l-NAME Sites Research Articles

Inhibition of nuclear factor-κB activation improves non-nitric oxide-mediated cutaneous microvascular function in reproductive-aged healthy women.

The transcriptional regulator nuclear factor-κB (NF-κB) is a mediator of endothelial dysfunction. Inhibiting NF-κB with salsalate is used to investigate inflammatory mechanisms contributing to accelerated cardiovascular disease risk. However, in the absence of disease, inhibition of NF-κB can impact redox mechanisms, resulting in paradoxically decreased endothelial function. This study aimed to measure microvascular endothelial function during inhibition of the transcriptional regulator NF-κB in reproductive-aged healthy women. In a randomized, single-blind, crossover, placebo-controlled design, nine healthy women were randomly assigned oral salsalate (1,500 mg, twice daily) or placebo treatments for 5 days. Subjects underwent graded perfusion with the endothelium-dependent agonist acetylcholine (ACh, 10-10 to 10-1 M, 33°C) alone and in combination with 15 mM NG-nitro-l-arginine methyl ester [l-NAME; nonselective nitric oxide (NO) synthase inhibitor] through intradermal microdialysis. Laser-Doppler flux was measured over each microdialysis site, and cutaneous vascular conductance (CVC) was calculated as flux divided by mean arterial pressure and normalized to site-specific maximum (CVC%max; 28 mM sodium nitroprusside + 43°C). The l-NAME sensitive component was calculated as the difference between the areas under the dose-response curves. During the placebo and salsalate treatments, the l-NAME sites were reduced compared with the control sites (both P < 0.0001). Across treatments, there was a significant difference between the control and l-NAME sites, where both sites shifted upward following salsalate treatment (both P < 0.0001), whereas the l-NAME-sensitive component was not different (P = 0.94). These data demonstrate that inhibition of the transcriptional regulator NF-κB improves cutaneous microvascular function in reproductive-aged healthy women through non-NO-dependent mechanisms.NEW & NOTEWORTHY The transcription factor nuclear factor-κB (NF-κB) regulates multiple aspects of innate and adaptive immunity by encoding for genes that participate in inflammation and impact endothelial function following NF-κB inhibition with salsalate treatment. Our results show that cutaneous microvascular function is increased through non-nitric oxide (NO)-dependent mechanisms following salsalate treatment in reproductive-aged healthy women.

Insulin-Mediated Capillary Recruitment Is Absent in the Cutaneous Microvasculature of Women with a History of Gestational Diabetes Mellitus

Women with a history of gestational diabetes mellitus (GDM) have a significantly greater risk of developing cardiovascular disease and type II diabetes compared to women who had an uncomplicated pregnancy (HC). Although the mechanisms underlying this increased risk are unclear, emerging evidence suggests that microvascular endothelial dysfunction, mediated via reduced nitric oxide (NO)-dependent dilation, persists after pregnancy complicated by GDM. Whether this microvascular dysfunction reduces insulin-mediated responses in these women is unexplored. We hypothesized that insulin-mediated capillary recruitment would be attenuated in women with a history of GDM via NO-dependent mechanisms. 24 women (n=12/group; HC: 35±1 years, 24±4 months post-partum; GDM: 33±1 years, 21±3 months postpartum) underwent intradermal microdialysis, which involved the placement of 2 fibers in the ventral forearm for the local delivery of 10−4M insulin alone or 10−4M insulin+15mM NG-nitro-L-arginine methyl ester (L-NAME, NO-synthase inhibitor). The cutaneous blood flow response to post-occlusive reactive hyperemia (PORH; 5 minutes) was assessed before and during insulin perfusion. Red blood cell flux was measured continuously over each microdialysis site by laser-Doppler flowmetry. Cutaneous vascular conductance was calculated (CVC=flux/mean arterial pressure), normalized to maximum (%CVCmax), and presented as PORH area under the curve (AUC, %CVCmax• sec−1). Microvascular capillary recruitment was assessed as ΔAUC (pre-insulin AUC - insulin AUC). Insulin perfusion increased PORH in HC (pre-insulin: 4066±490 vs. insulin: 7848±978 AUC; p=0.03) but had no effect in GDM (pre-insulin: 3765±293 vs. insulin: 6196±779 AUC; p=0.26). Insulin perfusion did not increase PORH at the L-NAME site in either group (p&gt;0.05). Within groups, L-NAME perfusion reduced insulin-mediated capillary recruitment in HC (control: 3781±1109 vs. L-NAME: 470±945 ΔAUC; p=0.04) but not GDM (control: 2431±796 vs. L-NAME: -41±1014 ΔAUC, p=0.11). These data suggest that insulin-mediated capillary recruitment assessed by PORH in the cutaneous circulation is mediated by NO-dependent mechanisms in healthy women with a history of uncomplicated pregnancy and attenuated in healthy women with a history of GDM. This reduction in insulin-mediated microvascular responses may contribute to the increased risk of cardiovascular disease and type II diabetes in these women. UI Fraternal Order of Eagles Diabetes Research Center Catalyst Grant; NIH HL169201. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Divergent Vasodilator Mechanisms to Endothelial-Dependent Agonists with Advancing Age

Introduction: Microvascular function is maintained by a number of vasodilating factors, with nitric oxide (NO) and endothelial hyperpolarizing factors (EDHF) predominating. Advancing age is an independent risk factor in the development of cardiovascular disease, and NO-mediated vasodilation declines, or is unchanged, depending on the vascular bed examined, number of cardiovascular risk factors, or specific endothelial-dependent agonist used. Mechanisms contributing to cutaneous microvascular dysfunction with age have been attributed largely to reductions in NO signaling, but the role of EDHF-mediated dilation, particularly to acetylcholine (ACh), remains unclear. The purpose of this study was to determine the endothelial mechanisms contributing to age-induced cutaneous microvascular function and compare cutaneous microvascular function responses to those from isolated adipose arteriole preparations. We hypothesized that cutaneous microvascular vasodilation to ACh in older adults will be mediated primarily by EDHFs relative to young adults and that isolated adipose arteriole preparations will corroborate these findings. Methods: Four intradermal microdialysis fibers were placed in the skin of the ventral forearm of 9 young (3F/6M; 29±7 yrs) and 3 older adults (3F/0M; 59±11 yrs) for infusion of Ringer’s solution; L-NAME (10 mM), an NO synthase inhibitor; tetraethylammonium chloride (TEA; 50 mM), a non-specific K+ channel inhibitor; and a combination of L-NAME and TEA. Laser Doppler flowmetry and brachial mean arterial pressure were measured during infusion of ACh (10 mM and 100 mM) and in response to local heating to 39°C. Maximal vasodilation was assessed with heating to 43°C and infusion of sodium nitroprusside (28 mM). Adipose resistance arterioles for pressure myography were obtained from a gluteal biopsy or from surgical discard tissue, which were pre-constricted (ET-1), and ACh responses (10−9–10−4 M) were assessed in the presence of the above inhibitors. Cutaneous vascular conductance (CVC) and isolated arteriole responses are presented as % maximum vasodilation. Results: In response to local heating in young adults, L-NAME reduced vasodilator magnitude (Ringers 69.0±18.1% vs. L-NAME 30.3±12.1%; p&lt; 0.05) while TEA did not have any effect (69±18% vs. 49±28%; p=0.20). Combined L-NAME and TEA reduced vasodilation (69±18% vs. 19±6%; p&lt;0.05). Conversely, vasodilator magnitude to ACh was not different across the Ringers, L-NAME, or TEA sites (p=0.93-0.94), while combined inhibition of NOS and K+ channels with L-NAME and TEA, respectively, reduced vasodilator responsiveness (p&lt;0.05). In isolated resistance arterioles, there were no differences in %max dilation to ACh across inhibitor sites (p=0.19) in young adults. In older adults, neither independent nor combined inhibition of NOS or K+ channels impacted vasodilator responses to local heating, while vasodilator magnitude to ACh was most impacted by combined inhibition with L-NAME and TEA (83±14% vs. 32±15%; p&lt;0.05). Conclusion: Our data demonstrate that vasodilator plasticity in response to local heating is divergent with advancing age, relying on non-NO or K+ mechanisms relative to young adults, while NO and K+ mechanisms are suffcient to maintain ACh-mediated vasodilation. These findings further support the concept of diversity in vasodilator mechanisms within the microvasculature. NHLBI K99-HL161491 (WEH), K08-HL150340 (JJM), R01-HL160752 (JKF), R01-HL133029 (AB), and NICHD R01-HD099340 (MJD). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oxidative stress does not influence local sweat rate during high-intensity exercise.

What is the central question of this study? We evaluated whether oxidative stress attenuates the contribution of nitric oxide to sweating during high-intensity exercise. What is the main finding and its importance? In contrast to our previous report of an oxidative stress-mediated reduction in nitric oxide-dependent cutaneous vasodilatation in this cohort during intense exercise, we demonstrated no influence of local ascorbate administration on the sweating response during moderate- (∼51% peak oxygen uptake) or high-intensity exercise (∼72% peak oxygen uptake). These new findings provide important mechanistic insight into how exercise-induced oxidative stress impacts sudomotor activity. Nitric oxide (NO)-dependent sweating is diminished during high- but not moderate-intensity exercise. We evaluated whether this impairment stems from increased oxidative stress during high-intensity exercise. On two separate days, 11 young (24±4years) men cycled in the heat (35°C) at a moderate [500W; 52±6% peak oxygen uptake (V̇O2 peak )] or high (700W; 71±5% V̇O2 peak ) rate of metabolic heat production. Each session included two 30min exercise bouts separated by a 20min recovery period. Local sweat rate was monitored at four forearm skin sites continuously perfused via intradermal microdialysis with the following: (i) lactated Ringer solution (Control); (ii) 10mm ascorbate (Ascorbate; non-selective antioxidant); (iii) 10mm NG -nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor); or (iv) 10mm ascorbate plus 10mm l-NAME (Ascorbate + l-NAME). During moderate exercise, sweat rate was attenuated at the l-NAME and Ascorbate + l-NAME sites (both ∼1.0mgmin-1 cm-2 ; all P<0.05) but not at the Ascorbate site (∼1.1mgmin-1 cm-2 ; both P≥0.28) in comparison to the Control site (∼1.1mgmin-1 cm-2 ). However, no differences were observed between treatment sites (∼1.4mgmin-1 cm-2 ; P=0.75) during high-intensity exercise. We conclude that diminished NO-dependent sweating during intense exercise occurs independent of oxidative stress.

Cutaneous reactive hyperaemia is unaltered by dietary nitrate supplementation in healthy humans.

The purpose of this study was to determine whether nitrate supplementation augments cutaneous reactive hyperaemia. Seven participants were tested pre- and postnitrate supplementation (25ml beetroot juice); participants consumed one shot per day for 3days. Participants were instrumented with two microdialysis fibres: control (Ringer's solution) and NO synthase inhibition (20mM L-NAME). Skin blood flow was measured via laser-Doppler flowmetry (LDF). A blood pressure cuff was placed on the experimental arm and inflated to 250 mmHg for 5mins to occlude arterial inflow. The cuff was released, and the resultant reactive hyperaemia was measured. Blood pressure was continuously measured via plethysmography from a finger on the non-experimental arm. Cutaneous vascular conductance was calculated (LDF/MAP) and normalized to maximal vasodilatation (%CVCmax ). Only diastolic blood pressure was reduced following nitrate supplementation (71±2 vs. 66±1mmHg; P<0·05). There was no effect of nitrate supplementation on peak reactive hyperaemia at control (Pre: 52±3 vs. Post: 57±2%CVCmax ) or L-NAME (Pre: 52±2 vs. Post: 59±4%CVCmax ) sites. There was no effect of nitrate supplementation on total reactive hyperaemia at either control (Pre: 4197±943 vs. Post: 4523±1040%CVCmax * sec) or L-NAME (Pre: 5108±997 vs. Post: 5694±1002%CVCmax * sec) sites. These data suggest cutaneous reactive hyperaemia is unaffected by dietary nitrate supplementation in healthy humans.

Do nitric oxide synthase and cyclooxygenase contribute to sweating response during passive heating in endurance-trained athletes?

The aim of our study was to determine if habitual endurance training can influence the relative contribution of nitric oxide synthase (NOS) and cyclooxygenase (COX) in the regulation of sweating during a passive heat stress in young adults. Ten trained athletes and nine untrained counterparts were passively heated until oral temperature (as estimated by sublingual temperature, Tor) increased by 1.5°C above baseline resting. Forearm sweat rate (ventilated capsule) was measured at three skin sites continuously perfused with either lactated Ringer's solution (Control), 10 mmol/L NG‐nitro‐L‐arginine methyl ester (L‐NAME, non‐selective NOS inhibitor), or 10 mmol/L ketorolac (Ketorolac, non‐selective COX inhibitor) via intradermal microdialysis. Sweat rate was averaged for each 0.3°C increase in Tor. Sweat rate at the L‐NAME site was lower than Control following a 0.9 and 1.2°C increase in Tor in both groups (all P ≤ 0.05). Relative to the Control site, NOS‐inhibition reduced sweating similarly between the groups (P = 0.51). Sweat rate at the Ketorolac site was not different from the Control at any levels of Tor in both groups (P > 0.05). Nevertheless, a greater sweat rate was measured at the end of heating in the trained as compared to the untrained individuals (P ≤ 0.05). We show that NOS contributes similarly to sweating in both trained and untrained individuals during a passive heat stress. Further, no effect of COX on sweating was measured for either group. The greater sweat production observed in endurance‐trained athletes is likely mediated by factors other than NOS‐ and COX‐dependent mechanisms.

The interactive contributions of Na+/K+‐ATPase and nitric oxide synthase to sweating and cutaneous vasodilatation during exercise in the heat

Nitric oxide synthase (NOS) contributes to sweating and cutaneous vasodilatation during exercise in the heat. Similarly, reports show that Na(+) /K(+) -ATPase activation can modulate sweating and microvascular circulation. In light of the fact that NO can activate Na(+) /K(+) -ATPase, we evaluated whether there is an interaction between Na(+) /K(+) -ATPase and NOS in the regulation of heat loss responses during an exercise-induced heat stress. We demonstrate that Na(+) /K(+) -ATPase and NOS do not synergistically influence local forearm sweating during moderate intensity (fixed rate of metabolic heat production of 500W) exercise in the heat (35°C). Conversely, we show an interactive role between NOS and Na(+) /K(+) -ATPase in the modulation of cutaneous vasodilatation. These findings provide novel insight regarding the mechanisms underpinning the control of sweating and cutaneous vasodilatation during exercise in the heat. Given that ouabain may be prescribed as a cardiac glycoside in clinical settings, potential heat loss impairments with ouabain administration should be explored. Nitric oxide (NO) synthase (NOS) contributes to the heat loss responses of sweating and cutaneous vasodilatation. Given that NO can activate Na(+) /K(+) -ATPase, which also contributes to sweating and microvasculature regulation, we evaluated the separate and combined influence of Na(+) /K(+) -ATPase and NOS on sweating and cutaneous vasodilatation. Thirteen young (23±3years) males performed two 30min semi-recumbent cycling bouts in the heat (35°C) at a fixed rate of metabolic heat production (500W) followed by 20 and 40min recoveries, respectively. Local sweat rate (LSR) and cutaneous vascular conductance (CVC) were measured at four forearm skin sites continuously perfused via intradermal microdialysis with either: (1) lactated Ringer solution (Control); (2) 6mᴍ ouabain (Ouabain), a Na(+) /K(+) -ATPase inhibitor; (3) 10mᴍ l-N(G) -nitroarginine methyl ester (l-NAME), a NOS inhibitor; or (4) 6mᴍ ouabain and 10mᴍ l-NAME (Ouabain+l-NAME). At the end of both exercise bouts relative to Control, LSR was attenuated with Ouabain (54-60%), l-NAME (12-13%) and Ouabain+l-NAME (68-74%; all P < 0.05). Moreover, the sum of attenuations from Control induced by independent administration of Ouabain and l-NAME was similar to the combined infusion of Ouabain+l-NAME (both P ≥ 0.74). Compared to Control, CVC at the end of both exercise bouts was similar with Ouabain (both P ≥ 0.30), but attenuated with l-NAME (%CVCmax reduction from Control, 24-25%). Furthermore, CVC at the Ouabain+l-NAME site (38-39%; all P < 0.01) was attenuated compared to Control and did not differ from baseline resting values (both P ≥ 0.81). We show that Na(+) /K(+) -ATPase and NOS do not synergistically mediate sweating, whereas they influence cutaneous blood flow in an interactive manner during exercise in the heat.

Do nitric oxide synthase and cyclooxygenase contribute to the heat loss responses in older males exercising in the heat?

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 performed 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 N(G) -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.

Oxidative stress impairs NO‐dependent cutaneous vasodilation but not NO‐dependent sweating in young adults during high intensity exercise

Our recent work showed that nitric oxide (NO)‐dependent sweating and cutaneous vasodilation are diminished in young males during intermittent exercise at high (700 W) relative to moderate (400 W) rates of metabolic heat production. We tested the hypothesis that this impairment results from increased oxidative stress during high intensity exercise. On separate days, nine young (24 ± 2 years) healthy males performed two 30 min bouts of semi‐recumbent cycling in the heat (35°C, 20% RH) at a rate of metabolic heat production of i) 500 W or ii) 700 W. The first and second exercise bouts were followed by 20 and 40 min of recovery, respectively. Forearm local sweat rate (LSR) and cutaneous vascular conductance (CVC) were measured at 4 skin sites that were continuously perfused via intradermal microdialysis with: 1) lactated Ringer's solution (Control), 2) 10 mM ascorbate (ASC, antioxidant), 3) 10 mM L‐NG‐Nitroarginine methyl ester (L‐NAME, non‐selective NO synthase inhibitor), or 4) 10 mM ASC + 10 mM L‐NAME. During moderate intensity exercise (i.e., 500 W), L‐NAME and ASC + L‐NAME reduced LSR and CVC compared to the Control site (all P &lt; 0.05), while no effect of ASC was observed (all P &gt; 0.05). Conversely, high intensity exercise performed at 700 W did not result in differences in LSR between treatment sites (P = 0.85). However, compared to Control, CVC was elevated at the ASC (all P &lt; 0.05) while it was reduced at the L‐NAME (all P &lt; 0.05) and ASC + L‐NAME (all P &lt; 0.05) sites. Altogether these results suggest that oxidative stress impairs NO‐dependent cutaneous vasodilation but not LSR during intense exercise. Support: Natural Sciences and Engineering Research Council of Canada.

Adenosine receptor inhibition attenuates the decrease in cutaneous vascular conductance during whole‐body cooling from hyperthermia

Adenosine has both vasodilatory and vasoconstrictive properties, yet its influence on cutaneous vascular conductance (CVC) during whole-body cooling remains unknown. The present study evaluated the influence of adenosine on reflex cutaneous vasoconstriction. Four microdialysis probes were inserted into the dorsal forearm skin of eight subjects and infused with the following solutions: (i) lactated Ringer solution (CON); (ii) 4 mm theophylline (Theo), a non-selective adenosine receptor antagonist; (iii) 10 mm l-NAME, an inhibitor of nitric oxide synthase; and (iv) combined 4 mm theophylline and 10 mm l-NAME (Theo + l-NAME). Subjects subsequently donned a water-perfusion garment. Following a thermoneutral baseline period, the suit was perfused with water at 10°C for 20 min (Cooling 1). The suit was then perfused with water at 49°C for 45 min (Heating), followed by a second cooling period of 20 min using 10°C water (Cooling 2). Cutaneous blood flow (laser-Doppler) was measured over each microdialysis probe and used to calculate CVC as a percentage of the maximum determined by sodium nitroprusside infusion and local heating. Cutaneous vascular conductance was significantly elevated at the Theo site relative to CON following Cooling 1 (18 ± 6 versus 8 ± 2%; P = 0.01) and Cooling 2 (27 ± 11 versus 14 ± 5%; P = 0.022). Likewise, CVC at the Theo + l-NAME site remained greater compared with l-NAME after Cooling 1 (13 ± 4 versus 7 ± 3%; P = 0.030) and Cooling 2 (15 ± 3 versus 9 ± 2%; P = 0.009). The present findings demonstrate that non-selective antagonism of adenosine receptors attenuates the decrease in cutaneous vascular conductance during whole-body cooling from hyperthermia.

No independent, but an interactive, role of calcium-activated potassium channels in human cutaneous active vasodilation

In human cutaneous microvasculature, endothelium-derived hyperpolarizing factors (EDHFs) account for a large portion of vasodilation associated with local stimuli. Thus we sought to determine the role of EDHFs in active vasodilation (AVD) to passive heating in two protocols. Whole body heating was achieved using water-perfused suits (core temperature increase of 0.8-1.0°C), and skin blood flow was measured using laser-Doppler flowmetry. In the first protocol, four sites were perfused continuously via microdialysis with: 1) control; 2) tetraethylammonium (TEA) to block calcium-activated potassium (KCa) channels, and thus the actions of EDHFs; 3) N-nitro-l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase (NOS); and 4) TEA + l-NAME (n = 8). Data are presented as percent maximal cutaneous vascular conductance (CVC). TEA had no effect on AVD (CVC during heated plateau: control 57.4 ± 4.9% vs. TEA 63.2 ± 5.2%, P = 0.27), indicating EDHFs are not obligatory. l-NAME attenuated plateau CVC to 33.7 ± 5.4% (P < 0.01 vs. control); while TEA + l-NAME augmented plateau CVC compared with l-NAME alone (49.7 ± 5.3%, P = 0.02). From these data, it appears combined blockade of EDHFs and NOS necessitates dilation through other means, possibly through inward rectifier (KIR) and/or ATP-sensitive (KATP) potassium channels. To test this second hypothesis, we measured AVD at the following sites (n = 8): 1) control, 2) l-NAME, 3) l-NAME + TEA, and 4) l-NAME + TEA + barium chloride (BaCl2; KIR and KATP blocker). The addition of BaCl2 to l-NAME + TEA reduced plateau CVC to 32.7 ± 6.6% (P = 0.02 vs. l-NAME + TEA), which did not differ from the l-NAME site. These data combined demonstrate a complex interplay between vasodilatory pathways, with cross-talk between NO, KCa channels, and KIR and/or KATP channels.

Dietary nitrate supplementation augments cutaneous reflex vasodilation in healthy humans

Beetroot juice (BRJ) has been shown to increase NO‐dependent dilation through both NOS‐dependent and independent pathways. We hypothesized BRJ supplementation would augment reflex cutaneous active vasodilation. Subjects were equipped with two microdialysis fibers on the forearm and randomly assigned as control (Ringer's) or NOS inhibition (20mM L‐NAME). Whole‐body heating was achieved via water‐perfused suits to raise core temperature (Tc; ingestible telemetric pill) 0.8°C. Maximal cutaneous vasodilation was reached by administering 54mM SNP and local heating to 43°C. Skin blood flow was measured via laser‐Doppler flowmetry and mean arterial pressure was measured; cutaneous vascular conductance (CVC) was calculated and expressed as %CVCmax. Subjects underwent heat stress pre‐ and post‐nitrate supplementation (3 days of BRJ: 5mM, .45g nitrates per day). BRJ increased the plateau CVC at control (pre: 55±3 vs. post: 77±5 %CVCmax) and L‐NAME (pre: 27±3 vs. post: 41±6 %CVCmax; p&lt;0.05 for all conditions) sites. The %NO contribution increased from pre‐ (47±2 %CVCmax) to post‐BRJ (71±4 %CVCmax; p&lt;0.05). These data suggest BRJ augments NO‐dependent component of reflex cutaneous vasodilation.

Role of nitric oxide synthase on local sweat rate in older adults during intermittent exercise in the heat

Studies suggest that nitric oxide (NO) has a role in sweating. Older adults have a lower production of and sensitivity to NO which may explain why older adults have lower local sweat rates (LSR) compared with their younger counterparts during exercise. We examined the role of NO synthase (NOS) on LSR during intermittent exercise in the heat. Ten young (Y) and ten older (O) participants completed 3 × 15 min of cycling (rate of metabolic heat production of 600 W) separated by 15 min recovery in the heat (35ºC, 30% RH). Intradermal microdialysis probes were inserted in the forearm and perfused with either 0.9% saline (CON) or 10 mM L‐NAME (a NOS inhibitor). LSR was measured directly over each membrane. LSR was similar between Y and O during the exercise bouts at the CON (average of the 3 exercise bouts: Y; 0.96 ± 0.20 vs. O; 0.87 ± 0.25 mg·min−1·cm−2 p=0.327) and the LNAME (Y: 0.74 ± 0.18 vs. O: 0.83 ± 0.27 mg·min−1·cm−2 p=0.327) sites. However, the relative contribution of NO to the sweating response was reduced in Y (CON: 0.96 ± 0.20 vs. L‐NAME: 0.74 ± 0.18 mg·min−1·cm−2, p=0.017), but not O (CON: 0.87 ± 0.25 vs. L‐NAME: 0.83 ± 0.27 mg·min−1·cm−2, p=0.965). We conclude that the exercise‐induced increase in sweating is influenced by NO in younger adults. However, it appears that the level of influence is blunted in older individuals during high intensity exercise in the heat.This research was funded by Natural Sciences and Engineering Research Council (RGPIN‐298159–2009, held by Dr. Kenny).

Sensory nerves and nitric oxide contribute to reflex cutaneous vasodilation in humans

We tested the hypothesis that inhibition of cutaneous sensory nerves would attenuate reflex cutaneous vasodilation in response to an increase in core temperature. Nine subjects were equipped with four microdialysis fibers on the forearm. Two sites were treated with topical anesthetic EMLA cream for 120 min. Sensory nerve inhibition was verified by lack of sensation to a pinprick. Microdialysis fibers were randomly assigned as 1) lactated Ringer (control); 2) 10 mM nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide synthase; 3) EMLA + lactated Ringer; and 4) EMLA + L-NAME. Laser-Doppler flowmetry was used as an index of skin blood flow, and blood pressure was measured via brachial auscultation. Subjects wore a water-perfused suit, and oral temperature was monitored as an index of core temperature. The suit was perfused with 50°C water to initiate whole body heat stress to raise oral temperature 0.8°C above baseline. Cutaneous vascular conductance (CVC) was calculated and normalized to maximal vasodilation (%CVC(max)). There was no difference in CVC between control and EMLA sites (67 ± 5 vs. 69 ± 6% CVC(max)), but the onset of vasodilation was delayed at EMLA compared with control sites. The L-NAME site was significantly attenuated compared with control and EMLA sites (45 ± 5% CVC(max); P < 0.01). Combined EMLA + L-NAME site (25 ± 6% CVC(max)) was attenuated compared with control and EMLA (P < 0.001) and L-NAME only (P < 0.01). These data suggest cutaneous sensory nerves contribute to reflex cutaneous vasodilation during the early, but not latter, stages of heat stress, and full expression of reflex cutaneous vasodilation requires functional sensory nerves and NOS.

Impaired acetylcholine-induced cutaneous vasodilation in young smokers: roles of nitric oxide and prostanoids

Cigarette smoking attenuates acetylcholine (ACh)-induced cutaneous vasodilation in humans, but the underlying mechanisms are unknown. We tested the hypothesis that smokers have impaired nitric oxide (NO)- and cyclooxygenase (COX)-dependent cutaneous vasodilation to ACh infusion. Twelve young smokers, who have smoked more than 5.2 ± 0.7 yr with an average daily consumption of 11.4 ± 1.2 cigarettes, and 12 nonsmokers were tested. Age, body mass index, and resting mean arterial pressure were similar between the groups. Cutaneous vascular conductance (CVC) was evaluated as laser-Doppler flux divided by mean arterial pressure, normalized to maximal CVC (local heating to 43.0°C plus sodium nitroprusside administration). We evaluated the increase in CVC from baseline to peak (CVCΔpeak) and area under the curve of CVC (CVCAUC) during a bolus infusion (1 min) of 137.5 μM ACh at four intradermal microdialysis sites: 1) Ringer (control), 2) 10 mM N(G)-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor), 3) 10 mM ketorolac (COX inhibitor), and 4) combination of l-NAME + ketorolac. CVCΔpeak and CVCAUC at the Ringer site in nonsmokers were greater than in smokers (CVCΔpeak, 42.9 ± 5.1 vs. 22.3 ± 3.5%max, P < 0.05; and CVCAUC, 8,085 ± 1,055 vs. 3,145 ± 539%max·s, P < 0.05). In nonsmokers, CVCΔpeak and CVCAUC at the l-NAME site were lower than the Ringer site (CVCΔpeak, 29.5 ± 6.2%max, P < 0.05; and CVCAUC, 5,377 ± 1,109%max·s, P < 0.05), but in smokers, there were no differences between the Ringer and l-NAME sites (CVCΔpeak, 16.8 ± 4.3%max, P = 0.11; and CVCAUC, 2,679 ± 785%max·s, P = 0.30). CVCΔpeak and CVCAUC were reduced with ketorolac in nonsmokers (CVCΔpeak, 13.3 ± 3.6%max, P < 0.05; and CVCAUC, 1,967 ± 527%max·s, P < 0.05) and smokers (CVCΔpeak, 7.8 ± 1.8%max, P < 0.05; and CVCAUC, 1,246 ± 305%max·s, P < 0.05) and at the combination site in nonsmokers (CVCΔpeak, 15.9 ± 3.1%max, P < 0.05; and CVCAUC, 2,660 ± 512%max·s, P < 0.05) and smokers (CVCΔpeak, 11.5 ± 2.6%max, P < 0.05; and CVCAUC, 1,693 ± 409%max·s, P < 0.05), but the magnitudes were greater in nonsmokers (P < 0.05). These results suggest that impaired ACh-induced skin vasodilation in young smokers is related to diminished NO- and COX-dependent vasodilation.

No direct role for A1/A2 adenosine receptor activation to reflex cutaneous vasodilatation during whole-body heat stress in humans

The precise mechanisms underlying reflex cutaneous vasodilatation during hyperthermia remain unresolved. The purpose of this study was to investigate a potential contribution of adenosine A1/A2 receptor activation to reflex cutaneous vasodilatation. Eight subjects were equipped with four microdialysis fibres on the left forearm, and each fibre was randomly assigned one of four treatments: (1) lactated Ringer's (control); (2) 4 mm of the non-selective A1/A2 adenosine receptor antagonist theophylline; (3) 10 mm L-NAME to inhibit nitric oxide (NO) synthase; and (4) combined 4 mm theophylline and 10 mm L-NAME. Laser-Doppler flowmetry (LDF) was used as an index of skin blood flow, and blood pressure was measured beat-by-beat via photoplethysmography and verified via brachial auscultation. Whole-body heat stress to raise oral temperature 0.8 °C above baseline was induced via water-perused suits. Cutaneous vascular conductance (CVC) was calculated as LDF/mean arterial pressure and normalized to maximal (%CVC max) via infusion of 28 mm nitroprusside and local heating to 43 °C. There was no difference between control (65 ± 5%CVC max) and theophylline (63 ± 5%CVC max) sites. L-NAME (44 ± 4%CVC max) and theophylline + L-NAME (32 ± 3%CVC max) sites were significantly attenuated compared to both control and theophylline only sites (P<0.05), and combined theophylline + L-NAME sites were significantly reduced compared to L-NAME only sites (P<0.05). These data suggest A1/A2 adenosine receptor activation does not directly contribute to cutaneous active vasodilatation; however, a role for A1/A2 adenosine receptor activation is unmasked when NO synthase is inhibited.

Intradermal microdialysis of hypertonic saline attenuates cutaneous vasodilatation in response to local heating

We tested the hypothesis that microdialysis of hypertonic saline would attenuate the skin blood flow response to local heating. Seventeen healthy subjects (23 ± 1 years old) were studied. In one group (n = 9), four microdialysis fibres were placed in the forearm skin and infused with the following: (1) Ringer solution; (2) normal saline (0.9% NaCl); (3) hypertonic saline (3% NaCl); and (4) 10 mm l-NAME. A second group (n = 8) was infused with the following: (1) normal saline; (2) hypertonic saline; (3) normal saline + l-NAME; and (4) hypertonic saline + l-NAME. Red blood cell flux was measured via laser Doppler flowmetry during local heating to 42°C. Site-specific maximal vasodilatation was determined by infusing 28 mm sodium nitroprusside while the skin was heated to 43°C. Data were expressed as the percentage of maximal cutaneous vascular conductance (%CVC(max)). The local heating response at the Ringer solution and normal saline sites did not differ (n = 9; initial peak Ringer solution, 69 ± 6 versus normal saline, 66 ± 2%CVC(max); plateau Ringer solution, 89 ± 4 versus normal saline, 89 ± 5%CVC(max)). Hypertonic saline reduced the initial peak (n = 9; normal saline, 66 ± 2 versus hypertonic saline, 54 ± 4%CVC(max); P < 0.05) and plateau (normal saline, 89 ± 5 versus hypertonic saline, 78 ± 2%CVC(max); P < 0.05) compared with normal saline. Plateau %CVC(max) was attenuated to a similar value at the normal saline + l-NAME and hypertonic saline + l-NAME sites (n = 8; normal saline + l-NAME, 39 ± 6 and hypertonic saline + l-NAME, 39 ± 5%CVC(max)). The nitric oxide contribution (plateau %CVC(max) - l-NAME plateau %CVC(max)) was lower at the hypertonic saline site (normal saline, 55 ± 6 versus hypertonic saline, 35 ± 4; P < 0.01). These data suggest an effect of salt on the cutaneous response to local heating, which may be mediated through a decreased production and/or availability of nitric oxide.

Altered thermal hyperaemia in human skin by prior desensitization of neurokinin-1 receptors.

The neuropeptides substance P (SP) and calcitonin gene-related peptide are believed to be involved in the axon reflex-mediated component of cutaneous thermal hyperaemia, but no studies have specifically addressed this issue. The purpose of this study was to determine whether neurokinin-1 receptors, which preferentially bind SP, contribute to the axon reflex component of cutaneous thermal hyperaemia. Nine subjects were equipped with four microdialysis fibres, and each fibre received one of the following four treatments: (1) lactated Ringer's solution (control); (2) 10 mml-NAME to inhibit nitric oxide synthase; (3) 10 μm SP; or (4) 10 μm SP + 10 mml-NAME. Skin blood flow was monitored via laser-Doppler flowmetry, and local skin temperature was controlled using local heating devices. Sites 3 and 4 were perfused with 10 μm SP for 15 min at a rate of 4 μl min(-1), and the ensuing vasodilatation was allowed to return to baseline. Following SP-induced vasodilatation, all skin sites were locally heated from a baseline temperature of 33°C to 42°C at a rate of 0.5°C every 5 s. Cutaneous vascular conductance (CVC) was calculated as skin blood flow/mean arterial pressure and normalized to the maximal value (%CVC(max)) via 28 mm nitroprusside and local heating to 43°C. The initial peak did not differ between control (79 ± 3%CVC(max)) and SP-only sites (79 ± 2%CVC(max)). The initial peaks at the l-NAME (43 ± 3%CVC(max)) and SP + l-NAME sites (53 ± 3%CVC(max)) were significantly reduced compared with both the control and the SP-only sites (P < 0.001 for both), and l-NAME sites were attenuated compared with SP + l-NAME sites (P < 0.01). There was no observable nadir response at sites pretreated with SP. Compared with control sites (57 ± 4%CVC(max)), the nadirs at the l-NAME (14 ± 2%CVC(max)) and the SP + l-NAME sites (31 ± 5%CVC(max)) were significantly reduced (P < 0.01 for all conditions). l-NAME significantly reduced the nadir compared with SP + l-NAME (P < 0.01). Plateau CVC values did not differ between control (86 ± 3%CVC(max)) and SP sites (91 ± 1%CVC(max)). At l-NAME (36 ± 4%CVC(max)) and SP +l-NAME sites (56 ± 6%CVC(max)), plateau CVC was significantly reduced compared with control and SP-only sites (P < 0.01 for all conditions). The plateau at l-NAME sites was significantly reduced compared with SP + l-NAME sites (P < 0.01). These data suggest that neurokinin-1 receptors contribute to both the axon reflex component and the secondary plateau phase of cutaneous thermal hyperaemia.

TRPV‐1 Channels Contribute to Cutaneous Active Vasodilation in Humans

We tested the hypothesis that inhibition of TRPV-1 channels would attenuate cutaneous active vasodilation (AVD). Six subjects were equipped with four microdialysis fibers on the ventral forearm and each site was randomly assigned a drug treatment: 1) 90% propylene glycol + 10% lactated Ringer's (vehicle control); 2)10 mM L-NAME to inhibit NO synthase; 3) 20 mM capsazepine (CPZ) to inhibit TRPV-1 channels; 4) combined L-NAME + CPZ. Whole body heating (WBH) was achieved via water perfused suits to raise oral temperature 0.8°C above baseline. Systemic arterial pressure (SAP) was monitored and skin blood flow was measured via laser-Doppler flowmetry (LDF). Maximal skin blood flow was achieved by local heating to 43°C and infusion of 28mM nitroprusside. Cutaneous vascular conductance (CVC) was calculated as SAP/LDF and normalized to maximal. During WBH, CPZ sites were significantly reduced compared to CON sites (51±2% vs. 65±3% CVCmax, respectively; p < 0.05). L-NAME (32±1%CVCmax) sites were attenuated compared to CPZ. There was no difference between L-NAME and combined L-NAME + CPZ. These data suggest: 1)TRPV-1 receptors directly contribute to AVD; and 2) TRPV-1 channels account for a portion of the NO component.

Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans

The initial, rapid increase in skin blood flow in response to direct application of heat is thought to be mediated by an axon reflex, which is dependent on intact cutaneous sensory nerves. We tested the hypothesis that inhibition of transient receptor potential vanilloid type 1 (TRPV-1) channels, which are putative channels located on sensory nerves, would attenuate the skin blood flow response to local heating in humans. Ten subjects were equipped with four microdialysis fibres which were randomly assigned one of four treatments: (1) vehicle control (90% propylene glycol + 10% lactated Ringer solution); (2) 20 mm capsazepine to inhibit TRPV-1 channels; (3) 10 mm l-NAME to inhibit NO synthase; and (4) combined 20 mm capsazepine + 10 mm l-NAME. Following baseline measurements, the temperature of skin heaters was increased from 33°C to 42°C at a rate of 1.0°C every 10 s and local temperature was held at 42°C for 20-30 min until a stable plateau in skin blood flow was achieved. An index of skin blood flow was measured directly over each microdialysis site via laser-Doppler flowmetry (LDF). Beat-by-beat blood pressure was measured via photoplethysmography and verified via automated brachial auscultation. At the end of the local heating protocol, temperature of the heaters was increased to 43°C and 28 mm nitroprusside was infused to achieve maximal vasodilatation. Cutaneous vascular conductance (CVC) was calculated as LDF/mean arterial pressure and normalized to maximal values (%CVCmax). Initial peak in capsazepine (44 ± 4%CVCmax), l-NAME (56 ± 4%CVCmax) and capsazepine + l-NAME (32 ± 6%CVCmax) sites was significantly attenuated compared to control (87 ± 5%CVCmax; P < 0.001 for all conditions). The plateau phase of thermal hyperaemia was significantly attenuated in capsazepine (73 ± 6%CVCmax), l-NAME (47 ± 5%CVCmax) and capsazepine + l-NAME (31 ± 7%CVCmax) sites compared to control (92 ± 5%CVCmax; P < 0.001 for all conditions). These data suggest TRPV-1 channels contribute substantially to the initial peak and modestly to the plateau phases of thermal hyperaemia. These data further suggest a portion of the NO component of thermal hyperaemia may be due to activation of TRPV-1 channels.