Experimental Physiology

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1 578 Exp Physiol 12.5 (17) pp Research Paper Research Paper Prostacyclin does not affect sweating but induces skin vasodilatation to a greater extent in older versus younger women: roles of NO and K Ca channels Naoto Fujii 1,2, Brendan D. McNeely 1, Takeshi Nishiyasu 2 and Glen P. Kenny 1 1 Human and Environmental Physiology Research Unit, University of Ottawa, Ottawa, Ontario, Canada 2 Institute of Health and Sport Sciences, University of Tsukuba, Tsukuba City, Japan Edited by: Philip Atherton Experimental Physiology New Findings What is the central question of this study? It remains unknown whether ageing modulates prostacyclin-induced cutaneous vasodilatation in women. What is the main finding and its importance? Prostacyclin induced cutaneous vasodilatation, albeit the magnitude of increase at lower concentrations of prostacyclin was greater in older relative to young women. This response was associated with greater contributions of nitric oxide synthase and calcium-activated potassium channels. Our results suggest that administration of prostacyclin might be an effective therapy to reverse microvascular hypoperfusion, especially in older women. We previously reported that prostacyclin induces cutaneous vasodilatation but not sweating in younger and older men. Furthermore, we demonstrated that nitric oxide synthase and calcium-activated potassium (K Ca ) channels contribute to the prostacyclin-induced cutaneous vasodilatation in younger men, although these contributions are diminished in older men. Given that the effects of ageing might differ between men and women, the above results cannot simply be applied to women. In this study, cutaneous vascular conductance and sweat rate were evaluated in younger (mean ± SD, 22 ± 3 years old) and older (55 ± 7 years old) women (1 per group) at four intradermal forearm skin sites treated as follows: (i) lactated Ringer solution without any drug (control); (ii) 1 mm N G -nitro-l-arginine (l-nna), a non-specific nitric oxide synthase inhibitor; (iii) 5 mm tetraethylammonium (TEA), a non-specific K Ca channel blocker; or (iv) 1 mml-nna plus 5 mm TEA. All four sites were co-administered with prostacyclin in an incremental manner (.4,.4, 4, 4 and 4 µm, each for 25 min). Surprisingly, increases in cutaneous vascular conductance in response to.4 4 µm prostacyclin were greater in older relative to younger women (all P.5), and these age-related differences were diminished when both l-nna and TEA were administered simultaneously (all P >.5). No effect on sweat rate was observed in either group (all concentrations, P >.5). We show that although prostacyclin does not mediate sweating, it induces cutaneous vasodilatation, and this response elicited by lower concentrations of prostacyclin is greater in older relative to younger women. This greater cutaneous vasodilatation in older women is likely to be attributable to nitric oxide synthase- and K Ca channel-dependent mechanisms. (Received 7 February 17; accepted after revision 2 March 17; first published online 7 March 17) Corresponding author G. P. Kenny: University of Ottawa, School of Human Kinetics, 125 University Private, Room 367, Montpetit Hall, Ottawa, ON, Canada K1N 6N5. gkenny@uottawa.ca DOI: /EP86297

2 Exp Physiol 12.5 (17) pp Ageing modulates prostacyclin-induced skin vasodilatation in women 579 Introduction We recently demonstrated that intradermal administration of prostacyclin induced cutaneous vasodilatation in a similar dose-dependent manner in younger and older men in normothermic resting conditions (Fujii et al. 16b). However, no influence on the sweating response was observed. Furthermore, we showed that both nitric oxide synthase (NOS) and calcium-activated potassium (K Ca ) channels contributed to the prostacyclin-induced cutaneous vasodilatation in younger men, but these contributions were diminished in older men (Fujii et al. 16b). It is unclear whether a similar response is observed in women. There is increasing evidence demonstrating that cutaneous vascular and sweating responses differ between men and women (Kellogg et al. 1; Gagnon & Kenny, 12; Quinton et al. 12; Gagnon et al. 13; Greaney et al. 14; Inoue et al. 14; Stanhewicz et al. 14; Fujii et al. 15a). Furthermore, although no study is currently available in humans in vivo, in vitro studies suggest that the ageing effect might affect men and women in different ways, including age-related vascular dysfunction (Barsha et al. 16; Costa et al. 16; Penna et al. 16). Therefore, the cutaneous vascular and sweating responses to prostacyclin observed in younger and older men of our previous work (Fujii et al. 16b) mightnotbesimply applied to younger and older women. We sought to evaluate the role of prostacyclin in the regulation of cutaneous vasodilatation and sweating in younger and older women. We evaluated the hypothesis that intradermal administration of prostacyclin elicits cutaneous vasodilatation to a similar magnitude in younger and older women, but the contributions of NOS and K Ca channels to the vasodilatation are attenuated in older women. We also hypothesized that prostacyclin does not increase sweating in both groups. Methods Ethical approval This study was conducted at the Human and Environmental Physiology Research Unit of the University of Ottawa and was approved by the University of Ottawa Health Sciences and Science Research Ethics Board in accordance with the standards outlined in the Declaration of Helsinki. All volunteers provided their verbal and written informed consent before their participation in the study. Participants Ten younger (mean ± SD, 22 ± 3 years old) and older (55 ± 7 years old) physically active (1 6 days per week, 3 min of exercise per day) women volunteered for one screening and one experimental session conducted Table 1. Participant characteristics Characteristic Younger Older Number of participants 1 1 Age (years) 22 ± 3 55 ± 7 Height (m) 1.63 ± ±.5 Body mass (kg) 6.6 ± ± 19.5 Arterial blood pressure (mmhg) Systolic 17 ± 6 1 ± 11 Diastolic 72 ± 9 78 ± 7 Mean 84 ± 7 92 ± 8 All values are expressed as means ± SD. P.5 versus older. on separate days. All participants were as follows: (i) no history of specific medical health conditions (cystic fibrosis transmembrane conductance regulator mutations, skin disorders, uncontrolled hypertension, heart disease, diabetes or autonomic disorders); (ii) not taking prescription medication; and (iii) not having smoked cigarettes for the past 1 years (including persons who had never experienced cigarette smoking). All younger women and four premenopausal older women (the other six older women were postmenopausal) were tested during their early follicular phase (within 6 days from the beginning of menstruation) or during the placebo pill phase if taking oral contraceptives. During the preliminary session, body height was assessed using an eye-level physician stadiometer (model 2391; Detecto Scale Company, Webb City, MO, USA) and body mass determined using a digital weight scale platform (model CBU15X; Mettler Toledo Inc., Columbus, OH, USA) with a weighing terminal (model IND56; Mettler Toledo Inc). Resting arterial blood pressure was obtained by manual auscultation using a mercury column sphygmomanometer (Baumanometer StandbyModel;W.A.BaumCo.Inc.,Copiague,NY,USA). The participant characteristics are summarized in Table 1. Experimental session The experimental session in this study was similar to our previous study conducted in younger and older men (Fujii et al. 16b). Before the experimental day, all participants refrained from taking over-the-counter medications for >48 h (e.g. non-steroidal anti-inflammatory drugs, vitamins and minerals), alcohol or caffeine and strenuous physical activity for >12 h and any food for >2 h. Participants rested in a semi-recumbent position on a bed (23 25 C), during which time a 25 gauge needle was inserted into the non-anaesthetized intradermal skin (left dorsal forearm) using an aseptic technique. The entry and exit points were separated by 2.5 cm. A microdialysis fibre (3 kda cut-off, 1 mm membrane; MD; Bioanalytical Systems, West Lafayette, IN, USA) wasthenintroducedthroughthelumenoftheneedle;

3 58 N. Fujii and others Exp Physiol 12.5 (17) pp thereafter, the needle was withdrawn. Four fibres were placed in the skin, each separated by 4 cmbetween sites. Each fibre was connected to a liquid switch to facilitate continuous agent perfusion (model 11; CMA Microdialysis AB, Kista, Sweden). Approximately 1 min after the fibre insertion, pharmacological agents were continuously administered in a counterbalanced manner to each skin site, as follows: (i) lactated Ringer solution (control; Baxter, Deerfield, IL, USA); (ii) 1 mm N ω -nitro-l-arginine (L-NNA; Sigma-Aldrich, St Louis, MO, USA), a non-selective NOS inhibitor; (iii) 5 mm tetraethylammonium (TEA), a non-specific K Ca channel blocker; or (iv) 1 mm L-NNA plus 5 mm TEA. Perfusion at each of the skin sites was maintained at a rate of 4. μl min 1 that was precisely regulated by a micro infusion pump (model 44; CMA Microdialysis AB). The concentrations of L-NNA (Medow et al. 8; Brunt et al. 15, 16) and TEA (Lorenzo & Minson, 7; Brunt et al. 15; Kutz et al. 15; Fujii et al. 16a) were determined according to previous studies using human intradermal microdialysis. The L-NNA and TEA were perfused for a minimal duration of 6 min before commencing data collection and throughout the protocol until the time the maximal cutaneous blood flow protocol began (see next paragraph). After the localized tissue trauma associated with fibre insertion had subsided ( 7 9 min after the fibre insertion), baseline measurements were acquired for at least 1 min (Baseline), after which the five incremental administrations of prostacyclin (Cayman Chemical, Ann Arbor, MI, USA;.4,.4, 4, 4 and 4 μm, each perfused for 25 min) were commenced at a rate of 4. μlmin 1 in accordance with our previous work (Fujii et al. 16b). After this incremental protocol, 5 mm of sodium nitroprusside (Sigma-Aldrich) was administered for 3 min at each microdialysis site at a rate of 6. μl min 1 to elicit maximal cutaneous blood flow, defined as a stable plateau for >2min. Measurements. A sweat capsule (1.1 cm 2 ) designed for use with intradermal microdialysis (Meade et al. 16) was attached to the intradermal microdialysis skin site using a topical skin glue (Collodion HV; Maiden Medical products, Lake Worth, FL, USA). The integrated laser-doppler flowmetry probe (model 413; Perimed, Stockholm, Sweden) was secured over the microdialysis fibre membrane through a form-fitted hole at the top of the ventilated capsule, allowing simultaneous measurements of cutaneous blood flow and sweating at each microdialysis site. Each laser-doppler probe was connected to the main controller (PeriFlux System 5; Perimed). Cutaneous red blood cell flux, an index of cutaneous blood flow expressed in perfusion units, was obtained at a sampling rate of 32 Hz. Cutaneous vascular conductance (CVC) was evaluated by cutaneous red blood cell flux divided by mean arterial pressure (diastolic arterial pressure plus one-third of the difference between systolic and diastolic pressures). Manual auscultation was performed using a mercury column sphygmomanometer (W. A. Baum Co. Inc.) to obtain blood pressures every 1 15 min. The CVC data were presented as a percentage of maximal blood flow (%max; Johnson et al. 14). To obtain the sweat rate, dry compressed air (equilibrated to room temperature) was supplied to each capsule at a rate of.2 l min 1. High-precision dew point hygrometers (model 473; RH Systems, Albuquerque, NM, USA) were used to measure the water content of the effluent air from the sweat capsule every 5 s at each measurement site. This information was used to derive the local forearm sweat rate per capsule area (in milligrams per minute per square centimetre). Data analysis. All CVC and sweat rate values used for data analysis were obtained by averaging values over the last 5 min of each stage (baseline and.4,.4, 4, 4 and 4 μm of prostacyclin, each 25 min). The maximal CVC elicited by sodium nitroprusside administration was obtained by averaging >2min ofdata. Ateach prostacyclin concentration, the change in CVC from the control site induced by antagonist (L-NNA and/or TEA) was calculated. Additionally, the prostacyclin-induced increase in CVC from baseline was evaluated at each site in order to adjust for any baseline differences. In this study, we were unabletoevaluateec 5, which is the agonist concentration that elicits half of the maximal response and is often evaluated in pharmacological studies. This is because the lowest dose of prostacyclin (i.e..4 μm) induced a substantial cutaneous vasodilatation (near 5% maximal response) at the control site in most of the older women, which did not allow us to evaluate EC 5. Statistical analysis. All statistical analyses in the present study were conducted using SPSS 24 software (IBM, Armonk, NY, USA). The CVC and sweat rate data were analysed with a three-way, mixed-model ANOVA with the repeated factors of treatment site (control, NOS inhibitor, K Ca channel blocker or combination) and stage (baseline,.4,.4, 4, 4 or 4 μm of prostacyclin), and non-repeated factor of age (younger or older). A two-way, mixed-model ANOVA with the factors of treatment site and age was used for maximal CVC (in perfusion units per millimetre of mercury). When detecting a significant interaction, simple main effect tests were performed. After identifying any main effects, post hoc comparisons were carried out using Student s paired or unpaired t tests. For between-site and between-stage comparisons, the P value was adjusted with the modified Bonferroni procedure (i.e. Hochberg s procedure; Hochberg, 1988) as a multiple comparison. Student s unpaired t tests were used for

4 Exp Physiol 12.5 (17) pp Ageing modulates prostacyclin-induced skin vasodilatation in women 581 the between-group difference in participant characteristics (Table 1). The level of significance for all analyses was set at P.5. All values are reported with a mean ± 95% confidence interval, with the exception of participant characteristics presented as means ± SD (Table 1). Results Cutaneous vascular conductance Cutaneous blood flow increased in response to prostacyclin administration in a dose-dependent fashion (see individual data examples presented in Fig. 1). Cutaneous vascular conductance at the control site during administration of.4 4 μm prostacyclin was greater in older in comparison to younger women (all P.5; Fig. 2). However, this difference was not observed at the NOS inhibitor (.4 μm prostacyclin), K Ca channel blocker (.4 4 μm prostacyclin) and Combination (.4 4 μm prostacyclin) sites (all P >.5; Fig. 2). In younger women, separate administration of either a NOS inhibitor or a K Ca channel blocker did not affect CVC during administration of any dose of prostacyclin (all P >.5, Fig. 2A), with the exception that the K Ca channel blocker attenuated CVC at.4 μm prostacyclin (P.5; Fig. 2A). In contrast, combined infusion of both the NOS inhibitor and the K Ca channel blocker reduced CVC during prostacyclin administration at all doses tested in younger women (all P.5; Fig. 2A). In older women, the NOS inhibitor or the K Ca channel blocker alone lowered CVC at.4 4 μm prostacyclin, and combined infusion reduced CVC at all doses tested (all P.5; Fig. 2B). A similar pattern of response remained intact even when considering baseline differences by evaluating the relative change in CVC from baseline for each site (Table 2). The antagonist-induced reduction in CVC relative to the control site was greater when a NOS inhibitor and ak Ca channel blocker were administered simultaneously compared with their separate infusion in both younger and older women (Fig. 3). Maximal CVC did not differ between sites and groups (P >.5 for main effects of age and treatment site and for an interaction between age and treatment site; Table 3). A Younger.4 μm.4 μm 4 μm 4 μm 4 μm Cutaneous blood flow (%max) B 6 4 Baseline Older.4 μm.4 μm 4 μm 4 μm 4 μm 6 4 Baseline Time (min) Figure 1. Data example of cutaneous blood flow during prostacyclin administration Cutaneous blood flow response to incremental doses of prostacyclin measured at the lactated Ringer solution site (control site) from a representative younger woman (A) and older woman (B).

5 582 N. Fujii and others Exp Physiol 12.5 (17) pp Sweating Prostacyclin administration did not affect sweat rate at any treatment site in both groups (all P >.5). Discussion We report, for the first time, that although prostacyclin does not elicit sweating, it induces cutaneous vasodilatation, and the vasodilatation elicited by lower doses of prostacyclin is greater in older relative to younger women. We also discovered that this modulation is a result of differences in NOS- and K Ca channel-dependent mechanisms between the two groups. Cutaneous vasodilatation Interestingly, our results showed that prostacyclininduced cutaneous vasodilatation was greater in older relative to younger women when lower doses of prostacyclin were administered (Fig. 2). Thus, it appears that ageing augments cutaneous vascular responsiveness to lower concentrations of prostacyclin in women. We do not know the exact mechanisms by which ageing augments prostacyclin-induced cutaneous vasodilatation, but ageing might increase the sensitivity and/or number of prostacyclin receptors. Importantly, we also investigated downstream mechanisms that are involved in the age-related augmentation of prostacyclin-induced cutaneous vasodilatation. Specifically, we showed that NOS and K Ca channels are responsible for the age-related difference in the prostacyclin-mediated cutaneous vasodilatation (Fig. 2). This is based on our observation that a greater cutaneous vasodilatation in response to prostacyclin measured at the control site in older women was diminished when inhibiting the actions of NOS and/or K Ca channels (Fig. 2). Although we showed that the separate inhibition of either NOS or K Ca channels had a negligible effect on the prostacyclin-induced cutaneous vasodilatation in younger women, their combined inhibition greatly attenuated this response (Fig. 2A). This observation suggests that NOS and K Ca channels are likely to mediate the prostacyclin-induced cutaneous vasodilatation in an interactive manner. It is plausible that when NOS is inhibited, the contribution of K Ca channels is increased, thereby minimizing the suppressive influence of NOS A Cutaneous vascular conductance (%max) Younger women (n=1) Control NOS inhibitor K Ca channel blocker Combination Baseline B Older women (n=1) * Baseline Figure 2. Cutaneous vasodilatation in response to prostacyclin administration Cutaneous vascular conductance at baseline and during prostacyclin administration in younger (A) and older women (B). All values are expressed as means ± 95% confidence interval. The four intradermal microdialysis skin sites were perfused with the following: (i) lactated Ringer solution (control); (ii) a non-specific nitric oxide synthase (NOS) inhibitor; (iii) a non-specific calcium-activated potassium (K Ca ) channel blocker; or (iv) a combination of an NOS inhibitor and a K Ca channel blocker (Combination). P.5, control versus NOS inhibitor. P.5 versus older. P.5, control versus combination. P.5, control versus K Ca channel blocker.

6 Exp Physiol 12.5 (17) pp Ageing modulates prostacyclin-induced skin vasodilatation in women 583 Table 2. Change in cutaneous vascular conductance from baseline (as a percentage of maximum) during the administration of prostacyclin Conditions Younger (n = 1) Control 3 ± 4 1 ± 8 27 ± 9 49 ± ± 12 NOS inhibitor 3 ± 3 8 ± 3 23 ± 7 43 ± ± 13 K Ca channel blocker 1 ± 2 4 ± 4 19 ± 1 41 ± ± 7 Combination ± 1 1 ± 1 6 ± 3 17 ± 6 47 ± 1 Older (n = 1) Control 21 ± ± ± 15 6 ± ± 7 NOS inhibitor 5 ± 4 16 ± 8 35 ± 1 65 ± 1 77 ± 7 K Ca channel blocker ± 4 7 ± 7 27 ± ± ± 7 Combination 2 ± 2 4 ± 2 1 ± 4 33 ± ± 11 All values are expressed as means ± 95% confidence interval. Abbreviations: Combination, a combination of a NOS inhibitor and ak Ca channel blocker; K Ca, calcium-activated potassium; and NOS, nitric oxide synthase. P.5 versus control; P.5 versus older. inhibition on cutaneous vasodilatory response. Likewise, an increased contribution of NOS might occur when K Ca channels are blocked. A comparable compensatory mechanism has been proposed in the cutaneous vasculature of mice, wherein a smaller NOS contribution accompanies a greater K Ca channel input when evaluating endothelium-dependent vasodilatation (Gaubert et al. 7). In contrast to younger women, we observed that both the separate and combined inhibition of NOS and K Ca channels greatly attenuated prostacyclin-induced cutaneous vasodilatation in older women (Fig. 2B). Therefore, it is possible that ageing might attenuate the Change in cutaneous vascular conductance from Control (%max) A Younger women (n = 1) B Older women (n = 1) * * ** ** ** ** * * NOS inhibitor K Ca channel blocker 6 Combination ** Figure 3. Reduction in prostacyclin-induced cutaneous vasodilatation as a result of each pharmacological treatment Change in cutaneous vascular conductance associated with the administration of a non-specific nitric oxide synthase (NOS) inhibitor, a non-specific calcium-activated potassium (K Ca ) channel blocker and a combination of both (Combination) relative to lactated Ringer solution site (control). All values are expressed as means ± 95% confidence interval. A and B exhibit data from younger and older women, respectively. Data are presented at.4 4 μm of prostacyclin. P.5 versus combination. P.5 versus older.

7 584 N. Fujii and others Exp Physiol 12.5 (17) pp Table 3. Maximal cutaneous vascular conductance (in perfusion units per millimetre of mercury) at the four forearm skin sites Conditions Younger (n = 1) Older (n = 1) Control 1.7 ± ±.41 NOS inhibitor 1.7 ± ±.71 K Ca channel blocker 1.48 ± ±.62 Combination 1.94 ± ±.35 All values are expressed as means ± 95% confidence interval. Abbreviations: Combination, a combination of a NOS inhibitor and a K Ca channel blocker; K Ca, calcium-activated potassium; and NOS, nitric oxide synthase. P >.5 for main effects of age and treatment site and for an interaction between age and treatment site. interactive or compensatory influence between NOS and K Ca channels. Further studies are warranted to evaluate the mechanisms underpinning this response. We showed that the combined inhibition of NOS and K Ca channels greatly suppressed cutaneous vasodilatation in response to all doses of prostacyclin in both younger and older women (Fig. 2). Of importance, however, we showed that this suppression was smaller at the highest dose of prostacyclin used (i.e. 4 μm; Fig. 2). Therefore, it is likely that other mechanisms independent of NOS and K Ca channels contribute to cutaneous vasodilatation at high doses of prostacyclin (i.e. 4 μm). Activation of prostacyclin receptors located on vascular smooth muscle cells can activate adenylyl cyclase that in turn elevates the level of camp. This elevated level of camp can relax vascular smooth muscle cells, thereby inducing vasodilatation. Alternatively, prostacyclin might activate K + channels other than K Ca channels solely, as in vitro studies have demonstrated that prostacyclin can activate voltage-gated (Dong et al. 1998) and ATP-sensitive K + channels (Jacksonet al. 1993). Further study is warranted to evaluate these possibilities. As noted above, we observed a negligible effect of either NOS inhibition or K Ca channel blockade alone on prostacyclin-induced cutaneous vasodilatation in younger women (Fig. 2A). These findings contrast our previous observations in younger men, wherein separate inhibition of either NOS or K Ca channels attenuated prostacyclin-induced cutaneous vasodilatation (Fujii et al. 16b). Furthermore, the age-related augmentation of prostacyclin-induced cutaneous vasodilatation observed in women of the present study differs markedly from our observations in men, wherein we reported no ageing effect on the magnitude of prostacyclin-induced cutaneous vasodilatation (Fujii et al. 16b). Taken together, our findings might suggest that the mechanisms underpinning prostacyclin-induced cutaneous vasodilatation, and how ageing may modulate them, differ between men and women. However, given that the present study was not designed to elucidate sex-related differences, future studies aimed specifically to examine these differences are warranted. Sweating Our results indicated that prostacyclin does not mediate sweat secretion in non-heat stress conditions in both younger and older women. This response is consistent with our findings in younger and older men (Fujii et al. 16b). Given our observation of a prostacyclin-mediated increase in cutaneous vasodilatation in both younger and older men (Fujii et al. 16b)andwomen(presentstudy), it can be concluded that the influence of prostacyclin differs depending on the target end-organ in the skin (i.e. cutaneous vessels versus sweat glands). In the present study, we can assess the activation of the sweat gland based only on the measurement of sweat gland output. It is possible that the activation of the sweat gland might have occurred without a measurable change in sweat output. In this case, the sweat produced in the lumenofthesweatglandmightbereabsorbedsuchthat net sweat output measured at the skin surface remains unchanged. Thus, our sweating data should be interpreted with caution given that we can measure changes in sweat output only at the skin surface. Clinical significance Our results demonstrated that prostacyclin can increase cutaneous perfusion in a dose-dependent manner in both younger and older women. This vasodilatory effect is robust such that the response is comparable to that elicited by other well-known vasodilators, such as NO (Fujii et al. 16a), ATP (Wingo et al. 1; Fujii et al. 15b) and cholinergic agents (Lee & Mack, 6; Medow et al. 8; Bruning et al. 12; Fujii et al. 14; Patik et al. 16). Importantly, older women exhibited greater cutaneous vasodilatation in response to prostacyclin compared with younger women. Therefore, our results indicate that prostacyclin therapy might be more effective in improving microvascular hypoperfusion in older relative to younger women. Finally, in the present study we also identified key factors that can mediate the prostacyclin-induced increases in skin perfusion (i.e. NOS and K Ca channels). This information could be used to develop more effective prostacyclin analogues that might induce a greater vasodilatory effect. Conclusion We show that cutaneous vasodilatation induced by lower doses of prostacyclin is greater in older relative to younger women, and this response appears to be a result of NOSand K Ca channel-dependent mechanisms. Furthermore,

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9 586 N. Fujii and others Exp Physiol 12.5 (17) pp Stanhewicz AE, Greaney JL, Kenney WL & Alexander LM (14). Sex- and limb-specific differences in the nitric oxide-dependent cutaneous vasodilation in response to local heating. Am J Physiol Regul Integr Comp Physiol 37, R914 R919. Wingo JE, Brothers RM, Del Coso J & Crandall CG (1). Intradermal administration of ATP does not mitigate tyramine-stimulated vasoconstriction in human skin. Am J Physiol Regul Integr Comp Physiol 298, R1417 R14. Additional information Competing interests None declared. Author contributions All experiments took place at the Human and Environmental Physiology Research Unit located at the University of Ottawa. N.F. and G.P.K. conceived and designed experiments. N.F. and B.D.M. contributed to data collection. N.F. performed data analysis. N.F., B.D.M., T.N. and G.P.K. interpreted the experimental results. N.F. drafted the manuscript. N.F., B.D.M., T.N. and G.P.K. 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. Funding This study was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery grant, RGPIN and Discovery Grants Program, Accelerator Supplement, RGPAS ; funds held by G.P.K.). G.P.K. is supported by a University of Ottawa Research Chair Award. N.F. was supported by the Human and Environmental Physiology Research Unit. B.D.M. is supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology. Acknowledgements We greatly appreciate all of the volunteers for giving their time to participate in this study. Author s present address N. Fujii: University of Tsukuba, Institute of Health and Sport Sciences, Tsukuba City, Japan.