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The Journal of Physiology logoLink to The Journal of Physiology
. 2016 Mar 11;594(12):3439–3452. doi: 10.1113/JP271735

Endothelin‐1 modulates methacholine‐induced cutaneous vasodilatation but not sweating in young human skin

Lyra Halili 1, Maya Sarah Singh 1, Naoto Fujii 1, Lacy M Alexander 2, Glen P Kenny 1,✉
PMCID: PMC4908015  PMID: 26846374

Abstract

Key points

  • Endothelin‐1 (ET‐1) is a potent endothelial‐derived vasoconstrictor that may modulate cholinergic cutaneous vascular regulation. Endothelin receptors are also expressed on the human eccrine sweat gland, although it remains unclear whether ET‐1 modulates cholinergic sweating.

  • We investigated whether ET‐1 attenuates cholinergic cutaneous vasodilatation and sweating through a nitric oxide synthase (NOS)‐dependent mechanism. Our findings show that ET‐1 attenuates methacholine‐induced cutaneous vasodilatation through a NOS‐independent mechanism.

  • We also demonstrate that ET‐1 attenuates cutaneous vasodilatation in response to sodium nitroprusside, suggesting that ET‐1 diminishes the dilatation capacity of vascular smooth muscle cells.

  • We show that ET‐1 does not modulate methacholine‐induced sweating at any of the administered concentrations.

  • Our findings advance our knowledge pertaining to the peripheral control underpinning the regulation of cutaneous blood flow and sweating and infer that ET‐1 may attenuate the heat loss responses of cutaneous blood flow, but not sweating.

Abstract

The present study investigated the effect of endothelin‐1 (ET‐1) on cholinergic mechanisms of end‐organs (i.e. skin blood vessels and sweat glands) for heat dissipation. We evaluated the hypothesis that ET‐1 attenuates cholinergic cutaneous vasodilatation and sweating through a nitric oxide synthase (NOS)‐dependent mechanism. Cutaneous vascular conductance (CVC) and sweat rate were assessed in three protocols: in Protocol 1 (n = 8), microdialysis sites were perfused with lactated Ringer solution (Control), 40 pm, 4 nm or 400 nm ET‐1; in Protocol 2 (n = 11) sites were perfused with lactated Ringer solution (Control), 400 nm ET‐1, 10 mm NG‐nitro‐l‐arginine (l‐NNA; a NOS inhibitor) or a combination of 400 nm ET‐1 and 10 mm l‐NNA; in Protocol 3 (n = 8), only two sites (Control and 400 nm ET‐1) were utilized to assess the influence of ET‐1 on the dilatation capacity of vascular smooth muscle cells (sodium nitroprusside; SNP). Methacholine (MCh) was co‐administered in a dose‐dependent manner (0.0125, 0.25, 5, 100, 2000 mm, each for 25 min) at all skin sites. ET‐1 at 400 nm (P < 0.05) compared to lower doses (40 pm and 4 nm) (all P > 0.05) significantly attenuated increases in CVC in response to 0.25 and 5 mm MCh. A high dose of ET‐1 (400 nm) co‐infused with l‐NNA further attenuated CVC during 0.25, 5 and 100 mm MCh administration relative to the ET‐1 site (all P < 0.05). Cutaneous vasodilatation in response to SNP was significantly blunted after administration of 400 nm ET‐1 (P < 0.05). We show that ET‐1 attenuates cutaneous vasodilatation through a NOS‐independent mechanism, possibly through a vascular smooth muscle cell‐dependent mechanism, and methacholine‐induced sweating is not altered by ET‐1.

Key points

  • Endothelin‐1 (ET‐1) is a potent endothelial‐derived vasoconstrictor that may modulate cholinergic cutaneous vascular regulation. Endothelin receptors are also expressed on the human eccrine sweat gland, although it remains unclear whether ET‐1 modulates cholinergic sweating.

  • We investigated whether ET‐1 attenuates cholinergic cutaneous vasodilatation and sweating through a nitric oxide synthase (NOS)‐dependent mechanism. Our findings show that ET‐1 attenuates methacholine‐induced cutaneous vasodilatation through a NOS‐independent mechanism.

  • We also demonstrate that ET‐1 attenuates cutaneous vasodilatation in response to sodium nitroprusside, suggesting that ET‐1 diminishes the dilatation capacity of vascular smooth muscle cells.

  • We show that ET‐1 does not modulate methacholine‐induced sweating at any of the administered concentrations.

  • Our findings advance our knowledge pertaining to the peripheral control underpinning the regulation of cutaneous blood flow and sweating and infer that ET‐1 may attenuate the heat loss responses of cutaneous blood flow, but not sweating.

Abbreviations

ACh

acetylcholine

BL

baseline

CI

confidence interval

COX

cyclooxygenase

CVC

cutaneous vascular conductance

EDHF

endothelium‐dependent hyperpolarizing factor

ET‐1

endothelin‐1

l‐NNA

NG‐nitro‐l‐arginine

MCh

methacholine

NO

nitric oxide

NOS

nitric oxide synthase

SNP

sodium nitroprusside

VSMC

vascular smooth muscle cell

Introduction

The control mechanisms of skin blood flow and sweating are crucial for regulating human heat exchange. An important action that can mediate thermoregulatory heat loss responses is the release of the neurotransmitter acetylcholine (ACh) from cholinergic nerves (Kellogg et al. 1995; Machado‐Moreira et al. 2012). The peripheral function of these responses is commonly assessed by ACh and/or methacholine (MCh; an ACh mimetic)‐induced sweating (Inoue et al. 1999; Kimura et al. 2007; Metzler‐Wilson et al. 2014) and cutaneous vasodilatation (Kellogg et al. 2005; Kimura et al. 2007; Medow et al. 2008; Bruning et al. 2012; Fujii et al. 2014). Currently, the precise factors and associated mechanisms that modulate cholinergic cutaneous vasodilatation and sweating are not well understood.

An important peptide that may modulate cholinergic cutaneous vascular and sweating responses is endothelin‐1 (ET‐1). ET‐1 is synthesized in the vascular endothelial cells and is a potent vasoconstrictor (Yanagisawa et al. 1988) that has been associated with the pathophysiology of several vascular diseases (Cardillo et al. 2000; Bohm et al. 2002 a). It is important to note that although plasma ET‐1 levels are maintained within a normal limit in young, healthy, control subjects relative to clinical populations (Yokoi et al. 2012; Cooke et al. 2015), elevated plasma ET‐1 levels have been reported in individuals with hypercholesterolaemia (Haak et al. 1994) and enhanced vascular activity of endogenous ET‐1 has been observed in obese hypertension (Cardillo et al. 2004). Moreover, compromised arterial compliance in hypertension (Ergul et al. 2006) has been associated with elevated plasma ET‐1 levels and increased activity of ET‐1 may be involved in the development of early stages of atherosclerosis (Ihling et al. 2001). Furthermore, there are two major classes of ET‐1 receptor subtypes (ETA and ETB), both of which can functionally contribute to cutaneous vascular regulation (Lipa et al. 1999; Kellogg et al. 2001; Wenner et al. 2013). The presence of both ETA and ETB receptors have been confirmed on the human eccrine sweat glands (Knock et al. 1993), however, the implications of these receptors on eccrine sweating are presently unknown.

Infusion of ET‐1 in the human brachial artery has been reported to cause marked decreases in endothelium‐dependent vasodilatation as induced by ACh administration (Bohm et al. 2002 b). Currently, the mechanism responsible for this attenuated vasodilatation is an ET‐1‐mediated reduction of nitric oxide (NO) bioavailability (Chen et al. 2003; Dong et al. 2005; Wedgwood & Black, 2005; Ramzy et al. 2006). In addition, it is notable that NO is a well‐known modulator of both cutaneous blood flow and sweating in humans (Kellogg et al. 1998; Shastry et al. 2000; Lee & Mack, 2006; Welch et al. 2009; McNamara et al. 2014; Meade et al. 2015). Therefore, ET‐1 may attenuate cholinergic cutaneous vasodilatation and sweating through NO synthase (NOS)‐dependent mechanisms, although this possibility remains to be directly elucidated.

The primary objective of the present study was to investigate whether and how ET‐1 modulates pharmacologically induced cholinergic (MCh) cutaneous vasodilatation and sweating in humans in vivo. We hypothesized that ET‐1 would attenuate MCh‐induced cutaneous vasodilatation and sweating, and that this response would be mediated through NOS‐dependent mechanisms.

Methods

Ethical approval

The present study conforms to the guidelines set out by the Declaration of Helsinki. Approval by the University of Ottawa Health Sciences and Science Research Ethics Board was attained. Verbal and written informed consent was obtained from all volunteers prior to their participation in the study. The experimental protocol as well as the risks and discomfort associated with participating in the study were thoroughly explained.

Participants

Twenty‐seven, habitually active (1–7 days per week, ≥30 min per day) young adults (12 females and 15 males) participated in one or more of the three experimental protocols. In Protocol 1 (4 females and 4 males), age, body mass and height were (mean ± SD): 20 ± 2 years, 72.9 ± 19.1 kg and 1.71 ± 0.10 m, respectively. In Protocol 2 (5 females and 6 males), age, body mass and height were (mean ± SD): 22 ± 3 years, 68.2 ± 9.7 kg and 1.71 ± 0.07 m, respectively. In Protocol 3 (3 females and 5 males), age, body mass and height were (mean ± SD): 22 ± 5 years, 69.6 ± 13.9 kg and 1.71 ± 0.07 m, respectively. Four of the 12 females were using contraceptives (3 females were using oral contraceptives and 1 female was using an intra‐uterine device). All female subjects participated in the experimental session during the early follicular phase (within 6 days of starting menstruation) or during the placebo stage, if using contraceptives. The previously reported effects of female sex hormones on the regulation of cutaneous vasodilatation and sweating (Kuwahara et al. 2005; Brunt et al. 2011) were accounted for by controlling for menstrual cycle in all three protocols.

Experimental design

All participants volunteered for an experimental session. It was requested that participants refrain from taking over‐the‐counter medication (i.e. non‐steroidal anti‐inflammatory drugs, allergy medication and vitamins) for at least 48 h before experimentation. Participants refrained from strenuous physical activity, caffeine and alcohol consumption for at least 12 h prior to taking part in the study. Approximately 2 h before the trial, participants were permitted to consume a regular meal. Body mass was measured using a digital weight scale platform (Model CBU150X, Mettler Toledo Inc., Columbus, OH, USA) accompanied by a weighing terminal (Model IND560, Mettler Toledo) and height was acquired using an eye‐level stadiometer (Model 2391, Detecto Scale Co., Webb City, MO, USA). Participants were then asked to rest on a bed in a semi‐recumbent position. Thereafter, four (Protocols 1 and 2) or two (Protocol 3) intradermal microdialysis fibres (30 kDa cutoff, 10 mm membrane; MD2000, Bioanalytical Systems, West Lafayette, IN, USA) were inserted in the dermal layer of the skin on the dorsal side of the left forearm. Fibre insertions were performed under aseptic conditions; firstly, a 25 gauge needle was inserted into the unanaesthetized skin and then exited ∼2.5 cm away from the insertion site. Secondly, a microdialysis fibre was threaded through the lumen of the needle and thereafter both the needle and the fibre were simultaneously pulled away from the exit site. The fibre remained intact within the skin and the needle was safely discarded. Both ends of the fibre were secured with surgical tape. Each insertion was separated by a minimum of 3.0 cm and the ends of the fibres were connected to the outlet port of a liquid switcher (Model 110, CMA Microdialysis AB, Kista, Sweden). Approximately 15 min later, lactated Ringer solution (Baxter, Deerfield, IL, USA) was perfused at a rate of 4.0 μl min−1 (60 to 120 min) in order for hyperaemic trauma to subside.

Protocol 1

This protocol aimed to determine which concentration of ET‐1 (Sigma Aldrich, St Louis, MO, USA) would induce a substantial reduction in MCh‐induced cutaneous vasodilatation and/or sweating. Following the completion of baseline (BL) (∼10 min), pharmacological agents were perfused through the microdialysis probes. The fibres were assigned in a randomized manner to receive either: (1) lactated Ringer solution (Control), or three different concentrations of ET‐1: (2) 40 pm, (3) 4 nm and (4) 400 nm. Upon completion of BL, a new pharmacological baseline with ET‐1 treatment (Treatment BL) was acquired for a minimum of 30 min. During Treatment BL, lactated Ringer solution and the three concentrations of ET‐1 were continuously perfused at all four randomized sites at a rate of 4.0 μl min−1.

Upon completion of Treatment BL measurements, each microdialysis fibre was perfused incrementally for 25 min with MCh (0.0125, 0.25, 5, 100 and 2000 mm; Sigma‐Aldrich) at a rate of 4.0 μl min−1. These concentrations of MCh were chosen based on our previous work (Fujii et al. 2015), suggesting that an all‐encompassing range of MCh concentrations is necessary to induce low to high levels of cutaneous vascular and sweating responses. A 25 min perfusion period for each stage of MCh administration was necessary to ensure that a stable plateau in cutaneous vascular conductance (CVC) and sweat rate was observed for a minimum of 2 min prior to administering subsequent MCh concentrations. To evaluate maximal CVC, 50 mm of sodium nitroprusside (SNP; Sigma‐Aldrich) was administered at a rate of 6.0 μl min−1 after completing the final concentration of MCh (2000 mm). SNP at 50 mm was used for maximal cutaneous vasodilatation since our pilot work confirmed that this concentration induces maximal cutaneous vasodilatation comparable to what is observed during local heating at 44°C. Administration of SNP continued for ∼30 to 60 min until a stable plateau for a minimum of 2 min was observed. Blood pressure was then measured approximately every 5 to 10 min using manual auscultation to quantify maximal CVC.

Protocol 2

Protocol 2 was employed to determine whether the ET‐1‐mediated attenuation of MCh‐induced cutaneous vasodilatation resulted from NOS‐dependent mechanisms. The procedure followed for Protocol 2 was identical to Protocol 1 with the exception that the four sites in Protocol 2 were continuously perfused with either (1) lactated Ringer solution (Control), (2) 400 nm ET‐1 (ET‐1), (3) 10 mm NG‐nitro‐l‐arginine (l‐NNA; Sigma‐Aldrich) to non‐selectively inhibit NOS, or (4) combined perfusion of 400 nm ET‐1 and 10 mm l‐NNA (ET‐1 and l‐NNA). Treatment BL for Protocol 2 included perfusion of lactated Ringer solution, 10 mm l‐NNA, 400 nm ET‐1 and a combination of 10 mm l‐NNA and 400 nm ET‐1 at four intradermal microdialysis sites. The concentration of l‐NNA was determined based on a previous study that utilized l‐NNA in the human skin using intradermal microdialysis (Medow et al. 2008).

Protocol 3

This protocol was developed to evaluate the effect of ET‐1 on cutaneous vascular smooth muscle cell (VSMC) function. Two skin sites were perfused with either: (1) lactated Ringer solution (Control) or (2) 400 nm ET‐1 (ET‐1). Prior to the perfusion of ET‐1 and MCh (referred to as the ‘Pre’ stage), 50 mm SNP was administered at both skin sites for 1 min at a rate of 100 μl min−1. Thereafter, approximately 60 to 120 min perfusion of Ringer solution was necessary to void both sites of SNP. Subsequently, Treatment BL and the dose‐dependent perfusion of MCh commenced as described in Protocols 1 and 2 (above), and were followed by a final SNP administration (referred to as the ‘Post’ stage).

Measurements

Cutaneous red blood cell flux (perfusion units mmHg−1) was measured locally as an index of cutaneous blood flow. A sampling rate of 32 Hz with laser Doppler flowmetry (PeriFlux System 5000, Perimed, Stockholm, Sweden) was used. In all three protocols, integrated laser Doppler flowmetry probes with a seven‐laser array (Model 413, Perimed) were situated directly above and in the centre of each sweat capsule over each microdialysis fibre. This allowed for concurrent measurement of both cutaneous red blood cell flux and local forearm sweat rate at four (Protocols 1 and 2) or two (Protocol 3) microdialysis sites. For all three protocols, manual auscultation was performed using a validated mercury column sphygmomanometer (Baumanometer Standby Model, WA Baum Co., Copiague, NY, USA) so as to obtain blood pressures at 5 to 10 min intervals. Mean arterial pressure was calculated using diastolic arterial pressure plus one‐third the difference between systolic and diastolic pressures (pulse pressure). To quantify CVC, cutaneous red blood cell flux was divided by mean arterial pressure. The data presented for CVC are displayed as a percentage of maximum (%CVCmax).

Local sweat production was measured using a ventilated sweat capsule technique. The sweat capsules covered the microdialysis membrane and were secured to the skin using adhesive rings and topical skin glue (Collodian HV, Mavidon Medical Products, Lake Worth, FL, USA). Dry compressed air maintained at room temperature (∼25°C) was used to ventilate each sweat capsule. Using high precision dew point mirrors (Model 473, RH Systems, Albuquerque, NM, USA) water content of the effluent air from the sweat capsule was measured. To connect the sweat capsules to the gas tanks and the dew point mirrors, long vinyl tubes were used, ensuring that internal gas temperature remained equilibrated to room temperature. Local forearm sweat rate was calculated every 5 s using the difference in water content between influent and effluent air. This difference was multiplied by flow rate and then normalized for the skin surface area under the capsule (mg min–1 cm–2).

Data analysis

Data from both men and women was combined for analysis, as there were no observed differences in response between sexes. For Protocols 1 and 2, the calculated CVC for each treatment site was expressed as a percentage of maximum CVC (%CVCmax). Absolute CVC was used for Protocol 3 to compare CVC during SNP administration at both Pre and Post stages (as described above). Presented in Figs 1 and 2 are the last 5 min averages of each stage (BL, Treatment BL, 0.0125, 0.25, 5, 100 and 2000 mm MCh) of drug perfusion. In Protocol 2, the change in CVC (∆CVC) relative to the Control site was evaluated at the ET‐1, l‐NNA and ET‐1 + l‐NNA sites (Fig. 3). In addition, in Protocol 2 ET‐1‐dependent vasoconstriction in the absence of NOS inhibition was evaluated as the difference in CVC between the Control and the ET‐1 skin sites. Similarly, ET‐1‐dependent vasoconstriction in the presence of NOS inhibition was also evaluated as the difference in CVC between l‐NNA and ET‐1 + l‐NNA. Peak values during SNP administration (Pre and Post) are presented as absolute CVC in Fig. 4. The values for the final 5 min of sweat rate data for each stage were used and presented in Figs 5 and 6. For all three protocols, a 12 min time delay was considered for data analysis. This delay represents the time taken for the pharmacological agents to elicit cutaneous vasodilatation and/or sweating after the start of infusion.

Figure 1. Cutaneous vascular conductance during methacholine administration .

Figure 1

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A, cutaneous vascular conductance (CVC, represented as %CVCmax) at Baseline (BL, with and without treatment) and during methacholine (MCh) administration from 0.0125 to 2000 mm at four intradermal microdialysis sites. The four sites were perfused with either (1) lactated Ringer solution (Control, circles), (2) 40 pm ET‐1 (squares), (3) 4 nm ET‐1 (triangles) or (4) 400 nm ET‐1 (diamonds). Values are presented as mean ± 95% CI (n = 8). †Significant differences relative to Control at the 400 nm site (P < 0.05). B, boxplots display medians and quartiles for individual changes (n = 8) in CVC from Control at the 40 pm, 4 nm and 400 nm ET‐1 sites during administration of 5 mm MCh. Using a Tukey analysis, outliers represented by crosses show individual points that are below Q1 (first quartile, 25th percentile) – 1.5 × IQR (interquartile range, Q3 – Q1) or above Q3 (third quartile, 75th percentile) + 1.5 × IQR.

Figure 2. Cutaneous vascular conductance during methacholine administration .

Figure 2

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A, cutaneous vascular conductance (CVC, represented as %CVCmax) at Baseline (BL, with and without treatment) and during methacholine (MCh) administration from 0.0125 to 2000 mm at four intradermal microdialysis sites. Sites were perfused with either (1) lactated Ringer solution (Control, circles), (2) 400 nm ET‐1 (ET‐1, squares), (3) 10 mm N G‐nitro‐l‐arginine (l‐NNA, triangles) or (4) a combination of 400 nm ET‐1 and 10 mm l‐NNA (ET‐1 + l‐NNA, diamonds). Values are presented as mean ± 95% CI (n = 11). †Significant differences relative to Control at the ET‐1, l‐NNA and ET‐1 + l‐NNA sites for the three concentrations (0.25, 5 and 100 mm) of MCh (P < 0.05). ‡Significant differences between the ET‐1 and the ET‐1 + l‐NNA sites at the same three concentrations of MCh (P < 0.05). B, boxplots display medians and quartiles for individual changes (n = 11) in CVC from Control at the ET‐1, l‐NNA and ET‐1 + l‐NNA sites during administration of 5 mm MCh. Using a Tukey analysis, outliers represented by crosses show individual points that are below Q1 (first quartile, 25th percentile) – 1.5 × IQR (interquartile range, Q3 – Q1) or above Q3 (third quartile, 75th percentile) + 1.5 × IQR.

Figure 3. Change in cutaneous vascular conductance during methacholine administration .

Figure 3

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A, change in cutaneous vascular conductance (∆CVC, represented as %CVCmax) between sites during 0.25, 5 and 100 mm methacholine (MCh) administration. The left and right columns represent the differences for Control vs. ET‐1, and l‐NNA vs. ET‐1 + l‐NNA, respectively, at the three displayed concentrations of MCh. The values are presented as mean ± 95% CI (n = 11). The P‐values represent the level of significance between the two comparisons (Control vs. ET‐1, and l‐NNA vs. ET‐1 + l‐NNA) for each concentration of MCh. All ∆CVC values presented are significantly different from each zero (i.e. reference) value (P < 0.05) with the exception of the ∆CVC between the l‐NNA and the ET‐1 + l‐NNA site (P = 0.06). B, boxplots display medians and quartiles for individual changes (n = 11) in CVC for Control vs. ET‐1, and l‐NNA vs. ET‐1 + l‐NNA, at the three concentrations of MCh. Using a Tukey analysis, outliers represented by crosses show individual points that are below Q1 (first quartile, 25th percentile) – 1.5 × IQR (interquartile range, Q3 – Q1) or above Q3 (third quartile, 75th percentile) + 1.5 × IQR.

Figure 4. Cutaneous vascular conductance during Pre and Post administration of sodium nitroprusside .

Figure 4

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A, absolute cutaneous vascular conductance (CVC, represented in perfusion units mmHg−1) for Protocol 3, during Pre (before ET‐1 and methacholine (MCh) administration) and Post (after completion of the 2000 mm MCh stage) 50 mm sodium nitroprusside (SNP) administration at two intradermal microdialysis sites. Values are presented as mean ± 95% CI (n = 8). †Significance at 400 nm ET‐1 between the Pre and Post stage (P = 0.001). P‐values are presented above the columns during the Pre and Post stage to represent the level of significance between the two sites (400 nm ET‐1 and Control) during Pre (P = 0.995) and Post (P = 0.002) SNP administration. B, boxplots display medians and quartiles for individual (n = 8) absolute CVC between ET‐1 and Control, during Pre and Post SNP administration. Using a Tukey analysis, outliers represented by crosses show individual points that are below Q1 (first quartile, 25th percentile) – 1.5 × IQR (interquartile range, Q3–Q1) or above Q3 (third quartile, 75th percentile) + 1.5 × IQR.

Figure 5. Sweat rate during methacholine administration .

Figure 5

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A, sweat rate (represented as mg min−1 cm−2) at Baseline (BL, with and without treatment) and during five concentrations of methacholine (MCh) (0.0125–2000 mm administration) at four intradermal microdialysis sites. Sites were perfused with either (1) lactated Ringer solution (Control, circles), (2) 40 pm ET‐1 (squares), (3) 4 nm ET‐1 (triangles) or (4) 400 nm ET‐1 (diamonds). Values are presented as mean ± 95% CI (n = 8). B, boxplots display medians and quartiles for individual changes in sweat rate from Control at the 40 pm, 4 nm and 400 nm ET‐1 sites during administration of 100 mm MCh. Using a Tukey analysis, outliers represented by crosses show individual points that are below Q1 (first quartile, 25th percentile) – 1.5 × IQR (interquartile range, Q3 – Q1) or above Q3 (third quartile, 75th percentile) + 1.5 × IQR.

Figure 6. Sweat rate during methacholine administration .

Figure 6

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A, sweat rate (represented as mg min−1 cm−2) at Baseline (BL, with and without treatment) and during five concentrations of methacholine (MCh) (0.0125–2000 mm administration) at four intradermal microdialysis sites. Sites were perfused with either (1) lactated Ringer solution (Control, circles), (2) 400 nm ET‐1 (ET‐1, squares), (3) 10 mm N G‐nitro‐l‐arginine (l‐NNA, triangles) or (4) a combination of 400 nm ET‐1 and 10 mm l‐NNA (ET‐1 + l‐NNA, diamonds). Values are presented as mean ± 95% CI (n = 8). B, boxplots display medians and quartiles for individual changes in sweat rate from Control at the ET‐1, l‐NNA and ET‐1 + l‐NNA sites during administration of 100 mm MCh. Using a Tukey analysis, outliers represented by crosses show individual points that are below Q1 (first quartile, 25th percentile) – 1.5 × IQR (interquartile range, Q3–Q1) or above Q3 (third quartile, 75th percentile) + 1.5 × IQR.

Statistical analysis

The numeric outputs from the software package SPSS 22.0 for Windows (IBM, Armonk, NY, USA) were used to evaluate the statistical values for data obtained in Protocols 1 to 3. CVC and sweat rate (Protocols 1 to 3) measured at each stage (seven levels: BL, Treatment BL, 0.0125, 0.25, 5, 100, 2000 mm MCh) were analysed using a two‐way repeated‐measures ANOVA with two factors of (1) treatment site (four levels: Control, ET‐1 concentrations of 40 pm, 4 nm and 400 nm ET‐1 for Protocol 1; Control, 400 nm ET‐1, 10 mm l‐NNA and 400 nm ET‐1 + 10 mm l‐NNA for Protocol 2; two levels: Control and 400 nm ET‐1 for Protocol 3) and (2) stage of perfusion (seven levels: BL, Treatment BL, 0.0125, 0.25, 5, 100, 2000 mm MCh). For Protocol 3, absolute CVC measured at each stage (two levels: Control and 400 nm ET‐1) was analysed using a two‐way repeated‐measures ANOVA with two factors of (1) treatment site (two levels: Control and 400 nm ET‐1) and (2) stage of perfusion (two levels: Pre SNP administration and Post SNP administration). The assumption of circularity was met for the data set as confirmed by Mauchly's test. If a significant interaction or a main effect of treatment site was observed, post hoc comparisons were conducted using Student's paired t‐tests. Three (Protocol 1: Control vs. 40 pm ET‐1, Control vs. 4 nm ET‐1 and Control vs. 400 nm ET‐1) or four (Protocol 2: Control vs. l‐NNA, Control vs. ET‐1, Control vs. ET‐1 + l‐NNA and ET‐1 vs. ET‐1 + l‐NNA) a priori between‐site comparisons were conducted with P‐values corrected for multiple comparisons using a Holm–Bonferroni procedure. For Protocol 2, two‐tailed Student's paired t‐tests were used to compare the ET‐1‐dependent vasoconstriction in the absence and presence of NOS inhibition, as well as whether the ∆CVC at each treatment site was significantly different from each zero (i.e. reference) value (Fig. 3). For Protocol 3, between‐ and within‐site comparisons were assessed with Student's paired t tests as follows: Pre vs. Post SNP administration at the 400 nm ET‐1 site; Pre vs. Post SNP administration at Control; Pre SNP administration between 400 nM ET‐1 site and Control; and Post SNP administration between 400 nm ET‐1 site and Control.

The data presented were normally distributed and the level of significance was set at P ≤ 0.05. The 95% confidence interval (CI) was calculated using 1.96 × SEM and all values are presented as mean ± 95% CI.

Results

Cutaneous vascular response

ET‐1‐mediated modulation of MCh‐induced cutaneous vasodilatation

The cutaneous blood flow responses to incremental concentrations of ET‐1 are illustrated in Fig. 1. CVC at the 400 nm ET‐1 site was lower relative to Control during 0.25 (40 ± 13 vs. 75 ± 10 %CVCmax, P = 0.018) and 5 mm (55 ± 11 vs. 91 ± 11 %CVCmax, P = 0.010) MCh administration. Furthermore, CVC at the Control site did not differ across all treatment sites during BL (all P > 0.80) and Treatment BL (all P > 0.56) (Fig. 1).

For Protocol 2, there was no difference in CVC at the Control site during BL and Treatment BL (all P > 0.90) across all treatment sites. During administration of three concentrations of MCh (0.25, 5 and 100 mm), CVC values at the ET‐1, l‐NNA and ET‐1 + l‐NNA sites were lower relative to Control (all P < 0.05; Fig. 2). Furthermore, CVC values were greater at the ET‐1 site than at the ET‐1 + l‐NNA site during 0.25 mm (43 ± 14 vs. 23 ± 9 %CVCmax, P = 0.03), 5 mm (61 ± 12 vs. 35 ± 10 %CVCmax, P = 0.005) and 100 mm (70 ± 10 vs. 43 ± 12 %CVCmax, P = 0.001) MCh. The magnitude of an ET‐1 component was assessed by calculating and comparing the delta changes in CVC between the Control and ET‐1 sites to those between the l‐NNA and ET‐1 + l‐NNA sites (Fig. 3). The calculated ET‐1 contribution was as follows during 0.25 mm (18 ± 15 vs. 13 ± 12 %CVCmax, P = 0.496), 5 mm (24 ± 13 vs. 14 ± 13 %CVCmax, P = 0.272) and 100 mm (19 ± 12 vs. 19 ± 14 %CVCmax, P = 0.987) MCh administration.

In Protocol 3, absolute CVC values during Pre relative to Post SNP administration were higher (perfusion units mmHg−1: 1.66 ± 0.30 vs. 0.80 ± 0.17, P = 0.001) at the ET‐1 site (Fig. 4). CVC in response to SNP administration was significantly blunted after co‐infusion of 400 nm ET‐1 and MCh at the ET‐1 site (P < 0.05). Additionally, there were no within‐site differences in absolute CVC between the Control and ET‐1 sites (perfusion units mmHg−1: 1.66 ± 0.43 vs. 1.66 ± 0.30) during SNP administration prior to ET‐1 and MCh perfusion (Pre) (P = 0.995). However, after completion of the 2000 mm MCh stage during the Post SNP administration, we observed a within‐site difference in absolute CVC between Control and ET‐1 (perfusion units mmHg−1: 0.80 ± 0.17 vs. 2.17 ± 0.40, P = 0.002) in which the ET‐1 site was significantly lower than Control (Fig. 4). Similar to the results obtained in Protocols 1 and 2, CVC at the ET‐1 site was significantly lower than Control during MCh administration (all P < 0.05).

Maximal absolute CVC

In Protocols 1, 2, and 3, maximal CVC at Control was similar across all skin sites (e.g. Protocol 1, P = 0.266 for main effect of treatment site; perfusion units mmHg−1: Control, 1.69 ± 0.29; 40 pm ET‐1, 1.30 ± 0.22; 4 nm ET‐1, 1.48 ± 0.24; 400 nm ET‐1, 1.52 ± 0.34).

Sweat rate

Sweat rate was collected for all three protocols. Across all treatment sites, there were no differences in sweat rate for Protocols 1 (Fig. 5), 2 (Fig. 6) and 3 (all P > 0.567 for main effect of treatment site and interaction between treatment site and stage of perfusion).

Discusion

In the present study we investigated the role of ET‐1 in cholinergic cutaneous vasodilatation and sweating in humans in vivo. In accordance with our hypothesis, we show that a high concentration of ET‐1 (400 nm) attenuated MCh‐induced cutaneous vasodilatation (0.25 and 5 mm), and that this reduction was mediated through a NOS‐independent mechanism. Furthermore, we observed that ET‐1 attenuates cutaneous vasodilatation in response to SNP, suggesting that ET‐1 diminishes or competes against the dilatation capacity of VSMCs. In contrast to our observed influences of ET‐1 on cutaneous vasodilatation, we show that cholinergic sweating was not altered at any of the administered concentrations of ET‐1. Collectively, our findings demonstrate that ET‐1 attenuates cholinergic vasodilatation through a NOS‐independent mechanism, and it does not modulate cholinergic sweating.

Cutaneous vascular response

ET‐1‐mediated vasoconstriction

In Protocol 1, we show that increases in CVC during administration of low (0.25 and 5 mm) concentrations of MCh were markedly attenuated by the highest dose of ET‐1 (Fig. 1). Importantly, both ETA and ETB receptors are located on the VSMC and previous studies suggest that activation of ETA receptors mediates the primary ET‐1 vasoconstrictor effect, both under non‐heat stressed conditions (Lipa et al. 1999) and during local skin heating (Wenner et al. 2013). Therefore, ET‐1‐mediated reductions in MCh‐induced cutaneous vasodilatation may be mediated by activation of ETA receptors. We also found that NO contributes to MCh‐induced cutaneous vasodilation (Fig. 2), which is consistent with previous studies using MCh (Fujii et al. 2014) and ACh (Kellogg et al. 2005; Medow et al. 2008; Bruning et al. 2012). In view of this, it is possible that ET‐1 administration causes diminished cholinergic cutaneous vasodilation as a result of NOS‐dependent mechanisms. This is further supported by numerous reports demonstrating that ET‐1 can decrease NO bioavailability (Chen et al. 2003; Dong et al. 2005; Wedgwood & Black, 2005; Ramzy et al. 2006), including in the human brachial artery (Bohm et al. 2002 b). However, unlike the human brachial artery, our findings demonstrate that ET‐1 administration attenuates cholinergic vasodilatation in the presence of NOS inhibition (Fig. 2) in human skin.

In addition to NOS, muscarinic receptor (M3) activation can induce vasodilatation through other major endothelium‐dependent pathways, which includes cyclooxygenase (COX) and endothelium‐dependent hyperpolarizing factors (EDHFs). With regard to COX mechanisms, ET‐1 appears to attenuate COX‐dependent vasodilatation as evidenced by reduced prostaglandin E2‐mediated increases in cutaneous blood flow (Crossman et al. 1991). However, as MCh‐induced cutaneous vasodilatation occurs independently of COX (Fujii et al. 2014), it is unlikely that ET‐1‐mediated attenuation of cholinergic cutaneous vasodilation is explained by reductions in COX‐derived vasodilator prostanoids. EDHFs, which can contribute to the regulation of cutaneous vascular control (Lorenzo & Minson, 2007; Brunt & Minson, 2012; Cracowski et al. 2013), may be involved in the ET‐1‐mediated modulation of cholinergic cutaneous vasodilatation. As such, it is possible that ET‐1 may be involved in attenuating VSMC hyperpolarization.

NOS‐independent mechanism

In contrast to a previous study (Bohm et al. 2002 b), which showed that ET‐1 administration did not alter SNP‐induced vasodilatation in the human brachial artery, we found that cutaneous vasodilatation to SNP was higher prior to ET‐1 and MCh administration (Fig. 4). These data suggest impaired VSMC function, which may be associated with ET‐1‐induced activation of RhoA/Rho‐kinase (ROCK) (Miao et al. 2002), subsequently causing vasoconstriction. Alternatively, ET‐1 may attenuate hyperpolarization in VSMCs. This proposed mechanism for our findings is supported by the fact that ETA receptor stimulation by ET‐1 can attenuate large conductance Ca2+‐activated K+ (BKCa) channels (Betts & Kozlowski, 2000), which are primarily located on the VSMCs and can cause hyperpolarization (Archer et al. 2003). Lastly, ET‐1 binding with ETA receptors on the VSMCs may stimulate an increase in intracellular Ca2+, thereby mediating vasoconstriction. In line with this, as an agonist, endogenous ET‐1 can increase cytosolic Ca2+ (Bourque et al. 2011), thereby inducing VSMC contraction.

Effect of ET‐1 on Baseline CVC

Interestingly, our findings indicate that ET‐1 does not affect CVC during Baseline conditions (Figs 1 and 2), which is in contrast to a previous study in which ET‐1 caused marked cutaneous vasoconstriction (Brain et al. 1992). Although the precise reasons underlying this discrepancy remain unknown, it is possible that this can be associated with the fact that the previous study by Brain and colleagues directly injected ET‐1 into the skin whereas we continuously delivered ET‐1 through a microdialysis technique. Additionally, ET‐1 can cause both vasodilatation (via ETB receptors) (Wenner et al. 2013) and vasoconstriction (via ETA receptors) (Lipa et al. 1999). As intradermal administration of ET‐1 may activate both receptor subtypes, it is possible that a compensatory or balancing effect is induced, which can result in minimal or no change in CVC. In contrast to baseline CVC, our results demonstrate that ET‐1 can reduce CVC during MCh administration. This may be explained by the functional activation of ETB receptors, which can induce the synthesis and release of NO (Hirata et al. 1993). Given that NO bioavailability is low during basal CVC, as evidenced by a lack of an effect of NOS inhibition during treatment baseline, it is possible that ETB receptor activation can increase CVC by increasing NO bioavailability and counteracting the vasoconstrictor effect of ETA receptor activation. However, this ETB receptor‐induced vasodilation may not be able to cause substantial vasodilation during MCh administration, when NO bioavailability is already at a high level, as evidenced by a clear reduction in CVC by l‐NNA. Accordingly, CVC may decline as a result of ETA receptor‐induced vasoconstriction. It is also possible that dose‐dependent differences in responses may occur whereby the vasoconstrictor effect of ETA receptor activation is augmented by higher concentrations of ET‐1. It is therefore plausible that the use of higher concentrations of ET‐1 (> 400 nm) may effectively over‐stimulate ETA receptors, resulting in vasoconstriction during basal CVC in conjunction with MCh administration.

ET‐1 and sweating

Both ET‐1 receptor subtypes (ETA and ETB) are expressed on the human eccrine sweat glands (Knock et al. 1993). Despite these histological findings, we demonstrate that ET‐1 does not directly modulate MCh‐induced sweating in humans in vivo (Fig. 5). As such, the influence of ET‐1 differs between the two thermoeffector organs (i.e. skin blood vessels and sweat glands), which are of critical importance for human heat exchange. Therefore, it may be that the ET‐1 receptors located on the sweat glands are not functionally involved in the process of human sweat secretion. A role for NOS in MCh‐induced sweating was not confirmed in this study, given that sweat rate did not differ between the Control and l‐NNA sites (Fig. 6). This is in contrast to a previous study by Lee & Mack (2006), which demonstrated that NOS does influence cholinergic sweating. However, the current results are consistent with our recent study in which NOS inhibition did not modulate cholinergic sweating during administration of high doses of MCh (1–2000 mm) (Fujii et al. 2014). The discrepancy between the results of the present study and those by Lee & Mack (2006) are unclear. However, one possible explanation is that the present study employed a different NOS inhibitor, i.e. l‐NNA, whereas the previous study employed NG‐nitro‐l‐arginine methyl ester and NG‐monomethyl‐l‐arginine.

Sex‐related differences

The present study did not observe clear sex differences in ET‐1‐mediated modulation of CVC and sweat rate during Baseline and MCh administration. In contrast to our findings, Kellogg et al. (2001) demonstrated that using a specific ETB receptor antagonist (BQ‐788), tonic vasoconstriction is mediated by ETB receptors in males whereas tonic vasodilatation is mediated by the same receptors in females. Our results reflect no sex differences in a net effect of ETA and ETB receptor activation by ET‐1. However, had we employed specific ET‐1 receptor antagonists, differences in responses between males and females in modulating cutaneous vasodilatation and sweating may have been observed. Investigating these differences is interesting and merits future scrutiny.

Perspectives and significance

Previous work has shown that ageing (Westby et al. 2011) and chronic health disorders such as diabetes (Cardillo et al. 2002; Mather et al. 2002) are associated with increased activity of the endothelin system. Recent evidence shows that older adults exhibit impaired sweat gland function (Stapleton et al. 2014 b), specifically mediated by a diminished NO component (Stapleton et al. 2014 a). Given that ET‐1 has been associated with reduced NO bioavailability, it is possible that elevated plasma ET‐1 levels may partly explain these impairments alongside the physiologically reduced ability for older adults to dissipate heat. Future studies are warranted to evaluate the role of ET‐1 and the associated underlying mechanisms of cutaneous vascular and sweating responses among older adults and those with chronic health conditions, when exposed to heat stress conditions such as exercising in the heat.

Conclusion

Our findings demonstrate that while ET‐1 attenuates MCh‐induced cutaneous vasodilatation through a NOS‐independent, possibly VSMC‐dependent mechanism, there was no observed effect of ET‐1 on cholinergic eccrine sweating.

Additional information

Competing interests

None.

Author contributions

L.H., N.F. and G.P.K. conceived and designed the experiments. L.H., M.S.S. and N.F. contributed to data collection. L.H. and M.S.S. performed data analysis and assembly. L.H., M.S.S., N.F., L.M.A. and G.P.K. interpreted the experimental results. L.H. prepared the figures and drafted the manuscript. L.H., M.S.S., N.F., L.M.A. and G.P.K. edited and revised the manuscript. All authors approved the final version of the manuscript. All experiments took place at the Human and Environmental Physiology Research Unit located at the University of Ottawa.

Funding

This study was supported by the Natural Sciences and Engineering Research Council of Canada (Discover grant, RGPIN‐06313‐2014; Discovery Grants Program ‐ Accelerator Supplement, RGPAS‐462252‐2014), and by Canada Foundation for Innovation (Leaders Opportunity Fund, 22529) (funds held by G.P.K.). G.P.K. was supported by a University of Ottawa Research Chair Award. L.H., M.S.S. and N.F. were supported by the Human and Environmental Physiology Research Unit.

Acknowledgements

We would like to sincerely thank all the volunteers for taking their time to participate in this study.

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