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Zhou X, Bohlen HG, Unthank JL, Miller SJ. Abnormal nitric oxide production in aged rat mesenteric arteries is mediated by NAD(P)H oxidase-derived peroxide. Am J Physiol Heart Circ Physiol 297: H2227-H2233, 2009. First published September 25, 2009; doi: 10.1152/ajpheart.00325.2009.--Previous work in our laboratory showed increased basal periarterial nitric oxide (NO) and [H.sub.2][O.sub.2] concentrations in the spontaneously hypertensive rat, characterized by oxidant stress, as well as impaired flow-mediated NO production that was corrected by a reduction of periarterial [H.sub.2][O.sub.2]. Aging is also associated with an increase in vascular reactive oxygen species and results in abnormal vascular function. The current study was designed to assess the role of [H.sub.2][O.sub.2] in regulating NO production during vascular aging. In vivo, real-time NO and [H.sub.2][O.sub.2] concentrations were measured by microelectrodes in mesenteric arteries of retired breeder (aged; 8-12 mo) and young (2 to 3 mo) Wistar-Kyoto rats under conditions of altered flow. The results in aged rats revealed elevated basal NO (1,611 [+ or -] 286 vs. 793 [+ or -] 112 nM, P < 0.05) and [H.sub.2][O.sub.2] concentrations (16 [+ or -] 2 vs. 9 [+ or -] 1 [micro]M, P < 0.05) and a flow- mediated increase in [H.sub.2][O.sub.2] but not NO production. Pretreatment of aged rats with the antioxidant apocynin lowered both basal [H.sub.2]02 (8 [+ or -] 1 [micro]M) and NO (760 [+ or -] 102 nM) to young levels and restored flow-mediated NO production. Similar results were obtained with the NAD(P)H oxidase inhibitor gp91ds-tat. In addition, acute incubation with topical polyethylene-glycolated catalase lowered the baseline NO concentration and restored flow-mediated NO production. Taken together, the data indicate that elevated baseline and suppressed flow-mediated NO production in aged Wistar-Kyoto rats are mediated by NAD(P)H oxidase-derived [H.sub.2][O.sub.2]. apocynin; flow mediated; nicotinamide adenine dinucleotide phosphate oxidase inhibition; oxidative stress; Wistar-Kyoto rats doi: 10.1152/ajpheart.00325.2009

Multiple investigators have shown interdependence of lymphatic contractions on nitric oxide (NO) activity by pharmacological and traumatic suppression of endothelial NO synthase (eNOS). We demonstrated that lymphatic diastolic relaxation is particularly sensitive to NO from the lymphatic endothelium. The predicted mechanism is shear forces produced by the lymph flow during phasic pumping, activating eNOS in the lymphatic endothelium to produce NO. We measured [NO] during phasic contractions using microelectrodes on in situ mesenteric lymphatics in anesthetized rats under basal conditions and with an intravenous saline bolus (0.5 ml/100 g) or infusion (0.5 ml x 100 [g.sup.-1] x [h.sup.-1]). Under basal conditions, [NO] measured on the tubular portions of the lymphatics was ~200-250 nM, slightly higher than in the adjacent adipocyte microvasculature, whereas [NO] measured on the lymphatic bulb surface was ~400 nM. Immunohistochemistry of eNOS in isolated lympathics indicated a much greater expression in the lymph valves and surrounding bulb area than in the tubular regions. During phasic lymphatic contractions, the valve and tubular [NO] increased with each contraction, and during intravenous saline infusion, [NO] increased in proportion to the contraction frequency and, presumably, lymph flow. The partial blockade of eNOS over ~1 cm length with [N.sup.[omega]]-nitro-L-arginine methyl ester lowered the [NO]. These in vivo data document for the first time that both valvular and tubular lymphatic segments increase NO generation during each phasic contraction and that [NO] summated with increased contraction frequency. The combined data predict regional variations in eNOS and [NO] in the tubular and valve areas, plus the summated NO responses dependent on contraction frequency provide for a complex relaxation mechanism involving NO. lymph flow; frequency contraction doi: 10.1152/ajpheart.00039.2009

Endogenous periadventitial adipose-derived factors have been shown to contribute to coronary vascular regulation by impairing endothelial function through a direct inhibition of endothelial nitric oxide synthase (eNOS). However, our understanding of the underlying mechanisms remains uncertain. Accordingly, this study was designed to test the hypothesis that periadventitial adipose tissue releases agents that attenuate coronary endothelial nitric oxide production via a protein kinase C (PKC)-[beta]dependent mechanism. Isometric tension studies were conducted on isolated canine circumflex coronary arteries with and without natural amounts of periadventitial adipose tissue. Adipose tissue significantly diminished coronary endothelial-dependent vasodilation and nitric oxide production in response to bradykinin and acetylcholine. The selective inhibition of endothelial PKC-[beta] with ruboxistaurin (1 [micro]M) abolished the adipose-induced impairment of hradykinin-mediated coronary vasodilation and the endothelial production of nitric oxide. Western blot analysis revealed a significant increase in eNOS phosphorylation at the inhibitory residue [Thr.sup.495] in arteries exposed to periadventitial adipose tissue. This site-specific phosphorylation of eNOS was prevented by the inhibition of PKC-[beta]. These data demonstrate that periadventitial adipose-derived factors impair coronary endothelial nitric oxide production via a PKC-[beta]-dependent, site-specific phosphorylation of eNOS at [Thr.sup.495]. coronary circulation; adipokine; endothelial nitric oxide synthase; protein kinase C-[beta]

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and [H.sub.2][O.sub.2] were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from ~50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and [H.sub.2][O.sub.2] were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and [H.sub.2][O.sub.2] concentrations (2,572 [+ or -] 241 vs. 1,059 [+ or -] 160 nM, P < 0.01; and 26 [+ or -] 7 vs. 7 [+ or -] 1 [micro]M, P < 0.05, respectively). With flow elevation, [H.sub.2][O.sub.2] but not NO increased in SHR; NO but not [H.sub.2][O.sub.2] was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and [H.sub.2][O.sub.2] to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR [H.sub.2][O.sub.2] to WKY levels. Addition of topical [H.sub.2][O.sub.2] to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR. hypertension; oxidative stress; flow mediated; NAD(P)H oxidase inhibition; spontaneously hypertensive rats

[Na.sup.+] cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the [Na.sup.+]/[Ca.sup.2+] exchanger, bumetanide for the [Na.sup.+]- [Ksup.+]-2C1- cotransporter, and amiloride for [Na.sup.+]/ [H.sup.+] cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanidesensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The [Na.sup.+]/[Ca.sup.+] exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive [Na.sup.+]/ [H.sup.+] cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to [Na.sup.+]-[K.sup.+]2Cl-cotransporter and [Na.sup.+]/ [Ca.sup.2+] exchanger functions are key to cerebral vascular oxygen responses. neuronal nitric oxide synthase; endothelial nitric oxide synthase; brain

Endothelial (eNOS) and neuronal nitric oxide synthase (nNOS) are implicated as important contributors to cerebral vascular regulation through nitric oxide (NO). However, direct in vivo measurements of NO in the brain have not been used to dissect their relative roles, particularly as related to oxygenation of brain tissue. We found that, in vivo, rat cerebral arterioles had increased NO concentration ([NO]) and diameter at reduced periarteriolar oxygen tension ([Po.sub.2]) when either bath oxygen tension or arterial pressure was decreased. Using these protocols with highly selective blockade of nNOS, we tested the hypothesis that brain tissue nNOS could donate NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. The decline in periarteriolar [Po.sub.2] by bath manipulation increased [NO] and vessel diameter comparable with responses at similarly decreased [Po.sub.2] during hypotension. To determine whether the nNOS provided much of the vascular wall NO, nNOS was locally suppressed with the highly selective inhibitor N-(4S)-(4-amino-5-[aminoethyl]aminopentyl)-[N.sup.']-nitroguanidine. After blockade, resting [NO], [Po.sub.2], and diameters decreased, and the increase in [NO] during reduced [Po.sub.2] or hypotension was completely absent. However, flow-mediated dilation during occlusion of a collateral arteriole did remain intact after nNOS blockade and the vessel wall [NO] increased to ~80% of normal. Therefore, nNOS predominantly increased NO during decreased periarteriolar oxygen tension, such as that during hypotension, but eNOS was the dominant source of NO for flow shear mechanisms. nitric oxide; brain; arterioles; in vivo; microelectrode; neuronal nitric oxide synthase

In cultured endothelial cells, 70-95% of extracellular L-arginine uptake has been attributed to the cationic amino acid transporter-1 protein (CAT-1). We tested the hypothesis that extracellular e-arginine entry into endothelial cells via CAT-1 plays a crucial role in endothelial nitric oxide (NO) production during in vivo conditions. Using L-lysine, the preferred amino acid transported by CAT-1, we competitively inhibited extracellular L-arginine transport into endothelial cells during conditions of NaCl hyperosmolarity, low oxygen, and flow increase. Our prior studies indicate that each of these perturbations causes NO-dependent vasodilation. The perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature. Suppression of extracellular L-arginine transport significantly and strongly inhibited increases in vascular [NO] and intestinal blood flow during NaCl hyperosmolarity, lowered oxygen tension, and increased flow. These results suggest that L-arginine from the extracellular space is accumulated by CAT-1. When CAT-1-mediated transport of extracellular L-arginine into endothelial cells was suppressed, the endothelial cell NO response to a wide range of physiological stimuli was strongly depressed. intestine; blood flow; arterioles; L-lysine; oxygen; hyperosmolarity; shear

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM. respectively, before and alter application of bumetanide ([Na.sup.+] -[K.sup.+] -2[Cl.sup.-] cotransporter inhibitor) or amiloride ([Na.sup.+]/[H.sup.+] exchange channel inhibitor). Suppressing amiloride-sensitive [Na.sup.+]/[H.sup.+] exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of [Na.sup.+] -[K.sup.+] -2[Cl.sup.-] channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the [Na.sup.+]/[Ca.sup.2+] exchanger extrudes [Na.sup.+] in exchange for [Ca.sup.2+], thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the [Na.sup.+]/[Ca.sup.2+] exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943: complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through [Na.sup.+] -[K.sup.+] -2[Cl.sup.-] channels. The [Na.sup.+]/[Ca.sup.2+] exchanger then extrudes [Na.sup.+] and increases endothelial [Ca.sup.2+]. The increase in endothelial [Ca.sup.2+] causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow. endothelial nitric oxide synthase; sodium/calcium exchanger; sodium-potassium-chloride cotransporter; sodium-hydrogen exchanger 1

Kidney glomeruli are important targets of diabetic nephropathy. We hypothesized a high concentration of glucose could suppress glomerular endothelial nitric oxide synthase (eNOS) by a protein kinase C (PKC) mechanism, as has been found in other tissues. Mouse kidney slices (150-200 [micro]m) were bathed in Hanks' solution with 100 [micro]M L-arginine and exposed to either 5 or 20-30 mM D-glucose. Immunofluorescence identified only eNOS in normal mouse glomeruli. Measurements of glomemlar NO concentration with NO-sensitive fluorescent dye (4,5-diaminofluorescein diacetate) using confocal microscopy and NO-sensitive microelectrodes verified that resting glomeruli had active production of NO that was inhibited by [N.sup.G]-nitro-L-arginine methyl ester. High-concentration (20-30 mM) D-glucose inhibited 60-70% of the NO production within 15-30 min; L-glucose at the same concentration did not have any effect. Inhibition of PKC-[beta] with 100 nM ruboxistaurin prevented eNOS suppression in high-glucose media. Activation of PKC with 100 nM phorbol ester also suppressed the glomerular NO concentration. We concluded that eNOS in the renal glomerular capillary endothelial cells is suppressed by activity of PKC at high-glucose concentrations comparable to those in diabetic animals and humans. The consequence is a rapid decline in the generation of NO in the glomerular endothelial cells in the presence of a high concentration of glucose. microelectrode; hyperglycemia; confocal imaging; protein kinase C; endothelial nitric oxide synthase

In severe obesity, microvascular endothelial regulation of nitric oxide (NO) formation is compromised in response to muscarinic stimulation, and major arteries have suppressed flow-mediated dilation. Because normal microvessels are highly dependent on flow-mediated stimulation of NO generation and are responsive to intra--and extravascular oxygen availability, they are likely a major site of impaired endothelial regulation. This study evaluated the blood flow and oxygen-dependent aspects of intestinal microvascular regulation and NO production in Zucker obese rats just before the onset of hyperglycemia. Ruboxistaurin (LY-333531) was used to inhibit PKC-[beta]11 to determine whether flow or oxygen-related NO regulation was improved. Blood flow velocity was increased by forcing arterioles to perfuse ~50% larger tissue areas by occlusion of nearby arterioles, and oxygen tension in the bath was lowered to create a modest oxygen depletion. When compared with lean Zucker rats, the periarteriolar NO concentration ([NO]) for obese rats was ~30% below normal. At elevated shear rates, the [NO] for arterioles of obese animals was 20-30% below those in the arterioles of lean rats, and the NO response to decreased oxygen was about half normal in obese rats. All of these regulatory problems were essentially corrected in obese rats by PKC blockade with only minor changes in the microvascular behavior in lean rats. Therefore, activation of PKC-[beta]II in endothelial cells during obesity suppressed NO regulation both at rest and in response to increased flow velocity and decreased oxygen availability. shear rate; ruboxistaurin; arteriole; intestine