Gábor Raffai

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.

Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries

The goals of this study were to 1) determine the acute effect of ANG-(1-7) on vascular tone in isolated middle cerebral arteries (MCAs) from Sprague-Dawley rats fed a normal salt (NS; 0.4% NaCl) diet, 2) evaluate the ability of chronic intravenous infusion of ANG-(1-7) (4 ng·kg(-1)·min(-1)) for 3 days to restore endothelium-dependent dilation to acetylcholine (ACh) in rats fed a high-salt (HS; 4% NaCl) diet, and 3) determine whether the amelioration of endothelial dysfunction by ANG-(1-7) infusion in rats fed a HS diet is different from the protective effect of low-dose ANG II infusion in salt-fed rats. MCAs from rats fed a NS diet dilated in response to exogenous ANG-(1-7) (10(-10)-10(-5) M). Chronic ANG-(1-7) infusion significantly reduced vascular superoxide levels and restored the nitric oxide-dependent dilation to ACh (10(-10)-10(-5) M) that was lost in MCAs of rats fed a HS diet. Acute vasodilation to ANG-(1-7) and the restoration of ACh-induced dilation by chronic ANG-(1-7) infusion in rats fed a HS diet were blocked by the Mas receptor antagonist [D-ALA(7)]-ANG-(1-7) or the ANG II type 2 receptor antagonist PD-123319 and unaffected by ANG II type 1 receptor blockade with losartan. The restoration of ACh-induced dilation in MCAs of HS-fed rats by chronic intravenous infusion of ANG II (5 ng·kg(-1)·min(-1)) was blocked by losartan and unaffected by d-ALA. These findings demonstrate that circulating ANG-(1-7), working via the Mas receptor, restores endothelium-dependent vasodilation in cerebral resistance arteries of animals fed a HS diet via mechanisms distinct from those activated by low-dose ANG II infusion.

Angiotensin-(1-7) Augments Endothelium-dependent Relaxations of Porcine Coronary Arteries to Bradykinin by Inhibiting Angiotensin-converting Enzyme 1

Abstract: Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II to angiotensin-(1-7) that activates Mas receptors, inhibits ACE1, and modulates bradykinin receptor sensitivity. This in vitro study compared the direct and indirect effects of angiotensin-(1-7), the ACE1 inhibitor captopril, and diminazene aceturate (DIZE) an alleged ACE2 activator in rings of porcine coronary arteries, by measuring changes of isometric tension. Angiotensin-(1-7), captopril, and DIZE did not cause significant changes in tension before or after desensitization of bradykinin receptors in preparations contracted with U46619. Bradykinin caused concentration-dependent and endothelium-dependent relaxations that were not affected by DIZE but were potentiated to a similar extent by angiotensin-(1-7) and captopril, given alone or in combination. Bradykinin responses potentiated by angiotensin-(1-7) and captopril were not affected by the BK1 antagonist SSR240612 and remained augmented in the presence of either N&ohgr;-nitro-L-arginine methyl ester hydrochloride plus indomethacin or TRAM-34 plus UCL-1684. ACE2 was identified in the coronary endothelium by immunofluorescence, but its basal activity was not influenced by DIZE. These results suggest that in coronary arteries, angiotensin-(1-7) and captopril both improves NO bioavailability and enhances endothelium-dependent hyperpolarization to bradykinin solely by ACE1 inhibition. Endothelial ACE2 activity cannot be increased by DIZE to produce local adequate amounts of angiotensin-(1-7) to influence vascular tone.

Dahl Salt-Sensitive Rats Are Protected Against Vascular Defects Related to Diet-Induced Obesity

Obesity increases plasma renin activity and angiotensin II levels, leading to vascular damage, elevated blood pressure, diabetes mellitus, and renal damage. Because genetic deletion of crucial parts of the renin-angiotensin system protect against obesity-related cardiovascular defects, we hypothesized that Dahl salt-sensitive (SS) rats, a model of chronically low plasma renin activity and angiotensin II levels, would be protected against vascular defects during diet-induced obesity compared with SS.13BN consomic rats showing normal renin-angiotensin system regulation. We evaluated vascular function in middle cerebral arteries of SS or SS.13BN rats fed high-fat (45% kcal from fat) versus normal-fat diet for 15 to 20 weeks from weaning. Endothelium-dependent relaxation in response to acetylcholine (10−8 to 10−4 mol/L) was restored in middle cerebral arteries of high-fat SS rats versus normal-fat diet controls, whereas vasodilation to acetylcholine was dramatically reduced in high-fat SS 13BN rats versus normal-fat diet controls. These findings support the hypothesis that physiological levels of angiotensin II play an important role in maintaining normal vascular relaxation in cerebral arteries and suggest that the cerebral vasculature of the SS rat model is genetically protected against endothelial dysfunction in diet-induced obesity.

Experimental orthostasis elicits sustained hypertension, which can be prevented by sympathetic blockade in the rat.

Incidence of orthostatic hypertension is estimated at 5% but is even more prevalent in borderline hypertension and autonomic neuropathies. The aim of this study was to develop a potential model to investigate orthostatic hypertension. We used normotensive and hypertensive Wistar rats to analyze responses and diurnal variations of arterial blood pressure, heart rate, temperature, and locomotor activity by telemetry. Orthostatic tests were carried out during 45° head-up tilt (R, repeated 3 times for 5 minutes; or S, sustained for 120 minutes). Hypertension was induced by blockade of nitric oxide synthesis. In normotensives, horizontal control blood pressure was R115.4 ± 1.4/S113.7 ± 1.6 mm Hg and heart rate R386.4 ± 7.0/S377.9 ± 8.8 bpm. Head-up tilt increased blood pressure by R4.5/S8.4 mm Hg, including a 3.8 mm Hg hydrostatic component. The sustained hypertensive response was prevented by prazosin (10 mg/kgbw) and augmented by a subanesthetic dose of chloralose (26 mg/kgbw). In NO-deprived hypertension, horizontal control blood pressure and heart rate were R138.4 ± 2.6/S140.3 ± 2.7 mm Hg and R342.1 ± 12.0/S346.0 ± 8.3 bpm, respectively. Tilt increased blood pressure further by R4.2/S9.4 mm Hg. In both normo- and hypertensives, variables exhibited similar diurnal rhythms except for nighttime locomotor activity, reduced from 3.7 ± 0.4 to 2.8 ± 0.3 counts/s. These data demonstrate that conscious rats respond to sustained orthostasis with hypertension, probably as a result of increased sympathetic output. Decreasing stress using a subanesthetic dose of chloralose increased this response, reducing the inhibitory effect on hypertensive responses.

Inverse-orthostasis may induce elevation of blood pressure due to sympathetic activation.

Microgravity and simulated microgravity may cause cardiovascular deconditioning, but mechanisms of instantaneous responses to inverse-orthostasis are not studied. Hence, we investigated transient and steady state cardiovascular changes by combining the tilt technique with cardiovascular telemetry. Normotensive and NO-deprived hypertensive Wistar rats were used to analyze responses of mean arterial blood pressure, heart rate, contractility, spontaneous baroreflex sensitivity (sBRS), and autonomic balance. Inverse-orthostasis tests were carried out by 45° head-down tilting (repeated 3 × 5 mins “R”, or sustained for 120 mins “S”). In normotensive rats, horizontal control blood pressure was R111.3 ± 1.7/S110.4 ± 2.3 mm Hg and heart rate was R385.2 ± 5.9/S371.1 ± 6.1 BPM. Head-down tilt induced an increase in blood pressure by R5.9/S10.6 mm Hg, while heart rate, contractility, sBRS, and autonomic balance did not change. The hypertensive response was sustained, could be prevented by prazosin (10 mg/kgbw), and augmented by subanesthetic doses of chloralose (26 and 43 mg/kgbw). In NO-suppressed hypertension, control blood pressure and heart rate were R132.4 ± 2.9/S130.0 ± 4.1 mm Hg and R339.2 ± 7.9/S307.2 ± 23.6 BPM, respectively. Head-down tilt further increased blood pressure by R5.1/S10.5 mm Hg. These data demonstrate that conscious rats respond to inverse-orthostasis by sustained elevation of blood pressure independent of NO synthesis. This response is neither due to increased contractility and altered sBRS, nor due to non-specific stress, but probably due to sympathetic activation elicited by gravity-related reflexes, which increase peripheral resistance.

Calcium entry is regulated by Zn2+ in relation to extracellular ionic environment in human airway epithelial cells.

The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). This elevation of [Ca(2+)](i) and activation of Ca(2+)-dependent Cl(-) channels represent a possible therapeutic approach in cystic fibrosis (CF). We investigated the changes of [Ca(2+)](i) in different external ionic environment, and P2X purinergic receptors (P2XRs) expression in the control and CF airway epithelial cells. The parallel removal of Na(+) and alkalinization of the extracellular solution increased the amplitude of sustained ATP-induced Ca(2+) signals independent of wild-type or mutant CFTR expression. The ATP-induced Ca(2+) entry was either inhibited or stimulated by Zn(2+) depending on the extracellular Na(+) concentration. In Na(+)-free environment, Zn(2+) and other divalent cations elicited a biphasic Ca(2+) signal. Immunohistochemical data suggest that, multiple subtypes of P2XRs are expressed in these airway epithelial cells. In conclusion, Ca(2+) entry is finely regulated by external ionic environment. Therefore, we speculate that properly compiled aerosols could influence efficacy of zinc-based therapy in CF.

Modulation by Cytochrome P450-4A ω-Hydroxylase Enzymes of Adrenergic Vasoconstriction and Response to Reduced PO2 in Mesenteric Resistance Arteries of Dahl Salt-Sensitive Rats

Please cite this paper as: Raffai, Wang, Roman, Anjaiah, Weinberg, Falck and Lombard (2010). Modulation by Cytochrome P450‐4A ω‐Hydroxylase Enzymes of Adrenergic Vasoconstriction and Response to Reduced PO2 in Mesenteric Resistance Arteries of Dahl Salt‐Sensitive Rats. Microcirculation17(7), 525–535.

Selective Suppression of an Endothelin and Platelet-Derived Growth Factor Containing Vesicular System in Endothelium of Rat Saphenous Vein by Long-Term Orthostasis

Electron-dense vesicles were observed in rat vascular endothelium. The purpose of this study was to characterize their content(s), venous-arterial distribution and response to chronic orthostatic stress in extremity vessels. Saphenous and brachial vessels – saphenous vein (SV), saphenous artery (SA), brachial vein, brachial artery – were prepared for electron microscopy to quantitate the vesicle area within the endothelium following immunohistochemical and immunocytochemical identification. The effect of long-term orthostasis was assessed by exposure to head-up tilt for 2 weeks. The vesicular area in relation to the total cross-sectional area of the endothelial cells in the SV and SA of normal and confined control groups was 3.88 ± 0.38 versus 0.89 ± 0.06% (p < 0.05) and 4.92 ± 0.25 versus 1.09 ± 0.47% (p < 0.05), respectively. Head-up tilt suppressed the vesicle content of the SV to 2.26 ± 0.39% (p < 0.05), but it remained low in the SA (1.29 ± 0.45%), brachial vein (0.45 ± 0.12%) and brachial artery (0.59 ± 0.17%). Endothelin and platelet-derived growth factor, but not acidic phosphatase activity or lipid content, could be identified in the vesicles. Plasma endothelin levels were unchanged. We conclude that dense vesicles in the endothelium of extremity vessels are not cell degradation products. They may represent a vesicular secretory or storage system for endothelin and platelet-derived growth factor which participates in regional vascular adaptation to long-term orthostatic load.

Amelioration of salt-induced vascular dysfunction in mesenteric arteries of Dahl salt-sensitive rats by missense mutation of extracellular superoxide dismutase

Superoxide dismutase (SOD) enzymes, including extracellular SOD (ecSOD), are important for scavenging superoxide radicals (O2(·-)) in the vasculature. This study investigated vascular control in rats [SS-Sod(3m1Mcwi) (ecSOD(E124D))] with a missense mutation that alters a single amino acid (E124D) of ecSOD that produces a malfunctioning protein in the salt-sensitive (Dahl SS) genetic background. We hypothesized that this mutation would exacerbate endothelial dysfunction due to elevated vascular O2(·-) levels in SS, even under normal salt (NS; 0.4% NaCl) conditions. Aortas of ecSOD(E124D) rats fed standard rodent chow showed enhanced sensitivity to phenylephrine and reduced relaxation to acetylcholine (ACh) vs. SS rats. Endothelium-dependent dilation to ACh was unaffected by the mutation in small mesenteric arteries of ecSOD(E124D) rats fed NS diet, and mesenteric arteries of ecSOD(E124D) rats were protected from endothelial dysfunction during short-term (3-5 days) high-salt (HS; 4% NaCl) diet. ACh-induced dilation of mesenteric arteries of ecSOD(E124D) rats and SS rats fed NS diet was inhibited by N(G)-nitro-l-arginine methyl ester and/or by H2O2 scavenging with polyethylene glycol-catalase at higher concentrations of ACh. Total SOD activity was significantly higher in ecSOD(E124D) rats vs. SS controls fed HS diet, most likely reflecting a compensatory response to loss of a functional ecSOD isoform. These findings indicate that, contrary to its effect in the aorta, this missense mutation of ecSOD in the SS rat genome has no negative effect on vascular function in small resistance arteries, but instead protects against salt-induced endothelial dysfunction, most likely via compensatory mechanisms involving an increase in total SOD activity.