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 Ca 2ϩ ([Ca 2ϩ ]i) in response to methacholine (10 M) and histamine (10 M) were significantly attenuated in aortic endothelial cells from rats fed a HS diet, whereas thapsigargin (10 M)-induced increases in [Ca 2ϩ ]i were unaffected. Methacholineinduced nitric oxide (NO) production was eliminated in endothelial cells of aortas from rats fed a HS diet. Low-dose ANG II infusion (5 ng ⅐ kg Ϫ1 ⅐ min Ϫ1 iv) for 3 days prevented impaired [Ca 2ϩ ]i signaling response to methacholine and histamine and restored methacholineinduced NO production in aortas from rats on a HS diet. Adding Tempol (500 M) to the tissue bath to scavenge superoxide anions increased NO release and caused N -nitro-L-arginine methyl estersensitive vascular relaxation in aortas from rats fed a HS diet but had no effect on methacholine-induced Ca 2ϩ responses. Chronic treatment with Tempol (1 mM) in the drinking water restored NO release, augmented vessel relaxation, and increased methacholine-induced Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ signaling in the vascular endothelial cells; and 3) reduced circulating ANG II levels during elevated dietary salt lead to elevated superoxide levels, impaired endothelial Ca 2ϩ signaling, and reduced NO production in the endothelium. endothelium; sodium; dietary salt intake; vascular relaxation; nitric oxide A VARIETY OF AGENTS, including muscarinic agonists such as ACh, dilate blood vessels by stimulating the synthesis of nitric oxide (NO) by endothelial NO synthase. Endothelial cell activation in response to agonists and autacoids leads to NO formation via receptor-mediated increases in intracellular calcium ion concentration ([Ca 2ϩ ] i ). However, emerging evidence indicates that endothelial NO synthase activity can be affected by a variety of other mechanisms that are independent of [Ca 2ϩ ] i (7,12,21,26,27). This raises the possibility that changes in NO production under some physiological conditions may be independent of changes in endothelial [Ca 2ϩ ] i . Endothelium-dependent relaxation in response to ACh is impaired in the aorta, resistance arteries, and microvessels of rats fed a high-salt (HS) diet (2-5, 15, 16, 18, 28). Elevated dietary salt intake causes suppression of plasma ANG II levels (8, 9), and previous studies (18,30,31) have demonstrated that the impaired vascular relaxation in Sprague-Dawley rats fed a HS diet can be prevented by a continuous intravenous infusion of a low dose of ANG II to maintain normal circulating levels of ANG II.In the present study, we tested the hypothesis that receptormediated increases in [Ca 2ϩ ] i and/or...

Objective: This study determined the mechanisms and time-course of recovery of vascular relaxation in middle cerebral arteries (MCA) of salt-fed Sprague-Dawley rats returned to low salt (LS) diet (0.4% NaCl) or infused with low-dose angiotensin II (ANG II).Methods: Rats were fed high salt (HS) diet (4% NaCl) for 3 days or 4 weeks before return to LS diet for various periods. Other rats fed HS diet (HS + ANG II) received a chronic (3 days) i.v. infusion of a low dose of ANG II (5 ng·kg −1 ·min −1 ) to prevent salt-induced ANG II suppression.Results: HS diet eliminated the increase in cerebral blood flow in response to acetylcholine (ACh) infusion and the relaxation of MCA in response to ACh, iloprost, cholera toxin, and reduced PO 2 . Recovery of vascular relaxation was slow, requiring at least 2 weeks of LS diet, regardless of the duration of exposure to HS diet. Hypoxic dilation was mediated by cyclooxygenase metabolites and ACh-induced dilation was mediated via NO in LS rats and in HS rats returned to LS diet or receiving ANG II infusion. Conclusions:Return to LS diet for 2 weeks or chronic 3-day ANG II infusion restore the mechanisms that normally mediate cerebral vascular relaxation.

Objectives We investigated the effect of suppressing plasma angiotensin II (ANG II) levels on arteriolar relaxation in the hamster cheek pouch. Methods Arteriolar diameters were measured via television microscopy during short-term (3–6 days) high salt (HS; 4% NaCl) diet and angiotensin converting enzyme (ACE) inhibition with captopril (100 mg/kg/day). Results ACE inhibition and/or HS diet eliminated endothelium-dependent arteriolar dilation to acetylcholine, endothelium-independent dilation to the NO donor sodium nitroprusside, the prostacyclin analogues carbacyclin and iloprost, and the KATP channel opener cromakalim; and eliminated arteriolar constriction during KATP channel blockade with glibenclamide. Scavenging of superoxide radicals and low dose ANG II infusion (25 ng/kg/min, subcutaneous) reduced oxidant stress and restored arteriolar dilation in arterioles of HS-fed hamsters. Vasoconstriction to topically-applied ANG II was unaffected by HS diet while arteriolar responses to elevation of superfusion solution PO2 were unaffected (5% O2, 10% O2) or reduced (21% O2) by HS diet. Conclusions These findings indicate that sustained exposure to low levels of circulating ANG II leads to widespread dysfunction in endothelium-dependent and independent vascular relaxation mechanisms in cheek pouch arterioles by increasing vascular oxidant stress, but does not potentiate O2- or ANG II-induced constriction of arterioles in the distal microcirculation of normotensive hamsters.

This study identified, on the integrative level, two components of the ANG II signaling pathway that lay downstream from the ANG II type 1 (AT(1)) receptor and are critically involved in maintaining vascular relaxation in cerebral resistance arteries. In these experiments, the relaxation of isolated middle cerebral arteries (MCA) in response to ACh (10(-9)-10(-5) M), iloprost (10(-16)-10(-11) g/ml), and reduced PO(2) was lost and the ratio of phospho-ERK/ERK1/2 was significantly reduced in aortas of male Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) diet to suppress plasma ANG II levels. In salt-fed rats, relaxation of MCA in response to these vasodilator stimuli was restored by chronic (3 days) intravenous infusion of either ANG II (5 ngxkg(-1)xmin(-1)) or epidermal growth factor (EGF; 2 microg/h). The protective effect of ANG II infusion to restore vascular relaxation was eliminated by coinfusion of either the EGF receptor kinase inhibitor AG-1478 (20 microg/h), the ERK1/2 inhibitor PD-98059 (10 microg/h), or the protein synthesis inhibitor cycloheximide (5 microg/h). In rats fed a low-salt (0.4% NaCl) diet, MCA relaxation in response to ACh, reduced PO(2), and iloprost was eliminated by intravenous infusion of AG-1478, PD-98059, or cycloheximide. In ANG II-infused rats fed HS diet, and in rats fed LS diet, vasodilator responses to reduced PO(2) and iloprost were unaffected by the p38 MAP kinase inhibitor SB-203580 and the phosphatidylinositol 3-kinase inhibitor wortmannin. These findings indicate that maintenance of normal vascular relaxation mechanisms by ANG II in rat MCA requires activation of the EGF receptor kinase and ERK1/2.

Donor-specific CD4+CD127−CD25+FOXP3+ regulatory T cells (AgTregs) have the potential to induce clinical transplant tolerance; however, their expansion ex vivo remains challenging. We optimized a novel expansion protocol to stimulate donor-specific Tregs using soluble 4-trimer CD40 ligand (CD40L)-activated donor B cells that expressed mature antigen-presenting cell markers. This avoided the use of CD40L-expressing stimulator cells that might otherwise result in potential cellular contamination. Purified allogeneic “recipient” CD4+CD25+ Tregs were stimulated on days 0 and 7 with expanded “donor” B cells in the presence of IL-2, TGFβ and sirolimus (SRL). Tregs were further amplified by polyclonal stimulation with anti-CD3/CD28 beads on day 14 without SRL, and harvested on day 21, with extrapolated fold expansion into the thousands. The expanded AgTregs maintained expression of classical Treg markers including demethylation of the Treg-specific demethylated region (CNS2) and also displayed constricted TcR repertoire. We observed AgTregs more potently inhibited MLR than polyclonally expanded Tregs and generated new Tregs in autologous responder cells (a measure of infectious tolerance). Thus, an optimized and more clinically applicable protocol for the expansion of donor-specific Tregs has been developed.