NO-mediated activation of KATP channels contributes to cutaneous thermal hyperemia in young adults
Abstract
Local skin heating to 42°C causes cutaneous thermal hyperemia largely via nitric oxide (NO) synthase (NOS)-related mechanisms. We assessed the hypothesis that ATP-sensitive K+ (KATP) channels interact with NOS to mediate cutaneous thermal hyperemia. In 13 young adults (6 women, 7 men), cutaneous vascular conductance (CVC) was measured at four intradermal microdialysis sites that were continuously perfused with 1) lactated Ringer solution (control), 2) 5 mM glibenclamide (KATP channel blocker), 3) 20 mM NG-nitro-l-arginine methyl ester (NOS inhibitor), or 4) a combination of KATP channel blocker and NOS inhibitor. Local skin heating to 42°C was administered at all four treatment sites to elicit cutaneous thermal hyperemia. Thirty minutes after the local heating, 1.25 mM pinacidil (KATP channel opener) and subsequently 25 mM sodium nitroprusside (NO donor) were administered to three of the four sites (each 25–30 min). The local heating-induced prolonged elevation in CVC was attenuated by glibenclamide (19%), but the transient initial peak was not. However, glibenclamide had no effect on the prolonged elevation in CVC in the presence of NOS inhibition. Pinacidil caused an elevation in CVC, but this response was abolished at the glibenclamide-treated skin site, demonstrating its effectiveness as a KATP channel blocker. The pinacidil-induced increase in CVC was unaffected by NOS inhibition, whereas the increase in CVC elicited by sodium nitroprusside was partly (15%) inhibited by glibenclamide. In summary, we showed an interactive effect of KATP channels and NOS for the plateau of cutaneous thermal hyperemia. This interplay may reflect a vascular smooth muscle cell KATP channel activation by NO.
INTRODUCTION
Local skin heating to 42°C causes an increase in cutaneous perfusion that is commonly referred to as cutaneous thermal hyperemia. The response to rapid, nonpainful local heating is characterized by a transient initial peak, followed by a brief nadir and then a sustained plateau phase (22, 31, 33). This local heat-induced vasodilator response has been widely employed 1) as a highly sensitive test of both neurovascular (i.e., initial peak) and endothelium-dependent (i.e., sustained plateau) cutaneous microvascular dysfunction and 2) to assess therapies aimed at improving microvascular reactivity (1, 5, 14, 36, 37). The transient initial peak is primarily initiated by a local sensory nerve-mediated axon reflex that is partly associated with the release of nitric oxide (NO) during this early phase (31). The sustained plateau is mostly mediated by the nitric oxide synthase (NOS) pathway (13, 14, 27, 39, 41) but also involves other mediators such as transient receptor potential vanilloid type 1 (TRPV1) channels (39), adenosine receptors (13), xanthine oxidase (29), H2O2 (29), and K+ channels (6, 15). However, despite our growing knowledge of the mechanisms underpinning cutaneous thermal hyperemia, significant gaps in our understanding of this response still exist.
With respect to the influence of K+ channels, Ca2+-activated K+ (KCa) channels, which mainly elicit vasodilation through endothelium-dependent hyperpolarization, have been shown to modulate cutaneous thermal hyperemia during both the initial peak and sustained plateau phases (6). Conversely, other K+ channels such as voltage-gated (KV) and ATP-sensitive (KATP) K+ channels do not appear to cause endothelium-dependent hyperpolarization (18). However, we recently reported that KV channels also contribute to both the initial peak and sustained plateau phases in association with NOS (15). It remains unclear whether KATP channels contribute to cutaneous thermal hyperemia. KATP channels appear to exist on vascular smooth muscle (21) and may also exist on the endothelium (3). This is supported by previous data showing that KATP channels have been implicated in both endothelium-dependent and endothelium-independent cutaneous vasodilation induced by local exogenous acetylcholine and sodium nitroprusside administration, respectively, in human skin (20). Furthermore, in rat sensory neurons, KATP channels play an important role in mediating membrane excitability and neurotransmitter release (23). Taken together, these data support the possibility that KATP channels may contribute directly to the local cutaneous thermal hyperemia response, a response that must be confirmed.
A contribution of KATP channels to the regulation of cutaneous thermal hyperemia, if any, would likely be associated with the activation of NOS. NOS has been implicated in the modulation of both the transient initial peak (31) and the sustained secondary plateau phases (13, 14, 27, 39, 41) of this response. Two possibilities exist in the context of a potential interplay between KATP channels and NOS in the regulation of cutaneous thermal hyperemia. If NOS is initially activated, this would elicit increases in NO that would subsequently activate KATP channels located on vascular smooth muscle cells, inducing a vasodilatory response. In line with this, in rat sensory neurons administration of S-nitroso-N-acetylpenicillamine, a NO donor, has been shown to directly activate KATP channels (23). Furthermore, a previous work by Hojs et al. (20) showed that cutaneous vasodilation induced by the exogenous NO donor sodium nitroprusside was partly attenuated by the KATP channel blocker glibenclamide in human skin. Alternatively, if endothelial KATP channels are initially activated, this would cause endothelial hyperpolarization, which facilitates an increase in the driving force for Ca2+ entry (7). Elevations in endothelial Ca2+ would in turn activate NOS (8), thereby increasing NO and causing vasodilation. In the context of the latter possibility, a vasodilation elicited by KATP channel openers such as pinacidil should be attenuated by the inhibition of NOS, a response confirmed in a nonhuman study (25).
Thus the purpose of the present study was to elucidate a possible role for KATP channels in the regulation of the initial peak and plateau phases of cutaneous thermal hyperemia during local heating to 42°C. We evaluated the hypothesis that KATP channels may in part mediate the initial peak and plateau phases of cutaneous thermal hyperemia in humans in vivo through NOS-dependent mechanisms. Furthermore, to delineate the possible mechanism(s) underlying the interplay between KATP channels and NO in the regulation of cutaneous thermal hyperemia, we conducted secondary tests in which the NO donor sodium nitroprusside and the KATP channel opener pinacidil were employed. We hypothesized that a NO-mediated cutaneous vasodilation, as induced by delivery of the NO donor sodium nitroprusside, would be partly mediated by KATP channel-dependent mechanisms. In contrast, KATP channel opener pinacidil-mediated cutaneous vasodilation would be unaffected by NOS. Consequently, we would expect that activation of NOS would increase NO, which would subsequently activate KATP channels on vascular smooth muscle cells, thereby mediating cutaneous thermal hyperemia.
MATERIALS AND METHODS
Ethical approval.
The present study was approved by the Human Subjects Committee of the University of Tsukuba (no. 29-24), in agreement with the Declaration of Helsinki. Written informed consent was obtained from all participants before their involvement in the present study.
Participants.
Thirteen healthy young adults (6 women, 7 men) participated in this study. Their mean (±SD) body mass, height, body mass index, age, and mean arterial pressure were 62.5 ± 13.7 kg, 1.67 ± 0.09 m, 22.1 ± 3.4 kg/m2, 26 ± 3 yr, and 80 ± 11 mmHg (104 ± 14 and 68 ± 11 mmHg for systolic and diastolic blood pressures, respectively), respectively. They were free of cystic fibrosis transmembrane conductance regulator (CFTR) mutations, skin disorders, hypertension, heart disease, diabetes, autonomic disorders, cigarette smoking, and prescription medications. All women were tested during their self-reported early follicular phase (≤6 days from the beginning of menstruation). None of the women was on contraceptives.
Microdialysis and antagonist preparation.
Before the experimental trial, all participants were told to abstain from 1) over-the-counter medications including nonsteroidal anti-inflammatory drugs and nutritional supplements (e.g., vitamins and minerals) >48 h before, 2) alcohol and caffeine consumption >12 h before, and 3) intense exercise the night before. On the day of the experimental session, participants fasted 2 h before and throughout the experiment. Upon arrival, they voided their bladder, after which body mass was measured with a weight scale platform. Participants were then placed on a semirecumbent bed where they rested in a room regulated to ~25°C. Once in position the microdialysis probes were inserted intradermally. First, a 25-gauge needle (Terumo, Tokyo, Japan) was aseptically inserted into the dermal layer of the unanesthetized left dorsal forearm skin. The needle was then advanced 2–2.5 cm and exited the skin. A microdialysis fiber made in house was threaded through the lumen of the needle, and the needle was then withdrawn, leaving the 10-mm regenerated cellulose membrane of the microdialysis fiber [0.22 mm outer diameter (OD), 0.20 mm inner diameter (ID)] within the skin. The cutoff of this membrane in the presence of continuous fluid perfusion is ~1,000 Da. Each end of the 10-mm membrane was connected with a polyimide tube (0.165 mm OD, 0.127 mm ID). A total of four microdialysis fibers were placed, each separated by >2 cm to avoid potential between-site contamination from the different pharmacological agents administrated at the respective sites.
Approximately 5 min after placement of the four fibers, infusion of the pharmacological agents was commenced at all four skin sites. Sites were treated with 1) lactated Ringer solution (Fuso Pharmaceutical Industries, Osaka, Japan), serving as a vehicle control site; 2) 5 mM glibenclamide (mol wt: 494.00; Sigma-Aldrich, St. Louis, MO), a KATP channel blocker [also known to inhibit CFTR and activate transient receptor potential ankyrin type 1 (TRPA1)]; 3) 20 mM NG-nitro-l-arginine methyl ester (mol wt: 269.69; Nacalai Tesque, Kyoto, Japan), a nonselective NOS inhibitor; or 4) a combination of KATP channel blocker and NOS inhibitor. The concentrations of drugs used in the present study were determined on the basis of previous work using intradermal microdialysis [NG-nitro-l-arginine methyl ester (28, 34, 40); glibenclamide (16, 24, 26)]. Glibenclamide is water insoluble; thus a combination of 5% dimethyl sulfoxide (Sigma-Aldrich) and high pH (~11) was used to maximally increase solubility of this drug. The pH was adjusted with sodium hydroxide (Sigma-Aldrich). We previously reported that a combination of 5% dimethyl sulfoxide and pH of ~11 had no effect on cutaneous vascular responses (16). However, all site-specific drugs were dissolved in lactated Ringer solution with 5% dimethyl sulfoxide and pH of ~11 to offset any potential influence of this combination on cutaneous vascular responses. All drugs were continuously perfused at a rate of 4.0 µL/min with a microinfusion pump (BASi Bee Hive controller and Baby Bee syringe drive; Bioanalytical Systems, West Lafayette, IN) throughout the experiment to ensure continuous blockade of KATP channels and/or inhibition of NOS. At least 60 min after the initiation of drug administration, a 10-min baseline measurement period was commenced. This time lag (≥60 min) has been shown to be of sufficient duration to allow the occurrence of hyperemia caused by the insertion of fibers to subside (2).
Local heating and agonist tests.
After a minimum 60-min period of KATP channel blocker and/or NOS inhibitor administration, a 10-min baseline was initiated during which time local skin temperature was fixed at 33°C as regulated by a local heater (PF 5020; Perimed, Stockholm, Sweden). Thereafter, local skin temperature was increased to 42°C at a rate of 0.1°C/s at all four skin sites to induce cutaneous thermal hyperemia. Once cutaneous blood flow reached a plateau, which typically occurred 30–35 min after initiation of heating, skin temperature was reduced to 33°C to allow cutaneous blood flow to return back to preheating baseline levels (~30 min). This skin temperature (33°C) was maintained throughout the remainder of the experiment until the protocol to induce maximal cutaneous vasodilation (i.e., increasing local skin temperature to 44°C in combination with administration of sodium nitroprusside) was initiated (see below). KATP channel blocker and/or NOS inhibitor administration was continued during this recovery period as well as after the second baseline measurement period of 10-min duration. Thereafter, a 30-min administration of 1.25 mM pinacidil (mol wt: 263.34; Santa Cruz Biotechnology, Dallas, TX), a KATP channel opener, was initiated (n = 11; 4 women, 7 men) at three skin sites treated with 1) lactated Ringer solution, 2) 5 mM glibenclamide, or 3) 20 mM NG-nitro-l-arginine methyl ester. Pinacidil was dissolved in each site-specific solution with 5% dimethyl sulfoxide plus pH of ~11. Our pilot work demonstrated that 5 mM diazoxide (mol wt: 230.7; Cayman Chemical, Ann Arbor, MI), another KATP channel opener, did not increase cutaneous blood flow; thus pinacidil was employed in the present study. After the 30-min pinacidil administration, 25 mM sodium nitroprusside (mol wt: 297.95; Sigma-Aldrich), a NO donor, was administered (n = 9; 4 women, 5 men) to the same three skin sites for 25–30 min. Sodium nitroprusside was dissolved in each site-specific solution with 5% dimethyl sulfoxide plus pH of ~11. Upon completion of sodium nitroprusside administration, local skin temperature was increased to 44°C at a rate of 0.1°C/s with the simultaneous administration of 50 mM sodium nitroprusside, which was maintained at a rate of 4.0 µL/min. This combined application continued for 20–30 min until maximum cutaneous vasodilation occurred.
Supplementary experimental trial.
As mentioned above, it is possible that glibenclamide can inhibit CFTR and activate TRPA1 channels. To obtain some insights into whether CFTR and TRPA1 channels modulate cutaneous vascular tone in humans in vivo, we conducted a supplementary experimental trial in which 10 healthy young adults (4 women, 6 men) were recruited. Their body mass, height, body mass index, age, and mean arterial pressure were 67.2 ± 8.5 kg, 1.70 ± 0.08 m, 23.1 ± 2.1 kg/m2, 25 ± 3 yr, and 74 ± 6 mmHg (99 ± 10 and 62 ± 5 mmHg for systolic and diastolic blood pressures, respectively), respectively. As with the main experimental session, all participants were free of CFTR mutations, skin disorders, hypertension, heart disease, diabetes, autonomic disorders, cigarette smoking, and prescription medications. All women were tested during their self-reported early follicular phase. In this session, one microdialysis fiber was inserted into the forearm. It was subsequently perfused with lactated Ringer solution at a rate of 4.0 µL/min with a microinfusion pump. After a minimum 60-min recovery period, a 10-min baseline measurement period was commenced. Then 5 mM 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO, mol wt: 231.08; Tocris, Ellisville, MO), a CFTR agonist, was administered through the fiber for 30 min. To maximize the solubility of DCEBIO (5 mM), and thus activation of CFTR, a combination of 5% dimethyl sulfoxide and high pH (~11) was used as a solvent. After completion of the 30-min DCEBIO administration, 98% cinnamaldehyde (mol wt: 132.16; Wako Pure Chemical Industries, Osaka, Japan), a TRPA1 activator, was administered for 30 min. Thereafter, maximum cutaneous vasodilation was elicited by the administration of 50 mM sodium nitroprusside in combination with local heating to 44°C as performed in the main experimental session. Because of technical difficulties, cutaneous vascular conductance was not obtained in one and two participants during DCEBIO and cinnamaldehyde administration, respectively.
Measurements.
Laser-Doppler flowmetry (PeriFlux System 5000; Perimed, Stockholm, Sweden) was employed to provide an index of local cutaneous blood flow as assessed by cutaneous red blood cell flux (expressed in perfusion units) with a sampling rate of 32 Hz. The integrated laser-Doppler flowmetry probes (PeriFlux probe 413) were housed in the center of local heaters (PeriFlux Heater PF 450). Systolic and diastolic arterial pressures were recorded at 5-min intervals with an automated sphygmomanometer (TM-2580; A&D, Tokyo, Japan) placed on the right arm. Mean arterial pressure was estimated as diastolic pressure plus one-third of the difference between systolic and diastolic arterial blood pressures. To minimize the influence of changes in perfusion pressure, cutaneous vascular conductance was evaluated as cutaneous red blood cell flux divided by mean arterial pressure.
Data analysis.
Cutaneous vascular conductance was presented as a percentage of the maximum conductance as assessed with the combination of 44°C local skin heating and the administration of 50 mM sodium nitroprusside (%max). Cutaneous vascular conductances for baseline, the plateau of cutaneous thermal hyperemia, each agonist administration (1.25 mM pinacidil, 25 mM sodium nitroprusside, 5 mM DCEBIO, 98% cinnamaldehyde), and maximum vasodilation were determined by averaging values over the last 5 min of each period. The cutaneous vascular conductances at the initial peak and nadir during local heating (Fig. 1) were determined by averaging the cutaneous vascular conductance over 30 s. We elected to use a brief period of 30 s for the initial peak and nadir responses because they were relatively transient (Fig. 1).
Statistical analysis.
The minimal sample size was calculated on the basis of previous data evaluating cutaneous thermal hyperemia (14). We determined that for 80% power and α set to 0.05, a minimum of eight participants would be required. Statistical software package SPSS 25 for Windows (IBM, Armonk, NY) was used for all statistical analyses. Cutaneous vascular conductance (%max) was analyzed with a two-way repeated-measures analysis of variance with the factors of treatment site and stage. A one-way repeated-measures analysis of variance was used for absolute baseline and maximum (perfusion units/mmHg) cutaneous vascular conductance with a factor of treatment site. For the supplementary experimental session, a one-way repeated-measures analysis of variance was also used for cutaneous vascular conductance (%max) with a factor of phase. When a significant interaction or main effect was detected, post hoc comparisons were performed with a modified Bonferroni correction, the Holm procedure (19). The level of significance for all analyses was set at 0.05. All values are reported as means ± 95% confidence interval unless otherwise indicated.
RESULTS
We did not observe any significant sex-related differences in any of the responses (data not shown).
We observed a similar pattern of response in cutaneous perfusion at all skin sites during local heating (i.e., 33–42°C), defined as a transient initial peak followed by a brief nadir and a subsequent plateau phase of cutaneous thermal hyperemia (Fig. 1). Main effects of treatment site and stage and their interaction were all significant (all P < 0.01). Baseline, initial peak, and nadir cutaneous vascular conductance were unaffected by KATP channel blockade relative to the control site (Fig. 2, A–C). However, they were reduced both with NOS inhibition alone and with combined NOS inhibition and KATP channel blockade. Cutaneous vascular conductance measured at baseline, initial peak, and nadir did not differ between the NOS inhibition and combined KATP channel blockade and NOS inhibition sites (Fig. 2, A–C). The sustained plateau in cutaneous vascular conductance was reduced from the control site (75 ± 6%max) with KATP channel blockade (61 ± 10%max), with NOS inhibition (28 ± 5%max), and with a combination of both (27 ± 7%max) (all P < 0.02, Fig. 2D). Based on the above data, the KATP channel component of the plateau response, as assessed by the difference between the control and KATP channel blockade sites, was 14 ± 9%max. The plateau response did not differ between skin sites treated with NOS inhibition alone and a combination of KATP channel blockade and NOS inhibition (Fig. 2D).
Fig. 1.A representative cutaneous thermal hyperemia response in 1 participant. Local heating was initiated at time 0. The response was obtained at 4 skin sites treated with lactated Ringer solution (control), ATP-sensitive K+ (KATP) channel blockade, nitric oxide synthase (NOS) inhibition, or combined KATP channel blockade + NOS inhibition (combination).
Fig. 2.Averaged values for each phase of the cutaneous thermal hyperemia response. Cutaneous vascular conductances at baseline (A) and during cutaneous thermal hyperemia characterized by the initial peak (B), nadir (C), and sustained plateau (D) phases are presented. Responses were assessed at 4 skin sites treated with lactated Ringer solution (control), ATP-sensitive K+ (KATP) channel blockade, nitric oxide synthase (NOS) inhibition, or combined KATP channel blockade + NOS inhibition (combination). Values are expressed as means ± 95% confidence interval (n = 13 for A–D).
Main effects of treatment site and stage and their interaction on cutaneous vascular conductance obtained during the agonist tests were all significant (all P < 0.01; Figs. 3 and 4). Cutaneous vascular conductance during the second baseline period following the local-heating protocol was reduced at the NOS inhibition skin site (9 ± 2%max) relative to the control site (25 ± 6%max), with no influence of KATP channel blockade (19 ± 5%max) (Fig. 3, Fig. 4A). Administration of the KATP channel opener pinacidil similarly increased cutaneous vascular conductance at the control (53 ± 10%max) and NOS inhibition (46 ± 10%max) sites, but this response was not observed at the KATP channel blockade site (16 ± 4%max) (Fig. 4B). The pinacidil-mediated increase in cutaneous vascular conductance from baseline was 28 ± 7%max at the control site. The elevation in cutaneous vascular conductance in response to the administration of the NO donor was attenuated by KATP channel blockade only (69 ± 3%max, P = 0.03), with no effect of NOS inhibition (78 ± 4%max, P = 0.29), relative to the control site (81 ± 7%max) (Fig. 4C).
Fig. 3.Representative cutaneous vascular responses to pinacidil [ATP-sensitive K+ (KATP) channel opener] and sodium nitroprusside [nitric oxide (NO) donor] in 1 participant. Cutaneous vascular conductance in response to the KATP channel opener (infusion started at time 0) and the subsequent NO donor was assessed at 3 skin sites treated with lactated Ringer solution (control), KATP channel blockade, or nitric oxide synthase (NOS) inhibition.
Fig. 4.Averaged cutaneous vascular responses to pinacidil [ATP-sensitive K+ (KATP) channel opener] and sodium nitroprusside [nitric oxide (NO) donor]. Cutaneous vascular conductances at baseline (A) and during administration of the KATP channel opener (B) and the NO donor (C) were assessed at 3 skin sites treated with lactated Ringer solution (control), KATP channel blockade, or nitric oxide synthase (NOS) inhibition. Values are expressed as means ± 95% confidence interval (n = 11 for baseline and KATP channel opener; n = 9 for NO donor).
There were no between-site differences in absolute baseline and maximum cutaneous vascular conductance (Table 1).
In the supplementary experiment, although CFTR activation had no effect on cutaneous vascular conductance, activation of TRPA1 channels substantially increased cutaneous vascular conductance (Figs. 5 and 6).
Fig. 5.Representative cutaneous vascular responses to 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one [DCEBIO, cystic fibrosis transmembrane conductance regulator (CFTR) activator] and cinnamaldehyde [transient receptor potential ankyrin type 1(TRPA1) channel activator] in 1 participant. Cutaneous vascular conductance in response to the CFTR activator (infusion started at time 0) and the TRPA1 channel activator was assessed at 1 skin site treated with lactated Ringer solution.
Fig. 6.Averaged cutaneous vascular responses to 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one [DCEBIO, cystic fibrosis transmembrane conductance regulator (CFTR) activator] and cinnamaldehyde [transient receptor potential ankyrin type 1(TRPA1) channel activator]. Cutaneous vascular conductance at baseline (n = 10) and during administration of the CFTR activator DCEBIO (n = 9) and the TRPA1 channel activator cinnamaldehyde (n = 8) was assessed at 1 site treated with lactated Ringer solution. Values are expressed as means ± 95% confidence interval.
DISCUSSION
We assessed the role of KATP channels in the regulation of cutaneous thermal hyperemia and whether KATP channels interact with NOS in mediating this response. We showed that application of the KATP channel blocker glibenclamide did not influence the initial vasodilatory peak, although it did attenuate the sustained plateau. Conversely, the NOS inhibitor NG-nitro-l-arginine methyl ester attenuated both the initial peak and plateau phases of the cutaneous thermal hyperemia response. Importantly, blockade of KATP channels did not affect the plateau response in the presence of NOS inhibition, as evidenced by the lack of a difference in cutaneous vascular conductance between NOS inhibition alone and the combined administration of the KATP channel blocker and NOS inhibitor. These results suggest that KATP channels and NOS interact in mediating the plateau response of cutaneous thermal hyperemia in young adults. Furthermore, our agonist experiments demonstrated that the KATP channel opener pinacidil induced cutaneous vasodilation, which was unaffected by NOS inhibition. In contrast, NO donor-induced cutaneous vasodilation via sodium nitroprusside was attenuated by KATP channel blockade. Thus, in support of our hypothesis during local heating, NOS appears to be initially activated, increasing NO production that subsequently activates KATP channels located on vascular smooth muscle cells, partly mediating the plateau response.
Cutaneous thermal hyperemia.
We showed that KATP channels had no measurable influences on the initial peak and nadir responses (Fig. 2, B and C). In light of the fact that the initial peak response is largely mediated through a local sensory nerve-mediated axon reflex (31), our results are in contrast to findings in rodent sensory neurons, where NO has been shown to directly activate KATP channels (23). Intuitively, an effect of KATP channel blockade would be expected given that NOS was involved in all three phases of cutaneous thermal hyperemia (Fig. 2, B–D) and NO can activate KATP channels, thereby inducing cutaneous vasodilation (Fig. 4C). It is plausible, however, that there is a NO threshold above which NO activates KATP channels. Future studies are required to assess the role of KATP channels in cutaneous vasodilation, and this could be best achieved in response to low to medium doses of NO donor.
We showed that cutaneous vasodilation associated with the sustained plateau phase during local heating was reduced by the application of the KATP channel blocker glibenclamide (Fig. 1 and Fig. 2D), suggesting that KATP channels contribute to this response in addition to previously identified factors such as NOS (13, 14, 27, 39, 41), TRPV1 channels (39), adenosine receptors (13), xanthine oxidase (29), H2O2 (29), and KCa and KV channels (6, 15) (Fig. 7). In keeping with our observation in the present study, we previously reported that cutaneous vasodilation elicited by whole body heat stress is also in part mediated by KATP channels (26). Therefore, our results suggest that both local and whole body heat stress can cause cutaneous vasodilation in part via the activation of KATP channels.
Fig. 7.Illustrative summary of the mechanisms underpinning the plateau phase of cutaneous thermal hyperemia. KATP, ATP-sensitive K+ channel; KCa, Ca2+-activated K+ channel; KV, voltage-gated K+ channel; NO, nitric oxide; NOS, nitric oxide synthase; sGC-cGMP, soluble guanylyl cyclase-cyclic guanosine monophosphate; TRPV1, transient receptor potential vanilloid type 1 channel.
Consistent with several previous studies (13, 14, 27, 39, 41), we demonstrated that NOS inhibition substantially blunted the plateau response (Fig. 2D). In the present study, we further assessed whether NOS interacts with KATP channels in mediating the plateau response. The KATP channel blocker-induced attenuation of the plateau response was not observed when the influence of NOS was removed. This was indicated by our observation that cutaneous vascular conductance measured at the plateau phase did not differ between the NOS inhibition site and the combined KATP channel blockade and NOS inhibition site (Fig. 2D). Our results suggest that KATP channels and NOS interact in mediating the plateau phase of the cutaneous thermal hyperemia response.
Although our findings demonstrate that there is an interaction between KATP channels and NOS in mediating the plateau response (Figs. 1 and 2), we cannot discern the mechanisms underpinning this interactive effect. Nonhuman studies suggest that KATP channel openers cause vasodilation by activating NOS (25). Thus, although KATP channels are generally thought to exist on vascular smooth muscle cells (thus it appears to mediate endothelium-independent vasodilation), they may also be found on the endothelium (3). Consequently, the activation of endothelial KATP channels may in turn activate NOS, mediating vasodilation in human skin. To evaluate this possibility, we compared cutaneous vasodilation elicited by the KATP channel opener pinacidil with and without NOS inhibition. Using this approach, we showed no differences in cutaneous vascular conductance between the two skin sites (Fig. 4B). Our observations therefore suggest that KATP channels may not exist in the endothelium of human cutaneous vessels or, if they are present, it is likely they have a limited interaction with NOS. In contrast, a previous study by Hojs et al. (20) reported that cutaneous vasodilation in response to the NO donor sodium nitroprusside administered via iontophoresis was partly blunted by microinjection of 8 mM glibenclamide. This indicates that NO can directly activate vascular smooth muscle KATP channels, eliciting cutaneous vasodilation, independent of any effects on the endothelium. Using intradermal microdialysis, we demonstrated similar results with sodium nitroprusside-induced cutaneous vasodilation, a response that was partly attenuated by the simultaneous administration of 5 mM glibenclamide (Fig. 4C). Taken together, our findings suggest that NOS-derived elevations in NO during local heating activate KATP channels (likely on vascular smooth muscle), partly modulating the plateau phase of cutaneous thermal hyperemia in humans in vivo (Fig. 7).
We found that the KATP channel opener pinacidil increased cutaneous vascular conductance to 53 ± 10%max (Fig. 4B), a level comparable to that observed during an exercise-induced heat stress (17, 38). Our results therefore suggest that KATP channel activation alone can elicit cutaneous vasodilation to levels that can alter heat exchange in humans. However, the difference in plateau cutaneous vascular conductance between the control and KATP channel blockade sites was 14 ± 9%max (Fig. 2D), which was half of that achieved by the administration of pinacidil (by 28 ± 7%max). Similarly, we showed a difference in cutaneous vascular conductance between the control and KATP channel blockade sites of ~15%max during whole body heating in our previous work (26). Assuming that 1.25 mM pinacidil maximally opened KATP channels in human skin in the present study, local and whole body heating may induce ~50% of the KATP channels’ maximum capacity to mediate cutaneous vasodilation.
Validity of antagonists.
Previously, we and others have administered glibenclamide via intradermal microdialysis (16, 24, 26) or microinjection (20) to understand the role of KATP channels in the regulation of cutaneous blood flow. However, to the best of our knowledge, no study to date has confirmed whether glibenclamide completely blocks KATP channels in human skin. In the present study, we found that the KATP channel opener pinacidil induced a marked cutaneous vasodilator response, which was abolished by coadministration of 5 mM glibenclamide (Fig. 4B). Therefore, 5 mM glibenclamide with intradermal microdialysis appears to effectively block KATP channels in human skin.
Although glibenclamide is a KATP channel blocker, it may also inhibit CFTR; additionally, it may potentially activate TRPA1 channels. However, we showed that the CFTR activator DCEBIO (we employed a high concentration of 5 mM for this drug to ensure maximum activation of CFTR) had no effect on cutaneous vascular conductance (Figs. 5 and 6). Consequently, if any modulation of CFTR associated with glibenclamide occurred, it seemed to have a minimum influence on cutaneous vascular responses. In contrast, we showed that the TRPA1 channel agonist cinnamaldehyde caused a substantial cutaneous vasodilation (Figs. 5 and 6). Thus, if glibenclamide did activate TRPA1 channels, it could potentially modulate cutaneous vascular responses during local heating. However, given that glibenclamide had no effect on cutaneous vascular conductance relative to the control site during the baseline period, any effect of glibenclamide-induced activation of TRPA1 channels on cutaneous vascular conductance appears to be negligible. If in fact glibenclamide-induced activation of TRPA1 channels occurred and caused some vasodilatory effects during the plateau phase of local heating, it is possible that we may have underestimated the contribution of KATP channels on the plateau response, a response that can be delineated by evaluating the difference between the control and KATP channel blockade sites. However, most importantly, we still observed an effect of KATP channel blockade on the plateau response, indicating that our conclusions remain valid.
NG-nitro-l-arginine methyl ester is widely used as a NOS inhibitor. However, some studies have suggested that the drug may cause side effects such as an antimuscarinic effect (9) and/or modulate vascular smooth muscle cell function (10, 12). As for the latter, we found that NO donor-induced cutaneous vasodilation, which reflects cutaneous vascular smooth muscle responsiveness to NO, did not differ between the control site and the site treated with NG-nitro-l-arginine methyl ester (Fig. 4C). Thus our findings indicate that a concentration of 20 mM NG-nitro-l-arginine methyl ester administered via intradermal microdialysis does not modulate cutaneous vascular smooth muscle function, validating the use of this drug as a NOS inhibitor in human nonglabrous skin.
Limitations.
A previous report showed that prior local heating alters the contribution of adrenergic nerves to cutaneous thermal hyperemia, though it has no effect on the contribution of NOS (11). As such, we cannot determine whether local heating to 42°C affected the subsequent cutaneous vasodilation elicited by pinacidil and sodium nitroprusside in the present study. Furthermore, we infused pinacidil and sodium nitroprusside for an extended period of 30 min, ensuring a stable plateau level of cutaneous vasodilation. However, although a longer duration of infusion may cause a further augmentation in cutaneous blood flow, this elevation is likely to be relatively minor. Moreover, to limit the duration of the experimental trial, which was already quite long for the participants (~5.5 h), we did not include a washout period between pinacidil and sodium nitroprusside administration. Consequently, we were not able to directly assess the extent to which sodium nitroprusside caused cutaneous vasodilation. To overcome the vasodilator effect of pinacidil, we infused a high dose of sodium nitroprusside. However, had we employed lower doses of sodium nitroprusside we might have seen different results.
Perspectives and Significance
The plateau phase of cutaneous thermal hyperemia is highly NOS dependent and is thought to reflect microvascular endothelial function. Previous studies have reported that the NOS-dependent plateau response of cutaneous thermal hyperemia is impaired with aging (32), chronic cigarette smoking (14), or postural tachycardia syndrome (35). This attenuation in general appears to be due to reduced NO availability in the skin. In the present study, our results suggest that NOS partly interacts with KATP channels in mediating the plateau response, which appears to be due to NO-induced activation of KATP channels located on vascular smooth muscle cells (Fig. 7). In this context, vascular smooth muscle KATP channel function could in part determine the NOS-dependent component of the plateau phase of cutaneous thermal hyperemia, a response that would occur independently of NO bioavailability. Thus the aforementioned reduced NOS-dependent component associated with aging (32), chronic cigarette smoking (14), or postural tachycardia syndrome (35) might be in part due to KATP channel dysfunction rather than reduced NO bioavailability. Therefore, assessing the conditions under which vascular smooth muscle KATP channel function is impaired is an important step in advancing our understanding of this response, as malfunction of vascular smooth muscle KATP channels could lead to cardiovascular dysregulation (4, 30).
Conclusions.
We show that NOS interacts with KATP channels, partly contributing to the sustained plateau phase of the cutaneous thermal hyperemia response in young adults. Our results also suggest that this interaction occurs via NO-induced activation of vascular smooth muscle KATP channels.
GRANTS
This study was supported by the grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan (JSPS KAKENHI; grant no. 17H04753).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
N.F. conceived and designed research; N.F. performed experiments; N.F. analyzed data; N.F., G.W.M., G.P.K., T.A., Y.H., N.K., and T.N. interpreted results of experiments; N.F. prepared figures; N.F. drafted manuscript; N.F., G.W.M., G.P.K., T.A., Y.H., N.K., and T.N. edited and revised manuscript; N.F., G.W.M., G.P.K., T.A., Y.H., N.K., and T.N. approved final version of manuscript.
ACKNOWLEDGMENTS
We greatly appreciate all of the volunteers for taking the time to participate in this study. All experiments took place at the University of Tsukuba.
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