Rebecca S. Bruning

Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin

Local skin heating is used to assess microvascular function in clinical populations because NO is required for full expression of the response; however, controversy exists as to the precise NO synthase (NOS) isoform producing NO. Human aging is associated with attenuated cutaneous vasodilation but little is known about the middle aged, an age cohort used for comparison with clinical populations. We hypothesized that endothelial NOS (eNOS) is the primary isoform mediating NO production during local heating, and eNOS-dependent vasodilation would be reduced in middle-aged skin. Vasodilation was induced by local heating (42°C) and during acetylcholine dose-response (ACh-DR: 0.01, 0.1, 1.0, 5.0, 10.0, 50.0, 100.0 mmol/l) protocols. Four microdialysis fibers were placed in the skin of 24 men and women; age cohorts were 12 middle-aged (53 ± 1 yr) and 12 young (23 ± 1 yr). Sites served as control, nonselective NOS inhibited [N(G)-nitro-l-arginine methyl ester (l-NAME)], inducible NOS (iNOS) inhibited (1400W), and neuronal NOS (nNOS) inhibited (N(ω)-propyl-l-arginine). After full expression of the local heating response, l-NAME was perfused at all sites. Cutaneous vascular conductance was measured and normalized to maximum (%CVC(max): Nitropress). l-NAME reduced %CVCmax at baseline, all phases of the local heating response, and at all ACh concentrations compared with all other sites. iNOS inhibition reduced the initial peak (53 ± 2 vs. 60 ± 2%CVC(max); P < 0.001); however, there were no other differences between control, nNOS-, and iNOS-inhibited sites during the phases of local heating or ACh-DR. When age cohorts were compared, NO-dependent vasodilation during local heating (52 ± 6 vs. 68 ± 4%CVC(max); P = 0.013) and ACh perfusion (50 mmol/l: 83 ± 3 vs. 93 ± 2%CVC(max); 100 mmol/l: 83 ± 4 vs. 92 ± 3%CVC(max); both P = 0.03) were reduced in middle-aged skin. There were no differences in NOS isoform expression obtained from skin biopsy samples between groups (all P > 0.05). These data suggest that eNOS mediates the production of NO during local heating and that cutaneous vasodilation is attenuated in middle-aged skin.

Upregulation of inducible nitric oxide synthase contributes to attenuated cutaneous vasodilation in essential hypertensive humans.

Essential hypertension is a proinflammatory, proconstrictor disease coinciding with endothelial dysfunction and inward vessel remodeling. Using the skin circulation, our aim was to determine whether inducible NO synthase (iNOS) upregulation attenuates NO-dependent cutaneous vasodilation in hypertensive humans. We hypothesized that, with hypertension, localized iNOS inhibition would restore vasodilation in response to NO-dependent stimuli, and iNOS expression would be increased and phosphorylated vasodilator-stimulated phosphoprotein would be decreased. For, in vivo protocols, 4 intradermal microdialysis fibers were placed in 9 hypertensive and 10 normotensive men and women (systolic blood pressure: 146±4 versus 113±2 mm Hg; P<0.001). Microdialysis fibers served as control, iNOS inhibited (1400 W), neuronal NO synthase inhibited (N&ohgr;-propyl-L-arginine), and nonselective NOS inhibited (NG-nitro-L-arginine methyl ester). Cutaneous vascular conductance was calculated (percentage of sodium nitroprusside) during standardized local heating (42°C) and acetylcholine dose-response protocols (0.01, 0.10, 1.00, 5.00, 10.00, 50.00, 100.00 mmol/L). The NO-dependent local heating response was attenuated at control (95±2% versus 76±2% cutaneous vascular conductance; P<0.05) and neuronal NO synthase–inhibited sites (94±4% versus 77±3% cutaneous vascular conductance; P<0.01) in hypertensives. iNOS inhibition augmented the NO-dependent local heating response (93±2% versus 89±10% cutaneous vascular conductance). Acetylcholine-induced vasodilation was attenuated in control sites at doses ≥0.1 mmol/L of acetylcholine in hypertensives and was restored with iNOS inhibition (0.1 mmol/L, P<0.05; 1, 5, and 10 mmol/L, P<0.001; 50 and 100 mmol/L, P<0.01). In vitro iNOS expression was increased (P=0.006) and phosphorylated vasodilator-stimulated phosphoprotein was decreased in skin from hypertensive humans (P=0.04). These data suggest that iNOS is upregulated in essential hypertensive humans and contributes to reduced NO-dependent cutaneous vasodilation.

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.

Aspirin and Clopidogrel Alter Core Temperature and Skin Blood Flow during Heat Stress

UNLABELLED Antithrombotic therapy with oral aspirin (ASA) or clopidogrel (CLO) (Plavix; Bristol-Myers Squibb, Bridgewater, NJ) is associated with an attenuated skin vasodilator response and a greater rate of rise in core temperature in healthy, middle-age individuals during passive heating in a water perfused suit. PURPOSE The present double-blind, crossover study examined the functional consequences of 7 d of low-dose ASA (81 mg·d) versus CLO (75 mg·d) treatment in 14 healthy, middle-age (50-65 yr) men and women during passive heating in air (40 min at 30°C, 40% relative humidity) followed by exercise (60% V˙O2peak). METHODS Oral temperature (Tor) was measured in the antechamber (23.0°C ± 0.1°C) before entering a warm environmental chamber. After 40 min of rest, subjects cycled on a recumbent cycle ergometer for up to 120 min. Esophageal temperature (Tes) and laser Doppler flux were measured continuously, and the latter was normalized to maximal cutaneous vascular conductance (%CVCmax). RESULTS Before entry into the environmental chamber there were no differences in Tor among treatments; however, after 40 min of rest in the heat, Tes was significantly higher for ASA and CLO versus placebo (37.2°C ± 0.1°C, 37.3°C ± 0.1°C, vs 37.0°C ± 0.1°C, both P < 0.001), a difference that persisted throughout exercise (P < 0.001 vs placebo). The mean body temperature thresholds for the onset of cutaneous vasodilation were shifted to the right for both ASA and CLO during exercise (P < 0.05). CONCLUSION ASA and CLO resulted in elevated core temperatures during passive heat stress and shifted the onset of peripheral thermoeffector mechanisms toward higher body temperatures during exercise heat stress.

Blood pressure regulation III: what happens when one system must serve two masters: temperature and pressure regulation?

When prolonged intense exercise is performed at high ambient temperatures, cardiac output must meet dual demands for increased blood flow to contracting muscle and to the skin. The literature has commonly painted this scenario as a fierce competition, wherein one circulation preserves perfusion at the expense of the other, with the regulated maintenance of blood pressure as the ultimate goal. This review redefines this scenario as commensalism, an integrated balance of regulatory control where one circulation benefits with little functional effect on the other. In young, healthy subjects, arterial pressure rarely falls to any great extent during either extreme passive heating or prolonged dynamic exercise in the heat, nor does body temperature rise disproportionately due to a compromised skin blood flow. Rather, it often takes the superimposition of additional stressors—e.g., dehydration or simulated hemorrhage—upon heat stress to substantially impact blood pressure regulation.

Altered skin flowmotion in hypertensive humans

Essential hypertensive humans exhibit attenuated cutaneous nitric oxide (NO)-dependent vasodilation. Using spectral analysis (fast Fourier transformation) we aimed to characterize the skin flowmotion contained in the laser-Doppler flowmetry recordings during local heating-induced vasodilation before and after concurrent pharmacological inhibition of nitric oxide synthase (NOS) in hypertensive and age-matched normotensive men and women. We hypothesized that hypertensive subjects would have lower total power spectral densities (PSDs), specifically in the frequency intervals associated with intrinsic endothelial and neurogenic control of the microvasculature. Furthermore, we hypothesized that NOS inhibition would attenuate the endothelial frequency interval. Laser-Doppler flowmetry recordings during local heating experiments from 18 hypertensive (MAP: 108±2mmHg) and 18 normotensive (MAP: 88±2mmHg) men and women were analyzed. Within site NO-dependent vasodilation was assessed by perfusion of a non-specific NOS inhibitor (N(G)-nitro-l-arginine methyl ester; l-NAME) through intradermal microdialysis during the heating-induced plateau in skin blood flow. Local heating-induced vasodilation increased total PSD for all frequency intervals (all p<0.001). Hypertensives had a lower total PSD (p=0.03) and absolute neurogenic frequency intervals (p<0.01) compared to the normotensives. When normalized as a percentage of total PSD, hypertensives had reduced neurogenic (p<0.001) and augmented myogenic contributions (p=0.04) to the total spectrum. NOS inhibition decreased total PSD (p<0.001) for both groups, but hypertensives exhibited lower absolute endothelial (p<0.01), neurogenic (p<0.05), and total PSD (p<0.001) frequency intervals compared to normotensives. These data suggest that essential hypertension results in altered neurogenic and NOS-dependent control of skin flowmotion and support the use of spectral analysis as a non-invasive technique to study vasoreactivity.

Oral clopidogrel improves cutaneous microvascular function through EDHF-dependent mechanisms in middle-aged humans

Platelet P₂Y₁₂-ADP and COX-1 receptor inhibition with oral clopidogrel (CLO) and low-dose aspirin (ASA), respectively, attenuates reflex-mediated cutaneous vasodilation, but little is known about how these medications affect local vasodilatory signaling. Reactive hyperemia (RH) results in vasodilation that is mediated by sensory nerves and endothelium-derived hyperpolarization factors (EDHF) through large-conductance calcium-activated potassium channels, whereas slow local heating (LH) elicits vasodilation largely through the production of nitric oxide (NO). We hypothesized that CLO and ASA would attenuate locally mediated cutaneous vasodilation assessed by RH and LH (0.5°C/min). In a randomized, cross-over, double-blind placebo-controlled study, nine healthy men and women (56 ± 1 yr) took CLO (75 mg), ASA (81 mg), and placebo for 7 days. Skin blood flow was measured (laser-Doppler flowmetry, LDF) and cutaneous vascular conductance (CVC) was calculated (LDF/mean arterial pressure) and normalized to maximal CVC (%CVCmax: 43°C and 28 mM sodium nitroprusside). RH response parameters, including area under the curve (AUC), total hyperemic response (THR), and the decay constant tau (λ) were calculated. NO-dependent vasodilation during LH was assessed by calculating the difference in %CVCmax between a control site and an NO synthase-inhibited site (10 mM l-NAME: intradermal microdialysis). CLO augmented the AUC and THR (AUCclo = 3,783 ± 342; THRclo = 2,306 ± 266% CVCmax/s) of the RH response compared with ASA (AUCASA = 3,101 ± 325; THRASA = 1,695 ± 197% CVCmax/s) and placebo (AUCPlacebo = 3,000 ± 283; THRPlacebo = 1,675 ± 170% CVCmax/s; all P < 0.0001 vs. CLO). There was no difference in the LH response or calculated NO-dependent vasodilation among treatments (all P > 0.05). Oral CLO treatment augments vasodilation during RH but not LH, suggesting that CLO may improve cutaneous microvascular function.