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1Noll Laboratory, 2Department of Kinesiology, Graduate Physiology Program, The Pennsylvania State University, University Park, Pennsylvania
Submitted 8 March 2007 ; accepted in final form 30 April 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-hydroxy-nor-L-arginine]. Oral temperature was increased by 0.8°C via a water-perfused suit. NG-nitro-L-arginine was then ultimately perfused through all MD sites to quantify the change in VD due to NO. Red blood cell flux was measured by laser-Doppler flowmetry over each skin MD site, and cutaneous vascular conductance (CVC) was calculated (CVC = flux/MAP) and normalized to maximal CVC (%CVCmax; 28 mM sodium nitroprusside + local heating to 43°C). During the plateau in skin blood flow (
Tor = 0.8°C), cutaneous VD was attenuated in HT skin (NT: 42 ± 4, HT: 35 ± 3 %CVCmax; P < 0.05). Asc and Asc+A-I augmented cutaneous VD in HT (Asc: 57 ± 5, Asc+A-I: 53 ± 6 %CVCmax; P < 0.05 vs. control) but not in NT. %CVCmax after NOS-I in the Asc- and Asc+A-I-treated sites was increased in HT (Asc: 41 ± 4, Asc+A-I: 40 ± 4, control: 29 ± 4; P < 0.05). Compared with the control site, the change in %CVCmax within each site after NOS-I was greater in HT (Asc: –19 ± 4, Asc+A-I: –17 ± 4, control: –9 ± 2; P < 0.05) than in NT. Antioxidant supplementation alone or combined with arginase inhibition augments attenuated reflex cutaneous VD in hypertensive skin through NO- and non-NO-dependent mechanisms.
skin blood flow; essential hypertension; oxidative stress; temperature regulation; vitamin C
30–40% to the total vasodilatory response (20, 30). Essential hypertension is associated with attenuated cutaneous vasodilation during local (6) and systemic thermal stress (16, 22). We (16) have recently shown that NO-dependent reflex cutaneous vasodilation is attenuated in hypertensive skin and that increasing L-arginine availability through acute arginase inhibition augments reflex vasodilation. Arginase catalyzes the conversion of L-arginine to L-ornithine and urea in the final step of the urea cycle and is capable of limiting NO production by competing for the common NO synthase substrate L-arginine (13). In addition to upregulated arginase contributing to decreased NO production, there are likely additional mechanisms impairing NO bioavailability in hypertensive cutaneous vasculature.
One potential mechanism contributing to the reduced NO bioavailability implicated in the pathogenesis of hypertensive vascular pathology is increased oxidative stress (35). In essential hypertensive vasculature, there is increased production of reactive oxygen species (ROS) coupled with decreased degradation of ROS through dysfunctional enzymatic antioxidant defense mechanisms (25). Furthermore, augmented ROS (superoxide) production reduces NO bioavailability by readily reacting with newly synthesized NO-forming peroxynitrite, which further potentiates superoxide production by uncoupling endothelial NO synthase (eNOS) (10). eNOS uncoupling can result from peroxynitrite-mediated oxidization of the essential NOS cofactor tetrahydrobiopterin (BH4) and/or inadequate L-arginine availability (28). Together, these data suggest that there may be a mechanistic link between hypertension-associated increased arginase activity decreasing L-arginine availability and potentially contributing to increased oxidant stress through uncoupled eNOS.
In vivo human studies examining vascular oxidant stress mechanisms commonly utilize high-dose antioxidant supplementation for measurement of functional NO-dependent vasodilation. To this end, acute L-ascorbate (Asc) supplementation in the forearm muscle vasculature in humans with essential hypertension increases endothelium-dependent vasodilation through NO-dependent mechanisms (31). In these studies, Asc may increase NO bioavailability directly through antioxidant capabilities and/or indirectly by stabilization of the essential NOS cofactor BH4, enabling NOS to "recouple" and produce functional NO (15, 32). The purpose of the present study was to determine the relative role of oxidant stress and augmented arginase activity on attenuated NO-dependent vasodilation in human hypertensive cutaneous vasculature. We sought to control for age-related increases in oxidant stress and arginase activity (17) by matching subjects with unmedicated essential hypertension to age- and sex-matched healthy normotensive control subjects. We hypothesized that 1) local acute antioxidant (Asc) supplementation would modestly augment NO-dependent reflex cutaneous vasodilation and 2) arginase inhibition (to increase L-arginine availability, enabling NOS to recouple and produce functional NO) combined with Asc supplementation would further enhance NO-dependent reflex vasodilation over Asc supplementation alone.
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METHODS |
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Instrumentation and measurements. All protocols were performed in a thermoneutral laboratory with the subject in the semisupine position with the experimental arm at heart level. After arrival to the laboratory, subjects were instrumented with four intradermal microdialysis fibers as previously described (16–19) (MD 2000, Bioanalytical Systems) (10-mm, 30-kDa cutoff membrane) in the skin on the right ventral forearm. The microdialysis fibers were taped in place and perfused with lactated Ringer solution during the insertion trauma resolution period at a rate of 2.0 µl/min (Bee Hive controller and Baby Bee microinfusion pumps; Bioanalytical Systems) for 60–90 min.
To obtain an index of skin blood flow, cutaneous red blood cell flux was measured with an integrated laser-Doppler flowmeter probe placed in a local heater (MoorLAB, Temperature Monitor SH02; Moor Instruments, Devon, UK) on the skin directly above each microdialysis membrane. Cutaneous vascular conductance (CVC) was calculated as red blood cell flux divided by mean arterial pressure (MAP). MAP was calculated as diastolic pressure plus one-third pulse pressure.
Whole body temperature was controlled and whole body heating was conducted with a water-perfused suit as previously described (16–19). The subject's ECG was monitored throughout the protocol, and blood pressure was measured via brachial auscultation every 5 min. Oral temperature (Tor) was continuously monitored during baseline and throughout whole body heating with a thermistor placed in the sublingual sulcus as an index of body core temperature. The subjects were instructed to keep the thermistor in the same location in the sublingual sulcus and not to open their mouths or speak during the protocol. After a 0.8°C rise in Tor was reached, skin temperature and Tor were clamped by perfusing the suit with 46°C for the remainder of the heating protocol. Local skin temperature over each microdialysis site was maintained at 33°C during baseline and whole body heating (Moor Instruments SHO2).
Experimental protocol.
After the insertion trauma resolution period (60–90 min), microdialysis sites were randomly assigned to receive 1) lactated Ringer solution to serve as control, 2) 10.0 mM NG-nitro-L-arginine (L-NAME; Calbiochem, San Diego, CA) to competitively inhibit NO production by NOS, 3) 10 mM ASC (Sigma, St. Louis, MO) to supplement antioxidants (17), or 4) the combination of 10 mM ASC and 5.0 mM (S)-(2-boronoethyl)-L-cysteine-HCl and 5.0 mM N-hydroxy-nor-L-arginine to supplement antioxidants and to inhibit arginase (Asc+A-I: Calbiochem) (16, 18). Our group (16–18) has previously used these doses of Asc and arginase inhibitors in conjunction with intradermal microdialysis in an aged population with known endothelial dysfunction. All pharmacological solutions were mixed just before usage, dissolved in lactated Ringer solution, sterilized with the use of syringe microfilters (Acrodisc, Pall, Ann Arbor, MI), and wrapped in foil.
All microdialysis sites were perfused continuously with assigned pharmacological agents for at least 60 min before the start of the baseline and throughout baseline (20 min) and whole body heating at a rate of 2.0 µl/min. After the baseline data collection period, whole body heating was conducted to raise Tor by 0.8°C. After a 0.8°C rise in Tor, body temperature was clamped for 30–40 min. After 10 min of stable laser-Doppler flux values, 10 mM L-NAME was perfused through all microdialysis fibers at a rate of 2.0 µl/min to inhibit NOS within each treatment site. L-NAME infusions were discontinued after laser-Doppler flux decreased to a stable plateau (Fig. 1). At the end of the whole body heating protocol, each microdialysis site was perfused with 28.0 mM sodium nitroprusside (SNP; Nitropress, Abbott Laboratories, Chicago, IL) at a rate of 4.0 µl/min to achieve maximal CVC. Local heating of the skin to 43°C was conducted simultaneously with SNP infusion to ensure that maximal CVC had been obtained.
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%CVCmax in the control, Asc, and Asc+A-I sites is the difference between the plateau values in %CVCmax before and after NOS inhibition. Maximal CVC was calculated as the average of the stable 10-min plateau in laser-Doppler flux values divided by MAP during SNP and local heating.
Student's unpaired t-tests were used to determine significant differences between the groups for physical characteristics. Two-way repeated-measures ANOVA was conducted to detect differences from blood pressure and pharmacological treatment on maximal CVC and on the within-site %CVCmax. A mixed model, three-way repeated-measures ANOVA was conducted to detect differences between subject groups at the pharmacological treatment sites over the rise in Tor (SAS, version 8.01). Tukey's post hoc tests were performed when appropriate to determine where differences between groups and drug treatments occurred. The level of significance was set at 
= 0.05. Values are presented as means ± SE.
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RESULTS |
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Figure 2 shows the %CVCmax responses for all sites to the rise in body core temperature in both subject groups. %CVCmax in the control site was attenuated in the hypertensive subjects starting with change in Tor (Tor) 
0.6°C compared with the normotensive group (P < 0.05). NOS inhibition throughout whole body heating attenuated %CVCmax in both subject groups starting at Tor 0.3°C in the hypertensive group and Tor 0.2°C in the normotensive group (both P < 0.05). Asc and Asc+A-I significantly increased %CVCmax in the hypertensive group at Tor 0.4°C (P < 0.05 vs. control) but did not alter %CVCmax in the normotensive group (P > 0.05 vs. control). There was not a significant difference between the Asc- and Asc+A-I-treated sites in either subject group (P > 0.05).
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Tor = 0.8°C. NOS inhibition within the control site decreased %CVCmax in both subject groups (P < 0.05). Furthermore, there was not a significant difference between the groups in %CVCmax in the control site after NOS inhibition (%CVCmax: 29 ± 4 for hypertensive vs. 34 ± 4 for normotensive; P > 0.05). Asc and Asc+A-I increased %CVCmax after NOS inhibition in the hypertensive group (%CVCmax: 41 ± 4 Asc and 40 ± 4 for Asc+A-I; P < 0.05 vs. control) but not in the normotensive group (%CVCmax: 29 ± 3 for Asc, 30 ± 4 for Asc+A-I; P > 0.05 vs. control).
Figure 3 summarizes the %CVCmax responses in all of the treatment sites during the plateau in skin blood flow with a 0.8°C rise in body core temperature before and after within-site NOS inhibition. Compared with the normotensive group, vasodilation at the control site was attenuated in the hypertensive group (P < 0.001), and there was a blunted decrease with NOS inhibition (hypertensive: –8 ± 1 vs. normotensive: –14 ± 2 %CVCmax; P < 0.001). Compared with their respective control sites, NOS inhibition in the Asc and combined Asc+A-I sites caused a greater %CVCmax in the hypertensive group (Asc: –19 ± 4, Asc+A-I: –17 ± 4; P < 0.05 vs. control) than in the normotensive subject group (Asc: –11 ± 3, Asc+A-I: –15 ± 3; P > 0.05 vs. control) (Fig. 3, inset).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Our data confirm earlier reports (22) that reflex cutaneous vasodilation is attenuated in subjects with essential hypertension. In the present study, cutaneous vasodilation at the control site was only modestly reduced when expressed as a percentage of maximal vasodilation; however, absolute maximal CVC was significantly attenuated. We chose to represent the data as a %CVCmax to account for variability in capillary density under each laser-Doppler flowmeter, allowing for comparisons between microdialysis treatment sites (4). Others (6) have consistently observed a reduction in maximal CVC using both venous occlusion plethysmography to measure absolute and laser-Doppler flowmetry to measure relative changes in skin blood flow during whole arm local heating. Together, these data suggest that reflex cutaneous vasodilation is significantly attenuated with hypertension with the reduction in maximal CVC, suggesting structural maladaptations of the cutaneous vessels that limit absolute vasodilator capacity. However, we also observed a clear reduction in NO-dependent vasodilation and alterations in vascular signaling that contribute to attenuated reflex cutaneous vasodilation in humans with essential hypertension.
We found that localized Asc administration in humans with essential hypertension augmented reflex cutaneous vasodilation through NO- and non-NO-dependent mechanisms, independent of arginase inhibition. In sites treated with Asc, there was a greater decrease in skin blood flow (%CVCmax) with NOS inhibition during established hyperthermia, and the resulting post-L-NAME plateau (%CVCmax) was increased compared with control sites (Figs. 2 and 3), demonstrating an increase in NO- and non-NO-dependent cutaneous vasodilation, respectively.
A prooxidant vascular environment and resulting decreased NO bioavailability are implicated in the pathogenesis of hypertensive vascular disease (35). NO bioavailability is dictated by the balance of NO and oxidant production and clearance. With essential hypertension oxidant production, particularly superoxide, in the vasculature increases predominantly through NAD(P)H oxidase, xanthine oxidase, and uncoupled eNOS (35). In this deleterious cycle, superoxide decreases functional NO by reacting to form peroxinitrite, which then oxidizes the critical NOS cofactor BH4. BH4 deficiency can then ultimately decrease functional NO production through uncoupled NOS (14, 23, 24, 33). In the present study, we chose to directly supplement antioxidants to the cutaneous vasculature to examine a functional link between hypertension-associated increased oxidant stress and reduced NO-dependent vasodilation. However, Asc is a nonspecific antioxidant and may have increased functional NO bioavailability through a variety of mechanisms, including 1) direct scavenging of superoxide and 2) stabilization of the essential NOS cofactor BH4, enabling recoupling of NOS and resulting in functional NO production (15, 32). However, with the present data, we are unable to delineate which of these mechanisms led to the increase in NO-dependent vasodilation.
Other mechanisms, including inadequate L-arginine availability, can also lead to eNOS uncoupling (34). We have recently shown that acute inhibition of arginase augments vasodilation in hypertensive skin (16), suggesting that arginase activity is upregulated in hypertensive cutaneous vasculature. Together, these data suggest that hypertension-associated upregulated arginase activity may cause decreased NO production and may contribute to increased superoxide synthesis through uncoupled eNOS. We hypothesized that concurrent inhibition of arginase and Asc supplementation would result in a greater increase in NO-dependent vasodilation than Asc alone; however, our data indicate that the resulting cutaneous vasodilation from the combined treatments was not additive. One possible explanation for this finding is that there is redundancy in the underlying cellular mechanisms of action of these treatments, where both treatments likely facilitated recoupling of eNOS, thereby increasing functional NO synthesis and decreasing superoxide production. Alternatively, the individual treatments (Asc or Asc+A-I) may have maximized the capacity of the hypertensive cutaneous vessels to vasodilate reaching a "ceiling" effect with this given hyperthermic stimulus (Tor = 0.8°C). It is possible, but not likely, that a further increase in cutaneous vasodilation with the combined treatments could be unmasked with a more significant hyperthermic stimulus.
An unexpected finding from the present study was that Asc supplementation alone and combined with arginase inhibition increased the resulting %CVCmax after NOS inhibition in hypertensive skin (Fig. 3). These data suggest that Asc supplementation augmented cutaneous vasodilation in hypertensive vasculature by non-NO-dependent as well as NO-dependent mechanisms. The results may be due to 1) hypertension-associated increased ROS augmenting vasoconstrictor tone or 2) possible nonspecific effects of Asc on the putative neurotransmitter pathways and/or 3) potential synergistic interactions between NO and these pathways involved in reflex cutaneous vasodilation.
In human cutaneous vascular smooth muscle cells, ROS act as signaling molecules to mediate cold-induced vasoconstriction through Rho kinase-dependent mechanisms (2). Because upregulated Rho kinase signaling is implicated in hypertensive vascular pathology (26), it is possible that ROS mediate enhanced vasoconstriction in the absence of a cold stimulus through Rho kinase-dependent mechanisms. Thus providing antioxidants would inhibit the ROS-mediated enhanced vasoconstriction unmasking augmented non-NO-dependent vasodilation.
Asc may also have mediated the increase in non-NO-dependent vasodilation through nonspecific effects on the putative neurotransmitter pathways implicated in reflex vasodilation. In vitro studies suggest that Asc enhances histamine 1 receptor sensitivity (7) and presynaptically increase VIP synthesis and release (5). Alternatively, increased NO itself may have augmented non-NO-dependent vasodilation through potential synergistic effects. In human skin, NO is capable of mediating cutaneous vasodilation synergistically with sympathetic cotransmitters, resulting in a combined vasodilation that is greater than the sum of the individual contributions (36). The synergistic effect may occur downstream through cross-talk between NO-mediated cGMP-dependent and sympathetic neurotransmitter(s)-mediated cAMP-dependent mechanisms (8, 9), or NO may prejunctionally enhance the release of sympathetic neurotransmitter(s) (12).
The precise effects of Asc on the mechanisms and potential interactions between NO and sympathetic neurotransmitter(s) involved in active cutaneous vasodilation remains elusive. However, our data suggest that augmenting NO bioavailability in a clinical population with compromised endothelial function can also increase non-NO-dependent vasodilation.
In conclusion, NO-dependent reflex cutaneous vasodilation is attenuated in humans with essential hypertension, and antioxidant supplementation alone or combined with arginase inhibition augments cutaneous vasodilation during hyperthermia through NO- and non-NO dependent mechanisms. These data suggest that oxidant stress is increased in hypertensive cutaneous vasculature, which decreases functional NO bioavailability. Finally, in subjects with hypertension, the resulting vasodilation with concurrent Asc supplementation and arginase inhibition was not greater than the vasodilation with Asc alone, suggesting redundancy in underlying cellular mechanisms through uncoupled eNOS and/or that Asc maximized the cutaneous vasodilatory capacity as this level of hyperthermia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) and a hypertensive (
) subject. Whole body heating started at minute 10, and NG-nitro-L-arginine (L-NAME) was perfused through the microdialysis sites after oral temperature increased 0.8°C (minute 65 in this example).
P < 0.001, significant vs. control site age-matched normotensive subject group; 
P < 0.001, significant vs. control site hypertensive subject group.