INTRODUCTION

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DURING BODY HEATING IN HUMANS, there is neurally mediated vasodilation in nonacral skin, occurring at about the same time that sweating is seen (15, 16). However, the nature of the substance(s) released by the dilating nerves is unknown. Atropine given to one forearm before heating can cause a brief delay and modest (20-30%) reductions in the rise in cutaneous blood flow (CBF) seen during body heating and can also eliminate sweating in the treated area (6, 11, 16). This indicates that some substance released by sweat glands, or perhaps ACh spillover from sudomotor nerves, can contribute to cutaneous dilation during body heating. Additionally, local administration of botulinum toxin, which acts presynaptically to block cholinergic nerve transmission, eliminates both sweating and dilation at the treated site (11). These results indicate that most of the neurally mediated dilation is caused by cholinergic nerves via corelease of an unknown transmitter or vasodilating substance.

One substance that has emerged as a possible mediator of cutaneous vasodilation during body heating is nitric oxide (NO). Studies in the rabbit have demonstrated that infusion of a nitric oxide synthase (NOS) inhibitor via the auricular artery abolished the active dilation in the ear seen during whole body heating (20). However, later studies on active dilation in the rabbit ear indicated that NO itself is not the dilating substance but that it plays a permissive role in the dilation (4, 5).

An early study in humans suggested that NO did not contribute to the rise in CBF seen during body heating (2). However, subsequent studies have demonstrated that NOS inhibitors, given via a brachial artery catheter or through microdialysis to the forearm skin, were able to blunt the increase in cutaneous vasodilation by up to 30% in some subjects (9, 18). These observations led to the conclusion that NO can contribute to cutaneous vasodilation during whole body heating in humans (9, 18). It should also be noted that NOS inhibitors can blunt the increase in cutaneous vascular conductance (CVC) induced with local heating by as much as 50% (10).

Interestingly, the magnitude of the effect of NOS inhibitors on CBF in the whole body heating studies was similar to the previously discussed observations made with atropine (11, 16). Using this information as a background, we hypothesized that, during heating, NO release is a by-product of increased metabolic activity linked to sweating (6, 8). Alternatively, NO might be released when muscarinic receptors on endothelial cells are stimulated by ACh spillover from the sudomotor nerves (8). In the present study, we sought to investigate the roles of sweating, ACh spillover, and NO after cutaneous vasodilation was established during body heating in humans.

	METHODS

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Subjects. The study was approved by the Institutional Review Board, and written informed consent was obtained from the subjects. Six subjects (3 men, 3 women) between the ages of 22 and 31 yr participated in the study. The female subjects each had a negative serum pregnancy test within the 48-h time period before the study. None of the subjects was taking medications of significance to the study. Smokers and individuals with chronic medical conditions were excluded.

Subject monitoring. During the study, arterial pressure and heart rate were monitored by using the pressure signal from a brachial catheter (see Drug preparation and infusions). A thermocouple was inserted under the tongue to measure body temperature.

Body heating. The subjects were heated with a water-perfused suit worn under an impermeable vinyl garment (2, 7, 9). To raise core temperature and evoke an increase in CBF, the suit was perfused with warm water (42-46°C). The suit did not cover the subject's forearms, which were exposed to ambient temperature (~23°C) to ensure that the increase in CBF was through neurogenic, not local, mechanisms.

CBF. CBF was measured with a laser-Doppler flowmeter (BPM 403, TSI, St. Paul, MN) that was placed over the midportion of the nondominant forearm (2, 7). CVC calculations were made by dividing the laser-Doppler flow signals by mean arterial pressure.

Sweat onset. Sweat onset was estimated with an evaporative water loss unit (model Z1885, Demco R & D, Lansing, MI). Sweat capsules were placed near the laser-Doppler flowmeter. Dry nitrogen was passed over 5 cm² of skin to evaporate the sweat, and sweat onset was estimated by observing the difference in relative humidity between the inlet gas and the outflow gas. Because sweating is neurally mediated and occurs at approximately the same time as active vasodilation of the skin, sweat onset was used as a temporal index of the increase in neurogenic vasodilator outflow to the skin that occurs when temperature increases (14, 15). Due to the high rates of sweating seen during body heating, however, the sweat capsules in our system became saturated and were no longer able to respond. Therefore, after sweat onset, the capsules were removed.

Sweat rate and sweat collection. Sweat rate was assessed by use of the Macroduct sweat collection system (Wescor, Logan, UT). After the arm was thoroughly dried, Macroduct devices were placed in approximately the same area as the sweat evaporative capsules and remained attached for a period of 5-7 min. These devices use capillary action to draw sweat from a ~6-cm² area of skin into a thin plastic tube with a volume of 2.7 µl/cm. The amount of sweat accumulation was then marked on the tube. All sweat rate values were compared with values obtained after heating but before administration of atropine.

Drug preparation and infusions. Atropine was obtained from American Regent Labs (Shirley, NY). The arginine analog N^G-nitro-L-arginine methyl ester (L-NAME) was obtained from Calbiochem (La Jolla, CA) and was administered under US Food and Drug Administration IND no. 53,169 (9). Drugs were dissolved in normal saline and infused through a brachial artery catheter (2). A 20-gauge, 5-cm Teflon arterial catheter was inserted into the brachial artery with the use of aseptic technique after local anesthesia. The catheter was connected to a pressure transducer and continuously flushed with heparinized saline at 3 ml/h. A three-port connector was placed in series with the catheter-transducer system (2). One port was used to measure arterial pressure, whereas the other two ports were used for drug infusions. L-NAME was infused with a mechanical syringe pump at 5 mg/min. Atropine was infused at a rate of 80 µg/min. For both drugs, the rate of infusion was 5 ml/min. This infusion rate has been found in our laboratory to have no impact on baseline blood flow (1-3). We chose not to use an ACh test of NOS inhibition because previous studies have shown little correlation between the ability of NOS inhibitors to blunt vasodilator responses and their ability to inhibit ACh-mediated dilation (3, 8, 14). Also, because the arm had received atropine prior to L-NAME, use of an ACh test of NOS blockade was not feasible (2). However, the dose of L-NAME used was similar to or greater than the dose of another NOS inhibitor, N^G-monomethyl-L-arginine (L-NMMA), that was used in our previous studies (1-3, 18). It is also believed that L-NAME may be a more potent NOS inhibitor (9).

Protocol. The subjects abstained from caffeine for 6 h before the study. On arrival at the laboratory, subjects were fitted with the water-perfused suit. The nondominant forearm was instrumented with a brachial artery catheter and also instrumented to measure CBF and sweat onset. Resting CBF values were obtained. The subjects were then heated while CBF data were collected at 5-min intervals. When core temperature had increased ~0.8-1.0°C and a marked rise in CBF had occurred, sweat was collected with the Macroduct device. Atropine was then infused for 5 min. The total dose of atropine given was 0.4 mg over the 5-min period. Another sweat rate assessment was conducted. After 10 min, L-NAME was infused for a period of 10 min. Supplemental atropine was infused for 1.25 min (0.1 mg total) to ensure that the arm receiving L-NAME remained atropinized. After infusion of the supplemental atropine, a second infusion of L-NAME was given for 10 min. CBF was measured continuously from the start of the first atropine infusion to 10 min after the second L-NAME infusion. The total dose of L-NAME was 100 mg, given over a period of 21 min. The last sweat measurement was then performed.

Microdialysis studies. We conducted a set of studies using microdialysis to verify the CVC responses observed during atropine and L-NAME infusions because the response to L-NAME was so variable among subjects. The rationale for this study was that larger and more consistent reductions in CVC have been demonstrated in studies using microdialysis (9). Four subjects (3 women, 1 man) between the ages of 20 and 26 yr participated in these studies. Arterial pressure and heart rate were monitored with the Finapres device (Ohmeda, Englewood, CO). A thermocouple was inserted under the tongue to estimate body temperature. Subjects were instrumented with two intradermal microdialysis probes (Bioanalytical Systems, West Lafayette, IN) placed at different sites on the ventral portion of one forearm (9). Probes were placed ~5 cm apart so that perfusion at one site did not affect the other. Entry and exit points of each probe were ~2-3 cm apart. The probes used were double-lumen probes with a molecular weight cutoff of 20. CBF was measured with laser-Doppler flowmeters (PF 5010, Perimed, Stockholm, Sweden) placed directly over the microdialysis sites. After an interval of 60-80 min while waiting for insertion trauma to subside, we performed a 3-min period of whole body cooling to verify active vasoconstrictor function at the sites. Subjects were then heated. As in the brachial artery protocol, subjects were heated (and cooled) with a water-perfused suit. When oral temperature had increased ~1°C and a marked rise in CBF was observed, one microdialysis probe was perfused with a 58 µM solution of atropine for 30 min. Pilot studies from our laboratory showed that this dose of atropine was adequate to inhibit the maximal dilation induced by 5.5 mM ACh. The other probe was perfused continuously with Ringer solution at a rate of 2 µl/min. After atropine, the experimental site was perfused with a 10 mM solution of L-NAME for 30 min. In one subject, the experimental site was perfused first with L-NAME for 30 min and then atropine for 30 min.

Data collection and analysis. Data were collected on computer and digitized at 100 Hz and then analyzed off-line using signal-processing software (WinDaq, Dataq Instruments, Akron, OH). CBF was derived from the laser Doppler signal. In the brachial artery protocol, mean arterial pressure and heart rate were derived from the arterial pressure waveform. During the microdialysis studies, mean arterial pressure was derived from the Finapres signal.

Statistics. Each subject was able to serve as his or her own control. In both the brachial artery and microdialysis studies, the effects of the drug treatment were compared with the responses in the same forearm just before drug administration. Paired t-tests were used to compare CVC responses before and after drug administrations. In the brachial artery protocol, repeated-measures ANOVA tests were used to compare systemic responses across events and to compare sweating responses. Significance was set at P < 0.05 for all comparisons. Significant effects were further analyzed by using the Student-Newman-Keuls test. Data are reported as means ± SE.

	RESULTS

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Systemic and sweating responses (brachial artery protocol). In response to body heating, both body temperature and heart rate increased significantly (P < 0.05). Mean arterial pressure remained constant. The time from initiation of heating to sweat onset was variable among subjects. Systemic responses are displayed in Table 1.

Cutaneous vascular conductance (brachial infusions). CVC comparisons were made between values obtained just before and after drug infusions. Values obtained after each drug infusion are expressed as a percentage of the values obtained before that drug infusion.

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Data from brachial artery protocol. Individual cutaneous vascular conductance (CVC) responses in treated arm, expressed as a percentage of preatropine values from this arm. Subject 6 showed a decrease in CVC during atropine administration.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Data from brachial artery protocol. Individual CVC responses in treated arm expressed as a percentage of values from this arm before administration of NG-nitro-L-arginine methyl ester (L-NAME). Subjects 1, 3, and 6 showed no changes in CVC in treated arm. Subjects 4 and 5 exhibited minimal declines of <15%, and subject 2 showed a decrease of 36%.

CVC (microdialysis). CVC comparisons were made between values obtained before and during drug administration. During atropine perfusion, CVC remained constant in the experimental (111 ± 9%, P > 0.05 vs. preatropine values) and control sites (110 ± 11%, P > 0.05 vs. preatropine values). With L-NAME perfusion, CVC decreased significantly to 63 ± 4% of pre-L-NAME values (P < 0.05, Fig. 3A). The CVC values in the control site remained constant (95 ± 2%, P > 0.05 vs. pre-L-NAME values, Fig. 3A). Subjects demonstrated reductions in CVC ranging from 27% to 47% (Fig. 3B). Figure 4 is an individual record from one subject. CVC is expressed as a percentage of the peak response to heating before any drug administration. This subject demonstrates a marked fall in CVC with L-NAME perfusion in the experimental site, although CVC remained constant during atropine administration.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   CVC responses to L-NAME in microdialysis studies. A: summary of CVC responses to Ringer solution (control) and L-NAME perfusion (experimental). Experimental site demonstrated a decline in CVC to 63 ± 4% of pre-L-NAME values (n = 4, * P < 0.05). No change was detected in control site (95 ± 2%, P > 0.05). B: individual CVC responses to Ringer solution and L-NAME perfusion, expressed as a percentage of pre-L-NAME values. Decreases in responses ranged from 27% to 47%.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Individual record from microdialysis study, showing changes in CVC (expressed as %peak response to heating) throughout study in response to heating and drug infusions. This subject showed an ~40% drop in CVC in response to L-NAME. No change in CVC was seen during atropine infusion.

	DISCUSSION

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The purpose of this study was to investigate the roles of sweating, ACh spillover from the sudomotor nerves, and NO in the neurally mediated cutaneous vasodilation during body heating in humans. Our findings are consistent with the results from our previous study (18) and the study performed by Kellogg and colleagues (9), which showed that NO contributes to cutaneous dilation in humans. However, atropine failed to blunt the elevated CVC levels during heating in five of six subjects and failed to blunt CVC levels in all four of the subjects in the microdialysis study. These observations indicate that neither sweating nor NO release through muscarinic receptor activation is critical to sustain cutaneous vasodilation during body heating in humans.

Sweating. Roddie et al. (16) were able to show that application of atropine before body heating both abolished sweating and also delayed and blunted the degree of vasodilation seen by up to 30%. These observations, which were later confirmed by Kellogg et al. (11), were important in leading to the hypothesis that a substance released by sweat glands contributed to the cutaneous vasodilation seen during body heating (6). In the brachial artery protocol of this study, atropine administration abolished the sweating response in the treated arms of all subjects, although vasodilation remained unchanged. These findings suggest that sweating itself during whole body heating is not critical to sustain the cutaneous vasodilation once it has been initiated, although it is possible that sweat gland function may still play a role. Additionally, after atropine administration, the forearm underwent a period of little or no sweating that lasted at least 30 min. This suggests that sweating does not have a time-dependent effect on the dilation during heating.

Role of NO. We initially hypothesized that atropine administration after heating would blunt the cutaneous dilation to a degree similar to that previously seen with NOS inhibitors (9, 18). An alternative hypothesis was that L-NAME infusion would result in a greater blunting of the cutaneous dilation than was seen with atropine administration. Our observations from both the brachial artery protocol and the microdialysis studies are consistent with the latter hypothesis, although the magnitude of the contribution of NO can be variable. One possible way that NO could be released is through a flow-induced mechanism (13, 17, 19). An unidentified vasodilating substance could augment blood flow, increasing the shear stress to which the endothelial cells are exposed. This shear stress could then stimulate NO release from these cells. It is also possible that NO could be coreleased by an autonomic nerve population other than sudomotor nerves. However, the findings by Kellogg et al. (11) indicate that these nerves would also be sensitive to botulinum toxin.

ACh spillover. Our observations with atropine, showing that the sweat response was abolished in the treated arm, indicate that we were able to adequately block the muscarinic receptors on the cutaneous vessels. Additionally, our pilot studies showed that the dose of atropine used for microdialysis was sufficient to block the maximal dilator response induced by ACh, thus verifying the inhibition of muscarinic receptors. Therefore, ACh would not have been able to bind to these muscarinic receptors and cause NO release. Because Kellogg et al. (11) showed that cutaneous active dilation cannot be mediated by activation of nonmuscarinic cholinergic receptors, it is unlikely that ACh spillover from sudomotor nerves acting on any population of vascular receptors is responsible for NO release in the cutaneous vasodilation during body heating in humans. This hypothesis is consistent with our observations from subjects in the microdialysis study. In three of the four subjects, atropine was infused before L-NAME, and no change in CVC was observed during atropine infusion. In the other subject, L-NAME was infused first. After a marked drop in CVC (~40%) was seen, atropine was infused. CVC did not fall further but remained at post-L-NAME levels.

Limitations. One limitation of the brachial artery protocol is the inconsistency of the response to L-NAME. In our previous study, we also found that CVC responses to brachial infusions of L-NMMA were not consistent (18). We used L-NAME in the present study because it appeared to have a greater impact on CVC in the study by Kellogg and colleagues (9). In this context, our inconsistent results in comparison to those obtained by Kellogg et al. probably reflect the fact that higher and more reliable concentrations of the inhibitor can be delivered to the skin by using microdialysis than by brachial artery drug administrations. Our observations with microdialysis, showing a more consistent decrease in CVC in response to L-NAME in all four subjects, confirm this interpretation.