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. 2012 Jun 6;590(Pt 15):3523–3534. doi: 10.1113/jphysiol.2012.236398

KCa channels and epoxyeicosatrienoic acids: major contributors to thermal hyperaemia in human skin

Vienna E Brunt 1, Christopher T Minson 1
PMCID: PMC3547267  PMID: 22674719

Abstract

While it is accepted that NO is responsible for ∼60% of the plateau in cutaneous thermal hyperaemia, a large portion of the response remains unknown. We sought to determine whether the remaining ∼40% could be attributed to EDHF-mediated activation of KCa channels, and whether the epoxyeicosatrienoic acids (EETs), derived via cytochrome P450, were the predominant EDHF active in the response. Four microdialysis fibres were placed in the forearm skin of 20 subjects. In Protocol 1 (n = 10): (1) Control, (2) NG-nitro-l-arginine methyl ester (l-NAME), (3) a KCa channel inhibitor, tetraethylammonium (TEA), and (4) TEA +l-NAME. In Protocol 2 (n = 10): (1) Control, (2) l-NAME, (3) a cytochrome P450 inhibitor, sulfaphenazole, and (4) sulfaphenazole +l-NAME. Local heating to 42°C was performed and skin blood flow was measured with laser Doppler flowmetry. Data are presented as the percentage of maximal cutaneous vascular conductance (CVC). All drug sites attenuated plateau CVC from the control site (86 ± 1%) to 79 ± 3% with sulfaphenazole (P = 0.02 from control), 71 ± 3% with TEA (P = 0.01 from control), and further to 38 ± 2% with l-NAME (P < 0.001 from control, P < 0.001 from TEA). Plateau was largely attenuated with sulfaphenazole +l-NAME (24 ± 2%; P = 0.002 from l-NAME), and nearly abolished with l-NAME + TEA (13 ± 2%; P = 0.001 from sulfaphenazole +l-NAME), which was not different from baseline (P = 0.14). Furthermore, the initial peak was just 17 ± 2% with TEA +l-NAME (P < 0.001 from l-NAME). These data suggest EDHFs are responsible for a large portion of initial peak and the remaining 40% of the plateau phase, as administration of TEA in combination with l-NAME abolished the majority of hyperaemia. These data also suggest EETs contribute to about half of the EDHF response.

Key points

  • The increased blood flow associated with local heating of the skin is ∼60% dependent on nitric oxide. The remaining ∼40% is unknown.

  • Endothelial-derived hyperpolarizing factors (EDHFs), a class of vasodilators, are known to contribute to increases in blood flow in other vascular beds.

  • In the present study, we showed the drug tetraethylammonium (which blocks the channels involved in EDHF-mediated vasodilatation), when given in combination with nitric oxide synthase inhibition, blocked the majority of hyperaemia to local heat, indicating that EDHFs are responsible for the majority of the remaining ∼40% of hyperaemia.

  • We also showed that about half of the EDHF-component is attributed to a specific type of EDHF, epoxyeicosatrienoic acid (EET), as evidenced using the cytochrome P450 inhibitor sulfaphenazole.

  • These findings help further our understanding of the mechanisms behind cutaneous thermal hyperaemia.

Introduction

Local heating of the skin elicits a biphasic rise in skin blood flow (SkBF): a rapid initial peak vasodilatation, followed by a prolonged rise in SkBF, which reaches a plateau within 20–30 min after the onset of local heating. The initial peak is predominantly mediated by a sensory nerve axon reflex (Minson et al. 2001). The sensory nerves are thought to release calcitonin gene-related peptide and substance P, although this has not been confirmed experimentally (Wallengren et al. 1987). TRPV-1 receptors, located primarily on the sensory nerves, also play a substantial role in the initial peak (Wong & Fieger, 2010).

Nitric oxide was shown to account for approximately 60% of plateau SkBF over a decade ago (Kellogg et al. 1999; Minson et al. 2001). Since then, several other factors have also been shown to play roles. These factors, which include adenosine receptors (Fieger & Wong, 2010), TRPV-1 receptors (Wong & Fieger, 2010), and reactive oxygen species (Medow et al. 2011), largely act through altering the availability of NO, although slight NO-independent effects on plateau SkBF have been observed. The COX pathway does not appear to be involved in the local heating response (McCord et al. 2006). Taken together, these findings indicate approximately 40% of the plateau is NO and COX independent. This constitutes a substantial portion of the cutaneous thermal hyperaemia response of which the mechanisms have yet to be discovered.

NO- and COX-independent vasodilatation exists in numerous vascular beds, and is typically attributed to endothelial-derived hyperpolarizing factors (EDHFs), a class of factors that cause vasodilatation through hyperpolarization of the vascular smooth muscle. While other mechanisms exist, the majority of EDHFs act through stimulating calcium-activated potassium (KCa) channels. Large conducting KCa (BKCa) channels are found on the smooth muscle membrane and directly hyperpolarize the smooth muscle, while intermediate (IKCa) and small (SKCa) KCa channels are found on the endothelial cell membrane. Hyperpolarization of the endothelial cells spreads through gap junctions to the smooth muscle, thus also contributing to vasodilatation (Félétou & Vanhoutte, 2009). Possible EDHFs include the epoxyeicosatrienoic acids (EETs), the lipoxygenase (LOX) derivatives 12-(S)-hydroxyeicosatetraenoic acid (12-S-HETE) and 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), and H2O2 (Félétou & Vanhoutte, 2009).

There is evidence to suggest KCa channels exist in the skin, although few studies have explored their effects on the cutaneous vasculature. KCa channels are involved in the cutaneous response to reactive hyperaemia, contributing approximately 45% of the hyperaemia (Lorenzo & Minson, 2007). Furthermore, BKCa channels are important in the cutaneous vasodilatory response to increases in external pressure (Garry et al. 2005). Together, these findings indicate a key role of KCa channels in the skin microvasculature.

Therefore, our primary goal was to determine whether EDHF-mediated activation of KCa channels contributes to the local heating response (Protocol 1). To explore this, we delivered tetraethylammonium (TEA), a non-specific KCa channel inhibitor, to the skin via microdialysis. NO production was inhibited in another site with NG-nitro-l-arginine methyl ester (l-NAME), and a third experimental site was used to observe the effect of combined inhibition of KCa channels and NO synthesis. We hypothesized that infusion of TEA would result in an attenuation of both the initial peak and plateau phases compared to the control site, and that infusion of TEA and l-NAME in combination would result in an attenuation of the plateau beyond that seen with l-NAME alone.

In the second part of the study, we sought to determine which EDHFs were responsible for the activation of the KCa channels in the local heating response. Of the various EDHFs, the epoxyeicosatrienoic acids (EETs) have been shown to cause vasodilatation in humans in other vascular beds (Hillig et al. 2003; Bellien et al. 2006; Ozkor et al. 2011). EET-induced vasodilatation is produced predominantly through KCa channels, activating both the SKCa and IKCa channels on the endothelium and the BKCa channels on the smooth muscle (Campbell et al. 1996) through inducing calcium sparks from the endoplasmic reticulum via TRPV4 channels (Earley et al. 2005). EETs are synthesized in the endothelium from arachadonic acid, a conversion catalysed by cytochrome P450. CYP2C9, a human subtype of cytochrome P450, catalyses the conversion of EET isoforms 11,12-EET and 14,15-EET from arachadonic acid, and is the predominant CYP subtype involved in EDHF-dependent vasodilatation (Michaelis & Fleming, 2006). Therefore, we tested the role of the EETs in local skin heating through selective inhibition of CYP2C9 with sulfaphenazole (Protocol 2). We hypothesized that sulfaphenazole would attenuate both peak and plateau blood flow, and, in combination with l-NAME, would attenuate plateau blood flow beyond l-NAME alone.

In addition, we studied a subset of subjects (Protocol 3) in order to confirm that, in the skin, EETs elicit vasodilatation through stimulation of KCa channels, as has been shown in other vascular beds (Earley et al. 2005). We infused TEA and TEA in combination with sulfaphenazole, both with and without l-NAME, in order to test our hypothesis that blocking EETs had no further effect on hyperaemia once KCa channels are blocked, thus confirming that EETs act in series with KCa channels.

Methods

Ethical approval

Prior to participation in the study, all subjects gave oral and written informed consent as set forth by the Declaration of Helsinki. All protocols were approved by the Institutional Review Board of the University of Oregon.

Subjects

Twenty-seven subjects participated in the study (14 male, 13 female). Four subjects participated in more than one protocol. All subjects were young (18–30 years of age), did not have any history of cardiovascular disease, and were not taking any medications, with the exception of contraceptives. All subjects refrained from alcohol and caffeine for 12 h prior to participation in the study, and from over-the-counter medications, including vitamins, for 24 h prior to participation in the study. Subjects fasted for 4 h prior. In order to minimize the effects of the female sex hormones, all female subjects were studied during menstruation or during the placebo phase if taking contraceptives.

Instrumentation

Throughout the study, subjects rested in a thermoneutral room and in a partially supine position, with the experimental arm to the side at the level of the heart. Blood pressure was monitored on the non-experimental arm throughout the study via brachial oscillation (Dinamap ProCare 100; GE Medical Systems, Tampa, FL, USA).

Four microdialysis fibres (MD 2000; Bioanalytical Systems, West Lafeyette, IN, USA) were placed at least 5 cm apart in the skin of the forearm of the non-dominant arm. Fibres were placed via insertion of a 25-gauge needle through the skin, with the entry and exit points approximately 2.5 cm apart. Following needle insertion, the fibres were threaded through the lumen of the needle. The needles were then removed leaving the fibre in place with the membrane (1 cm in length) under the skin, centred between the entry and exit points. A lactated Ringer solution was infused through all fibres at a rate of 2μl min−1 (CMA 102 Syringe Pump; CMA Microdialysis AB, Solna, Sweden) until infusion of study drugs began.

Local heating of the skin was achieved using local heaters (Skin Heater/Temperature Monitor SH02; Moor Instruments, Axminster, UK) centred over each of the four microdialysis sites, covering ∼0.78 cm2 of tissue. Red blood cell flux was measured continuously to give an index of skin blood flow using single-point laser-Doppler flowmetry (DRT-4 and moorLab; Moor Instruments). Laser-Doppler probes were seated in the centre of the local heaters.

Protocol

A period of 60–90 min was allowed for recovery from the trauma associated with needle insertion. Following recovery, baseline SkBF was measured for 10 min with the local heaters set to 33°C. Drug infusions then began at the four sites, which continued through the duration of the study. Drugs infused are described in the following section. Drugs were infused for 60 min before the start of the local heating. This time period was shown to allow for maximal efficacy of TEA in pilot studies. Drug baseline was taken as the last 5 min of this time period.

Local heating was achieved by raising the temperature of the local heaters at a rate of 0.1°C s−1 up to 42°C. The local heaters were held constant at 42°C for at least 30 min, or until SkBF reached a plateau for at least 5 min in all four sites. Following plateau, 56 mm sodium nitroprusside (SNP; Nitropress, Ciba Pharmaceuticals, East Hanover, NJ, USA) was infused at all four sites in order to elicit maximal SkBF in an endothelium-independent manner. In addition, the local heaters were raised to a temperature of 44°C, again at a rate of 0.1°C s−1.

Drug protocols

Subjects underwent one of two drug protocols. Protocol 1 explored the role of KCa channels, and Protocol 2 explored the role of CYP2C9-derived EETs. Both protocols had four microdialysis sites. In Protocol 1, the four sites were (1) Control, (2) 20 mm l-NAME, (3) 50 mm TEA, and (4) 50 mm TEA + 20 mm l-NAME. In Protocol 2, the four sites were (1) Control, (2) 20 mm l-NAME, (3) 1 mm sulfaphenazole, and (4) 1 mm sulfaphenazole + 20 mm l-NAME.

Protocol 1

Ten subjects (5 male, 5 female) were studied in Protocol 1. All drugs were dissolved in a lactated Ringer solution. The control site received a continuous infusion of lactated Ringer solution.

Nitric oxide synthase (NOS) inhibition was achieved with 20 mm l-NAME. NOS inhibition was used in order to separate the NO-dependent and NO-independent portion of thermal hyperaemia. In a set of pilot subjects, the non-specific COX inhibitor ketorolac (Keto; Sigma-Aldrich Co., St Louis, MO, USA) was also infused in the l-NAME and TEA +l-NAME sites. No additional effects were seen with COX inhibition and thus ketorolac was not used in the subjects reported in this study. This finding was consistent with previous findings that indicate no role of prostanoids in non-painful local heating (McCord et al. 2006).

Tetraethylammonium chloride (Sigma-Aldrich) was selected as a KCa channel inhibitor. A dose of 50 mm was used as higher doses have no further effect on blocking hyperaemia in the skin (Lorenzo & Minson, 2007) and it is not high enough to also inhibit other types of potassium channels, as has been observed with doses exceeding 100 mm (Bellien et al. 2008). Specific BKCa channel inhibitors, such as iberiotoxin and charybdotoxin, were not used as they are toxic to humans (Pickkers et al. 1998).

The combination site was included in order to determine the combined effects of both NOS inhibition and KCa channel blockade. EDHF mechanisms can be masked by NO as there is cross-talk between the two pathways (Zhou & Raij, 2003). We also wanted to determine if there was an additional portion of thermal hyperaemia that was NO, COX and KCa independent.

Protocol 2

Ten subjects (5 male, 5 female) participated in Protocol 2. EET production was inhibited with 1 mm sulfaphenazole (Spectrum Chemical Mfg Group, Gardena, CA, USA) a selective inhibitor of CYP 2C9. CYP 2C9 is the predominant CYP isoform present in human endothelial cells and catalyses the conversion of arachadonic acid to 11,12-EET and 14,15-EET. Sulfaphenazole is insoluble in water, which presented difficulties in delivering the drug through microdialysis fibres in pilot studies. Dimethyl sulfoxide (DMSO) can be effective in dissolving water-insoluble compounds (Shastry & Joyner, 2002; Kellogg et al. 2007). Thus, we mixed sulfaphenazole in a 5% DMSO–lactated Ringer solution solution, showing more favourable results. However, DMSO has slight vasodilatory effects in normothermia (Kellogg et al. 2007). These effects are addressed in the limitations section. Although much weaker concentrations of sulfaphenazole have typically been used, we selected 1 mm for use in microdialysis as smaller doses did not elicit as great an attenuation of thermal hyperaemia in pilot studies, and 1 mm was the strongest concentration that could be successfully brought into solution with DMSO. Stronger concentrations might be possible using organic solvents, however, such solvents are not suitable for use in humans.

In order to be able to attribute inter-site differences to l-NAME and/or sulfaphenazole, and not to DMSO, 5% DMSO-Ringer solution was used as the solvent in all sites in Protocol 2. This also allowed us to make comparisons between the two protocols and assess the effects of DMSO while using different pharmacological interventions. Infusion with 5% DMSO-Ringer solution began following baseline measurements at 33°C. DMSO was infused for 20 min and ‘DMSO baseline’ was taken during the last 5 min of this time period. The control site was then continuously infused with 5% DMSO-Ringer solution while the other sites were infused with the drug solutions. As in Protocol 1, drugs were infused for 60 min before the start of the heating protocol.

Protocol 3

The combined effects of TEA and sulfaphenazole were explored in a subset of subjects (n = 7, 4 male, 3 female). Specifically, we sought to determine whether any additional attenuation of skin blood flow could be seen when giving sulfaphenazole in combination with TEA, as compared to TEA alone, thus determining whether EETs produce vasodilatation through activation of KCa channels, or through a KCa-independent mechanism. We tested this combination with and without NOS inhibition with l-NAME. The four drug sites were: (1) 50 mm TEA, (2) 50 mm TEA + 1 mm sulfaphenazole, (3) 20 mm l-NAME + 50 mm TEA, and (4) 20 mm l-NAME + 50 mm TEA + 1 mm sulfaphenazole. All drugs were dissolved in a 5% DMSO-Ringer solution and were infused for 60 min before the start of the heating protocol. DMSO was infused for 20 min prior to drug infusions in order to record ‘DMSO baseline’.

Data analysis

Cutaneous vascular conductance (CVC) was calculated as red blood cell flux divided by mean arterial pressure (MAP). All values are presented as a percentage of maximal CVC (%CVCmax), as determined during SNP infusion. The local heating response was characterized by an initial peak and prolonged secondary plateau, with a brief nadir between the two phases. In sites where these phases were indiscernible (namely the TEA +l-NAME site), nadir and plateau CVC were taken at the same time as they occurred in the control site.

Statistical analysis

For each protocol, initial peak, nadir, and plateau CVC at each of the four microdialysis sites were compared using one-way repeated measures ANOVA. Multiple pairwise comparisons were made using Student–Newman–Keul's post hoc test. Student's unpaired t test was used to compare TEA to sulfaphenazole and TEA +l-NAME to sulfaphenazole +l-NAME across protocols 1 and 2, to determine the effects of DMSO during local heating by comparing the control and l-NAME sites between Protocol 1 (Ringer solution) and Protocol 2 (5% DMSO-Ringer solution), and to compare baseline before and after drug infusions in each site. For all statistical analyses, P values <0.05 were considered to be statistically significant.

Results

All subjects were young (age = 23 ± 1 years) and healthy (BMI = 24 ± 1 kg m−2; resting MAP = 83 ± 1 mmHg). There were no significant group differences in age, BMI, or blood pressure across protocols.

Local heating

Local heating of the skin resulted in an initial peak in blood flow, occurring within the first 5 min following the onset of heating, and a prolonged secondary plateau which was reached after approximately 20–30 min of heating. A brief nadir was seen between the two phases. Figures 1A and 2A give a representative tracing from one subject from Protocols 1 and 2, respectively. There were no differences in maximal flux between microdialysis sites (P = 0.41 Protocol 1, P = 0.41 Protocol 2, P = 0.27 Protocol 3).

Figure 1. Summary of the results of Protocol 1.

Figure 1

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All data are presented as percentage of maximal cutaneous vascular conductance (CVC), and are means ± SEM; statistical significance is defined as P < 0.05. A, a representative tracing from one subject during local heating of the skin, showing all four drug sites: control (lactated Ringer solution), tetraethylammonium (TEA), NG-nitro-l-arginine methyl ester(l-NAME), and TEA +l-NAME. B, comparison of initial peak CVC between the four sites. C, comparison of the plateau CVC between the four sites. In both panels B and C, the dotted line indicates the average pre-drug baseline across the four sites. *P < 0.05 from control site; †P < 0.05 from l-NAME site; ‡P < 0.05 from TEA site.

Figure 2. Summary of the results of Protocol 2.

Figure 2

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All data are presented as percentage of maximal cutaneous vascular conductance (CVC), and are means ± SEM; statistical significance is defined as P < 0.05. A, a representative tracing from one subject during local heating of the skin, showing all four drug sites: control (5% DMSO-Ringer solution), sulfaphenazole, NG-nitro-l-arginine methyl ester(l-NAME), and sulfaphenazole +l-NAME. B, comparison of initial peak CVC between the four sites. C, comparison of the plateau CVC between the four sites. In both panels B and C, the dotted line indicates the average pre-drug baseline across the four sites. *P < 0.05 from control site; †P < 0.05 from l-NAME site; ‡P < 0.05 from sulfaphenazole site.

The effects of the drugs in Protocol 1 on the initial peak (Fig. 1B) and plateau (Fig. 1C) phases are summarized in Fig. 1. l-NAME attenuated both initial peak and plateau CVC, consistent with previous findings. TEA attenuated the initial peak to the same extent as l-NAME, and TEA +l-NAME abolished the majority of the peak, indicating a large role of KCa channels in the axon-reflex portion of local heating. TEA also affected nadir, reducing it from 46.8 ± 6.1% in the control site to 20.1 ± 3.2% with TEA (P < 0.001). TEA +l-NAME reduced nadir to 7.6 ± 1.1% (P < 0.001 from control, P = 0.05 from TEA). TEA attenuated plateau CVC from the control site. The combination of TEA +l-NAME abolished almost the entirety of plateau CVC, which was not significantly different from baseline (before drug infusions) in that site, indicating the majority of the response in CVC to the plateau phase of local heating can be attributed to the combination of NO and KCa channels.

The effects of the drugs in Protocol 2 on the initial peak (Fig. 2B) and plateau (Fig. 2C) phases are summarized in Fig. 2. Initial peak, nadir, and plateau in both the control and l-NAME sites were not different from those in Protocol 1. Sulfaphenazole showed a slight attenuation of initial peak, but not nadir (control: 44.8 ± 4.1%vs. sulfaphenazole: 41.2 ± 4.1%, P = 0.49). Sulfaphenazole +l-NAME showed only a very slight further attenuation of peak and nadir from the l-NAME site, and this difference was not significant (nadir –l-NAME: 17.6 ± 2.3%vs. sulfaphenazole +l-NAME: 15.5 ± 2.1%, P = 0.44). Together, these results indicate a minimal role of EETs in the axon-reflex portion of local heating. Sulfaphenazole significantly attenuated plateau from the control site, and sulfaphenazole +l-NAME attenuated plateau from the l-NAME site, indicating a role of EETs in the plateau phase. However, the contribution of EETs is not as great as KCa channels, neither with nor without NOS inhibition. In both cases, TEA significantly attenuated plateau beyond what sulfaphenazole did (TEA: 69.0 ± 2.3%vs. sulfaphenazole: 78.9 ± 2.5%, P = 0.01; l-NAME + TEA: 13.3 ± 2.2%vs. l-NAME + sulfaphenazole: 24.2 ± 1.6%, P = 0.001), indicating that EDHFs other than EETs are also involved in thermal hyperaemia.

In protocol 3, there were no differences in initial peak, nadir, or plateau between TEA and TEA + sulfaphenazole nor between l-NAME + TEA and l-NAME + TEA + sulfaphenazole, indicating that EETs produce vasodilatation through activation of KCa channels. Table 1 summarizes these data.

Table 1.

Thermal hyperaemia data from Protocol 3

TEA TEA + sulfaphenazole P-value
Without l-NAME
 Initial peak 35.3 ± 3.3% 32.4 ± 4.4% 0.54
 Plateau 61.0 ± 5.7% 58.9 ± 7.4% 0.76
With l-NAME
 Initial peak 21.6 ± 2.3% 26.9 ± 2.8% 0.25
 Plateau 18.4 ± 1.4% 26.5 ± 3.4% 0.24
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Average data from Protocol 3. Data are presented as percentage maximal cutaneous vascular conductance, and are means ± SEM. Drugs include NG-nitro-l-arginine methyl ester(l-NAME), tetraethylammonium (TEA) and sulfaphenazole.

Baseline effects

Initial baseline CVC (before drug infusions) was not significantly different between the four sites in any protocol (Protocol 1, P = 0.10; Protocol 2, P = 0.97; Protocol 3, P = (0.78). After 60 min of drug infusion, both l-NAME and TEA showed significant attenuation of baseline. These results are summarized in Table 2.

Table 2.

Effects of study drugs on baseline at 33oC

Before drug infusions 60 min into drug infusions P-value
Protocol 1
 Control 5.8 ± 0.8% 6.8 ± 1.2% 0.39
 l-NAME 4.8 ± 0.9% 3.4 ± 0.8%* 0.02
 TEA 6.7 ± 1.4% 5.0 ± 1.3%* 0.03
 TEA +l-NAME 8.6 ± 1.8% 2.7 ± 0.6%* 0.01
Protocol 2
 Control 13.2 ± 2.5% 12.3 ± 2.6% 0.28
 l-NAME 14.6 ± 2.5% 10.2 ± 1.6%* 0.02
 Sulfaphenazole 13.1 ± 1.8% 11.7 ± 2.0% 0.15
 Sulfaphenazole +l-NAME 11.9 ± 1.2% 7.9 ± 0.8%* 0.001
Protocol 3
 TEA 15.7 ± 2.4% 8.7 ± 1.2%* 0.02
 TEA + sulfaphenazole 16.1 ± 3.5% 10.4 ± 2.2% 0.19
 TEA +l-NAME 13.5 ± 3.0% 3.3 ± 0.6%* 0.006
 TEA + sulfaphenazole +l-NAME 18.4 ± 4.3% 3.5 ± 0.6%* 0.005
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Data are presented as percentage maximal cutaneous vascular conductance, and are means ± SEM. Drugs include NG-nitro-l-arginine methyl ester(l-NAME), tetraethylammonium (TEA), and sulfaphenazole. *P < 0.05 from baseline in that site.

DMSO

As a 5% DMSO-Ringer solution was used in all sites in Protocol 2 and Protocol 3, we were able to analyse the effects of DMSO on SkBF during both baseline and heating. DMSO increased baseline SkBF by ∼7% of CVCmax, but had no effect on SkBF during heating, which is consistent with previous findings (Kellogg et al. 2007). In addition, DMSO had no effect on the response of the l-NAME site during heating; although, a slight, but non-significant attenuation was seen in the TEA sites. Table 3 summarizes our findings on the effects of DMSO.

Table 3.

Effects of DMSO on cutaneous vascular conductance under baseline conditions and during local heating

Ringer solution 5% DMSO-Ringer P value
Baseline (33°C) 7.3 ± 0.7% 14.5 ± 1.3%* < 0.001
Initial Peak
 Control Site 61.8 ± 3.5% 63.6 ± 5.3% 0.78
 l-NAME Site 45.9 ± 4.0% 40.1 ± 3.7% 0.31
 TEA site 44.5 ± 3.1% 35.3 ± 3.3% 0.06
Plateau
 Control Site 84.1 ± 1.6% 86.6 ± 2.5% 0.41
 l-NAME Site 39.5 ± 4.1% 37.8 ± 3.3% 0.76
 TEA site 69.0 ± 2.3% 61.0 ± 5.7% 0.22
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Data are presented as percentage maximal cutaneous vascular conductance, and are means ± SEM. Data is included for control, NG-nitro-l-arginine methyl ester(l-NAME), and tetraethylammonium (TEA) sites during heating. DMSO, dimethyl sufoxide. Control and l-NAME sites are compared between Protocol 1 (n = 10) and Protocol 2 (n = 10). TEA sites are compared between Protocol 1 and Protocol 3 (n = 7). Baseline is compared in all sites that received DMSO, before and after DMSO infusion (n = 36).

*

P < 0.05 from Ringer solution only condition.

Discussion

A major finding of the present study is that EDHF-mediated activation of KCa channels plays a major role in cutaneous thermal hyperaemia. Infusion of TEA in combination with l-NAME abolished nearly all hyperaemia above baseline levels. Furthermore, EETs appear to contribute approximately half of the EDHF-dependent portion of thermal hyperaemia when NO is simultaneously blocked. These results necessitate a new model of the mechanisms behind cutaneous thermal hyperaemia to include EDHFs.

Initial peak

Minson et al. (2001) showed the initial peak to be predominantly the result of a sensory nerve axon reflex, through blockade of the sensory nerves with EMLA cream. The sensory nerves are thought to release substance P and/or CGRP (Wallengren et al. 1987), which then presumably act on the endothelium and/or smooth muscle to produce vasodilatation. The results of the present study indicate that this axon reflex likely results in vasodilatation through activation of the KCa channels. Observations made by Lorenzo & Minson (2007) support this hypothesis, as the rise in SkBF associated with reactive hyperaemia was substantially reduced by both TEA and EMLA cream, demonstrating involvement of EDHFs in the downstream actions of the sensory nerves. Our results also indicate that KCa channels are activated to an extent beyond that which can be blocked with sensory nerve inhibition. Initial peak in the TEA +l-NAME site in the present study was 17% of CVCmax versus 31% of CVCmax with EMLA cream +l-NAME in the study conducted by Minson et al. (2001). Assuming a complete blockade with EMLA was achieved in those studies, our results suggest an additional factor, not requiring activation of the sensory nerves, may also activate KCa channels during this phase.

Sulfaphenazole slightly attenuated initial peak, but not to the extent of TEA, and showed no effect on nadir. Additionally, sulfaphenazole +l-NAME did not attenuate initial peak further than l-NAME alone. This suggests that EETs may play a role in the axon reflex, but only a minimal one. Other EDHFs are likely involved. It is also possible that we were not able to fully block EET production in all subjects. There was a great deal of variability in the response to sulfaphenazole, which may be explained by the challenges involved in using the drug (see ‘Sulfaphenazole’ section below).

Plateau

It is well accepted that the plateau phase of cutaneous thermal hyperaemia is dependent on NO. The present study shows that KCa channels are equally important in this phase. TEA +l-NAME almost entirely abolished the plateau. Furthermore, this double-blocked plateau was not significantly different from the initial baseline CVC, although it was slightly elevated from baseline CVC following 60 min of infusion with TEA +l-NAME.

EETs also contribute to the plateau phase, but not to the extent of KCa channels. We conducted Protocol 3 in order to confirm that the hyperaemia attributable to EETs was occurring through stimulation of KCa channels. When combining administration of sulfaphenazole with TEA, we saw no further attenuation of plateau beyond that seen with TEA alone. When exploring the same combination in the presence of NOS inhibition, the l-NAME + TEA + sulfaphenazole site actually showed a slightly higher CVC than the TEA + sulfaphenazole site, but this difference was not significant. Thus, from these results, it appears that EETs are working in series with KCa channels, which is consistent with studies done in animals and in vitro.

When comparing our results from Protocols 1 and 2, it appears that EETs contribute approximately half of the KCa-dependent portion of the plateau, indicating that other EDHFs must contribute the remaining half. The lipoxygenase (LOX) pathway produces a variety of products that affect the blood vessels in different ways. Those that cause vasodilatation are 12-S-HETE and 11,12,15-THETA, produced by the isoforms 12- and 15-LOX, respectively. 12-S-HETE is known to act on KCa channels (Zink et al. 2001) and both products have been shown to be involved in endothelial-dependent relaxation in animal models (Tang et al. 2006). Another possibility is H2O2. Although H2O2 is typically considered a vasoconstrictor and a product of oxidative stress, it can act as a dilator at low concentrations through stimulating KCa channels (Lucchesi et al. 2005). Medow et al. (2011) reported H2O2 scavenging with ebselen to attenuate plateau CVC by about 20% of CVCmax. However, ebselen also has effects on the LOX pathway. It may also be possible to activate KCa channels by heat, without an EDHF mediator. TRPV channels are known to be heat sensitive (Caterina 2007), although it is not clear whether the channels themselves are the direct heat sensors or whether intermediates are required. For example, TRPV-4 channels (also activated by EETs) can activate KCa channels, and may contribute to some of the KCa-dependent vasodilatation, partially explaining the difference between the sulfaphenazole and TEA sites.

Although TEA +l-NAME plateau was not different from pre-drug baseline in that site, it was different from baseline following the 60 min drug infusion with TEA +l-NAME. There are a couple explanations for this difference. First, it is possible there is an additional portion of hyperaemia that is NO, COX, and KCa independent. KATP channels may be responsible for this hyperaemia. KATP channels have not yet been explored in human skin, but do contribute to hyperaemia in other vascular beds. In addition, they have been shown to be activated by EETs (Ye et al. 2005). Hydrogen sulfide (H2S) is also considered to be an EDHF, and can cause vasodilatation by activating KATP channels (Zhao et al. 2001).

The second possibility is that adrenergic constrictor tone was present in the baseline measurements, contributing to the lower CVC. If this were the case, once heating occurred and removed adrenergic constrictor tone, TEA +l-NAME would have been successful in blocking all vasodilatation. Minson et al. (2001) showed proximal blockade of the cutaneous nerves via the antecubital fossa to increase baseline CVC by 6% of CVCmax; however Hodges et al. (2009) saw no effect of adrenergic blockade via bretylium on baseline CVC, so, given the conflicting data, it is questionable as to whether this possibility is likely.

Cross talk between NO pathway and EDHFs

Neither TEA nor sulfaphenazole showed as great an effect on SkBF when given alone as compared to when administered in combination with l-NAME. This observation can be explained by the cross-talk between the two pathways.

NO has been shown to activate KCa channels (Bolotina et al. 1994; Wellman et al. 1996), which may involve TRPV4 channels (also activated by EETs), as TRPV4 knockout mice are more sensitive to NOS inhibition compared to wild-type mice (Earley et al. 2009). CYP2C9 has been shown to contribute to production of reactive oxygen species in some animal models (Fleming et al. 2001; Elmi et al. 2008) and human patient populations (Fichtlscherer et al. 2004), which would result in reductions in NO bioavailability. Inhibition of CYP2C9 with sulfaphenazole may thus increase NO bioavailability and CVC during local heating. In addition, increases in intracellular [Ca2+] lead to activation of both KCa channels and eNOS. Thus, the NO pathway is likely to be upregulated when KCa channels are inhibited, and vice versa. For this reason, it follows that the combination of TEA (or sulfaphenazole) and l-NAME results in a greater attenuation of the response to local heating than the sum of the attenuation seen in the sites with a single inhibitor, demonstrating a synergistic effect of the two drugs.

Limitations

DMSO

Sulfaphenazole is insoluble in water, and thus we had great difficulty delivering the drug to the skin when lactated Ringer solution was used as the solvent. DMSO is considered an irritant, and has an effect on blood flow. DMSO has been shown to increase baseline SkBF, but it does not seem to have an effect when the skin is heated (Kellogg et al. 2007). Our data are consistent with these findings. Plateau SkBF was similar between the two protocols in both the Control and l-NAME sites, suggesting a minimal effect, if any, of DMSO during local heating. The TEA site was slightly attenuated with DMSO compared to without DMSO. This difference was not statistically significant for the plateau, but was nearing significance (P = 0.06) for the initial peak. Thus, it is reasonable to assume DMSO also did not affect CVC in the sulfaphenazole and sulfaphenazole +l-NAME sites, although it should be recognized that we cannot confirm DMSO did not modestly impact our results with sulfaphenazole.

Sulfaphenazole

Based on our results, the EETs appear to contribute approximately half of the EDHF-dependent vasodilatation. The remainder of the plateau that was blocked with TEA can be attributed to other EDHFs that act through KCa channels, including the LOX derivatives and H2O2. However, it is possible EETs contribute to a greater portion of the KCa-dependent vasodilatation than we were able to show. Although we used a concentration of sulfaphenazole exceeding that shown to be maximally effective in previous studies, we cannot be certain that enough of the drug actually did reach the skin vessels to entirely inhibit EET production. The technique of microdialysis requires substances to be dissolved thoroughly in order to ensure delivery, and, upon delivery, substances become further diluted in the interstitium. A concentration of 1 mm may not be great enough to elicit a full inhibition when using microdialysis. Unfortunately, 1 mm was the strongest concentration we were able to dissolve, even using DMSO. Therefore, it is possible EETs may contribute to thermal hyperaemia to a greater extent than we have shown.

Multiple drug complications

There was a higher level of variability in these sites compared to the first two protocols, particularly in the sulfaphenazole sites (both with and without l-NAME). Despite the variability, the means were very similar between TEA and TEA + sulfaphenazole sites (for both initial peak and plateau, and with and without l-NAME), thus the variability did not falsely indicate no difference between the responses. It is possible that when giving double and triple blockades, individual subjects may have differing responses. Furthermore, there may be inter-site differences depending on the distribution of the sensory nerves. When blocking pathways pharmacologically, other pathways may be upregulated, thus producing responses that are not consistent with normal physiology.

Perspectives

The results of the present study show EDHFs to be important in both the initial peak and plateau phases of local heating. These results explain a large portion of thermal hyperaemia, whose mechanisms have previously been unknown. However, not only do the results of this study further our knowledge of the mechanisms controlling cutaneous blood flow, but they also fit well with the scheme that the cutaneous vasculature is reflective of other vascular beds. There is growing evidence that cutaneous thermal hyperaemia can be used to assess globalized microvascular function (Minson, 2010), and the roles we have shown EDHFs and EETs to play in the skin support this claim, as both EDHFs and EETs are important contributors to vasodilatation in most vascular beds, to various stimuli.

Furthermore, performing the pharmacological studies to determine the mechanisms behind hyperaemia are especially important in translation to disease states. Cutaneous thermal hyperaemia is attenuated in various pathological conditions, including advanced age (Minson et al. 2002), hypertension (Smith et al. 2011), postural tachycardia syndrome (Stewart et al. 2007; Stewart et al. 2009), diabetes (Wigington et al. 2004; Colberg et al. 2005), and renal failure (Stewart et al. 2004; Kruger et al. 2006). Knowing the importance of EDHF pathways in this response offers possible mechanisms for the impaired hyperaemia, and perhaps an avenue for developing therapeutics targeted at improving microvascular function in these patients.

Acknowledgments

The authors would like to acknowledge the subjects who participated in this study for their time and effort. Funding for this study was provided by the National Institutes of Health Grant HL081671.

Glossary

BKCa

large conducting calcium-activated potassium

CGRP

calcitonin gene-related peptide

COX

cyclooxygenase

CVC

cutaneous vascular conductance

CYP

cytochrome P450

DMSO

dimethyl sulfoxide

EDHFs

endothelial-derived hyperpolarizing factors

EETs

epoxyeicosatrienoic acids

EMLA

eutectic mixture of local anaesthetics

eNOS

endothelial nitric oxide synthase

IKCa

intermediate conducting calcium-activated potassium

KATP

ATP-activated potassium channel

KCa

calcium-activated potassium

l-NAME

NG-nitro-l-arginine methyl ester

LOX

lipoxygenase

MAP

mean arterial pressure

NO

nitric oxide

NOS

nitric oxide synthase

SkBF

skin blood flow

SKCa

small conducting calcium-activated potassium

SNP

sodium nitroprusside

TEA

tetraethylammonium

TRPV

transient receptor potential vanilloid

Author contributions

V.E.B. contributed to the following aspects of the study: conception and design of the experiments; collection, analysis and interpretation of data; drafting the article and revising it critically for important intellectual content. C.T.M. contributed to the following aspects of the study: conception and design of the experiments; interpretation of data; revising the article critically for important intellectual content. Both authors approved the final version of the manuscript.

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