Hepatoprotective and cardioprotective effects of empagliflozin in spontaneously hypertensive rats fed a high-fat diet.

A combination of liver and heart dysfunction worsens the prognosis of human survival. The aim of this study was to investigate whether empagliflozin (a sodium-glucose transporter-2 inhibitor) has beneficial effects not only on cardiac and renal function but also on hepatic function. Adult (6-month-old) male spontaneously hypertensive rats (SHR) were fed a high-fat diet (60% fat) for four months to induce hepatic steatosis and mild heart failure. For the last two months, the rats were treated with empagliflozin (empa, 10 mg.kg-1.day-1 in the drinking water). Renal function and oral glucose tolerance test were analyzed in control (n=8), high-fat diet (SHR+HF, n=10), and empagliflozin-treated (SHR+HF+empa, n=9) SHR throughout the study. Metabolic parameters and echocardiography were evaluated at the end of the experiment. High-fat diet feeding increased body weight and visceral adiposity, liver triglyceride and cholesterol concentrations, and worsened glucose tolerance. Although the high-fat diet did not affect renal function, it significantly worsened cardiac function in a subset of SHR rats. Empagliflozin reduced body weight gain but not visceral fat deposition. It also improved glucose sensitivity and several metabolic parameters (plasma insulin, uric acid, and HDL cholesterol). In the liver, empagliflozin reduced ectopic lipid accumulation, lipoperoxidation, inflammation and pro-inflammatory HETEs, while increasing anti-inflammatory EETs. In addition, empagliflozin improved cardiac function (systolic, diastolic and pumping) independent of blood pressure. The results of our study suggest that hepatoprotection plays a decisive role in the beneficial effects of empagliflozin in preventing the progression of cardiac dysfunction induced by high-fat diet feeding.

Lipidized PrRP Analog Exhibits Strong Anti-Obesity and Antidiabetic Properties in Old WKY Rats with Obesity and Glucose Intolerance.

Prolactin-releasing peptide (PrRP) is an anorexigenic neuropeptide that has potential for the treatment of obesity and its complications. Recently, we designed a palmitoylated PrRP31 analog (palm11-PrRP31) that is more stable than the natural peptide and able to act centrally after peripheral administration. This analog acted as an anti-obesity and glucose-lowering agent, attenuating lipogenesis in rats and mice with high-fat (HF) diet-induced obesity. In Wistar Kyoto (WKY) rats fed a HF diet for 52 weeks, we explored glucose intolerance, but also prediabetes, liver steatosis and insulin resistance-related changes, as well as neuroinflammation in the brain. A potential beneficial effect of 6 weeks of treatment with palm11-PrRP31 and liraglutide as comparator was investigated. Liver lipid profiles, as well as urinary and plasma metabolomic profiles, were measured by lipidomics and metabolomics, respectively. Old obese WKY rats showed robust glucose intolerance that was attenuated by palm11-PrRP31, but not by liraglutide treatment. On the contrary, liraglutide had a beneficial effect on insulin resistance parameters. Despite obesity and prediabetes, WKY rats did not develop steatosis owing to HF diet feeding, even though liver lipogenesis was enhanced. Plasma triglycerides and cholesterol were not increased by HFD feeding, which points to unincreased lipid transport from the liver. The liver lipid profile was significantly altered by a HF diet that remained unaffected by palm11-PrRP31 or liraglutide treatment. The HF-diet-fed WKY rats revealed astrogliosis in the brain cortex and hippocampus, which was attenuated by treatment. In conclusion, this study suggested multiple beneficial anti-obesity-related effects of palm11-PrRP31 and liraglutide in both the periphery and brain.

Empagliflozin Is Not Renoprotective in Non-Diabetic Rat Models of Chronic Kidney Disease.

Gliflozins (sodium-glucose transporter-2 inhibitors) exhibited renoprotective effects not only in diabetic but also in non-diabetic patients with chronic kidney disease (CKD). Controversial results were reported in experimental non-diabetic models of CKD. Therefore, we examined empagliflozin effects in three CKD models, namely, in fawn-hooded hypertensive (FHH) rats, uninephrectomized salt-loaded (UNX + HS) rats, and in rats with Goldblatt hypertension (two-kidney, one-clip 2K1C) that were either untreated or treated with empagliflozin (10 mg/kg/day) for eight weeks. Plethysmography blood pressure (BP) was recorded weekly, and renal parameters (proteinuria, plasma urea, creatinine clearance, and sodium excretion) were analyzed three times during the experiment. At the end of the study, blood pressure was also measured directly. Markers of oxidative stress (TBARS) and inflammation (MCP-1) were analyzed in kidney and plasma, respectively. Body weight and visceral adiposity were reduced by empagliflozin in FHH rats, without a significant effect on BP. Experimentally induced CKD (UNX + HS and 2K1C) was associated with a substantial increase in BP and relative heart and kidney weights. Empagliflozin influenced neither visceral adiposity nor BP in these two models. Although empagliflozin increased sodium excretion, suggesting effective SGLT-2 inhibition, it did not affect diuresis in any experimental model. Unexpectedly, empagliflozin did not provide renoprotection because proteinuria, plasma urea, and plasma creatinine were not lowered by empagliflozin treatment in all three CKD models. In line with these results, empagliflozin treatment did not decrease TBARS or MCP-1 levels in either model. In conclusion, empagliflozin did not provide the expected beneficial effects on kidney function in experimental models of CKD.

Beneficial Effects of Empagliflozin Are Mediated by Reduced Renal Inflammation and Oxidative Stress in Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein.

Gliflozins (inhibitors of sodium-glucose cotransporter 2) show many beneficial actions beyond their antidiabetic effects. The underlying mechanisms of these additional protective effects are still not well understood, especially under non-diabetic conditions. Therefore, we analyzed the effects of empagliflozin in young (3-month-old) and adult (12-month-old) male spontaneously hypertensive rats (SHR) expressing human C-reactive protein (CRP) in the liver. SHR-CRP rats are a non-diabetic model of metabolic syndrome, inflammation, and organ damage. Empagliflozin was given in a daily dose of 10 mg/kg body weight for 8 weeks. Both age groups of SHR-CRP rats treated with empagliflozin had lower body weight, decreased weight of fat depots, reduced ectopic fat accumulation in the liver and kidneys, and decreased levels of plasma insulin and ß-hydroxybutyrate. Empagliflozin effectively reduced ectopic renal fat accumulation, and was associated with decreased inflammation. Exclusively in young rats, decreased microalbuminuria after empagliflozin treatment was accompanied by attenuated oxidative stress. In adult animals, empagliflozin also improved left ventricle function. In conclusion, in young animals, the beneficial renoprotective effects of empagliflozin could be ascribed to reduced lipid deposition in the kidney and the attenuation of oxidative stress and inflammation. In contrast, hepatic lipid metabolism was ameliorated in adult rats.

Palmitoylated prolactin-releasing peptide treatment had neuroprotective but not anti-obesity effect in fa/fa rats with leptin signaling disturbances.

BACKGROUND/OBJECTIVE: Anorexigenic palmitoylated prolactin-releasing peptide (palm11-PrRP) is able to act centrally after peripheral administration in rat and mouse models of obesity, type 2 diabetes mellitus and/or neurodegeneration. Functional leptin and intact leptin signaling pathways are necessary for the body weight reducing and glucose tolerance improving effect of palm11-PrRP. We have previously shown that palm11-PrRP31 had glucose-lowering properties but not anti-obesity effect in Koletsky rats with leptin signaling disturbances, so improvements in glucose metabolism appear to be completely independent of leptin signaling. The purpose of this study was to describe relationship between metabolic and neurodegenerative pathologies and explore if palm11-PrRP31 could ameliorate them in obese fa/fa rat model with leptin signaling disruption. SUBJECT/METHODS: The fa/fa rats and their age-matched lean controls at the age 32 weeks were used for this study. The rats were infused for 2 months with saline or palm11-PrRP31 (n = 7-8 per group) at a dose of 5 mg/kg per day using Alzet osmotic pumps. During the dosing period food intake and body weight were monitored. At the end of experiment the oral glucose tolerance test was performed; plasma and tissue samples were collected and arterial blood pressure was measured. Then, markers of leptin and insulin signaling, Tau phosphorylation, neuroinflammation, and synaptogenesis were measured by western blotting and immunohistochemistry. RESULTS: Fa/fa rats developed obesity, mild glucose intolerance, and peripheral insulin resistance but not hypertension while palm11-PrRP31 treatment neither lowered body weight nor attenuated glucose tolerance but ameliorated leptin and insulin signaling and synaptogenesis in hippocampus. CONCLUSION: We demonstrated that palm11-PrRP31 had neuroprotective features without anti-obesity and glucose lowering effects in fa/fa rats. This data suggest that this analog has the potential to exert neuroprotective effect despite of leptin signaling disturbances in this rat model.

Both central sympathoexcitation and peripheral angiotensin II-dependent vasoconstriction contribute to hypertension development in immature heterozygous Ren-2 transgenic rats.

Recently, we demonstrated that chronic blockade of the renin-angiotensin system (RAS) lowered the blood pressure (BP) of adult Ren-2 transgenic rats (TGR) mainly through the attenuation of central sympathoexcitation. However, the participation of central and peripheral mechanisms in the development of high BP in immature TGR remains unclear. In the present study, 6-week-old heterozygous TGR males were chronically treated with intracerebroventricular (ICV) or intraperitoneal (IP) infusions of the AT1 receptor inhibitor losartan (1 or 2 mg/kg/day) for 4 weeks. The influence of these treatments on sympathetic- and angiotensin II-dependent BP components (BP response to pentolinium or captopril, respectively) as well as on BP response to exogenous angiotensin II were determined to evaluate the participation of central and peripheral RAS in hypertension development. Chronic IP losartan administration (1 or 2 mg/kg/day) lowered the BP of immature TGR by reducing both sympathetic and angiotensin II-dependent BP components. The central action of IP-administered losartan was indicated by a reduced BP response to acute ICV angiotensin II injection. Chronic ICV administration of a lower losartan dose (1 mg/kg/day) reduced only the sympathetic BP component, whereas a higher ICV administered dose (2 mg/kg/day) was required to influence the angiotensin II-dependent BP component. Accordingly, chronic ICV losartan administration of 2 mg/kg/day (but not 1 mg/kg/day) attenuated the BP response to acute intravenous angiotensin II application. In conclusion, central sympathoexcitation seems to play an important role in hypertension development in immature TGR. Central sympathoexcitation is highly susceptible to inhibition by low doses of RAS-blocking agents, whereas higher doses also affect peripheral angiotensin II-dependent vasoconstriction.

Antihypertensive and metabolic effects of empagliflozin in Ren-2 transgenic rats, an experimental non-diabetic model of hypertension.

The new antidiabetic drugs, gliflozins, inhibit sodium-glucose transporter-2 in renal proximal tubules promoting glucose and sodium excretion. This leads not only to a significant improvement of glucose control but also to the reduction of blood pressure and body weight in both diabetic patients and experimental models. We examined whether these beneficial effects can also be achieved in a non-diabetic hypertensive model, namely in Ren-2 transgenic rats (TGR). Adult 6-month-old hypertensive TGR and their normotensive controls (Hannover Sprague-Dawley rats), were either untreated or treated with empagliflozin (10 mg/kg/day) for two months. Telemetric blood pressure monitoring, renal parameters as well as cardiac function via echocardiography were analyzed during the experiment. At the end of the study, the contribution of major vasoactive systems to blood pressure maintenance was studied. Metabolic parameters and markers of oxidative stress and inflammation were also analyzed. Empagliflozin had no effect on plasma glucose level but partially reduced blood pressure in TGR. Although food consumption was substantially higher in empagliflozin-treated TGR compared to the untreated animals, their body weight and the amount of epididymal and perirenal fat was decreased. Empagliflozin had no effect on proteinuria, but it decreased plasma urea, attenuated renal oxidative stress and temporarily increased urinary urea excretion. Several metabolic (hepatic triglycerides, non-esterified fatty acids, insulin) and inflammatory (TNF-α, leptin) parameters were also improved by empagliflozin treatment. By contrast, echocardiography did not reveal any effect of empagliflozin on cardiac function. In conclusion, empagliflozin exerted beneficial antihypertensive, anti-inflammatory and metabolic effects also in a non-diabetic hypertensive model.

Effects of systemic and renal intramedullary endothelin-1 receptor blockade on tissue NO and intrarenal hemodynamics in normotensive and hypertensive rats.

Endothelin 1 (ET-1) seems essential in salt-dependent hypertension, and activation of ETA receptors causes renal vasoconstriction. However, the response in the renal medulla and the role of tissue NO availability has never been adequately explored in vivo. We examined effects of ETA and ETB receptor blockade (atrasentan and BQ788) on blood pressure (MAP), medullary blood flow (MBF) and medullary tissue NO. Effects of systemic and intramedullary blocker application were compared in anesthetized normotensive ET-1-pretreated Sprague-Dawley rats (S-D), in salt-dependent hypertension (HS/UNX) and in spontaneously hypertensive rats (SHR). Total renal blood flow (RBF) was measured using a Transonic renal artery probe, MBF as laser-Doppler flux, and tissue NO signal using selective electrodes. In normotensive rats ET-1 significantly increased MAP, decreased RBF (-20%) and renal medullary NO. In HS/UNX rats atrasentan decreased MAP and increased medullary NO, earlier and more profoundly with intravenous infusion. In SHR atrasentan decreased MAP, more effectively with intravenous infusion; the increase in tissue NO (∼10%) was similar with both routes; however, only intramedullary atrasentan increased MBF. No consistent responses to BQ788 were seen. We confirmed dominant role of ETA receptors in regulation of blood pressure and renal hemodynamics in normotensive and hypertensive rats and provided novel evidence for the role of ETA in control of intrarenal NO bioavailability in salt-dependent and spontaneous hypertension. Under conditions of activation of the endothelin system ETB stimulation preserved medullary perfusion.

Renoprotection Provided by Additional Diuretic Treatment in Partially Nephrectomized Ren-2 Transgenic Rats Subjected to the Combined RAS and ETA Blockade.

OBJECTIVE: Our previous study in heterozygous Ren-2 transgenic rats (TGR) demonstrated that long-term treatment with endothelin receptor A (ETA) blocker atrasentan added to the renin-angiotensin system (RAS) blockade had renoprotective effects in a model of chronic kidney disease (CKD) induced by partial nephrectomy. Since ETA blockade is known to cause edema, we were interested whether diuretic treatment added to this therapy would be beneficial. DESIGN AND METHODS: Partial nephrectomy (NX) was performed at the age of 3 months in TGR rats which were subjected to: (i) RAS blockade alone (angiotensin receptor blocker losartan and angiotensin converting enzyme inhibitor trandolapril), (ii) combined RAS (losartan and trandolapril) and ETA receptor blockade (atrasentan), or (iii) diuretic (hydrochlorothiazide) added to the combined RAS + ETA blockade for 50 weeks following NX. RESULTS: At the end of the study systolic blood pressure and cardiac hypertrophy were similarly decreased in all treated groups. Survival was significantly improved by ETA receptor blockade added to RAS blockade with no further effects of diuretic treatment. However, additional diuretic treatment combined with RAS + ETA blockade decreased body weight and had beneficial renoprotective effects - reductions of both kidney weight and kidney damage markers. Proteinuria gradually increased in rats treated with RAS blockade alone, while it was substantially lowered by additional ETA blockade. In rats treated with additional diuretic, proteinuria was progressively reduced throughout the experiment. CONCLUSION: A diuretic added to the combined RAS and ETA blockade has late renoprotective effects in CKD induced by partial nephrectomy in Ren-2 transgenic rats. The diuretic improved: renal function (evaluated as proteinuria and creatinine clearance), renal morphology (kidney mass, glomerular volume), and histological markers of kidney damage (glomerulosclerosis index, tubulointerstitial injury).

Role of angiotensin II in chronic blood pressure control of heterozygous Ren-2 transgenic rats: Peripheral vasoconstriction versus central sympathoexcitation.

Our previous studies demonstrated that chronic systemic blockade of renin-angiotensin system (RAS) lowered blood pressure (BP) of Ren-2 transgenic rats (TGR) by the attenuation of both angiotensin II-dependent and sympathetic vasoconstriction. Since systemic RAS blockade also inhibits brain RAS, we were interested which effects on these two types of vasoconstriction will have the central RAS blockade in hypertensive TGR rats. Adult male heterozygous TGR rats and their Hannover Sprague Dawley (HanSD) controls were subjected to chronic systemic or intracerebroventricular administration of either angiotensin type 1 receptor blocker losartan or direct renin inhibitor aliskiren for 4 weeks. Additional groups of TGR and HanSD rats were used for the evaluation of acute peripheral and brain effects of angiotensin II. Both chronic systemic and intracerebroventricular administrations of losartan or aliskiren normalized BP of TGR animals. BP effect of brain RAS blockade was based solely on the reduced sympathetic vasoconstriction, while systemic RAS blockade attenuated both angiotensin II-dependent and sympathetic vasoconstriction. Surprisingly, neither peripheral nor central pressor effects of acute angiotensin II administration were enhanced in TGR compared to HanSD rats. In conclusion, sympathoinhibition represents the main mechanism of BP reduction in heterozygous TGR rats subjected to chronic brain or systemic RAS blockade, while peripheral attenuation of angiotensin II-dependent vasoconstriction during systemic RAS blockade is less important. Our data suggest that the participation of angiotensin II in BP control of adult heterozygous TGR rats is shifted from peripheral vasoconstriction to central sympathoexcitation. Similar mechanisms cannot be excluded in human essential hypertension.

Exaggerated blood pressure response to fasudil or nifedipine in hypertensive Ren-2 transgenic rats: role of altered baroreflex.

Basal calcium sensitization is decreased in spontaneously hypertensive rats, although their blood pressure (BP) response to acute Rho-kinase inhibition is enhanced. Using fasudil (Rho-kinase inhibitor) or nifedipine (L-VDCC blocker), we evaluated the contribution of calcium sensitization and calcium entry to BP maintenance in hypertensive transgenic Ren-2 rats (TGR) focusing on the influence of major vasoactive systems and/or baroreflex efficiency on BP responses to these two drugs. Homozygous TGR and normotensive Hannover Sprague-Dawley (HanSD) control rats aged 5, 11, or 22 weeks were used. The acute BP-lowering effects of fasudil or nifedipine were studied in intact rats, nitric oxide-deficient L-NAME-pretreated rats and rats subjected to combined blockade of the renin-angiotensin system (RAS), sympathetic nervous system (SNS) and nitric oxide synthase (NOS). Fasudil- or nifedipine-induced BP reduction increased during hypertension development in TGR. By contrast, the nifedipine-induced BP response decreased, whereas the fasudil-induced BP response increased with age in HanSD controls. Our data indicated a major contribution of nifedipine-sensitive calcium entry and relative attenuation of calcium sensitization in hypertensive rats compared with normotensive controls. The BP responses to fasudil or nifedipine were enhanced by NOS inhibition and combined blockade in normotensive HanSD rats but not in hypertensive TGR. In conclusion, calcium  sensitization is attenuated by endogenous nitric oxide in normotensive HanSD rats but not in hypertensive TGR. Moreover, BP reduction elicited by acute Rho-kinase inhibition is partially compensated by enhanced sympathetic vasoconstriction. The decreased compensation in hypertensive rats with impaired baroreflex efficiency explains their greater BP response to fasudil than in normotensive animals.

High-fat diet induces emergence of brown-like adipocytes in white adipose tissue of spontaneously hypertensive rats.

Obesity has a profound adverse impact on health. In this study, we present evidence for high-fat diet (HFD)-induced emergence of brown-like adipocytes in white adipose tissue (WAT) of the spontaneously hypertensive rat (SHR). We studied adult males fed a HFD or normal diet (ND) for 12 weeks. At the end of the 12-week dietary intervention, HFD compared with ND rats showed significantly higher whole-body energy expenditure. HFD vs. ND rats also showed higher expression of genes involved in fatty acid oxidation, mitochondrial biogenesis and brown fat adipogenesis, as well as augmented mitochondrial mass in WAT but not in the liver or skeletal muscle. Consistent with the molecular changes, in HFD but not in ND rats, histological and immunohistochemistry-based analyses of WAT demonstrated the presence of small multilocular cells staining positively for uncoupling protein 1, indicating the emergence of brown-like adipocytes in WAT. Our results suggest that SHR may have the capacity to increase energy expenditure in response to a chronic HFD that may be linked to the emergence of brown-like adipocytes in WAT. Thus, the SHR may be an important genetic model to uncover novel mechanisms of resistance to dietary obesity.

Genetic locus on rat chromosome 20 regulates diet-induced adipocyte hypertrophy: a microarray gene expression study.

Obesity is a leading cause of diabetes mellitus and hypertension. Molecular signals produced by adipose tissue may contribute to the pathogenesis of these two disorders. We showed previously that a specific segment of rat chromosome 20 (RNO20) contains a gene(s) regulating the degree of obesity, glucose intolerance, and hypertension in response to a chronic high-fat diet (HFD). Here we examined microarray gene expression profiles and cellular morphology of adipose tissues and whole body energy expenditure in this model. Adult male spontaneously hypertensive rats (SHR) and a congenic strain (SHR.1N) that differs from SHR by the above-mentioned segment of RNO20 were fed for 12 wk with HFD or a normal diet. At the end of this period, whole body energy expenditure was measured with indirect calorimetry. In response to HFD, body weight, fat pad weights, adipocyte size, and serum leptin levels increased significantly more in SHR.1N than SHR. Microarray gene expression profiles [Affymetrix, 15,923 genes and expressed sequence tags (ESTs)] showed that multiple genes of molecular pathways involved in lipogenesis were downregulated to a similar level in both strains, whereas genes involved in fatty acid oxidation and energy dissipation were upregulated less in SHR.1N than SHR. This was associated with lower whole body energy expenditure in SHR.1N than SHR at the end of the 12-wk HFD. Our results suggest that a gene(s) within the RNO20 segment regulate(s) HFD-induced increases in adiposity, and that this effect may be mediated, at least in part, by the impact of that gene(s) on fat burning and energy expenditure.

Regression of L-NAME-induced hypertension: the role of nitric oxide and endothelium-derived constricting factor.

N(G)-Nitro-L-arginine-methyl ester (L-NAME)-induced hypertension is a well established model of experimental hypertension. Although regression experiments are effective at approximating a clinical setting the reversal of already established L-NAME hypertension has not been intensively researched. We investigated whether spontaneous regression of L-NAME hypertension after discontinuing the drug administration was associated with recovery of endothelial dysfunction. Special attention was devoted to NO signaling and endothelium-derived constricting factor (EDCF) formation in various parts of the vascular tree. Male adult Wistar rats were divided into 4 groups: an L-NAME (5 weeks), a spontaneous recovery (5 weeks L-NAME + 3 weeks of recovery) and two age-matched control groups (a 5- and 8-week control group). The NO-mediated and EDCF-mediated components of acetylcholine-induced responses were evaluated in preconstricted small mesenteric and femoral arteries. The activity, mRNA and protein expression of NO synthase together with the mRNA expression of cyclooxygenase were determined in the aorta. L-NAME administration caused hypertension, impaired NO signaling (as indicated by the reduced NO component of acetylcholine-induced relaxation and decreased NO synthase activity) in all arteries investigated and reduced the inner diameter of the femoral artery. Moreover, we observed enhanced cyclooxygenase-dependent EDCF formation in the femoral arteries and enhanced cyclooxygenase-2 expression in the aortas of L-NAME-treated rats. During spontaneous recovery a functional restoration of NO signaling took place in all parts of the vascular tree. However, the increases in systolic blood pressure, EDCF formation, and cyclooxygenase expression and the reduction in femoral artery diameter were not completely restored. We conclude that impaired NO signaling was improved after the cessation of L-NAME administration. However, persisting arterial structural alterations and enhanced EDCF formation may decelerate blood pressure reduction even after the restoration of NO synthase activity.

The participation of brain NO synthase in blood pressure control of adult spontaneously hypertensive rats.

Increased blood pressure (BP) in genetic hypertension is usually caused by high activity of sympathetic nervous system (SNS) which is enhanced by central angiotensin II but lowered by central nitric oxide (NO). We have therefore evaluated NO synthase (NOS) activity as well as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) protein expression in brainstem and midbrain of adult spontaneously hypertensive rats (SHR) characterized by enhanced sympathetic vasoconstriction. We also studied possible participation of brain NO in antihypertensive effects of chronic captopril treatment of adult SHR. NOS activity was increased in midbrain of SHR compared to Wistar-Kyoto (WKY) rats. This could be ascribed to enhanced iNOS expression, whereas nNOS expression was unchanged and eNOS expression was reduced in this brain region. In contrast, no significant changes of NOS activity were found in brainstem of SHR in which nNOS and iNOS expression was unchanged, but eNOS expression was increased. Chronic captopril administration lowered BP of adult SHR mainly by attenuation of sympathetic tone, whereas the reduction of angiotensin II-dependent vasoconstriction and the decrease of residual BP (amelioration of structural remodeling of resistance vessels) were less important. This treatment did not affect significantly either NOS activity or expression of any NOS isoform in the two brain regions. Our data do not support the hypothesis that altered brain NO formation contributes to sympathetic hyperactivity and high BP of adult SHR with established hypertension.