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.

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.

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(ω)-propyl-l-arginine), and nonselective NOS inhibited (N(G)-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.

Acute localized administration of tetrahydrobiopterin and chronic systemic atorvastatin treatment restore cutaneous microvascular function in hypercholesterolaemic humans.

Elevated oxidized low-density lipoproteins (LDL) are associated with vascular dysfunction in the cutaneous microvasculature, induced in part by upregulated arginase activity and increased globalized oxidant stress. Since tetrahydrobiopterin (BH(4)) is an essential cofactor for endothelial nitric oxide synthase (NOS3), decreased bioavailability of the substrate l-arginine and/or BH(4) may contribute to decreased NO production with hypercholesterolaemia. We hypothesized that (1) localized administration of BH(4) would augment NO-dependent vasodilatation in hypercholesterolaemic human skin, which would be further increased when combined with arginase inhibition and (2) the improvement induced by localized BH(4) would be attenuated after a 3 month oral atorvastatin intervention (10 mg). Four microdialysis fibres were placed in the skin of nine normocholesterolaemic (NC: LDL = 95 ± 4 mg dl(-1)) and nine hypercholesterolaemic (HC: LDL = 177 ± 6 mg dl(-1)) men and women before and after 3 months of systemic atorvastatin. Sites served as control, NOS inhibited, BH(4), and arginase inhibited + BH(4) (combo). Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilatation. After the established plateau l-NAME was perfused in all sites to quantify NO-dependent vasodilatation (NO). Data were normalized to maximum cutaneous vascular conductance (CVC). Vasodilatation at the plateau and NO-dependent vasodilatation were reduced in HC subjects (plateau HC: 70 ± 5% CVC(max) vs. NC: 95 ± 2% CVC(max); NO HC: 45 ± 5% CVC(max) vs. NC: 64 ± 5% CVC(max); both P < 0.001). Localized BH(4) alone or combo augmented the plateau (BH(4): 93 ± 3% CVC(max); combo 89 ± 3% CVC(max), both P < 0.001) and NO-dependent vasodilatation in HC (BH(4): 74 ± 3% CVC(max); combo 76 ± 3% CVC(max), both P < 0.001), but there was no effect in NC subjects (plateau BH(4): 90 ± 2% CVC(max); combo 95 ± 3% CVC(max); NO-dependent vasodilatation BH(4): 68 ± 3% CVC(max); combo 58 ± 4% CVC(max), all P > 0.05 vs. control site). After the atorvastatin intervention (LDL = 98 ± mg * dl(-1)) there was an increase in the plateau in HC (96 ± 4% CVC(max), P < 0.001) and NO-dependent vasodilatation (68 ± 3% CVC(max), P < 0.001). Localized BH(4) alone or combo was less effective at increasing NO-dependent vasodilatation after the drug intervention (BH(4): 60 ± 5% CVC(max); combo 58 ± 2% CVC(max), both P < 0.001). These data suggest that decreased BH(4) bioavailability contributes in part to cutaneous microvascular dysfunction in hypercholesterolaemic humans and that atorvastatin is an effective systemic treatment for improving NOS coupling mechanisms in the microvasculature.

Oral atorvastatin therapy increases nitric oxide-dependent cutaneous vasodilation in humans by decreasing ascorbate-sensitive oxidants.

Elevated low-density lipoproteins (LDL) are associated with cutaneous microvascular dysfunction partially mediated by increased arginase activity, which is decreased following a systemic atorvastatin therapy. We hypothesized that increased ascorbate-sensitive oxidant stress, partially mediated through uncoupled nitric oxide synthase (NOS) induced by upregulated arginase, contributes to cutaneous microvascular dysfunction in hypercholesterolemic (HC) humans. Four microdialysis fibers were placed in the skin of nine HC (LDL = 177 ± 6 mg/dl) men and women before and after 3 mo of a systemic atorvastatin intervention and at baseline in nine normocholesterolemic (NC) (LDL = 95 ± 4 mg/dl) subjects. Sites served as control, NOS inhibited, L-ascorbate, and arginase-inhibited+L-ascorbate. Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilation. After the established plateau in all sites, 20 mM ≪ngname≫ was infused to quantify NO-dependent vasodilation. Data were normalized to maximum cutaneous vascular conductance (CVC) (sodium nitroprusside + 43°C). The plateau in vasodilation during local heating (HC: 78 ± 4 vs. NC: 96 ± 2% CVC(max), P < 0.01) and NO-dependent vasodilation (HC: 40 ± 4 vs. NC: 54 ± 4% CVC(max), P < 0.01) was reduced in the HC group. Acute L-ascorbate alone (91 ± 5% CVC(max), P < 0.001) or combined with arginase inhibition (96 ± 3% CVC(max), P < 0.001) augmented the plateau in vasodilation in the HC group but not the NC group (ascorbate: 96 ± 2; combo: 93 ± 4% CVC(max), both P > 0.05). After the atorvastatin intervention NO-dependent vasodilation was augmented in the HC group (HC postatorvastatin: 64 ± 4% CVC(max), P < 0.01), and there was no further effect of ascorbate alone (58 ± 4% CVC(max,) P > 0.05) or combined with arginase inhibition (67 ± 4% CVC(max,) P > 0.05). Increased ascorbate-sensitive oxidants contribute to hypercholesteromic associated cutaneous microvascular dysfunction which is partially reversed with atorvastatin therapy.

Changes in the control of skin blood flow with exercise training: where do cutaneous vascular adaptations fit in?

Heat is the most abundant byproduct of cellular metabolism. As such, dynamic exercise in which a significant percentage of muscle mass is engaged generates thermoregulatory demands that are met in part by increases in skin blood flow. Increased skin blood flow during exercise adds to the demands on cardiac output and confers additional circulatory strain beyond that associated with perfusion of active muscle alone. Endurance exercise training results in a number of physiological adaptations which ultimately reduce circulatory strain and shift thermoregulatory control of skin blood flow to higher levels of blood flow for a given core temperature. In addition, exercise training induces peripheral vascular adaptations within the cutaneous microvasculature indicative of enhanced endothelium-dependent vasomotor function. However, it is not currently clear how (or if) these local vascular adaptations contribute to the beneficial changes in thermoregulatory control of skin blood flow following exercise training. The purpose of this Hot Topic Review is to synthesize the literature pertaining to exercise training-mediated changes in cutaneous microvascular reactivity and thermoregulatory control of skin blood flow. In addition, we address mechanisms driving changes in cutaneous microvascular reactivity and thermoregulatory control of skin blood flow, and pose the question: what (if any) is the functional role of increased cutaneous microvascular reactivity following exercise training?

Oral atorvastatin therapy restores cutaneous microvascular function by decreasing arginase activity in hypercholesterolaemic humans.

Elevated low-density lipoproteins (LDLs) are associated with vascular dysfunction evident in the cutaneous microvasculature. We hypothesized that uncoupled endothelial nitric oxide synthase (NOS3) through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic (HC) humans and that a statin intervention would decrease arginase activity. Five microdialysis fibres were placed in the skin of nine normocholesterolaemic (NC: LDL level 95±4 mg dl⁻¹) and nine hypercholesterolaemic (HC: LDL: 177±6 mg dl⁻¹) men and women before and after 3 months of systemic atrovastatin. Sites served as control, NOS inhibited, arginase inhibited, L-arginine supplemented and arginase inhibited plus L-arginine supplemented. Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilatation. L-NAME was infused after the established plateau in all sites to quantify NO-dependent vasodilatation. Data were normalized to maximum cutaneous vascular conductance (CVC(max)). Skin samples were obtained to measure total arginase activity and arginase I and arginase II protein. Vasodilatation was reduced in hyperocholesterolaemic subjects (HC: 76±2 vs. NC: 94±3%CVC(max), P < 0.001) as was NO-dependent vasodilatation (HC: 43±5 vs. NC: 62±4%CVC(max), P < 0.001). The plateau and NO-dependent vasodilatation were augmented in HC with arginase inhibition (92±2, 67±2%CVC(max), P < 0.001), L-arginine (93±2, 71±5%CVC(max), P < 0.001) and combined treatments (94±4, 65±5%CVC(max), P < 0.001) but not in NC. After statin intervention (LDL: 98±5 mg dl⁻¹) there was no longer a difference between control sites (88±4, 61±5%CVC(max)) and localized microdialysis treatment sites (all P > 0.05). Arginase activity and protein were increased in HC skin (P < 0.05 vs. NC) and activity decreased with atrovastatin treatment (P < 0.05). Reduced NOS3 substrate availability through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic humans, which is corrected with atorvastatin therapy.

Tetrahydrobiopterin does not affect end-organ responsiveness to norepinephrine-mediated vasoconstriction in aged skin.

We have recently demonstrated that tetrahydrobiopterin (BH(4)) augments reflex vasoconstriction (VC) in aged skin. Although this appears to occur through its role in norepinephrine (NE) biosynthesis, the extent with which vascular mechanisms are affected are unknown. We hypothesized that localized BH(4) supplementation would not affect the VC response to exogenous NE when sympathetic nerves were blocked. Two microdialysis fibers were placed in bretylium tosylate pretreated (presynaptically blocks neurotransmitter release from sympathetic adrenergic nerve terminals; iontophoresis, 200 µA for 20 min) 3-cm(2) forearm skin of 10 young (Y) and 10 older (O) subjects for perfusion of 1) Ringer (control) and 2) 5 mM BH(4). While local skin temperature was clamped at 34°C, six concentrations of NE (10(-12), 10(-10), 10(-8), 10(-6), 10(-4), 10(-2) M) were infused at each drug-treated site. Cutaneous vascular conductance (CVC) was calculated (CVC = laser Doppler flux/mean arterial pressure) and normalized to baseline (%ΔCVC(base)). Despite prejunctional adrenergic blockade, NE-mediated VC was blunted in aged skin at each NE dose (10(-12): -12 ± 2 vs. -21 ± 2; 10(-10): -15 ± 2 vs. -27 ± 1; 10(-8): -22 ± 2 vs. -32 ± 2; 10(-6): -27 ± 2 vs. -38 ± 1; 10(-4): -52 ± 3 vs. -66 ± 5; 10(-2): -62 ± 3 vs. -75 ± 4%ΔCVC(base); P < 0.01), and this response was not affected by pretreatment with BH(4) (P > 0.05). Localized BH(4) did not affect end-organ responsiveness to exogenous NE, suggesting that the effects of BH(4) on cutaneous VC are primarily isolated to the NE biosynthetic pathway.

Peripheral mechanisms of thermoregulatory control of skin blood flow in aged humans.

Human skin blood flow is controlled via dual innervation from the sympathetic nervous system. Reflex cutaneous vasoconstriction and vasodilation are both impaired with primary aging, rendering the aged more vulnerable to hypothermia and cardiovascular complications from heat-related illness. Age-related alterations in the thermoregulatory control of skin blood flow occur at multiple points along the efferent arm of the reflex, including 1) diminished sympathetic outflow, 2) altered presynaptic neurotransmitter synthesis, 3) reduced vascular responsiveness, and 4) impairments in downstream (endothelial and vascular smooth muscle) second-messenger signaling. This mechanistic review highlights some of the recent findings in the area of aging and the thermoregulatory control of skin blood flow.

Systemic low-dose aspirin and clopidogrel independently attenuate reflex cutaneous vasodilation in middle-aged humans.

Chronic systemic platelet cyclooxygenase (COX) inhibition with low-dose aspirin [acetylsalicylic acid (ASA)] significantly attenuates reflex cutaneous vasodilation in middle-aged humans, whereas acute, localized, nonisoform-specific inhibition of vascular COX with intradermal administration of ketorolac does not alter skin blood flow during hyperthermia. Taken together, these data suggest that platelets may be involved in reflex cutaneous vasodilation, and this response is inhibited with systemic pharmacological platelet inhibition. We hypothesized that, similar to ASA, specific platelet ADP receptor inhibition with clopidogrel would attenuate reflex vasodilation in middle-aged skin. In a double-blind crossover design, 10 subjects (53+/-2 yr) were instrumented with four microdialysis fibers for localized drug administration and heated to increase body core temperature [oral temperature (Tor)] 1 degrees C during no systemic drug (ND), and after 7 days of systemic ASA (81 mg) and clopidogrel (75 mg) treatment. Skin blood flow (SkBF) was measured using laser-Doppler flowmetry over each site assigned as 1) control, 2) nitric oxide synthase inhibited (NOS-I; 10 mM NG-nitro-L-arginine methyl ester), 3) COX inhibited (COX-I; 10 mM ketorolac), and 4) NOS-I+COX-I. Data were normalized and presented as a percentage of maximal cutaneous vascular conductance (%CVCmax; 28 mM sodium nitroprusside+local heating to 43 degrees C). During ND conditions, SkBF with change (Delta) in Tor=1.0 degrees C was 56+/-3% CVCmax. Systemic low-dose ASA and clopidogrel both attenuated reflex vasodilation (ASA: 43+/-3; clopidogrel: 32+/-3% CVCmax; both P<0.001). In all trials, localized COX-I did not alter SkBF during significant hyperthermia (ND: 56+/-7; ASA: 43+/-5; clopidogrel: 35+/-5% CVCmax; all P>0.05). NOS-I attenuated vasodilation in ND and ASA (ND: 28+/-6; ASA: 25+/-4% CVCmax; both P<0.001), but not with clopidogrel (27+/-4% CVCmax; P>0.05). NOS-I+COX-I was not different compared with NOS-I alone in either systemic treatment condition. Both systemic ASA and clopidogrel reduced the time required to increase Tor 1 degrees C (ND: 58+/-3 vs. ASA: 45+/-2; clopidogrel: 39+/-2 min; both P<0.001). ASA-induced COX and specific platelet ADP receptor inhibition attenuate reflex vasodilation, suggesting platelet involvement in reflex vasodilation through the release of vasodilating factors.

Localized tyrosine or tetrahydrobiopterin supplementation corrects the age-related decline in cutaneous vasoconstriction.

The attenuated reflex vasoconstriction in aged skin may be partly mediated by oxidant-induced reduction in functional substrate and cofactor availability for noradrenaline biosynthesis. We hypothesized that localized supplementation of tyrosine and tetrahydrobiopterin (BH(4)) in aged human skin could augment reflex- (whole-body cooling) and pharmacologically (tyramine, which displaces noradrenaline from axon terminals) induced vasoconstriction. Four microdialysis fibres were placed in the forearm skin of 10 young and 10 older subjects for infusion of (1) Ringer solution (control), (2) 0.5 mm L-tyrosine, (3) 5 mm BH(4), and (4) BH(4) + L-tyrosine. Cutaneous vascular conductance (CVC) was calculated (laser Doppler flux/mean arterial pressure) and normalized to baseline (% Delta CVC(base)). Vasoconstriction was attenuated at the control site in the older subjects during both whole-body cooling (young: 39 +/- 3, older: 17 +/- 3% Delta CVC(base); P < 0.01) and tyramine infusion (young: 41 +/- 3, older: 21 +/- 4% Delta CVC(base); P < 0.01). BH(4) (cold, young: 37 +/- 3, older: 36 +/- 3; tyramine, young: 41 +/- 2, older: 36 +/- 3% Delta CVC(base)) and tyrosine (cold, young: 37 +/- 4, older: 34 +/- 4; tyramine, young: 40 +/- 4, older: 45 +/- 4% Delta CVC(base)) both resolved the age-related decrease in cutaneous vasoconstriction, but BH(4) + tyrosine did not further augment vasoconstriction (cold, young: 38 +/- 4, older: 31 +/- 3; tyramine, young: 36 +/- 3, older: 36 +/- 5 Delta %CVC(base)). These data are consistent with the concept that reduced bioavailability of BH(4) and/or tyrosine may impair noradrenaline synthesis and contribute to the attenuated vasoconstrictor response in aged skin.

Aging and the control of human skin blood flow.

Human exposure to cold and heat stimulates cutaneous vasoconstriction and vasodilation via distinct sympathetic reflex and locally mediated pathways. The mechanisms mediating cutaneous vasoconstriction and vasodilation are impaired with primary aging, rendering the aged more vulnerable to hypothermia and cardiovascular complications from heat-related illness, respectively. This paper highlights recent findings discussing how age-related decrements in sympathetic neurotransmission contribute directly to thermoregulatory impairments, whereas changes in local intracellular signaling suggest a more generalized age-associated vascular dysfunction.

Reflex vasoconstriction in aged human skin increasingly relies on Rho kinase-dependent mechanisms during whole body cooling.

Primary human aging may be associated with augmented Rho kinase (ROCK)-mediated contraction of vascular smooth muscle and ROCK-mediated inhibition of nitric oxide synthase (NOS). We hypothesized that the contribution of ROCK to reflex vasoconstriction (VC) is greater in aged skin. Cutaneous VC was elicited by 1) whole body cooling [mean skin temperature (T(sk)) = 30.5 degrees C] and 2) local norepinephrine (NE) infusion (1 x 10(-6) M). Four microdialysis fibers were placed in the forearm skin of eight young (Y) and eight older (O) subjects for infusion of 1) Ringer solution (control), 2) 3 mM fasudil (ROCK inhibition), 3) 20 mM N(G)-nitro-l-arginine methyl ester (NOS inhibition), and 4) both ROCK + NOS inhibitors. Red cell flux was measured by laser-Doppler flowmetry over each site. Cutaneous vascular conductance (CVC) was calculated as flux/mean arterial pressure and normalized to baseline CVC (%DeltaCVC(baseline)). VC was reduced at the control site in O during cooling (Y, -34 + or - 3; and O, -18 + or - 3%DeltaCVC(baseline); P < 0.001) and NE infusion (Y, -53 + or - 4, and O, -41 + or - 9%DeltaCVC(baseline); P = 0.006). Fasudil attenuated VC in both age groups during mild cooling; however, this reduction remained only in O but not in Y skin during moderate cooling (Y, -30 + or - 5; and O, -7 + or - 1%DeltaCVC(baseline); P = 0.016) and was not altered by NOS inhibition. Fasudil blunted NE-mediated VC in both age groups (Y, -23 + or - 4; and O, -7 + or - 3%DeltaCVC(baseline); P < 0.01). Cumulatively, these data indicate that reflex VC is more reliant on ROCK in aged skin such that approximately half of the total VC response to whole body cooling is ROCK dependent.

Ketorolac alters blood flow during normothermia but not during hyperthermia in middle-aged human skin.

In young healthy humans full expression of reflex cutaneous vasodilation is dependent on cyclooxygenase (COX)- and nitric oxide synthase (NOS)-dependent mechanisms. Chronic low-dose aspirin therapy attenuates reflex cutaneous vasodilation potentially through both platelet and vascular COX-dependent mechanisms. We hypothesized the contribution of COX-dependent vasodilators to reflex cutaneous vasodilation during localized acute COX inhibition would be attenuated in healthy middle-aged humans due to a shift toward COX-dependent vasoconstrictors. Four microdialysis fibers were placed in forearm skin of 13 middle-aged (53 +/- 2 yr) normotensive healthy humans, serving as control (Ringer), COX-inhibited (10 mM ketorolac), NOS-inhibited (10 mM N(G)-nitro-l-arginine methyl ester), and combined NOS- and COX-inhibited sites. Red blood cell flux was measured over each site by laser-Doppler flowmetry as reflex vasodilation was induced by increasing oral temperature (T(or)) 1.0 degrees C using a water-perfused suit. Cutaneous vascular conductance was calculated (CVC = flux/mean arterial pressure) and normalized to maximal CVC (CVC(max); 28 mM sodium nitroprusside). CVC(max) was not affected by localized microdialysis drug treatment (P > 0.05). Localized COX inhibition increased baseline (18 +/- 3%CVC(max); P < 0.001) compared with control (9 +/- 1%CVC(max)), NOS-inhibited (7 +/- 1%CVC(max)), and combined sites (10 +/- 1%CVC(max)). %CVC(max) in the COX-inhibited site remained greater than the control site with DeltaT(or) < or = 0.3 degrees C; however, there was no difference between these sites with DeltaT(or) > or = 0.4 degrees C. NOS inhibition and combined COX and NOS inhibition attenuated reflex vasodilation compared with control (P < 0.001), but there was no difference between these sites. Localized COX inhibition with ketorolac significantly augments baseline CVC but does not alter the subsequent skin blood flow response to hyperthermia, suggesting a limited role for COX-derived vasodilator prostanoids in reflex cutaneous vasodilation and a shift toward COX-derived vasoconstrictors in middle-aged human skin.

Local tetrahydrobiopterin administration augments cutaneous vasoconstriction in aged humans.

Reflex vasoconstriction (VC) is attenuated in aged skin resulting in greater skin blood flow and heat loss during cold exposure. We hypothesized that adrenergic function is compromised due to depletion of tetrahydrobiopterin (BH(4)), an essential cofactor required for catecholamine synthesis, and therefore local BH(4) supplementation would functionally augment reflex and pharmacologically induced VC elicited by gradual whole-body cooling (skin temperature (T(sk)) = 30.5 degrees C) and tyramine infusion, respectively. Four microdialysis (MD) fibres were placed in the forearm skin of 11 young (Y) and 11 older (O) human subjects for infusion of (1) Ringer solution (control), (2) 5 mM BH(4), (3) 5 mM BH(4) + 10 mM ascorbate, and (4) 5 mM BH(4) + adrenoreceptor blockade (5 mM yohimbine + 1 mM propranolol). Laser Doppler flux (LDF) was measured over each MD site and cutaneous vascular conductance was calculated as CVC = LDF/MAP and expressed as per cent change from baseline (% DeltaCVC(base)). The VC response was lower at the control site in O during cooling (Y: -34 +/- 2% DeltaCVC(base), O: -17 +/- 2% DeltaCVC(base); P < 0.001) and tyramine infusion (Y: - 33 +/- 4% DeltaCVC(base), O: -15 +/- 3% DeltaCVC(base); P < 0.001). BH(4) infusion normalized O to Y values during both cooling (Y: -34 +/- 4% DeltaCVC(base), O: -34 +/- 2% DeltaCVC(base); P < 0.001) and tyramine (Y: -38 +/- 4% DeltaCVC(base), O: -35 +/- 3% DeltaCVC(base); P < 0.001), however, adding adrenoreceptor blockade abolished VC in aged skin indicating that BH(4) acts through adrenergic, not cotransmitter, mechanisms. Local BH(4) supplementation augments reflex and tyramine-induced VC in aged skin, suggesting that reduced BH(4) bioavailability may contribute to attenuated VC during whole-body cooling.

Chronic low-dose aspirin therapy attenuates reflex cutaneous vasodilation in middle-aged humans.

Full expression of reflex cutaneous vasodilation is dependent on cyclooxygenase- (COX) and nitric oxide synthase- (NOS) dependent mechanisms. Low-dose aspirin therapy is widely prescribed to inhibit COX-1 in platelets for atherothrombotic prevention. We hypothesized that chronic COX inhibition with daily low-dose aspirin therapy (81 mg) would attenuate reflex vasodilation in healthy human skin. Two microdialysis fibers were placed in forearm skin of seven middle-aged (57 +/- 3 yr), normotensive, healthy humans with no preexisting cardiovascular disease, taking daily low-dose aspirin therapy (aspirin: 81 mg), and seven unmedicated, healthy, age-matched control (no aspirin, 55 +/- 3 yr) subjects, with one site serving as a control (Ringer) and the other NOS inhibited (NOS inhibited: 10 mM N(G)-nitro-l-arginine methyl ester). Red cell flux was measured over each site by laser-Doppler flowmetry, as reflex vasodilation was induced by increasing core temperature (oral temperature) 1.0 degrees C using a water-perfused suit. Cutaneous vascular conductance (CVC) was calculated (CVC = flux/mean arterial pressure) and normalized to maximal CVC (CVC(max); 28 mM sodium nitroprusside). CVC(max) was not affected by either aspirin or NOS inhibition. The plateau in cutaneous vasodilation during heating (change in oral temperature = 1.0 degrees C) was significantly attenuated in the aspirin group (aspirin: 25 +/- 3% CVC(max) vs. no aspirin: 50 +/- 7% CVC(max), P < 0.001 between groups). NOS inhibition significantly attenuated %CVC(max) in both groups (aspirin: 17 +/- 2% CVC(max), no aspirin: 23 +/- 3% CVC(max); P < 0.001 vs. control), but this attenuation was less in the no-aspirin treatment group (P < 0.001). This is the first observation that chronic low-dose aspirin therapy attenuates reflex cutaneous vasodilation through both COX- and NOS-dependent mechanisms.