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Research Paper
Augmented reflex cutaneous vasodilatation following short-term dietary nitrate supplementation in humans Erica L. Levitt1 , Jeremy T. Keen1 and Brett J. Wong1,2 1
Experimental Physiology
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Department of Kinesiology, Kansas State University, Manhattan, KS, USA Department of Kinesiology and Health, Georgia State University, Atlanta, GA, USA
New Findings r What is the central question of this study? Nitrate supplementation via beetroot juice has been shown to have several benefits in healthy humans, including reduced blood pressure and increased blood flow to exercising muscle. Whether nitrate supplementation can improve blood flow to the skin in heat-stressed humans has not been investigated. r What is the main finding and its importance? Similar to previous studies, we found that nitrate supplementation reduces blood pressure. Nitrate supplementation increased vasodilatation in the skin of heat-stressed humans but did not directly increase skin blood flow.
Nitrate supplementation has been shown to increase NO-dependent vasodilatation through both NO synthase (NOS)-dependent and NOS-independent pathways. We hypothesized that nitrate supplementation would augment reflex cutaneous active vasodilatation. Subjects were equipped with two microdialysis fibres on the forearm randomly assigned as control (Ringer solution) or NOS inhibition (20 mm l-NAME). Whole-body heating was performed to raise core temperature by 0.8°C above baseline core temperature. Maximal cutaneous vasodilatation was achieved via 54 mm sodium nitroprusside and local heating to 43°C. Skin blood flow (measured by laser-Doppler flowmetry) and blood pressure were measured. Cutaneous vascular conductance (CVC) was calculated as skin blood flow divided by mean arterial pressure (MAP) and expressed as a percentage of maximal CVC (%CVCmax ). Subjects underwent heat stress before and after nitrate supplementation (3 days of beetroot juice; 5 mm, 0.45 g nitrates per day). During heat stress, MAP was reduced following nitrate supplementation compared with the control conditions (before 88 ± 3 mmHg versus after 78 ± 2 mmHg; P < 0.05); however, resting MAP was not different between conditions (before 88 ± 3 mmHg versus after 83 ± 2 mmHg; P = 0.117). Nitrate supplementation increased plateau CVC at control sites (before 67 ± 2%CVCmax versus after 80 ± 5%CVCmax ; P = 0.01) but not at l-NAME-treated sites (before 45 ± 4%CVCmax versus after 40 ± 5%CVCmax ; P = 0.617). There was no change in the calculated percentage of NOS-dependent vasodilatation before and after supplementation (before 59 ± 4% versus after 64 ± 6%; P = 0.577). These data suggest that nitrate supplementation augments CVC and reduces MAP during heat stress. (Received 8 January 2015; accepted after revision 30 March 2015; first published online 31 March 2015) Corresponding author B. J. Wong: Department of Kinesiology & Health, Georgia State University, PO Box 3975, Atlanta, GA 30302-3975, USA. Email: [email protected]
DOI: 10.1113/EP085061
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Introduction In humans, the onset of heat stress initiates an increase in core temperature and is associated with an increase in cutaneous vasodilatation and sweating (Edholm et al. 1957). The cutaneous circulation is regulated by two branches of the sympathetic nervous system, namely the adrenergic vasoconstrictor nerves and the cholinergic active vasodilator nerves (Edholm et al. 1957; Kellogg et al. 1995). With an increase in core body temperature, the sympathetic cholinergic vasodilator nerves reflexively mediate increases in skin blood flow (Edholm et al. 1957; Kellogg et al. 1995). The cotransmission theory (Kellogg et al. 1995) of active vasodilatation suggests that the release of acetylcholine and an unknown substance(s) are responsible for the sweat response and cutaneous vasodilatation, respectively (Kellogg et al. 1995). A multitude of potential vasodilators and vasodilator pathways have been suggested to mediate at least a portion of the reflex cutaneous vasodilatation (Wong et al. 2004; McCord et al. 2006; Wong & Minson, 2006; Kellogg et al. 2010; Wong & Fieger, 2012; Brunt et al. 2013; Wong, 2013). Nitric oxide (Dietz et al. 1994; Kellogg et al. 1998, 2008a,b, 2009; Shastry et al. 1998; Wilkins et al. 2003) has been shown to contribute 35-45% to cutaneous active vasodilatation, and several of the suggested vasodilators have been shown to include a substantial NO component. Data have also demonstrated that NO has both a direct vasodilator role and may also work synergistically with the unknown transmitter(s) (Wilkins et al. 2003). Recent evidence from Kellogg et al. (2008b) suggests the specific nitric oxide synthase (NOS) isoform responsible for active vasodilatation to be neuronal nitric oxide synthase (nNOS). The classical pathway used for NO generation through the conversion of L-arginine to L-citrulline and NO has been shown to be NOS dependent (Moncada & Higgs, 1993). Conversely, the reduction of nitrate to nitrite and, subsequently, to NO has been shown to be an NOS-independent pathway for NO generation (Govoni et al. 2008; Lundberg et al. 2008; Lundberg & Weitzberg, 2009). Several studies have demonstrated that consumption of dietary nitrates has positive health benefits, including an increase in exercising skeletal muscle blood flow (Ferguson et al. 2013; Casey et al. 2015), reduced oxygen cost of exercise (Larsen et al. 2007; Bailey et al. 2009), improved exercise tolerance in patients with peripheral arterial disease (Kenjale et al. 2011) and lower blood pressure (Larsen et al. 2006; Webb et al. 2008). It has been suggested that most of these effects of nitrate consumption are due in large part to an increase in NOS-independent NO production. Researchers in our laboratory have recently shown that acute dietary nitrate supplementation augments cutaneous vascular C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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conductance (CVC) to local heating, primarily due to a reduction in mean arterial pressure (MAP) and not due to a direct increase in skin blood flow (Keen et al. 2015); however, it is unknown what effects short-term dietary nitrate supplementation has on the cutaneous vascular response to whole-body heat stress. The purpose of the present investigation was to determine whether short-term dietary nitrate supplementation would augment the cutaneous vascular response to wholebody heat stress via NOS-dependent or NOS-independent mechanisms. Based on our previous findings (Keen et al. 2015), we hypothesized that nitrate supplementation would augment cutaneous reflex vasodilatation via NOSindependent mechanisms in healthy humans.
Methods Ethical approval
The Institutional Review Board at Kansas State University approved all protocols of this study. A written informed consent was reviewed and signed by each participant prior to participation in this experiment. All procedures and protocols were performed in conjunction with the standards set forth by the Declaration of Helsinki.
Participants
Seven participants (seven men; age 22 ± 1 years, height 179 ± 8 cm, body mass 80 ± 14 kg and body mass index 25 ± 4 kg m−2 ) participated in this study. All were healthy, non-smokers, taking no medications and were free of cardiovascular, respiratory and metabolic disease as determined by medical history questionnaire. Participants were asked to refrain from exercise, consumption of abnormal amounts (>300 g day−1 ) of leafy green vegetables (e.g. spinach), consumption of alcohol and the use of mouthwash products containing alcohol during the 3 day nitrate supplementation period. Exercise and consumption of large quantities of leafy green vegetables can increase systemic nitrates, while alcohol and alcohol-based mouthwash can kill the bacteria required to reduce nitrate to nitrite and subsequently to nitric oxide (Govoni et al. 2008; Bailey et al. 2009). The postsupplementation test was conducted 7–10 days after the presupplementation test to allow the microdialysis sites to heal. During this time, participants were asked to maintain their normal exercise and diet routine (i.e. not to begin a new exercise regimen or diet) and to continue avoiding consumption of excessive quantities of leafy green vegetables. Participants were asked to refrain from caffeine for at least 12 h prior to the study. Testing was administered in a thermoneutral environment with a room temperature
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of 23–24°C. All experimental protocols were conducted between the months of August and December. Dietary intervention
Upon completion of pre-intervention testing, participants received 3 days of nitrate-rich beetroot juice (70 ml shots; 5 mM of nitrates day−1 ; 0.45 g of nitrates; Beet It Sport; James White Drinks, Ipswich, UK). Participants were instructed to drink one 70 ml shot for three consecutive days, with the last shot consumed in the laboratory approximately 1.5–2 h prior to beginning the whole-body heating protocol. We have shown previously that this duration of nitrate consumption reduces MAP and increases CVC in response to local heating of the skin (Keen et al. 2015). The peak circulating plasma concentration of nitrites (range 200–600 nM) has been shown to occur approximately 1.5–2 h after consumption of nitrates (Govoni et al. 2008; Webb et al. 2008; Bailey et al. 2009; Vanhatalo et al. 2010). All studies were completed in a fixed order, where the pre-intervention testing was always performed first and postintervention testing was conducted on the final day of nitrate supplementation (Keen et al. 2015). Instrumentation of subjects
Testing was conducted with the participant resting in the supine position, with the experimental arm at heart level. To avoid the use of anaesthetics, ice was used to numb the desired location prior to placement of microdialysis fibres (Hodges et al. 2009). Fibre placement was approximately 3–5 cm apart. Microdialysis fibres 10 mm in length with a 55 kDa molecular mass cut-off (CMA 31 Linear Probe; CMA Microdialysis, Kista, Sweden) were placed by first threading a 23 gauge needle through the intradermal layer of the skin on the ventral aspect of the left forearm (Fieger & Wong, 2010, 2012; Wong & Fieger, 2010, 2012; Wong, 2013; McNamara et al. 2014; Keen et al. 2015). A fibre was then threaded through the lumen of the needle, and the needle was then removed, leaving the membrane in place. Approximately 45–90 min was allowed for resolution of the trauma response following microdialysis fibre placement before the continuation of the protocol. During this time, all fibres were perfused with lactated Ringer solution at a rate of 2 μl min−1 . Blood pressure (systolic, diastolic and mean) was monitored beat by beat via photoplethysmography (NexfinHD; BMEYE, Amsterdam, The Netherlands) and verified every 5 min via automated brachial auscultation (S/5 Light Monitor; GE Healthcare, Madison, WI, USA). Heart rate was monitored via three-lead ECG (S/5 Light Monitor). The core temperature (TC ) was measured via telemetric ingestible pill (CorTemp Data Recorder,
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CorTemp Temperature Sensor; Wireless Sensing Systems and Design, Palmetto, FL, USA). Participants were instructed to ingest the pill approximately 2–3 h prior to beginning data collection on the day of each experimental test. Core temperatures were recorded at least every minute during baseline, the period of whole-body heating and the cooling period. Red blood cell flux was used as an index of skin blood flow via laser-Doppler flowmetry (PeriFlux 5010 laser-Doppler perfusion monitor; Perimed, Jarfalla, Sweden). Local heating units (PF5020 local heating units and PeriFlux 5020 Temperature Unit; Perimed) were placed on the skin directly over each microdialysis membrane, and an integrated laser-Doppler probe (Probe 413; Perimed) was placed in the centre of each local heating unit to measure red blood cell flux directly over each microdialysis site. Local heaters at each microdialysis site were set and clamped at 33°C during baseline and the whole-body heating protocol until the temperature was raised at the end of the protocol to elicit maximal vasodilatation. Drugs administered
Lactated Ringer solution was administered at the control site. A 20 mM concentration of the L-arginine analogue L-NAME was used to inhibit NOS and has been shown previously to inhibit all isoforms of NOS non-selectively in human cutaneous microvasculature (Hodges et al. 2008; Smith et al. 2011; Bruning et al. 2012; Brunt et al. 2013; McNamara et al. 2014; Keen et al. 2015). At the end of the protocol, sodium nitroprusside (54 mM) was administered while simultaneously heating the skin locally to 43°C to elicit maximal vasodilatation (Holowatz et al. 2005; Smith et al. 2011; McNamara et al. 2014; Keen et al. 2015). All drugs were infused through the microdialysis fibres at a rate of 2 μl min−1 via microinfusion pumps (Bee Hive controller and Baby Bee Syringe Pumps; Bioanalytical Systems, West Lafayette, IN, USA) to each randomly assigned site. Whole-body heating protocol
Baseline skin blood flow and blood pressure were collected for 10 min following resolution of the trauma period associated with placement of the microdialysis fibres. Subsequently, one of the following two treatments was infused at random to each microdialysis site: (i) lactated Ringer solution (control site); or (ii) 20 mM L-NAME (non-selective NOS inhibitor). The L-NAME was infused for at least 45 min before and for the duration of the whole-body heating protocol. Participants wore a water-perfused suit to control whole-body temperature (Allen Vanguard, Ottawa, C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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Ontario, Canada) that covered the entire body except for the hands, feet, head, face and the experimental forearm. The water-perfused suit was covered with a water-impermeable rain suit to limit evaporative heat loss during whole-body heating. Whole-body heat stress was initiated by pumping water at 50°C through the water-perfused suit. Whole-body heating continued until participants’ core temperature was raised by 0.8–1.0°C above baseline. Core temperature was maintained at this level until a 5–10 min plateau in skin blood flow for both sites was achieved. Once a plateau in skin blood flow was achieved, 20 mM L-NAME was infused into the control site until skin blood flow decreased and reached a plateau. Once a new plateau was achieved at the control site in response to L-NAME (post-L-NAME drop), participants were cooled by pumping thermoneutral water through the suit, and the plastic rain suit was removed. Maximal skin blood flow was elicited via sodium nitroprusside and local heating to 43°C as described in the previous subsection. Data collection and analysis
Data were digitized and stored at 100 Hz on a personal computer. Data were analysed offline using signal-processing software (Windaq; Data Instruments, Akron, OH, USA). Blood flow measurements were normalized to MAP and expressed as CVC, calculated as the ratio of red blood cell flux to MAP (i.e. red blood cell flux/MAP). Cutaneous vascular conductance values were normalized as a percentage of maximal vasodilatation (%CVCmax ) via sodium nitroprusside infusion and local heating to 43°C. All data were analysed with SPSS 20 (IBM Corporation). All values are presented as means ± SEM, and P < 0.05 was considered to be statistically significant. Resting baseline data were taken as the average over a 3–5 min period immediately prior to initiating whole-body heat stress; the plateau data were taken as the average over 2–3 min immediately prior to infusing L-NAME at the control site; and the end of the protocol data were taken as the average over 1–2 min immediately prior to terminating the experiment when participants’ core temperature had returned to thermoneutral and when maximal vasodilatation had been achieved. To determine whether nitrate supplementation directly augmented skin blood flow, raw skin blood flow data (in arbitrary units) were analysed at baseline and during the plateau of heat stress pre- and postnitrate supplementation, and the percentage increase from baseline was calculated (Keen et al. 2015). Cutaneous vascular conductance data were also analysed for a given increase in core temperature (TC ) at each microdialysis site during the 30–60 s of each 0.1°C increase in core temperature from baseline (TC = 0.0°C) until the end of the heat stress protocol (TC = 0.8°C). The onset of reflex cutaneous C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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vasodilatation was defined as the core temperature at which there was a robust and sustained increase in %CVCmax above baseline. Data were analysed using one-way repeated-measures ANOVA, two-way repeated-measures ANOVA and Student’s paired t tests as appropriate. For all ANOVAs, post hoc Student’s paired t tests were used to determine where significant differences occurred, and Sidak correction for multiple pairwise comparisons was employed as appropriate. The percentage NO contribution was calculated at the control sites as: [(Whole Body Heating (WBH) control plateau−Post-LNAME drop control)/WBH control plateau] × 100 where the post-L-NAME drop is the plateau in CVC following the infusion of L-NAME during hyperthermia. The reduction in CVC in response to the infusion of L-NAME during the plateau was calculated as the difference between the plateau CVC and post-L-NAME plateau and presented as CVC. Results Heart rate and blood pressure data (Table 1) and core temperature responses
Heart rate and blood pressure data for both pre- and postnitrate supplementation are shown in Table 1. There was no effect of nitrate supplementation on resting heart rate (before 59 ± 5 beats min−1 versus after 63 ± 3 beats min−1 ; P = 0.599). At the plateau of whole-body heating, there was a statistically significant increase in HR compared with rest for both pre- (86 ± 7 beats min−1 ; P < 0.001) and postnitrate (93 ± 3 beats min−1 ; P < 0.001) supplementation; however, the increase in HR during heat stress was not statistically significant between pre- and postnitrate supplementation (P = 0.466). There was no difference between the baseline HR and the HR at the end of the protocol for either set of conditions (P > 0.05 for both sets of conditions). There was no effect of nitrate supplementation on core temperature either at baseline (thermoneutral; before 37.18 ± 0.20°C versus after 37.09 ± 0.14°C) or during heat stress (before 38.03 ± 0.15°C versus after 37.95 ± 0.27°C; P = 1.000 for all conditions). Core temperature during heat stress was significantly increased compared with baseline (P < 0.05 for all conditions). Following nitrate supplementation, there was a statistically significant decrease in resting (baseline) systolic blood pressure (before 128 ± 4 mmHg versus after 116 ± 4 mmHg; P < 0.05), but there was no effect of nitrate supplementation on resting diastolic blood pressure (before 68 ± 3 mmHg versus after 66 ± 2 mmHg; P = 0.415) or mean arterial blood pressure (before 88 ± 3 mmHg versus
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Table 1. Heart rate and blood pressure data Before nitrate supplementation Parameter
Rest (baseline) min−1 )
Heart rate (beats Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean arterial pressure (mmHg)
59 ± 5 128 ± 4 68 ± 3 88 ± 3
Plateau 7†
86 ± 131 ± 5 66 ± 2 88 ± 3
End 64 ± 5 122 ± 4 74 ± 2† 93 ± 1
After nitrate supplementation Rest (baseline) 63 ± 3 116 ± 4∗ 66 ± 2 83 ± 2
Plateau 93 106 63 78
3†
± ± 4∗† ± 2 ± 2∗†
End 70 114 63 78
± ± ± ±
5 2 3∗ 4∗
Data are means ± SEM. ‘Plateau’ is the plateau in skin blood flow during heat stress. ‘End’ is the end of the protocol during maximal vasodilatation and when core temperature had returned to baseline. ∗ P < 0.05 versus before nitrate supplementation; † P < 0.05 versus baseline within the same supplement conditions.
after 83 ± 2 mmHg; P = 0.117). At the plateau of wholebody heating, nitrate supplementation resulted in a statistically significant decrease in both systolic blood pressure (before 131 ± 5 mmHg versus after 106 ± 4 mmHg; P = 0.029) and mean arterial blood pressure (before 88 ± 3 mmHg versus after 78 ± 2 mmHg; P < 0.05), but there was no effect on diastolic blood pressure (before 66 ± 2 mmHg versus after 63 ± 2 mmHg; P = 0.112). There was no effect of heat stress or supplement on diastolic arterial pressure or MAP compared with baseline (P > 0.193 for all conditions), but postnitrate supplement the systolic blood pressure during heat stress was reduced compared with baseline (P < 0.05). At the end of the protocol, when the participants’ core temperature returned to preheat-stress levels and while maximal vasodilatation was being elicited, nitrate supplementation resulted in a statistically significant reduction in diastolic blood pressure (before 74 ± 2 mmHg versus after 63 ± 3 mmHg; P = 0.016) and MAP (before 93 ± 1 mmHg versus after 78 ± 4 mmHg; P = 0.007), but there was no effect on systolic blood pressure (before 122 ± 4 mmHg versus after 114 ± 2 mmHg; P = 0.142). The blood pressure data are summarized in Table 1.
Raw skin blood flow data (Table 2)
There was no effect of nitrate supplementation on baseline skin blood flow at control (before 29 ± 5 a.u. versus after 28 ± 3 a.u.; P = 1.000) or L-NAME sites (before 22 ± 3 a.u. versus after 17 ± 1 a.u.; P = 0.644). There was no difference in baseline skin blood flow between control and L-NAME sites prenitrate supplementation (P = 0.178). There was a trend towards a greater baseline skin blood flow at control compared with L-NAME sites postnitrate supplementation, but this did not reach statistical significance (P = 0.06). These data are shown in Table 2. Similar to baseline skin blood flow, there was no effect of nitrate supplementation on skin blood flow during the plateau of heat stress at control (before 89 ± 5 a.u. versus after 87 ± 7 a.u.; P = 1.000) or L-NAME sites (before 63 ± 9 a.u. versus after 51 ± 6 a.u.; P = 1.000). The
L-NAME attenuated plateau skin blood flow compared with control sites for both pre- (P < 0.05) and postnitrate supplementation (P = 0.007). Plateau skin blood flow data are shown in Table 2. There was no effect of nitrate supplementation on the percentage increase in skin blood flow from baseline at control (before 67 ± 6% versus after 68 ± 2%; P = 0.751) or L-NAME sites (before 59 ± 9% versus after 65 ± 4%; P = 0.477). There was no statistical difference in the percentage increase from baseline between control and L-NAME sites for either set of conditions (P = 0.788 for both sets of conditions). These data are shown in Table 2.
Cutaneous vascular conductance data
There was no effect of nitrate supplementation on baseline CVC at either the control (before 17 ± 2%CVCmax versus after 21 ± 2%CVCmax ; P = 1.000) or L-NAME sites (before 14 ± 2%CVCmax versus after 13 ± 1%CVCmax ; P = 1.000). Baseline CVC did not differ between control or L-NAME sites for either pre- or postnitrate supplementation conditions (P > 0.111 for all conditions). There was an increase in the plateau phase during whole-body heating following nitrate supplementation; however, the magnitude of the post-L-NAME drop was not different between pre- and postnitrate supplementation periods. As the magnitude of the post-L-NAME drop was similar between conditions, there was no difference in the calculated percentage NOS-dependent vasodilatation (see CVC data below). Figure 1 shows the CVC response to whole-body heating (plateau) at control and L-NAME sites for pre- and postnitrate supplementation periods. The plateau CVC at the control site was significantly greater after 3 days of nitrate supplementation (80 ± 5%CVCmax ) compared with the control site before nitrate supplementation (67 ± 2%CVCmax ; P < 0.01). At L-NAME-treated sites, there was no statistical difference between plateau CVC prenitrate (45 ± 4%CVCmax ) and postnitrate supplementation (40 ± 5%CVCmax ; P = 0.617). Plateau CVC at L-NAME sites for both pre- and postnitrate conditions C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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Table 2. Raw skin blood flow data Site and conditions Control site Before nitrate supplementation After nitrate supplementation L-NAME-treated site Before nitrate supplementation After nitrate supplementation
Baseline
Plateau
Percentage increase from baseline
29 ± 5 28 ± 3
89 ± 5 87 ± 7
67 ± 6 68 ± 2
22 ± 3 17 ± 1
63 ± 9∗ 51 ± 6∗
59 ± 9 65 ± 4
Data are means ± SEM. Raw skin blood flow units are arbitrary units. ∗ P < 0.05 versus control within same conditions.
Cutaneous Vascular Conductance (% Maximal)
were significantly attenuated compared with plateau CVC at control sites for both pre- and postnitrate conditions (P < 0.02 for all conditions). Figure 2 shows the CVC responses as a function of core temperature. The onset of cutaneous vasodilatation was not affected by nitrate supplementation at either control (TC = 0.3°C) or L-NAME sites (TC = 0.4°C). The individual whole-body heating plateau responses are shown in Fig. 3A (control) and B (L-NAME). The CVC responses to L-NAME infusion (i.e. reduction in CVC) during the plateau of heat stress were not statistically significant between pre(43 ± 3%CVCmax ) and postnitrate (51 ± 2%CVCmax ; P = 0.125). Maximal CVC values at control sites were not different from pre- (1.98 ± 0.26) mV/mmHg to postnitrate supplementation (1.90 ± 0.20; P = 0.523) mV/mmHg. Likewise, maximal CVC at L-NAME sites was not affected by nitrate supplementation (before 1.92 ± 0.30 versus after 1.95 ± 0.33; P = 1.000) mV/mmHg. There was no
difference in maximal CVC between control and L-NAME sites for any conditions. Nitric oxide synthase-dependent vasodilatation
Figure 4 shows the group mean data for the calculated contribution of NOS-dependent vasodilatation to reflex cutaneous vasodilatation at control sites for preand postnitrate supplementation conditions. There was no statistical difference in the percentage of NOSdependent vasodilatation between prenitrate (59 ± 4% NOS-dependent vasodilatation) and postnitrate conditions (64 ± 6%NOS-dependent vasodilatation; P = 0.577). Discussion The primary finding of this investigation suggests that nitrate supplementation augments reflex cutaneous vasodilatation. Physiologically, the augmented CVC
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Figure 1. Cutaneous vascular conductance responses to whole-body heat stress Data are shown for control and l-NAME microdialysis sites for both pre- and postnitrate supplementation. Cutaneous vascular conductance was augmented following nitrate supplementation at the control sites only. Values are shown as means + SEM. ∗ P < 0.05 versus prenitrate. †P < 0.05 versus control within same conditions.
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appeared to be due to increased vasodilatation of the cutaneous microvasculature, while the calculated (mathematical) increase in CVC was driven largely by the decrease in MAP, with no change in skin blood flow per se. We also observed a significant augmentation of the plateau phase of reflex cutaneous vasodilatation at the control but not the L-NAME-treated site after 3 days of nitrate supplementation. Collectively, these findings suggest that short-term dietary nitrate supplementation may improve the CVC response to whole-body heat stress in young, healthy participants. The increase in CVC was most probably driven by increased cutaneous vasodilatation. Based on the hydraulic resistance equation, it appears that after nitrate supplementation there was significant vasodilatation (decreased vascular resistance) of the cutaneous vasculature despite decreases in MAP and no change in skin blood flow. The calculated (mathematical) increase in CVC during heat stress after nitrate supplementation can be explained by the reduction in MAP during heat stress after nitrate supplementation. As shown in Table 2, there was no significant increase in raw skin blood flow at either baseline or during heat stress from pre- to postnitrate supplementation, suggesting that the increase in calculated CVC was driven largely by the reduction in MAP. Our conclusion that CVC was augmented owing to NOS-independent mechanisms is based on our data showing an increase in the skin blood flow plateau during heat stress at control sites yet no change in the calculated
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percentage NOS-dependent vasodilatation. In order to determine the percentage NOS-dependent vasodilatation, we infused the non-selective NOS inhibitor, L-NAME, through the microdialysis fibres at the control site once skin blood flow had reached a plateau during heat stress. The difference between the initial plateau during heat stress and the plateau following L-NAME infusion (post-L-NAME drop) yields the calculated percentage NOS-dependent vasodilatation. Despite an augmented plateau at control sites following nitrate supplementation, there was no significant increase in the calculated percentage NOS-dependent vasodilatation owing to a similar magnitude of reduction following L-NAME infusion. It would thus appear that the augmented CVC during heat stress is associated with NOS-independent mechanisms. While our data suggest no effect of nitrate supplementation on NOS-dependent vasodilatation, we cannot rule out the possibility that the increase in reflex cutaneous vasodilatation was NO dependent. Inasmuch as nitrate is eventually reduced to NO independent of NOS activity (McKnight et al. 1997; Lundberg et al. 2008; Lundberg & Weitzberg, 2009), it is possible that the enhanced cutaneous vascular response to heat stress was due to an increase in NO that was independent of functional NOS activity and would thus not be affected by NOS inhibition. Our present finding that nitrate supplementation does not augment vascular conductance at the L-NAME site is in contrast to what we have recently observed during local heating of the skin (Keen et al. 2015). During local
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ΔTc (°C) Figure 2. Cutaneous vascular conductance as a function of core temperature (Tc ) Cutaneous vascular conductance data are presented as a function of increasing core temperature in 0.1°C increments. Abbreviation: BRJ, beetroot juice. Data are means ± SEM. ∗ P < 0.05 versus pre-BRJ control site. † P < 0.05 versus pre- and post-BRJ control site.
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heating of the skin, we found that nitrate supplementation augmented the plateau phase at both control and L-NAME sites. Yet, similar to the present data, there was no increase in skin blood flow from pre- to postnitrate supplementation, which suggests that the majority of the increase in vascular conductance in response to local heating of the skin was driven by a decrease in MAP. It is possible that high levels of skin blood flow are a prerequisite for enhanced NO-dependent vasodilatation after nitrate supplementation. That is, elevated shear stress may be required to increase NO production after nitrate supplementation, and the attenuated skin blood flow with
continuous L-NAME infusion may prevent this requisite level of shear stress. While this possible explanation is intriguing, the importance of shear stress-induced NO production in the cutaneous vasculature is unclear (Wong et al. 2003; Zhao et al. 2004). Nevertheless, it appears that short-term dietary nitrate supplementation augments NOS-independent increases in CVC during both whole-body heating and local heating of the skin in young, healthy humans, and these increases in CVC are largely due to reduced MAP. We also observed significant reductions in both systolic arterial blood pressure and MAP after 3 days
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Figure 3. Individual and group cutaneous vascular conductance responses pre- and postnitrate supplementation Individual and group responses for the control sites (A) and L-NAME sites (B). Individual responses are depicted as thin dashed lines; group responses are shown as the thick black line.
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of nitrate supplementation at baseline and during heat stress. Although our participants were normotensive, reductions in both systolic arterial blood pressure and MAP are consistent with previous studies after nitrate supplementation (Larsen et al. 2006; Webb et al. 2008; Kapil et al. 2010; Vanhatalo et al. 2010). Although we found no changes in baseline (thermoneutral) MAP between conditions as in our previous study (Keen et al. 2015), there was a trend towards a decrease in baseline (thermoneutral) MAP postnitrate supplementation that may not have reached statistical significance owing to greater variability in the present study. It has been suggested that nitrate supplementation reduces blood pressure owing to an increase in NO production (Lundberg et al. 2008, 2011; Webb et al. 2008; Lundberg & Weitzberg, 2009; Kapil et al. 2010). The increase in NO production via reduction of nitrate to nitrite and, eventually, to NO is independent of NOS and thus would not be in conflict with our CVC conclusions. Our data showing no effect of nitrate supplementation on heart rate responses is consistent with previous literature in both humans and animals and suggests that nitrate supplementation has minimal direct effect on the heart (Webb et al. 2008; Ferguson et al. 2013). Limitations
A potential limitation to this study is that we did not quantify serum levels of plasma nitrate and nitrite from pre- to postnitrate supplementation. Measuring changes in plasma nitrate and nitrite levels would have allowed us to determine whether changes in blood pressure were correlated with peak nitrite levels preto postsupplement intervention as previously discussed and presented in current mechanistic studies with nitrate
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supplementation (Webb et al. 2008). Although indirect, our data demonstrating a reduction in both systolic arterial blood pressure and MAP would appear to confirm that 3 days of nitrate supplementation was adequate in increasing plasma nitrate and nitrite concentrations. Significance
Net heat transfer from core to skin is a function of skin blood flow. In young, healthy participants, there was no significant increase in skin blood flow per se after nitrate supplementation, suggesting no improved thermoregulatory benefit. Despite our finding that skin blood flow did not change, it is possible that in populations with a diminished skin blood flow response to heat stress, such as an ageing population, nitrate supplementation may directly improve skin blood flow and thus improve the thermoregulatory response to heat stress. Conclusions
To our knowledge, this is the first study to identify a role for dietary nitrate supplementation via beetroot juice in the skin blood flow response to whole-body heat stress. Our data suggest that dietary nitrate supplementation augmented reflex cutaneous vasodilatation in young, healthy subjects by the following mechanisms: (i) vasodilatation of the cutaneous vasculature despite decreases in MAP during heat stress; and (ii) NOS-independent vasodilatation, as evidenced by an increase in CVC from pre- to postnitrate supplementation in the plateau phase at control sites, with no change in the calculated percentage of NOS-dependent vasodilatation. References
80 60 40 20 0 Pre-Nitrate
Post-Nitrate
Figure 4. Calculated percentage of nitric oxide synthase (NOS)-dependent vasodilatation pre- and postnitrate supplementation There was no difference in the calculated percentage of NOS-dependent vasodilatation between pre- and postnitrate supplementation. Values are shown as means + SEM.
Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, Benjamin N & Jones AM (2009). Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol 107, 1144–1155. Bruning RS, Santhanam L, Stanhewicz AE, Smith CJ, Berkowitz DE, Kenney WL & Holowatz LA (2012). Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin. J Appl Physiol 112, 2019–2026. Brunt VE, Fujii N & Minson CT (2013). No independent, but an interactive, role of calcium-activated potassium channels in human cutaneous active vasodilation. J Appl Physiol 115, 1290–1296. Casey DP, Treichler DP, Ganger CT 4th, Schneider AC & Ueda K (2015). Acute dietary nitrate supplementation enhances compensatory vasodilation during hypoxic exercise in older adults. J Appl Physiol 118, 176–186. C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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Dietz NM, Rivera JM, Warner DO & Joyner MJ (1994). Is nitric oxide involved in cutaneous vasodilation during body heating in humans? J Appl Physiol 76, 2047–2053. Edholm OG, Fox RH & Macpherson RK (1957). Vasomotor control of the cutaneous blood vessels in the human forearm. J Physiol 139, 455–465. Ferguson SK, Hirai DM, Copp SW, Holdsworth CT, Allen JD, Jones AM, Musch TI & Poole DC (2013). Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control in rats. J Physiol 591, 547–557. Fieger SM & Wong BJ (2010). Adenosine receptor inhibition with theophylline attenuates the skin blood flow response to local heating in humans. Exp Physiol 95, 946–954. Fieger SM & Wong BJ (2012). No direct role for A1/A2 adenosine receptor activation to reflex cutaneous vasodilatation during whole-body heat stress in humans. Acta Physiol (Oxf) 205, 403–410. Govoni M, Jansson EA, Weitzberg E & Lundberg JO (2008). The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide 19, 333–337. 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 106, 1112–1118. Hodges GJ, Kosiba WA, Zhao K & Johnson JM (2008). The involvement of norepinephrine, neuropeptide Y, and nitric oxide in the cutaneous vasodilator response to local heating in humans. J Appl Physiol 105, 233–240. 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. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S, MacAllister R, Hobbs AJ, Webb AJ & Ahluwalia A (2010). Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO. Hypertension 56, 274–281. Keen JT, Levitt EL, Hodges GJ & Wong BJ (2015). Short-term dietary nitrate supplementation augments cutaneous vasodilatation and reduces mean arterial pressure in healthy humans. Microvasc Res 98, 48–53. Kellogg DL Jr, Crandall CG, Liu Y, Charkoudian N & Johnson JM (1998). Nitric oxide and cutaneous active vasodilation during heat stress in humans. J Appl Physiol 85, 824–829. Kellogg DL Jr, P´ergola PE, Piest KL, Kosiba WA, Crandall CG, Grossmann M & Johnson JM (1995). Cutaneous active vasodilation in humans is mediated by cholinergic nerve cotransmission. Circ Res 77, 1222–1228. Kellogg DL Jr, Zhao JL & Wu Y (2008a). Endothelial nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo. Am J Physiol Heart Circ Physiol 295, H123–H129. Kellogg DL Jr, Zhao JL & Wu Y (2008b). Neuronal nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo. J Physiol 586, 847–857. Kellogg DL Jr, Zhao JL & Wu Y (2009). Roles of nitric oxide synthase isoforms in cutaneous vasodilation induced by local warming of the skin and whole body heat stress in humans. J Appl Physiol 107, 1438–1444. C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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Kellogg DL Jr, Zhao JL, Wu Y & Johnson JM (2010). VIP/PACAP receptor mediation of cutaneous active vasodilation during heat stress in humans. J Appl Physiol 109, 95–100. Kenjale AA, Ham KL, Stabler T, Robbins JL, Johnson JL, Vanbruggen M, Privette G, Yim E, Kraus WE & Allen JD (2011). Dietary nitrate supplementation enhances exercise performance in peripheral arterial disease. J Appl Physiol 110, 1582–1591. Larsen FJ, Ekblom BR, Sahlin K, Lundberg JO & Weitzberg E (2006). Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med 355, 2792–2793. Larsen FJ, Weitzberg E, Lundberg JO & Ekblom B (2007). Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol (Oxf) 191, 59–66. Lundberg JO & Weitzberg E (2009). NO generation from inorganic nitrate and nitrite: Role in physiology, nutrition and therapeutics. Arch Pharm Res 32, 1119–1126. Lundberg JO, Weitzberg E & Gladwin M (2008). The nitrate–nitrite–nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Disc 7, 156–167. Lundberg JO, Carlstr¨om M, Larsen FJ & Weitzberg E (2011). Roles of dietary inorganic nitrate in cardiovascular health and disease. Cardiovasc Res 89, 525–532. McCord GR, Cracowski J-L & Minson CT (2006). Prostanoids contribute to cutaneous active vasodilation in humans. Am J Physiol Regul Integr Comp Physiol 291, R596–R602. McKnight GM, Smith LM, Drummond RS, Duncan CW, Golden M & Benjamin N (1997). Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans. Gut 40, 211–214. 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. Moncada S & Higgs A (1993). The L-arginine-nitric oxide pathway. N Engl J Med 329, 2002–2012. Shastry S, Dietz NM, Halliwill JR, Reed AS & Joyner MJ (1998). Effects of nitric oxide synthase inhibition on cutaneous vasodilation during body heating in humans. J Appl Physiol 85, 830–834. Smith CJ, Santhanam L, Bruning RS, Stanhewicz A, Berkowitz DE & Holowatz LA (2011). Upregulation of inducible nitric oxide synthase contributes to attenuated cutaneous vasodilation in essential hypertensive humans. Hypertension 58, 935–942. Vanhatalo A, Bailey SJ, Blackwell JR, DiMenna FJ, Pavey TG, Wilkerson DP, Benjamin N, Winyard PG & Jones AM (2010). Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise. Am J Physiol Reg Integ Comp Physiol 299, R1121–R1131. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, Deanfield J, Benjamin N, MacAllister R, Hobbs AJ & Ahluwalia A (2008). Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension 51, 784–790.
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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 (2013). Sensory nerves and nitric oxide contribute to reflex cutaneous vasodilation in humans. Am J Physiol Reg Integ Comp Physiol 304, R651–R656. Wong BJ & Fieger SM (2010). Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans. J Physiol 588, 4317–4326. 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. Wong BJ, Wilkins BW, Holowatz LA & Minson CT (2003). Nitric oxide synthase inhibition does not alter the reactive hyperemic response in the cutaneous circulation. J Appl Physiol 95, 504–510. Wong BJ, Wilkins BW & Minson CT (2004). H1 but not H2 histamine receptor activation contributes to the rise in skin blood flow during whole body heating in humans. J Physiol 560, 941–948. Zhao JL, Pergola PE, Roman LJ & Kellogg DL (2004). Bioactive nitric oxide concentration does not increase during reactive hyperemia in human skin. J Appl Physiol 96, 628–632.
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Additional information Competing interests None declared. Author contributions This study was completed in the Department of Kinesiology at Kansas State University in Manhattan, KS, USA. This study was completed by E.L.L. in part fulfilment of the degree of MSc in the Department of Kinesiology at Kansas State University. E.L.L. was responsible for experimental design, recruitment of participants, data collection, analysis and interpretation, and drafting and editing all versions of the manuscript. J.T.K. was responsible for data collection and interpretation, and editing drafts of the manuscript. B.J.W. was responsible for experimental design, data analysis and interpretation, and drafting and editing all versions of the manuscript. Funding None declared. Acknowledgements The authors are grateful to all the participants for their time and willingness to participate in this study. The authors would also like to thank Ms Jena K. Eder for her assistance with data collection.
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Research Paper
Augmented reflex cutaneous vasodilatation following short-term dietary nitrate supplementation in humans Erica L. Levitt1 , Jeremy T. Keen1 and Brett J. Wong1,2 1
Experimental Physiology
2
Department of Kinesiology, Kansas State University, Manhattan, KS, USA Department of Kinesiology and Health, Georgia State University, Atlanta, GA, USA
New Findings r What is the central question of this study? Nitrate supplementation via beetroot juice has been shown to have several benefits in healthy humans, including reduced blood pressure and increased blood flow to exercising muscle. Whether nitrate supplementation can improve blood flow to the skin in heat-stressed humans has not been investigated. r What is the main finding and its importance? Similar to previous studies, we found that nitrate supplementation reduces blood pressure. Nitrate supplementation increased vasodilatation in the skin of heat-stressed humans but did not directly increase skin blood flow.
Nitrate supplementation has been shown to increase NO-dependent vasodilatation through both NO synthase (NOS)-dependent and NOS-independent pathways. We hypothesized that nitrate supplementation would augment reflex cutaneous active vasodilatation. Subjects were equipped with two microdialysis fibres on the forearm randomly assigned as control (Ringer solution) or NOS inhibition (20 mm l-NAME). Whole-body heating was performed to raise core temperature by 0.8°C above baseline core temperature. Maximal cutaneous vasodilatation was achieved via 54 mm sodium nitroprusside and local heating to 43°C. Skin blood flow (measured by laser-Doppler flowmetry) and blood pressure were measured. Cutaneous vascular conductance (CVC) was calculated as skin blood flow divided by mean arterial pressure (MAP) and expressed as a percentage of maximal CVC (%CVCmax ). Subjects underwent heat stress before and after nitrate supplementation (3 days of beetroot juice; 5 mm, 0.45 g nitrates per day). During heat stress, MAP was reduced following nitrate supplementation compared with the control conditions (before 88 ± 3 mmHg versus after 78 ± 2 mmHg; P < 0.05); however, resting MAP was not different between conditions (before 88 ± 3 mmHg versus after 83 ± 2 mmHg; P = 0.117). Nitrate supplementation increased plateau CVC at control sites (before 67 ± 2%CVCmax versus after 80 ± 5%CVCmax ; P = 0.01) but not at l-NAME-treated sites (before 45 ± 4%CVCmax versus after 40 ± 5%CVCmax ; P = 0.617). There was no change in the calculated percentage of NOS-dependent vasodilatation before and after supplementation (before 59 ± 4% versus after 64 ± 6%; P = 0.577). These data suggest that nitrate supplementation augments CVC and reduces MAP during heat stress. (Received 8 January 2015; accepted after revision 30 March 2015; first published online 31 March 2015) Corresponding author B. J. Wong: Department of Kinesiology & Health, Georgia State University, PO Box 3975, Atlanta, GA 30302-3975, USA. Email: [email protected]
DOI: 10.1113/EP085061
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Introduction In humans, the onset of heat stress initiates an increase in core temperature and is associated with an increase in cutaneous vasodilatation and sweating (Edholm et al. 1957). The cutaneous circulation is regulated by two branches of the sympathetic nervous system, namely the adrenergic vasoconstrictor nerves and the cholinergic active vasodilator nerves (Edholm et al. 1957; Kellogg et al. 1995). With an increase in core body temperature, the sympathetic cholinergic vasodilator nerves reflexively mediate increases in skin blood flow (Edholm et al. 1957; Kellogg et al. 1995). The cotransmission theory (Kellogg et al. 1995) of active vasodilatation suggests that the release of acetylcholine and an unknown substance(s) are responsible for the sweat response and cutaneous vasodilatation, respectively (Kellogg et al. 1995). A multitude of potential vasodilators and vasodilator pathways have been suggested to mediate at least a portion of the reflex cutaneous vasodilatation (Wong et al. 2004; McCord et al. 2006; Wong & Minson, 2006; Kellogg et al. 2010; Wong & Fieger, 2012; Brunt et al. 2013; Wong, 2013). Nitric oxide (Dietz et al. 1994; Kellogg et al. 1998, 2008a,b, 2009; Shastry et al. 1998; Wilkins et al. 2003) has been shown to contribute 35-45% to cutaneous active vasodilatation, and several of the suggested vasodilators have been shown to include a substantial NO component. Data have also demonstrated that NO has both a direct vasodilator role and may also work synergistically with the unknown transmitter(s) (Wilkins et al. 2003). Recent evidence from Kellogg et al. (2008b) suggests the specific nitric oxide synthase (NOS) isoform responsible for active vasodilatation to be neuronal nitric oxide synthase (nNOS). The classical pathway used for NO generation through the conversion of L-arginine to L-citrulline and NO has been shown to be NOS dependent (Moncada & Higgs, 1993). Conversely, the reduction of nitrate to nitrite and, subsequently, to NO has been shown to be an NOS-independent pathway for NO generation (Govoni et al. 2008; Lundberg et al. 2008; Lundberg & Weitzberg, 2009). Several studies have demonstrated that consumption of dietary nitrates has positive health benefits, including an increase in exercising skeletal muscle blood flow (Ferguson et al. 2013; Casey et al. 2015), reduced oxygen cost of exercise (Larsen et al. 2007; Bailey et al. 2009), improved exercise tolerance in patients with peripheral arterial disease (Kenjale et al. 2011) and lower blood pressure (Larsen et al. 2006; Webb et al. 2008). It has been suggested that most of these effects of nitrate consumption are due in large part to an increase in NOS-independent NO production. Researchers in our laboratory have recently shown that acute dietary nitrate supplementation augments cutaneous vascular C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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conductance (CVC) to local heating, primarily due to a reduction in mean arterial pressure (MAP) and not due to a direct increase in skin blood flow (Keen et al. 2015); however, it is unknown what effects short-term dietary nitrate supplementation has on the cutaneous vascular response to whole-body heat stress. The purpose of the present investigation was to determine whether short-term dietary nitrate supplementation would augment the cutaneous vascular response to wholebody heat stress via NOS-dependent or NOS-independent mechanisms. Based on our previous findings (Keen et al. 2015), we hypothesized that nitrate supplementation would augment cutaneous reflex vasodilatation via NOSindependent mechanisms in healthy humans.
Methods Ethical approval
The Institutional Review Board at Kansas State University approved all protocols of this study. A written informed consent was reviewed and signed by each participant prior to participation in this experiment. All procedures and protocols were performed in conjunction with the standards set forth by the Declaration of Helsinki.
Participants
Seven participants (seven men; age 22 ± 1 years, height 179 ± 8 cm, body mass 80 ± 14 kg and body mass index 25 ± 4 kg m−2 ) participated in this study. All were healthy, non-smokers, taking no medications and were free of cardiovascular, respiratory and metabolic disease as determined by medical history questionnaire. Participants were asked to refrain from exercise, consumption of abnormal amounts (>300 g day−1 ) of leafy green vegetables (e.g. spinach), consumption of alcohol and the use of mouthwash products containing alcohol during the 3 day nitrate supplementation period. Exercise and consumption of large quantities of leafy green vegetables can increase systemic nitrates, while alcohol and alcohol-based mouthwash can kill the bacteria required to reduce nitrate to nitrite and subsequently to nitric oxide (Govoni et al. 2008; Bailey et al. 2009). The postsupplementation test was conducted 7–10 days after the presupplementation test to allow the microdialysis sites to heal. During this time, participants were asked to maintain their normal exercise and diet routine (i.e. not to begin a new exercise regimen or diet) and to continue avoiding consumption of excessive quantities of leafy green vegetables. Participants were asked to refrain from caffeine for at least 12 h prior to the study. Testing was administered in a thermoneutral environment with a room temperature
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of 23–24°C. All experimental protocols were conducted between the months of August and December. Dietary intervention
Upon completion of pre-intervention testing, participants received 3 days of nitrate-rich beetroot juice (70 ml shots; 5 mM of nitrates day−1 ; 0.45 g of nitrates; Beet It Sport; James White Drinks, Ipswich, UK). Participants were instructed to drink one 70 ml shot for three consecutive days, with the last shot consumed in the laboratory approximately 1.5–2 h prior to beginning the whole-body heating protocol. We have shown previously that this duration of nitrate consumption reduces MAP and increases CVC in response to local heating of the skin (Keen et al. 2015). The peak circulating plasma concentration of nitrites (range 200–600 nM) has been shown to occur approximately 1.5–2 h after consumption of nitrates (Govoni et al. 2008; Webb et al. 2008; Bailey et al. 2009; Vanhatalo et al. 2010). All studies were completed in a fixed order, where the pre-intervention testing was always performed first and postintervention testing was conducted on the final day of nitrate supplementation (Keen et al. 2015). Instrumentation of subjects
Testing was conducted with the participant resting in the supine position, with the experimental arm at heart level. To avoid the use of anaesthetics, ice was used to numb the desired location prior to placement of microdialysis fibres (Hodges et al. 2009). Fibre placement was approximately 3–5 cm apart. Microdialysis fibres 10 mm in length with a 55 kDa molecular mass cut-off (CMA 31 Linear Probe; CMA Microdialysis, Kista, Sweden) were placed by first threading a 23 gauge needle through the intradermal layer of the skin on the ventral aspect of the left forearm (Fieger & Wong, 2010, 2012; Wong & Fieger, 2010, 2012; Wong, 2013; McNamara et al. 2014; Keen et al. 2015). A fibre was then threaded through the lumen of the needle, and the needle was then removed, leaving the membrane in place. Approximately 45–90 min was allowed for resolution of the trauma response following microdialysis fibre placement before the continuation of the protocol. During this time, all fibres were perfused with lactated Ringer solution at a rate of 2 μl min−1 . Blood pressure (systolic, diastolic and mean) was monitored beat by beat via photoplethysmography (NexfinHD; BMEYE, Amsterdam, The Netherlands) and verified every 5 min via automated brachial auscultation (S/5 Light Monitor; GE Healthcare, Madison, WI, USA). Heart rate was monitored via three-lead ECG (S/5 Light Monitor). The core temperature (TC ) was measured via telemetric ingestible pill (CorTemp Data Recorder,
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CorTemp Temperature Sensor; Wireless Sensing Systems and Design, Palmetto, FL, USA). Participants were instructed to ingest the pill approximately 2–3 h prior to beginning data collection on the day of each experimental test. Core temperatures were recorded at least every minute during baseline, the period of whole-body heating and the cooling period. Red blood cell flux was used as an index of skin blood flow via laser-Doppler flowmetry (PeriFlux 5010 laser-Doppler perfusion monitor; Perimed, Jarfalla, Sweden). Local heating units (PF5020 local heating units and PeriFlux 5020 Temperature Unit; Perimed) were placed on the skin directly over each microdialysis membrane, and an integrated laser-Doppler probe (Probe 413; Perimed) was placed in the centre of each local heating unit to measure red blood cell flux directly over each microdialysis site. Local heaters at each microdialysis site were set and clamped at 33°C during baseline and the whole-body heating protocol until the temperature was raised at the end of the protocol to elicit maximal vasodilatation. Drugs administered
Lactated Ringer solution was administered at the control site. A 20 mM concentration of the L-arginine analogue L-NAME was used to inhibit NOS and has been shown previously to inhibit all isoforms of NOS non-selectively in human cutaneous microvasculature (Hodges et al. 2008; Smith et al. 2011; Bruning et al. 2012; Brunt et al. 2013; McNamara et al. 2014; Keen et al. 2015). At the end of the protocol, sodium nitroprusside (54 mM) was administered while simultaneously heating the skin locally to 43°C to elicit maximal vasodilatation (Holowatz et al. 2005; Smith et al. 2011; McNamara et al. 2014; Keen et al. 2015). All drugs were infused through the microdialysis fibres at a rate of 2 μl min−1 via microinfusion pumps (Bee Hive controller and Baby Bee Syringe Pumps; Bioanalytical Systems, West Lafayette, IN, USA) to each randomly assigned site. Whole-body heating protocol
Baseline skin blood flow and blood pressure were collected for 10 min following resolution of the trauma period associated with placement of the microdialysis fibres. Subsequently, one of the following two treatments was infused at random to each microdialysis site: (i) lactated Ringer solution (control site); or (ii) 20 mM L-NAME (non-selective NOS inhibitor). The L-NAME was infused for at least 45 min before and for the duration of the whole-body heating protocol. Participants wore a water-perfused suit to control whole-body temperature (Allen Vanguard, Ottawa, C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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Ontario, Canada) that covered the entire body except for the hands, feet, head, face and the experimental forearm. The water-perfused suit was covered with a water-impermeable rain suit to limit evaporative heat loss during whole-body heating. Whole-body heat stress was initiated by pumping water at 50°C through the water-perfused suit. Whole-body heating continued until participants’ core temperature was raised by 0.8–1.0°C above baseline. Core temperature was maintained at this level until a 5–10 min plateau in skin blood flow for both sites was achieved. Once a plateau in skin blood flow was achieved, 20 mM L-NAME was infused into the control site until skin blood flow decreased and reached a plateau. Once a new plateau was achieved at the control site in response to L-NAME (post-L-NAME drop), participants were cooled by pumping thermoneutral water through the suit, and the plastic rain suit was removed. Maximal skin blood flow was elicited via sodium nitroprusside and local heating to 43°C as described in the previous subsection. Data collection and analysis
Data were digitized and stored at 100 Hz on a personal computer. Data were analysed offline using signal-processing software (Windaq; Data Instruments, Akron, OH, USA). Blood flow measurements were normalized to MAP and expressed as CVC, calculated as the ratio of red blood cell flux to MAP (i.e. red blood cell flux/MAP). Cutaneous vascular conductance values were normalized as a percentage of maximal vasodilatation (%CVCmax ) via sodium nitroprusside infusion and local heating to 43°C. All data were analysed with SPSS 20 (IBM Corporation). All values are presented as means ± SEM, and P < 0.05 was considered to be statistically significant. Resting baseline data were taken as the average over a 3–5 min period immediately prior to initiating whole-body heat stress; the plateau data were taken as the average over 2–3 min immediately prior to infusing L-NAME at the control site; and the end of the protocol data were taken as the average over 1–2 min immediately prior to terminating the experiment when participants’ core temperature had returned to thermoneutral and when maximal vasodilatation had been achieved. To determine whether nitrate supplementation directly augmented skin blood flow, raw skin blood flow data (in arbitrary units) were analysed at baseline and during the plateau of heat stress pre- and postnitrate supplementation, and the percentage increase from baseline was calculated (Keen et al. 2015). Cutaneous vascular conductance data were also analysed for a given increase in core temperature (TC ) at each microdialysis site during the 30–60 s of each 0.1°C increase in core temperature from baseline (TC = 0.0°C) until the end of the heat stress protocol (TC = 0.8°C). The onset of reflex cutaneous C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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vasodilatation was defined as the core temperature at which there was a robust and sustained increase in %CVCmax above baseline. Data were analysed using one-way repeated-measures ANOVA, two-way repeated-measures ANOVA and Student’s paired t tests as appropriate. For all ANOVAs, post hoc Student’s paired t tests were used to determine where significant differences occurred, and Sidak correction for multiple pairwise comparisons was employed as appropriate. The percentage NO contribution was calculated at the control sites as: [(Whole Body Heating (WBH) control plateau−Post-LNAME drop control)/WBH control plateau] × 100 where the post-L-NAME drop is the plateau in CVC following the infusion of L-NAME during hyperthermia. The reduction in CVC in response to the infusion of L-NAME during the plateau was calculated as the difference between the plateau CVC and post-L-NAME plateau and presented as CVC. Results Heart rate and blood pressure data (Table 1) and core temperature responses
Heart rate and blood pressure data for both pre- and postnitrate supplementation are shown in Table 1. There was no effect of nitrate supplementation on resting heart rate (before 59 ± 5 beats min−1 versus after 63 ± 3 beats min−1 ; P = 0.599). At the plateau of whole-body heating, there was a statistically significant increase in HR compared with rest for both pre- (86 ± 7 beats min−1 ; P < 0.001) and postnitrate (93 ± 3 beats min−1 ; P < 0.001) supplementation; however, the increase in HR during heat stress was not statistically significant between pre- and postnitrate supplementation (P = 0.466). There was no difference between the baseline HR and the HR at the end of the protocol for either set of conditions (P > 0.05 for both sets of conditions). There was no effect of nitrate supplementation on core temperature either at baseline (thermoneutral; before 37.18 ± 0.20°C versus after 37.09 ± 0.14°C) or during heat stress (before 38.03 ± 0.15°C versus after 37.95 ± 0.27°C; P = 1.000 for all conditions). Core temperature during heat stress was significantly increased compared with baseline (P < 0.05 for all conditions). Following nitrate supplementation, there was a statistically significant decrease in resting (baseline) systolic blood pressure (before 128 ± 4 mmHg versus after 116 ± 4 mmHg; P < 0.05), but there was no effect of nitrate supplementation on resting diastolic blood pressure (before 68 ± 3 mmHg versus after 66 ± 2 mmHg; P = 0.415) or mean arterial blood pressure (before 88 ± 3 mmHg versus
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Table 1. Heart rate and blood pressure data Before nitrate supplementation Parameter
Rest (baseline) min−1 )
Heart rate (beats Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean arterial pressure (mmHg)
59 ± 5 128 ± 4 68 ± 3 88 ± 3
Plateau 7†
86 ± 131 ± 5 66 ± 2 88 ± 3
End 64 ± 5 122 ± 4 74 ± 2† 93 ± 1
After nitrate supplementation Rest (baseline) 63 ± 3 116 ± 4∗ 66 ± 2 83 ± 2
Plateau 93 106 63 78
3†
± ± 4∗† ± 2 ± 2∗†
End 70 114 63 78
± ± ± ±
5 2 3∗ 4∗
Data are means ± SEM. ‘Plateau’ is the plateau in skin blood flow during heat stress. ‘End’ is the end of the protocol during maximal vasodilatation and when core temperature had returned to baseline. ∗ P < 0.05 versus before nitrate supplementation; † P < 0.05 versus baseline within the same supplement conditions.
after 83 ± 2 mmHg; P = 0.117). At the plateau of wholebody heating, nitrate supplementation resulted in a statistically significant decrease in both systolic blood pressure (before 131 ± 5 mmHg versus after 106 ± 4 mmHg; P = 0.029) and mean arterial blood pressure (before 88 ± 3 mmHg versus after 78 ± 2 mmHg; P < 0.05), but there was no effect on diastolic blood pressure (before 66 ± 2 mmHg versus after 63 ± 2 mmHg; P = 0.112). There was no effect of heat stress or supplement on diastolic arterial pressure or MAP compared with baseline (P > 0.193 for all conditions), but postnitrate supplement the systolic blood pressure during heat stress was reduced compared with baseline (P < 0.05). At the end of the protocol, when the participants’ core temperature returned to preheat-stress levels and while maximal vasodilatation was being elicited, nitrate supplementation resulted in a statistically significant reduction in diastolic blood pressure (before 74 ± 2 mmHg versus after 63 ± 3 mmHg; P = 0.016) and MAP (before 93 ± 1 mmHg versus after 78 ± 4 mmHg; P = 0.007), but there was no effect on systolic blood pressure (before 122 ± 4 mmHg versus after 114 ± 2 mmHg; P = 0.142). The blood pressure data are summarized in Table 1.
Raw skin blood flow data (Table 2)
There was no effect of nitrate supplementation on baseline skin blood flow at control (before 29 ± 5 a.u. versus after 28 ± 3 a.u.; P = 1.000) or L-NAME sites (before 22 ± 3 a.u. versus after 17 ± 1 a.u.; P = 0.644). There was no difference in baseline skin blood flow between control and L-NAME sites prenitrate supplementation (P = 0.178). There was a trend towards a greater baseline skin blood flow at control compared with L-NAME sites postnitrate supplementation, but this did not reach statistical significance (P = 0.06). These data are shown in Table 2. Similar to baseline skin blood flow, there was no effect of nitrate supplementation on skin blood flow during the plateau of heat stress at control (before 89 ± 5 a.u. versus after 87 ± 7 a.u.; P = 1.000) or L-NAME sites (before 63 ± 9 a.u. versus after 51 ± 6 a.u.; P = 1.000). The
L-NAME attenuated plateau skin blood flow compared with control sites for both pre- (P < 0.05) and postnitrate supplementation (P = 0.007). Plateau skin blood flow data are shown in Table 2. There was no effect of nitrate supplementation on the percentage increase in skin blood flow from baseline at control (before 67 ± 6% versus after 68 ± 2%; P = 0.751) or L-NAME sites (before 59 ± 9% versus after 65 ± 4%; P = 0.477). There was no statistical difference in the percentage increase from baseline between control and L-NAME sites for either set of conditions (P = 0.788 for both sets of conditions). These data are shown in Table 2.
Cutaneous vascular conductance data
There was no effect of nitrate supplementation on baseline CVC at either the control (before 17 ± 2%CVCmax versus after 21 ± 2%CVCmax ; P = 1.000) or L-NAME sites (before 14 ± 2%CVCmax versus after 13 ± 1%CVCmax ; P = 1.000). Baseline CVC did not differ between control or L-NAME sites for either pre- or postnitrate supplementation conditions (P > 0.111 for all conditions). There was an increase in the plateau phase during whole-body heating following nitrate supplementation; however, the magnitude of the post-L-NAME drop was not different between pre- and postnitrate supplementation periods. As the magnitude of the post-L-NAME drop was similar between conditions, there was no difference in the calculated percentage NOS-dependent vasodilatation (see CVC data below). Figure 1 shows the CVC response to whole-body heating (plateau) at control and L-NAME sites for pre- and postnitrate supplementation periods. The plateau CVC at the control site was significantly greater after 3 days of nitrate supplementation (80 ± 5%CVCmax ) compared with the control site before nitrate supplementation (67 ± 2%CVCmax ; P < 0.01). At L-NAME-treated sites, there was no statistical difference between plateau CVC prenitrate (45 ± 4%CVCmax ) and postnitrate supplementation (40 ± 5%CVCmax ; P = 0.617). Plateau CVC at L-NAME sites for both pre- and postnitrate conditions C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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Table 2. Raw skin blood flow data Site and conditions Control site Before nitrate supplementation After nitrate supplementation L-NAME-treated site Before nitrate supplementation After nitrate supplementation
Baseline
Plateau
Percentage increase from baseline
29 ± 5 28 ± 3
89 ± 5 87 ± 7
67 ± 6 68 ± 2
22 ± 3 17 ± 1
63 ± 9∗ 51 ± 6∗
59 ± 9 65 ± 4
Data are means ± SEM. Raw skin blood flow units are arbitrary units. ∗ P < 0.05 versus control within same conditions.
Cutaneous Vascular Conductance (% Maximal)
were significantly attenuated compared with plateau CVC at control sites for both pre- and postnitrate conditions (P < 0.02 for all conditions). Figure 2 shows the CVC responses as a function of core temperature. The onset of cutaneous vasodilatation was not affected by nitrate supplementation at either control (TC = 0.3°C) or L-NAME sites (TC = 0.4°C). The individual whole-body heating plateau responses are shown in Fig. 3A (control) and B (L-NAME). The CVC responses to L-NAME infusion (i.e. reduction in CVC) during the plateau of heat stress were not statistically significant between pre(43 ± 3%CVCmax ) and postnitrate (51 ± 2%CVCmax ; P = 0.125). Maximal CVC values at control sites were not different from pre- (1.98 ± 0.26) mV/mmHg to postnitrate supplementation (1.90 ± 0.20; P = 0.523) mV/mmHg. Likewise, maximal CVC at L-NAME sites was not affected by nitrate supplementation (before 1.92 ± 0.30 versus after 1.95 ± 0.33; P = 1.000) mV/mmHg. There was no
difference in maximal CVC between control and L-NAME sites for any conditions. Nitric oxide synthase-dependent vasodilatation
Figure 4 shows the group mean data for the calculated contribution of NOS-dependent vasodilatation to reflex cutaneous vasodilatation at control sites for preand postnitrate supplementation conditions. There was no statistical difference in the percentage of NOSdependent vasodilatation between prenitrate (59 ± 4% NOS-dependent vasodilatation) and postnitrate conditions (64 ± 6%NOS-dependent vasodilatation; P = 0.577). Discussion The primary finding of this investigation suggests that nitrate supplementation augments reflex cutaneous vasodilatation. Physiologically, the augmented CVC
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Figure 1. Cutaneous vascular conductance responses to whole-body heat stress Data are shown for control and l-NAME microdialysis sites for both pre- and postnitrate supplementation. Cutaneous vascular conductance was augmented following nitrate supplementation at the control sites only. Values are shown as means + SEM. ∗ P < 0.05 versus prenitrate. †P < 0.05 versus control within same conditions.
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appeared to be due to increased vasodilatation of the cutaneous microvasculature, while the calculated (mathematical) increase in CVC was driven largely by the decrease in MAP, with no change in skin blood flow per se. We also observed a significant augmentation of the plateau phase of reflex cutaneous vasodilatation at the control but not the L-NAME-treated site after 3 days of nitrate supplementation. Collectively, these findings suggest that short-term dietary nitrate supplementation may improve the CVC response to whole-body heat stress in young, healthy participants. The increase in CVC was most probably driven by increased cutaneous vasodilatation. Based on the hydraulic resistance equation, it appears that after nitrate supplementation there was significant vasodilatation (decreased vascular resistance) of the cutaneous vasculature despite decreases in MAP and no change in skin blood flow. The calculated (mathematical) increase in CVC during heat stress after nitrate supplementation can be explained by the reduction in MAP during heat stress after nitrate supplementation. As shown in Table 2, there was no significant increase in raw skin blood flow at either baseline or during heat stress from pre- to postnitrate supplementation, suggesting that the increase in calculated CVC was driven largely by the reduction in MAP. Our conclusion that CVC was augmented owing to NOS-independent mechanisms is based on our data showing an increase in the skin blood flow plateau during heat stress at control sites yet no change in the calculated
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percentage NOS-dependent vasodilatation. In order to determine the percentage NOS-dependent vasodilatation, we infused the non-selective NOS inhibitor, L-NAME, through the microdialysis fibres at the control site once skin blood flow had reached a plateau during heat stress. The difference between the initial plateau during heat stress and the plateau following L-NAME infusion (post-L-NAME drop) yields the calculated percentage NOS-dependent vasodilatation. Despite an augmented plateau at control sites following nitrate supplementation, there was no significant increase in the calculated percentage NOS-dependent vasodilatation owing to a similar magnitude of reduction following L-NAME infusion. It would thus appear that the augmented CVC during heat stress is associated with NOS-independent mechanisms. While our data suggest no effect of nitrate supplementation on NOS-dependent vasodilatation, we cannot rule out the possibility that the increase in reflex cutaneous vasodilatation was NO dependent. Inasmuch as nitrate is eventually reduced to NO independent of NOS activity (McKnight et al. 1997; Lundberg et al. 2008; Lundberg & Weitzberg, 2009), it is possible that the enhanced cutaneous vascular response to heat stress was due to an increase in NO that was independent of functional NOS activity and would thus not be affected by NOS inhibition. Our present finding that nitrate supplementation does not augment vascular conductance at the L-NAME site is in contrast to what we have recently observed during local heating of the skin (Keen et al. 2015). During local
Cutaneous Vascular Conductance (% Maximal)
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Control Pre-BRJ
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L-NAME Post-BRJ
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20 † 0 0
0.1
0.2
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ΔTc (°C) Figure 2. Cutaneous vascular conductance as a function of core temperature (Tc ) Cutaneous vascular conductance data are presented as a function of increasing core temperature in 0.1°C increments. Abbreviation: BRJ, beetroot juice. Data are means ± SEM. ∗ P < 0.05 versus pre-BRJ control site. † P < 0.05 versus pre- and post-BRJ control site.
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heating of the skin, we found that nitrate supplementation augmented the plateau phase at both control and L-NAME sites. Yet, similar to the present data, there was no increase in skin blood flow from pre- to postnitrate supplementation, which suggests that the majority of the increase in vascular conductance in response to local heating of the skin was driven by a decrease in MAP. It is possible that high levels of skin blood flow are a prerequisite for enhanced NO-dependent vasodilatation after nitrate supplementation. That is, elevated shear stress may be required to increase NO production after nitrate supplementation, and the attenuated skin blood flow with
continuous L-NAME infusion may prevent this requisite level of shear stress. While this possible explanation is intriguing, the importance of shear stress-induced NO production in the cutaneous vasculature is unclear (Wong et al. 2003; Zhao et al. 2004). Nevertheless, it appears that short-term dietary nitrate supplementation augments NOS-independent increases in CVC during both whole-body heating and local heating of the skin in young, healthy humans, and these increases in CVC are largely due to reduced MAP. We also observed significant reductions in both systolic arterial blood pressure and MAP after 3 days
A
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Control Post-Nitrate
B
Cutaneous Vascular Conductance (% Maximal)
100
80
60
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0 L-NAME Pre-Nitrate
L-NAME Post-Nitrate
Figure 3. Individual and group cutaneous vascular conductance responses pre- and postnitrate supplementation Individual and group responses for the control sites (A) and L-NAME sites (B). Individual responses are depicted as thin dashed lines; group responses are shown as the thick black line.
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of nitrate supplementation at baseline and during heat stress. Although our participants were normotensive, reductions in both systolic arterial blood pressure and MAP are consistent with previous studies after nitrate supplementation (Larsen et al. 2006; Webb et al. 2008; Kapil et al. 2010; Vanhatalo et al. 2010). Although we found no changes in baseline (thermoneutral) MAP between conditions as in our previous study (Keen et al. 2015), there was a trend towards a decrease in baseline (thermoneutral) MAP postnitrate supplementation that may not have reached statistical significance owing to greater variability in the present study. It has been suggested that nitrate supplementation reduces blood pressure owing to an increase in NO production (Lundberg et al. 2008, 2011; Webb et al. 2008; Lundberg & Weitzberg, 2009; Kapil et al. 2010). The increase in NO production via reduction of nitrate to nitrite and, eventually, to NO is independent of NOS and thus would not be in conflict with our CVC conclusions. Our data showing no effect of nitrate supplementation on heart rate responses is consistent with previous literature in both humans and animals and suggests that nitrate supplementation has minimal direct effect on the heart (Webb et al. 2008; Ferguson et al. 2013). Limitations
A potential limitation to this study is that we did not quantify serum levels of plasma nitrate and nitrite from pre- to postnitrate supplementation. Measuring changes in plasma nitrate and nitrite levels would have allowed us to determine whether changes in blood pressure were correlated with peak nitrite levels preto postsupplement intervention as previously discussed and presented in current mechanistic studies with nitrate
Percentage NOS-dependent vasodilatation
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supplementation (Webb et al. 2008). Although indirect, our data demonstrating a reduction in both systolic arterial blood pressure and MAP would appear to confirm that 3 days of nitrate supplementation was adequate in increasing plasma nitrate and nitrite concentrations. Significance
Net heat transfer from core to skin is a function of skin blood flow. In young, healthy participants, there was no significant increase in skin blood flow per se after nitrate supplementation, suggesting no improved thermoregulatory benefit. Despite our finding that skin blood flow did not change, it is possible that in populations with a diminished skin blood flow response to heat stress, such as an ageing population, nitrate supplementation may directly improve skin blood flow and thus improve the thermoregulatory response to heat stress. Conclusions
To our knowledge, this is the first study to identify a role for dietary nitrate supplementation via beetroot juice in the skin blood flow response to whole-body heat stress. Our data suggest that dietary nitrate supplementation augmented reflex cutaneous vasodilatation in young, healthy subjects by the following mechanisms: (i) vasodilatation of the cutaneous vasculature despite decreases in MAP during heat stress; and (ii) NOS-independent vasodilatation, as evidenced by an increase in CVC from pre- to postnitrate supplementation in the plateau phase at control sites, with no change in the calculated percentage of NOS-dependent vasodilatation. References
80 60 40 20 0 Pre-Nitrate
Post-Nitrate
Figure 4. Calculated percentage of nitric oxide synthase (NOS)-dependent vasodilatation pre- and postnitrate supplementation There was no difference in the calculated percentage of NOS-dependent vasodilatation between pre- and postnitrate supplementation. Values are shown as means + SEM.
Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, Benjamin N & Jones AM (2009). Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol 107, 1144–1155. Bruning RS, Santhanam L, Stanhewicz AE, Smith CJ, Berkowitz DE, Kenney WL & Holowatz LA (2012). Endothelial nitric oxide synthase mediates cutaneous vasodilation during local heating and is attenuated in middle-aged human skin. J Appl Physiol 112, 2019–2026. Brunt VE, Fujii N & Minson CT (2013). No independent, but an interactive, role of calcium-activated potassium channels in human cutaneous active vasodilation. J Appl Physiol 115, 1290–1296. Casey DP, Treichler DP, Ganger CT 4th, Schneider AC & Ueda K (2015). Acute dietary nitrate supplementation enhances compensatory vasodilation during hypoxic exercise in older adults. J Appl Physiol 118, 176–186. C 2015 The Authors. Experimental Physiology C 2015 The Physiological Society
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Additional information Competing interests None declared. Author contributions This study was completed in the Department of Kinesiology at Kansas State University in Manhattan, KS, USA. This study was completed by E.L.L. in part fulfilment of the degree of MSc in the Department of Kinesiology at Kansas State University. E.L.L. was responsible for experimental design, recruitment of participants, data collection, analysis and interpretation, and drafting and editing all versions of the manuscript. J.T.K. was responsible for data collection and interpretation, and editing drafts of the manuscript. B.J.W. was responsible for experimental design, data analysis and interpretation, and drafting and editing all versions of the manuscript. Funding None declared. Acknowledgements The authors are grateful to all the participants for their time and willingness to participate in this study. The authors would also like to thank Ms Jena K. Eder for her assistance with data collection.
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