Regional contributions of nitric oxide synthase to cholinergic cutaneous vasodilatation and sweating in young men
Funding information:
This study was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN‐06313‐2014 and RGPAS‐462252‐2014; funds held by G.P.K.); G.P.K is supported by a University of Ottawa Research Chair Award. G.W.M. is supported by the Human and Environmental Physiology Research Unit. R.G. was supported by the Natural Sciences and Engineering Research Council of Canada.
Edited by: Robert Brothers
Abstract
New Findings
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What is the central question of this study?
We evaluated whether regional variations exist in NO‐dependent cutaneous vasodilatation and sweating during cholinergic stimulation.
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What is the main finding and its importance?
Peak cutaneous vasodilatation and sweating were greater on the torso than the forearm. Furthermore, we found that NO was an important modulator of cholinergic cutaneous vasodilatation, but not sweating, across body regions, with a greater contribution of NO to cutaneous vasodilatation in the limb compared with the torso. These findings advance our understanding of the mechanisms influencing regional variations in cutaneous vasodilator and sweating responses to pharmacological stimulation.
Abstract
Regional variations in cutaneous vasodilatation and sweating exist across the body. Nitric oxide (NO) is an important modulator of these heat loss responses in the forearm. However, whether regional differences in NO‐dependent cutaneous vasodilatation and sweating exist remain uncertain. In 14 habitually active young men (23 ± 4 years of age), cutaneous vascular conductance (CVC%max) and local sweat rates were assessed at six skin sites. On each of the dorsal forearm, chest and upper back (trapezius), sites were continuously perfused with either lactated Ringer solution (control) or 10 mm Nω‐nitro‐l‐arginine (l‐NNA; an NO synthase inhibitor) dissolved in Ringer solution, via microdialysis. At all sites, cutaneous vasodilatation and sweating were induced by co‐administration of the cholinergic agonist methacholine (1, 10, 100, 1000 and 2000 mm; 25 min per dose) followed by 50 mm sodium nitroprusside (20–25 min) to induce maximal vasodilatation. The l‐NNA attenuated CVC%max relative to the control conditions for all regions (all P < 0.05), and NO‐dependent vasodilatation was greater at the forearm compared with the back and chest (both P < 0.05). Furthermore, maximal vasodilatation was higher at the back and chest relative to the forearm (both P < 0.05). Conversely, l‐NNA had negligible effects on sweating across the body (all P > 0.05). Peak local sweat rate was higher at the back relative to the forearm (P < 0.05), with a similar trend observed for the chest. In habitually active young men, NO‐dependent cholinergic cutaneous vasodilatation varied across the body, and the contribution to cholinergic sweating was negligible. These findings advance our understanding of the mechanisms influencing regional variations in cutaneous vasodilatation and sweating during pharmacological stimulation.
1 INTRODUCTION
Cutaneous vasodilatation and sweating are important for regulating core temperature during heat stress. Locally, these heat loss responses are modulated by acetylcholine released from cholinergic nerves, which stimulates muscarinic receptors on sweat glands, and an unknown cotransmitter that modulates cutaneous vasodilatation (Kellogg et al., 1995). Although it is well established that cutaneous vasodilator and sweating responses vary across the body, the functional mechanisms underlying regional variations in these heat loss responses remain unresolved (Hertzman & Randall, 1948; Kondo et al., 1998; Smith & Havenith, 2011).
On the forearm, NO is an important modulator of cholinergic cutaneous vasodilatation, although its importance to cholinergic sweating remains uncertain (Fujii et al., 2014a; Lee & Mack, 2006). Although previous work did not demonstrate regional variations in cutaneous vasodilatation or sweating in response to acetylcholine (Smith, Kenney, & Alexander, 2013), the modulatory effects of NO on these responses might still vary across the body. Indeed, limb differences (between the arm and the leg) in the contribution of NO to basal cutaneous perfusion and during the plateau phase of local skin heating have been reported previously (Del Pozzi, Carter, Collins, & Hodges, 2013; Stanhewicz, Greaney, Kenney, & Alexander, 2014), indicating that NO‐dependent cholinergic cutaneous vasodilatation might also display regional heterogeneity. Unlike cholinergic sweating, NO‐dependent forearm sweating during moderate‐intensity exercise is well established in young men (Fujii et al., 2014b, 2016; Welch, Foote, Hansen, & Mack, 2009); an effect that is more pronounced in individuals with higher sweat rates (Amano, Fujii, Louie, Meade, & Kenny, 2017). Therefore, despite mixed results for NO‐dependent cholinergic sweating on the forearm, it remains possible that modulation of cholinergic sweating by NO is more pronounced in regions with higher sweat rates, such as the back and chest (Smith & Havenith, 2011).
As such, the purpose of this study was to compare NO‐dependent cutaneous vasodilatation and sweating across the forearm, upper back and chest during incremental doses of methacholine delivered via intradermal microdialysis. It was hypothesized that the NO‐dependent contributions of these heat‐loss responses would be greater at the chest and back, where cutaneous vasodilator and sweating responses are higher than at the forearm.
2 METHODS
2.1 Ethical approval
This study was approved by the University of Ottawa Health Sciences and Science Research Ethics Board (H04‐17‐01) in accordance with the guidelines set forth by the Declaration of Helsinki, with the exception that registration in a database was not done. Verbal and written informed consent were obtained from all volunteers before participation.
2.2 Participants
Fourteen young men participated in this study. All participants were habitually active (i.e. they performed ≥30 min of structured physical activity at least two times per week) as determined by a standardized questionnaire (Kohl, Blair, Paffenbarger, Macera, & Kronenfeld, 1988). The mean (SD) for age, height, body mass, body mass index, relative peak oxygen uptake and resting mean arterial pressure for the participants were 23 ± 4 years, 1.74 ± 0.06 m, 76.6 ± 10.0 kg, 25.3 ± 2.3 kg m−2, 44.0 ± 6.8 ml kg−1 min−1 and 93 ± 7 mmHg, respectively. Participants were excluded if they had a history of skin, cardiorespiratory and autonomic disorders or were smokers. No participants were taking prescription medications, and all abstained from over‐the‐counter medications, including non‐steroidal anti‐inflammatory agents and vitamins, for ≥48 h before the experiment. Intense exercise was avoided for ≥24 h before the experiment, alcohol and caffeine consumption for ≥12 h and food consumption for 2 h before and throughout the experiment.
2.3 Experimental design
All participants completed one screening and one experimental session. During screening, body mass was measured using a digital weight scale platform (model CBU150X; Mettler Toledo Inc., Columbus, OH, USA). Height was measured using an eye‐level physician stadiometer (model 2391; Detecto Scale Company, Webb City, MO, USA). Peak oxygen uptake was determined via an incremental cycling exercise protocol. Participants were seated on a semi‐recumbent cycling ergometer and were asked to maintain a consistent pedalling rate between 60 and 100 r.p.m. The resistance was set to 60 W, which was then increased by 20 W min−1 until volitional fatigue. Ventilation and metabolic data were collected with an automated indirect calorimetry system (Medgraphic Ultima; Medical Graphic, St Paul, MN, USA).
The experimental session was performed ≥48 h after screening. Upon arrival, participants voided their bladders, after which pretrial body mass was assessed (model CBU150X; Mettler Toledo Inc.). Next, participants sat in a semi‐recumbent position on a medical bed in a temperate environment (26°C). A total of six microdialysis fibres (30 kDa cut‐off, 10 mm membrane; MD2000; Bioanalytical Systems, West Lafayette, IN, USA) were inserted into the dermal layer of the skin on the left side of the body, including two fibres at each of the proximal half of the dorsal forearm, upper back (trapezius, ∼3 cm medial to the acromion and superior to the spine of the scapula) and chest (∼3 cm superior to the nipple line). A foam pad was placed between the participant and the bed to avoid measurement interference on the back. At each site, a 25‐gauge needle was inserted subcutaneously. (∼2.5 cm in length), and the microdialysis fibre was subsequently threaded through the lumen of the needle, after which the needle was removed, leaving the fibre embedded in the skin. All fibres were secured with surgical tape, and in each region the two fibres were separated by ≥4 cm to avoid between‐site drug interference. At this time, perfusion of pharmacological agents was initiated at all six sites at a rate of 4 μl min−1 with a microinfusion pump (model 400; CMA Microdialysis, Solna, Sweden). In each region, the two sites were perfused with either lactated Ringer solution [CTRL, control (Baxter, Missisauga, ON, Canada)] or 10 mm Nω‐nitro‐l‐arginine [l‐NNA, NO synthase (NOS) inhibitor (Sigma‐Aldrich, St Louis, MO, USA)]. Drug infusion continued for ≥60 min to ensure that inflammation from fibre insertion had subsided (Hodges, Chiu, Kosiba, Zhao, & Johnson, 2009).
After an initial 10 min baseline period, five incremental doses of methacholine were co‐infused through all six microdialysis fibres (1, 10, 100, 1000 and 2000 mm; 25 min per dose) while they continued to be infused with either Ringer solution or l‐NNA throughout the experiment. Finally, 50 mm sodium nitroprusside (Sigma‐Aldrich) was infused for 20–25 min at all six sites at a rate of 6 μl min−1 until maximal values for cutaneous perfusion were achieved for ≥2 min.
2.4 Measurements
Cutaneous red blood cell flux (expressed in perfusion units) was measured at all six sites using laser‐Doppler flowmetry (PeriFlux System 5000; Perimed, Stockholm, Sweden) at a sampling rate of 32 Hz. Integrated seven‐laser array laser‐Doppler probes (model 413; Perimed) were housed in a sweat capsule positioned directly over the centre of each microdialysis membrane to allow for simultaneous measurements of local cutaneous red blood cell flux and sweat rate (Meade et al., 2016). Cutaneous vascular conductance (CVC) was then calculated as perfusion units divided by mean arterial pressure and expressed as a percentage of maximal conductance (CVC%max). Blood pressure was determined manually every 5 min using a mercury column sphygmomanometer (Baumonometer standby model; WA Baum, Copiague, NY, USA), and mean arterial pressure was calculated as diastolic arterial pressure plus one‐third of the difference between systolic and diastolic pressures.
Local sweat rate (LSR) was evaluated simultaneously at each site with a ventilated capsule (1.1 cm2) positioned directly over the centre of the microdialysis membrane (Meade et al., 2016). Sweat capsules were secured to the skin surface using adhesive rings and topical glue (Collodion HV; Mavidon Medical Products, Lake Worth, FL, USA). Anhydrous air equilibrated to room temperature was passed through each capsule at a constant rate of 0.4 l min−1 and monitored with a flow‐rate monitor (Omega FMA‐A2307; Omega Engineering, Stamford, CT, USA). The water content of the effluent air exiting the ventilated capsule was measured using capacitance hygrometers (model HMT333; Vaisala, Helsinki, Finland). The LSR was calculated every 5 s using the difference in water content between effluent and influent air multiplied by flow rate and normalized for skin surface area beneath the capsule (in milligrams per minute per square centimetre).
Aural canal temperature was measured as an index of core temperature. Skin temperature was measured continuously at four sites (calf, quadriceps, chest and biceps) using thermocouple discs (Concept Engineering, Old Saybrook, CT, USA) attached to the skin with adhesive rings and surgical tape. Mean skin temperature was then estimated as the weighted mean of the local skin temperatures from the calf (20%), quadriceps (20%), chest (30%) and biceps (30%) (Ramanathan, 1964). Skin temperature data were recorded using a data‐acquisition module (model 34970A; Agilent Technologies Canada, Mississauga, ON, Canada) and were displayed and recorded using LabVIEW software (National Instruments, Austin, TX, USA). Heart rate was measured continuously using a Polar coded WearLink and transmitter, Polar RS400 Interface, and Polar Trainer 5 software (Polar Electro, Kempele, Finland).
2.5 Data analysis
For all variables, a 5 min average was taken during the final 5 min of the 10 min baseline rest period and during the final 5 min of each methacholine dose. Maximal CVC was defined as the highest consecutive 2 min interval during sodium nitroprusside infusion. Blood pressure was recorded manually every 5 min throughout the protocol. For CVC%max, values for one participant were removed completely, and values for a second participant were removed on each of the forearm, back and chest. For LSR, values for two participants were removed owing to negligible sweating responses during methacholine infusion. For an additional participant, the chest site was removed owing to technical problems.
2.6 Statistical analysis
Responses for CVC%max and LSR were evaluated using linear mixed models to account for missing values as described above. Within each region (forearm, back and chest) the effects of treatment (two elements: control and l‐NNA) and methacholine dose (six levels: 0, 1, 10, 100, 1000 and 2000 mm methacholine) on CVC%max and LSR were examined using two‐way mixed models. Comparisons of control or l‐NNA sites across regions were each made using two‐way mixed models for region (three elements: forearm, back and chest) by dose (six elements). The contributions of NOS (l‐NNA minus control) to CVC and LSR across regions were examined using two‐way mixed models for region (three elements) by dose (six elements). Absolute maximal CVC was evaluated using a mixed model to compare region (three elements) by treatment site (two elements).
All secondary variables (skin and core temperatures, heart rate and mean arterial pressure) were analysed using a one‐way repeated‐measures ANOVA (six elements: 0, 1, 10, 100, 1000 and 2000 mm methacholine). When significant main effects or interactions were detected, multiple comparisons tests were performed using Tukey's or Dunnett's tests where appropriate. Significance was set at P < 0.05 for all analyses. Statistical analyses were conducted using GraphPad v.8.2.1 (GraphPad Software Inc., San Diego, CA, USA).
3 RESULTS
3.1 Body temperatures and cardiovascular responses
Mean skin temperature was significantly different across methacholine doses (P < 0.05), whereas core temperature did not change (P > 0.05). Heart rate was significantly different across methacholine doses (P < 0.05), whereas mean arterial pressure did not change (P > 0.05; Table 1).
| Methacholine (mM) | ||||||
|---|---|---|---|---|---|---|
| Parameter | Baseline | 1 | 10 | 100 | 1000 | 2000 |
| Core temperature (°C) | 36.65 (0.25) | 36.63 (0.24) | 36.66 (0.23) | 36.61 (0.26) | 36.64 (0.32) | 36.61 (0.32) |
| Mean skin temperature (°C) | 31.86 (0.49) | 31.80 (0.49) | 31.67 (0.52)** P < 0.05 versus baseline. |
31.60 (0.54)** P < 0.05 versus baseline. |
31.62 (0.54) | 31.71 (0.48) |
| Heart rate (beats min−1) | 63 (9) | 64 (8) | 64 (9) | 63 (8) | 65 (9) | 67 (9) |
| Mean arterial pressure (mmHg) | 93 (7) | 94 (8) | 95 (7) | 95 (6) | 94 (6) | 93 (6) |
- All values are presented as the mean (SD); n = 14. All values represent the average over each time interval.
- * P < 0.05 versus baseline.
3.2 Cutaneous vascular responses
3.2.1 Within region
At the forearm, there were significant main effects for dose and treatment site and for the dose‐by‐treatment site interaction (all P < 0.05) such that l‐NNA‐treated sites were significantly reduced relative to control from 1 to 1000 mm methacholine (all P < 0.05; Figure 1a). At the back, there were significant main effects for dose and treatment site and for the dose‐by‐treatment site interaction (all P < 0.05) such that l‐NNA‐treated sites were significantly reduced relative to control from 1 to 100 mm methacholine (all P < 0.05; Figure 1b). At the chest, there were significant main effects for dose and treatment site (both P < 0.05; Figure 1c).
3.2.2 Between regions
At the control sites, there were significant main effects for dose and body region (both P < 0.05) such that all regions were significantly different from each other (all P < 0.05). For l‐NNA‐treated sites, there were significant main effects for dose and treatment site (P < 0.05) such that the chest was greater than the forearm and back (both P < 0.05). For the NOS contribution, there were significant main effects for dose and body region (both P < 0.05; Figure 1d). The contribution of NOS was increased compared with baseline from 1 to 1000 mm methacholine (all P < 0.05), with a greater effect at the forearm compared with the back and chest skin sites (both P < 0.05).
3.2.3 Maximal cutaneous vasodilatation
For maximal absolute CVC, there was a significant main effect for region (P < 0.05) such that maximal cutaneous vasodilatation was higher at the back and chest compared with the forearm (both P < 0.05; Figure 2).
3.3 Sweating responses
3.3.1 Within region
At both the forearm and back, there were significant main effects for dose only (both P < 0.05; Figure 3a and b, respectively). At the chest, there were significant main effects for dose and the dose‐by‐treatment site interaction (both P < 0.05; Figure 3c). However, analysis of multiple comparisons failed to identify significant differences between sites at any dose.
3.3.2 Between regions
At the control sites, there were significant main effects for dose and the dose‐by‐treatment site interaction (both P < 0.05) such that sweating at the back was significantly higher than that for the forearm at 1000 and 2000 mm methacholine (both P < 0.05), with a similar trend for the chest. At l‐NNA‐treated sites, there were significant main effects for dose and the dose‐by‐treatment site interaction (both P = 0.05). However, analysis of multiple comparisons failed to identify significant differences between sites at any dose. For the contribution of NOS, there was a significant main effect for the dose‐by‐region interaction (P < 0.05; Figure 3d). However, analysis of multiple comparisons failed to identify significant differences between sites at any dose.
4 DISCUSSION
In the present study, we have shown that NO contributes to cholinergic cutaneous vasodilatation at the forearm, upper back and chest. Furthermore, the contribution of NO to cutaneous vasodilatation was greater on the forearm than on the back and chest. Conversely, NO played a negligible role in mediating cholinergic sweating across all regions. Finally, maximal cutaneous vasodilator and peak sweating responses were both greater on the torso compared with the forearm.
4.1 Cutaneous vascular conductance
There was a NO‐dependent cutaneous vasodilator effect for all regions during cholinergic stimulation, with a greater contribution of NO on the forearm compared with the back and chest. During local skin heating, differences between limbs in NO‐dependent cutaneous vasodilatation have also been observed, with a greater contribution on the forearm than on the calf (Stanhewicz et al., 2014). The mechanisms contributing to regional variability in NO‐dependent cutaneous vasodilatation remain unclear. Chronic differences in exposure to shear stress between the limb and torso might contribute to this effect. However, there were no regional differences in basal NO bioavailability, as evidenced by a negligible contribution of NO to resting cutaneous vasodilator tone across all regions. Differences in cutaneous microvessel density might also exist between non‐glabrous skin of the forearm and torso, resulting in varying functional responses to NO inhibition. In support of this, maximal cutaneous vascular conductance was higher on the back and chest compared with the forearm. Finally, the reduced contribution of NO to cutaneous vasodilatation at the torso sites might reflect a larger role of NO‐independent mechanisms in mediating cholinergic cutaneous vasodilatation at the back and chest relative to the forearm. Further research is necessary to delineate the structural and/or biochemical underpinnings of this regional variation in cutaneous vasodilatation.
4.2 Sweating
Peak cholinergic sweating was greater on the upper back and chest than on the forearm, consistent with previous work examining regional sweat rates during exercise (Smith & Havenith, 2011). However, this contrasts with a prior study showing similar cholinergic sweating responses across the forearm, thigh, abdomen and lower back during administration of acetylcholine (Smith et al., 2013). This discrepancy might be attributable to differences in the body regions examined, although regional variations in the former work were still anticipated (Smith & Havenith, 2011). We also used the acetylcholine analogue methacholine, which is more resistant to breakdown by acetylcholinesterase (Kimura, Low, Keller, Davis, & Crandall, 2007), combined with longer durations of each dose (25 versus 10 min each) and a higher maximal dose (2000 versus 1000 mm) compared with previous work (Smith et al., 2013).
There is an intrinsic NOS system in human eccrine sweat glands, which might contribute to augmenting sweat output (Shimizu, Sakai, Umemura, & Ueda, 1997a, b; Zancanaro, Merigo, Crescimanno, Orlandini, & Osculati, 1999). Importantly, the functional relevance of this was subsequently supported by the finding that NO contributes to local methacholine‐induced sweating in human forearm skin (Lee & Mack, 2006). However, this effect is inconsistent with our present and earlier work using higher doses of methacholine compared with Lee and Mack (2006), indicating that the effects of NOS on cholinergic sweating might be overridden at higher sweat rates, as previously discussed (Fujii et al., 2014a). In contrast, the contribution of NO to forearm sweating is well established in young men during moderate‐intensity exercise (Fujii et al., 2014b, 2016; Welch et al., 2009), and this effect is more pronounced in individuals with higher sweat rates for a given heat load (Amano et al., 2017). Therefore, despite mixed results for NO‐dependent cholinergic sweating on the forearm, we deemed it plausible that NO modulation might be more evident in body regions with higher sweat rates, such as the back and chest, relative to the forearm. However, although sweating was indeed greater on the torso than on the forearm, the contribution of NO to this response was negligible across all regions.
4.3 Considerations
During heat exposure, cutaneous vasodilatation and sweating are crucial for preventing dangerous increases in core temperature. Importantly, these heat‐loss responses vary across the body, and when evaluated locally they do not always respond consistently during heat acclimatization (Poirier, Gagnon, & Kenny, 2016) or in pathological states, such as diabetes (Petrofsky, Lee, Patterson, Cole, & Stewart, 2005). Although previous studies have examined regional variations in skin blood flow and sweating, to our knowledge this is the first to examine the potential underlying mechanisms contributing to such variation directly. Although NO is a well‐established mediator of cutaneous vasodilatation and sweating in young men, prior work has largely focused on the forearm. Development of a better understanding of regional variations in the contribution of NO to these heat‐loss responses in young men provides an important starting point for understanding the mechanisms contributing to augmented cutaneous end‐organ responses after heat acclimatization in addition to reductions in heat tolerance at the whole‐body level that may occur with ageing and chronic disease, such as diabetes.
4.4 Conclusion
We demonstrated a greater contribution of NO to cholinergic cutaneous vasodilatation at the forearm compared with the upper back and chest, although there was a negligible contribution of NO to cholinergic sweating across all examined regions. Finally, peak cholinergic cutaneous vasodilatation and sweating responses were greater at the back and chest compared with the forearm.
ACKNOWLEDGEMENTS
The authors would like to thank the participants for their involvement in the study.
COMPETING INTERESTS
None declared.
AUTHOR CONTRIBUTIONS
All experiments took place at the Human and Environmental Physiology Research Unit, located at the University of Ottawa. G.W.M., N.F. and G.P.K. conceived and designed the experiments. G.W.M. and R.G. performed data collection and analysis. All authors interpreted the results. G.W.M. prepared figures and drafted the manuscript. All authors edited and revised the manuscript. All authors approved the final version of the 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.
