INTRODUCTION

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TWO BRANCHES of the sympathetic nervous system work in concert to regulate skin blood flow: an adrenergic vasoconstrictor system and an active vasodilator system (19). As core body temperature begins to rise, skin blood flow increases initially by the release of tonic adrenergic vasoconstrictor tone. With further increases in core temperature, a threshold is reached, whereupon sweating and cutaneous active vasodilation (AVD) occur (19). Current evidence suggests that AVD is mediated by the corelease of a neurotransmitter(s) with acetylcholine from cholinergic nerves in the skin (8). However, the precise mechanism of action, and even the neurotransmitter(s) itself, are unknown.

With advanced age there is a reduction in the ability to raise skin blood flow during heat stress (9, 10, 13, 17, 21). This may contribute to increased rates of heat-related illness and death in people over the age of 65 (14, 22). Evidence from Kenney (9) and colleagues has shown that the attenuated cutaneous reflex vasodilation is due to either decreased sensitivity of the AVD system or decreased skin vascular responsiveness to a given level of stimulation.

In young healthy subjects, ~30% of AVD is mediated by nitric oxide (NO)-dependent mechanisms (6, 23). There is evidence to suggest that NO may be diminished with advanced age, including data showing a reduction in the NO precursor L-arginine and the NO metabolites nitrate and nitrite (18). Our laboratory recently demonstrated (16) that NO-dependent vasodilation is reduced in the skin of older subjects during local heating. However, the vasodilatory mechanisms invoked by local heating are distinct from those arising reflexively from an increased core temperature. Therefore, the goal of the present study was to examine the contribution of NO to reflex cutaneous vasodilation during passive whole body heating in older subjects. We hypothesized that NO-dependent vasodilation would be diminished in older subjects and that this reduction would contribute to the attenuated rise in skin blood flow in older subjects during hyperthermia.

	METHODS

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Subjects. Studies were performed on seven older (71 ± 6 yr) and seven young (23 ± 2 yr) men. Experimental protocols were approved by the Institutional Review Boards at the University of Oregon and Sacred Heart Medical Center. Verbal and written consent was voluntarily obtained from all subjects before their participation. Each subject was interviewed for a history of cardiovascular disease, including hypercholesterolemia and hypertension. All subjects were normally active, healthy nonsmokers who were not currently taking medications that had the potential to impact the cardiovascular or thermoregulatory variables of interest. One older subject was taking 81 mg of aspirin, and one older subject was taking Pamelor. The subject taking aspirin abstained from this medication for 72 h before participation in the study. These data were included in the older subject group data because there was no difference between their individual data and the group data. Older subjects underwent a physician-supervised maximal graded exercise test at least 1 wk before participating in the study to ensure that they did not have any underlying cardiovascular disease. Subject characteristics are summarized in Table 1.

Instrumentation. On arrival in the laboratory, subjects were instrumented with two intradermal microdialysis fibers (MD 2000, Bioanalytical Systems) with a 10-mm, 20-kDa cutoff membrane on the ventral side of the nondominant forearm. Placement of the microdialysis probes was accomplished at each site by first inserting a 25-gauge needle through the skin. The entry and exit points were ~2.5 cm apart. The microdialysis probe was then threaded through the lumen of the needle. The needle was withdrawn, leaving the probe in place. The microdialysis fibers were taped in place, and lactated Ringer solution was perfused though the fibers at a rate of 2 µl/min.

Measurements. To obtain an index of skin blood flow, cutaneous red blood cell (RBC) flux was measured with an integrated laser-Doppler flowmeter probe (Moor DRT4) placed over each microdialysis site. Cutaneous vascular conductance was calculated as RBC flux divided by mean arterial pressure (CVC = RBC flux/MAP). Blood pressure was obtained by using a beat-to-beat noninvasive measuring device (Ohmeda Portapress) and verified by auscultation. Electrocardiogram and respiration were continuously monitored and recorded throughout the protocol (Cardiocap Datex-Ohmeda). An index of core body temperature (T_or) was measured continuously with a thermistor placed in the sublingual sulcus. The subjects were instructed to keep the thermistor in the same location in the sublingual sulcus and not to open their mouth or speak during the protocol. Mean skin temperature (_sk) was calculated as the unweighted average of five copper-constantan thermocouples placed on the chest, middle back, abdomen, thigh, and calf.

Protocol. After placement of the microdialysis fibers, RBC flux over each microdialysis site was monitored to ensure that the initial hyperemia caused by the insertion trauma had resolved before the study started. One microdialysis site was randomly assigned to receive 10 mM N^G-nitro-L-arginine methyl ester (L-NAME; Calbiochem, San Diego, CA) dissolved in lactated Ringer solution to inhibit NO production by nitric oxide synthase (NOS), and the other site received only lactated Ringer solution. These infusions were maintained throughout the baseline and heating periods. Our laboratory previously showed (16) that this dose of L-NAME is sufficient to maximally inhibit NO production in both subject groups. The microdialysis fibers were perfused at a rate of 2.0 µl/min for at least 30 min to ensure adequate NOS inhibition before starting data collection. A 10-min baseline period of measuring RBC flux by laser-Doppler flowmetry was obtained. After baseline measurements, 50°C water was circulated through the water-perfused suit to raise T_or. All subjects were heated until T_or had increased by 1.0°C. The subjects were then cooled to their baseline T_or by circulating 32°C water though the water-perfused suit. After cooling, the NO donor sodium nitroprusside (SNP, 28 mM; Nitropres, Ciba Pharmaceuticals) was infused through both microdialysis fibers for 30 min at a rate of 4.0 µl/min to obtain maximal CVC at both sites. We previously determined (16) that 28 mM SNP maximally vasodilates the skin of both groups of subjects.

Data acquisition and analysis. Data were digitized and stored on a computer at 150 Hz. Data were analyzed off-line with signal processing software (Windaq; Dataq Instruments, Akron, OH). CVC values were determined by averaging values over a stable 2-min period for a given rise in T_or. In general, the change in T_or from baseline (T_or) was used to compare the groups and for graphic display. However, we also analyzed the absolute T_or thresholds for reflex vasodilation. A reviewer blinded to the age of the subjects visually identified the absolute T_or at which the threshold for reflex cutaneous vasodilation was initiated in both microdialysis sites. All data are presented as a percentage of maximal CVC (%CVC_max). The relative percent NO contribution to CVC for a given rise in T_or was calculated as [(control CVC  NOS-inhibited CVC)/control CVC] × 100.

Statistical analyses. Student's t-tests were used to determine significant differences between the young and older groups for physical characteristics, baseline T_or, baseline _sk, absolute T_or at the threshold for reflex vasodilation, and relative percent NO contribution. Two-way repeated-measures analysis of variance (age × T_or) was performed for CVC on the control site (Ringer infusion) and the experimental site (L-NAME infusion) from baseline throughout heating. Tukey's post hoc analyses were performed when significance was achieved. The level of significance was set at P < 0.05. Values are means ± SE.

	RESULTS

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The physical characteristics of the subjects are presented in Table 1. Older (O) and younger (Y) men differed in age by ~50 yr. The older men had a significantly higher body mass index than the younger men (O: 26.1 ± 0.7, Y: 23.1 ± 0.6 kg/m²; P < 0.05). There was no significant statistical difference in resting MAP between the groups, although the older subjects tended to have higher resting MAP.

The older subjects began the protocol at a significantly lower T_or (O: 36.2 ± 0.1, Y: 36.5 ± 0.1°C) and lower _sk (O: 33.8 ± 0.3, Y: 35.3 ± 0.9°C) (both P < 0.05). A 1.0°C increase in T_or from baseline was achieved during heating in all subjects. Representative tracings of a young subject and an older subject at the control site and the NOS-inhibited site are presented in Fig. 1, A and B, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   A: representative tracing of the cutaneous vascular conductance (CVC) response during whole body heating at the control site (Ringer infusion) and the nitric oxide (NO) synthase (NOS)-inhibited site [NG-nitro-L-arginine methyl ester (L-NAME) infusion] in a young subject. B: representative tracing of the CVC response during whole body heating at the control site (Ringer infusion) and the NOS-inhibited site (L-NAME infusion) in an older subject.

Group mean data for the control and the NOS-inhibited sites for the subject groups are depicted in Fig. 2, A (young) and B (older). In the young subjects, CVC at the control site was significantly elevated from baseline after T_or had risen 0.3°C (P < 0.05). However, CVC in the young subjects at the NOS-inhibited site did not increase significantly from baseline until T_or had risen 0.6°C (P < 0.05; Fig. 2A). The threshold for onset of reflex cutaneous vasodilation in the young subjects was 36.5 ± 0.2°C in the control site and 36.7 ± 0.1°C in the NOS inhibited site (P = 0.45). In the older subjects, CVC at the control site did not significantly differ from baseline until T_or had risen 0.6°C (P < 0.05) whereas at the NOS-inhibited site CVC did not become significantly elevated above baseline until T_or had risen 0.9°C (P < 0.05) (Fig. 2B). The threshold for onset of reflex cutaneous vasodilation in the older subjects was 36.6 ± 0.1°C and 36.8 ± 0.1°C for the control site and the NOS-inhibited site, respectively (P = 0.21). There was no difference between the groups for absolute temperature thresholds for the onset of reflex cutaneous vasodilation in either site (P = 0.67 control site, P = 0.44 NOS-inhibited site).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A: mean ± SE subject responses for young men (18-26 yr) during passive whole body heating in the control (Ringer infusion only) and NOS-inhibited (L-NAME infusion) microdialysis sites. B: mean ± SE subject responses for older men (65-81 yr) during passive whole body heating in the control (Ringer infusion only) and NOS-inhibited (L-NAME infusion) microdialysis sites. * CVC significantly increased from baseline values (P < 0.05).

Group mean data for the control site of both groups are shown in Fig. 3A. Baseline CVC at the control site was similar between groups. There were significant main effects in CVC for age (P = 0.001) and for the change in T_or at the control site (P < 0.001). Post hoc analyses on the interaction effect revealed that significant differences in CVC across age and T_or occurred with a T_or rise from 0.3°C and 0.9°C above baseline. CVC at the control site rose to 75 ± 6%CVC_max in the young subjects and 64 ± 5%CVC_max in the older subjects with a 1.0°C rise in T_or.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   A: mean ± SE subject responses by age group for young (18-26 yr) and older (65-81 yr) men during passive whole body heating in the control microdialysis site (Ringer infusion only). B: mean ± SE subject responses by age group for young (18-26 yr) and older (65-81 yr) men during passive whole body heating in the NOS-inhibited microdialysis site (L-NAME infusion throughout). * Significant difference between young and older subject groups.

Group mean data for the NOS-inhibited site for both groups are presented in Fig. 3B. There was no difference in CVC between the groups at baseline in the NOS-inhibited site. There was a significant main effect in CVC for age (P < 0.001) and for the change in T_or at the NOS-inhibited site (P < 0.001). Post hoc analyses on the interaction effect revealed that differences in CVC across age and T_or occurred after T_or had risen 0.5°C above baseline. When T_or had risen 1.0°C, the younger subjects' CVC was 53 ± 3%CVC_max and the older subjects' CVC was 29 ± 2%CVC_max (P < 0.001).

The relative percent NO contribution to CVC in the control site for a given rise in T_or above baseline is presented in Fig. 4. With a 0.3°C rise in T_or, younger subjects had significantly greater relative percent NO contribution to CVC compared with older subjects (O: 20 ± 8%, Y: 40 ± 6%; P < 0.05). There was no significant difference in the relative percent NO contribution to CVC between the older and young subjects with a 0.5°C rise in T_or. In terms of percent maximal vasodilation, NOS inhibition decreased CVC by 35%CVC_max in the older subjects and by 22%CVC_max in the young subjects. The relative percent NO contribution to CVC was greater in the older subjects with a 1.0°C rise in core temperature (O: 60 ± 3%, Y: 23 ± 4%; P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Mean ± SE relative % contribution of NO to CVC {[(control CVC  NOS-inhibited CVC)/control CVC] × 100} for a given rise in core temperature (Tor). Young subjects showed a significantly greater relative % contribution of NO to CVC with a 0.3°C rise in Tor, with a 0.5°C rise in Tor there was no difference between the age groups, and when Tor was increased 1.0°C older individuals had a greater NO contribution to skin blood flow. * Significant difference between young and older subject groups.

	DISCUSSION

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It has been well established that older individuals have an attenuated rise in skin blood flow during whole body heating (9, 10, 17, 21). The major finding of the present study is that NO-mediated pathways contributed more to the total vasodilatory response of the older men at high T_or. In fact, there was very little cutaneous vasodilation in the absence of functional NO in older subjects. Therefore, the attenuated reflex vasodilation in older subjects is primarily due to diminished sympathetic mediated vasodilation by an unknown active vasodilator(s).

These findings were unexpected in light of previous studies suggesting that advanced age impacts NO-mediated vasodilation (16, 18). Reckelhoff and colleagues (18) reported that there is an age-related reduction in L-arginine, the NO precursor, and its metabolites nitrate and nitrite. Furthermore, our laboratory recently demonstrated (15, 16) an attenuated NO-dependent vasodilation during local heating of the skin in older subjects. In light of the present data, a better explanation for the age-related decline in reflex cutaneous vasodilation directly involves the unidentified neurotransmitter(s) and/or the cutaneous vascular response to this activated sympathetic pathway.

There are a number of mechanisms by which the relationship between the neural thermoregulatory stimulus and AVD may be altered with aging. First, attenuated reflex cutaneous vasodilation in the older subjects might be explained by an age-related reduction in efferent cutaneous neural outflow for a given rise in T_or. This would suggest a centrally mediated alteration in AVD with advanced age. Second, there may be less neurotransmitter released for a given neural outflow. If this were true, it would take a greater stimulus (i.e., a greater increase in T_or) for the onset of reflex cutaneous vasodilation. Finally, the efferent signal may not be altered but vascular sensitivity to the unknown neurotransmitter may be decreased, such as a reduction or desensitization of the receptors or secondary messengers mediating AVD. Although these possibilities could explain attenuated reflex cutaneous vasodilation in older subjects, we are unable to distinguish between these possibilities with the present data and our limited understanding of the cutaneous active vasodilator system.

One problem in interpreting age-related changes in reflex cutaneous vasodilation stems from the fact that the role of NO in this response is poorly understood. Studies in the rabbit ear suggest that NO may play a permissive role with the unknown neurotransmitter(s) to mediate AVD (4, 5). That is, there is a convergence in the molecular pathways between NO and the unknown neurotransmitter(s) such that neurotransmitter-mediated vasodilation is dependent on the presence of NO. In this model, NO maintains basal levels of cGMP and the unknown neurotransmitter(s) mediate vasodilation via a cAMP pathway that is dependent on phosphodiesterase inhibition by cGMP (5). A study by Crandall and McLean (3) supports this role for NO in humans, because they were not able to measure an increase in metabolites of NO during whole body heating. In this context, older subjects may have a diminished ability to produce or respond to NO but may be able to maintain a sufficient basal level of NO during whole body heating. However, we (26) and others (7) have preliminary data to argue against a purely permissive role for NO in AVD.

An alternative to the foregoing hypotheses regarding the role of NO during reflex vasodilation is that NO may be released via flow-mediated mechanisms. In this construct, the unknown active vasodilator substance(s) increase flow to a sufficient degree to stimulate NO release, possibly through an increase in shear stress. Consistent with this hypothesis, the contribution of NO to reflex cutaneous vasodilation was greater with a smaller rise in T_or in the young subjects, whereas the contribution of NO to reflex cutaneous vasodilation was greater in the older subjects once the unknown vasodilator(s) had increased skin blood flow significantly above baseline (Figs. 3A and 4). That is, in older subjects it takes a greater rise in T_or for reflex cutaneous vasodilation to develop, so little NO-dependent vasodilation is seen with small increases in T_or (T_or 0.0-0.5°C). However, once CVC is significantly elevated by the unknown vasodilator(s), the contribution of NO is critical for full expression of reflex cutaneous vasodilation. It is important to note that although the percent contribution of NO to reflex cutaneous vasodilation is greater in older subjects with large increases in T_or, the overall skin blood flow at these elevated T_or values is significantly attenuated in older subjects (10, 16, 20). That is, NO contributes more to a lesser overall vasodilation.

Shibasaki and colleagues (25) recently reported that the initial rise in skin blood flow during reflex cutaneous vasodilation, but not the sustained rise in skin blood flow, is mediated by acetylcholine-stimulated NO production. This finding is consistent with other studies showing that the initial rise in skin blood flow during reflex vasodilation is diminished with atropine (12), but atropine has little effect once vasodilation has been established in hyperthermia (24). Diminished acetylcholine-mediated NO release early in heating with aging may partially explain the rightward shift to a greater rise in T_or for the onset of reflex cutaneous vasodilation in older subjects, as observed by Kenney et al. (10) and shown in Fig. 3A. Consistent with this hypothesis, the rise in skin blood flow during acetylcholine iontophoresis is diminished in older subjects (1). However, it is unknown whether the NO-mediated portion of dilation to acetylcholine iontophoresis is attenuated with advanced age.

The rightward shift in reflex vasodilation with age brings to light questions regarding baseline T_or and threshold for thermoregulatory reflexes. In our study, the older subjects' baseline T_or was lower than the young subjects', but absolute T_or at threshold for vasodilation was similar between groups. Kenney and colleagues (10, 11) also showed that the thresholds for reflex vasodilation do not change with age. This is different from what is observed during classic "resetting" of thermoregulatory responses, such as that observed with oral contraceptive use. During the course of oral contraceptive use, baseline T_or is higher during the active pill phase than during the placebo pill phase. However, the threshold for vasodilation is likewise shifted to a higher temperature during active pill phase (2). This suggests that a central shift in overall thermoregulatory responses occurs along with a shift in baseline T_or during oral contraceptive use. In contrast, our data suggest that thermoregulatory reflexes are not shifted with age but advanced age results in only a lower baseline T_or. This baseline temperature shift without concomitant shifts in reflex vasodilation thresholds may be due to nonthermoregulatory influences on reflex vasodilation thresholds such as changes in plasma volume and blood volume with advanced age (10, 11). More studies are needed to determine whether "classic" resetting occurs with advanced age but the shifts in reflex vasodilation are blunted because of nonthermoregulatory reflexes.

In summary, we found that the attenuated reflex cutaneous vasodilation in older subjects is likely due to diminished release of, or vascular responsiveness to, the unknown vasodilator(s) mediating active vasodilation. Furthermore, our data suggest that NO is critical for the increase in skin blood flow in older subjects during heat stress. More research is needed to understand the mechanisms of AVD in humans and how they may be altered with advanced age.