Reflex Cutaneous Vasodilation Research Articles

Thermoregulatory reflex control of cutaneous vasodilation in healthy aging

ABSTRACT Reflex cutaneous vasodilation during heating is attenuated in healthy human aging secondary to blunted increases in efferent skin sympathetic nervous system activity (SSNA) and reductions in end-organ sensitivity. Whether age-related alterations in the mean body temperature ( b) threshold for increasing SSNA and/or the sensitivity of responses are evident with aging have not been examined. We tested the hypotheses that the Tb threshold for SSNA and cutaneous vascular conductance (CVC) would be increased, but the sensitivity would be reduced, with aging. Reflex vasodilation was induced in 13 young (23 ± 3 y) and 13 older (67 ± 7 y) adults using a water-perfused suit to systematically increase mean skin and esophageal temperatures. SSNA (peroneal microneurography) and red cell flux (laser Doppler flowmetry) in the innervated dermatome were continuously measured. SSNA was normalized to baseline; CVC was normalized as a percentage of maximal CVC. Baseline b was lower in older adults (36.0 ± 0.4°C vs 36.4 ± 0.3°C; p = 0.005). During passive heating, the ∆ b thresholds for increasing SSNA and CVC were greater (1.3 ± 0.4°C vs 0.9 ± 0.3°C; p = 0.007 and 1.3 ± 0.4°C vs 0.8 ± 0.3°C; p = 0.002, respectively) in older adults. The slope of the relation between both SSNA (0.31 ± 0.23 vs 0.13 ± 0.10 V⋅s⋅°C −1; p = 0.01) and CVC (87.5 ± 50.1 vs 32.4 ± 18.1%max⋅°C−1; p = 0.002) vs b was lower in older adults. The relative b threshold for activation of SSNA and the initiation of reflex cutaneous vasodilation is higher in older adults, and once activated, the sensitivity of both responses is diminished, supporting the concept that the efferent component of the thermoregulatory reflex arc is impaired in healthy aging. Abbreviations: CI: confidence interval; CVC: cutaneous vascular conductance; SSNA: skin sympathetic nervous system activity; b: mean body temperature; Tes: esophageal temperature; sk: mean skin temperature.

Reliability of Reflex Cutaneous Vasodilation on the Forearm Measured Using Laser‐Doppler Flowmetry During Whole‐body Passive Heating

Laser‐Doppler flowmetry (LDF) quantifies changes in localized microvascular perfusion in response to thermal and non‐thermal stimuli. Despite its widespread use, the reliability of the measurement during whole‐body heating is unknown. This presents a key knowledge gap that is critical for experimental design, statistical analysis, and interpretation. The purpose of this study was therefore to assess the reliability of LDF‐derived cutaneous vasodilation between separate days (between‐day) and between forearm sites (within‐day) during incremental whole‐body heat stress. On three occasions (~1 week apart), 11 healthy men (25 [SD 5] years) were passively heated (whole‐body heating using a water perfused suit) to increase esophageal temperature (Teso) by +0.7°C [0.11] (low heat stress, LHS) and +1.3°C [0.14] (moderate heat stress, MHS). Forearm skin blood flow (arbitrary perfusion units; PU) was measured in three adjacent sites on the mid‐dorsal forearm using LDF (Perimed) while local skin temperature was clamped (34°C). Maximal vasodilation was achieved by heating each skin site to 44°C (for ≥ 20 min), but without the use of SNP, while Teso was clamped at +1.8°C [0.1]. Beat‐to‐beat mean arterial pressure (MAP) was measured continuously. Cutaneous vascular conductance (CVC) at each skin site was calculated as PU/MAP. Data were presented as absolute CVC and percentage of maximal vasodilation (%CVC44). Reliability was assessed at baseline (no heat stress, NHS), LHS, and MHS using the intra‐class correlation coefficient (ICC), with values ≥0.7 considered acceptable for research purposes. Between days, CVC increased from 0.3 [0.2] to 2.0 PU [0.8] at LHS, and then to 2.1 PU [0.8] at MHS. Expressed as %CVC44 this equated to increases from 12 [5] to 68% [15] and then 73% [13]. Within‐day (i.e., between sites), average CVC increased from 0.3 [0.2] to 1.9 PU [0.7] at LHS, and 2.0 PU [0.7] at MHS, which related to an increase from 13 [6] to 71% [15] at LHS, and to 76% [12] at MHS when expressed as %CVC44. At NHS, the between‐ and within‐day reliability of these measurements did not exceed the 0.7 ICC threshold when expressed as CVC or %CVC44 at NHS (range: 0.00 to 0.45). Similarly, at LHS and MHS the between‐day reliability did not exceed 0.7 when expressed as CVC (0.57 and 0.69, respectively) or %CVC44 (both 0.57). While within‐day reliability of %CVC44 at LHS and MHS was not acceptable (0.54 and 0.64, respectively), it was when expressed as CVC (0.74 and 0.71, respectively). We show that the between‐day reliability of this technique was poor, irrespective of the level of whole‐body heat stress, but within‐day reliability was acceptable during heating, but not for resting measurements. This has important implications for studies assessing reactivity of skin vasculature to passive whole‐body hyperthermia, particularly with respect to the expected difference between the signal (i.e., expected change) and noise (i.e., measurement and biological variability), and the need to ensure that repeated measurements reflect the same microvascular environment.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada

The Nitric Oxide Contribution to Reflex Cutaneous Vasodilation During Intradermal Microdialysis is Similar When Quantified Either Within or Between Sites

Nitric oxide (NO) is an important component of the reflex cutaneous vasodilatory response during whole body heating (WBH). The NO contribution to reflex cutaneous vasodilation can be quantified via local intradermal microdialysis delivery of the endothelial NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME), either (1) within a single site by perfusing L‐NAME after a set rise in oral temperature (ΔTor) is reached, or (2) between two sites, one of which acts as a control while the other is perfused with L‐NAME throughout WBH. It is unclear, however, whether these two methods of quantifying the NO contribution to reflex cutaneous vasodilation yield similar results.PurposeTo compare the NO contribution to reflex cutaneous vasodilation when calculated either within a site or between two sites. We hypothesized that the NO contribution to reflex cutaneous vasodilation would be similar when quantified either within or between sites.MethodsRetrospective analysis of skin blood flow responses during whole‐body heat stress (water‐perfused suit) was performed for 11 young (22 ± 1 yr) and 48 middle‐aged/older (64 ± 5 yr) adults. Two intradermal microdialysis fibers placed in the ventral skin of the forearm were perfused with either lactated Ringer’s (control) or 20 mM L‐NAME during WBH. After ΔTor = 1°C was reached, mean body temperature was clamped and 20 mM L‐NAME was added to the control site until a post‐L‐NAME plateau was established. Red cell flux was measured continuously at each site (laser‐Doppler flowmetry) and cutaneous vascular conductance (CVC = flux/MAP) was expressed as a percentage of maximum (%CVCmax; 28mM sodium nitroprusside + 43°C). The NO contribution to reflex vasodilation was calculated in two ways for each subject: (1) as the difference in %CVCmax between the ΔTor = 1°C and post‐L‐NAME plateau within the control site, and (2) as the difference in %CVCmax between the control and L‐NAME sites at ΔTor = 1°C.ResultsWhen analyzed as a single group, there were no differences between methods of quantifying the NO contribution to reflex vasodilation (17.3 ± 1.6% vs. 21.7 ± 2.9%, p = 0.09). Similarly, there were no differences between methods when the young (26.1 ± 4.9% vs. 36.0 ± 8.5%, p = 0.20) and middle‐aged/older (15.2 ± 1.5% vs. 18.3 ± 2.9%, p = 0.26) groups were analyzed separately. Maximal CVC was similar between the young (1.85 ± 0.19) and middle‐aged/older (1.66 ± 0.14) groups (p = 0.53). There was a negative correlation between age and NO contribution (R2 = 0.42; p = 0.02) with no difference in slope (p = 0.46) or intercept (p = 0.18) between the two methods.ConclusionQuantification of the NO contribution to reflex cutaneous vasodilation yields similar results when calculated either within a site or between two sites. Importantly, the well‐documented age‐related reduction in the NO contribution to reflex vasodilation is evident regardless of the method used. These data suggest that either of these two methods are effective for quantifying the NO contribution to reflex cutaneous vasodilation, regardless of the magnitude of the overall vasodilatory response.Support or Funding InformationSupported by NIH T‐32 Grant #5T32AG049676‐03

Thermoregulation in the Aging Population and Practical Strategies to Overcome a Warmer Tomorrow.

As global temperatures continue to rise, improving thermal tolerance in the aged population is crucial to counteract age-associated impairments in thermoregulatory function. Impairments in reflex cutaneous vasodilation and sweating response can augment the vulnerability of older adults to heat-related injuries following exposure to heat stress. Mechanisms underlying a compromised cutaneous vasodilation are suggested to include reduced sympathetic neural drive, diminished cholinergic co-transmitter contribution, and altered second messenger signaling events. On the other hand, impairments in sweating response are ascribed to reduced sweat gland cholinergic sensitivity and altered cyclooxygenase and nitric oxide signaling. Several practical mitigation strategies such as exercise, passive heating, and behavioral adaptations are proposed as means to overcome heat stress and improve thermal tolerance in the aged. Aerobic exercise training is shown to be amongst the most effective ways to enhance thermoregulatory function. However, in elderly with limited exercise capability due to chronic diseases and mobility issues, passive heating can serve as a functional alternative as it has been shown to confer similar benefits to that of exercise training. Supplementary to exercise training and passive heating, behavioral adaptations can be applied to further enhance the heat-preparedness of the aged.

Chronic statin therapy is associated with enhanced cutaneous vascular responsiveness to sympathetic outflow during passive heat stress.

Impairments in both central sympathetic and peripheral microvascular function contribute to blunted reflex cutaneous vasodilatation during heat stress in healthy older adults. Hypercholesterolaemia is associated with decrements in neurovascular function; however, little is known about the impact of hypercholesterolaemia on the integrated responses to heat stress. Further, whether chronic statin therapy alters skin sympathetic outflow or its relation to cutaneous vascular conductance during heat stress is unknown. We demonstrate that reflex cutaneous vasodilatation is impaired in older hypercholesterolaemic adults but not in formerly hypercholesterolaemic adults currently treated with a statin compared to age-matched controls. Additionally, chronic statin treatment-induced improvements in reflex vasodilatation are mediated, in part, by increases in end-organ responsiveness to efferent sympathetic outflow during whole-body heating. These data add to the growing body of literature substantiating the beneficial pleiotropic neurovascular effects of chronic statin treatment and provide further support for the use of statins to confer additional cardioprotective benefits in older adults. Attenuated reflex cutaneous vasodilatation in healthy human ageing is mediated by alterations in both central (sympathetic outflow) and peripheral (microvascular endothelial) function. Hypercholesterolaemia is associated with further impairments in neurovascular function. HMG-CoA reductase inhibitors (statins) improve cutaneous endothelium-dependent dilatation; however, whether statin therapy alters skin sympathetic nervous system activity (SSNA) or its relation to cutaneous vascular conductance (CVC) during passive heat stress is unknown. We hypothesized that (1) hypercholesterolaemic older adults would demonstrate blunted increases in both SSNA and CVC during passive heating and (2) chronic statin treatment would improve the response range and sensitivity of the SSNA:CVC relation. Reflex vasodilatation in response to a 1.0°C rise in oral temperature (Tor ; water perfused suit) was induced in 13 healthy normocholesterolaemic adults (62±2years; LDL=113±7mg/dl), 10 hypercholesterolaemic adults (60±1years; LDL=183±2mg/dl), and 10 previously hypercholesterolaemic adults (64±1years; LDL=102±2mg/dl) treated with lipophilic statin (10-40mg daily). SSNA (peroneal microneurography) and red cell flux (laser-Doppler flowmetry) in the innervated dermatome (dorsum of foot) were continuously measured. Reflex vasodilatation was blunted in hypercholesterolaemic adults, but not in statin-treated adults, compared to normocholesterolaemic adults (at ∆Tor =1.0°C: normal=36±1%CVCmax , high=32±1%CVCmax , statin=38±1%CVCmax ; P<0.01). ∆SSNA was not different (at ∆Tor =1.0°C: normal: ∆=393±96%, high: ∆=311±120%, statin: ∆=256±90%; P=0.11). The slope of the SSNA:CVC relation was blunted in hypercholesterolaemic adults (0.02±0.03%CVCmax /%baseline ) compared to both normocholesterolaemic (0.09±0.02%CVCmax /%baseline ; P=0.024) and statin-treated (0.12±0.05%CVCmax /%baseline ; P=0.03) adults. Chronic statin treatment improves reflex cutaneous vasodilatation in formerly hypercholesterolaemic older adults by increasing end-organ responsiveness to sympathetic outflow during passive heat stress.

Statin Therapy Enhances Reflex Cutaneous Vasodilation in Formerly Hypercholesterolemic Older Adults by Improving Neurovascular Coupling

Reflex cutaneous vasodilation is attenuated in healthy human aging and is mediated by alterations in both central (sympathetic outflow) and peripheral (microvascular endothelial) function. Hypercholesterolemia, a primary risk factor for cardiovascular disease, is associated with increased sympathetic nervous system activity and further impairments in endothelial function. Independent of their lipid‐lowering effect, HMG‐CoA reductase inhibitors (statins) – the most commonly prescribed medication in the U.S. – reduce sympathetic outflow and augment endothelium‐dependent dilation. However, whether statin therapy improves the relation between skin sympathetic nervous system activity (SSNA) and cutaneous vascular conductance (CVC) during passive heat stress is unknown. We hypothesized that (1) hypercholesterolemic older adults would demonstrate blunted increases in both SSNA and CVC during passive heating and (2) chronic statin treatment would improve the response range and sensitivity of the SSNA:CVC relation. Reflex vasodilation was induced in 13 healthy normocholesterolemic older adults [62±2 yrs; low‐density lipoprotein (LDL) 113±7 mg/dl), 10 hypercholesterolemic older adults (60±1 yrs; LDL 183±2 mg/dl), and 10 previously hypercholesterolemic older adults now treated with a lipophilic statin (10–40 mg daily) to normalize LDL (64±1 yrs; LDL 102±2 mg/dl) using a water‐perfused suit to elevate oral temperature (Tor) by 1.0°C. SSNA (peroneal microneurography) and red cell flux (laser Doppler flowmetry) in the innervated dermatome (the dorsum of foot) were continuously measured. SSNA was normalized to, and expressed as, a percentage of baseline and CVC was calculated as flux/mean arterial pressure and expressed as a percentage of maximal CVC (local heating to 43°C). Mean arterial pressure, baseline CVC, and maximal CVC were not significantly different among groups (all p&gt;0.05). During passive heating, the increase in SSNA was not different among groups (normal: Δ=393±96%, high: Δ=311±120%, and statin: Δ=256±90%; p=0.11). Reflex vasodilation was blunted in hypercholesterolemic adults, but not in statin‐treated adults, compared to normocholesterolemic adults (normal: 36±1%CVCmax, high: 32±1%CVCmax, and statin: 38±1%CVCmax respectively, at ΔTor=1.0°C; p&lt;0.01). There were no differences among groups for the slope of the ΔSSNA:ΔCVC relation (normal: 0.06±0.00, high: 0.07±0.01, and statin: 0.07±0.01, p=0.91); however, this relation was shifted upward in statin‐treated adults (y‐intercept; normal: 3.1±0.9, high: 4.0±0.9, and statin: 7.3±1.3; p=0.001). These data demonstrate that chronic statin treatment improves reflex cutaneous vasodilation in formerly hypercholesterolemic older adults, in part via increases in end‐organ responsiveness to sympathetic outflow during passive heat stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Folic acid supplementation increases cutaneous vasodilator sensitivity to sympathetic nerve activity in older adults.

During heat stress, blunted increases in skin sympathetic nervous system activity (SSNA) and reductions in end-organ vascular responsiveness contribute to the age-related reduction in reflex cutaneous vasodilation. In older adults, folic acid supplementation improves the cutaneous vascular conductance (CVC) response to passive heating; however, the influence of folic acid supplementation on SSNA:CVC transduction is unknown. Fourteen older adults (66 ± 1 yr, 8 male/6 female) ingested folic acid (5 mg/day) or placebo for 6 wk in a randomized, double-blind, crossover design. In protocol 1, esophageal temperature (Tes) was increased by 1.0°C (water-perfused suit) while SSNA (peroneal microneurography) and red cell flux in the innervated dermatome (laser Doppler flowmetry; dorsum of the foot) were continuously measured. In protocol 2, two intradermal microdialysis fibers were placed in the skin of the lateral calf for graded infusions of acetylcholine (ACh; 10-10 to 10-1 M) with and without nitric oxide synthase (NOS) blockade (20 mM nitro-l-arginine methyl ester). Folic acid improved reflex vasodilation (46 ± 4% vs. 31 ± 3% CVCmax for placebo; P < 0.001) without affecting the increase in SSNA (Δ506 ± 104% vs. Δ415 ± 73% for placebo; NS). Folic acid increased the slope of the SSNA-to-CVC relation (0.08 ± 0.02 vs. 0.05 ± 0.01 for placebo; P < 0.05) and extended the response range. Folic acid augmented ACh-induced vasodilation (83 ± 3% vs. 66 ± 4% CVCmax for placebo; P = 0.002); however, there was no difference between treatments at the NOS-inhibited site (53 ± 4% vs. 52 ± 4% CVCmax for placebo; NS). These data demonstrate that folic acid supplementation enhances reflex vasodilation by increasing the sensitivity of skin arterioles to central sympathetic nerve outflow during hyperthermia in aged human subjects.

Spectral analysis of reflex cutaneous vasodilatation during passive heat stress

Previous work has demonstrated that spectral analysis is a useful tool to non-invasively ascertain the mechanisms of control of the cutaneous circulation. The majority of work using spectral analysis has focused on local control mechanisms, with none examining reflex control. Skin blood flow was analysed using spectral analysis on the dorsal aspect of the forearm of 7 males and 7 females during passive heat stress, with mean forearm and local temperature at the site of measurement maintained at thermoneutral (33°C) to minimize the effect of local control mechanisms. Participants were passively heated to ~1.2±0.1°C above baseline rectal temperature (d=4.0, P<0.001) using a water-perfused, tube lined suit, with skin blood flow assessed using a laser-Doppler probe with an integrated temperature monitor. Spectral analysis was performed using a Morlet wavelet on the entire data set, with median power extracted during 20min of data during baseline (normothermia) and hyperthermia. Passive heat stress significantly increased laser-Doppler flux above baseline (d=4.7, P<0.001). Spectral power of the endothelial nitric oxide-independent (0.005–0.01Hz; d=1.1, P=0.004), neurogenic (0.2–0.05Hz; d=0.6, P=0.025), myogenic (0.05–0.15Hz; d=1.5, P=0.002), respiratory (0.15–0.4Hz; d=1.4 P=0.002), and cardiac (0.4–2.0Hz; d=1.1, P=0.012) frequency intervals increased with passive heat stress. In contrast, the endothelial nitric oxide-dependent frequency interval did not change (0.01–0.02Hz; d=0.3, P=0.09) with passive heat stress. These data suggest that cutaneous reflex vasodilatation is neurogenic in origin and not mediated by endothelial-nitric oxide synthase, and are congruent with invasive examinations of reflex cutaneous vasodilatation.

Blunted increases in skin sympathetic nerve activity are related to attenuated reflex vasodilation in aged human skin.

Reflex cutaneous vasodilation in response to passive heating is attenuated in human aging. This diminished response is mediated, in part, by age-associated reductions in endothelial function; however, the contribution of altered skin sympathetic nervous system activity (SSNA) is unknown. We hypothesized that 1) healthy older adults would demonstrate blunted SSNA responses to increased core temperature compared with young adults and 2) the decreased SSNA response would be associated with attenuated cutaneous vasodilation. Reflex vasodilation was elicited in 13 young [23 ± 1 (SE) yr] and 13 older (67 ± 2 yr) adults using a water-perfused suit to elevate esophageal temperature by 1.0°C. SSNA (peroneal microneurography) and red cell flux (laser Doppler flowmetry) in the innervated dermatome (the dorsum of foot) were continuously measured. SSNA was normalized to, and expressed as, a percentage of baseline. Cutaneous vascular conductance (CVC) was calculated as flux/mean arterial pressure and expressed as a percentage of maximal CVC (local heating, 43°C). Reflex vasodilation was attenuated in older adults (P < 0.001). During heating, SSNA increased in both groups (P < 0.05); however, the response was significantly blunted in older adults (P = 0.01). The increase in SSNA during heating was linearly related to cutaneous vasodilation in both young (R2 = 0.87 ± 0.02, P < 0.01) and older (R2 = 0.76 ± 0.05, P < 0.01) adults; however, slope of the linear regression between ΔSSNA and ΔCVC was reduced in older compared with young (older: 0.05 ± 0.01 vs. young: 0.08 ± 0.01; P < 0.05). These data demonstrate that age-related impairments in reflex cutaneous vasodilation are mediated, in part, by blunted efferent SSNA during hyperthermia.

Augmented reflex cutaneous vasodilatation following short-term dietary nitrate supplementation in humans.

What is the central question of this study? Nitrate supplementation via beetroot juice has been shown to have several benefits in healthy humans, including reduced blood pressure and increased blood flow to exercising muscle. Whether nitrate supplementation can improve blood flow to the skin in heat-stressed humans has not been investigated. What is the main finding and its importance? Similar to previous studies, we found that nitrate supplementation reduces blood pressure. Nitrate supplementation increased vasodilatation in the skin of heat-stressed humans but did not directly increase skin blood flow. Nitrate supplementation has been shown to increase NO-dependent vasodilatation through both NO synthase (NOS)-dependent and NOS-independent pathways. We hypothesized that nitrate supplementation would augment reflex cutaneous active vasodilatation. Subjects were equipped with two microdialysis fibres on the forearm randomly assigned as control (Ringer solution) or NOS inhibition (20mm l-NAME). Whole-body heating was performed to raise core temperature by 0.8°C above baseline core temperature. Maximal cutaneous vasodilatation was achieved via 54mm sodium nitroprusside and local heating to 43°C. Skin blood flow (measured by laser-Doppler flowmetry) and blood pressure were measured. Cutaneous vascular conductance (CVC) was calculated as skin blood flow divided by mean arterial pressure (MAP) and expressed as a percentage of maximal CVC (%CVCmax ). Subjects underwent heat stress before and after nitrate supplementation (3days of beetroot juice; 5mm, 0.45g nitrates per day). During heat stress, MAP was reduced following nitrate supplementation compared with the control conditions (before 88±3mmHg versus after 78±2mmHg; P<0.05); however, resting MAP was not different between conditions (before 88±3mmHg versus after 83±2mmHg; P=0.117). Nitrate supplementation increased plateau CVC at control sites (before 67±2%CVCmax versus after 80±5%CVCmax ; P=0.01) but not at l-NAME-treated sites (before 45±4%CVCmax versus after 40±5%CVCmax ; P=0.617). There was no change in the calculated percentage of NOS-dependent vasodilatation before and after supplementation (before 59±4%versus after 64±6%; P=0.577). These data suggest that nitrate supplementation augments CVC and reduces MAP during heat stress.

Endothelial nitric oxide synthase mediates the nitric oxide component of reflex cutaneous vasodilatation during dynamic exercise in humans.

Recent data suggests neuronal nitric oxide synthase (nNOS) mediates the NO component of reflex cutaneous vasodilatation with passive heat stress. We tested the hypothesis that nNOS inhibition would attenuate reflex cutaneous vasodilatation during sustained dynamic exercise in young healthy humans. All subjects first performed an incremental V̇O2, peak test to exhaustion on a custom-built supine cycle ergometer. On a separate day, subjects were instrumented with four intradermal microdialysis fibres on the forearm and each randomly assigned as: (1) lactated Ringer's (control); (2) 20mm Nω-nitro-l-arginine methyl ester hydrochloride (non-selective NOS inhibitor); (3) 5mm N-propyl-l-arginine (nNOS inhibitor); and (4) 10mm N(5)-(1-iminoethyl)-l-ornithine dihydrochloride [endothelial NOS (eNOS) inhibitor]. Following microdialysis placement, subjects performed supine cycling with the experimental arm at heart level at 60% V̇O2, peak for a period sufficient to raise core temperature 0.8°C. At the end of cycling, all microdialysis sites were locally heated to 43°C and sodium nitroprusside was perfused to elicit maximal vasodilatation. Mean arterial pressure, skin blood flow via laser-Doppler flowmetry and core temperature via ingestible telemetric pill were measured continuously; cutaneous vascular conductance (CVC) was calculated as laser-Doppler flowmetry/mean arterial pressure and normalized to maximum. There was no significant difference between control (58±2%CVCmax) and nNOS-inhibited (56±3%CVCmax) sites in response to exercise-induced hyperthermia. The increase in CVC at eNOS-inhibited (41±3%CVCmax) and non-selective NOS-inhibited (40±4%CVCmax) sites were significantly attenuated compared to control and nNOS-inhibited (P<0.001 all conditions) but there was no difference between eNOS-inhibited and non-selective NOS-inhibited sites. These data suggest eNOS, not nNOS, mediate NO synthesis during reflex cutaneous vasodilatation with sustained dynamic exercise.

Oral sapropterin acutely augments reflex vasodilation in aged human skin through nitric oxide-dependent mechanisms

Functional constitutive nitric oxide synthase (NOS) and its cofactor tetrahydrobiopterin (BH4) are required for full reflex cutaneous vasodilation and are attenuated in primary aging. Acute, locally administered BH4 increases reflex vasodilation through NO-dependent mechanisms in aged skin. We hypothesized that oral sapropterin (Kuvan, shelf-stable pharmaceutical formulation of BH4) would augment reflex vasodilation in aged human skin during hyperthermia. Nine healthy human subjects (76 ± 1 yr) ingested sapropterin (10 mg/kg) or placebo in a randomized double-blind crossover design. Venous blood samples were collected prior to, and 3 h following, ingestion of sapropterin for measurement of plasma BH4. Three intradermal microdialysis fibers were placed in the forearm skin for local delivery of 1) lactated Ringer's solution, 2) 10 mM BH4, and 3) 20 mM N(G)-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS. Red cell flux was measured at each site by laser-Doppler flowmetry (LDF) as reflex vasodilation was induced using a water-perfused suit. At 1°C rise in oral temperature, mean body temperature was clamped and 20 mM l-NAME was perfused at each site. Cutaneous vascular conductance was calculated (CVC = LDF/MAP) and expressed as a percentage of maximum (%CVCmax 28 mM sodium nitroprusside and local heat 43°C). Plasma concentrations of BH4 were significantly elevated 3 h after ingestion of sapropterin (0 h: 19.1 ± 2 pmol/ml vs. 3 h: 43.8 ± 3 pmol/ml; P < 0.001). Sapropterin increased NO-dependent vasodilation at control site (placebo: 14 ± 1 %CVCmax vs. sapropterin: 25 ± 4 %CVCmax; P = 0.004). Local BH4 administration increased NO-dependent vasodilation compared with control in placebo trials only (control: 14 ± 1 %CVCmax vs. BH4-treated: 24 ± 3 %CVCmax; P = 0.02). These data suggest oral sapropterin increases bioavailable BH4 in aged skin microvasculature sufficiently to increase NO synthesis through NOS and that sapropterin may be a viable intervention to increase skin blood flow during hyperthermia in healthy aged humans.

Dietary nitrate supplementation augments cutaneous reflex vasodilation in healthy humans

Beetroot juice (BRJ) has been shown to increase NO‐dependent dilation through both NOS‐dependent and independent pathways. We hypothesized BRJ supplementation would augment reflex cutaneous active vasodilation. Subjects were equipped with two microdialysis fibers on the forearm and randomly assigned as control (Ringer's) or NOS inhibition (20mM L‐NAME). Whole‐body heating was achieved via water‐perfused suits to raise core temperature (Tc; ingestible telemetric pill) 0.8°C. Maximal cutaneous vasodilation was reached by administering 54mM SNP and local heating to 43°C. Skin blood flow was measured via laser‐Doppler flowmetry and mean arterial pressure was measured; cutaneous vascular conductance (CVC) was calculated and expressed as %CVCmax. Subjects underwent heat stress pre‐ and post‐nitrate supplementation (3 days of BRJ: 5mM, .45g nitrates per day). BRJ increased the plateau CVC at control (pre: 55±3 vs. post: 77±5 %CVCmax) and L‐NAME (pre: 27±3 vs. post: 41±6 %CVCmax; p&lt;0.05 for all conditions) sites. The %NO contribution increased from pre‐ (47±2 %CVCmax) to post‐BRJ (71±4 %CVCmax; p&lt;0.05). These data suggest BRJ augments NO‐dependent component of reflex cutaneous vasodilation.

Acute oral sapropterin (Kuvan®) augments NO‐dependent reflex vasodilation in aged human skin

Nitric oxide‐synthase (NOS) and its essential cofactor tetrahydrobiopterin (BH4) are required for full expression of reflex cutaneous vasodilation (VD). BH4 bioavailability is reduced with aging, as is VD. We hypothesized that oral sapropterin (S; BH4 sold as Kuvan®) would augment NO‐dependent VD in aged skin. Seven subjects (76±3 yrs) ingested S (10mg/kg) or placebo (P) in a randomized double‐blind crossover study design. Three microdialysis fibers were placed in the forearm skin for local delivery of Ringer's solution (control), 10mM BH4, or 20mM L‐NAME (NOS inhibitor). Three h later, red cell flux was measured over each site by laser‐Doppler flowmetry (LDF) as reflex VD was induced using a water perfused suit. At a 1°C rise in oral temperature (Tor) body temperature was clamped and L‐NAME was perfused at each site. Cutaneous vascular conductance was calculated (CVC = LDF/MAP) and normalized to maximum (sodium nitroprusside). S increased VD at the control site (S: 50±4 vs. P: 33±2%CVCmax; p&lt;0.001). Locally adding additional BH4 increased VD in the P trial only (P: 49±5 vs. S: 49±6%CVCmax; p=0.86). At a 1°C rise in Tor S increased NOS‐dependent VD in the control site (S: 27±4 vs. P: 14±2 %CVCmax; p=0.008). Acute oral sapropterin increases NO‐dependent reflex VD in aged skin, suggesting that oral BH4 becomes bioavailable in aged skin microvasculature sufficiently to increase NO synthesis through NOS. NIH RO1‐AG07004–25

Sensory nerves and nitric oxide contribute to reflex cutaneous vasodilation in humans

We tested the hypothesis that inhibition of cutaneous sensory nerves would attenuate reflex cutaneous vasodilation in response to an increase in core temperature. Nine subjects were equipped with four microdialysis fibers on the forearm. Two sites were treated with topical anesthetic EMLA cream for 120 min. Sensory nerve inhibition was verified by lack of sensation to a pinprick. Microdialysis fibers were randomly assigned as 1) lactated Ringer (control); 2) 10 mM nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide synthase; 3) EMLA + lactated Ringer; and 4) EMLA + L-NAME. Laser-Doppler flowmetry was used as an index of skin blood flow, and blood pressure was measured via brachial auscultation. Subjects wore a water-perfused suit, and oral temperature was monitored as an index of core temperature. The suit was perfused with 50°C water to initiate whole body heat stress to raise oral temperature 0.8°C above baseline. Cutaneous vascular conductance (CVC) was calculated and normalized to maximal vasodilation (%CVC(max)). There was no difference in CVC between control and EMLA sites (67 ± 5 vs. 69 ± 6% CVC(max)), but the onset of vasodilation was delayed at EMLA compared with control sites. The L-NAME site was significantly attenuated compared with control and EMLA sites (45 ± 5% CVC(max); P < 0.01). Combined EMLA + L-NAME site (25 ± 6% CVC(max)) was attenuated compared with control and EMLA (P < 0.001) and L-NAME only (P < 0.01). These data suggest cutaneous sensory nerves contribute to reflex cutaneous vasodilation during the early, but not latter, stages of heat stress, and full expression of reflex cutaneous vasodilation requires functional sensory nerves and NOS.

Transient receptor potential vanilloid type 1 channels contribute to reflex cutaneous vasodilation in humans

Mechanisms underlying the cutaneous vasodilation in response to an increase in core temperature remain unresolved. The purpose of this study was to determine a potential contribution of transient receptor potential vanilloid type 1 (TRPV-1) channels to reflex cutaneous vasodilation. Twelve subjects were equipped with four microdialysis fibers on the ventral forearm, and each site randomly received 1) 90% propylene glycol + 10% lactated Ringer (vehicle control); 2) 10 mM l-NAME; 3) 20 mM capsazepine to inhibit TRPV-1 channels; 4) combined 10 mM l-NAME + 20 mM capsazepine. Whole body heating was achieved via water-perfused suits sufficient to raise oral temperature at least 0.8°C above baseline. Maximal skin blood flow was achieved by local heating to 43°C and infusion of 28 mM nitroprusside. Systemic arterial pressure (SAP) was measured, and skin blood flow was monitored via laser-Doppler flowmetry (LDF). Cutaneous vascular conductance (CVC) was calculated as LDF/SAP and normalized to maximal vasodilation (%CVC(max)). Capsazepine sites were significantly reduced compared with control (50 ± 4%CVC(max) vs. 67 ± 5%CVC(max), respectively; P < 0.05). l-NAME (33 ± 3%CVC(max)) and l-NAME + capsazepine (30 ± 4%CVC(max)) sites were attenuated compared with control (P < 0.01) and capsazepine (P < 0.05); however, there was no difference between l-NAME and combined l-NAME + capsazepine. These data suggest TRPV-1 channels participate in reflex cutaneous vasodilation and TRPV-1 channels may account for a portion of the NO component. TRPV-1 channels may have a direct neural contribution or have an indirect effect via increased arterial blood temperature. Whether the TRPV-1 channels directly or indirectly contribute to reflex cutaneous vasodilation remains uncertain.

ENOS and nNOS contribution to reflex cutaneous vasodilation during dynamic exercise in humans

Recent data suggests nNOS mediates NO‐component of reflex cutaneous vasodilation with passive heat stress. Our hypothesis was nNOS but not eNOS inhibition would attenuate reflex cutaneous vasodilation during dynamic exercise. Protocol 1: subjects performed a VO2 peak test on a supine cycle ergometer. Protocol 2: with experimental arm at heart level subjects cycled in supine posture at 60% VO2 peak to raise core temperature (Tc) 0.8–1.0°C (35–45 min). In protocol 2 subjects were equipped with 4 microdialysis fibers on the forearm and each randomly assigned as: 1) lactated Ringer's (control); 2) 5mM NPLA (nNOS inhibition); 3) 10mM L‐NIO (eNOS inhibition); and 4) 20mM L‐NAME (non‐selective NOS inhibition). At the end of protocol 2 all sites were locally heated to 43°C and infused with SNP to elicit maximal dilation. Mean arterial pressure (MAP), skin blood flow via laser‐Doppler flowmetry (LDF), and Tc via ingestible telemetric pill were measured; cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to maximum. In protocol 2 there was no significant difference between control (62±5 %CVCmax) and NPLA (61±6 %CVCmax). L‐NIO (38±4 %CVCmax) and L‐NAME (41±7 %CVCmax) significantly attenuated CVC compared to control and NPLA (p&lt;0.001 all conditions). There was no difference between L‐NIO and L‐NAME. We conclude eNOS, not nNOS, contribute to reflex cutaneous vasodilation during dynamic exercise.

No direct role for A1/A2 adenosine receptor activation to reflex cutaneous vasodilatation during whole-body heat stress in humans

The precise mechanisms underlying reflex cutaneous vasodilatation during hyperthermia remain unresolved. The purpose of this study was to investigate a potential contribution of adenosine A1/A2 receptor activation to reflex cutaneous vasodilatation. Eight subjects were equipped with four microdialysis fibres on the left forearm, and each fibre was randomly assigned one of four treatments: (1) lactated Ringer's (control); (2) 4 mm of the non-selective A1/A2 adenosine receptor antagonist theophylline; (3) 10 mm L-NAME to inhibit nitric oxide (NO) synthase; and (4) combined 4 mm theophylline and 10 mm L-NAME. Laser-Doppler flowmetry (LDF) was used as an index of skin blood flow, and blood pressure was measured beat-by-beat via photoplethysmography and verified via brachial auscultation. Whole-body heat stress to raise oral temperature 0.8 °C above baseline was induced via water-perused suits. Cutaneous vascular conductance (CVC) was calculated as LDF/mean arterial pressure and normalized to maximal (%CVC max) via infusion of 28 mm nitroprusside and local heating to 43 °C. There was no difference between control (65 ± 5%CVC max) and theophylline (63 ± 5%CVC max) sites. L-NAME (44 ± 4%CVC max) and theophylline + L-NAME (32 ± 3%CVC max) sites were significantly attenuated compared to both control and theophylline only sites (P<0.05), and combined theophylline + L-NAME sites were significantly reduced compared to L-NAME only sites (P<0.05). These data suggest A1/A2 adenosine receptor activation does not directly contribute to cutaneous active vasodilatation; however, a role for A1/A2 adenosine receptor activation is unmasked when NO synthase is inhibited.

Local tetrahydrobiopterin administration augments reflex cutaneous vasodilation through nitric oxide-dependent mechanisms in aged human skin

Functional constitutive nitric oxide synthase (NOS) is required for full expression of reflex cutaneous vasodilation that is attenuated in aged skin. Both the essential cofactor tetrahydrobiopterin (BH(4)) and adequate substrate concentrations are necessary for the functional synthesis of nitric oxide (NO) through NOS, both of which are reduced in aged vasculature through increased oxidant stress and upregulated arginase, respectively. We hypothesized that acute local BH(4) administration or arginase inhibition would similarly augment reflex vasodilation in aged skin during passive whole body heat stress. Four intradermal microdialysis fibers were placed in the forearm skin of 11 young (22 ± 1 yr) and 11 older (73 ± 2 yr) men and women for local infusion of 1) lactated Ringer, 2) 10 mM BH(4), 3) 5 mM (S)-(2-boronoethyl)-l-cysteine + 5 mM N(ω)-hydroxy-nor-l-arginine to inhibit arginase, and 4) 20 mM N(G)-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS. Red cell flux was measured at each site by laser-Doppler flowmetry (LDF) as reflex vasodilation was induced. After a 1.0°C rise in oral temperature (T(or)), mean body temperature was clamped and 20 mM l-NAME was perfused at each site. Cutaneous vascular conductance was calculated (CVC = LDF/mean arterial pressure) and expressed as a percentage of maximum (%CVC(max); 28 mM sodium nitroprusside and local heat, 43°C). Vasodilation was attenuated at the control site of the older subjects compared with young beginning at a 0.3°C rise in T(or). BH(4) and arginase inhibition both increased vasodilation in older (BH(4): 55 ± 5%; arginase-inhibited: 47 ± 5% vs. control: 37 ± 3%, both P < 0.01) but not young subjects compared with control (BH(4): 51 ± 4%CVC(max); arginase-inhibited: 55 ± 4%CVC(max) vs. control: 56 ± 6%CVC(max), both P > 0.05) at a 1°C rise in T(or). With a 1°C rise in T(or), local BH(4) increased NO-dependent vasodilation in the older (BH(4): 31.8 ± 2.4%CVC(max) vs. control: 11.7 ± 2.0%CVC(max), P < 0.001) but not the young (BH(4): 23 ± 4%CVC(max) vs. control: 21 ± 4%CVC(max), P = 0.718) subject group. Together these data suggest that reduced BH(4) contributes to attenuated vasodilation in aged human skin and that BH(4) NOS coupling mechanisms may be a potential therapeutic target for increasing skin blood flow during hyperthermia in older humans.

Local tetrahydrobiopterin supplementation augments reflex cutaneous vasodilaton in aged human skin

NO-synthase (NOS) is necessary for full expression of reflex cutaneous vasodilation (VD). Tetrahydrobiopterin (BH4) is an essential cofactor for NOS. Reduced BH4 bioavailability is associated with aging. We hypothesized that acute local BH4 supplementation would augment NO-dependent VD in aged skin during hyperthermia. Three intradermal microdialysis fibers were placed in the forearm skin of 10 young (Y, 24 ± 6 years) and 7 older (O, 72 ± 6 years) human subjects for local infusion of Ringers solution (control), L-NAME (NOS-inhibition) and 10mM BH4, respectively. Red cell flux was measured at each site by laser-Doppler flowmetry (LDF) as reflex VD was induced using a water perfused suit. After a 1°C rise in oral temperature, mean body temperature was clamped and L-NAME was perfused at each site. Cutaneous vascular conductance was calculated (CVC = LDF/MAP) and expressed as percent of maximum (%CVCmax: infusion of 28mM sodium nitroprusside). VD was attenuated at control site in O (O: 35 ± 6 vs. Y: 54 ± 7 %CVCmax; p<0.05). BH4 supplementation augmented VD in O (54 ± 11 %CVCmax; p<0.05) but not Y (57 ± 8 %CVCmax; p<0.05). NOS inhibition equally attenuated VD at the BH4 site in both groups (O: 38 ± 4 vs. Y: 24 ± 5 %CVCmax; p<0.05). Acute local BH4 supplementation augments reflex NO-dependant VD in aged skin, suggesting that reduced BH4 bioavailability contributes to the attenuated VD in older humans. Supported by: NIH AG007-004-20