanisms critically dependent on NOS.

The 5-Hydroxytryptamine2A Receptor Is Involved in (+)-Norfenfluramine-Induced Arterial Contraction and Blood Pressure Increase in Deoxycorticosterone Acetate-Salt Hypertension

The highly effective anorexigen (+)-fenfluramine was widely used to control body weight until the association with primary pulmonary hypertension and valvular heart disease. (+)-Norfenfluramine is the major hepatic metabolite of (+)-fenfluramine and is primarily responsible for the anorexic effect as well as side effects. We reported that (+)-norfenfluramine causes vasoconstriction and a blood pressure increase in rats with normal blood pressure via the 5-hydroxytryptamine (5-HT)2A receptor. With the knowledge that (+)-norfenfluramine also has affinity for 5-HT2B receptors and that arterial 5-HT2B receptor expression is up-regulated in deoxycorticosterone acetate (DOCA)-salt hypertension, we tested the hypothesis that (+)-norfenfluramine-induced vasoconstriction and pressor effects are potentiated in DOCA-salt hypertensive rats in a 5-HT2 receptor-dependent manner. Contractions of arteries were measured using an isolated tissue bath system or myograph. Mean arterial blood pressure was measured in chronically instrumented conscious rats. Effects of (+)-norfenfluramine in stimulating arterial contraction (leftward shift versus SHAM, aorta, 5.13-fold; renal artery, 1.95-fold; mesenteric resistance artery, 1.77-fold) and raising blood pressure were significantly enhanced in hypertension. In arteries from both normotensive and hypertensive rats, (+)-norfenfluramine-induced contraction in aorta was inhibited by 5-HT2A receptor antagonists, ketanserin and LY53857 (4-isopropyl-7-methyl-9-(2-hydroxy-1-meth ylpropoxycarbonyl)4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline), but not by the 5-HT2B receptor antagonist, LY272015 [6-chloro-5-methyl-N-(5-quinolinyl)-2,3-dihydro-1H-indole-1-carboxamide]. Ketanserin (3 mg/kg) reduced (+)-norfenfluramine-induced pressor response in both SHAM and DOCA rats. Our results demonstrate that (+)-norfenfluramine-induced arterial contraction and blood pressure increases are potentiated in DOCA-salt hypertensive rats. However, it is the 5-HT2A receptor and not the 5-HT2B receptor that participates in these effects.

Interaction of the heme oxygenase system with the renin‐angiotensin system in DOCA‐salt hypertensive rats

Heme analogues are known to lower blood pressure in a variety of hypertensive models, including angiotensin-II-infused-, phenylephrine-induced- and deoxycorticosterone acetate (DOCA)-salt-induced hypertensive models. In DOCA-salt rats, elevated activity of the renin-angiotensin system (RAS) leads to oxidative stress, inflammation and hypertrophy. Whether the application of a heme oxygenase inducer like hemin will attenuate RAS activity and suppress kidney hypertrophy needs clarification. Moreover, the interaction of the heme oxygenase system with the RAS in DOCA rats is not fully understood. In this study, the administration of hemin (30 mg/kg daily, i.p.) for four weeks to DOCA-salt, significantly lower blood pressure from 189.9±3.6 vs.136.6±1.3 mmHg (p<0.01, n=6) and attenuated kidney hypertrophy from 7.6 ±0.4 vs. 6.0±0.3 g/kg body weight (p< 0.01, n=6). This was accompanied by significant reduction of plasma angiotensin-II (254.8±66.5 vs. 13.6±1.5 pg/ml/protein; p<0.05, n=4) and aldosterone (16.4±0.75 vs. 10.9±2.9 pg/ml/protein; p <0.05, n=4) as well as reduced inflammatory activity, assessed as NF-κβ expression. In DOCA control rats (sham), blood pressure (118.5±0.2 mmHg) was unaffected by hemin. Similarly, hemin had no effect on normotensive Sprague Dawley rats (123.6±1.6 vs. 123±0.7 mmHg, n=5). Our studies unveils that the heme oxygenase system directly modulates angiotensin-II and aldosterone activities in DOCA rats to suppress hypertension and secondarily-induced damages like hypertrophy and inflammation. (This work is supported by Heart & Stroke Foundation of Saskatchewan, Canada)

Phorbol ester-induced contraction through p38 mitogen-activated protein kinase is diminished in aortas from DOCA-salt hypertensive rats

The role of mitogen-activated protein kinase (MAPK) in the decreased contractile response to phorbol ester in aortic smooth muscle strips from deoxycorticosterone acetate (DOCA)-salt hypertensive rats was examined. Norepinephrine (NE) evoked greater contractility in aortic strips from DOCA rats than in those of sham-operated rats. 12-Deoxyphorbol 13-isobutyrate (DPB) induced contraction in Ca2+-free medium, which was diminished in strips from DOCA rats compared to sham-operated rats. Vasoconstrictions induced by these stimulants were inhibited by SB203580 and PD098059, inhibitors of p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2, respectively, in both strips. The phosphorylation of p38 MAPK and ERK1/2 induced by NE was greater in strips from DOCA rats compared to those from sham-operated rats, and this phosphorylation was inhibited by the kinase inhibitors. DPB increased the phosphorylation of p38 MAPK and ERK1/2 in strips from both animals, and the increment of p38 MAPK phosphorylation by the stimulant was diminished in strips from DOCA rats compared to sham-operated rats. These findings suggest that the Ca2+-independent contraction evoked by DPB results from the activation of MAPKs in rat aortic smooth muscle and that the attenuated contractility by DPB in DOCA rat appears to be associated with diminished p38 MAPK activity.

Pleiotropic Effects of Hydrogen Peroxide in Arteries and Veins From Normotensive and Hypertensive Rats

Hydrogen peroxide causes vascular contraction and relaxation and contributes to the pathogenesis of hypertension. We hypothesized that the contractile state of blood vessels governs whether H2O2 causes contraction or relaxation. Hydrogen peroxide (1 micromol/L to 1 mmol/L) concentration-dependently contracted thoracic aorta and vena cava from sham normotensive and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The maximal contraction to H2O2 was 3 times greater in DOCA aorta compared with sham aorta but unchanged in DOCA vena cava compared with sham vena cava. In prostaglandin F2alpha (20 micromol/L)-contracted aorta and vena cava from sham and DOCA rats, H2O2 (1 micromol/L to 1 mmol/L) induced a concentration-dependent relaxation that was impaired in DOCA aorta but not DOCA vena cava. In contrast, in KCl (30 mmol/L)-contracted vessels, maximal H2O2-induced contraction was enhanced 15-fold in sham aorta and 5-fold in DOCA aorta but only 2-fold in sham vena cava. Tetraethylammonium (10 mmol/L), BAY K 8644 (100 nmol/L), and ouabain (1 mmol/L) all enhanced maximal aortic H2O2-induced contraction, whereas only ouabain enhanced venous H2O2-induced contraction. The removal of extracellular Ca2+ reduced H2O2-induced contraction in KCl-contracted aorta, whereas maximal venous H2O2-induced contraction (under basal conditions) was unchanged. Our data suggest that differences in arterial and venous K+ channel activity and extracellular Ca2+ influx are responsible for differences in arterial and venous contraction to H2O2. In DOCA-salt hypertension, arterial but not venous contraction to H2O2 is enhanced, and relaxation to H2O2 is reduced.

Dual angiotensin-converting enzyme/neutral endopeptidase inhibition on cardiac and renal fibrosis and inflammation in DOCA-salt hypertensive rats

The relative roles of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) inhibition on cardiac and renal fibrosis in deoxycorticosterone acetate (DOCA)-salt hypertensive rats were studied. The ACE/NEP inhibitor omapatrilat (40 mg/kg per day), the ACE inhibitor enalapril (10 mg/kg per day) and the NEP inhibitor CGS 25462(100 mg/kg per day) were administrated for 3 weeks to DOCA rats. Collagen was stained with Sirius red, and mediators of inflammation were identified by immunolabeling (vascular cell adhesion molecule, nuclear factor-kappaB, infiltrating ED-1-positive macrophages and monocyte chemotactic protein-1) or by western blot (platelet-endothelial cell adhesion molecule-1). Elevated systolic blood pressure of DOCA rats was significantly reduced (P < 0.05) by omapatrilat and CGS 25462. Omapatrilat and CGS 25462 significantly (P < 0.05) decreased interstitial collagen density in the left ventricle of DOCA rats compared with untreated DOCA rats. Enalapril only decreased the subepicardial collagen of DOCA rats. Omapatrilat significantly (P < 0.05) decreased renal mesangial collagen deposition in DOCA rats. Cardiac and renal expression of surface adhesion molecules, nuclear factor-kappaB, monocyte chemotactic protein and ED-1-positive cells were decreased in omapatrilat-treated DOCA rats compared with untreated DOCA rats. Enalapril and CGS 25462 did not alter mesangial collagen of DOCA rats. Dual ACE/NEP inhibition was more effective than ACE or NEP inhibition in decreasing inflammatory mediators, and improving cardiac and renal fibrosis. This suggests a role for NEP inhibition added to blockade of the renin-angiotensin system that may explain the greater efficacy of omapatrilat.

Nitric oxide generated by nNOS in the macula densa regulates the afferent arteriolar diameter in rat kidney

Nitric oxide (NO) derived from neuronal NO synthase (nNOS) in the macula densa is a modulator of tubuloglomerular feedback. However, little is known about the regulation of the afferent arteriolar diameter by NO from the macula densa in salt-sensitive hypertension. We investigated the relationship between nNOS in the macula densa and the afferent arteriolar diameter in deoxycorticosterone acetate (DOCA)-salt hypertensive rats treated with angiotensin converting enzyme (ACE) inhibitor or thiazide for 5 weeks. DOCA rats had reduced nNOS expression in the macula densa compared to controls and reduction of NO production evaluated with 4,5-diaminofluorescein diacetate in the juxtaglomerular apparatus (JGA). Treatment with ACE inhibitor and thiazide increased nNOS and NO production in the JGA. The diameter of the afferent arteriole observed by SEM using microvascular casts was smaller in DOCA rats compared to control rats, and ACE inhibitor or thiazide dilated the afferent arteriole with a positive correlation to nNOS immunoreactivity in the macula densa. In conclusion, the afferent arteriolar diameter might be regulated by NO derived from nNOS in the macula densa in DOCA-salt hypertensive rats.

Effect of a methionine-supplemented diet on the blood pressure of Sprague-Dawley and deoxycorticosterone acetate-salt hypertensive rats.

The objectives of the present study were to evaluate the effects of a methionine-supplemented diet on systolic blood pressure (BP) and vasomotor functions in Sprague-Dawley (SD) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. SD and DOCA rats were fed a normal or a methionine (8 g/kg)-supplemented diet for 10 weeks. Systolic BP was monitored and plasma homocysteine, methionine and cysteine levels were determined at the end of the experiment. Vasoconstriction and vasodilatation of aortic rings were measured. The methionine-supplemented diet induced a greater increase in homocysteinaemia concentration in DOCA rats than in SD rats and an increase in plasma cysteine concentration in DOCA rats. This diet was associated with an increase in systolic BP in SD rats and with a lesser development of DOCA-salt hypertension. An enhanced aortic constriction and a decreased responsiveness to acetylcholine, bradykinin and sodium nitroprusside in the SD rats fed the methionine-rich diet were consistent with the elevated systolic BP. In DOCA rats the increased responsiveness to bradykinin was in accordance with the systolic BP-lowering effect. In conclusion, the methionine-enriched diet cannot simply be considered as model of hyperhomocysteinaemia, since other metabolites and mechanisms seemed to be implicated in these complex interactions. The differential vasopressive effect of the methionine supplementation in SD and DOCA rats, and in particular the lowering of systolic BP obtained with a greater degree of hyperhomocysteinaemia in DOCA rats, suggest that more complex interactions exist between hyperhomocysteinaemia and BP than the simple positive association described previously.