What is the central question of this study?

What is the role of nicotinic receptors in the regulation of normothermic cutaneous blood flow and cutaneous vasodilatation and sweating during whole‐body heating induced following resting in a non‐heat‐stress condition?
What is the main finding and its importance?

Nicotinic receptors modulated cutaneous vascular tone during rest in a non‐heat‐stress condition and in the early stage of heating, but they had a limited role in mediating cutaneous vasodilatation when core temperature increased >0.4°C. Further, the contribution of nicotinic receptors to sweating was negligible during whole‐body heating. Our findings provide new insights into the role of nicotinic receptors in end‐organ function of skin vasculature and sweat glands in humans.

Abstract

Nicotinic receptors are present in human skin including cutaneous vessels and eccrine sweat glands as well as peripheral nerves. We tested the hypothesis that nicotinic receptors do not contribute to the control of cutaneous vascular tone in the normothermic state, but are involved in mediating cutaneous vasodilatation and sweating during a whole‐body passive heat stress in humans. We first performed a nicotinic receptor blocker verification protocol in six young adults (one female) wherein increases in cutaneous vascular conductance and sweating elicited by 10 mm nicotine were blocked by administration of 500 µm hexamethonium to confirm effective blockade. Thereafter, 12 young males participated in a passive heating protocol. After an instrumentation period in a non‐heat‐stress condition, participants rested for a 10 min baseline period. Thereafter, oesophageal temperature was increased by 1.0°C using water‐perfusion suits. Cutaneous vascular conductance, sweat rate, active sweat gland density and sweat output per individual gland were assessed with and without 500 µm hexamethonium administered via intradermal microdialysis. Hexamethonium reduced cutaneous vascular conductance by 22–34% during normothermia and the early stage of heating. However, this effect was diminished as oesophageal temperature increased >0.4°C. Active sweat gland density was reduced by hexamethonium when oesophageal temperature was elevated by 0.4–0.6°C above baseline resting. However, this was paralleled by a marginal increase in sweat gland output. Consequently, sweat rate remained unchanged. We showed that nicotinic receptors modulate cutaneous perfusion during normothermia and the early stage of heating, but not when core temperature increases >0.4°C. Additionally, they play a limited role in mediating sweating during heating.

1 INTRODUCTION

Heat stress caused by higher ambient temperatures increases the requirement for the body to dissipate heat to maintain core temperature within safe limits. An increase in whole‐body heat loss is achieved by the activation of the heat loss responses of cutaneous vasodilatation and sweating. However, the mechanisms responsible for the activation of heat loss responses are complex and involve the interplay of different factors that modulate the response (Johnson, Minson, & Kellogg, 2014; Smith & Johnson, 2016; Wong & Hollowed, 2017). Of these, sympathetic cholinergic nerves have been shown to play a critical role in the regulation of cutaneous vasculature and sweating responses as evidenced by the observation that presynaptic cholinergic nerve blockade with botulinum toxin type A abolishes the heat‐induced activation of cutaneous vasodilatation and sweating (Kellogg et al., 1995). However, the mechanisms underlying the postsynaptic response of sympathetic cholinergic nerves in the regulation of heat loss responses remain incomplete.

During whole‐body heating, sympathetic cholinergic nerves release acetylcholine (and cotransmitters), which has been shown to activate muscarinic receptors (G‐protein‐coupled receptors) (Pappano, 2011). Studies using the muscarinic receptor blocker atropine (Kellogg et al., 1995; Roddie, Shepherd, & Whelan, 1957; Shastry, Minson, Wilson, Dietz, & Joyner, 2000) or the acetylcholinesterase inhibitor neostigmine (Shibasaki, Wilson, Cui, & Crandall, 2002) indicate that muscarinic receptors partly mediate cutaneous vasodilatation during the early stages of heating, albeit some studies suggest that atropine has no effect (Bennett, Johnson, Stephens, Saad, & Kellogg, 2003) or augments cutaneous vasodilatation (Kolka, Stephenson, Allan, & Rock, 1989) during heat stress. In addition to activating muscarinic receptors, acetylcholine can open nicotinic receptors (ligand‐gated ion channels) (Pappano, 2011). Recent work demonstrates that one of the major mechanisms underlying the nicotinic receptor‐mediated response appears to be the cholinergic axon reflex (Fujii et al., 2017a) wherein nicotinic receptors located on cholinergic axons are initially activated, which leads to the antidromic propagation of action potentials toward the axon branch. Thereafter, action potentials are redirected orthodromically to cholinergic nerve terminals, ultimately stimulating release of acetylcholine, thereby activating muscarinic receptors of cutaneous vasculature. The axon reflex of cutaneous sensory nerves also could contribute to nicotinic cutaneous vasodilatation (Izumi & Karita, 1992) in which activation of nicotinic receptors located on cutaneous sensory nerves antidromically stimulates release of vasodilator substances such as calcitonin‐gene‐related peptides and substance P from sensory nerve terminals. Importantly, however, it remains to be determined if nicotinic receptors are also involved in the regulation of cutaneous vascular tone, and therefore cutaneous vasodilatation during heat stress in humans in vivo.

Heat‐induced sweating is thought to be exclusively regulated via acetylcholine‐induced activation of muscarinic receptors in the eccrine sweat glands. This is evidenced by the consistent observations that muscarinic receptor blockade with atropine abolishes sweating during whole‐body heating (Buono et al., 2010; Kellogg et al., 1995; Machado‐Moreira et al., 2012). Acetylcholine can also activate nicotinic receptors located on sudomotor cholinergic axons, causing the release of more acetylcholine from cholinergic nerves and in turn augmenting muscarinic receptor‐mediated sweating (the so called sudomotor cholinergic axon reflex) (Schlereth, Brosda, & Birklein, 2005). While these findings suggest that nicotinic receptors may play a role in the modulation of sweating during heat stress, no study to date has confirmed a role for nicotinic receptors in the regulation of the sweating response during heat stress.

In the present study, we evaluated the hypothesis that nicotinic receptors contribute to the regulation of cutaneous vasodilatation and sweating during heat stress induced by whole‐body heating in humans in vivo. We also hypothesized that nicotinic receptors do not modulate cutaneous vascular tone under normothermic conditions since acetylcholine release from cholinergic nerves appears to be negligible under these conditions. Before testing our hypotheses, we assessed if nicotinic receptor blockade induced by the administration hexamethonium attenuates nicotine‐induced cutaneous vasodilatation and sweating. This procedure was done to verify the blocking efficacy of the agent. Given the known sex‐related differences in the regulation of cutaneous vascular and sweating responses (Fujii, Halili, Singh, Meade, & Kenny, 2015; Gagnon, Crandall, & Kenny, 2013; Greaney, Stanhewicz, Kenney, & Alexander, 2014), we evaluated young males only in the present study.

2 METHODS

2.1 Ethical approval

This study was approved by the Human Subjects Committee of the University of Tsukuba (no. 29‐24), in agreement with the Declaration of Helsinki, except for registration in a database. All participants provided written informed consent prior to participating in the study.

2.2 Participants

Six young adults (one female) participated in an initial verification protocol to test the efficacy of the nicotinic receptor blocker. Thereafter 12 young males participated in the main passive heating protocol (see below for details of each protocol). Age, body mass, height and body mass index (mean ± SD) were 25 ± 2 years, 71.8 ± 9.3 kg, 1.70 ± 0.06 m, and 25.0 ± 3.2 kg m⁻², respectively, in the blocker verification protocol, and 24 ± 3 years, 74.8 ± 8.1 kg, 1.74 ± 0.05 m and 24.8 ± 3.0 kg m⁻², respectively, in the passive heating protocol. All participants were healthy and physically active with no history of any medical conditions. All participants were non‐smokers and were not taking prescribed medications. Prior to both protocols, participants were asked to refrain from taking over‐the‐counter medications for >48 h, alcohol and caffeine for 12 h, and any food for >2 h. Strenuous physical activity was also not allowed for >12 h prior to both sessions.

2.3 Blocker verification protocol

Upon arrival at the laboratory, participants rested on a semi‐recumbent chair in a non‐heat‐stress environment (∼25°C) after which a 25‐gauge needle was aseptically inserted into the dermal layer of the unanaesthetized left dorsal forearm skin. The entry and exit points were separated by ∼2.5 cm. A custom‐made microdialysis fibre (50 kDa cutoff, 10 mm regenerated cellulose membrane with 0.22 mm o.d., 0.20 mm i.d.) was passed through the lumen of the needle, and then the needle was withdrawn, leaving the membrane of the microdialysis fibre in the skin. The second fibre was placed similarly in the skin. The two fibres were separated by >4 cm to avoid any potential between‐site interference of drug administration. Approximately 10 min after the fibre placements, continuous administration of 500 µm hexamethonium (LKT Laboratories, Minneapolis, MN, USA), a nicotinic receptor blocker (known as a ganglionic blocker or Nn antagonist), dissolved in lactated Ringer solution (Fuso Pharmaceutical Industries, Osaka, Japan) was administered to one of the two microdialysis fibres. The remaining fibre was continuously perfused with lactated Ringer solution only and served as the control site. Each fibre was continuously perfused at a rate of 4.0 µl min⁻¹ using a micro‐infusion pump (BASi Bee Hive controller and Baby Bee syringe drive; Bioanalytical Systems, West Lafayette, IN, USA) for a minimum of 60 min to ensure nicotinic receptor blockade before commencing baseline measurements. This duration (i.e. ≥60 min) has also been shown to permit the trauma evoked by fibre insertion to subside (Anderson, Andersson, & Wardell, 1994). While maintaining drug perfusion, baseline measurements were recorded for an additional minimum 10 min period. Thereafter, a solution of 10 mm nicotine (Nacalai Tesque, Kyoto, Japan), a nicotinic receptor agonist, was simultaneously administered at a rate of 4.0 µl min⁻¹ in both microdialysis fibres for 20 min. We previously reported that this dose of nicotine induced substantial sweating and cutaneous vasodilatation (Fujii et al., 2017b). Ten millimolar nicotine was dissolved in site‐specific solutions (i.e. lactated Ringer solution or 500 µm hexamethonium). The pH of the nicotine solution was adjusted with sodium hydroxide (Sigma‐Aldrich, St Louis, MO, USA) to 6.5, a pH that is similar to the lactated Ringer solution. After completing the infusion of the 10 mm nicotine solution, administration of 50 mm sodium nitroprusside (Nacalai Tesque), a known potent vasodilator, was commenced at the two skin sites to induce maximal cutaneous blood flow over a minimum 25 min evaluation period.

2.4 Passive heating protocol

Upon arrival at the laboratory, participants donned a water‐perfusion suit. The forearm skin sites used for the insertion of the microdialysis probes were left uncovered. Participants then rested on a semi‐recumbent bed in a non‐heat‐stress condition (ambient temperature of ∼25°C) during which time the water‐perfusion suit was continuously perfused with 35°C water (Variable Flow Chemical Pump, Thermo Fisher Scientific, Waltham, MA, USA) to maintain the normothermic state (as defined by a stable resting body temperature response). During this time, four microdialysis fibres were inserted in pre‐selected forearm skin sites using the same procedure outlined above. Two of the microdialysis fibres were perfused with 500 µm hexamethonium, a nicotinic receptor blocker, while the other two microdialysis fibres were perfused with lactated Ringer solution (control sites). One of the control sites and one of the hexamethonium‐treated sites were used for the combined measurement of cutaneous blood flow and sweat rate (see description of specialized sweat capsule system below), whereas the remaining two sites were used for the assessment of active sweat gland density. After ≥60 min of continuous administration of site‐specific solutions (i.e. lactated Ringer solution or 500 µm hexamethonium), a 10 min baseline measurement was performed. Thereafter, the temperature of the water circulating through the water‐perfusion suits was increased to 48–50°C to induce an increase in body temperature. The water‐perfusion suit was covered with a plastic insulated garment to minimize the evaporation of sweat (which would cool the body) and facilitate a progressive increase in body temperature to the required level of hyperthermia. Heating continued until body temperature as estimated from the measurement of oesophageal temperature increased by 1.0°C above baseline resting levels (∼60–90 min). Following cessation of heating, the temperature of the circulating water was reduced to ∼30°C to cool the body and restore body temperature at or near baseline resting levels. During this time, 50 mm of sodium nitroprusside was administered for ∼30 min to one control and one hexamethonium‐treated site employed for the combined assessment of cutaneous blood flow and sweat rate to obtain maximal cutaneous blood flow.

2.5 Measurements

Cutaneous blood flow was estimated by cutaneous red blood cell flux (presented in perfusion units) measured by laser Doppler flowmetry (PeriFlux System 5000, Perimed, Stockholm, Sweden) at a sampling rate of 32 Hz. The integrated laser Doppler flowmetry probes with seven‐laser array (Model 413, Perimed) were put in the centre of each sweat capsule (see below), which covered the microdialysis membrane area, for the simultaneous measurement of forearm local cutaneous blood flow and sweat rate. Arterial blood pressures (systolic and diastolic) of the right brachial artery were obtained via an automated sphygmomanometer (TM‐2580, A&D Company, Ltd, Tokyo, Japan) at 5 min intervals. Mean arterial pressure was calculated as diastolic pressure plus one‐third of the difference between systolic and diastolic pressures. Cutaneous vascular conductance was evaluated as cutaneous red blood cell flux divided by mean arterial pressure. Cutaneous vascular conductance was presented as a percentage of the maximum conductance.

Custom‐made oval‐shaped sweat capsules, designed for use with intradermal microdialysis fibres (Meade et al., 2016), were attached on the forearm intradermal microdialysis skin sites with 5% collodion (Nacalai Tesque), covering a surface area of 1.1 cm². Dry compressed N₂ gas equilibrated to room temperature was supplied from gas tanks to each capsule at a rate of 1.0 l min⁻¹ regulated by a mass flow controller (DF‐200, Kofloc, Kyoto, Japan). A capacitance hygrometer (HMT330, Vaisala, Helsinki, Finland) was used to measure the relative humidity and temperature in the effluent air from the sweat capsule every 1 s, and stored in a data logger (GM10, Yokogawa Electric Corp., Tokyo, Japan). Long vinyl tubes were used for connections between the N₂ gas tank and the sweat capsule (inlet), and between the sweat capsule and the capacitance hygrometer (outlet). The N₂ flow rate, area under the capsule, and measured relative humidity and temperature were used to calculate local sweat rate presented in mg min⁻¹ cm⁻².

Active sweat gland density (glands cm⁻²) was determined by the iodine paper technique (Gagnon et al., 2012; Inoue et al., 2014), the details of which are described elsewhere (Ichinose‐Kuwahara et al., 2010). This measurement was performed at two microdialysis sites (one control and one hexamethonium‐treated site) each covering 2 cm² (1 cm × 2 cm). Sweat output per individual gland (ng min⁻¹ cm⁻²) was assessed as local sweat rate divided by active sweat gland density.

Oesophageal and skin temperatures were measured every 1 s using copper‐constant thermocouples and stored in the data logger (GM10, Yokogawa Electric Corp.). For the measurement of oesophageal temperature, a thermocouple probe was inserted through the nasal passage to a distance equal to approximately one‐fourth of the participant's height. The location of the probe within the oesophagus is estimated to be posterior to the lower border of the left atrium (Wenger & Roberts, 1980). Skin temperature was measured at seven sites: chest, upper back, lower back, abdomen, thigh, calf and forearm. Mean skin temperature was calculated using a method described by Taylor, Johnson, O'Leary, and Park (1984). A forearm thermocouple probe was attached near the microdialysis sites to monitor local temperature at these sites. Due to technical difficulties, forearm skin temperature was not obtained in 3 out of 12 participants, and oesophageal temperature data in 1 out of 12 participants could not be saved to the data logger.

2.6 Data analysis

In both protocols, baseline cutaneous vascular conductance and sweat rate were assessed by averaging the last 5 min of values during the baseline period. Maximum cutaneous vascular conductance was obtained by averaging conductance values over the last 5 min of sodium nitroprusside administration. In the blocker verification protocol, nicotine administration transiently increased cutaneous vascular conductance and sweat rate (Figure 1a,c). The highest values averaged over 5 min during nicotine administration were evaluated and were presented as changes from baseline. In the passive heating protocol, cutaneous vascular conductance, sweat rate, body temperatures and blood pressures during whole‐body heating were assessed by averaging values over 5 min when oesophageal temperature was elevated by 0.2, 0.4, 0.6, 0.8, and 1.0°C above normothermic baseline levels. Changes in cutaneous vascular conductance and sweat rate from normothermic baseline levels were also assessed. Active sweat gland density and sweat output per individual gland were assessed at normothermia and each 0.2°C increment in oesophageal temperature. Sweat output per individual gland was assessed only after sweat rate was elevated above non‐heat‐stress baseline resting values (i.e. this was not assessed in all participants at baseline, and 3 and 1 participants at +0.2 and +0.4°C oesophageal temperature above normothermic levels, respectively). The oesophageal temperature threshold for the activation of thermoeffectors was evaluated using a previously reported method (Cheuvront et al., 2009) with minor modifications as follows. Two regression lines were drawn from the relationship between oesophageal temperature and cutaneous vascular conductance using 1 min averaged data with the least squares method; then, oesophageal temperature at which the two regression lines crossed was defined as the oesophageal temperature threshold for cutaneous vasodilatation (GraphPad Prism 7.04; GraphPad Software Inc., La Jolla, CA, USA). Similarly, the oesophageal temperature threshold for sweating was also assessed from the relationship between oesophageal temperature and sweat rate.

**Figure 1**
Open in figure viewer PowerPoint

Cutaneous vascular conductance and sweat rate data from blocker verification protocol. An example of the response of cutaneous vascular conductance (a) and sweat rate (c) during 10 mm nicotine administration is presented for a representative participant. Responses were evaluated at two forearm skin sites continuously treated with (1) lactated Ringer solution (control) or (2) 500 µm hexamethonium, a nicotinic receptor blocker. Averaged data ± 95% confidence intervals are presented for nicotine induced changes in cutaneous vascular conductance (b) and sweat rate (d) from baseline (n = 6)

2.7 Statistical analysis

Based on our previous work wherein cutaneous vascular conductance (Fujii, Notley, Minson, & Kenny, 2016) and sweat rate (Stapleton et al., 2014) were compared between the control and nitric oxide synthase inhibition sites, a minimal sample size of n = 9 for cutaneous vascular conductance and n = 8 for sweat rate was determined with 80% power and a significance level of 0.05. In the blocker verification protocol, cutaneous vascular conductance and sweat rate were assessed by a two‐way repeated‐measures analysis of variance with factors of treatment site (two levels: control and hexamethonium‐treated sites) and phase (two levels: baseline and nicotine administration). In the passive heating protocol, cutaneous vascular conductance, sweat rate and active sweat gland density were analysed using a two‐way repeated‐measures analysis of variance with factors of treatment site (two levels) and oesophageal temperature (six levels: normothermic baseline and increases in oesophageal temperature of 0.2, 0.4, 0.6, 0.8 and 1.0°C above baseline levels). Body temperatures and blood pressures were analysed with a one‐way repeated‐measures analysis of variance with a factor of oesophageal temperature (six levels). When detecting a significant interaction, or a main effect in both protocols, a modified version of the Bonferroni correction (i.e. Holm's method) was used for multiple comparisons. Analysis of variance was not performed for sweat output per individual gland due to lower sample size during the early stage of whole‐body heating, and only a between‐site comparison was performed using Student's paired t test at each level of oesophageal temperature. A paired t test was also used for comparing maximum cutaneous vascular conductance between the control and hexamethonium‐treated sites. The level of significance for all analyses was set at ≤0.05. All values are reported as means ± 95% confidence intervals (1.96 × standard error of the mean). Statistical analyses were conducted using SPSS Statistics 25 (IBM Corp., Armonk, NY, USA).

3 RESULTS

3.1 Blocker verification protocol

Nicotine administration increased cutaneous vascular conductance and sweat rate at the control site (both P < 0.006), whereas these responses were not observed at the hexamethonium‐treated site (both P ≥ 0.57) (Figure 1a,c). In parallel, nicotine‐induced changes in cutaneous vascular conductance and sweat rate at the hexamethonium‐treated site were lower than at the control site (both P ≤ 0.006, Figure 1b,d). Nicotine administration did not affect mean arterial pressure (P = 0.81). Maximum cutaneous vascular conductance was similar between the control and hexamethonium‐treated sites (2.21 ± 0.41 vs. 2.81 ± 0.83 perfusion units mmHg⁻¹, P = 0.07).

3.2 Passive heating protocol

Red blood cell flux and absolute cutaneous vascular conductance are presented in Table 1. Individual and averaged cutaneous vascular conductance data are presented in Figure 2a and b, respectively. Whole‐body heating gradually increased cutaneous vascular conductance at both the control and hexamethonium‐treated sites (all P ≤ 0.05). A main effect of treatment site on cutaneous vascular conductance was significant (P = 0.02) whereas an interaction between treatment site and oesophageal temperature was not (P = 0.76). Cutaneous vascular conductance before and during the early stage of heating was lower at the skin site treated with hexamethonium relative to the control site (P = 0.001). However, this between‐site difference was diminished during the late stage of heating as oesophageal temperature increased above 37.0°C (all P ≥ 0.12). The changes in cutaneous vascular conductance from baseline were similar between the control and hexamethonium‐treated sites at all levels of oesophageal temperature (P ≥ 0.50, for a main effect of treatment site and interaction between treatment site and oesophageal temperature). Oesophageal temperature threshold for cutaneous vasodilatation was also not different between the control and hexamethonium‐treated sites (36.78 ± 0.22 vs. 36.77 ± 0.20°C, P = 0.90). Maximum cutaneous vascular conductance did not differ between the control and hexamethonium‐treated sites (2.77 ± 0.72 vs. 3.01 ± 1.08 perfusion unit mmHg⁻¹, P = 0.62).

Table 1. Red blood cell flux and absolute cutaneous vascular conductance during baseline resting and whole‐body heating

		Whole‐body heating
Parameter	Baseline	+0.2°C	+0.4°C	+0.6°C	+0.8°C	+1.0°C
Red blood cell flux (perfusion unit)
Control	53 ± 15	77 ± 40	104 ± 63	128 ± 61Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	144 ± 61Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	157 ± 52Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.
Hexamethonium	36 ± 9	47 ± 14Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	73 ± 27Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	104 ± 30Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	127 ± 36Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	143 ± 34Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.
Cutaneous vascular conductance (perfusion unit mmHg⁻¹)
Control	0.64 ± 0.20	0.97 ± 0.53	1.25 ± 0.73	1.52 ± 0.68Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	1.70 ± 0.70Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	1.94 ± 0.69Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.
Hexamethonium	0.44 ± 0.13	0.58 ± 0.19Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	0.88 ± 0.32Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	1.23 ± 0.36Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	1.52 ± 0.49Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.	1.78 ± 0.53Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.

Values are presented as means ± 95% confidence interval (n = 12). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^*P ≤ 0.05 vs. Baseline. P > 0.252 for a main effect of treatment site and an interaction between treatment site and oesophageal temperature for both parameters.

**Figure 2**
Open in figure viewer PowerPoint

Cutaneous vascular conductance before and during whole‐body heating. Cutaneous vascular conductance was evaluated at two forearm skin sites continuously treated with (1) lactated Ringer solution (control) or (2) 500 µm hexamethonium, a nicotinic receptor blocker. Individual (a) and averaged data ± 95% confidence intervals (b) are presented (n = 12). *P ≤ 0.05 vs. normothermic baseline; †P ≤ 0.05 control vs. hexamethonium treated sites

Individual (Figure 3b,d,f) and averaged (Figure 3c,e,g) data are presented for active sweat gland density assessed at a skin area of 2 cm² (example presented in Figure 3a), sweat output per individual gland and sweat rate. Active sweat gland density and sweat rate (Figure 3b,c,f,g) were elevated in parallel to the increases in oesophageal temperature (all P ≤ 0.05). Active sweat gland density was reduced by hexamethonium relative to the control site at oesophageal temperature elevations of 0.4–0.6°C above baseline resting levels (both P ≤ 0.05, Figure 3b,c). By contrast, sweat output per individual gland tended to increase at the hexamethonium‐treated site in comparison to the control site at oesophageal temperature elevation of 0.6°C above baseline resting levels (P = 0.07, Figure 3d,e). Consequently, sweat rate did not differ between the two skin sites throughout the whole‐body heating protocol (P ≥ 0.34 for a main effect of treatment site and interaction between treatment site and oesophageal temperature, Figure 3f,g). The changes in sweat rate from baseline were similar between the control and hexamethonium‐treated sites throughout heating (P ≥ 0.36, for a main effect of treatment site and interaction between treatment site and oesophageal temperature). Oesophageal temperature threshold for sweating did not differ between the control and hexamethonium‐treated sites (36.81 ± 0.20 vs. 36.78 ± 0.21°C, P = 0.19). During the administration of 50 mm sodium nitroprusside, sweat rate remained at normothermic baseline levels at both of the control and hexamethonium‐treated sites (both 0.09 ± 0.03 mg min⁻¹ cm⁻²).

**Figure 3**
Open in figure viewer PowerPoint

Sweating responses before and during whole‐body heating. (a) Schematic diagram demonstrating the active sweat gland density at a skin area of 2 cm². (b–g) Individual and averaged data ± 95% confidence intervals (n = 12) for active sweat gland density (b,c), sweat output per individual gland (d,e) and sweat rate (f,g). Responses were evaluated at two forearm skin sites continuously treated with (1) lactated Ringer solution (control) or (2) 500 µm hexamethonium, a nicotinic receptor blocker. Sweat output per individual gland was not assessed in all participants at normothermic baseline, and 3 and 1 participants at a +0.2 and +0.4°C oesophageal temperature above baseline resting levels, respectively. *P ≤ 0.05 vs. normothermic baseline; †P ≤ 0.05 control vs. hexamethonium treated sites. P‐value provided in (c) is for between‐site comparison

Oesophageal, mean skin and forearm skin temperatures were all elevated during whole‐body heating in comparison to baseline (all P ≤ 0.05) with no change in blood pressure (P = 0.44 for a main effect of oesophageal temperature, Table 2).

Table 2. Body temperature and cardiovascular responses during baseline resting and whole‐body heating

		Whole‐body heating
Parameter	Baseline	+0.2°C	+0.4°C	+0.6°C	+0.8°C	+1.0°C
Oesophageal temperature (°C) (n = 11)	36.54 ± 0.13	36.76 ± 0.12Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	36.96 ± 0.12Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	37.16 ± 0.12Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	37.37 ± 0.13Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	37.59 ± 0.10Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.
Mean skin temperature (°C)	35.11 ± 0.35	38.55 ± 0.37Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	38.72 ± 0.39Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	38.81 ± 0.35Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	38.96 ± 0.34Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	38.84 ± 0.35Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.
Forearm skin temperature (°C) (n = 9)	31.72 ± 0.73	32.32 ± 0.81	32.69 ± 0.74	32.83 ± 0.70Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	33.20 ± 0.72Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.	33.35 ± 0.63Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^P ≤ 0.05 vs. Baseline.
Systolic arterial pressure (mmHg)	117 ± 10	118 ± 7	118 ± 6	121 ± 7	123 ± 6	121 ± 7
Diastolic arterial pressure (mmHg)	71 ± 6	66 ± 4	68 ± 5	67 ± 4	67 ± 4	64 ± 4
Mean arterial pressure (mmHg)	86 ± 7	83 ± 4	84 ± 5	85 ± 5	86 ± 4	83 ± 5

Values are presented as means ± 95% confidence interval (n = 12 otherwise indicated). +0.2°C, +0.4°C, +0.6°C, +0.8°C and +1.0°C; represent the level of body temperature increase induced during whole‐body heating as measured by an increase in oesophageal temperature of 0.2°C, 0.4°C, 0.6°C, 0.8°C and 1.0°C above resting levels, respectively. ^*P ≤ 0.05 vs. Baseline.

4 DISCUSSION

We evaluated role of nicotinic receptors in the regulation of cutaneous blood flow and sweating during normothermia and passive heating at rest using hexamethonium, a nicotinic receptor blocker. We first verified the effectiveness of hexamethonium by demonstrating that this drug abolished nicotine‐induced cutaneous vasodilatation and sweating in human skin. In the subsequent main experiment, we showed that nicotinic receptors modulate cutaneous vascular tone under non‐heat‐stress conditions and during the early stage of heating (≤0.4°C elevation in oesophageal temperature) as evidenced by a reduction in cutaneous vascular conductance of 22–34%. In contrast, no effects were observed when oesophageal temperature increased further. Moreover, while we demonstrated that nicotinic receptors can modulate sweat gland activation and sweat production per individual gland, they have a negligible influence on the regulation of sweating during a heat stress induced by whole‐body passive heating.

4.1 Blocker verification protocol

Administration of 10 mm nicotine increased cutaneous vascular conductance and sweat rate by ∼40%max and ∼0.55 mg min⁻¹ cm⁻², respectively (Figure 1b,d), which is consistent with the magnitude of increase we previously observed using the same dose of nicotine (Fujii et al., 2017b). Importantly, the levels of cutaneous vasodilatation and sweating achieved by nicotine administration were similar to those observed when oesophageal temperature was elevated by 0.6 −0.8°C above baseline resting levels (Figures 2 and 3). Our observations indicate that nicotinic receptor activation alone can have a modulating influence on the regulation of cutaneous vasodilatation and sweating. In order to directly assess roles of nicotinic receptors in the regulation of heat loss responses of cutaneous vasodilatation and sweating, we employed hexamethonium, a nicotinic receptor antagonist that was previously shown to abolish nicotine‐induced increases in blood flow in human forearm and hand (i.e. blood flow in both skin and skeletal muscle) (Fewings, Rand, Scroop, & Whelan, 1966). In the blocker verification protocol, we demonstrated that hexamethonium abolished nicotine‐induced cutaneous vasodilatation and sweating (Figure 1), validating the use of this drug as a nicotinic receptor blocker in human skin.

4.2 Cutaneous vascular response during normothermia and whole‐body heating

During baseline resting in a non‐heat‐stress condition, hexamethonium reduced cutaneous vascular conductance relative to the control site (Figure 2) indicating that nicotinic receptors play a role in the regulation of cutaneous vascular tone under this condition. Activation of nicotinic receptors of cholinergic axons can cause cholinergic axon reflex, increasing acetylcholine release from cholinergic nerves, subsequently activating muscarinic receptors of cutaneous vessels and thus cutaneous vasodilatation (Fujii et al., 2017a). Together, these observations lend support to the hypothesis that muscarinic mechanisms primarily explain the contribution of nicotinic receptors to the regulation of cutaneous vascular tone under non‐heat‐stress conditions. However, this possibility is unlikely given that previous studies showed that muscarinic receptor blockade with atropine had no effect on cutaneous blood flow under non‐heat‐stress normothermic resting states (Kellogg et al., 1995; Shastry et al., 2000). Further, we recently reported that nicotinic receptor activation elicited by a high dose of nicotine (≥33 mm) can cause cutaneous vasodilatation independently of muscarinic receptors (Fujii et al., 2017a). Consequently, the nicotinic receptor contribution to the regulation of cutaneous vascular tone during rest in a non‐heat‐stress condition may be explained by cutaneous sensory nerve axon reflex caused by nicotinic receptor activation on this nerve (Izumi & Karita, 1992) and/or activation of nicotinic receptors located on endothelial cells of cutaneous vessels (Hagforsen, Edvinsson, Nordlind, & Michaelsson, 2002) independently of muscarinic receptors.

During whole‐body heating, cutaneous vascular conductance gradually increased (Figure 2), which is indicative of heat‐induced cutaneous vasodilatation (Johnson et al., 2014; Smith & Johnson, 2016; Wong & Hollowed, 2017). We found that the reduced cutaneous vascular conductance associated with the administration of hexamethonium observed under normothermic resting states was gradually diminished as oesophageal temperature increased by >0.4°C above baseline resting levels (Figure 2, all P ≥ 0.12). Furthermore, the changes in cutaneous vascular conductance from normothermic baseline at any oesophageal temperature levels and oesophageal temperature threshold for cutaneous vasodilatation were not different between the control and hexamethonium‐treated sites. Thus, our results suggest that nicotinic receptors likely have a limited role in the regulation of cutaneous vasodilatation during whole‐body heating. However, the mechanism(s) underlying the attenuation of nicotinic receptor contribution to cutaneous blood flow during whole‐body heating cannot be elucidated from the present study. It is possible that acetylcholine released from cholinergic nerves would also increase as body temperature increases, which would in turn cause greater activation of muscarinic receptors of cutaneous vessels. Consequently, this would induce a greater muscarinic cutaneous vasodilatation, overriding the influence of nicotinic receptors. In support of this possibility, we recently reported that nicotinic cutaneous vasodilatation is diminished when muscarinic cutaneous vasodilatation is enhanced (Fujii et al., 2017b). Alternatively, the lack of a clear nicotinic receptor contribution to heat‐induced cutaneous vasodilatation may be due to the fact that nicotinic receptor‐mediated cutaneous vasodilatation subsides over time as shown in our current (Figure 1a) and previous work (Fujii et al., 2017b). Since oesophageal temperature was gradually elevated by 1.0°C over a 60–90 min heating period in the present study, vasodilatation induced by the activation of nicotinic receptors may be blunted during the mid‐to‐late stages of whole‐body heating. Nicotinic receptors may contribute to a more rapid increase in cutaneous vasodilatation that would typically occur with conditions that elicit a rapid increase in body temperature such as during immersion in very warm water or an exercise heat stress. Further studies are required to investigate this possibility.

4.3 Sweating during whole‐body heating

Sweat rate and active sweat gland density increased along with elevations in oesophageal temperature (Figure 3b,c,f,g), indicating a thermally induced activation of eccrine sweating. We found that active sweat gland density was reduced by hexamethonium during whole‐body heating at relatively low‐to‐moderate elevations in oesophageal temperature of 0.4–0.6°C (Figure 3b,c). It is possible therefore that nicotinic receptors contribute to increase the number of activated sweat glands in the initial exposure to a heat stress. This is consistent with previous work demonstrating that activation of nicotinic receptors located in cholinergic axons can cause a cholinergic axon reflex, resulting in an increase in the number of active sweat glands (Schlereth et al., 2005). However, although not statistically significant, sweat output per gland during whole‐body heating tended to be greater at the skin site treated with hexamethonium relative to the control site (Figure 3d,e). Therefore, the nicotinic receptor‐induced increase in number of activated sweat glands could be to some extent offset by a concomitant reduction in individual sweat output, resulting in no measurable effects on sweat rate (Figure 3f,g). Along these lines, the changes in sweat rate from normothermic baseline at any level of increase in oesophageal temperature and the oesophageal temperature threshold for the onset of sweating did not differ between the control and hexamethonium‐treated sites.

Our observation of a lack of an influence of nicotinic receptors on sweat rate is in contrast to our previous work wherein we reported that activation of nicotinic receptors increased sweat gland sensitivity to muscarinic receptor agonists such that nicotinic receptor activation lowers the muscarinic receptor agonist threshold for sweating under non‐heat‐stress conditions (Fujii et al., 2017b). It is noteworthy, however, that in our previous work, nicotine‐induced sensitization of muscarinic sweating was not observed at higher sweat rates (Fujii et al., 2017b). Therefore, the lack of a clear effect of nicotinic receptors on sweat rate in the present study may be due to greater sweat rate associated with whole‐body heating. Alternatively, since sweating induced by the administration of nicotine is relatively transient as demonstrated in our current (Figure 1c) and previous work (Fujii et al., 2017b), the effect of nicotinic receptor activation on sweat rate, if any, may be gradually diminished during the mid‐to‐late stages of whole‐body heating in the present study.

4.4 Limitations

There are several limitations to note. We tested young men only in the present study. Thus, our results cannot be simply generalized to other populations such as young women, older men and women, and those with chronic disease conditions. Also, our results were obtained during mild hyperthermia under a resting state (≤1.0°C oesophageal temperature). Hence results might differ at greater increases in oesophageal temperature (i.e. >1.0°C) or when heating manoeuvres cause a faster rate of increase in oesophageal temperature (e.g. warm water immersion). We assessed cutaneous vascular and sweating responses on the forearm that was exposed to room air maintained at ∼25°C (local forearm temperature was 33.35 ± 0.63°C with a corresponding elevation in oesophageal temperature of +1.0°C). Thus, our results may not reflect the responses under ambient heat stress conditions wherein local skin temperature would be higher than that measured in the present study. Our sample size was determined for the primary outcome of our study, which was the assessment of cutaneous vascular conductance and sweat rate (see ‘Statistical analysis’ section). Consequently, it is possible that a greater sample may have been required to evaluate the secondary measures of active sweat gland density and sweat output per individual gland. Lastly, sweat rate and active sweat gland density were assessed from different skin sites. Thus, sweat output per individual gland, which was evaluated as local sweat rate divided by active sweat gland density, could be influenced by the placement of the sweat capsule relative to the insertion of the microdialysis probe on the forearm skin site.

4.5 Perspectives and significance

Our results demonstrated that nicotinic receptors play a limited role, if any, in the heat loss responses of cutaneous vasodilatation and sweating in passively heated resting humans. However, we found that nicotinic receptors contribute to the regulation of cutaneous vascular tone under a resting non‐heat‐stress condition and during the early stage of heating resulting in a relatively small increase in core temperature of 0.4°C. Given acetylcholine, a ubiquitous neurotransmitter throughout the body, can stimulate nicotinic receptors (Pappano, 2011), acetylcholine may be available in the skin even without a substantial elevation in core temperature, activating nicotinic receptors and contributing to the regulation of cutaneous perfusion and therefore dry heat exchange, ultimately influencing body temperature regulation under these conditions. This possibility requires further assessment.

5 CONCLUSION

We showed that nicotinic receptors modulated cutaneous perfusion under normothermia and during the early stage of heating, but no effect of these receptors was observed during whole‐body heating with >0.4°C elevation in core temperature. Further, while we demonstrated that the blockade of nicotinic receptors reduced the number of active sweat glands during whole‐body heating, it was paralleled by a concomitant increase in sweat output per individual gland such that sweat rate remained unchanged.

ACKNOWLEDGEMENTS

We thank all volunteers for participating in this study.

COMPETING INTERESTS

None.

AUTHOR CONTRIBUTIONS

All experiments took place at the Faculty of Health and Sport Sciences, University of Tsukuba. N.F. conceived and designed the experiments. N.F. contributed to data collection. N.F. performed data analysis. N.F., T.A, G.P.K, Y.H., N.K. and T.N. interpreted the experimental results. N.F. drafted the manuscript. N.F., T.A, G.P.K, Y.H., N.K., and T.N. edited and revised the manuscript. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

REFERENCES

Anderson, C., Andersson, T., & Wardell, K. (1994). Changes in skin circulation after insertion of a microdialysis probe visualized by laser Doppler perfusion imaging. Journal of Investigative Dermatology, 102, 807– 811.
Crossref CAS PubMed Web of Science®Google Scholar
Bennett, L. A., Johnson, J. M., Stephens, D. P., Saad, A. R., & Kellogg, D. L., Jr. (2003). Evidence for a role for vasoactive intestinal peptide in active vasodilatation in the cutaneous vasculature of humans. Journal of Physiology, 552, 223– 232.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Buono, M. J., Gonzalez, G., Guest, S., Hare, A., Numan, T., Tabor, B., & White, A. (2010). The role of in vivo β‐adrenergic stimulation on sweat production during exercise. Autonomic Neuroscience, 155, 91– 93.
Crossref CAS PubMed Web of Science®Google Scholar
Cheuvront, S. N., Bearden, S. E., Kenefick, R. W., Ely, B. R., Degroot, D. W., Sawka, M. N., & Montain, S. J. (2009). A simple and valid method to determine thermoregulatory sweating threshold and sensitivity. Journal of Applied Physiology, 107, 69– 75.
Crossref PubMed Web of Science®Google Scholar
Fewings, J. D., Rand, M. J., Scroop, G. C., & Whelan, R. F. (1966). The action of nicotine on the blood vessels of the hand and forearm in man. British Journal of Pharmacology and Chemotherapy, 26, 567– 579.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Fujii, N., Halili, L., Singh, M. S., Meade, R. D., & Kenny, G. P. (2015). Intradermal administration of ATP augments methacholine‐induced cutaneous vasodilation but not sweating in young males and females. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 309, R912– R919.
Crossref CAS PubMed Web of Science®Google Scholar
Fujii, N., Louie, J. C., McNeely, B. D., Amano, T., Nishiyasu, T., & Kenny, G. P. (2017a). Mechanisms of nicotine‐induced cutaneous vasodilation and sweating in young adults: Roles for K_Ca, K_ATP, and K_V channels, nitric oxide, and prostanoids. Applied Physiology, Nutrition and Metabolism, 42, 470– 478.
Crossref CAS PubMed Web of Science®Google Scholar
Fujii, N., Louie, J. C., McNeely, B. D., Zhang, S. Y., Tran, M. A., & Kenny, G. P. (2017b). Nicotinic receptor activation augments muscarinic receptor‐mediated eccrine sweating but not cutaneous vasodilatation in young males. Experimental Physiology, 102, 245– 254.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Fujii, N., Notley, S. R., Minson, C. T., & Kenny, G. P. (2016). Administration of prostacyclin modulates cutaneous blood flow but not sweating in young and older males: Roles for nitric oxide and calcium‐activated potassium channels. Journal of Physiology, 594, 6419– 6429.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Gagnon, D., Crandall, C. G., & Kenny, G. P. (2013). Sex‐differences in post‐synaptic sweating and cutaneous vasodilation. Journal of Applied Physiology, 114, 394– 401.
Crossref CAS PubMed Web of Science®Google Scholar
Gagnon, D., Ganio, M. S., Lucas, R. A., Pearson, J., Crandall, C. G., & Kenny, G. P. (2012). Modified iodine‐paper technique for the standardized determination of sweat gland activation. Journal of Applied Physiology, 112, 1419– 1425.
Crossref PubMed Web of Science®Google Scholar
Greaney, J. L., Stanhewicz, A. E., Kenney, W. L., & Alexander, L. M. (2014). Lack of limb or sex differences in the cutaneous vascular responses to exogenous norepinephrine. Journal of Applied Physiology, 117, 1417– 1423.
Crossref CAS PubMed Web of Science®Google Scholar
Hagforsen, E., Edvinsson, M., Nordlind, K., & Michaelsson, G. (2002). Expression of nicotinic receptors in the skin of patients with palmoplantar pustulosis. British Journal of Dermatology, 146, 383– 391.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Ichinose‐Kuwahara, T., Inoue, Y., Iseki, Y., Hara, S., Ogura, Y., & Kondo, N. (2010). Sex differences in the effects of physical training on sweat gland responses during a graded exercise. Experimental Physiology, 95, 1026– 1032.
Wiley Online Library PubMed Web of Science®Google Scholar
Inoue, Y., Ichinose‐Kuwahara, T., Funaki, C., Ueda, H., Tochihara, Y., & Kondo, N. (2014). Sex differences in acetylcholine‐induced sweating responses due to physical training. Journal of Physiological Anthropology, 33, 13.
Crossref PubMed Web of Science®Google Scholar
Izumi, H., & Karita, K. (1992). Axon reflex flare evoked by nicotine in human skin. Japanese Journal of Physiology, 42, 721– 730.
Crossref CAS PubMed Web of Science®Google Scholar
Johnson, J. M., Minson, C. T., & Kellogg, D. L., Jr. (2014). Cutaneous vasodilator and vasoconstrictor mechanisms in temperature regulation. Comprehensive Physiology, 4, 33– 89.
Wiley Online Library PubMed Web of Science®Google Scholar
Kellogg, D. L., Jr., Pergola, P. E., Piest, K. L., Kosiba, W. A., Crandall, C. G., Grossmann, M., & Johnson, J. M. (1995). Cutaneous active vasodilation in humans is mediated by cholinergic nerve cotransmission. Circulation Research, 77, 1222– 1228.
Crossref CAS PubMed Web of Science®Google Scholar
Kolka, M. A., Stephenson, L. A., Allan, A. E., & Rock, P. B. (1989). Atropine‐induced cutaneous vasodilation decreases esophageal temperature during exercise. American Journal of Physiology, 257, R1089– R1095.
CAS PubMed Web of Science®Google Scholar
Machado‐Moreira, C. A., McLennan, P. L., Lillioja, S., van Dijk, W., Caldwell, J. N., & Taylor, N. A. S. (2012). The cholinergic blockade of both thermally and non‐thermally induced human eccrine sweating. Experimental Physiology, 97, 930– 942.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Meade, R. D., Louie, J. C., Poirier, M. P., McGinn, R., Fujii, N., & Kenny, G. P. (2016). Exploring the mechanisms underpinning sweating: The development of a specialized ventilated capsule for use with intradermal microdialysis. Physiological Reports, 4, e12738.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Pappano, A. J. (2011). Cholinoceptor‐activating & cholinesterase‐inhibiting drugs. In B. G. Katzung, S. B. Masters, & A. J. Trevor (Eds.), Basic and Clinical Pharmacology ( 12th edn, pp. 97– 113). New York: McGraw‐Hill Medical.
Google Scholar
Roddie, I. C., Shepherd, J. T., & Whelan, R. F. (1957). The contribution of constrictor and dilator nerves to the skin vasodilation during body heating. Journal of Physiology, 136, 489– 497.
Wiley Online Library CAS PubMed Web of Science®Google Scholar
Schlereth, T., Brosda, N., & Birklein, F. (2005). Spreading of sudomotor axon reflexes in human skin. Neurology, 64, 1417– 1421.
Crossref PubMed Web of Science®Google Scholar
Shastry, S., Minson, C. T., Wilson, S. A., Dietz, N. M., & Joyner, M. J. (2000). Effects of atropine and L‐NAME on cutaneous blood flow during body heating in humans. Journal of Applied Physiology, 88, 467– 472.
Crossref CAS PubMed Web of Science®Google Scholar
Shibasaki, M., Wilson, T. E., Cui, J., & Crandall, C. G. (2002). Acetylcholine released from cholinergic nerves contributes to cutaneous vasodilation during heat stress. Journal of Applied Physiology, 93, 1947– 1951.
Crossref CAS PubMed Web of Science®Google Scholar
Smith, C. J., & Johnson, J. M. (2016). Responses to hyperthermia. Optimizing heat dissipation by convection and evaporation: Neural control of skin blood flow and sweating in humans. Autonomic Neuroscience, 196, 25– 36.
Crossref PubMed Web of Science®Google Scholar
Stapleton, J. M., Fujii, N., McGinn, R., McDonald, K., Carter, M., & Kenny, G. P. (2014). Age‐related differences in postsynaptic increases in sweating and skin blood flow postexercise. Physiological Reports, 2, e12078.
Wiley Online Library CAS PubMed Google Scholar
Taylor, W. F., Johnson, J. M., O'Leary, D., & Park, M. K. (1984). Effect of high local temperature on reflex cutaneous vasodilation. Journal of Applied Physiology, 57, 191– 196.
Crossref CAS PubMed Web of Science®Google Scholar
Wenger, C. B., & Roberts, M. F. (1980). Control of forearm venous volume during exercise and body heating. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology, 48, 114– 119.
Crossref CAS PubMed Web of Science®Google Scholar
Wong, B. J., & Hollowed, C. G. (2017). Current concepts of active vasodilation in human skin. Temperature, 4, 41– 59.
Crossref Google Scholar

Citing Literature

Volume104, Issue12

1 December 2019

Pages 1808-1818

Nicotinic receptors modulate skin perfusion during normothermia, and have a limited role in skin vasodilatation and sweating during hyperthermia