Nitric oxide concentration increases in the cutaneous interstitial space during heat stress in humans.

To examine the role of nitric oxide (NO) in cutaneous active vasodilation, we measured the NO concentration from skin before and during whole body heat stress in nine healthy subjects. A forearm site was instrumented with a NO-selective, amperometric electrode and an adjacent intradermal microdialysis probe. Skin blood flow (SkBF) was monitored by laser-Doppler flowmetry (LDF). NO concentrations and LDF were measured in normothermia and heat stress. After heat stress, a solution of ACh was perfused through the microdialysis probe to pharmacologically generate NO and verify the electrode's function. During whole body warming, both SkBF and NO concentrations began to increase at the same internal temperature. Both SkBF and NO concentrations increased during heat stress (402 +/- 76% change from LDF baseline, P < 0.05; 22 +/- 5% change from NO baseline, P < 0.05). During a second baseline condition after heat stress, ACh perfusion led to increases in both SkBF and NO concentrations (496 +/- 119% change from LDF baseline, P < 0.05; 16 +/- 10% change from NO baseline, P < 0.05). We conclude that NO does increase in skin during heat stress in humans, attendant to active vasodilation. This result suggests that NO has a role beyond that of a permissive factor in the process; rather, NO may well be an effector of cutaneous vasodilation during heat stress.     

Acetylcholine-induced vasodilation is mediated by nitric oxide and prostaglandins in human skin.

Acetylcholine (ACh) can effect vasodilation by several mechanisms, including activation of endothelial nitric oxide (NO) synthase and prostaglandin (PG) production. In human skin, exogenous ACh increases both skin blood flow (SkBF) and bioavailable NO levels, but the relative increase is much greater in SkBF than NO. This led us to speculate ACh may dilate cutaneous blood vessels through PGs, as well as NO. To test this hypothesis, we performed a study in 11 healthy people. We measured SkBF by laser-Doppler flowmetry (LDF) at four skin sites instrumented for intradermal microdialysis. One site was treated with ketorolac (Keto), a nonselective cyclooxygenase antagonist. A second site was treated with NG-nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase. A third site was treated with a combination of Keto and L-NAME. The fourth site was an untreated control site. After the three treated sites received the different inhibiting agents, ACh was administered to all four sites by intradermal microdialysis. Finally, sodium nitroprusside (SNP) was administered to all four sites. Mean arterial pressure (MAP) was monitored by Finapres, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF/MAP). For data analysis, CVC values for each site were normalized to their respective maxima as effected by SNP. The results showed that both Keto and L-NAME each attenuated the vasodilation induced by exogenous ACh (ACh control = 79 +/- 4% maximal CVC, Keto = 55 +/- 7% maximal CVC, L-NAME = 46 +/- 6% maximal CVC; P < 0.05, ACh vs. Keto or L-NAME). The combination of the two agents produced an even greater attenuation of ACh-induced vasodilation (31 +/- 5% maximal CVC; P < 0.05 vs. all other sites). We conclude that a portion of the vasodilation effected by exogenous ACh in skin is due to NO; however, a significant portion is also mediated by PGs.     

Decreased nitric oxide- and axon reflex-mediated cutaneous vasodilation with age during local heating.

Cutaneous vasodilation is reduced in healthy older vs. young subjects; however, the mechanisms that underlie these age-related changes are unclear. Our goal in the present study was to determine the role of nitric oxide (NO) and the axon reflexes in the skin blood flow (SkBF) response to local heating with advanced age. We placed two microdialysis fibers in the forearm skin of 10 young (Y; 22 +/- 2 yr) and 10 older (O; 77 +/- 5 yr) men and women. SkBF over each site was measured by laser-Doppler flowmetry (LDF; Moor DRT4). Both sites were heated to 42 degrees C for ~60 min while 10 mM N(G)-nitro-L-arginine methyl ester (L-NAME) was infused throughout the protocol to inhibit NO synthase (NOS) in one site and 10 mM L-NAME was infused after 40 min of local heating in the second site. Data were expressed as a percentage of maximal vasodilation (%CVC(max); 28 mM nitroprusside infusion). Local heating before L-NAME infusion resulted in a significantly reduced initial peak (Y: 61 +/- 2%CVC(max) vs. O: 46 +/- 4%CVC(max)) and plateau (Y: 93 +/- 2%CVC(max) vs. O: 82 +/- 5%CVC(max)) CVC values in older subjects (P < 0.05). When NOS was inhibited after 40 min of heating, CVC declined to the same value in the young and older groups. Thus the overall contribution of NO to the plateau phase of the SkBF response to local heating was less in the older subjects. The initial peak response was significantly lower in the older subjects in both microdialysis sites (Y: 52 +/- 4%CVC(max) vs. O: 38 +/- 5%CVCmax; P < 0.05). These data suggest that age-related changes in both axon reflex-mediated and NO-mediated vasodilation contribute to attenuated cutaneous vasodilator responses in the elderly.     

Bradykinin does not mediate cutaneous active vasodilation during heat stress in humans.

To test the hypothesis that bradykinin effects cutaneous active vasodilation during hyperthermia, we examined whether the increase in skin blood flow (SkBF) during heat stress was affected by blockade of bradykinin B(2) receptors with the receptor antagonist HOE-140. Two adjacent sites on the forearm were instrumented with intradermal microdialysis probes for local delivery of drugs in eight healthy subjects. HOE-140 was dissolved in Ringer solution (40 microM) and perfused at one site, whereas the second site was perfused with Ringer alone. SkBF was monitored by laser-Doppler flowmetry (LDF) at both sites. Mean arterial pressure (MAP) was monitored from a finger, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF/MAP). Water-perfused suits were used to control body temperature and evoke hyperthermia. After hyperthermia, both microdialysis sites were perfused with 28 mM nitroprusside to effect maximal vasodilation. During hyperthermia, CVC increased at HOE-140 (69 +/- 2% maximal CVC, P < 0.01) and untreated sites (65 +/- 2% maximal CVC, P < 0.01). These responses did not differ between sites (P > 0.05). Because the bradykinin B(2)-receptor antagonist HOE-140 did not alter SkBF responses to heat stress, we conclude that bradykinin does not mediate cutaneous active vasodilation.     

Endothelial nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo

Nitric oxide (NO) participates in locally mediated vasodilation induced by increased local skin temperature (T(loc)) and in sympathetically mediated vasodilation during whole body heat stress. We hypothesized that endothelial NOS (eNOS) participates in the former, but not the latter, response. We tested this hypothesis by examining the effects of the eNOS antagonist N(G)-amino-l-arginine (l-NAA) on skin blood flow (SkBF) responses to increased T(loc) and whole body heat stress. Microdialysis probes were inserted into forearm skin for drug delivery. One microdialysis site was perfused with l-NAA in Ringer solution and a second site with Ringer solution alone. SkBF [laser-Doppler flowmetry (LDF)] and blood pressure [mean arterial pressure (MAP)] were monitored, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF / MAP). In protocol 1, T(loc) was controlled with LDF/local heating units. T(loc) initially was held at 34 degrees C and then increased to 41.5 degrees C. In protocol 2, after a normothermic period, whole body heat stress was induced (water-perfused suits). At the end of both protocols, 58 mM sodium nitroprusside was perfused at both microdialysis sites to cause maximal vasodilation for data normalization. In protocol 1, CVC at 34 degrees C T(loc) did not differ between l-NAA-treated and untreated sites (P > 0.05). Local skin warming to 41.5 degrees C T(loc) increased CVC at both sites. This response was attenuated at l-NAA-treated sites (P < 0.05). In protocol 2, during normothermia, CVC did not differ between l-NAA-treated and untreated sites (P > 0.05). During heat stress, CVC rose to similar levels at l-NAA-treated and untreated sites (P > 0.05). We conclude that eNOS is predominantly responsible for NO generation in skin during responses to increased T(loc), but not during reflex responses to whole body heat stress.     

Cholinergic mechanisms of cutaneous active vasodilation during heat stress in cystic fibrosis.

To test the hypothesis that cutaneous active vasodilation in heat stress is mediated by a redundant cholinergic cotransmitter system, we examined the effects of atropine on skin blood flow (SkBF) increases during heat stress in persons with (CF) and without cystic fibrosis (non-CF). Vasoactive intestinal peptide (VIP) has been implicated as a mediator of cutaneous vasodilation in heat stress. VIP-containing cutaneous neurons are sparse in CF, yet SkBF increases during heat stress are normal. In CF, augmented ACh release or muscarinic receptor sensitivity could compensate for decreased VIP; if so, active vasodilation would be attenuated by atropine in CF relative to non-CF. Atropine was administered into skin by iontophoresis in seven CF and seven matched non-CF subjects. SkBF was monitored by laser-Doppler flowmetry (LDF) at atropine treated and untreated sites. Blood pressure [mean arterial pressure (MAP)] was monitored (Finapres), and cutaneous vascular conductance was calculated (CVC = LDF/MAP). The protocol began with a normothermic period followed by a 3-min cold stress and 30-45 min of heat stress. Finally, LDF sites were warmed to 42 degrees C to effect maximal vasodilation. CVC was normalized to its site-specific maximum. During heat stress, CVC increased in both CF and non-CF (P < 0.01). CVC increases were attenuated by atropine in both groups (P < 0.01); however, the responses did not differ between groups (P = 0.99). We conclude that in CF there is not greater dependence on redundant cholinergic mechanisms for cutaneous active vasodilation than in non-CF.     

Effect of local acetylcholinesterase inhibition on sweat rate in humans.

ACh is the neurotransmitter responsible for increasing sweat rate (SR) in humans. Because ACh is rapidly hydrolyzed by acetylcholinesterase (AChE), it is possible that AChE contributes to the modulation of SR. Thus the primary purpose of this project was to identify whether AChE around human sweat glands is capable of modulating SR during local application of various concentrations of ACh in vivo, as well as during a heat stress. In seven subjects, two microdialysis probes were placed in the intradermal space of the forearm. One probe was perfused with the AChE inhibitor neostigmine (10 microM); the adjacent membrane was perfused with the vehicle (Ringer solution). SR over both membranes was monitored via capacitance hygrometry during microdialysis administration of various concentrations of ACh (1 x 10(-7)-2 M) and during whole body heating. SR was significantly greater at the neostigmine-treated site than at the control site during administration of lower concentrations of ACh (1 x 10(-7)-1 x 10(-3) M, P < 0.05), but not during administration of higher concentrations of ACh (1 x 10(-2)-2 M, P > 0.05). Moreover, the core temperature threshold for the onset of sweating at the neostigmine-treated site was significantly reduced relative to that at the control site. However, no differences in SR were observed between sites after 35 min of whole body heating. These results suggest that AChE is capable of modulating SR when ACh concentrations are low to moderate (i.e., when sudomotor activity is low) but is less effective in governing SR after SR has increased substantially.     

Vasoconstriction during venous congestion: effects of venoarteriolar response, myogenic reflexes, and hemodynamics of changing perfusion pressure

We dissected the relative contribution of arteriovenous hemodynamics, the venoarteriolar response (VAR), and the myogenic reflex toward a decrease in local blood flow induced by venous congestion. Skin blood flow (SkBF) was measured in 12 supine subjects via laser-Doppler flowmetry 1) over areas of forearm and calf skin, in which the VAR was blocked by using eutectic mixture of local anesthetics (EMLA sites) and 2) over the contralateral forearm or calf skin (control sites), using two different techniques: limb dependency of 23-37 cm below the heart and cuff inflation to 40 mmHg. During limb dependency, SkBF decreased at the control sites, whereas it remained unchanged at the EMLA sites. In contrast, during cuff inflation, SkBF decreased at the control sites and also decreased at the EMLA sites. The percent change in SkBF from baseline was greater during cuff inflation than limb dependency at both the control sites and the EMLA sites. Estimated skin vascular resistance remained unchanged at the EMLA sites during cuff inflation, as well as limb dependency. Thus the decrease in SkBF during venous congestion with cuff inflation is not solely due to the cutaneous VAR but also to a reduction in local perfusion pressure. The VAR is therefore most specifically quantified by venous congestion induced by limb dependency, rather than cuff inflation. Finally, from both techniques, we calculated that during venous congestion induced by limb dependency (calf), approximately 45% of the nonbaroreflex vasoconstriction is induced by the VAR and approximately 55% by the myogenic reflex.     

Aerobic training and cutaneous vasodilation in young and older men.

To determine the effect and underlying mechanisms of exercise training and the influence of age on the skin blood flow (SkBF) response to exercise in a hot environment, 22 young (Y; 18-30 yr) and 21 older (O; 61-78 yr) men were assigned to 16 wk of aerobic (A; YA, n = 8; OA, n = 11), resistance (R; YR, n = 7; OR, n = 3), or no training (C; YC, n = 7; OC, n = 7). Before and after treatment, subjects exercised at 60% of maximum oxygen consumption (VO2 max) on a cycle ergometer for 60 min at 36 degrees C. Cutaneous vascular conductance, defined as SkBF divided by mean arterial pressure, was monitored at control (vasoconstriction intact) and bretylium-treated (vasoconstriction blocked) sites on the forearm using laser-Doppler flowmetry. Forearm vascular conductance was calculated as forearm blood flow (venous occlusion plethysmography) divided by mean arterial pressure. Esophageal and skin temperatures were recorded. Only aerobic training (functionally defined a priori as a 5% or greater increase in VO2 max) produced a decrease in the mean body temperature threshold for increasing forearm vascular conductance (36.89 +/- 0.08 to 36.63 +/- 0.08 degrees C, P < 0.003) and cutaneous vascular conductance (36.91 +/- 0.08 to 36.65 +/- 0.08 degrees C, P < 0.004). Similar thresholds between control and bretylium-treated sites indicated that the decrease was mediated through the active vasodilator system. This shift was more pronounced in the older men who presented greater training-induced increases in VO2 max than did the young men (22 and 9%, respectively). In summary, older men improved their SkBF response to exercise-heat stress through the effect of aerobic training on the cutaneous vasodilator system.     

Nitric oxide synthase inhibition does not alter the reactive hyperemic response in the cutaneous circulation.

Reactive hyperemia is the sudden rise in blood flow after release of an arterial occlusion. Currently, the mechanisms mediating this response in the cutaneous circulation are poorly understood. The purpose of this study was to 1). characterize the reactive hyperemic response in the cutaneous circulation and 2). determine the contribution of nitric oxide (NO) to reactive hyperemia. Using laser-Doppler flowmetry, we characterized reactive hyperemia after 3-, 5-, 10-, and 15-min arterial occlusions in 10 subjects. The total hyperemic response was calculated by taking the area under the curve (AUC) of the hyperemic response minus baseline skin blood flow (SkBF) [i.e., total hyperemic response = AUC - [baseline SkBF as %maximal cutaneous vascular conductance (CVC(max) x duration of hyperemic response in s]]. For the characterization protocol, the total hyperemic response significantly increased as the period of ischemia increased from 5 to 15 min (P < 0.05). However, the 3-min response was not significantly different from the 5-min response. In the NO contribution protocol, two microdialysis fibers were placed in the forearm skin of eight subjects. One site served as a control and was continuously perfused with Ringer solution. The second site was continuously perfused with 10 mM NG-nitro-l-arginine methyl ester (l-NAME) to inhibit NO synthase. CVC was calculated as flux/mean arterial pressure and normalized to maximal blood flow (28 mM sodium nitroprusside). The total hyperemic response in control sites was not significantly different from l-NAME sites after a 5-min occlusion (3261 +/- 890 vs. 2907 +/- 531% CVC(max. s). Similarly, total hyperemic responses in control sites were not different from l-NAME sites (9155 +/- 1121 vs. 9126 +/- 1088% CVC(max. s) after a 15-min arterial occlusion. These data suggest that NO does not directly mediate reactive hyperemia and that NO is not produced in response to an increase in shear stress in the cutaneous circulation.     

Acetylcholine released from cholinergic nerves contributes to cutaneous vasodilation during heat stress.

Nitric oxide (NO) contributes to active cutaneous vasodilation during a heat stress in humans. Given that acetylcholine is released from cholinergic nerves during whole body heating, coupled with evidence that acetylcholine causes vasodilation via NO mechanisms, it is possible that release of acetylcholine in the dermal space contributes to cutaneous vasodilation during a heat stress. To test this hypothesis, in seven subjects skin blood flow (SkBF) and sweat rate were simultaneously monitored over three microdialysis membranes placed in the dermal space of dorsal forearm skin. One membrane was perfused with the acetylcholinesterase inhibitor neostigmine (10 microM), the second membrane was perfused with the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME; 10 mM) dissolved in the aforementioned neostigmine solution (l-NAME(Neo)), and the third membrane was perfused with Ringer solution as a control site. Each subject was exposed to approximately 20 min of whole body heating via a water-perfused suit, which increased mean body temperature from 36.4 +/- 0.1 to 37.5 +/- 0.1 degrees C (P < 0.05). After the heat stress, SkBF at each site was normalized to its maximum value, identified by administration of 28 mM sodium nitroprusside. Mean body temperature threshold for cutaneous vasodilation was significantly lower at the neostigmine-treated site relative to the other sites (neostigmine: 36.6 +/- 0.1 degrees C, l-NAME(Neo): 37.1 +/- 0.1 degrees C, control: 36.9 +/- 0.1 degrees C), whereas no significant threshold difference was observed between the l-NAME(Neo)-treated and control sites. At the end of the heat stress, SkBF was not different between the neostigmine-treated and control sites, whereas SkBF at the l-NAME(Neo)-treated site was significantly lower than the other sites. These results suggest that acetylcholine released from cholinergic nerves is capable of modulating cutaneous vasodilation via NO synthase mechanisms early in the heat stress but not after substantial cutaneous vasodilation.     

Sex differences in salt sensitivity to nitric oxide dependent vasodilation in healthy young adults

Dietary sodium and blood pressure regulation differs between normotensive men and women, an effect which may involve endothelial production of nitric oxide (NO). Therefore, we tested the hypothesis that differences in the NO component of endothelium-dependent vasodilation between low and high dietary sodium intake depend on sex. For 5 days prior to study, healthy adults consumed a controlled low-sodium diet (10 mmol/day, n = 30, mean age ± SE: 30 ± 1 yr, 16 men) or high-sodium diet (400 mmol/day, n = 36, age 23 ± 1 yr, 13 men). Forearm blood flow (FBF, plethysmography) responses to brachial artery administration of acetylcholine (ACh, 4 μg·100 ml tissue(-1)·min(-1)) were measured before and after endothelial NO synthase inhibition with N(G)-monomethyl-l-arginine (l-NMMA, 50 mg bolus + 1 mg/min infusion). The NO component of endothelium-dependent dilation was calculated as the response to ACh before and after l-NMMA accounting for changes in baseline FBF: [(FBF ACh - FBF baseline) - (FBF ACh(L-NMMA) - FBF baseline(L-NMMA))]. This value was 5.7 ± 1.3 and 2.5 ± 0.8 ml·100 ml forearm tissue(-1)·min(-1) for the low- and high-sodium diets, respectively (main effect of sodium, P = 0.019). The sodium effect was larger for the men, with values of 7.9 ± 2.0 and 2.2 ± 1.4 for men vs. 3.1 ± 1.3 and 2.7 ± 1.0 ml·100 ml forearm tissue(-1)·min(-1) for the women (P = 0.034, sex-by-sodium interaction). We conclude that the NO component of endothelium-dependent vasodilation is altered by dietary sodium intake based on sex, suggesting that endothelial NO production is sensitive to dietary sodium in healthy young men but not women.     

Aging and aerobic fitness affect the contribution of noradrenergic sympathetic nerves to the rapid cutaneous vasodilator response to local heating

Sedentary aging results in a diminished rapid cutaneous vasodilator response to local heating. We investigated whether this diminished response was due to altered contributions of noradrenergic sympathetic nerves by assessing 1) the age-related decline and 2) the effect of aerobic fitness. Using laser-Doppler flowmetry, we measured skin blood flow (SkBF) in young (24 ± 1 yr) and older (64 ± 1 yr) endurance-trained and sedentary men (n = 7 per group) at baseline and during 35 min of local skin heating to 42°C at 1) untreated forearm sites, 2) forearm sites treated with bretylium tosylate (BT), which prevents neurotransmitter release from noradrenergic sympathetic nerves, and 3) forearm sites treated with yohimbine + propranolol (YP), which antagonizes α- and β-adrenergic receptors. SkBF was converted to cutaneous vascular conductance (CVC = SkBF/mean arterial pressure) and normalized to maximal CVC (%CVC(max)) achieved by skin heating to 44°C. Pharmacological agents were administered using microdialysis. In the young trained group, the rapid vasodilator response was reduced at BT and YP sites (P < 0.05); by contrast, in the young sedentary and older trained groups, YP had no effect (P > 0.05), but BT did (P > 0.05). Neither BT nor YP affected the rapid vasodilator response in the older sedentary group (P > 0.05). These data suggest that the age-related reduction in the rapid vasodilator response is due to an impairment of sympathetic-dependent mechanisms, which can be partly attenuated with habitual aerobic exercise. Rapid vasodilation involves noradrenergic neurotransmitters in young trained men and nonadrenergic sympathetic cotransmitters (e.g., neuropeptide Y) in young sedentary and older trained men, possibly as a compensatory mechanism. Finally, in older sedentary men, the rapid vasodilation appears not to involve the sympathetic system.     

Oral vitamin C enhances the adrenergic vasoconstrictor response to local cooling in human skin

Local administration of ascorbic acid (Asc) at a supraphysiological concentration inhibits the cutaneous vasoconstrictor response to local cooling (LC). However, whether orally ingesting Asc inhibits the LC-induced vasoconstrictor response remains unknown. The purpose of the present study was to examine the acute influence of oral Asc on the adrenergic vasoconstrictor response to LC in human skin. In experiment 1, skin blood flow (SkBF) was measured by laser-Doppler flowmetry at three sites (forearm, calf, palm). The three skin sites were locally cooled from 34 to 24°C at -1°C/min and maintained at 24°C for 20 min before (Pre) and 1.5 h after (Post) oral Asc (2-g single dose) or placebo supplementation. Cutaneous vascular conductance (CVC) was calculated as the ratio of SkBF to blood pressure and expressed relative to the baseline value before LC. Oral Asc enhanced (P < 0.05) the reductions in CVC in the forearm (Pre, -50.3 ± 3.3%; Post, -57.8 ± 2.2%), calf (Pre, -52.6 ± 3.7%; Post, -66.1 ± 4.3%), and palm (Pre, -46.2 ± 6.2%; Post, -60.4 ± 5.6%) during LC. The placebo did not change the responses at any site. In experiment 2, to examine whether the increased vasoconstrictor response caused by oral Asc is due to the adrenergic system, the release of neurotransmitters from adrenergic nerves in forearm skin was blocked locally by iontophoresis of bretylium tosylate (BT). Oral Asc enhanced (P < 0.05) the reductions in CVC at untreated control sites but did not change the responses at BT-treated sites during LC. In experiment 3, to further examine whether adrenergically mediated vasoconstriction is enhanced by oral Asc, 0.1 mM tyramine was administered using intradermal microdialysis in the forearm skin at 34°C in the Pre and Post periods. Oral Asc increased (P < 0.05) the tyramine-induced reduction in CVC. These findings suggest that oral Asc acutely enhances the cutaneous vasoconstrictor responses to LC through the modification of adrenergic sympathetic mechanisms.     

Effect of high local temperature on reflex cutaneous vasodilation

We examined the effect of high local forearm skin temperature (Tloc) on reflex cutaneous vasodilator responses to elevated whole-body skin (Tsk) and internal temperatures. One forearm was locally warmed to 42 degrees C while the other was left at ambient conditions to determine if a high Tloc could attenuate or abolish reflex vasodilation. Forearm blood flow (FBF) was monitored in both arms, increases being indicative of increases in skin blood flow (SkBF). In one protocol, Tsk was raised to 39-40 degrees C 30 min after Tloc in one arm had been raised to 42 degrees C. In a second protocol, Tsk and Tloc were elevated simultaneously. In protocol 1, the locally warmed arm showed little or no change in blood flow in response to increasing Tsk and esophageal temperature (average rise = 0.76 +/- 1.18 ml X 100 ml-1 X min-1), whereas FBF in the normothermic arm rose by an average of 8.84 +/- 3.85 ml X 100 ml-1 X min-1. In protocol 2, FBF in the normothermic arm converged with that in the warmed arm in three of four cases but did not surpass it. We conclude that local warming to 42 degrees C for 35-55 min prevents reflex forearm cutaneous vasodilator responses to whole-body heat stress. The data strongly suggest that this attenuation is via reduction or abolition of basal tone in the cutaneous arteriolar smooth muscle and that at a Tloc of 42 degrees C a maximum forearm SkBF has been achieved. Implicit in this conclusion is that local warming has been applied for a duration sufficient to achieve a plateau in FBF.     

Improved method for the routine analysis of acetylcholine release in vivo: quantitation in the presence and absence of esterase inhibitor

An improved high-performance liquid chromatographic (HPLC) method using electrochemical detection (ED) is described capable of routinely measuring the low levels of acetylcholine (ACh) typically found in rat brain microdialysis samples. Microdialysis was performed in the striatum of the urethane anesthetized rat using a 4-mm membrane length, high recovery (40% at 1.0 μl/min; ambient conditions), loop-design probe perfused with an artificial cerebrospinal fluid (aCSF) solution containing physiologically normal calcium levels (1.2 mM). The HPLC method utilizes a polymeric stationary phase to resolve choline (Ch) from ACh. These analytes are then converted to hydrogen peroxide (H₂O₂) by a solid-phase reactor (containing immobilized choline oxidase and acetylcholinesterase enzymes). The H₂O₂ is detected amperometrically and quantitated on a platinum (Pt) working electrode (+300 mV; with a unique analytical cell featuring a solid-state palladium reference electrode). Two designs of the Pt working electrode were examined, differing only in the support material used (Kel-F or PEEK). The Kel-F/Pt electrode had a limit of detection (LOD) for both analytes of <30 fmol per 10 μl with a signal-to-noise ratio of 3:1. Striatal microdialysis perfusates were monitored for ACh and Ch over a 0–1000 nM range of neostigmine (NEO) in the CSF perfusion medium. Using the 4-mm probe, basal ACh and Ch levels were detected with a NEO level as low as 10 nM and were found to be 37 ± 3 fmol and 22 ± 1 pmol per 10 μl (mean ± S.E.M., n = 6 replicates) respectively. In similar experiments using 3-mm concentric probes comparable (lower) levels of ACh were found with the 50 and 1000 nM NEO doses (n = 4–21 animals). ACh could not be reliably quantitated when animals were perfused with the 10 nM dose of NEO (n = 4). The PEEK/Pt electrode had an improved LOD of < 20 fmol per 10 μl due to a two- to three-fold decrease in the background noise component. Basal striatal levels of ACh in the absence of NEO approached the LOD and were found to be 15 ± 2 fmol per 10 μl; Ch was 5 ± 1 pmol per 10 μl (n = 2, mean of five basal samples). The analytical system requires very little maintenance; a simple electrochemical electrode cleaning step eliminates the need for routine polishing of the Pt electrode and the mobile phase is stable for up to one week. Both ACh and Ch are resolved in under 7 min making this method highly suitable for analysis of microdialysis samples.     

Nitric oxide and neurally mediated regulation of skin blood flow during local heating.

The mechanisms underlying the skin blood flow (SkBF) response to local heating are complex and poorly understood. Our goal was to examine the role of axon reflexes and nitric oxide (NO) in the SkBF response to a local heating protocol. We performed 40 experiments following a standardized heating protocol with different interventions, including blockade of the axon reflex (EMLA cream), antebrachial nerve blockade (0.5% bupivacaine injection), and NO synthase (NOS) inhibition (> or =10 mM N(G)-nitro-L-arginine methyl ester; microdialysis). Appropriate controls were performed to verify the efficacy of the various blocks. Values are expressed as a percentage of maximal SkBF (SkBF(max); 50 mM sodium nitroprusside). At the initiation of local heating, SkBF rose to an initial peak, followed by a brief nadir, and a secondary, progressive rise to a plateau. Axon reflex block decreased the initial peak from 75+3 to 32 +/- 2% SkBF(max) (P < 0.01 vs. control) but did not affect the plateau. NOS inhibition before and throughout local heating reduced the initial peak from 75 +/- 3 to 56 +/- 3% SkBF(max) (P < 0.01) and the plateau from 87 +/- 4 to 40 +/- 5%. NOS inhibition during axon reflex block did not further reduce the initial SkBF peak compared with axon reflex block alone. Antebrachial nerve block did not affect the local heating SkBF response. The primary finding of these studies is that there are at least two independent mechanisms contributing to the rise in SkBF during nonpainful local heating: a fast-responding vasodilator system mediated by the axon reflexes and a more slowly responding vasodilator system that relies on local production of NO.     

Interactions between local and reflex influences on human forearm skin blood flow.

A three-part experiment was designed to examine interactions between local and reflex influences on forearm skin blood flow (SkBF). In part I locally increasing arm skin temperature (Tsk) to 42.5 degrees C was not associated with increases in underlying forearm muscle blood flow, esophageal temperature (Tes), or forearm blood flow in the contralateral cool arm. In part II whole-body Tsk was held at 38 or 40 degrees C and the surface temperature of one arm held at 38 or 42 degrees C for prolonged periods. SkBF in the heated arm rose rapidly with the elevation in body Tsk and arm Tsk continued to rise as Tes rose. SkBF in the arm kept at 32 degrees C paralleled rising Tes. In six studies, SkBF in the cool arm ultimately converged with SkBF in the heated arm. In eight other studies, heated arm SkBF maintained an offset above cool arm SkBF throughout the period of whole-body heating. In part III, local arm Tsk of 42.5 degrees C did not abolish skin vasoconstrictor response to lower body negative pressure. We conclude that local and reflex influences to skin interact so as to modify the degree but not the pattern of skin vasomotor response.     

Role of nitric oxide in the vascular effects of local warming of the skin in humans.

Local warming of skin induces vasodilation by unknown mechanisms. To test whether nitric oxide (NO) is involved, we examined effects of NO synthase (NOS) inhibition with NG-nitro-L-arginine methyl ester (L-NAME) on vasodilation induced by local warming of skin in six subjects. Two adjacent sites on the forearm were instrumented with intradermal microdialysis probes for delivery of L-NAME and sodium nitroprusside. Skin blood flow was monitored by laser-Doppler flowmetry (LDF) at microdialysis sites. Local temperature (Tloc) of the skin at both sites was controlled with special LDF probe holders. Mean arterial pressure (MAP; Finapres) was measured and cutaneous vascular conductance calculated (CVC = LDF/MAP = mV/mmHg). Data collection began with a control period (Tloc at both sites = 34 degrees C). One site was then warmed to 41 degrees C while the second was maintained at 34 degrees C. Local warming increased CVC from 1.44 +/- 0.41 to 4.28 +/- 0.60 mV/mmHg (P < 0.05). Subsequent L-NAME administration reduced CVC to 2.28 +/- 0.47 mV/mmHg (P < 0.05 vs. heating), despite the continued elevation of Tloc. At a Tloc of 34 degrees C, L-NAME reduced CVC from 1.17 +/- 0.23 to 0.75 +/- 0.11 mV/mmHg (P < 0.05). Administration of sodium nitroprusside increased CVC to levels no different from those induced by local warming. Thus NOS inhibition attenuated, and sodium nitroprusside restored, the cutaneous vasodilation induced by elevation of Tloc; therefore, the mechanism of cutaneous vasodilation by local warming requires NOS generation of NO.     

Glutamate measured by 6-s resolution brain microdialysis: capillary electrophoretic and laser-induced fluorescence detection application

In the present experiment the combination of brain microdialysis and CZE–LIFD permitted the measurement of glutamate in 100 nl microdialysis samples collected every 5 or 6 s. Samples were collected every 6 s, in rats anesthetized with two different anesthetic agents (ketamine and sodium thiopental). A microdialysis probe was inserted in the cortex of an anesthetized rat in the territory irrigated by the middle cerebral artery. The artery was clamped for 30 s and then released. The samples were derivatized with fluorescein isothiocyanate I (FITC) by means of a continuous-flow reactor, collected and injected into a home-made CZE–LIFD instrument. Glutamate decreased immediately after clamping the artery in ketamine anesthetized rats and increased 1 min after the onset of the ischemia in sodium thiopental anesthetized rats. In another experiment a 60 mM KCl solution was injected through a microdialysis probe inserted in the hippocampus of an anesthetized rat. In the first 5 s after the KCl solution reached the tissue, glutamate increased but γ-aminobutytic acid and glutamine did not. The experiments show that time resolution of brain microdialysis can be reduced to a few seconds if the analytical technique is the proper one.