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Do nitric oxide synthase and cyclooxygenase contribute to the heat loss responses in older males exercising in the heat? Naoto Fujii1 , Gabrielle Paull1 , Robert D. Meade1 , Ryan McGinn1 , Jill M. Stapleton1 , Pegah Akbari1 and Glen P. Kenny1,2 1 2
Human and Environmental Physiology Research Unit, School of Human Kinetics, University of Ottawa, Ottawa, ON, Canada Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
Key points
The Journal of Physiology
r Studies show that nitric oxide synthase (NOS) and cyclooxygenase (COX) are involved in sweating and cutaneous vascular regulation in young adults in a potentially interactive manner.
r We evaluated the separate and interactive roles of NOS and COX in forearm sweating and r r
cutaneous vasodilatation in older adults during intermittent exercise in the heat performed at a moderate fixed rate of metabolic heat production (400 W, 48% VO2 max ). We demonstrated that neither NOS nor COX are functionally involved in the forearm sweating response in older adults during exercise, whereas only NOS contributed to cutaneous vasodilatation. These results provide valuable insight into the age-related changes in heat loss and suggest that COX inhibitors (i.e. non-steroidal anti-inflammatory drugs) may not impair core body temperature regulation during exercise in the heat in older adults.
Abstract This study evaluated the separate and combined roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in forearm sweating and cutaneous vasodilatation in older adults during intermittent exercise in the heat. Twelve healthy older (62 ± 7 years) males peformed two 30 min cycling bouts at a fixed rate of metabolic heat production (400 W) in the heat (35°C, 20% relative humidity). The exercise bouts were followed by 20 and 40 min of recovery, respectively. Forearm sweat rate (ventilated capsule) and cutaneous vascular conductance (CVC, laser Doppler perfusion units/mean arterial pressure) were evaluated at four skin sites that were continuously perfused via intradermal microdialysis with: (1) lactated Ringer solution (Control), (2) 10 mM ketorolac (non-selective COX inhibitor), (3) 10 mM NG -nitro-L-arginine methyl ester (L-NAME; non-selective NOS inhibitor) or (4) a combination of 10 mM ketorolac + 10 mM L-NAME. Sweating was not different between the four sites during either exercise bout (main effect P = 0.92) (average of last 5 min of second exercise, Control, 0.80 ± 0.06; ketorolac, 0.77 ± 0.09; L-NAME, 0.74 ± 0.07; ketorolac + L-NAME, 0.77 ± 0.09 mg min−1 cm−2 ). During both exercise bouts, relative to CVC evaluated at the Control site (average of last 5 min of second exercise, 69 ± 6%max), CVC was similar at the ketorolac site (P = 0.62; 66 ± 4%max) whereas it was attenuated to a similar extent at both the L-NAME (49 ± 8%max) and ketorolac + L-NAME (54 ± 8%max) sites (both P < 0.05). Thus, we demonstrate that NOS and COX are not functionally involved in forearm sweating whereas only NOS contributes to forearm cutaneous vasodilatation in older adults during intermittent exercise in the heat.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
DOI: 10.1113/JP270330
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(Received 6 February 2015; accepted after revision 18 March 2015; first published online 27 March 2015) Corresponding author G. P. Kenny: University of Ottawa, School of Human Kinetics, 125 University, Room 367, Montpetit Hall, Ottawa, Ontario, Canada, K1N 6N5. Email:
[email protected] Abbreviations COX, cyclooxygenase; CVC, cutaneous vascular conductance; L-NAME, NG -nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; NSAID, non-steroidal anti-inflammatory drug; SNP, sodium nitroprusside.
Introduction In humans, the evaporation of sweat is the primary avenue for heat loss when ambient temperature is similar to or above mean skin temperature (e.g. 35°C) (Gisolfi & Wenger, 1984). Previous work in young adults has indicated that both local inhibition of nitric oxide (NO) synthase (NOS) (Welch et al. 2009; Stapleton et al. 2014a) and cyclooxygenase (COX) (Fujii et al. 2014) impair sweat production during exercise in the heat. While it has recently been demonstrated that NOS does not contribute to sweating in older adults during intermittent exercise in the heat (Stapleton et al. 2014a), it has not been determined how COX-dependent sweating is impacted in these individuals. However, given that COX is reportedly not involved in cutaneous vasodilatation in older adults during whole-body passive heating (Holowatz et al. 2009) or exercise-induced hyperemia in the forearm (Schrage et al. 2007), COX-dependent sweating may be similarly impaired in older adults during exercise in the heat. In addition to their influence on sweating, both NOS and COX have also been shown to modulate heat dissipation via cutaneous vasodilatation in young adults during a passively induced heat stress at rest (Wilkins et al. 2003; McCord et al. 2006; Wong & Minson, 2006; Wong & Fieger, 2012). However, their respective contributions during whole-body heat stress are diminished in older adults such that no measurable contribution of COX (Holowatz et al. 2009) or a significant albeit smaller contribution of NOS (Stanhewicz et al. 2012) to cutaneous vasodilatation was detected in older adults. It is important to note however, that the underlying mechanisms governing cutaneous vasodilatation may differ between a passive- and exercise-induced heat stress (Fujii et al. 2014; McNamara et al. 2014). Indeed, in contrast to the finding by McCord et al. (2006) who employed whole-body passive heating, no functional role of COX in cutaneous vasodilatation was reported during exercise in the heat in young adults (Fujii et al. 2014). Therefore, it remains to be determined whether NOS and/or COX contribute to cutaneous vasodilatation in older adults exercising in the heat. Most studies to date have focused on the contribution of NOS and COX separately (Dalle-Ave et al. 2004; Welch et al. 2009; McGinn et al. 2014a; Stapleton et al. 2014a). However, the existence of a potential interaction between these pathways is proposed in young adults
for sweating (Fujii et al. 2014) and cutaneous vasodilatation (Medow et al. 2008; Fujii et al. 2013). This interaction is evidenced by the non-additive reduction in sweating and cutaneous vasodilatation as a result of the separate and combined inhibition of NOS and COX. As a consequence, inhibiting NOS and/or COX separately may lead to incomplete conclusions of their roles in modulating sweat production and cutaneous vasodilatation. Indeed, in older adults, COX inhibition has been shown to up-regulate NOS-dependent mechanisms as evaluated by resting cutaneous blood flow during normothermia (Holowatz et al. 2009). Although Holowatz et al. (2009) demonstrated that no interaction of NOS and COX pathways exist for cutaneous vasodilatation in older adults during whole-body passive heating, direct evaluation is necessary to clarify whether this is also true for the cutaneous vasodilatation and sweating responses during exercise in the heat. The effects of ageing on COX-dependent sweating and cutaneous vasodilatation are also of clinical importance given that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX, are more frequently prescribed to older rather than younger adults (Chiroli et al. 2003). Thus, the purpose of the current study was to evaluate the separate and combined influence of NOS and COX on forearm sweating and cutaneous vasodilatation in older adults during exercise in the heat. Assessing the contribution of these pathways in the context of recent reports that have demonstrated marked attenuations in heat dissipation in older adults, which remained intact with successive exercise bouts (Larose et al. 2013, 2014; Stapleton et al. 2014b), would provide valuable insight into the potential mechanism(s) underpinning this response. We hypothesized that in older adults both NOS and COX inhibition would not modulate local forearm sweat rate during exercise in the heat. Furthermore, we surmised that NOS would independently contribute to forearm cutaneous vasodilatation, and the aforementioned mechanisms would not differ between the first and second exercise bouts. Methods Ethical approval
This study was approved by the University of Ottawa Health Sciences and Science Research Ethics Board and C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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conformed to the guidelines set out in the Declaration of Helsinki. Verbal and written informed consent was obtained from all volunteers prior to their participation in the study. Subjects
Twelve older males ranging from 53 to 72 years in age participated in this study. All subjects were healthy and habitually active (2–6 days per week, 30 min of exercise per day). Subjects were excluded if they had a history of cystic fibrosis transmembrane conductance regulator mutations, skin disorders, hypertension, heart disease, diabetes, autonomic disorders or cigarette smoking. Mean (± SD) characteristics of the subjects as assessed during a preliminary session (see below) were: age, 62 ± 7 years; height, 1.73 ± 0.06 m; body mass, 72.5 ± 9.0 kg; body surface area, 1.86 ± 0.13 m2 ; and body fat, 23.0 ± 6.7%. Maximal oxygen consumption was 3.0 ± 0.5 l min−1 and when expressed relative to body mass it was 42.9 ± 9.3 ml O2 kg−1 min−1 . Resting mean arterial pressure was 93 ± 10 mmHg. Experimental procedures
The current study consisted of one preliminary and one experimental session. For both sessions, all subjects abstained from taking over-the-counter medications (including NSAIDs, vitamins and minerals) for at least 48 h prior to arriving at the laboratory (no subjects were taking prescribed medications). Subjects also refrained from alcohol, caffeine and heavy exercise at least 12 h before each session, and did not consume any food 2 h before and throughout each session. During the preliminary session, body height, mass, surface area and density as well as maximal oxygen consumption were determined. Body height was measured using an eye-level physician stadiometer (Model 2391, Detecto Scale, Webb City, MO, USA), while body mass was measured using a digital weight scale platform (Model CBU150X, Mettler Toledo, Schwerzenbach, Switzerland) with a weighing terminal (Model IND560, Mettler Toledo). Body surface area was subsequently calculated from the measurements of body height and mass (DuBois & DuBois, 1916). Body density was measured using the hydrostatic weighing technique, and used to estimate body fat percentage (Siri, 1956). To determine maximal oxygen consumption and heart rate, the subjects performed an incremental cycling protocol until exhaustion at a pedalling rate of 60–90 r.p.m. on a semi-recumbent cycle ergometer (Corival Recumbent, Lode, Groningen, Netherlands). The starting workload for the first 1 min was set at 80 W and was increased at a rate of 20 W min−1 until the subject could no longer maintain a pedalling rate of >50 r.p.m.. During C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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the incremental exercise, breath-by-breath oxygen uptake was estimated by an automated gas analyser (Medgraphics Ultima, Medical Graphics, St Paul, MN, USA) while a qualified technician continuously monitored the subjects via ECG. Maximal oxygen consumption was taken as the highest average oxygen uptake measured over 30 s. Shortly after arriving in the laboratory on the day of the experimental session, subjects changed into shorts and running shoes, provided a urine sample and voided the remainder of their bladder. Following a measurement of body mass, subjects were then seated in a semi-recumbent position in a thermoneutral room (23°C) and instrumented with four microdialysis fibres (30 kDa cutoff, 10 mm membrane) (MD2000, Bioanalytical Systems, West Lafayette, IN, USA) in the dermal layer of the skin on the dorsal side of the left forearm. A 25-gauge needle was first inserted into the unanaesthetized skin using the aseptic technique, with the entry and exit points separated by 2.5 cm. Ice or local anaesthetic cream used previously (Hodges et al. 2009) was not employed in this study, as it is unknown how these interventions influence the sweating response elicited by exercise in the heat. The microdialysis fibre was then threaded through the lumen of the needle after which the needle was withdrawn leaving the fibre in place. Each microdialysis fibre was secured with surgical tape and was separated from adjacent fibres by at least 4 cm. Thereafter, the subjects moved to a thermal chamber (Can-Trol Environmental Systems, Markham, ON, Canada) regulated to an ambient air temperature of 35°C and a relative humidity of 20%, and rested on a semi-recumbent cycle ergometer (Corival Recumbent). At least 20 min after the fibre placement, perfusion of the microdialysis fibres with the pharmacological agents began. Fibres were assigned in a counterbalanced manner to receive (1) lactated Ringer solution (Control); (2) 10 mM ketorolac, a non-selective COX inhibitor (Ketorolac, Sigma-Aldrich, St Louis, MO, USA); (3) 10 mM NG -nitro-L-arginine methyl ester (L-NAME, Sigma-Aldrich) to non-selectively inhibit NOS and thus to reduce NO bioavailability; or (4) a combination of 10 mM ketorolac and 10 mM L-NAME (Ketorolac + L-NAME). Ketorolac and L-NAME were dissolved in lactated Ringer solution. These concentrations were determined based on previous studies in which intradermal microdialysis was employed in human skin (Holowatz et al. 2005, 2009; Kellogg et al. 2005; McCord et al. 2006; Medow et al. 2008; Fujii et al. 2013, 2014). We chose to employ a 10 mM concentration of ketorolac to inhibit COX while avoiding a sustained increase in cutaneous blood flow, as has been seen with concentrations >10 mM (Holowatz et al. 2005; Kellogg et al. 2005). A microinfusion pump (Model 400, CMA Microdialysis, Solna, Sweden) was used to continuously perfuse each drug at
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a rate of 2.0 µl min−1 for at least 75 min to ensure the establishment of each blockade. This 75 min plus 20 min between fibre placement and the start of drug infusion was likely sufficient for the hyperemia associated with the placement of the fibres to subside, i.e. 90 min (Hodges et al. 2009). The drug perfusion continued for the entire experimental protocol until the maximal cutaneous vasodilatation procedure began (see below). After 10 min of baseline data collection, subjects performed two successive 30 min bouts of semi-recumbent cycling at a fixed rate of metabolic heat production of 400 W (equivalent to a percentage of maximal oxygen uptake of 48 ± 3% and requiring an external workload of 70 ± 3 W). The first and second bouts of exercise were followed by a 20 and 40 min recovery period, respectively. Absolute heat load was used in the current study to ensure a similar thermal drive for whole-body sweating in all subjects (Gagnon et al. 2013). Following the second 40 min recovery period, 50 mM sodium nitroprusside (SNP; Sigma-Aldrich) was administered at a rate of 3.0 µl min−1 for 20–30 min to elicit maximal cutaneous vasodilatation. After maximal cutaneous vasodilatation was achieved, as defined by a plateau in cutaneous blood flow for at least 2 min, body mass was measured and a final urine sample was collected.
Measurements
Sweat capsules, each covering an area of 3.8 cm2 , were placed directly over the centre of the microdialysis membranes and attached to the skin with adhesive rings and topical skin glue (Collodion HV, Mavidon Medical Products, Lake Worth, FL, USA). Dry compressed air from gas tanks located in the thermal chamber was supplied to each capsule at a rate of 1.0 l min−1 . The water content of the effluent air was measured with a capacitance hygrometer (Model HMT333, Vaisala, Helsinki, Finland). Long vinyl tubes were used for connections between the gas tank and the sweat capsule, and between the sweat capsule and the hygrometer, which allowed internal gas temperature to be equilibrated to near room temperature (35°C) before reaching the sweat capsule (inlet) and the hygrometer (outlet). Local forearm sweat rate was calculated every 5 s based on the difference in water content between influent and effluent air, multiplied by the flow rate, and normalized for the skin surface area under the capsule (expressed in mg min−1 cm−2 ). Cutaneous red blood cell flux (expressed in perfusion units), which is an index of cutaneous blood flow, was locally measured at a sampling rate of 32 Hz with laser Doppler flowmetry (PeriFlux System 5000, Perimed, Stockholm, Sweden). Integrated laser Doppler flowmetry probes with a seven-laser array (Model 413, Perimed) were housed in the centre of each sweat
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capsule directly over each microdialysis fibre, allowing for the simultaneous measurement of both local forearm sweat rate and cutaneous red blood cell flux at each skin site. Systolic and diastolic blood pressures as well as heart rate were measured every 5 min using an automated blood pressure monitor (Tango+, SunTech Medical, Morrisville, NC, USA) and verified by auditory inspection. Mean arterial pressure was evaluated as diastolic arterial pressure plus one-third the difference between systolic and diastolic pressures (i.e. pulse pressure). Cutaneous vascular conductance (CVC) was evaluated as cutaneous red blood cell flux divided by mean arterial pressure. CVC data were represented as percentage of maximum as evaluated during the maximal cutaneous vasodilatation procedure to minimize the effect of site-to-site heterogeneity in the level of cutaneous blood flow (Minson, 2010). Oesophageal temperature was measured with a general purpose thermocouple temperature probe (Mallinckrodt Medical, St Louis, MO, USA). The probe was inserted 40 cm past the entrance of the nostril while the subject sipped water (200 ml) through a straw. Skin temperature was measured at ten sites using thermocouples (Concept Engineering, Old Saybrook, CT, USA) attached to the skin with surgical tape. Mean skin temperature was calculated according to proportions determined by Hardy and Dubois (1938) based on local skin temperature measurements at the ten sites (forehead, 7%; upper back, 8.75%; chest, 8.75%; bicep, 9.5%; forearm, 9.5%; abdomen, 8.75%; lower back, 8.75%; quadriceps, 9.5%; hamstring, 9.5%; front calf, 20%). Core body and skin temperature data were collected at a sampling rate of 15 s using a data acquisition module (Model 34970A; Agilent Technologies Canada, Mississauga, ON, Canada) and simultaneously displayed and recorded in spreadsheet format on a personal computer with LabVIEW software (Version 7.0, National Instruments, Austin, TX, USA). Metabolic rate was determined using indirect calorimetry (Nishi, 1981). Expired gas was analysed for oxygen (error of ±0.01%) and carbon dioxide (error of ±0.02%) concentrations using electrochemical gas analysers (AMETEK model S-3A/1 and CD3A, Applied Electrochemistry, Pittsburgh, PA, USA). Approximately 20 min before the start of baseline data collection, gas mixtures of known concentrations were used to calibrate gas analysers and a 3 litre syringe was used to calibrate the turbine ventilometer. The subjects wore a full face mask (Model 7600 V2, Hans-Rudolph, Kansas City, MO, USA) attached to a two-way T-shape non-rebreathing valve (Model 2700, Hans-Rudolph). Oxygen uptake and respiratory exchange ratio were obtained every 30 s and were used to calculate metabolic rate (Nishi, 1981; Kenny & Jay, 2013). Metabolic heat load was estimated from metabolic rate minus external work (i.e. work rate during cycling). C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Urine specific gravity was evaluated from the urine samples obtained at the start and end of the experimental protocol using a hand-held total solids refractometer (Model TS400, Reichter, Depew, NY, USA). Data analysis
Baseline resting values were obtained by averaging measurements performed over >10 min. Values at the start of intermittent exercise (time 0) were obtained during the last 5 min before exercise commenced. Local forearm sweat rate and CVC as well as 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 difference in sweat rate from Control at the Ketorolac, L-NAME and Ketorolac + L-NAME sites were evaluated to determine the magnitude to which each treatment influenced sweating. Changes () from baseline resting were calculated for body temperatures at each time point. The heart rate and blood pressure data acquired during the exercise and recovery periods were obtained by averaging the two measurements made over each 10 min interval. Maximal CVC values induced with SNP administration at the end of the experimental protocol were determined from averaging CVC data over at least 2 min once a plateau was established. Statistical analysis
For statistical purposes, the exercise/recovery cycles were defined based on the following time periods: (1) 0–30 min: exercise 1, (2) 30–50 min: recovery 1, (3) 50–80 min: exercise 2, and (4) 80–120 min: recovery 2. Local forearm sweat rate and CVC were analysed using a two-way repeated-measures ANOVA with the factor of time (14 levels: rest, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 min) and of treatment site (four levels: Control, Ketorolac, L-NAME and Ketorolac + L-NAME). Body temperature and cardiovascular variables were analysed using a one-way repeated-measures ANOVA with the factor of time (six levels: rest, exercise 1 at 30 min, recovery 1 at 20 min, exercise 2 at 30 min, recovery 2 at 20 min and 40 min). Moreover, local forearm absolute maximal CVC (expressed in perfusion units mmHg−1 multiplied by 100) attained during the SNP infusion was analysed with a one-way repeated-measures ANOVA with the factor of treatment site (four levels: Control, Ketorolac, L-NAME and Ketorolac + L-NAME). When a significant main effect was observed, post hoc multiple comparisons were carried out using a Student–Newman–Keuls procedure. All values are reported as mean ± SEM unless otherwise indicated. As a secondary analysis, we assessed the relative influence of the separate and combined inhibition of NOS C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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and COX pathways on individual sweat rates. Specifically, Pearson’s product moment correlation coefficients were used to determine if the effect of the treatment was influenced by the level of sweating achieved during exercise (as measured at the Control site). Furthermore, a correlative analysis was also performed to determine if the magnitude of the difference in sweat rate from Control was similar between Ketorolac and L-NAME treatment sites. Two-tailed Student’s paired t tests were used to compare body mass as well as urine specific gravity before and after the trial. The level of significance for all analyses was set at P 0.05. Results Hydration status
Body mass was reduced from baseline levels by 1.3 ± 0.1% after the experiment (P < 0.01). Urine specific gravity following the session (1.019 ± 0.001) was elevated in comparison to pre-trial levels (1.013 ± 0.002, P < 0.01). The post-session urine specific gravity indicates that the subjects remained relatively euhydrated following two bouts of exercise in the heat according to the standard guidelines (Sawka et al. 2007). Core body and skin temperatures
There was a main effect of time on oesophageal temperature (P < 0.01). Specifically, oesophageal temperature was elevated during each exercise and recovery period relative to baseline resting and higher at the end of the second relative to the first exercise bout (Table 1). Furthermore, a main effect of time (P < 0.01) was measured for mean skin temperature such that it was elevated during each exercise bout compared to baseline resting (Table 1). Cardiovascular variables
A main effect of time was detected for heart rate and mean arterial pressure (both P < 0.01). In comparison to baseline resting (60 ± 3 b.p.m.), heart rate was elevated during intermittent exercise (last 10 min of each bout: 98 ± 3 and 102 ± 4 b.p.m., respectively) and the recovery period (minutes 10–20 of recovery 1: 66 ± 4 b.p.m.; minutes 10–20 and 30–40 of recovery 2: 69 ± 4 and 68 ± 5 b.p.m., respectively) (all P < 0.01). Likewise, mean arterial pressure was elevated from baseline resting (93 ± 2 mmHg) during the last 5 min of exercise 1 (100 ± 3 mmHg) and exercise 2 (100 ± 3 mmHg). However, mean arterial pressure during recovery 1 (10–20 min: 91 ± 2 mmHg) and recovery 2 (10–20 min: 89 ± 2 mmHg, 30–40 min: 90 ± 2 mmHg) did not differ from baseline resting (all P > 0.05).
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Table 1. Oesophageal and mean skin temperatures (°C) at rest and during the two exercise and recovery periods Exercise 1
Recovery 1
Exercise 2
Recovery 2
Rest
30 min
20 min
30 min
20 min
40 min
37.03 ± 0.06
37.59 ± 0.09∗ 0.56 ± 0.05
37.30 ± 0.06∗ 0.27 ± 0.03
37.75 ± 0.09∗,† 0.72 ± 0.05
37.38 ± 0.06∗ 0.35 ± 0.04
37.34 ± 0.05∗ 0.31 ± 0.05
34.96 ± 0.11
35.48 ± 0.12∗ 0.52 ± 0.10
35.08 ± 0.12 0.12 ± 0.10
35.45 ± 0.12∗ 0.49 ± 0.13
35.05 ± 0.12 0.09 ± 0.12
34.90 ± 0.11 –0.06 ± 0.12
Oesophageal temperature Change from Rest Mean skin temperature Change from Rest
Values are means ± SEM. Values represent an average of the final 5 min of the corresponding period. ∗ P < 0.05 vs. Rest. No statistical analysis was performed for the change in oesophageal and mean skin temperatures. † P < 0.05 Exercise 1 vs. Exercise 2 or Recovery 1 vs. Recovery 2.
baseline resting until the end of the second recovery period (Fig. 1). The change in the sweat rate induced by Ketorolac and L-NAME were negatively correlated with absolute sweat rate measured at the untreated Control site (Fig. 2). Similar results were also obtained at the combined Ketorolac + L-NAME site (R = −0.63 and P = 0.02 for exercise 1; R = −0.52 and P = 0.07 for exercise 2). Moreover, the patterns of response at the Ketorolac and L-NAME treatment sites were similar
Local forearm sweat rate
While there was no interaction of treatment site and time (P = 1.00), a main effect of time (P < 0.01) was observed for local forearm sweat rate. At all four sites, sweat rate measured at 10 min into exercise 2 was greater than it was at 10 min into exercise 1 (all P < 0.05). However, no main effect of treatment site was detected for local forearm sweat rate (P = 0.92) such that no differences in local forearm sweat rate were measured between the treatment sites from
Sweat rate (mg min−1cm−2)
1.0
Rest
Ex 1
Rec 1
Ex 2
Rec 2
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
120
Time (min) Control
Ketorolac
L-NAME
Ketorolac + L-NAME
Figure 1. Time-course changes in sweat rate during exercise performed at a fixed rate of metabolic heat production (400 W). Four skin sites continuously received either (1) lactated Ringer solution (Control, circles); (2) 10 mM ketorolac (Ketorolac, squares), a non-selective cyclooxygenase inhibitor; (3) 10 mM NG -nitro-L-arginine methyl ester (L-NAME, triangles), a non-specific nitric oxide synthase inhibitor; or (4) a combination of 10 mM ketorolac + 10 mM L-NAME (Ketorolac + L-NAME, diamonds). Values are means ± SEM. Each value during exercise and recovery represents the average of the last 5 min of each 10 min interval. Start of intermittent exercise (time 0) indicates resting values 5 min before exercise. Ex 1, first exercise; Rec 1, first recovery; Ex 2, second exercise; Rec 2, second recovery. Neither a main effect of treatment site (P = 0.92) nor an interaction of treatment site and time (P = 1.00) was detected. Thus, a between-site difference in sweat rate at each time point was not observed. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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(R = 0.79 and P < 0.01 for exercise 1; R = 0.76 and P < 0.01 for exercise 2).
Local forearm cutaneous vascular response
There was no interaction of treatment site and time (P = 0.46) measured for local forearm CVC (P < 0.01). However, a main effect of time was detected (P < 0.01), revealing that, irrespective of treatment site, CVC was elevated during the intermittent exercise bouts and returned to baseline resting within the first 20 min of both recovery periods. There was also a main effect of treatment site (P = 0.04) detected for CVC. During both exercise bouts, CVC was similar between Ketorolac and Control sites. However, CVC was lower at the L-NAME and Ketorolac + L-NAME sites relative to Control (Fig. 3). Similarly, relative to the Ketorolac site, CVC was lower at the L-NAME (10, 20 and 30 min into the first exercise and 30 min into the second exercise) and Ketorolac + L-NAME sites (20 and 30 min into the first exercise and 30 min into the second exercise) (all P 0.05). No between-site
Change in SR from Control (mg min-1 cm-2)
A
Discussion The present study examined the separate and combined influence of NOS and COX on forearm sweating and cutaneous vasodilatation in older adults during intermittent exercise (two exercise/recovery cycles) in the heat. Consistent with our hypothesis, we demonstrated that separate and combined inhibition of NOS and COX did not affect sweat rate during the first or second exercise bout. Furthermore, cutaneous vasodilatation was not influenced by COX inhibition; however, NOS blockade
1.0
0.5
0.5
0.0
0.0 R = −0.61 P = 0.01
−0.5
L-NAME
D
(first exercise)
L-NAME
1.0
0.5
0.5
0.0
0.0 −0.5
R = −0.80 P < 0.01
−1.0 0.0
0.5
R = -0.51 P = 0.08
−1.0 0.0 0.5 1.0 1.5 SR at Control (mg min-1 cm-2)
1.0
−0.5
Ketorolac (second exercise)
−0.5
−1.0 0.0 0.5 1.0 1.5 SR at Control (mg min-1 cm-2)
C Change in SR from Control (mg min-1 cm-2)
differences in CVC were found at baseline resting, at the start of intermittent exercise (time 0), or throughout both recovery periods (all P > 0.05). There was no main effect of treatment site (P = 0.10) detected for local forearm absolute maximal CVC. Thus, local forearm absolute maximal CVC (in perfusion units mmHg−1 multiplied by 100) did not differ between treatment sites (Control: 172 ± 13, Ketorolac: 192 ± 14, L-NAME: 183 ± 13, Ketorolac + L-NAME: 157 ± 20).
B
Ketorolac (first exercise)
1.0
1.0
1.5
SR at Control (mg min-1 cm-2)
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(second exercise)
R = −0.79 P < 0.01
−1.0 0.0
0.5
1.0
1.5
SR at Control (mg min-1 cm-2)
Figure 2. Relationship between sweat rate (SR) at the Control site and the change in sweat rate from the Control at skin sites receiving ketorolac and L-NAME Correlative relationship between SR measured at the lactated Ringer solution site (Control) and the change in sweat rate from the Control at skin sites receiving 10 mM ketorolac (Ketorolac, A and B), a non-selective cyclooxygenase inhibitor, or 10 mM NG -nitro-L-arginine methyl ester (L-NAME, C and D), a non-specific nitric oxide synthase inhibitor. Data represent the 5 min average of the first and second exercise bouts for each subject. Horizontal dotted line indicates 0 value. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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attenuated the response regardless of whether COX was inhibited simultaneously. In addition, we show that the sweating and cutaneous vascular responses were unaltered by the separate and combined inhibition of NOS and COX during each recovery period. Altogether, our findings demonstrate that the NOS and COX pathways do not functionally modulate forearm sweating whereas NOS contributes to forearm cutaneous vasodilatation independent of COX during intermittent exercise in the heat.
Sweating
Cutaneous vascular conductance (%max)
The current study reveals no functional role of NOS and COX in the sweating response during exercise-induced heat stress in older adults. We demonstrated that sweat rate during the first exercise bout did not differ between the Control and L-NAME sites (Fig. 1), confirming a previous study suggesting that NOS is not functionally involved in the sweating response in older adults during exercise in the heat (Stapleton et al. 2014a). Moreover, we did not observe a measurable NOS-dependent sweating in the second exercise bout despite the fact that core body
100
Rest
Ex 1
† *
80
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temperature was elevated relative to the first exercise bout. Hence, it appears that differences in level of core body temperature, and therefore thermal drive, do not alter the involvement of NOS in the mechanisms for sweating in older adults. Given that L-NAME has recently been shown to reduce sweating in young adults during an intermittent exercise protocol identical to that used in the current study (Fujii et al. 2014), our results imply an age-related reduction in NOS-dependent sweating during successive exercise bouts in the heat. This is consistent with recent reports demonstrating that whole-body sweat rate is impaired in older adults, resulting in greater levels of body heat storage during intermittent exercise in the heat relative to their younger counterparts (Larose et al. 2013, 2014; Stapleton et al. 2014b). Clearly, the mechanisms underlying the impairment in sweating seen in older adults require further scrutiny. However, it is plausible that an age-related increase in reactive oxygen species such as superoxide, which has been shown to decrease NO bioavailability in the human skin (Holowatz et al. 2006), may in part be involved. In fact, greater concentrations of urinary malondialdehyde, a marker of oxidative stress, were correlated with a lower sweat response induced by electrophoresis of acetylcholine (Hoeldtke et al. 2011). Ex 2
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Figure 3. Time course changes in cutaneous vascular conductance during exercise performed at a fixed rate of metabolic heat production (400 W) Four skin sites continuously received either (1) lactated Ringer solution (Control, circles); (2) 10 mM ketorolac (Ketorolac, squares), a non-selective cyclooxygenase inhibitor; (3) 10 mM NG -nitro-L-arginine methyl ester (L-NAME, triangles), a non-specific nitric oxide synthase inhibitor; or (4) a combination of 10 mM ketorolac + 10 mM NG -nitro-L-arginine methyl ester (Ketorolac + L-NAME, diamonds). Values are means ± SEM. Each value during exercise and recovery represents the average of the last 5 min of each 10 min interval. Start of intermittent exercise (time 0) indicates resting values 5 min before exercise. Ex 1, first exercise; Rec 1, first recovery; Ex 2, second exercise; Rec 2, second recovery. ∗ Control significantly different from L-NAME (P < 0.05); † Control significantly different from Ketorolac + L-NAME (P < 0.05). C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Ageing and cyclooxygenase-dependent sweating
For the first time, we demonstrate that local administration of ketorolac does not modulate the sweating response in older adults during successive exercise bouts in the heat (Fig. 1). Given that a role for COX in the mechanisms underpinning sweat production has recently been elucidated in young adults (Fujii et al. 2014), our findings indicate that similar to NOS, COX-dependent sweating appears to be diminished in older adults even with repeated exercise bouts eliciting progressively greater levels of hyperthermia. The reason(s) for this age-related attenuation in COX-dependent sweating is/are currently unknown. However, in parallel with the impairments in NOS-dependent sweating, age-related increases in reactive oxygen species in the skin (Holowatz et al. 2006) may be involved. Specifically, superoxide can deactivate COX (Egan et al. 1976) resulting in a lower bioavailability of prostanoids at the level of the sweat gland. Alternatively, ageing may decrease the responsiveness of prostanoid receptor(s) to agonists, as indicated by Nicholson et al. (2009) who showed that ageing decreases prostanoid receptor sensitivity to prostacyclin- (a prostanoid) mediated forearm vasodilatation in humans. Altogether, diminished COX-dependent sweating in older adults may be explained by a lower bioavailability of prostanoids, reduced prostanoid receptor sensitivity or a combination of the two. Consistent with our results for the separate inhibition of both NOS and COX, we observed no clear effect of the simultaneous inhibition of these pathways on the sweating response (Fig. 1). Holowatz et al. (2009) highlighted the importance of the interaction between these pathways in the context of the regulation of cutaneous blood flow under normothermic conditions. Specifically, they reported that COX inhibition can up-regulate NOS-dependent mechanisms, thereby altering the level of cutaneous blood flow. However, we can discount the COX inhibition-mediated compensatory up-regulation of NOS-dependent mechanisms for sweating in the context of exercise in the heat in our study. Furthermore, while recent findings suggest that NOS and COX modulate sweating during exercise in an interactive manner in young adults (Fujii et al. 2014), the current findings are consistent with an age-related attenuation in the contribution of these pathways to the regulation of sweating during exercise in the heat. We observed a rapid decrease in sweating at the Control site after the first exercise and this response remained intact after a second exercise bout despite greater increases in core body temperature (Table 1). How this rapid and robust suppression of sweating occurs has not been completely elucidated, but it is postulated that several factors of non-thermal origin (i.e. baro-, metaboand/or osmoreceptors) play an important role (Kenny & Jay, 2013). Furthermore, the pronounced decrease in sweating measured at the Control site was similarly C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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observed with the separate and combined inhibition of NOS and COX such that responses between sites remained similar throughout each recovery period. In view of these observations, we can conclude that the post-exercise attenuation of sweating occurs independent of NOS- and COX-dependent mechanisms in older adults, a response consistent with our observations in young adults (Fujii et al. 2014). While our findings, when expressed as a group mean, indicate no clear influence of either pathway on the sweating response, upon closer examination we observed that the relative influence of NOS and COX differed markedly amongst subjects. Specifically, we showed that individuals who achieved a higher sweat rate at the untreated Control site exhibited a more pronounced reduction in sweat rate induced by inhibition of NOS and/or COX (Fig. 2) and that the pattern of response was similar between NOS and COX inhibition sites. While the mechanism underpinning this variation cannot be elucidated from the current data, it does reveal that the responsiveness of the NOS and COX pathways to the sweating response may be influenced by unknown factors which require further scrutiny.
Cutaneous vascular response
Until now, the mechanisms underlying the cutaneous blood flow response in older adults during exercise in the heat have remained largely unstudied. In the current study, we demonstrate that despite no measurable effect on sweating, NOS blockade attenuated CVC during both exercise bouts relative to Control (Fig. 3). Furthermore, NOS-dependent cutaneous vasodilatation remained intact with successive exercise bouts and the consequent increases in the level of hyperthermia. The preserved NOS-dependent cutaneous vasodilatation observed in the current study is in accordance with previous work reporting NOS-dependent vasodilatation during whole-body passive heating in older adults (Stanhewicz et al. 2012). Altogether, these findings show that NOS is an important contributor to the regulation of cutaneous blood flow in older adults when exposed to passively induced or exercise-induced heat stress. However, our disparate findings for a role of NOS between cutaneous blood flow and sweating during exercise suggest that distinct pathways are involved in the regulation of these heat loss mechanisms. Further studies should be conducted to advance our understanding of the pathways governing age-related changes of these heat loss responses given their importance in the regulation of heat exchange during exercise, especially in the heat. In the current study, we showed that inhibition of COX activity did not affect cutaneous vasodilatation during or following exercise irrespective of increases in
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the level of hyperthermia with successive exercise bouts. A similar pattern of response was reported by Holowatz et al. (2009), who also observed no functional role for COX in cutaneous vasodilatation in older adults undergoing a passive heat stress resulting in increases in core body temperature comparable to those measured in the current study (i.e. 0.5–0.7°C). Moreover, they showed that this response remained intact with elevations in core body temperature of up to 1.0°C. Furthermore in young adults, COX contribution to cutaneous vasodilatation was not observed during two successive bouts of exercise (Fujii et al. 2014). Hence, it appears that COX does not functionally contribute to the cutaneous vasodilatatory response in younger adults during exercise in the heat and this pattern of response is unchanged by ageing. In the current study, CVC returned to values similar to baseline resting levels at each treatment site by the 20 min time point of both recovery periods despite a greater increase in thermal drive as reflected by a higher core body temperature at the end of the second relative to first exercise bout (Table 1). In parallel, we observed that neither separate nor combined inhibition of NOS and COX modulated CVC during either recovery period. Thus, factors unrelated to NOS and COX are probably involved in cutaneous vascular regulation during post-exercise recovery in older adults. For instance, we recently identified in young adults that adenosine receptors are largely involved in the rapid suppression of cutaneous blood flow following exercise (McGinn et al. 2014a,b). Given that the role of adenosine receptors in the human forearm vascular response is preserved in older adults (as evaluated by adenosine-mediated vasodilatation) (Kirby et al. 2010), adenosine receptor activation may also play a major role in the post-exercise suppression of CVC in older adults.
Clinical perspectives
NSAIDs such as aspirin, which inhibit COX activity, are more frequently prescribed in older relative to younger adults as an analgesic and an anti-platelet agent (Chiroli et al. 2003). Hence an evaluation of how COX inhibitors impact heat loss responses during exercise in the heat has particular clinical relevance. In the current study, which was specifically designed to evaluate the local mechanisms governing forearm sweating and cutaneous vasodilatation, we demonstrated that there is no clear influence of COX on forearm sweating and cutaneous vasodilatator responses in older adults. It is important to acknowledge that COX is commonly prescribed by oral administration, which may influence both local and/or central mechanisms. In contrast to our results demonstrating that local COX inhibition did not affect the cutaneous blood flow response, Bruning et al. (2013) demonstrated that oral
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administration of low dose aspirin (81 mg daily for 7 days) attenuated cutaneous vasodilatation during exercise in the heat in middle-aged adults. Altogether these results seem to suggest that central mechanisms are involved in the aspirin-mediated attenuation of cutaneous vasodilatation observed by Bruning et al. (2013). Along these lines, COX is found in the preoptic area of the brain, which is responsible for body temperature regulation (Eskilsson et al. 2014). Thus, future studies should be conducted to determine if the effects of orally administered COX inhibitors resemble those observed during local administration via microdialysis and if oral doses of COX inhibitors can also influence the heat loss responses through centrally mediated pathways. Conclusion
We demonstrate that there is no clear role of NOS and COX in the forearm sweating response of older adults during exercise in the heat. On the other hand, NOS but not COX contributes to forearm cutaneous vasodilatation during exercise in the heat. Furthermore, we demonstrate that following exercise forearm sweating and cutaneous vascular responses occur without measurable involvements of NOS- and COX-dependent mechanisms. Finally, we show that all the above mechanisms remain unchanged with successive exercise bouts/recovery periods. References Bruning RS, Dahmus JD, Kenney WL & Alexander LM (2013). Aspirin and clopidogrel alter core temperature and skin blood flow during heat stress. Med Sci Sports Exerc 45, 674–682. Chiroli S, Chinellato A, Didoni G, Mazzi S & Lucioni C (2003). Utilisation pattern of nonspecific nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in a local health service unit in northeast Italy. Clin Drug Invest 23, 751–760. Dalle-Ave A, Kubli S, Golay S, Delachaux A, Liaudet L, Waeber B & Feihl F (2004). Acetylcholine-induced vasodilation and reactive hyperemia are not affected by acute cyclo-oxygenase inhibition in human skin. Microcirculation 11, 327–336. DuBois D & DuBois EF (1916). A formula to estimate the approximate surface area if height and weight be known. Arch Intern Med 17, 863–871. Egan RW, Paxton J & Kuehl FA, Jr (1976). Mechanism for irreversible self-deactivation of prostaglandin synthetase. J Biol Chem 251, 7329–7335. Eskilsson A, Tachikawa M, Hosoya K & Blomqvist A (2014). Distribution of microsomal prostaglandin E synthase-1 in the mouse brain. J Comp Neurol 522, 3229–3244. Fujii N, McGinn R, Stapleton JM, Paull G, Meade RD & Kenny GP (2014). Evidence for cyclooxygenase-dependent sweating in young males during intermittent exercise in the heat. J Physiol 592, 5327–5339. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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Fujii N, Reinke MC, Brunt VE & Minson CT (2013). Impaired acetylcholine-induced cutaneous vasodilation in young smokers: roles of nitric oxide and prostanoids. Am J Physiol Heart Circ Physiol 304, H667–H673. Gagnon D, Jay O & Kenny GP (2013). The evaporative requirement for heat balance determines whole-body sweat rate during exercise under conditions permitting full evaporation. J Physiol 591, 2925–2935. Gisolfi CV & Wenger CB (1984). Temperature regulation during exercise: old concepts, new ideas. Exerc Sport Sci Rev 12, 339–372. Hardy JD & Dubois EF (1938). The technic of measuring radiation and convection. J Nutr 15, 461–475. Hodges GJ, Chiu C, Kosiba WA, Zhao K & Johnson JM (2009). The effect of microdialysis needle trauma on cutaneous vascular responses in humans. J Appl Physiol (1985) 106, 1112–1118. Hoeldtke RD, Bryner KD & VanDyke K (2011). Oxidative stress and autonomic nerve function in early type 1 diabetes. Clin Auton Res 21, 19–28. Holowatz LA, Jennings JD, Lang JA & Kenney WL (2009). Ketorolac alters blood flow during normothermia but not during hyperthermia in middle-aged human skin. J Appl Physiol 107, 1121–1127. Holowatz LA, Thompson CS & Kenney WL (2006). Acute ascorbate supplementation alone or combined with arginase inhibition augments reflex cutaneous vasodilation in aged human skin. Am J Physiol Heart Circ Physiol 291, H2965–H2970. Holowatz LA, Thompson CS, Minson CT & Kenney WL (2005). Mechanisms of acetylcholine-mediated vasodilatation in young and aged human skin. J Physiol 563, 965–973. Kellogg DL, Zhao JL, Coey U & Green JV (2005). Acetylcholine-induced vasodilation is mediated by nitric oxide and prostaglandins in human skin. J Appl Physiol 98, 629–632. Kenny GP & Jay O (2013). Thermometry, calorimetry, and mean body temperature during heat stress. Compr Physiol 3, 1689–1719. Kirby BS, Crecelius AR, Voyles WF & Dinenno FA (2010). Vasodilatory responsiveness to adenosine triphosphate in ageing humans. J Physiol 588, 4017–4027. Larose J, Boulay P, Wright-Beatty HE, Sigal RJ, Hardcastle S & Kenny GP (2014). Age-related differences in heat loss capacity occur under both dry and humid heat stress conditions. J Appl Physiol (1985) 117, 69–79. Larose J, Wright HE, Stapleton J, Sigal RJ, Boulay P, Hardcastle S & Kenny GP (2013). Whole body heat loss is reduced in older males during short bouts of intermittent exercise. Am J Physiol Regul Integr Comp Physiol 305, R619–R629. McCord GR, Cracowski JL & Minson CT (2006). Prostanoids contribute to cutaneous active vasodilation in humans. Am J Physiol Regul Integr Comp Physiol 291, R596–R602. McGinn R, Fujii N, Swift B, Lamarche DT & Kenny GP (2014a). Adenosine receptor inhibition attenuates the suppression of postexercise cutaneous blood flow. J Physiol 592, 2667–2678. McGinn R, Paull G, Meade RD, Fujii N & Kenny GP (2014b). Mechanisms underlying the postexercise baroreceptor-mediated suppression of heat loss. Physiol Rep 2. pii: e12168. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
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McNamara TC, Keen JT, Simmons GH, Alexander LM & Wong BJ (2014). Endothelial nitric oxide synthase mediates the nitric oxide component of reflex cutaneous vasodilatation during dynamic exercise in humans. J Physiol 592, 5317–5326. Medow MS, Glover JL & Stewart JM (2008). Nitric oxide and prostaglandin inhibition during acetylcholine-mediated cutaneous vasodilation in humans. Microcirculation 15, 569–579. Minson CT (2010). Thermal provocation to evaluate microvascular reactivity in human skin. J Appl Physiol 109, 1239–1246. Nicholson WT, Vaa B, Hesse C, Eisenach JH & Joyner MJ (2009). Aging is associated with reduced prostacyclin-mediated dilation in the human forearm. Hypertension 53, 973–978. Nishi Y (1981). Measurement of thermal balance in man. In Bioengineering, Thermal Physiology and Comfort, ed. Cena KCJ, pp. 29–39. Elsevier, New York. Sawka MN, Burke LM, Eichner ER, Maughan RJ, Montain SJ & Stachenfeld NS (2007). American College of Sports Medicine position stand. Exercise and fluid replacement. Med Sci Sports Exerc 39, 377–390. Schrage WG, Eisenach JH & Joyner MJ (2007). Ageing reduces nitric-oxide- and prostaglandin-mediated vasodilatation in exercising humans. J Physiol 579, 227–236. Siri WE (1956). The gross composition of the body. Adv Biol Med Phys 4, 239–280. Stanhewicz AE, Bruning RS, Smith CJ, Kenney WL & Holowatz LA (2012). Local tetrahydrobiopterin administration augments reflex cutaneous vasodilation through nitric oxide-dependent mechanisms in aged human skin. J Appl Physiol (1985) 112, 791–797. Stapleton JM, Fujii N, Carter M & Kenny GP (2014a). Diminished nitric oxide-dependent sweating in older males during intermittent exercise in the heat. Exp Physiol 99, 921–932. Stapleton JM, Poirier MP, Flouris AD, Boulay P, Sigal RJ, Malcolm J & Kenny GP (2014b). Aging impairs heat loss, but when does it matter? J Appl Physiol (1985) 118, 299–309. Welch G, Foote KM, Hansen C & Mack GW (2009). Nonselective NOS inhibition blunts the sweat response to exercise in a warm environment. J Appl Physiol 106, 796–803. Wilkins BW, Holowatz LA, Wong BJ & Minson CT (2003). Nitric oxide is not permissive for cutaneous active vasodilatation in humans. J Physiol 548, 963–969. Wong BJ & Fieger SM (2012). Transient receptor potential vanilloid type 1 channels contribute to reflex cutaneous vasodilation in humans. J Appl Physiol 112, 2037–2042. Wong BJ & Minson CT (2006). Neurokinin-1 receptor desensitization attenuates cutaneous active vasodilatation in humans. J Physiol 577, 1043–1051.
Additional information Competing interests None.
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Author contributions N.F. and G.P.K. conceived and designed the experiments. N.F., G.P., R.D.M., R.M. and P.A. contributed to data collection. N.F. performed data analysis. N.F., G.P., R.D.M., R.M., P.A. and G.P.K. interpreted the experimental results. N.F. drafted the manuscript. N.F., G.P., R.D.M., R.M., J.M.S., P.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.
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Program - Accelerator Supplements (RGPAS-462252-2014), and by Leaders Opportunity Fund from the Canada Foundation for Innovation (Grant 22529) (Funds held by G.P.K.). G.P.K. was supported by a University of Ottawa Research Chair Award. N.F. and J.M.S. were supported by the Human and Environmental Physiology Research Unit. R.M. and R.D.M. were supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology. G.P. was supported by the Ontario Graduate Scholarship.
Acknowledgements Funding This study was supported by the Natural Sciences and Engineering Research Council Discovery Grant (RGPIN298159-2009 and RGPIN-06313-2014), Discovery Grants
We greatly appreciate all of the volunteers for taking their time to participate in this study and Martin Poirier for his technical assistance. We thank Michael Sabino of Can-Trol Environmental Systems Ltd (Markham, ON, Canada) for his support.
C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society
