INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

WHOLE BODY HEATING IN HUMANS causes a substantial increase in skin blood flow (SkBF) in nonglabrous skin initially because of withdrawal of sympathetic vasoconstrictor activity followed by increases in sympathetic active vasodilator activity (7, 10, 12, 17, 19, 20). The active vasodilator component mediates the majority of the reflex thermoregulatory increase in SkBF (12). However, the mechanism(s), including the neurotransmitter, responsible for elevating SkBF during a heat stress remains unknown.

Previously, we (15) identified that this neurotransmitter was coreleased from cholinergic nerves, because cutaneous active vasodilation was abolished when cholinergic neurotransmission was blocked via local administration of botulinum toxin, whereas blockade of muscarinic receptors, via local administration of atropine, only partially inhibited cutaneous vasodilation associated with a heat stress. Recently, we (13) and others (21, 22) reported that nitric oxide (NO) was required for the full manifestation of cutaneous active vasodilation during a heat stress. This finding was revealed after local inhibition of NO synthase (NOS) during a heat stress, either via microdialysis or intra-arterial administration of a NOS inhibitor, which reduced the elevation in cutaneous vascular conductance by ~30%.

In contrast to humans, blockade of NOS in the rabbit ear completely abolished elevations in SkBF during a heat stress (24). In a series of follow-up experiments, Farrell and Bishop (8, 9) revealed that NO-dependent cutaneous vasodilation in the rabbit ear did not result from an increased NO concentration associated with the heat stress. Rather, NO served in a permissive role, meaning that an unknown neurotransmitter was solely responsible for causing active vasodilation in heated rabbits but that it could due so only in the presence of a basal pool of NO.

Given these findings in the rabbit, the aforementioned observations that NOS inhibition in humans reduces the elevation in SkBF during a heat stress is insufficient to conclude that this perturbation increases NO concentration in the skin. Thus the purpose of this study was to test the hypothesis that whole body heating increases cutaneous interstitial NO concentration. This purpose was accomplished through the use of intradermal microdialysis to sample cutaneous interstitial fluid for changes in NO concentrations before a heat stress (normothermic conditions) and after SkBF was substantially elevated due to whole body heating.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Twelve subjects (7 women and 5 men) participated in the study. The subjects' average age was 31 ± 2 yr, and all were of normal weight (73 ± 4 kg) and height (173 ± 2 cm). Each subject was informed of the purpose and risks of this institutionally approved study before providing written consent.

Instrumentation. Each subject was instrumented for the measurement of sublingual temperature (T_sl) with a thermistor and mean skin temperature (T_sk) from the electrical average of six thermocouples attached to the skin. The subject then dressed in a tube-lined suit that permitted the control of T_sk by changing the temperature of the water perfusing the suit. The suit covered the entire body with the exception of the head, feet, and one arm. The arm not exposed to the tube-lined suits was used to assess SkBF during the heat stress. Thus any changes in SkBF during the heat stress were not related to mechanisms associated with local heating. Heart rate was obtained from the electrocardiogram via a cardiotachometer.

Experimental protocol. After instrumentation, the subjects rested for a minimum of 60-90 min before data collection to allow the hyperemic response associated with microdialysis placement to subside. Before whole body heating, dialysate draining both microdialysis membrane was collected in a single vial for a duration of 12 min. T_sk was then elevated to ~38.8°C by perfusing the tube-lined suit with warm water (46°C). Once sublingual temperature was elevated ~0.8-1.3°C, a second 12-min dialysate collection period ensued. T_sk was then returned to normothermic levels by initially perfusing the suit with cool water (~25°C) followed by perfusing the suit with neutral water (~34°C).

Data collection and analysis. Heart rate and temperature data were obtained during the 12-min periods in which dialysate was collected (pre-heat stress, heat stress, and post-heat stress). These data were sampled at 10 Hz through a data acquisition system (Biopac) and averaged over the last 3 min of each period. For each period, SkBF values over both membranes were averaged to a single value. All data were statistically analyzed using one-way analysis of variance followed by a Student-Newman-Keuls multiple-comparison test when significant main-factor differences were identified. Data are expressed as means ± SE. The level of statistical significance was set at P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic and temperature data are presented in Table 1. Whole body heating increased T_sk from 34.6 ± 0.2 to 38.8 ± 0.2°C and returned to 34.8 ± 0.6°C after the heat stress. The heat stress caused a significant elevation in T_sl from 36.4 ± 0.1 to 37.6 ± 0.1°C. During the heat stress, heart rate increased from 63 ± 5 to 93 ± 5 beats/min (P < 0.05) and then returned toward pre-heat stress levels after the end of heating (68 ± 6 beats/min). SkBF increased significantly during the heat stress from 21 ± 4 to 88 ± 10 perfusion units. Administration of 1 M acetylcholine through the microdialysis membrane further increased SkBF to 128 ± 6 perfusion units (P < 0.05 relative to preheat stress and heat stress values). Dialysate concentration of NO did not increase significantly during the heat stress (Fig. 1). However, administration of acetylcholine through the microdialysis membrane virtually doubled the concentration of NO in the dialysate (P < 0.05 relative to preheat stress and heat stress periods).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Nitric oxide concentration as measured via intradermal microdialysis during normothermia, heat stress, and administration of 1 M acetylcholine in the perfusate after the heat stress. Whole body heating did not increase the concentration of nitric oxide in the dialysate from microdialysis membranes placed in forearm skin. However, after the heat stress, in 6 subjects dialysate concentration of nitric oxide was significantly elevated because of the administration of acetylcholine relative to both the normothermic and heat stress periods (* P < 0.05). These data suggest that cutaneous interstitial nitric oxide concentration does not increase during whole body heating in humans. Values are means ± SE; n, no. of subjects.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The primary finding from this study is that cutaneous interstitial NO concentration, as detected by intradermal microdialysis, does not increase during whole body heating sufficiently to increase T_sl ~1.2°C and increase SkBF approximately fourfold. However, we are confident that increases in cutaneous interstitial NO concentration can be detected using intradermal microdialysis, as evidenced by an approximately twofold increase in dialysate NO concentration when NO production was stimulated with acetylcholine.

The use of intradermal microdialysis to quantify cutaneous interstitial NO concentration in normothermic humans was previously performed by Clough and colleagues (5, 6). They demonstrated that basal NO concentration in the dialysate was 0.5-0.6 µM and that this concentration was reduced by 31% when the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) was administered in the perfusate. Moreover, intradermal injection of histamine increased the concentration of NO in the dialysate by two- to threefold. In the present experiment, the basal concentration of NO in the dialysate was an order of magnitude greater than that observed by Clough and colleagues. Differences in basal concentration of NO in the dialysate between the present and cited studies could be due to differences in flow through the microdialysis membrane (2 vs. 5 µl/min, respectively), differences in the technique used to reduce nitrate and nitrite (reaction products of NO) to NO, and/or differences in the method of analysis (chemiluminescence vs. amperometry, respectively). Nevertheless, the relative increase in dialysate concentration of NO because of the addition of acetylcholine in the perfusate was similar to that observed by Clough and colleagues in studies in which histamine was injected intradermally near the microdialysis membrane. Taken together, it is clear that intradermal microdialysis is a useful tool in assessing cutaneous interstitial NO concentration and that this technique is useful in assessing increases in NO production during perturbations such as acetylcholine and histamine administration.

In both humans (3, 7, 10, 17) and rabbits (8, 9, 25), increases in SkBF during a heat stress are primary due to an active neurogenic mechanism because nerve blockade proximal to the area where SkBF is monitored abolishes the majority of cutaneous vasodilation. In addition, inhibition of NOS prevents the elevation in SkBF in the rabbit ear (8, 9, 24) while decreasing the magnitude of elevation in SkBF in the human (13, 21, 22) during a heat stress. These findings could lead to the conclusion that NO release is increased during a heat stress and, because of its vasodilatory properties, is responsible for or contributes to the elevation in SkBF. However, findings by Farrell and Bishop (8, 9) in the rabbit ear are counter to this hypothesis. In these studies, inhibition of NOS via intra-arterial administration of L-NAME in heated rabbits returned ear blood flow to pre-heat stress levels. Subsequent infusion of a small dose of the NO donor sodium nitroprusside while the rabbits remained heated elevated ear blood flow to levels observed before L-NAME administration. However, that same dose of sodium nitroprusside administered in normothermic rabbits was insufficient to increase ear blood flow. These investigators concluded that NO played a permissive role in active vasodilation, meaning that an unknown neurotransmitter was solely responsible for causing active vasodilation in heated rabbits, but it could do so only in the presence of a basal pool of NO (8).

In humans, we (13) and others (21, 22) showed that inhibition of NOS via intra-arterial or microdialysis administration of NOS antagonists reduces the magnitude of cutaneous vasodilation during a heat stress by ~30%. However, these data do not identify whether NO actively participates in cutaneous vasodilation or serves solely in a permissive role with an unknown neurotransmitter, as was shown in the rabbit (8, 9). The present observation of a lack of increase in cutaneous interstitial concentration of NO in heated humans, coupled with the aforementioned observation in heated rabbits (8, 9), strongly suggests a permissive role of NO in facilitating cutaneous active vasodilation during a heat stress in humans.

Cutaneous vascular conductance in humans during local heating is reduced by ~70% when NOS is inhibited via intradermally administered L-NAME (14). This is in contrast to ~30% reduction in the magnitude of cutaneous vasodilation in the unheated arm during a heat stress in humans in which NOS is inhibited (13, 21, 22). The mechanism resulting in cutaneous vasodilation during local heating is different from that observed during whole body heating because local heating-induced cutaneous vasodilation is preserved in botulinum toxin-treated skin, whereas active cutaneous vasodilation is abolished (15). It is currently unknown whether cutaneous interstitial NO concentration increases during local heating. If local heating increases NO concentration, being that in the present experiment T_sk over the majority of the body is locally heated to 38.8°C during the heat stress, it is possible that systemic NO concentrations increase during the heat stress. However, even if this were to occur, it is clear from the present study that the concentration of NO released would be insufficient to cause measurable increases in cutaneous interstitial NO concentration, or its metabolites, in nonheated but actively vasodilated skin. Thus it is unlikely that increases in systemic NO concentration, if present due to local heating under the water-perfused suit, contribute to cutaneous vasodilation in unheated skin.

Limitations to the interpretation of the data. Microdialysis is a viable method of assessing the concentration of substances in the cutaneous interstitium (1, 2, 4-6, 11, 16, 18). However, whereas 100% of the substance is not diffused into the membrane, estimates of the concentration of a substance in the interstitium cannot be made without identifying the relative recovery of that substance. In the present experiment, we did not quantify the relative recovery of NO and thus we do not know the absolute concentration of NO within the interstitium. Moreover, we assumed that the relative recovery of the microdialysis membrane did not change during the course of the experiment, which is consistent with the findings of others (2). Nevertheless, identification of relative recovery was not required to address the question of interest, since the hypothesis to be tested was whether cutaneous interstitial concentration of NO increases during a heat stress in nonheated skin. If interstitial NO concentration increased during the heat stress, this would result in an increase in NO concentration in the dialysate. Thus we are confident that cutaneous interstitial NO concentration within the area measured did not increase during a heat stress. Moreover, we are confident that, had cutaneous interstitial NO concentration increased, we would have been able to identify this increase because we were able to detect increases in interstitial NO concentration during acetylcholine administration.