High salt intake reduces endothelium-dependent dilation of mouse arterioles via superoxide anion generated from nitric oxide synthase

In skeletal muscle arterioles of normotensive rats fed a high salt diet, the bioavailability of endothelium-derived nitric oxide (NO) is reduced by superoxide anion. Because the impact of dietary salt on resistance vessels in other species is largely unknown, we investigated endothelium-dependent dilation and oxidant activity in spinotrapezius muscle arterioles of C57BL/6J mice fed normal (0.45%, NS) or high salt (7%, HS) diets for 4 wk. Mean arterial pressure in HS mice was not different from that in NS mice, but the magnitude of arteriolar dilation in response to different levels of ACh was 42-57% smaller in HS mice than in NS mice. Inhibition of nitric oxide synthase (NOS) with N(G) monomethyl L-arginine (L-NMMA) significantly reduced resting diameters and reduced responses to ACh (by 45-63%) in NS mice but not in HS mice. Arteriolar wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction or hydroethidine oxidation, was greater in HS mice than in NS mice. Exposure to the superoxide scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) + catalase reduced this oxidant activity to normal and restored normal arteriolar responsiveness to ACh in HS mice but had no effect in NS mice. L-NMMA also restored arteriolar oxidant activity to normal in HS mice. ACh further increased arteriolar oxidant activity in HS mice but not in NS mice, and this effect was prevented with L-NMMA. These data suggest that a high salt diet promotes increased generation of superoxide anion from NOS in the murine skeletal muscle microcirculation, thus impairing endothelium-dependent dilation through reduced NO bioavailability.     

High-salt diet depresses acetylcholine reactivity proximal to NOS activation in cerebral arteries

Rats were fed a low-salt (LS; 0.4% NaCl) or high-salt (HS; 4.0% NaCl) diet for 3 days, and the responses of isolated cerebral arteries to acetylcholine (ACh), the nitric oxide (NO)-dependent dilator bradykinin, and the NO donor 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex-anamine (NOC-9) were determined. ACh-induced vasodilation and NO release, assessed with the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2) diacetate, were eliminated with the HS diet. Inhibition of cyclooxygenase, cytochrome P-450 epoxygenase, and acetylcholinesterase did not alter ACh responses. Bradykinin and NOC-9 caused a similar dilation in cerebral arteries of all groups. Arteries from animals on LS or HS diets exhibited similar levels of basal superoxide (O(2)(-)) production, assessed by dihydroethidine fluorescence, and ACh responses were unaffected by O(2)(-) scavengers. Muscarinic type 3 receptor expression was unaffected by dietary salt intake. These results indicate that 1) a HS diet attenuates ACh reactivity in cerebral arteries by inhibiting NO release, 2) this attenuation is not due to production of a cyclooxygenase-derived vasoconstrictor or elevated O(2)(-) levels, and 3) alteration(s) in ACh signaling are located upstream from NO synthase.     

High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle

The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated, in part, by changes in local production of 20-HETE. In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle and the role of 20-HETE in this responsiveness. Rats were fed either a normal-salt (NS, 0.45%) or high-salt (HS, 4%) diet for 4-5 wk. There was no difference in steady-state tissue Po(2) between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po(2) to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the large-conductance Ca(2+)-activated potassium (K(Ca)) channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K(Ca) channels and that a high-salt diet impairs arteriolar responses to increased oxygen availability due to a reduction in vascular smooth muscle responsiveness to 20-HETE.     

Role of superoxide and angiotensin II suppression in salt-induced changes in endothelial Ca2+ signaling and NO production in rat aorta

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or changed to a high-salt (HS) diet (4% NaCl) for 3 days. Increases in intracellular Ca2+ ([Ca2+]i) in response to methacholine (10 microM) and histamine (10 microM) were significantly attenuated in aortic endothelial cells from rats fed a HS diet, whereas thapsigargin (10 microM)-induced increases in [Ca2+]i were unaffected. Methacholine-induced nitric oxide (NO) production was eliminated in endothelial cells of aortas from rats fed a HS diet. Low-dose ANG II infusion (5 ng x kg(-1) x min(-1) iv) for 3 days prevented impaired [Ca2+]i signaling response to methacholine and histamine and restored methacholine-induced NO production in aortas from rats on a HS diet. Adding Tempol (500 microM) to the tissue bath to scavenge superoxide anions increased NO release and caused N(omega)-nitro-L-arginine methyl ester-sensitive vascular relaxation in aortas from rats fed a HS diet but had no effect on methacholine-induced Ca2+ responses. Chronic treatment with Tempol (1 mM) in the drinking water restored NO release, augmented vessel relaxation, and increased methacholine-induced Ca2+ responses significantly in aortas from rats on a HS diet but not in aortas from rats on a LS diet. These findings suggest that 1) agonist-induced Ca2+ responses and NO levels are reduced in aortas of rats on a HS diet; 2) increased vascular superoxide levels contribute to NO destruction, and, eventually, to impaired Ca2+ signaling in the vascular endothelial cells; and 3) reduced circulating ANG II levels during elevated dietary salt lead to elevated superoxide levels, impaired endothelial Ca2+ signaling, and reduced NO production in the endothelium.     

Contribution of cytochrome P-450 omega-hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension

The present study evaluated the contribution of cytochrome P-450 omega-hydroxylase in modulating the reactivity of cremaster muscle arterioles in normotensive rats on high-salt (HS) and low-salt (LS) diet and in rats with reduced renal mass hypertension (RRM-HT). Changes in arteriolar diameter in response to ACh, sodium nitroprusside (SNP), ANG II, and elevated O(2) were measured via television microscopy under control conditions and following cytochrome P-450 omega-hydroxylase inhibition with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In normotensive rats on either LS or HS diet, resting tone was unaffected and arteriolar reactivity to ACh or SNP was minimally affected by cytochrome P-450 omega-hydroxylase inhibition. In RRM-HT rats, cytochrome P-450 omega-hydroxylase inhibition reduced resting tone and significantly enhanced arteriolar dilation to ACh and SNP. Treatment with 17-ODYA or DDMS inhibited arteriolar constriction to ANG II and O(2) in all the groups, although the degree of inhibition was greater in RRM-HT than in normotensive animals. These results suggest that metabolites of cytochrome P-450 omega-hydroxylase contribute to the altered reactivity of skeletal muscle arterioles to vasoconstrictor and vasodilator stimuli in RRM-HT.     

Elevated salt intake impairs dilation of rat skeletal muscle resistance arteries via ANG II suppression

Vasodilator responses were assessed in resistance arteries (100-200 microm) isolated from the gracilis muscle of normotensive rats after changes in dietary salt intake. Sprague-Dawley rats were maintained on either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 4-8 wk (chronic) or 3 days (short-term) with water ad libitum. One group of short-term HS rats received a continuous intravenous infusion of a low dose (5 ng x kg(-1) x min(-1)) of ANG II to prevent the ANG II suppression that occurs with HS diet. Short-term and chronic HS diet eliminated arterial dilation in response to ACh and reduced PO(2) (30-40 mmHg) and the stable prostacyclin analog iloprost. ANG II infusion preserved the response to these vasodilator stimuli in short-term HS animals. Dilator responses to sodium nitroprusside and forskolin were unaffected by HS diet. These findings suggest that ANG II suppression during HS diet impairs vascular relaxation mechanisms upstream from the cAMP and cGMP second messenger systems.     

Reinforcement of arteriolar myogenic activity by endogenous ANG II: susceptibility to dietary salt

The purpose of this study was to determine whether endogenous ANG II augments arteriolar myogenic behavior in striated muscle. Because circulating ANG II is decreased during high salt intake, we also investigated whether dietary salt could alter any influence of ANG II on myogenic behavior. Normotensive rats fed low-salt (0.45%, LS) or high-salt (7%, HS) diets were enclosed in a ventilated box with the spinotrapezius muscle exteriorized for intravital microscopy. Dietary salt did not affect resting arteriolar diameters. Microvascular pressure elevation by box pressurization caused greater arteriolar constriction in LS rats (up to 12 microm) than in HS rats (up to 4 microm). The ANG II-receptor antagonists saralasin and losartan attenuated myogenic responsiveness in LS rats but not HS rats. The bradykinin-receptor antagonist HOE-140 had no effect on myogenic responsiveness in LS rats but augmented myogenic responsiveness in HS rats. HOE-140 with the angiotensin-converting enzyme inhibitor captopril attenuated myogenic responsiveness to a greater extent in LS rats than in HS rats. We conclude that endogenous ANG II normally reinforces arteriolar myogenic behavior in striated muscle and that attenuated myogenic behavior associated with high salt intake is due to decreased circulating ANG II and increased local kinin levels.     

Effect of high-salt diet on NO release and superoxide production in rat aorta

Sprague-Dawley rats were fed either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 3 days. Nitric oxide (NO) and superoxide production were assessed in the thoracic aorta by evaluating the fluorescence signal intensity from 4,5-diaminofluorescein (DAF-2DA) and dihydroethidine, respectively. Methacholine caused increased NO release in the aortas from rats on a LS but not HS diet. The SOD mimetic tempol restored methacholine-induced NO release in aortas from rats on a HS diet. Methacholine also caused superoxide production in the aortas of rats on a HS diet but not in the aortas of rats on a LS diet. Tempol and N(G)-monomethyl-l-arginine eliminated methacholine-induced superoxide production in the aortas of rats on a HS diet. Aortic rings from rats on the HS diet showed impaired methacholine-induced relaxation, which was improved by tempol. Tempol alone caused a NO-dependent relaxation of norepinephrine-precontracted aortas that was significantly greater in the aortas of rats on the HS diet than in vessels from rats on the LS diet. These data suggest that a HS diet impairs endothelium-dependent relaxation via reduced NO levels and increased superoxide production.     

Arteriolar wall PO(2) and nitric oxide release during sympathetic vasoconstriction in the rat intestine

Endothelium-derived nitric oxide (NO) attenuates arteriolar constriction in the rat small intestine during periods of increased sympathetic nerve activity. This study was undertaken to test the hypothesis that a flow-dependent fall in arteriolar wall PO(2) serves as the stimulus for endothelial NO release under these conditions. Sympathetic nerve stimulation at 3-16 Hz induced frequency-dependent arteriolar constriction, with arteriolar wall O(2) tension (PO(2)) falling from 67 +/- 3 mmHg to as low as 41 +/- 6 mmHg. Arteriolar responses to nerve stimulation were enhanced after inhibition of NO synthase with N(G)-monomethyl-L-arginine (L-NMMA). Under a high-O(2) (20%) superfusate, the fall in wall PO(2) was significantly attenuated, arteriolar constrictions were increased by 57 +/- 9 to 66 +/- 12%, and these responses were no longer sensitive to L-NMMA. The high-O(2) superfusate had no effect on vascular smooth muscle responsiveness to NO (as judged by arteriolar responses to sodium nitroprusside) or on arteriolar wall oxidant activity (as determined by the reduction of tetranitroblue tetrazolium dye). These results indicate that a flow-dependent fall in arteriolar wall PO(2) may serve as a stimulus for the release of endothelium-derived NO during periods of increased sympathetic nerve activity.     

Regulation of adrenal atrial natriuretic hormone receptor subtypes.

Regulation of atrial natriuretic hormone (ANH) receptor binding and aldosterone suppression was studied in isolated adrenal glomerulosa cells from rats fed a high-salt (HS) or low-salt (LS) diet for 3 days. In plasma of HS rats, aldosterone levels were 5 times lower and immunoreactive ANH two times higher than in LS rats. Competitive binding studies showed the same affinity for human atrial natriuretic hormone (hANH) in both pools of cells, but receptor density was 50% higher on LS cells. A linear ANH analog that binds to non-guanylate-cyclase-coupled receptors did not show increased binding to LS cells. Cyclic GMP production in response to hANH was identical in both groups. The aldosterone-inhibitory effect of hANH on both groups of basal and angiotensin II-stimulated cells was also identical. Thus a short-term high-salt diet causes decreased density of ANH receptors in glomerulosa cells without changing biological activity of ANH. These results suggest that dietary salt content changes the number of ANH receptors and that non-guanylate-cyclase-coupled receptors contain at least two classes of receptors.     

Impaired NO-dependent dilation of skeletal muscle arterioles in hypertensive diabetic obese Zucker rats

This study determined alterations to nitric oxide (NO)-dependent dilation of skeletal muscle arterioles from obese (OZR) versus lean Zucker rats (LZR). In situ cremaster muscle arterioles from both groups were viewed via television microscopy, and vessel dilation was measured with a video micrometer. Arteriolar dilation to acetylcholine and sodium nitroprusside was reduced in OZR versus LZR, although dilation to aprikalim was unaltered. NO-dependent flow-induced arteriolar dilation (via parallel microvessel occlusion) was attenuated in OZR, impairing arteriolar ability to regulate wall shear rate. Vascular superoxide levels, as assessed by dihydroethidine fluorescence, were elevated in OZR versus LZR. Treatment of cremaster muscles of OZR with the superoxide scavengers polyethylene glycol-superoxide dismutase and catalase improved arteriolar dilation to acetylcholine and sodium nitroprusside and restored flow-induced dilation and microvascular ability to regulate wall shear rate. These results suggest that NO-dependent dilation of skeletal muscle microvessels in OZR is impaired due to increased levels of superoxide. Taken together, these data suggest that the development of diabetes and hypertension in OZR may be associated with an impaired skeletal muscle perfusion via an elevated vascular oxidant stress.     

Effects of high-salt diet on CYP450-4A omega-hydroxylase expression and active tone in mesenteric resistance arteries

This study investigated the role of changes in the expression of the cytochrome P-450 4A (CYP450-4A) enzymes that produce 20-hydroxyeicosatetraenoic acid (20-HETE) in modulating the responses of rat mesenteric resistance arteries to norepinephrine (NE) and reduced Po(2) after short-term (3-day) changes in dietary salt intake. The CYP450-4A2, -4A3, and -4A8 isoforms were all detected by RT-PCR in arteries obtained from rats fed a high-salt (HS, 4% NaCl) diet, whereas only the CYP450-4A3 isoform was detected in vessels from rats fed a low-salt (LS, 0.4% NaCl) diet. Expression of the 51-kDa CYP450-4A protein was significantly increased by a HS diet. Inhibiting 20-HETE synthesis with 30 muM N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) reduced the vasoconstrictor response to NE in arteries obtained from rats fed either a LS or HS diet, but NE sensitivity after DDMS treatment was significantly lower in vessels from rats on a HS diet. DDMS treatment also restored the vasodilator response to reduced Po(2) that was impaired in arteries from rats on a HS diet. These findings suggest that 1) a HS diet increases the expression of CYP450-4A enzymes in the mesenteric vasculature, 2) 20-HETE contributes to the vasoconstrictor response to NE in mesenteric resistance arteries, 3) the contribution of 20-HETE to the vasoconstrictor response to NE is greater in rats fed a HS diet than in rats fed a LS diet, and 4) upregulation of the production of 20-HETE contributes to the impaired dilation of mesenteric resistance arteries in response to hypoxia in rats fed a HS diet.     

Acute and chronic angiotensin-(1-7) restores vasodilation and reduces oxidative stress in mesenteric arteries of salt-fed rats

This study determined the effect of ANG-(1-7) on salt-induced suppression of endothelium-dependent vasodilatation in the mesenteric arteries of male Sprague-Dawley rats. Chronic intravenous infusion of ANG-(1-7), oral administration of the nonpeptide mas receptor agonist AVE-0991, and acute preincubation of the arteries with ANG-(1-7) and AVE-0991 all restored vasodilator responses to both ACh and histamine that were absent in the arteries of rats fed a high-salt (4% NaCl) diet. The protective effects of ANG-(1-7) and AVE-0991 were inhibited by acute or chronic administration of the mas receptor antagonist A-779, the ANG II type 2 (AT(2)) receptor blocker PD-123319, or N-nitro-l-arginine methyl ester, but not the ANG II type 1 receptor antagonist losartan. Preincubation with the antioxidant tempol or the nitric oxide (NO) donor diethylenetriamine NONOate and acute and chronic administration of the AT(2) receptor agonist CGP-42112 mimicked the protective effect of ANG-(1-7) to restore vascular relaxation. Acute preincubation with ANG-(1-7) and chronic infusion of ANG-(1-7) ameliorated the elevated superoxide levels in rats fed a high-salt diet, but the expression of Cu/Zn SOD and Mn SOD enzyme proteins in the vessel wall was unaffected by ANG-(1-7) infusion. These results indicate that both acute and chronic systemic administration of ANG-(1-7) or AVE-0991 restore endothelium-dependent vascular relaxation in salt-fed Sprague-Dawley rats by reducing vascular oxidant stress and enhancing NO availability via mas and AT(2) receptors. These findings suggest a therapeutic potential for mas/AT(2) receptor activation in preventing the vascular oxidant stress and endothelial dysfunction associated with elevated dietary salt intake.     

High-salt diet impairs hypoxia-induced cAMP production and hyperpolarization in rat skeletal muscle arteries

This study determined the effects of hypoxia on diameter, vascular smooth muscle (VSM) transmembrane potential (E(m)), and vascular cAMP levels for in vitro cannulated skeletal muscle resistance arteries (gracilis arteries) from Sprague-Dawley rats fed a low-salt (LS) or a high-salt (HS) diet. Arterial diameter and VSM E(m) were measured in response to hypoxia, iloprost, cholera toxin, forskolin, and aprikalim. In HS rats, arterial dilation and VSM hyperpolarization after hypoxia, iloprost, and cholera toxin were impaired versus responses in LS rats, whereas responses to forskolin and aprikalim were unaltered. Blockade of prostaglandin H(2) and thromboxane A(2) receptors had no effect on responses to hypoxia or iloprost in vessels from both rat groups, suggesting that inappropriate activation of these receptors does not contribute to the impaired hypoxic dilation with HS. Hypoxia, cholera toxin, and iloprost increased vascular cAMP levels in vessels of LS rats only, whereas forskolin increased cAMP levels in all vessels. These data suggest that reduced hypoxic dilation of skeletal muscle microvessels in rats on a HS diet may reflect an impaired ability of VSM to produce cAMP after exposure to prostacyclin.     

Effects of dietary salt intake on plasma arginine

Because L-arginine is degraded by hepatic arginase to ornithine and urea and is transported by the regulated 2A cationic amino acid y(+) transporter (CAT2A), hepatic transport may regulate plasma arginine concentration. Groups of rats (n = 6) were fed a diet of either low salt (LS) or high salt (HS) for 7 days to test the hypothesis that dietary salt intake regulates plasma arginine concentration and renal nitric oxide (NO) generation by measuring plasma arginine and ornithine concentrations, renal NO excretion, and expression of hepatic CAT2A, and arginase. LS rats had lower excretion of NO metabolites and cGMP, lower plasma arginine concentration (LS: 83 +/- 7 vs. HS: 165 +/- 10 micromol/l, P < 0.001), but higher plasma ornithine concentration (LS: 82 +/- 6 vs. HS: 66 +/- 4 micromol/l, P < 0.05) and urea excretion. However, neither the in vitro hepatic arginase activity nor the mRNA for hepatic arginase I was different between groups. In contrast, LS rats had twice the abundance of mRNA for hepatic CAT2A (LS: 3.4 +/- 0.4 vs. HS: 1.6 +/- 0.5, P < 0.05). The reduced plasma arginine concentration with increased plasma ornithine concentration and urea excretion during LS indicates increased arginine metabolism by arginase. This cannot be ascribed to changes in hepatic arginase expression but may be a consequence of increased hepatic arginine uptake via CAT2A.     

Effects of tempol on renal angiotensinogen production in Dahl salt-sensitive rats

We have recently reported that Dahl salt-sensitive rats (DS) on high salt diet (HS) have an inappropriate augmentation of intrarenal angiotensinogen. Recent studies also reported that the augmented superoxide anion formation plays important roles in this animal model of hypertension. This study was performed to address the hypothesis that an inappropriate augmentation of intrarenal angiotensinogen by HS is caused by the augmented reactive oxygen species. Male DS (200-220 g) were maintained on low salt diet LS (N = 7) or HS (N = 27) for 4 weeks. The HS group was subdivided into three subgroups to receive null (N = 12), superoxide dismutase mimetic, tempol (3 mmol/l, N = 8), or vasodilator, hydralazine (0.5 mmol/l, N = 7) in drinking water during the period. Systolic BP was significantly increased in the DS+HS group compared to the DS+LS group (184+/-7 mmHg vs. 107+/-5 at 4-week). Tempol or hydralazine treatment equivalently attenuated the hypertension (128+/-3 and 127+/-5 at 4-week, respectively). Urinary excretion of thiobarbituric acid reactive substances at 4-week was significantly increased in the DS+HS group compared to the DS+LS group (0.66+/-0.05 micromol/day vs. 0.14+/-0.01). Tempol treatment prevented this effect (0.24+/-0.04) but hydralazine treatment only partially prevented the effect (0.40+/-0.03). Kidney angiotensinogen levels, measured by Western blot analysis, were significantly increased in the DS+HS group compared to the DS+LS group (32+/-5 densitometric units vs. 21+/-1). Tempol (14+/-3) but not hydralazine (32+/-5) treatment prevented the intrarenal angiotensinogen augmentation. The evidence suggests that the enhanced intrarenal angiotensinogen in DS challenged with HS is associated with the augmented reactive oxygen species.     

High-salt diet impairs vascular relaxation mechanisms in rat middle cerebral arteries

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or a high-salt (HS) diet (4% NaCl) for 3 days or 4 wk. PO(2) reduction to 40-45 mmHg, the stable prostacyclin analog iloprost (10 pg/ml), and stimulatory G protein activation with cholera toxin (1 ng/ml) caused vascular smooth muscle (VSM) hyperpolarization, increased cAMP production, and dilation in cerebral arteries from rats on a LS diet. Arteries from rats on a HS diet exhibited VSM depolarization and constriction in response to hypoxia and iloprost, failed to dilate or hyperpolarize in response to cholera toxin, and cAMP production did not increase in response to hypoxia, iloprost, or cholera toxin. Low-dose angiotensin II infusion (5 ng x kg(-1) x min(-1) i.v.) restored normal responses to reduced PO(2) and iloprost in arteries from animals on a HS diet. These observations suggest that angiotensin II suppression with a HS diet leads to impaired relaxation of cerebral arteries in response to vasodilator stimuli acting at the cell membrane.     

Regression of Glomerular and Tubulointerstitial Injuries by Dietary Salt Reduction with Combination Therapy of Angiotensin II Receptor Blocker and Calcium Channel Blocker in Dahl Salt-Sensitive Rats

A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats.     

Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice

Changes in dietary sodium intake are associated with changes in vascular volume and reactivity that may be mediated, in part, by alterations in endothelial nitric oxide synthase (eNOS) activity. Caveolin-1 (Cav-1), a transmembrane anchoring protein in the plasma membrane caveolae, binds eNOS and limits its translocation and activation. To test the hypothesis that endothelial Cav-1 participates in the dietary sodium-mediated effects on vascular function, we assessed vascular responses and nitric oxide (NO)-mediated mechanisms of vascular relaxation in Cav-1 knockout mice (Cav-1-/-) and wild-type control mice (WT; Cav-1+/+) placed on a high-salt (HS; 4% NaCl) or low-salt (LS; 0.08% NaCl) diet for 16 days. After the systolic blood pressure was measured, the thoracic aorta was isolated for measurement of vascular reactivity and NO production, and the heart was used for measurement of eNOS expression and/or activity. The blood pressure was elevated in HS mice treated with NG-nitro-l-arginine methyl ester and more so in Cav-1-/- than WT mice and was significantly reduced during the LS diet. Phenylephrine caused vascular contraction that was significantly reduced in Cav-1-/- (maximum 0.25 +/- 0.06 g/mg) compared with WT (0.75 +/- 0.22 g/mg) on the HS diet, and the differences were eliminated with the LS diet. Also, vascular contraction in response to membrane depolarization by high KCl (96 mM) was reduced in Cav-1-/- (0.27 +/- 0.05 g/mg) compared with WT mice (0.53 +/- 0.12 g/mg) on the HS diet, suggesting that the reduced vascular contraction is not limited to a particular receptor. Acetylcholine (10(-5) M) caused aortic relaxation in WT mice on HS (23.6 +/- 3.5%) and LS (23.7 +/- 5.5%) that was enhanced in Cav-1-/- HS (72.6 +/- 6.1%) and more so in Cav-1-/- LS mice (93.6 +/- 3.5%). RT-PCR analysis indicated increased eNOS mRNA expression in the aorta and heart, and Western blots indicated increased total eNOS and phosphorylated eNOS in the heart of Cav-1-/- compared with WT mice on the HS diet, and the genotypic differences were less apparent during the LS diet. Thus Cav-1 deficiency during the HS diet is associated with decreased vasoconstriction, increased vascular relaxation, and increased eNOS expression and activity, and these effects are altered during the LS diet. The data support the hypothesis that endothelial Cav-1, likely through an effect on eNOS activity, plays a prominent role in the regulation of vascular function during substantial changes in dietary sodium intake.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.