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Proper function of the vascular endothelium is essential for cardiovascular health, in large part due to its anti-proliferative, anti-hypertrophic, and anti-inflammatory properties. Crucial to the protective role of the endothelium is the production and liberation of nitric oxide (NO), which not only acts as a potent vasodilator, but also reduces levels of reactive oxygen species (ROS), including superoxide anion (O2•-). Superoxide anion is highly injurious to the vasculature because it not only scavenges NO molecules, but has other damaging effects, including direct oxidative disruption of normal signaling mechanisms in the endothelium and vascular smooth muscle cells. The renin-angiotensin system plays a crucial role in the maintenance of normal blood pressure. This function is mediated via the peptide hormone angiotensin II (ANG II), which maintains normal blood volume by regulating Na+ excretion. However, elevation of ANG II above normal levels increases O2•- production, promotes oxidative stress and endothelial dysfunction, and plays a major role in multiple disease conditions. Elevated dietary salt intake also leads to oxidant stress and endothelial dysfunction, but these occur in the face of salt-induced ANG II suppression and reduced levels of circulating ANG II. While the effects of abnormally high levels of ANG II have been extensively studied, far less is known regarding the mechanisms of oxidant stress and endothelial dysfunction occurring in response to chronic exposure to abnormally low levels of ANG II. The current article focuses on the mechanisms and consequences of this less well understood relationship among salt, superoxide, and endothelial function. Proper function of the vascular endothelium is essential for cardiovascular health, in large part due to its anti- proliferative, anti-hypertrophic, and anti- inflammatory properties. Crucial to the protective role of the endothelium is the production and liberation of nitric oxide (NO), which not only acts as a potent vasodilator, but also reduces levels of reactive oxygen species (ROS), including superoxide anion (O2•-). Superoxide anion is highly injurious to the vasculature because it not only scavenges NO molecules, but has other damaging effects, including direct oxidative disruption of normal signaling mechanisms in the endothelium and vascular smooth muscle cells. The renin-angiotensin system plays a crucial role in the maintenance of normal blood pressure. This function is mediated via the peptide hormone angiotensin II (ANG II), which maintains normal blood volume by regulating Na+ excretion. However, elevation of ANG II above normal levels increases O2•- production, promotes oxidative stress and endothelial dysfunction, and plays a major role in multiple disease conditions. Elevated dietary salt intake also leads to oxidant stress and endothelial dysfunction, but these occur in the face of salt-induced ANG II suppression and reduced levels of circulating ANG II. While the effects of abnormally high levels of ANG II have been extensively studied, far less is known regarding the mechanisms of oxidant stress and endothelial dysfunction occurring in response to chronic exposure to abnormally low levels of ANG II. The current article focuses on the mechanisms and consequences of this less well understood relationship among salt, superoxide, and endothelial function.

Rationale: Hypertension prevalence is much higher among children and adolescents with low birth weight and greater postnatal weight gain than in individuals with normal birth weight. However, the cause and molecular mechanisms underlying this complication remain largely unknown. Our previous studies have shown that RGC-32 (response gene to complement 32)–deficient (RGC-32 −/− ) mice are born significantly smaller but grow faster than their WT (wild type) controls, which allows adult RGC-32 −/− mice to attain body weights similar to those of control mice. Objective: The objective of this study is to determine whether RGC-32 −/− mice develop hypertension, and if so, to elucidate the underlying mechanisms. Methods and Results: By using a radiotelemetry system, we found that RGC-32 −/− mice exhibit higher mean arterial pressure than WT mice (101±4 versus 119±5 mm Hg), which enabled us to use RGC-32 −/− mice to study the mechanisms underlying low birth weight–related hypertension. The increased blood pressure in RGC-32 −/− mice was associated with increased vascular tone and decreased distensibility of small resistance arteries. The increased vascular tone was because of an increase in the relative contribution of sympathetic versus parasympathetic activity and was linked to increased expression of AT1R (angiotensin II type I receptor) and α1-AdR (α1-adrenergic receptor) in arterial smooth muscles. Mechanistically, RGC-32 regulated AT1R gene transcription by interacting with Sp1 (specificity protein 1) transcription factor and further blocking its binding to the AT1R promoter, leading to suppression of AT1R expression. The attenuation of AT1R leads to reduction in α1-AdR expression, which was critical for the balance of sympathetic versus parasympathetic control of vascular tone. Of importance, downregulation of RGC-32 in arterial smooth muscles was also associated with low birth weight and hypertension in humans. Conclusions: Our results indicate that RGC-32 is a novel protein factor vital for maintaining blood pressure homeostasis, especially in individuals with low birth weight.

Within the last 25 years, it has become increasingly clear that high dietary salt intake represents a risk factor for the development of cardiovascular disease that is independent of its well-known ability to increase arterial pressure in some individuals. Studies in normotensive experimental animals and human subjects have revealed that a key feature of this pressure-independent effect of dietary salt is a profound reduction in vascular nitric oxide (NO) bioavailability that limits endothelium-dependent dilation. This reduction in NO is strongly associated with increased levels of reactive oxygen species (ROS) generated by NAD(P)H oxidase, xanthine oxidase or uncoupled endothelial NO synthase within the vascular wall, leading not only to scavenging of NO but also to disruption of some signaling pathways that mediate its production. The mechanistic link between high salt intake and elevated levels of enzymatically generated ROS in the peripheral vasculature is not clear, but a reduction in circulating angiotensin II may play a key role in this regard. Additional studies are needed to further elucidate the mechanisms, both at the systemic level and within the vascular wall, that trigger these salt-induced deficits in endothelial function, and to further clarify how the attendant loss of NO may disrupt tissue blood flow regulation and ultimately lead to adverse cardiovascular events.

High salt (HS) intake can change the arterial tone in mice, and the nitric oxide (NO) acts as a mediator to some of the receptors mediated vascular response. The main aim of this study was to explore the mechanism behind adenosine-induced vascular response in HS-fed eNOS(+/+) and eNOS(-/-) mice The modulation of vascular response by HS was examined using aortas from mice (eNOS(+/+) and eNOS(-/-)) fed 4% (HS) or 0.45% (NS) NaCl-diet through acetylcholine (ACh), NECA (adenosine-analog), CGS 21680 (A(2A) AR-agonist), MS-PPOH (CYP epoxygenase-blocker; 10(-5) M), AUDA (sEH-blocker; 10(-5) M), and DDMS (CYP4A-blocker; 10(-5) M). ACh-response was greater in HS-eNOS(+/+) (+59.3 ± 6.3%) versus NS-eNOS(+/+) (+33.3 ± 8.0%; P0.05). However, there was no response in both HS-eNOS(-/-) and NS-eNOS(-/-). NECA-response was greater in HS-eNOS(-/-) (+37.4 ± 3.2%) versus NS-eNOS(-/-) (+7.4.0 ± 3.8%; P0.05). CGS 21680-response was also greater in HS-eNOS(-/-) (+45.4 ± 5.2%) versus NS-eNOS(-/-)(+5.1 ± 5.0%; P0.05). In HS-eNOS(-/-), the CGS 21680-response was reduced by MS-PPOH (+7.3 ± 3.2%; P0.05). In NS-eNOS(-/-), the CGS 21680-response was increased by AUDA (+38.2 ± 3.3%; P0.05) and DDMS (+30.1 ± 4.1%; P0.05). Compared to NS, HS increased CYP2J2 in eNOS(+/+) (35%; P0.05) and eNOS(-/-) (61%; P0.05), but decreased sEH in eNOS(+/+) (74%; P0.05) and eNOS(-/-) (40%; P0.05). Similarly, CYP4A decreased in HS-eNOS(+/+) (35%; P0.05) and HS-eNOS(-/-) (34%; P0.05). These data suggest that NS causes reduced-vasodilation in both eNOS(+/+) and eNOS(-/-) via sEH and CYP4A. However, HS triggers possible A(2A)AR-induced relaxation through CYP epoxygenase in both eNOS(+/+) and eNOS(-/-).

Previous study suggests that with evolution of the metabolic syndrome, patterns of arteriolar reactivity are profoundly altered and may constrain functional hyperemia. This study investigated interactions between parameters of vascular reactivity at two levels of resistance arterioles in obese Zucker rats (OZR), translating these observations into perfusion regulation for in situ skeletal muscle. Dilation of isolated and in situ resistance arterioles from OZR to acetylcholine, arachidonic acid (AA), and hypoxia (isolated arterioles only) were blunted vs. lean Zucker rats (LZR), although dilation to adenosine was intact. Increased adrenergic tone (phenylephrine) or intralumenal pressure (ILP) impaired dilation in both strains (OZR>LZR). Treatment of OZR arterioles with Tempol (superoxide dismutase mimetic) or SQ-29548 (prostaglandin H2/thromboxane A2 receptor antagonist) improved dilator reactivity under control conditions and with increased ILP, but had minimal effect with increased adrenergic tone. Arteriolar dilation to adenosine was well maintained in both strains under all conditions. For in situ cremasteric arterioles, muscle contraction-induced elevations in metabolic demand elicited arteriolar dilations and hyperemic responses that were blunted in OZR vs. LZR, although distal parallel arterioles were characterized by heterogeneous dilator and perfusion responses. α-Adrenoreceptor blockade improved outcomes at rest but had minimal effect with elevated metabolic demand. Treatment with Tempol or SQ-29548 had minimal impact at rest, but lessened distal arteriolar perfusion heterogeneity with increased metabolic demand. In blood-perfused gastrocnemius of OZR, perfusion was constrained primarily by adrenergic tone, while myogenic activation and endothelium-dependent dilation did not appear to contribute significantly to ischemia. These results of this novel, integrated approach suggest that adrenergic tone and metabolic dilation are robust determinants of bulk perfusion to skeletal muscle of OZR, while endothelial dysfunction may more strongly regulate perfusion distribution homogeneity via the impact of oxidant stress and AA metabolism.

Previous studies from this laboratory suggest that during juvenile growth, structural changes in the arteriolar network are accompanied by changes in some of the mechanisms responsible for regulation of tissue blood flow. To test the hypothesis that arteriolar myogenic behavior is altered with growth, we studied gracilis muscle arterioles isolated from Sprague-Dawley rats at two ages: 21–28 and 42–49 days. When studied at their respective in vivo pressures, the myogenic index (instantaneous slope of the active pressure-diameter curve) of arterioles from 42–49-day-old rats was more negative than that of arterioles from 21–28-day-old rats, indicating greater myogenic responsiveness. Endothelial denudation, or prostaglandin H2(PGH2)/thromboxane A2(TxA2) receptor antagonism without denudation, significantly reduced the myogenic responsiveness of arterioles from the older rats over a wide range of pressures but had no consistent effects on the myogenic responsiveness of arterioles from the younger rats. The heme oxygenase inhibitor chromium (III) mesoporphyrin IX chloride had no effect on the myogenic activity of arterioles from either age group. These findings indicate that microvascular growth in young animals is accompanied by an increase in the myogenic behavior of arterioles, possibly because PGH2or TxA2assumes a role in reinforcing myogenic activity over this period. As a result, the relative contribution of myogenic activity to blood flow regulation in skeletal muscle may increase during rapid juvenile growth.

High dietary salt intake decreases the arteriolar dilation associated with skeletal muscle contraction. Because hydrogen peroxide (H2O2) can be released from contracting muscle fibers, this study was designed to assess the possible contribution of H2O2 to skeletal muscle functional hyperemia and its sensitivity to dietary salt.The authors investigated the effect of catalase treatment on arteriolar dilation and hyperemia in contracting spinotrapezius muscle of rats fed a normal salt (0.45%, NS) or high salt (4%, HS) diet for 4 weeks. Catalase-sensitive 2',7'-dichlorofluorescein (DCF) fluorescence was measured as an index of H2O2 formation, and the mechanism of arteriolar dilation to H2O2 was probed in each group using pharmacological inhibitors.DCF fluorescence increased with muscle contraction, but not if catalase was present. Catalase also reduced arteriolar dilation and hyperemia during contraction in both dietary groups. Exogenous H2O2 dilated arterioles in both groups, with greater responses in HS rats. Guanylate cyclase inhibition did not affect arteriolar responses to H2O2 in either group, but K(Ca) or KATP channel inhibition equally reduced these responses, and K(ATP) channel inhibition equally reduced functional hyperemia in both groups.These results indicate that locally produced H2O2 contributes to arteriolar dilation and hyperemia in contracting skeletal muscle, and that the effect of H2O2 on arteriolar tone in this vascular bed is mediated largely through K+ channel activation. High dietary salt intake does not reduce the contribution of H2O2 to active hyperemia, or alter the mechanism through which H2O2 relaxes arteriolar smooth muscle.

Previous studies from this laboratory suggest that during maturation, rapid microvascular growth is accompanied by changes in the mechanisms responsible for regulation of tissue blood flow. To further define these changes, we studied isolated gracilis muscle arterioles from weanling (∼25 days) and juvenile (∼44 days) Sprague-Dawley rats to test the hypothesis that endothelial mechanisms for the control of arteriolar tone are altered with growth. Responses to the endothelium-dependent dilator acetylcholine (ACh) were greater in weanling arterioles (WA) than in juvenile arterioles (JA), whereas there were no consistent differences between age groups in arteriolar responses to other endothelium-dependent agonists (A-23187, vascular endothelial growth factor, and simvastatin). Inhibition of nitric oxide synthase (NOS) with Nω-nitro-l-arginine methyl ester (l-NAME) attenuated ACh-induced dilation in JA but not in WA. In JA, combined inhibition of NOS and cyclooxygenase (with indomethacin) reduced the dilator responses to ACh and simvastatin by ∼90% and ∼70%, respectively, but had no effect in WA. Cytochrome P450 epoxygenase inhibition [with 2-(propargyloxyphenyl) hexanoic acid] had no effect on responses to ACh or simvastatin in either age group. Inhibition of Ca2+-activated or ATP-dependent potassium channels (with tetraethylammonium or glibenclamide, respectively) reduced these arteriolar responses in JA but not those in WA. These findings suggest that in fully grown microvascular networks, endothelium-dependent arteriolar dilation is mediated by the combined release of endothelial nitric oxide and vasodilator prostanoids, and in part through activation of Ca2+-activated and ATP-dependent potassium channels. However, during earlier microvascular growth, this dilation is mediated by other factors yet to be identified. This may have significant implications for the regulation of tissue perfusion during microvascular development.

Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigated the possible contribution of epithelial sodium channels (ENaC) to the myogenic behavior of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21-28 days (“weanlings”) and 42-49 days (“juveniles”). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and NG-monomethyl-L-arginine (L-NMMA). At physiological pressures, both steady-state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, β and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by L-NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth. This article is protected by copyright. All rights reserved.

We previously reported that in skeletal muscle arterioles of rats fed a very high salt (HS; 7%) diet, the bioavailability of endothelium-derived nitric oxide (NO) is reduced through scavenging by reactive oxygen species. Because arteriolar NO can play an important role in local blood flow control, we investigated whether arteriolar responses to increased tissue metabolism become compromised in skeletal muscle of salt-fed rats. Consumption of a HS (4%) diet for 4 weeks had no effect on arteriolar diameters, volume flow or shear stress in resting spinotrapezius muscle. Arteriolar responses to a modest elevation in metabolic demand (0.5 Hz contraction) were not different from those in rats fed a normal diet, but diameter responses to a greater elevation in metabolic demand (4 Hz contraction) were significantly less in HS rats than in rats fed a normal diet. In both groups, the NO synthase inhibitor NG-monomethyl-L-arginine reduced resting arteriolar diameters and flow by a similar amount and had little or no effect on arteriolar diameter or flow responses to muscle contraction. Arterioles in HS rats exhibited an increase in overall oxidant activity (tetranitroblue tetrazolium reduction) but not in superoxide activity (dihydroethidine oxidation). Reactive oxygen species scavengers (2,2,6,6-tetramethylpiperidine-N-oxyl and catalase) did not normalize the reduced arteriolar responses to muscle contraction in HS rats. These findings suggest that increased oxidant activity in the arteriolar network of salt-fed rats is not due to accumulation of superoxide anion and that neither this oxidant activity nor reduced NO availability can account for the blunted active arteriolar dilation in rats fed a 4% salt diet.

High dietary salt intake decreases the endothelium-dependent dilation of skeletal muscle arterioles by inhibiting local nitric oxide (NO) activity without changing vascular smooth muscle responsiveness to NO. Under these conditions, microvascular walls show evidence of oxidative stress, and scavengers of reactive oxygen species (ROS) abolish this oxidative stress and restore normal arteriolar responses to acetylcholine (ACh). We tested the hypothesis that the salt-dependent appearance of microvascular ROS, and accompanying reduction in endothelium-dependent dilation, is due to a decrease in antioxidant enzyme expression or activity. We studied spinotrapezius muscle microvessels in rats fed normal (NS) (0.45%) or high (HS) (7%) salt diets for 4–5 weeks. Western analysis of arteriolar and venular protein showed no difference between groups in the content of superoxide dismutase (Cu/Zn SOD), catalase, or glutathione peroxidase. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) increased arteriolar and venular oxidant activity (assessed by tetranitroblue tetrazolium reduction) by the same amount in both groups, suggesting similar levels of catalase activity. 3AT did not affect arteriolar responses to ACh in either group. The Cu/Zn SOD inhibitor diethyldithiocarbamate increased arteriolar and venular oxidant activity to a lesser extent in HS rats, suggesting reduced Cu/Zn SOD activity in this group. Cu/Zn SOD inhibition decreased arteriolar responses to ACh only in NS rats. These findings suggest that endogenous Cu/Zn SOD preserves the endothelium-dependent control of arteriolar tone in NS rats, and that a reduction in Cu/Zn SOD activity contributes to the loss of arteriolar NO activity in HS rats.

Previous reports by this laboratory have indicated that a flow-dependent fall in arteriolar wall PO2 may be a stimulus for the sustained release of endothelial nitric oxide (NO) during sympathetic vasoconstriction in the superfused rat intestine. In this study, we tested the hypothesis that locally formed adenosine serves as the link between the fall in local PO2 and NO synthesis under these conditions. Adenosine applied via pressurized micropipettes directly onto the wall or at a distance of 25 µm from the wall of first-order arterioles (resting diameter = 54 ± 1 µm) elicited dose-dependent dilations of 15–46% that were significantly reduced by the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10–4M). Arteriolar responses to sympathetic nerve stimulation were enhanced by 57–66% in the presence of L-NMMA or when tissue PO2 was prevented from falling under a high O2 superfusate. Adenosine deaminase (2.0 U/ml) or the selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (4 × 10–4M) completely blocked the enhancing effect of L-NMMA on sympathetic constriction. These results are consistent with the hypothesis that the fall in arteriolar wall and/or tissue PO2 that accompanies sympathetic arteriolar constriction in the rat intestine can lead to local adenosine production, which in turn preserves endothelial NO release.

In normotensive rats, an increase in dietary salt leads to decreased arteriolar responsiveness to acetylcholine (ACh) because of suppressed local nitric oxide (NO) activity. We evaluated the possibility that generation of reactive oxygen species in the arteriolar wall is responsible for this loss of NO activity. Arteriolar responses to iontophoretically applied ACh were examined in the superfused spinotrapezius muscle of Sprague-Dawley rats fed a low-salt (LS; 0.45%) or high-salt diet (HS; 7%) for 4–5 wk. Responses to ACh were significantly depressed in HS rats but returned to normal in the presence of the oxidant scavengers superoxide dismutase + catalase or 2,2,6,6-tetamethylpiperidine- N-oxyl (TEMPO) + catalase. Arteriolar responses to the NO donor sodium nitroprusside were similar in HS and LS rats. Arteriolar and venular wall oxidant activity, as determined by reduction of tetranitroblue tetrazolium, was significantly greater in HS rats than in LS rats. Exposure to TEMPO + catalase reduced microvascular oxidant levels to normal in HS rats. These data suggest that a high-salt diet leads to increased generation of reactive oxygen species in striated muscle microvessels, and this increased oxidative state may be responsible for decreased endothelium-dependent responses associated with high salt intake.

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 μm) than in HS rats (up to 4 μm). 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.

Ingestion of a high-salt diet has previously been shown to suppress the endogenous influence of nitric oxide (NO) on arteriolar tone in hypertension-resistant, salt-resistant Dahl (SR/Jr) rats. Because luminal blood flow can be an important stimulus for endothelial NO release, this study was undertaken to determine whether high salt intake can also lead to a deficit in the direct flow-dependent regulation of arteriolar diameter. The spinotrapezius muscle microvasculature was studied by in vivo microscopy in SR/Jr rats fed low (0.45%)- or high (7%)-salt diets for 2 wk, and arcade arteriole responses to increased luminal flow (via parallel vessel occlusion) were studied in both dietary groups. There was no significant difference between groups in arterial pressure or in resting arteriolar diameters, volume flows, or wall shear rates. In low-salt SR/Jr, a 36% increase in luminal flow produced an average arteriolar dilation of 38% that was significantly reduced by the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). In high-salt SR/Jr, a similar flow increase produced an average dilation of only 16% (P < 0.05 vs. low-salt SR/Jr), and this response was unaffected by L-NMMA. Inhibition of cyclooxygenase activity with meclofenamate had no effect on this response in either group. These findings suggest that NO release mediates a portion of flow-dependent arteriolar dilation in rat spinotrapezius muscle and that high salt intake, in the absence of hypertension, can attenuate this response via a suppression of NO activity.

Pulmonary particulate matter (PM) exposure has been epidemiologically associated with an increased risk of cardiovascular morbidity and mortality, but the mechanistic foundations for this association are unclear. Exposure to certain types of PM causes changes in the vascular reactivity of several macrovascular segments. However, no studies have focused upon the systemic microcirculation, which is the primary site for the development of peripheral resistance and, typically, the site of origin for numerous pathologies. Ultrafine PM--also referred to as nanoparticles, which are defined as ambient and engineered particles with at least one physical dimension less than 100 nm (Oberdorster et al. 2005)--has been suggested to be more toxic than its larger counterparts by virtue of a larger surface area per unit mass. The purpose of this study was fourfold: (1) determine whether particle size affects the severity of postexposure microvascular dysfunction; (2) characterize alterations in microvascular nitric oxide (NO) production after PM exposure; (3) determine whether alterations in microvascular oxidative stress are associated with NO production, arteriolar dysfunction, or both; and (4) determine whether circulating inflammatory mediators, leukocytes, neurologic mechanisms, or a combination of these play a fundamental role in mediating pulmonary PM exposure and peripheral microvascular dysfunction. To achieve these goals, we created an inhalation chamber that generates stable titanium dioxide (TiO2) aerosols at concentrations up to 20 mg/m3. TiO2 is a well-characterized particle devoid of soluble metals. Sprague Dawley and Fischer 344 (F-344) rats were exposed to fine or nano-TiO2 PM (primary count modes of approximately 710 nm and approximately 100 nm in diameter, respectively) at concentrations of 1.5 to 16 mg/m3 for 4 to 12 hours to produce pulmonary loads of 7 to 150 microg in each rat. Twenty-four hours after pulmonary exposure, the following procedures were performed: the spinotrapezius muscle was prepared for in vivo microscopy, blood samples were taken from an arterial line, and various tissues were harvested for histologic and immunohistochemical analyses. Some rats received a bolus dose of cyclophosphamide 3 days prior to PM exposure to deplete circulating neutrophils and bronchoalveolar lavage (BAL) was performed in separate groups of rats exposed to identical TiO2 loads. No significant differences in BAL fluid composition based on PM size or load were found in these rats. Plasma levels of interleukin (IL)-2, IL-18, IL-13, and growth-related oncogene (GRO) (also known as keratinocyte-derived-chemokine [KC]) were altered after PM exposure. In rats exposed to fine TiO2, endothelium-dependent arteriolar dilation was significantly decreased, and this dysfunction was robustly augmented in rats exposed to nano-TiO2. This effect was not related to an altered smooth-muscle responsiveness to NO because arterioles in both groups dilated comparably in response to the NO donor sodium nitroprusside (SNP). Endogenous microvascular NO production was similarly decreased after inhalation of either fine or nano-TiO2 in a dose-dependent manner. Microvascular oxidative stress was significantly increased among both exposure groups. Furthermore, treatment with antioxidants (2,2,6,6-tetramethylpiperdine-N-oxyl [TEMPOL] plus catalase), the myeloperoxidase (MPO) inhibitor 4-aminobenzoic hydrazide (ABAH), or the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) inhibitor apocynin partially restored NO production and normalized arteriolar function in both groups. Neutrophil depletion restored dilation in PM-exposed rats by as much as 42%. Coincubation of the spinotrapezius muscle with the fast sodium (Na+) channel antagonist tetrodotoxin (TTX) restored arteriolar dilation by as much as 54%, suggesting that sympathetic neural input may be affected by PM exposure. The results of these experiments indicate that (1) the size of inhaled PM dictates the intensity of systemic microvascular dysfunction; (2) this arteriolar dysfunction is characterized by a decreased bioavailability of endogenous NO; (3) the loss of bioavailable NO after PM exposure is at least partially caused by elevations in local oxidative stress, MPO activity, NADPH oxidase activity, or a combination of these responses; and (4) circulating neutrophils and sympathetic neurogenic mechanisms also appear to be involved in the systemic microvascular dysfunction that follows PM exposure. Taken together, these mechanistic studies support prominent hypotheses that suggest peripheral vascular effects associated with PM exposure are due to the activation of inflammatory mechanisms, neurogenic mechanisms, or both.

Nitric oxide (NO) mediates a major portion of arteriolar endothelium-dependent dilation in adults, but indirect evidence has suggested that NO contributes minimally to these responses in the young. Isolated segments of arterioles were studied in vitro to verify this age-related increase in NO release and investigate the mechanism by which it occurs. Directly measured NO release induced by ACh or the Ca2+ ionophore A-23187 was five- to sixfold higher in gracilis muscle arterioles from 42- to 46-day-old (juvenile) rats than in those from 25- to 28-day-old (weanling) rats. There were no differences between groups in arteriolar endothelial NO synthase (eNOS) expression or tetrahydrobiopterin levels, and arteriolar l-arginine levels were lower in juvenile vessels than in weanling vessels (104 ± 6 vs.126 ± 3 pmol/mg). In contrast, agonist-induced eNOS Thr495 dephosphorylation and eNOS Ser1177 phosphorylation (events required for maximal activity) were up to 30% and 65% greater, respectively, in juvenile vessels. Juvenile vessels did not show increased expression of enzymes that mediate these events [protein phosphatases 1 and 2A and PKA and PKB (Akt)] or heat shock protein 90, which facilitates Ser1177 phosphorylation. However, agonist-induced colocalization of heat shock protein 90 with eNOS was 34–66% greater in juvenile vessels than in weanling vessels, and abolition of this difference with geldanamycin also abolished the difference in Ser1177 phosphorylation between groups. These findings suggest that growth-related increases in arteriolar NO bioavailability may be due at least partially to changes in the regulation of eNOS phosphorylation and increased signaling activity, with no change in the abundance of eNOS signaling proteins.

Hypertension is frequently associated with insulin resistance. We evaluated the effects of pioglitazone, an agent that increases insulin sensitivity, on the development of hypertension in the Dahl salt-sensitive (Dahl-S) rat and in the one-kidney, one-clip Sprague-Dawley rat. We also evaluated the effects of pioglitazone on growth of cultured preglomerular renal arteriolar smooth muscle cells. In Dahl-S rats fed a 3% NaCl diet, tail systolic blood pressures and direct arterial pressures were lower (P < 0.05) in pioglitazone-treated (20 mg/kg daily by gavage for 3 wk) than in control rats (n = 10 rats/group). In one-kidney, one-clip Sprague-Dawley rats, systolic blood pressures were also lower in pioglitazone-treated animals (P < 0.0001). In vitro, proliferation of arteriolar smooth muscle cells was stimulated (P < 0.01) by insulin, epidermal growth factor (EGF), and fetal calf serum (FCS); pioglitazone (5 microM) reversibly inhibited (P < 0.01) insulin-, EGF-, and FCS-induced proliferation. Pioglitazone (0.01-100 microM) also inhibited insulin- (1 mU/ml), EGF- (100 ng/ml), and 5% FCS-induced [3H]thymidine incorporation in a concentration-dependent manner (P < 0.01). Thus pioglitazone attenuated the development of hypertension in the Dahl-S rat and the one-kidney, one-clip rat. The ability of pioglitazone to inhibit growth of vascular smooth muscle may contribute to its hypotensive effect.

This study evaluated the influence of high dietary salt intake on nitric oxide (NO) activity in the arteriolar network of rats resistant to salt-induced hypertension. The spinotrapezius muscle microvasculature was studied in inbred Dahl salt-resistant (SR/Jr) rats fed low (0.45%)- or high (7%)-salt diets for 4–5 wk. Arterial pressures were not different between groups at any time during the study. NO synthesis inhibition with NG-nitro-L-arginine-methyl ester (L-NAME) constricted arcade arterioles in low-salt SR/Jr and dilated arcade arterioles in high-salt SR/Jr. Arcade arteriole dilation to acetylcholine (ACh), but not sodium nitroprusside (SNP), was impaired in high-salt SR/Jr. In contrast, transverse and distal arteriole responses to L-NAME, ACh, and SNP were identical in high- and low-salt SR/Jr. These findings indicate that high salt intake, in the absence of increased arterial pressure, suppresses the influence of basal and evoked NO on vascular tone in arcading arterioles, but not in smaller transverse and distal arterioles. Unaltered SNP responses in high-salt SR/Jr suggest that this effect does not involve a change in arteriolar smooth muscle responsiveness to NO.

The aims of this study were (1) to determine if the establishment of hypertension in the Dahl salt-sensitive (DS) rat is accompanied by alterations in arteriolar vasomotion and (2) to explore the influence of endogenous nitric oxide on vasomotion in normotensive and hypertensive DS rats. Rhythmic diameter changes of arcading arterioles were studied in the superfused spinotrapezius muscle of DS fed high (4%) or low (0.45%) salt diets for 6 weeks. Mean arterial pressure for DS on high salt (166 +/- 7 mm Hg) was significantly greater than that for DS on low salt (131 +/- 9 mm Hg). There was no difference between hypertensive and normotensive DS in time-averaged arteriolar diameter, vasomotion frequency, or vasomotion cycle length. However, average vasomotion amplitude was 93% greater in hypertensive DS than in normotensive DS. Inhibition of nitric oxide synthesis with NG-Nitro-L-arginine methyl ester did not alter vasomotion in hypertensive DS, but increased vasomotion amplitude in normotensive DS to a level not different from that in hypertensive DS. L-Arginine had no effect on vasomotion in either group. Therefore, cyclic variations in arcade arteriole diameter are normally limited by basal nitric oxide, and the enhancement of these variations in animals with salt-induced hypertension may be attributable to the loss of this nitric oxide influence. The increased vasomotion amplitude and unchanged average diameter in hypertensive DS suggests a reduced hydraulic resistance within this particular segment of the arteriolar network.

Endothelial dysfunction can develop at an early age in children with risk factors for cardiovascular disease. A clear understanding of the nature of this dysfunction and how it can worsen over time requires detailed information on the normal growth-related changes in endothelial function on which the pathological changes are superimposed. This review summarizes our current understanding of these normal changes, as derived from studies in four different mammalian species. Although the endothelium plays an important role in controlling vascular tone from birth onward, the vasoactive molecules that mediate this control often change during postnatal or juvenile growth. The specifics of this transition to an adult endothelial cell phenotype can vary depending on the vascular bed. During growth, the contribution of nitric oxide to endothelium-dependent dilation generally increases in the lung, cerebral cortex, and skeletal muscle, but decreases in the intestine. Endothelial capacity for release of other vasoactive factors (e.g., cyclooxygenase products, hydrogen peroxide, carbon monoxide) can also increase or decrease during growth. Although these changes have been well documented, there is less information on their underlying cellular or molecular events. Further research is required to clarify these mechanisms, and to evaluate the functional significance of such shifts in endothelial phenotype.

High-salt intake can change the effect of adenosine on arterial tone in mice. The aim of this study was to clarify the mechanism by which this occurs. Using aortas from mice fed a 4% NaCl (HS) or 0.45% NaCl (NS) diet for 4–5 wks, concentration-response curves for ACh, 5′- N-ethylcarboxamidoadenosine (NECA; adenosine analog) and 2- p-(2-carboxyethyl)phenethylamino-5′- N-ethylcarboxamidoadenosine hydrochloride hydrate [CGS-21680; A2Aadenosine receptor (A2AAR) agonist] were obtained with Nω-nitro-l-arginine methyl ester (l-NAME; nitric oxide inhibitor, 10−4M), methylsulfonyl-propargyloxyphenylhexanamide [MS-PPOH; a CYP (cytochrome P-450) epoxygenase blocker, 10−5M including CYP2J2], 12-(3-adamantan-1-yl-ureido)dodecanoic acid [AUDA; soluble epoxide hydrolase (sEH) blocker, 10−5M], dibromo-dodecenyl-methylsulfimide [DDMS; CYP ω-hydroxylase (CYP4A blocker), 10−5M], glibenclamide (KATPchannel blocker; 10−5M) and 5-hydroxydecanoate (5-HD; mitochondrial-KATPchannel blocker, 10−4M). HS dose response to ACh (10−7− 10−5M) was not different from NS ( P > 0.05). Relaxation to 10−6M NECA was greater in the HS group (28.4 ± 3.9%) than in the NS group (4.1 ± 2.3%). Relaxation to 10−6M CGS-21680 was also greater in HS (27.9 ± 4.5%) than in NS (4.9 ± 2.2%). l-NAME was able to block the dose response of ACh (10−7− 10−5M) equally in both HS and NS ( P > 0.05), whereas l-NAME did not block CGS-21680-induced response in HS. In HS the CGS-21680 response was greatly reduced by MS-PPOH (to 4.7 ± 2.0%) and 5-HD (to 8.9 ± 2.2%), and also abolished by glibenclamide (−1.0 ± 5.9%). In NS, the CGS-21680 response was increased by AUDA (to 26.3 ± 3.4%) and DDMS (to 27.2 ± 3.0%). Compared with NS, HS vessels showed increased CYP2J2 and A2AAR expression (46 and 74% higher, respectively) but decreased sEH, CYP4A, and A1AR expression (75, 30, and 55% lower, respectively). These data suggest that in mice fed NS-containing diet, upregulation of arterial A1receptor causes vasoconstriction via increased sEH and CYP4A proteins. However, in mice fed HS-containing diet, upregulation of A2Areceptor protein triggers vascular relaxation through ATP-sensitive (K+) channels via upregulation of CYP2J2 enzyme.

Impaired endothelium-dependent arteriolar dilation in mice fed high salt (HS) is due to local oxidation of nitric oxide (NO) by superoxide anion (O(2) (-)). We explored the possibility that "uncoupled" endothelial nitric oxide synthase (eNOS) is the source of this O(2) (-).Levels of L-arginine (L-Arg), tetrahydrobiopterin (BH(4)), and O(2) (-) (hydroethidine oxidation) were measured in spinotrapezius muscle arterioles of mice fed normal salt (0.45%, NS) or (4%, HS) diets for 4 weeks, with or without dietary L-Arg supplementation. The contribution of NO to endothelium-dependent dilation was determined from the effect of N(omega)-nitro-L-arginine methyl ester (L-NAME) on responses to acetylcholine (ACh).Arterioles in HS mice had lower [BH(4)] and higher O(2) (-) levels than those in NS mice. ACh further increased arteriolar O(2) (-) in HS mice only. L-Arg supplementation prevented the reduction in [BH(4)] in arterioles of HS mice, and O(2) (-) was not elevated in these vessels. Compared to NS mice, arteriolar ACh responses were diminished and insensitive to L-NAME in HS mice, but not in HS mice supplemented with L-Arg.These findings suggest that eNOS uncoupling due to low [BH(4)] is responsible for O(2) (-) generation and reduced NO-dependent dilation in arterioles of mice fed a HS diet.

The aim of this study was to evaluate the influence of endogenous nitric oxide on resting microvascular tone in the Dahl salt-sensitive (DS) rat and to determine how this influence is altered in salt-induced hypertension. Intravital microscopy was used to examine the arteriolar network in the spinotrapezius muscle of DS rats maintained on low (0.45% NaCl) or high (4% NaCl) salt diets for 6-7 weeks. Mean arterial pressure for DS rats on high salt (163 +/- 3 mm Hg) was significantly greater than that for DS rats on low salt (128 +/- 4 mm Hg). Inhibition of microvascular nitric oxide synthesis with NG-nitro-L-arginine-methyl ester caused arteriolar constriction in normotensive DS but not in hypertensive DS rats. Application of L-arginine consistently caused arteriolar dilation in normotensive DS but not hypertensive DS rats. In contrast, arteriolar responses to iontophoretically applied acetylcholine and sodium nitroprusside were similar in both groups. These results indicate that basal release of nitric oxide, presumably from the endothelium, normally influences arteriolar tone in skeletal muscle of DS rats and that this influence is suppressed in established salt-induced hypertension. However, the normal arteriolar response to acetylcholine in hypertensive DS rats suggests that a generalized impairment of endothelial function may not occur in the microcirculation of these animals. Unaltered arteriolar responsiveness to sodium nitroprusside in hypertensive DS rats also suggests that salt-induced hypertension is not accompanied by a change in the responsiveness of arteriolar smooth muscle to nitric oxide.

We have shown that pulmonary nanoparticle exposure impairs endothelium dependent dilation in systemic arterioles. However, the mechanism(s) through which this effect occurs is/are unclear. The purpose of this study was to identify alterations in the production of reactive species and endogenous nitric oxide (NO) after nanoparticle exposure, and determine the relative contribution of hemoproteins and oxidative enzymes in this process. Sprague-Dawley rats were exposed to fine TiO2 (primary particle diameter approximately 1 microm) and TiO2 nanoparticles (primary particle diameter approximately 21 nm) via aerosol inhalation at depositions of 4-90 microg per rat. As in previous intravital experiments in the spinotrapezius muscle, dose-dependent arteriolar dilations were produced by intraluminal infusions of the calcium ionophore A23187. Nanoparticle exposure robustly attenuated these endothelium-dependent responses. However, this attenuation was not due to altered microvascular smooth muscle NO sensitivity because nanoparticle exposure did not alter arteriolar dilations in response to local sodium nitroprusside iontophoresis. Nanoparticle exposure significantly increased microvascular oxidative stress by approximately 60%, and also elevated nitrosative stress fourfold. These reactive stresses coincided with a decreased NO production in a particle deposition dose-dependent manner. Radical scavenging, or inhibition of either myeloperoxidase or nicotinamide adenine dinucleotide phosphate oxidase (reduced) oxidase partially restored NO production as well as normal microvascular function. These results indicate that in conjunction with microvascular dysfunction, nanoparticle exposure also decreases NO bioavailability through at least two functionally distinct mechanisms that may mutually increase local reactive species.

The purpose of this study was to determine the effect of salt-induced hypertension on the microvascular pressure profile in skeletal muscle. Measurements were made in the spinotrapezius muscle of anesthetized Dahl salt-sensitive (DS) rats fed either a high- (7%) or low-normal (0.45%)-NaCl diet for 4 wk. Age-matched Dahl salt-resistant (DR) rats on high- or low-normal salt diets were also studied. The high-salt diet had no effect on either arterial or microvascular pressures in DR rats. DS rats maintained on high salt developed arterial hypertension accompanied by a pressure increase of 49% in the feed arterioles and 31% in the transverse arterioles. Hypertensive DS rats exhibited a greater pressure drop across the small arteries, arcade arterioles, and distal arterioles, indicating that each of these segments contributes to increased whole organ resistance. Pressures in the collecting and draining venules were not elevated in DS on 7% NaCl, suggesting that increased precapillary resistance in salt-induced hypertension effectively shields the skeletal muscle capillary and venular networks from high hydrostatic pressures.

We hypothesize that A2Aadenosine receptors (A2AAR) promote aortic relaxation in mice through cytochrome P450 (CYP)-epoxygenases and help to avoid salt sensitivity. Aortas from male mice maintained on a high-salt (HS; 7% NaCl) or normal-salt (NS; 0.45% NaCl) diet for 4–5 wks were used. Concentration-response curves (10−11–10−5M) for 5′- N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine analog) and CGS 21680 (A2AAR agonist) were obtained with different antagonists including ZM 241385 (A2AAR antagonist; 10−6M), SCH 58261 (A2AAR antagonist; 10−6M), Nω-nitro-l-arginine methyl ester (l-NAME; endothelial nitric oxide synthase inhibitor; 10−4M) and inhibitors including methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; CYP epoxygenases inhibitor; 10−5M), 14,15-epoxyeicosa-5(z)-enoic acid (14,15-EEZE; EET antagonist; 10−5M), dibromo-dodecenyl-methylsulfimide (DDMS; CYP4A inhibitor; 10−5M), and HET0016 (20-HETE inhibitor; 10−5M). At 10−7M of NECA, significant relaxation in HS (+22.58 ± 3.12%) was observed compared with contraction in NS (−10.62 ± 6.27%, P < 0.05). ZM 241385 changed the NECA response to contraction ( P < 0.05) in HS. At 10−7M of CGS 21680, significant relaxation in HS (+32.04 ± 3.08%) was observed compared with NS (+10.45 ± 1.34%, P < 0.05). SCH 58261, l-NAME, MS-PPOH, and 14,15-EEZE changed the CGS 21680-induced relaxation to contraction ( P < 0.05) in HS. Interestingly, DDMS and HET0016 changed CGS 21680 response to relaxation ( P < 0.05) in NS; however, there was no significant difference found between DDMS, HET0016-treated HS and NS vs. nontreated HS group ( P > 0.05). CYP2C29 protein was 55% and 74% upregulated in HS vs. NS ( P < 0.05) mice aorta and kidney, respectively. CYP4A protein was 30.30% and 35.70% upregulated in NS vs. HS ( P < 0.05) mice aorta and kidneys, respectively. A1AR was downregulated, whereas A2AAR was upregulated in HS compared with NS. These data suggest that HS may activate CYP2C29 via A2AAR, causing relaxation, whereas NS may contribute to the upregulation of CYP4A causing contraction.

Background/Aims: Endothelium-dependent dilation of skeletal muscle arterioles is mediated by unknown factors in very young rats. We assessed the possible contribution of carbon monoxide (CO) to this dilation and to dilation in older animals. Methods: The effects of de-endothelialization or various pharmacological inhibitors on responses to CO or endothelium-dependent dilators were studied in gracilis muscle arterioles from rats at 3–4 weeks (‘weanlings’) and 6–7 weeks (‘juveniles’). Results: Exogenous CO constricted, rather than dilated, arterioles from both age groups. This constriction was reduced by endothelial removal or NOS inhibition in juvenile, but not weanling, arterioles. In contrast, this constriction was abolished by K+ channel inhibition in weanling, but not juvenile, arterioles. The heme precursor δ-aminolevulinic acid constricted juvenile arterioles but did not affect weanling arterioles. The heme oxygenase inhibitor chromium (III) mesoporphyrin IX abolished the endothelium-dependent dilation of juvenile arterioles to simvastatin, and reduced ACh- and simvastatin-induced dilations of weanling arterioles. Conclusion: These findings suggest that relatively high concentrations of exogenous CO can cause constriction by inhibiting endothelium-derived NO in juvenile arterioles and inhibiting K+ channels in weanling arterioles. Endogenous CO produced at lower concentrations can contribute to endothelium-dependent dilation in both age groups.

The endothelium-dependent dilation of skeletal muscle arterioles is mediated by factors that have not been identified in young rats, and partly mediated by an unidentified hyperpolarizing factor in maturing rats. This study was designed to determine if endogenous hydrogen peroxide (H2O2) contributes to this arteriolar dilation at either of these growth stages.Gracilis muscle arterioles were isolated from rats at ages 24-26 days ("weanlings") and 46-48 days ("juveniles"). We investigated the effects of catalase treatment on the endothelium-dependent dilation of these vessels to simvastatin and acetylcholine (ACh). Catalase-sensitive 2',7'-dichlorofluorescein (DCF) fluorescence also was measured as an index of H2O2 formation, and arteriolar dilation to exogenous H2O2 was pharmacologically probed in each age group.Responses to simvastatin and ACh were attenuated by catalase in juvenile, but not weanling, arterioles. Juvenile, but not weanling, arterioles also displayed catalase-sensitive DCF fluorescence that was increased by ACh. Exogenous H2O2 could induce dilation in juvenile, but not weanling, arterioles. In juvenile arterioles, this dilation was abolished by the K+ channel inhibitors TEA and glibenclamide, and attenuated by NOS inhibition or endothelial removal.These findings suggest that endogenous H2O2 contributes to endothelium-dependent arteriolar dilation in juvenile rats, but not in younger rats, and that H2O2 acts in juvenile rats by stimulating endothelial NO release and activating smooth muscle K+ channels.

The purpose of this study was to determine if structural rarefaction of arterioles occurs in the gracilis muscle of Dahl salt-sensitive (DS) rats with salt-induced hypertension. Arteriolar network architecture was studied in cleared muscles removed from DS fed either a high (7% NaCl) or low-normal (0.45% NaCl) salt diet for 4 weeks. Muscles removed from Dahl salt-resistant (DR) rats on high and low-normal salt diets served as controls. The 7% NaCl diet had no effect on arterial pressure in DR, but caused marked hypertension in DS. The density of arcade arterioles was significantly lower in DS than in DR (0.77 vs 1.26 segments/mg tissue, respectively) and was unrelated to either dietary salt content or mean arterial pressure in both strains. The number of transverse arterioles/mm parent vessel was 19% lower in DS on 7% NaCl than in DS on 0.45% NaCl and DR on either diet. These data indicate that compared to normotensive DR, the DS rat with salt-induced hypertension exhibits a lower vascular density within both the arcading and the transverse portions of the gracilis muscle arteriolar network. The lower arcade vessel density reflects an inherent characteristic of the DS strain, whereas the lower transverse arteriole density reflects a true structural rarefaction associated with salt-induced hypertension.

The purpose of this study was to identify microvascular alterations that could contribute to increased peripheral vascular resistance in the Dahl salt-sensitive rat with salt-induced hypertension. Intravital microscopy was used to study the spinotrapezius muscle arteriolar network in anesthetized salt-sensitive rats fed either a high salt (7% sodium chloride) or low-normal salt (0.45% sodium chloride) diet for 4 weeks. Age-matched Dahl salt-resistant rats on high and low-normal salt diets served as controls. The high salt diet had no effect on arterial pressure in salt-resistant rats but increased arterial pressure in salt-sensitive rats. Mean resting diameter of arcade arterioles in salt-sensitive rats on high salt diet was reduced by 25% compared with salt-sensitive rats on low salt or salt-resistant rats on either diet. After abolition of vascular tone with 10(-3) M adenosine, arcade diameters were comparable in all groups. No difference among groups was found in either resting or passive diameter of the more distal transverse arterioles. Measurement of vessel lengths and numbers in cleared muscle specimens revealed no differences among groups in the anatomic density of either arcade or transverse arterioles. These data suggest that a reduction in the resting diameter of arcade, but not transverse, arterioles may increase spinotrapezius muscle vascular resistance in hypertensive salt-sensitive rats. The similarity in vascular densities among groups indicates that structural rarefaction of arterioles does not contribute to any increase in spinotrapezius muscle resistance at this stage of salt-induced hypertension.

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 NG 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.

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 Po2 between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po2 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 Ca2+-activated potassium (KCa) 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 KCa 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.

The epidemiologic association between pulmonary exposure to ambient particulate matter (PM) and cardiovascular dysfunction is well known, but the systemic mechanisms that drive this effect remain unclear. We have previously shown that acute pulmonary exposure to PM impairs or abolishes endothelium-dependent arteriolar dilation in the rat spinotrapezius muscle. The purpose of this study was to further characterize the effect of pulmonary PM exposure on systemic microvascular function and to identify local inflammatory events that may contribute to these effects. Rats were intratracheally instilled with residual oil fly ash (ROFA) or titanium dioxide at 0.1 or 0.25 mg/rat 24 hr before measurement of pulmonary and systemic microvascular responses. In vivo microscopy of the spinotrapezius muscle was used to study systemic arteriolar responses to intraluminal infusion of the Ca2+ ionophore A23187 or iontophoretic abluminal application of the adrenergic agonist phenylephrine (PHE). Leukocyte rolling and adhesion were quantified in venules paired with the studied arterioles. Histologic techniques were used to assess pulmonary inflammation, characterize the adherence of leukocytes to systemic venules, verify the presence of myeloperoxidase (MPO) in the systemic microvascular wall, and quantify systemic microvascular oxidative stress. In the lungs of rats exposed to ROFA or TiO2, changes in some bronchoalveolar lavage markers of inflammation were noted, but an indication of cellular damage was not found. In rats exposed to 0.1 mg ROFA, focal alveolitis was evident, particularly at sites of particle deposition. Exposure to either ROFA or TiO2 caused a dose-dependent impairment of endothelium-dependent arteriolar dilation. However, exposure to these particles did not affect microvascular constriction in response to PHE. ROFA and TiO2 exposure significantly increased leukocyte rolling and adhesion in paired venules, and these cells were positively identified as polymorphonuclear leukocytes (PMNLs). In ROFA- and TiO2-exposed rats, MPO was found in PMNLs adhering to the systemic microvascular wall. Evidence suggests that some of this MPO had been deposited in the microvascular wall. There was also evidence for oxidative stress in the microvascular wall. These results indicate that after PM exposure, the impairment of endothelium-dependent dilation in the systemic microcirculation coincides with PMNL adhesion, MPO deposition, and local oxidative stress. Collectively, these microvascular observations are consistent with events that contribute to the disruption of the control of peripheral resistance and/or cardiac dysfunction associated with PM exposure.

To test the hypothesis that active hyperemia is reduced in skeletal muscle of old rats due to a decreased bioavailability of prostanoids, which in turn is due to increased oxidative stress.The microvasculature of the spinotrapezius muscle of 3-, 12-, and 24-month male Sprague-Dawley rats was examined using in vivo videomicroscopy. Arteriolar diameter and centerline red cell velocity were measured in resting and contracting muscle. The effect of prostanoids was examined using indomethacin (10 microM), and passive resting arteriolar diameters were determined using adenosine (100 microM). Lipid peroxidation was assessed ex vivo by measuring tissue levels of malondialdehyde.Arteriolar diameters and blood flow in resting muscle did not differ among the age groups, but increases in diameter and flow during muscle contraction in young rats were greater than in the two older age groups. Indomethacin did not affect resting arteriolar diameters and blood flow in 3- and 12-month rats, but significantly decreased both parameters in 24-month rats. Indomethacin had no effect on arteriolar diameter and blood flow responses during muscle contraction in any age group. Passive resting diameters of arterioles were significantly smaller in 12- and 24-month rats than in 3-month rats. Tissue levels of TBARS were not different among the three age groups.Arteriolar tone and blood flow in resting skeletal muscle of rats is not altered with age, whereas the increases in these variables that normally accompany muscle contraction are markedly impaired during aging. Neither cyclooxygenase metabolites nor lipid peroxidation appear to be involved in this impairment.

In young rats, skeletal muscle arterioles respond to acetylcholine, which elicits Ca2+-dependent endothelial nitric oxide (NO) release, but not to shear stress, which does not require increased intracellular Ca2+ for endothelial NO release. The aim of this study was to determine if, in general, signaling pathways for endothelial NO release that differ in their reliance on Ca2+ may be developing at different times during normal arteriolar network growth in skeletal muscle.Arteriolar responses to intraluminal infusion of the Ca2+ ionophore A23187, and the Ca2+-independent agonists vascular endothelial growth factor (VEGF) and simvastatin, were studied before and during NO synthase (NOS) inhibition with NG-monomethyl-L-arginine (L-NMMA) in the exteriorized spinotrapezius muscle of weanling (age 4-5 wks) and juvenile (7-8 wks) rats.There were no age-dependent differences in arteriolar responses to A23187 applied over a concentration range that dilated arterioles from 9 +/- 2 to 74 +/- 8% of maximum, and L-NMMA attenuated these responses by the same amount in both age groups. In juveniles, arterioles dilated up to 39+/-5% of maximum in response to VEGF, and up to 83 +/- 6% of maximum in response to simvastatin, with L-NMMA greatly reducing the responses to both agonists. In contrast, arterioles in weanlings did not dilate in response to either agonist.These findings suggest that some Ca2+-independent signaling pathways for endothelial NO release may not initially be operational in the arteriolar network, but quickly become established during juvenile growth. This is consistent with the idea that microvascular control mechanisms are not fixed at birth, but rather undergo progressive changes in concert with microvascular network growth and changes in tissue metabolic requirements.

Acute exposure to airborne pollutants, such as solid particulate matter (PM), increases the risk of cardiovascular dysfunction, but the mechanisms by which PM evokes systemic effects remain to be identified. The purpose of this study was to determine if pulmonary exposure to a PM surrogate, such as residual oil fly ash (ROFA), affects endothelium-dependent dilation in the systemic microcirculation. Rats were intratracheally instilled with ROFA at 0.1, 0.25, 1 or 2 mg/rat 24 hr before experimental measurements. Rats intratracheally instilled with saline or titanium dioxide (0.25 mg/rat) served as vehicle or particle control groups, respectively. In vivo microscopy of the spinotrapezius muscle was used to study systemic arteriolar dilator responses to the Ca2+ ionophore A23187, administered by ejection via pressurized micropipette into the arteriolar lumen. We used analysis of bronchoalveolar lavage (BAL) samples to monitor identified pulmonary inflammation and damage. To determine if ROFA exposure affected arteriolar nitric oxide sensitivity, sodium nitroprusside was iontophoretically applied to arterioles of rats exposed to ROFA. In saline-treated rats, A23187 dilated arterioles up to 72 +/- 7% of maximum. In ROFA- and TiO2-exposed rats, A23187-induced dilation was significantly attenuated. BAL fluid analysis revealed measurable pulmonary inflammation and damage after exposure to 1 and 2 mg ROFA (but not TiO2 or < 1 mg ROFA), as evidenced by significantly higher polymorphonuclear leukocyte cell counts, enhanced BAL albumin levels, and increased lactate dehydrogenase activity in BAL fluid. The sensitivity of arteriolar smooth muscle to NO was similar in saline-treated and ROFA-exposed rats, suggesting that pulmonary exposure to ROFA affected endothelial rather than smooth muscle function. A significant increase in venular leukocyte adhesion and rolling was observed in ROFA-exposed rats, suggesting local inflammation at the systemic microvascular level. These results indicate that pulmonary PM exposure impairs systemic endothelium-dependent arteriolar dilation. Moreover, because rats exposed to < 1 mg ROFA or TiO2 did not exhibit BAL signs of pulmonary damage or inflammation, it appears that PM exposure can impair systemic microvascular function independently of detectable pulmonary inflammation.

In many individuals with essential hypertension, dietary salt can further increase blood pressure by augmentation of an already elevated total peripheral resistance. There is little information on the microvascular changes that contribute to salt-sensitive hypertension in humans, but studies in the Dahl salt-sensitive rat have provided some knowledge of the microcirculation in this form of hypertension. These studies, most of which have used intravital microscopy or isolated vessel technology, are the focus of this review. The salt-induced exacerbation of hypertension in Dahl rats is due to a uniform increase in hemodynamic resistance throughout most of the peripheral vasculature. In the spinotrapezius muscle, this resistance increase is largely due to the intense constriction of proximal arterioles. The mechanisms responsible for this increased arteriolar tone include increased responsiveness to oxygen and a loss of tonic nitric oxide (NO) availability caused by reduced endothelial NO production and/or accelerated NO degradation by reactive oxygen species. Within the last decade, it has become increasingly clear that high salt intake can also lead to changes in microvascular structure and function in the absence of increased arterial pressure. This effect must also be considered when evaluating microvascular changes and their functional consequences in salt-sensitive hypertension.

Increased salt intake attenuates the endothelium-dependent dilation of skeletal muscle arterioles by abolishing local nitric oxide (NO) activity. There is evidence of oxidative stress in arteriolar and venular walls of rats fed a high-salt diet, and depressed arteriolar responses to acetylcholine (ACh) in these rats are reversed by scavengers of reactive oxygen species (ROS). In this study, we tested the hypothesis that this salt-dependent increase in microvascular ROS and the resulting attenuation of endothelium-dependent dilation are due to increased expression and/or activity of oxidant enzymes in the microvascular wall. Resting arteriolar and venular wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction, was consistently higher in the spinotrapezius muscle of rats fed a high-salt diet (7% NaCl, HS) for 4–5 wk than in those fed a normal diet (0.45% NaCl, NS) for the same duration. Western analysis of protein from isolated microvessels showed no difference between HS and NS rats in the expression of NAD(P)H oxidase or xanthine oxidase. Inhibition of NAD(P)H oxidase and/or xanthine oxidase with diphenyleneiodonium chloride and oxypurinol, respectively, reduced resting arteriolar wall oxidant activity to normal levels in HS rats but had no effect in NS rats, suggesting that the basal activities of NAD(P)H oxidase and xanthine oxidase are increased in HS microvessels. However, inhibition of these enzymes in HS rats did not restore normal arteriolar responses to ACh, suggesting that this stimulus activates an alternate source of ROS that eliminates the role of NO in the subsequent dilation.