Contribution of nitric oxide synthase to cutaneous vasodilatation and sweating in men of black‐African and Caucasian descent during exercise in the heat
Funding information:
This research was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN‐06313‐2014 and RGPAS‐462252‐2014; all funds held by G.P.K.). G.P.K. is supported by a University of Ottawa Research Chair. G.W.M., C.M.M. and M.D.S. are supported by the Human and Environmental Physiology Research Unit.
Edited by: Shigehiko Ogoh
Abstract
New Findings
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What is the central question of this study?
Nitric oxide modulates cutaneous vasodilatation and sweating during exercise‐induced heat stress in young men. However, it remains uncertain whether these effects are reduced in black‐African descendants, who commonly demonstrate reduced nitric oxide bioavailability. Therefore, we assessed whether black‐African descendants display reduced nitric oxide‐dependent cutaneous vasodilatation and sweating compared with Caucasians in these conditions.
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What is the main finding and its importance?
Nitric oxide‐dependent cutaneous vasodilatation and sweating were similar between groups, indicating that reduced nitric oxide bioavailability in black‐African descendants does not attenuate these heat‐loss responses during an exercise‐induced heat stress.
Abstract
Men of black‐African descent are at an increased risk of heat‐related illness relative to their Caucasian counterparts. This might be attributable, in part, to reduced cutaneous nitric oxide (NO) bioavailability in this population, which might alter local cutaneous vasodilatation and sweating. To evaluate this, we compared these heat‐loss responses in young men (18–30 years of age) of black‐African (n = 10) and Caucasian (n = 10) descent during rest, exercise and recovery in the heat. Participants were matched for physical characteristics and fitness, and they were all born and raised in the same temperate environment (i.e. Canada; second generation and higher). Both groups rested for 10 min and then performed 50 min of moderate‐intensity exercise at 200 W m−2, followed by 30 min of recovery in hot, dry heat (35°C, 20% relative humidity). Local cutaneous vascular conductance (CVC%max) and sweat rate (SR) were measured at two forearm skin sites treated with either lactated Ringer solution (control) or 10 mm NG‐nitro‐l‐arginine methyl ester (l‐NAME, a nitric oxide (NO) synthase inhibitor). l‐NAME significantly reduced CVC%max throughout rest, exercise and recovery in both groups (both P < 0.001). However, there were no significant main effects for the contribution of NO to CVC%max between groups (all P > 0.500). l‐NAME significantly reduced local SR in both groups (both P < 0.050). The contribution of NO to SR was similar between groups such that l‐NAME reduced SR relative to control at 40 and 50 min into exercise (both P < 0.05). We demonstrate that ethnicity per se does not influence NO‐dependent cutaneous vasodilatation and sweating in healthy young men of black‐African and Caucasian descent during exercise in dry heat.
1 INTRODUCTION
Heat‐related mortality is higher among men of black‐African descent compared with Caucasians (white‐Europeans; CDC, 2017). Although the reasons behind this are multifactorial, it might be attributed, in part, to a reduced capacity for heat dissipation via attenuated vasodilator and sweating responses at the cutaneous end‐organ level (i.e. cutaneous vasculature and eccrine sweat glands). Indeed, black‐African descendants demonstrate reduced forearm (Ozkor et al., 2014) and local cutaneous (Maley, House, Tipton, & Eglin, 2017) vasodilator responsiveness relative to Caucasians. Furthermore, sweat gland activation during passive heat exposure is lower in men of black‐African descent than in Caucasians (Kawahata & Adams, 1961) albeit, relative increases in sweat output in response to phamacological stimulation have also been reported for men of black‐African descent (Gibson & Shelley, 1948).
In young men, nitric oxide (NO) has been established as an important modulator of cutaneous vasodilatation and sweating during exercise‐induced heat stress (Fujii et al., 2016; McNamara, Keen, Simmons, Alexander, & Wong, 2014; Stapleton, Fujii, Carter, & Kenny, 2014; Welch, Foote, Hansen, & Mack, 2009). However, previous work was not designed to examine potential ethnic differences in the contribution of NO to these heat‐loss responses. Importantly, in vitro evidence indicates that NO bioavailability might be reduced in men of black‐African descent relative to Caucasian men, which has been linked to excess superoxide production in this population (Kalinowski, Dobrucki, & Malinski, 2004; Mason, Kalinowski, Jacob, Jacoby, & Malinski, 2005). In support of this, NO‐dependent cutaneous vasodilatation is also attenuated in young men of black‐African descent, which can be counteracted by local delivery of the superoxide dismutase mimetic, Tempol (Hurr, Patik, Kim, Christmas, & Brothers, 2018) or with l‐arginine supplementation (Kim, Hurr, Patik, & Brothers, 2018). Although other factors associated with reduced NO bioavailability, such as advanced age, are known to attenuate cutaneous vasodilatation and sweating during exercise (Fujii et al., 2016), it remains uncertain whether this translates into local impairments of these heat‐loss responses in young men of black‐African descent.
Therefore, we assessed NO‐dependent cutaneous vasodilator and sweating responses in young men of black‐African descent and Caucasian men during and after an exercise‐induced heat stress. We hypothesized that NO‐dependent cutaneous vasodilatation and sweating would be attenuated in young men of black‐African descent compared with Caucasians in these conditions.
2 METHODS
2.1 Ethical approval
This study was approved by the University of Ottawa Health Sciences and Science Research Ethics Board (H04‐17‐05) and conformed to the standards outlined in the Declaration of Helsinki, with the exception that registration in a database was not done in the present study. All volunteers provided verbal and written informed consent before participation.
2.2 Participants
Twenty healthy, non‐smoking young men of black‐African descent (AFD; n = 10) and Caucasian men (CAU; n = 10) matched for peak oxygen uptake (
) and physical characteristics participated (Table 1). Ethnicity was based on self‐classification, whereby participants reported their ethnicity and that of their parents. Participants were eligible if they were second or higher generation Canadians (i.e. born and raised in Canada), as defined by Statistics Canada (Dobson, Maheux, & Chui, 2018), and if both parents were of the same ethnicity.
| Parameter | Black‐African descent | Caucasian |
|---|---|---|
| Age (years) | 22 (3) | 23 (4) |
| Height (m) | 1.75 (0.05) | 1.77 (0.07) |
| Body mass (kg) | 78.21 (12.46) | 78.86 (8.10) |
| Body surface area (m2) | 1.94 (0.16) | 1.96 (0.12) |
| Body fat (%) | 13.47 (7.91) | 19.34 (4.72) |
| Peak oxygen uptake (ml kg−1 min−1) | 39.29 (7.55) | 40.81 (3.78) |
- Values are presented as the mean (SD); n = 10 participants in each group.
2.3 Experimental design
All participants completed one screening and one experimental session, separated by ≥48 h. For all sessions, all participants were instructed to consume ∼250–500 ml of water ∼2 h before the study and were advised to abstain from over‐the‐counter medications for 48 h, from strenuous exercise, alcohol and caffeine for 12 h, and from food for 2 h before and throughout the experiment.
During the screening session, body mass and height were measured using a digital weight scale platform (model CBU150X; Mettler‐Toledo International Inc., Columbus, OH, USA) and an eye‐level physician stadiometer (model 2391; Detecto Scale Company, Webb City, MO, USA), respectively. The 
was assessed using an automated indirect calorimetry system (Medgraphic Ultima; Medical Graphic, St. Paul, MN, USA) via a progressive incremental cycling exercise protocol on a semi‐recumbent cycling ergometer (Corival; Lode BV, Groningen, The Netherlands). The protocol began with 1 min of cycling at a workload of 80 W, which was then increased by 20 W min−1 until volitional fatigue. The participants maintained a consistent pedaling rate between 60 and 100 r.p.m.
Upon arrival for the experimental session, participants voided their bladders and, after confirming euhydration (urine specific gravity <1.025; Kenefick & Cheuvront, 2012), changed into athletic shorts, then measured the pre‐trial body mass on a weighing terminal (model CBU150X; Mettler‐Toledo International Inc.). Participants then sat in a semi‐recumbent position on a medical bed in a non‐heat‐stress environment (25°C) while two microdialysis fibres (30 kDa cut‐off, 10 mm membrane; MD2000; Bioanalytical Systems, West Lafayette, IN, USA) were inserted into the dermal layer of skin on the left dorsal forearm in aseptic conditions. Each skin site was initially wiped with an alcohol swab. At each forearm site, a 25‐gauge needle was inserted in the dermal layer of the skin (∼2.5 cm in length). The microdialysis fibre was subsequently threaded through the lumen of the needle, after which the needle was removed from the skin, leaving the fibre embedded under the skin. Each fibre was secured with surgical tape and separated by ≥4 cm. The process was repeated for the other skin site. After fibre insertions, participants were then transferred to an adjacent thermal chamber regulated at 35°C and 20% relative humidity (Can‐Trol Environmental Systems, Markham, ON, Canada) where they remained resting on a semi‐recumbent cycle ergometer for a minimum of 75 min. At the start of the resting period in the heat, perfusion of pharmacological agents was initiated at the two microdialysis sites at a rate of 4 μl min−1 with a microinfusion pump (model 400; CMA Microdialysis, Solna, Sweden). The sites were perfused with either lactated Ringer solution (CTRL; Baxter, Deerfield, IL, USA) or 10 mm NG‐nitro‐l‐arginine methyl ester (l‐NAME; Sigma‐Aldrich, St Louis, MO, USA, NO synthase (NOS) inhibitor), as previously described (Fujii et al., 2014). Drug infusion continued for ≥75 min before baseline resting measurements commenced to ensure that vasodilatation attributable to fibre insertion trauma had subsided (Anderson, Andersson, & Wardell, 1994).
Participants rested for 10 min, after which they performed 50 min of cycling at a fixed rate of metabolic heat production of 200 W m−2, equivalent to [mean (SD)] 47 (7) (AFD) and 44 (5)
(CAU), followed by 30 min of recovery. After recovery, 50 mm sodium nitroprusside (SNP; Sigma‐Aldrich) was infused for 20–25 min at both sites at a rate of 6 μl mim−1 until maximal cutaneous perfusion was achieved for ≥2 min. Afterwards, the participant's body mass was measured again.
2.4 Measurements
Local cutaneous red blood cell flux [expressed in perfusion units (PU)] and local sweat rate (SR; expressed in milligrams per minute per square centimetre) were measured simultaneously at both skin sites using laser‐Doppler flowmetry (PeriFlux System 5000; Perimed, Stockholm, Sweden) and custom‐designed ventilated capsules, as previously described (Meade et al., 2016). Cutaneous vascular conductance (CVC) was calculated as perfusion units divided by mean arterial pressure and expressed as a percentage of maximum during SNP infusion (CVC%max).
Blood pressure (Baumonometer Standby Model; WA Baum, Copiague, NY, USA), heart rate (Polar M400 model; Polar Electro, Kempele, Finland), aural canal temperature (an index of body core temperature; Braun Thermoscan Pro 6000; Welch Allyn, Skaneateles Falls, NY, USA), skin temperatures (thermocouple disks, Concept Engineering, Old Saybrook, CT, USA) and metabolic energy expenditure (AMETEK model S‐3A/1 and CD3A; Applied Electrochemistry, Pittsburgh, PA, USA) were all measured as previously described (Fujii et al., 2018).
2.5 Data analysis
Baseline resting values were obtained by averaging measurements performed over 10 min. Values at the start of exercise (time 0) were obtained during the last 5 min before exercise commenced. Local forearm SR and CVC, and core body and mean skin temperature data acquired during the exercise and recovery periods were obtained by averaging measurements made over the last 5 min of each 10 min interval. The maximal CVC was defined as the highest consecutive 2 min interval during SNP infusion.
2.6 Statistical analysis
The SR and CVC%max were analysed using a two‐way repeated‐measures ANOVA, with factors of time (10 levels: 10 min throughout baseline, and every 10 min during the 50 min exercise and 30 min recovery periods) and treatment site (two levels: CTRL and l‐NAME) separately performed in each group (AFD and CAU). Group differences in SR and CVC%max at the control sites were analysed using a two‐way mixed‐model ANOVA, with factors of time and group. Group differences in NO‐dependent SR and CVC%max were evaluated by examining the absolute differences between l‐NAME and control sites across time using a two‐way mixed‐model ANOVA, with factors of time and group. Body temperatures (i.e. core body and skin temperatures) and cardiovascular (mean arterial pressure and heart rate) variables were analysed using a two‐way mixed‐model ANOVA, with factors of time (three levels: baseline, end‐exercise and end‐recovery) and group (two levels: AFD and CAU). When a significant main effect or interaction was detected, multiple comparisons tests were performed using the Bonferroni method. Physical characteristics and hydration status were compared between groups using Student's unpaired, two‐tailed t tests. The value of α was set at P < 0.05 for all statistical comparisons. Values are reported as the mean [95% confidence interval (CI)] or the mean (SD), where appropriate. Statistical analyses were conducted using GraphPad Prism v.8.2 (GraphPad, La Jolla, CA, USA).
3 RESULTS
3.1 Cutaneous vascular conductance
For CVC%max, the treatment site‐by‐time interactions were not significantly different for either group (P = 0.647, AFD; and P = 0.751, CAU). However, both demonstrated significant main effects for time (P < 0.001, both groups) and treatment site (P = 0.001, AFD; and P = 0.007, CAU) such that l‐NAME significantly reduced CVC%max relative to control throughout the protocol (Figure 1a). At the control sites, CVC%max was similar between groups (P = 0.947 for group‐by‐time interaction, P = 0.916 for main effect of group, and P < 0.001 for main effect of time). The NO‐dependent effects on CVC%max were not significantly different between groups (P = 0.993 for the group‐by‐time interaction, P = 0.794 for main effect of group, and P = 0.477 for main effect of time).
For maximal absolute CVC for AFD [CTRL, 1.72 (0.65) PU mmHg−1; l‐NAME, 1.27 (0.67) PU mmHg−1] and CAU [CTRL, 1.81 (0.28) PU mmHg−1; l‐NAME, 1.80 (0.66) PU mmHg−1] there were no significant main effects for treatment site, group or the treatment site‐by‐group interaction (all P > 0.05).
3.2 Sweat rate
For SR, there was a significant treatment site‐by‐time interaction (P = 0.049) and a significant main effect of time (P < 0.001) but not treatment site (P = 0.073) for men of black‐African descent. For Caucasians, the treatment site‐by‐time interaction was not statistically significant (P = 0.202), but there were significant main effects for time (P < 0.001) and treatment site (P = 0.044; Figure 1b). At the control sites, SR was similar between groups (P = 0.254 for group‐by‐time interaction, P = 0.228 for main effect of group, and P < 0.001 for main effect of time). The NO‐dependent effects on SR were not significantly different between groups (P = 0.535 for the group‐by‐time interaction, P = 0.496 for main effect of group, and P = 0.028 for main effect of time). However, NOS‐dependent sweating was significantly different from baseline at 40 and 50 min into exercise (both P < 0.05).
3.3 Body temperatures and cardiovascular responses
For core body and skin temperatures, heart rates and mean arterial pressures, there were no significant effects of group or the group‐by‐time interaction between black‐African descendants and Caucasians (P > 0.05). For all variables, there were significant main effects for time (P < 0.05) such that all responses were elevated from baseline at end‐exercise and end‐recovery, and at end‐exercise compared with end‐recovery (all P < 0.05; Table 2).
| Parameter | Rest | End‐exercise | End‐recovery |
|---|---|---|---|
| Core temperature (°C) | |||
| AFD | 37.1 (0.3) | 37.8 (0.3) | 37.4 (0.3) |
| CAU | 37.1 (0.3) | 37.7 (0.3) | 37.4 (0.3) |
| Mean skin temperature (°C) | |||
| AFD | 34.4 (0.3) | 35.2 (0.4) | 34.5 (0.5) |
| CAU | 34.2 (0.5) | 35.3 (0.4) | 34.6 (0.5) |
| Heart rate (beats min−1) | |||
| AFD | 77 (11) | 135 (17) | 88 (16) |
| CAU | 76 (10) | 130 (19) | 90 (13) |
| Mean arterial pressure (mmHg) | |||
| AFD | 95 (6) | 106 (11) | 95 (5) |
| CAU | 93 (4) | 102 (12) | 93 (5) |
- Values are presented as the mean (SD); n = 10 subjects in each group. All values represent the average over the final 5 min of each time interval. Abbreviations: AFD, black‐African descendant; and CAU, Caucasian.
3.4 Hydration status and body weight loss
All participants were euhydrated, as confirmed by baseline urine specific gravity of 1.016 (0.01) (AFD) and 1.016 (0.01) (CAU). After the experiment, body mass was reduced by 1.1 (0.16) (AFD) and 1.2 (0.13) kg (CAU), respectively.
4 DISCUSSION
This is the first study to compare NO‐dependent cutaneous vasodilatation and sweating responses in young men of black‐African descent and Caucasian men during rest, exercise and recovery in dry heat. Consistent with previous work, we observed NO‐dependent attenuations in cutaneous vasodilatation and sweating responses in both groups. However, in contrast to our hypothesis, these effects were not attenuated in men of black‐African descent relative to Caucasians.
Consistent with previous work in young men of various ethnicities (Fujii et al., 2015; McNamara et al., 2014; Stapleton et al., 2014; Welch et al., 2009), we showed that NOS inhibition attenuated cutaneous vasodilatation throughout rest, exercise and recovery in the heat. Here we extend previous work by showing that NOS inhibition attenuated cutaneous vasodilatation to a similar extent in men of black‐African descent and Caucasian men, indicating that ethnicity does not influence NO‐dependent cutaneous vasodilatation in the conditions tested. Our present findings contrast with previously identified attenuations in NOS‐dependent cutaneous vasodilatation during local skin heating in young men of black‐African descent compared with Caucasian men (Hurr et al., 2018; Kim et al., 2018), which might be attributable to different mechanisms governing cutaneous vasodilatation during local heating and whole‐body exercise (Brunt, Fujii, & Minson, 2013; Louie, Fujii, Meade, McNeely, & Kenny, 2017). Additionally, this discrepancy might be attributable to higher levels of cutaneous perfusion achieved during these local heating studies (∼80–90 CVC%max) compared with the current protocol (∼55–60 CVC%max at end‐exercise). A greater increase in metabolic rate associated with a higher exercise intensity would augment the requirements for heat dissipation, a response that may be mediated by elevated cutaneous perfusion. As such, in exercise conditions eliciting higher levels of cutaneous perfusion, NO‐dependent cutaneous vasodilatation might be attenuated in young men of black‐African descent relative to Caucasians, but this remains to be confirmed.
Consistent with previous work in young men (Fujii et al., 2015; McNamara et al., 2014; Stapleton et al., 2014; Welch et al., 2009), we showed that NOS inhibition attenuated sweating, and this effect was most evident at end‐exercise, when the local sweat rates were at their highest. Here, we extend these previous findings by showing that NOS‐dependent sweating is similar between men of black‐African descent and Caucasian men in these conditions. Additionally, local forearm sweat rates at the untreated control sites were comparable between groups, which demonstrates that the overall capacity for sweating may not be different between men of black‐African descent and Caucasian men in these conditions. Nitric oxide synthase‐dependent sweating during exercise is more evident in the individuals who produce higher sweat rates for a given heat load, regardless of aerobic fitness (Amano, Fujii, Louie, Meade, & Kenny, 2017). As such, the similar sweating capacities observed between groups should have resulted in a comparable NOS contribution to this response, assuming that NO bioavailability is not compromised, which is consistent with our present observations. This, along with similar NOS‐dependent cutaneous vasodilator responses between groups, indicates that NO bioavailability was not compromised in the men of black‐African descent in the present conditions. Although we examined local forearm sweating rates, the magnitude of sweating differs across body regions in both ethnic groups (Thomson, 1954). However, there is no indication that the underlying mechanisms differ regionally. Therefore, we believe that the arm represents a valid location for comparison between ethnicities.
This is the first study designed to compare NO‐dependent cutaneous vasodilatation and sweating in young men of black‐African descent and Caucasian men during exercise in dry heat. As previously mentioned, one potential mechanism contributing to altered cutaneous vasodilatation and sweating responses in the young black‐African descendants might be decreased NO bioavailability, which is often ascribed to genotypic adaptations (Mata‐Greenwood & Chen, 2008). However, when examining ethnic groups who may reside in different climates (i.e. temperate versus tropical), phenotypic adaptations resulting from long‐term residence in a hot environment (i.e. heat acclimatization) may influence such comparisons, obscuring genotypic adaptations (Taylor, 2006). Therefore, in the present study, we selected men born and raised in the same temperate climate in Canada (i.e. second generation or higher) to minimize the effects of phenotypic adaptations resulting from environmental influences on NO‐dependent cutaneous vasodilatation and sweating.
In addition to isolating the genotypic effects of ethnicity, the groups were also matched for physical characteristics, and we used a design eliciting a similar fixed rate of metabolic heat production between groups, and therefore, the same heat loss required to attain heat balance. This approach minimized the influence of possible variables that could influence heat exchange, and therefore, allowed for isolation of the influence of ethnicity on NO‐dependent cutaneous vasodilatation and sweating between groups. Using this new approach, we demonstrated that ethnicity per se does not influence NO‐dependent cutaneous vasodilatation or sweating in healthy young men during rest, moderate‐intensity exercise and recovery in dry heat. That said, differences in cutaneous vasodilator responsiveness between men of black‐African and Caucasian descent have been observed in other experimental conditions (Hurr et al., 2018; Kim et al., 2018; Maley et al., 2017). This indicates that ethnicity should remain an important consideration when designing mechanistic studies to evaluate local thermoeffector control, depending on the modulator being investigated and the experimental conditions being used.
4.1 Considerations
Although this study provides new insights into the effects of ethnicity on NO‐dependent cutaneous vasodilatation and sweating, the results are limited to healthy young men of black‐African and Caucasian descent. Therefore, the results are not directly applicable to women. Women were excluded because oestrogen augments NOS expression (Kleinert et al., 1998), thereby promoting NO‐dependent vasodilatation (Charkoudian, Stephens, Pirkle, Kosiba, & Johnson, 1999), which might also influence sweating. Additionally, the results are not directly applicable to other ethnicities or older adults. Therefore, future studies are warranted in these populations who might demonstrate differences in these heat‐loss responses via reduced NO bioavailability compared with young men.
4.2 Conclusion
We show that inherited (i.e. genetic) adaptations associated with ethnicity do not influence NO‐dependent cutaneous vasodilatation and sweating during rest, exercise and recovery in the heat. Our findings provide valuable insights into the mechanisms regulating cutaneous vasodilatation and sweating in young men of black‐African descent, who might have a reduced capacity to dissipate heat during exercise, resulting in a greater risk of heat‐related injury relative to Caucasians.
ACKNOWLEDGEMENTS
We thank the participants for volunteering and Emma McCourt and Sam van de Sande for assistance with data collection.
COMPETING INTERESTS
None declared.
AUTHOR CONTRIBUTIONS
C.M.M., G.W.M., N.F. and G.P.K. conceptualized and designed the research. C.M.M., M.D.S. and G.W.M. contributed to data collection. G.W.M. prepared figures. C.M.M. and G.W.M. analysed the data and drafted the manuscript. All authors interpreted the results, edited the manuscript and approved the final version 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.
