J Appl Physiol 115: 1290 –1296, 2013.
First published August 22, 2013; doi:10.1152/japplphysiol.00358.2013.
No independent, but an interactive, role of calcium-activated potassium
channels in human cutaneous active vasodilation
Vienna E. Brunt, Naoto Fujii, and Christopher T. Minson
Department of Human Physiology, University of Oregon, Eugene, Oregon
Submitted 25 March 2013; accepted in final form 15 August 2013
endothelium-derived hyperpolarizing factors; inward rectifier potassium channels; nitric oxide; thermoregulation; whole body heating
IN ADDITION TO BEING SUBJECT to local signaling, the cutaneous
vasculature is under both vasoconstrictor and vasodilator neural control. Small rises in core temperature (⬃0.1– 0.2°C)
cause an increase in skin blood flow (approximately doubling
baseline values) due to removal of tonic vasoconstrictor tone,
mediated by sympathetic adrenergic nerves. Further increases
in core temperature produce robust dilation due to activation of
sympathetic vasodilator nerves, which release acetylcholine
and a cotransmitter (16), possibly vasoactive intestinal polypeptide (VIP) (2, 17), pituitary adenylate cyclase activating
peptide (PACAP) (17, 18), and/or substance P (48). Approximately 30 – 45% of the vasodilation to increased core temperature can be attributed to nitric oxide (NO) (15, 37, 45), for
Address for reprint requests and other correspondence: C. T. Minson, Dept.
of Human Physiology, Univ. of Oregon, Eugene, OR 97403-1240 (e-mail:
[email protected]).
1290
which neuronal NO synthase (nNOS) appears to be the predominant isoform responsible for its production (19, 20).
However, prostanoids (24) and H1 histamine receptors (49)
have been shown to play roles in active vasodilation, indicating
that the endothelium is likely still involved. Additionally,
heat-sensitive transient receptor potential vanilloid type 1
(TRPV-1) receptors located on the endothelium have been
shown to contribute to the response (47).
Despite a large amount of research in this field, a portion of
cutaneous active vasodilation remains unexplained. Endotheliumderived hyperpolarizing factors (EDHFs) are well known to
play a prominent role in other vascular beds, and recently our
lab has shown a robust role of EDHFs in the skin, in both the
responses to local thermal hyperemia (5) and reactive hyperemia (22). EDHFs elicit vasodilation by stimulating calciumactivated potassium (KCa) channels on the endothelium and
vascular smooth muscle. Potassium efflux through these channels then causes hyperpolarization, and thus relaxation of the
smooth muscle. Possible EDHFs include the epoxyeicosatrienoic acids (EETs), the lipoxygensase derivatives 12-Shydroxyeicosatetraenoic acid (12-S-HETE) and 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), and H2O2
(11). Our lab has recently shown EETs to be involved in
EDHF-mediated vasodilation in the skin (5). EDHF-mediated
vasodilation can be blocked with the nonspecific KCa channelinhibitor tetraethylammonium (TEA). In animal models, inhibition of KCa channels with TEA reduces VIP-induced relaxation (14, 21), lending to the possibility that the cotransmitters
involved in active vasodilation cause vasodilation in part by
stimulating the production of EDHFs.
Therefore, the purpose of our first experiment (protocol 1)
was to examine whether KCa channels play a role in active
vasodilation. We hypothesized that inhibition of KCa channels
with TEA would cause an attenuated response to passive
heating, and combined blockade of KCa channels and NO
synthase (NOS) with N-nitro-L-arginine methyl ester (LNAME) would reduce the response to a greater extent than
NOS inhibition alone.
To further explore observations from protocol 1, we designed protocol 2 as a follow-up study to determine the
involvement of inward rectifier (KIR) and/or ATP-sensitive
(KATP) potassium channels in active vasodilation. KIR channels
are found on both vascular endothelial (6, 42) and smooth
muscle cells (34, 36) and are thought to be involved in resting
membrane conductance and amplification of hyperpolarizing
stimuli (30), suggesting they may be involved with cross-talk
among the pathways controlling active vasodilation. KATP
channels are responsive to a large number of other agents,
including EETs (50), adenosine (8), prostacyclin (35, 39), and
NO (28), also making it likely for them to be involved in
cross-talk with KCa channels and/or NOS inhibition. Both KIR
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Brunt VE, Fujii N, Minson CT. No independent, but an interactive,
role of calcium-activated potassium channels in human cutaneous active
vasodilation. J Appl Physiol 115: 1290 –1296, 2013. First published
August 22, 2013; doi:10.1152/japplphysiol.00358.2013.—In human cutaneous microvasculature, endothelium-derived hyperpolarizing factors
(EDHFs) account for a large portion of vasodilation associated with
local stimuli. Thus we sought to determine the role of EDHFs in active
vasodilation (AVD) to passive heating in two protocols. Whole body
heating was achieved using water-perfused suits (core temperature
increase of 0.8 –1.0°C), and skin blood flow was measured using
laser-Doppler flowmetry. In the first protocol, four sites were perfused
continuously via microdialysis with: 1) control; 2) tetraethylammonium (TEA) to block calcium-activated potassium (KCa) channels,
and thus the actions of EDHFs; 3) N-nitro-L-arginine methyl ester
(L-NAME) to inhibit nitric oxide synthase (NOS); and 4) TEA ⫹
L-NAME (n ⫽ 8). Data are presented as percent maximal cutaneous
vascular conductance (CVC). TEA had no effect on AVD (CVC
during heated plateau: control 57.4 ⫾ 4.9% vs. TEA 63.2 ⫾ 5.2%,
P ⫽ 0.27), indicating EDHFs are not obligatory. L-NAME attenuated
plateau CVC to 33.7 ⫾ 5.4% (P ⬍ 0.01 vs. control); while TEA ⫹
L-NAME augmented plateau CVC compared with L-NAME alone
(49.7 ⫾ 5.3%, P ⫽ 0.02). From these data, it appears combined
blockade of EDHFs and NOS necessitates dilation through other
means, possibly through inward rectifier (KIR) and/or ATP-sensitive
(KATP) potassium channels. To test this second hypothesis, we measured AVD at the following sites (n ⫽ 8): 1) control, 2) L-NAME,
3) L-NAME ⫹ TEA, and 4) L-NAME ⫹ TEA ⫹ barium chloride
(BaCl2; KIR and KATP blocker). The addition of BaCl2 to L-NAME ⫹
TEA reduced plateau CVC to 32.7 ⫾ 6.6% (P ⫽ 0.02 vs. L-NAME ⫹
TEA), which did not differ from the L-NAME site. These data
combined demonstrate a complex interplay between vasodilatory
pathways, with cross-talk between NO, KCa channels, and KIR and/or
KATP channels.
KCa Channels and Cutaneous Active Vasodilation
(13, 40) and KATP (4, 25), channels can be inhibited by
extracellular barium ions, although this has only been shown to
be true of KATP channels in nonvascular smooth muscle. In
addition, the density of KATP channels is considerably lower
than KIR channels in vascular smooth muscle (35), making it
more likely that the effects of barium on the vasculature are the
result of KIR channel inhibition. Accordingly, we administered
barium chloride (BaCl2) to determine the role of KIR and/or
KATP channels in active vasodilation.
MATERIALS AND METHODS
Brunt VE et al.
1291
Drugs were infused for 60 min prior to the start of heating to achieve
full efficacy of TEA (5) and were continued at the same rate throughout heating. Postdrug baseline was recorded as the last 5 min of the
drug infusion period. During this time, water was circulated through
the water-perfused suit at 33°C.
Passive heating was achieved by circulating water at 50°C through
the water-perfused suit until Tor was increased by 0.8 –1.0°C. Heating
took approximately 45– 60 min. A plateau in skin blood flow of at
least 5 min was recorded at the highest temperature before ending heat
stress.
Upon completion of heating, subjects were cooled down by reducing the water temperature to 28°C. At this time, maximal skin blood
flux was attained by infusing 56 mM sodium nitroprusside (SNP;
Nitropress, Ciba Pharmaceuticals, East Hanover, NJ).
Pharmacological agents. We had two protocols in which we
passively heated subjects to measure cutaneous active vasodilation. In
each protocol, four microdialysis sites were established and continuously infused with the following drugs. All drugs were dissolved in a
lactated Ringer’s solution.
In protocol 1 (n ⫽ 8, 5 men, 3 women), the four sites were
1) control (lactated Ringer’s), 2) 50 mM TEA, 3) 20 mM L-NAME,
and 4) 20 mM L-NAME ⫹ 50 mM TEA. TEA (Sigma-Aldrich, St.
Louis, MO) was infused to block all subtypes of KCa channels. An
infusion concentration of 50 mM was selected as it was shown to be
the lowest concentration able to fully inhibit KCa channels in pilot
work associated with other studies from our laboratory (22). We also
performed pilot studies to ensure specificity of TEA at this concentration to KCa channels. In six subjects, 50 mM TEA had no effect on
the dilation observed when pharmacologically stimulating the opening
of KIR channels with potassium chloride, a response that was blocked
by BaCl2. Although studies in isolated cell models have shown a
concentration of 50 mM TEA to inhibit other types of potassium
channels, equilibration of the pharmacological agents infused with the
interstitium is unlikely to occur via microdialysis, particularly with
infusion rates as high as 2.0 ␮l/min. Thus a lower concentration than
50 mM is reaching the blood vessels. Specific inhibitors of BKCa and
SKCa channels, such as charybdotoxin and apamin, were not used as
these drugs are toxic to humans (33). L-NAME (Tocris Bioscience;
Minneapolis, MN) was infused through site 3 to inhibit NO synthase.
A concentration of 20 mM was selected for L-NAME based on
previous studies (5). NOS inhibition was used to separate the potential
NO-dependent and NO-independent effects of TEA, especially as the
full effects of EDHFs are commonly only observed in the presence of
NOS inhibition (51). Site 4 served as a combination site, in which both
50 mM TEA and 20 mM L-NAME were infused.
In protocol 2 (n ⫽ 8, 6 men, 2 women), the four microdialysis sites
were: 1) control (lactated Ringer’s), 2) 20 mM L-NAME, 3) 20 mM
L-NAME ⫹ 50 mM TEA, and 4) 20 mM L-NAME ⫹ 50 mM TEA ⫹
100 ␮M BaCl2 to block KIR and KATP channels. Sites 1 and 2 were
included in this protocol (despite having already been studied in
protocol 1) to be compared with the fourth site, in which we simultaneously blocked NO, KCa channels, and KIR and KATP channels.
BaCl2 was just added in the fourth site to investigate possible
cross-talk between the NO and hyperpolarization pathways. A concentration of 100 ␮M BaCl2 was selected based on pilot studies as the
concentration that gave the most effective inhibition of KIR-mediated
vasodilation to KCl (KIR channel opener). This concentration was
confirmed to completely block KCl-induced vasodilation up to concentrations of 10 mM KCl. Concentrations as low as 10 ␮M have been
shown to completely block conductance through both KIR channels in
isolated vascular smooth muscle cells (34); however, a higher concentration is required when delivered via microdialysis.
In two subjects in protocol 2 (1 man, 1 woman), we added a fifth
site which was infused with 100 ␮M BaCl2 to differentiate whether
KIR and/or KATP channels play a role in active vasodilation when all
other vasodilatory pathways are intact, versus only in the presence of
combined NOS and KCa channel inhibition.
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Subjects. Sixteen subjects participated in the study (10 men, 6
women). Two subjects participated in more than one protocol. All
subjects were young (18 –30 yr of age), healthy, nonsmokers, did not
have any history of cardiovascular disease, and were not taking any
medications, with the exception of oral contraceptives. Subjects reported to the laboratory on the study day having refrained from all
over-the-counter medications and heavy exercise for 24 h, and alcohol
and caffeine for 12 h, and having fasted for at least 4 h. All female
subjects were studied during menses, or during the placebo phase if
taking oral contraceptives, to minimize the effects of the female sex
hormones. Female subjects were also required to provide a negative
pregnancy test prior to participation in the study day. All studies were
conducted in a thermoneutral room (ambient temperature ⬃20°C)
with the subject resting in a supine position.
All subjects gave oral and written consent prior to participation in
the study, as set forth by the Declaration of Helsinki. All experimental
procedures were approved by the Institutional Review Board of the
University of Oregon.
Subject instrumentation. Subjects were instrumented with a fivelead electrocardiogram (CardioCap; Datex Ohmeda, Louisville, CO).
Beat-by-beat blood pressure was measured on the middle finger of the
nonexperimental arm with finger photoplethysmography (Nexfin;
BMEye, Amsterdam, The Netherlands) and was verified via brachial
auscultation (CardioCap) every 5 min. Subjects wore a water-perfused
suit to control whole body skin temperature, which covered all skin
surfaces except the experimental arm, face, hands, and feet, and a
water-impermeable plastic garment over the suit to prevent heat
evaporation. Oral temperature (Tor) was continuously monitored with
a thermistor placed in the sublingual sulcus, which was held in place
with tape on the cheek. While the thermistor was in place, subjects
were instructed to breathe through their nose and not to talk or open
their mouth. Mean skin temperature (Tsk) was calculated as the
weighted average of six copper-constantan thermocouples, placed on
the chest, abdomen, upper back, lower back, thigh, and calf (41).
Under aseptic conditions, four microdialysis fibers (30-kDa cutoff,
10-mm membrane; MD 2000; Bioanalytical Systems, West Lafayette,
IN) were placed at least 5 cm apart in the ventral skin of the left
forearm. Fibers were introduced using a 25-gauge needle, with entry
and exit points ⬃2.5 cm apart. Following needle insertion, fibers were
threaded through the lumen of the needle. The needle was then
removed, leaving just the fiber under the surface of the skin, with the
semipermeable membrane (1 cm in length) of the fiber centered
between the entry and exit points. A lactated Ringer’s solution was
infused through the fibers at a rate of 2 ␮l/min (CMA 102 Syringe
Pump; CMA Microdialysis AB, Solna, Sweden) until the infusion of
study drugs (see Pharmacological agents).
Laser-Doppler flowmetry (DRT-4 and MoorLab; Moor Instruments, Devon, UK) was used to measure red blood cell (RBC) flux,
and thus attain an index of skin blood flow, at each microdialysis site.
Study protocol. A period of 60 –90 min was allowed following
microdialysis fiber placement for the trauma associated with needle
insertion to subside. Once skin blood flow had stabilized, a 5-min
predrug baseline was recorded under thermoneutral conditions. Study
drugs were then infused through the fibers at a rate of 2.0 ␮l/min.
•
1292
KCa Channels and Cutaneous Active Vasodilation
RESULTS
All subjects studied were young (23.9 ⫾ 0.6 yr of age) and
healthy (body mass index 23.8 ⫾ 0.7 kg/m2; resting MAP
83.6 ⫾ 0.7 mmHg). Infusion of the study drugs resulted in an
attenuation of baseline CVC in the TEA, L-NAME ⫹ TEA, and
L-NAME ⫹ TEA ⫹ BaCl2 sites, but not in the L-NAME site.
These data are summarized in Table 1. There were no differences in maximal flux between sites (protocol 1: P ⫽ 0.25;
protocol 2: P ⫽ 0.69).
Passive heating (both protocols) increased Tor from 36.42 ⫾
0.06°C to 37.37 ⫾ 0.08°C. There were no significant differences between sites for Tor threshold at which active vasodilation was initiated when presented as absolute Tor or as ⌬Tor
from baseline (Table 2), although Tor threshold trended toward
being delayed in the L-NAME site (vs. control: protocol 1, P ⫽
0.10; protocol 2, P ⫽ 0.15).
Figure 1, A (protocol 1) and B (protocol 2), depicts the rise
in CVC across ⌬Tor from baseline. Data for the CVC plateau
at the end of passive heating are summarized in Fig. 2. NOS
inhibition with L-NAME attenuated the CVC response to
passive heating, consistent with previous studies (15, 18, 37,
45, 47). KCa channel blockade with TEA tended to augment
Table 1. Baseline data
Control
TEA
L-NAME
L-NAME ⫹ TEA
L-NAME ⫹ TEA ⫹ BaCl2
Predrug Infusion
Postdrug Infusion
8.4 ⫾ 1.3%
10.5 ⫾ 1.8%
6.3 ⫾ 0.8%
7.8 ⫾ 0.6%
7.8 ⫾ 1.1%
7.5 ⫾ 1.3%
4.7 ⫾ 0.6%
6.2 ⫾ 1.0%
4.1 ⫾ 0.5%
4.1 ⫾ 0.5%
P Value
n
0.10 16
⬍0.01
8
0.98 16
⬍0.001 16
⬍0.001 8
Data are presented as means ⫾ SE of % of maximal cutaneous vascular
conductance (CVC). Baseline CVC pre and post 60 min of infusion with the
study drugs are shown. Drugs include tetraethylammonium (TEA), N-nitro-Larginine methyl ester (L-NAME), and barium chloride (BaCl2). CVC was
significantly attenuated in the TEA, L-NAME ⫹ TEA, and L-NAME ⫹ TEA ⫹
BaCl2 sites.
Brunt VE et al.
Table 2. Tor threshold for active vasodilation
Protocol 1
Control
TEA
L-NAME
L-NAME ⫹ TEA
Protocol 2
Control
L-NAME
L -NAME ⫹ TEA
L-NAME ⫹ TEA ⫹ BaCl2
Absolute Tor at
Threshold, °C
⌬Tor at
Threshold, °C
36.55 ⫾ 0.07
36.50 ⫾ 0.08
36.60 ⫾ 0.07
36.61 ⫾ 0.09
0.20 ⫾ 0.05
0.14 ⫾ 0.05
0.25 ⫾ 0.04
0.27 ⫾ 0.05
36.57 ⫾ 0.10
36.71 ⫾ 0.12
36.59 ⫾ 0.14
36.61 ⫾ 0.10
0.13 ⫾ 0.03
0.27 ⫾ 0.06
0.15 ⫾ 0.05
0.17 ⫾ 0.04
Data are presented as means ⫾ SE in °C. Oral temperature (Tor) threshold
at which active vasodilation was initiated in both protocols, presented as both
absolute Tor and a change in Tor from normothermic baseline (⌬Tor). Drugs
include TEA, L-NAME, and barium chloride (BaCl2).
plateau CVC, although this effect was not significant (P ⫽
0.27). A power analysis (power ⫽ 0.80, ␣ ⫽ 0.05) revealed a
sample size of n ⫽ 67 would be needed to show a significant
difference between the control and TEA sites. Combined
blockade of NOS and KCa channels (TEA ⫹ L-NAME) did not
attenuate plateau CVC to as great an extent as L-NAME alone
and, in protocol 1, was not significantly different from the
control site (plateau CVC: P ⫽ 0.15). In protocol 2, the
addition of BaCl2 to TEA ⫹ L-NAME attenuated plateau CVC
compared with the TEA ⫹ L-NAME site back to the level of
the L-NAME site. In the two subjects who had a fifth fiber
infused with BaCl2 alone, there was no effect of BaCl2 compared with the control site. There were no differences in the
control, L-NAME, nor L-NAME ⫹ TEA sites between protocols 1 and 2.
DISCUSSION
The present study was designed to determine whether endothelium-derived hyperpolarization plays a role in the response
of skin blood flow to passive heating. Contrary to our hypotheses, blockade of KCa channels with TEA had no effect on
active vasodilation, and furthermore, blockade of KCa channels
in combination with NOS inhibition (with L-NAME) augmented active vasodilation compared with NOS inhibition
alone. Secondarily, we tested the follow-up hypothesis that this
augmentation of active vasodilation was the result of activation
of KIR and/or KATP channels, which was confirmed, as blockade of KIR and KATP channels (with BaCl2) in combination
with KCa channel and NOS inhibition returned active vasodilation back to the level of NOS inhibition alone. This is the first
time KCa channels have been studied in cutaneous active
vasodilation, and the first time KIR channels have been studied
in cutaneous tissue.
Protocol 1: role of EDHFs in active vasodilation. EDHFs
make up a class of vasodilators that elicit vasodilation via hyperpolarization of the vascular smooth muscle. Hyperpolarization is
achieved via stimulation of KCa channels on the endothelium and
smooth muscle. Common EDHFs include EETs and the lipoxygenase derivatives 12-S-HETE and 11,12,15-THETA (11),
among others. EDHFs, especially EETs, play an important role in
vasodilation in other vascular beds and are known to contribute to
vasodilation in the skin (5, 7).
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Data analysis. Data were digitized and stored on a computer at 20
Hz. Data were analyzed offline using signal-processing software
(Windaq; Dataq Instruments, Akron, OH). Cutaneous vascular conductance (CVC) was calculated as RBC flux divided by arterial blood
pressure. Blood pressure was attained from the Nexfin beat-by-beat
measurements and corrected to mean arterial pressure (MAP) attained
via brachial auscultation. All CVC data were normalized to 100% of
maximal CVC, as attained through infusion of SNP.
To quantify the skin blood flow response to passive heating, CVC
and Tsk were averaged over 5 min during the predrug baseline,
postdrug baseline, and plateau at the end of heat stress, which ranged
from a change of 0.8 to 1.0°C in ⌬Tor. CVC and Tsk were also
averaged over 1-min periods following each 0.1°C increase in Tor.
Threshold Tor for active vasodilation was determined by looking at the
CVC tracing over time and identified as the Tor at which a sustained
increase in CVC began after Tsk had increased to at least 38°C (18).
Threshold was identified by an investigator blinded to the drug at each
site and was confirmed by a second investigator.
Statistical analysis. Differences in baseline, plateau at the end of
heat stress, maximal flux, and Tor threshold for active vasodilation
were compared across drug sites using one-way repeated measures
analysis of variance (ANOVA). Pairwise interactions were analyzed
using the Student-Newman-Keuls post hoc test. Drug effects on
baseline (predrug baseline vs. postdrug baseline) and plateau CVC in
the same drug site across protocols were compared with Student’s
paired t-test.
•
KCa Channels and Cutaneous Active Vasodilation
Cutaneous Vascular Conductance
(% Maximal)
A
80
Control
TEA
L-NAME
L-NAME + TEA
70
60
50
*
*
*
40
*
30
*
20
*
10
*
*
*
*
*
*
*
0
Control
L-NAME
L-NAME + TEA
L-NAME + TEA + BaCl
80
70
*
50
40
*
*
30
*
*
*
*
20
*
10
*
*
*
*
0
C
2
60
*
*
*
*
*
*
*
*
*
*
40
39
Tsk
38
37
1293
Brunt VE et al.
and PACAP (17, 18), have been shown to act, in part, via
EDHFs in animal vessels (14, 21); however, it appears that
they do not in cutaneous active vasodilation. Vasodilation
induced by substance P, another possible cotransmitter (48), is
not affected by KCa channel inhibition, as shown in porcine
coronary arteries (31). To the best of our knowledge, no studies
have explored links between EDHFs and these neurotransmitters in humans. Our results also raise the question of what other
downstream vasodilatory agents are involved. Combined inhibition of NOS and prostanoids abolishes approximately twothirds of active vasodilation, (24); but the remainder is still
unknown. Furthermore, inhibition of NOS only blocks ⬃50%
of vasodilation to exogenous VIP (44); thus VIP must have
other effects in the skin. Perhaps the remaining dilation is the
result of VIP and/or other cotransmitter(s) acting directly on
smooth muscle. While this may be true of VIP, it is likely not
the case with PACAP as a recent study demonstrated that,
while blockade of PACAP receptors during whole body heating reduced CVC compared with the control site, combined
NOS and PACAP receptor inhibition did not attenuate CVC
beyond NOS inhibition alone (18), indicating PACAP works
solely in series with NOS.
Despite no role of EDHFs under normal conditions, when
TEA was infused in combination with L-NAME, active vasodilation was augmented compared with the NOS inhibitiononly site, and to an extent such that CVC was not statistically
different from that in the control site. These results were
opposite of what was hypothesized but still support the conclusion that KCa channels do not directly contribute to active
vasodilation. There are two possible explanations for this
observation. First, TEA may be acting on potassium channels
in the sympathetic cholinergic neurons. Although KCa channels
have never been shown to exist in cutaneous sympathetic
neurons, they are present in other types of neurons (12, 27).
TEA may also inhibit voltage-gated potassium (Kv) channels.
A concentration of 50 mM TEA (although likely lower than
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Fig. 1. A: response of cutaneous vascular conductance (CVC) to passive
heating relative to the rise in oral temperature (⌬Tor) for protocol 1. Infusion
of tetraethylammonium (TEA) had no effect on the CVC response compared
with the control site, whereas infusion with N-nitro-L-arginine methyl ester
(L-NAME) attenuated CVC. Combined infusion of TEA ⫹ L-NAME augmented the response compared with the L-NAME site. *P ⬍ 0.05 from the
control site; †P ⬍ 0.05 from the TEA site; ‡P ⬍ 0.05 from the L-NAME site.
B: response of CVC relative to ⌬Tor for protocol 2. The addition of barium
chloride (BaCl2) to L-NAME ⫹ TEA attenuated CVC compared with the
L-NAME ⫹ TEA, back to the level of the L-NAME alone site. *P ⬍ 0.05 from
the control site; †P ⬍ 0.05 from the L-NAME site; ‡P ⬍ 0.05 from the
L-NAME ⫹ TEA site. C: rise in mean skin temperature (Tsk) relative to
changes in ⌬Tor, averaged across subjects in protocols 1 and 2. Data are
presented as means ⫾ SE of %CVCmax.
In the present study, blockade of KCa channels with TEA
had no measurable effect on the cutaneous response to passive
heating, suggesting no role of EDHFs in active vasodilation
when other vasodilatory pathways are intact. In the context of
the current theory of cotransmission, this means that, normally,
the neurotransmitters released in response to increases in core
temperature do not cause vasodilation through stimulating the
production of EDHFs. The likely cotransmitters, VIP (2, 17)
Plateau Cutaneous Vascular Conducatance
(% Maximal)
36
80
Protocol 1
Protocol 2
60
*
*
40
*
*
20
0
Control
TEA
L-NAME
L-NAME
+ TEA
L-NAME
+ TEA
+ BaCl2
BaCl2
Fig. 2. The plateau in CVC observed at the end of heat stress for protocols 1
and 2. Heat stress resulted in rise in core temperature ranging from 0.8 to
1.0°C. Drugs include tetraethylammonium (TEA), N-nitro-L-arginine methyl
ester (L-NAME), and barium chloride (BaCl2). The dotted line represents the
average plateau CVC for the control site between the two protocols. Data are
presented as means ⫾ SE of %CVCmax. *P ⬍ 0.05 from the control site
within each protocol; †P ⬍ 0.05 between drug sites within each protocol.
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Cutaneous Vascular Conductance
(% Maximal)
B
•
1294
KCa Channels and Cutaneous Active Vasodilation
Brunt VE et al.
viding an excellent means of examining molecular pathways
relatively noninvasively, presents a greater challenge to dosing
as it is not possible to determine the exact concentration of the
drug once it reaches the interstitium.
The present study used TEA to examine the role of EDHFs
in active vasodilation. TEA has been reported to inhibit other
types of potassium channels (other than KCa) at concentrations
lower than what was used in the present study (9, 29). Although the concentration of the drug once it reached the blood
vessels would have been ⬍50 mM, our results should be
interpreted considering the possibility that TEA may have
inhibited other potassium channels; however, this does not
change the interpretation that KCa channels do not contribute to
active vasodilation. At a minimum, it appears unlikely that
TEA was also inhibiting barium-sensitive pathways.
We used BaCl2 to inhibit both KIR and KATP channels. A
more specific inhibitor of KIR channels is not available for use
in humans, and we did not specifically test the involvement of
KATP channels in this study. The commonly used KATPchannel inhibitor, glibenclamide, also has other effects, namely
on prostaglandin-induced vasodilation, the Na⫹-K⫹-ATPase
pump, and other potassium channels, although it does not
affect KCa or KIR channels (35), and so would not allow us to
conclude a role of only KATP channels in active vasodilation.
Last, it is important to recognize that the responses observed
when pharmacologically manipulating a system may not reflect
the true physiology of the intact system. For example, our
results suggest cross-talk between KCa channels and KIR and/or
KATP channels to be the cause of the augmentation of active
vasodilation when administering L-NAME ⫹ TEA relative to
the L-NAME only site. But perhaps drug interactions between
L-NAME and TEA, or even L-NAME and BaCl2, are (at least
partially) responsible for our results. Perhaps TEA prevents
L-NAME from effectively inhibiting NOS, thus allowing some
NO-dependent dilation to still occur. There is no way of testing
this in vivo, but the reader should be aware of the limitations
of our techniques.
Conclusions and perspectives: endothelium-derived hyperpolarization and the cutaneous microcirculation. To summarize, the present study has demonstrated that EDHFs and KCa
channels do not contribute to cutaneous active vasodilation
under normal conditions; however, combined inhibition of KCa
channels and NOS initiates cross-talk between these pathways
and other vasodilatory pathways, namely KIR and/or KATP
channels. Previously, EDHFs have been shown to significantly
contribute to the cutaneous responses to reactive hyperemia (7,
22) and local thermal hyperemia (5). Taken together with the
results of the present study, while EDHFs may represent a
predominant means of vasodilation in response to local stimuli,
their role when vasodilation is mediated by sympathetic cholinergic nerves is clearly very different.
ACKNOWLEDGMENTS
We graciously thank the subjects for participation.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-081671.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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this once in the interstitium) is high enough to inhibit some
types of Kv channels (23). Inhibition of neuronal KCa and/or Kv
channels would cause depolarization and increased neurotransmitter release, thus augmenting active vasodilation. However,
if this were occurring, we would expect to have also seen an
augmentation of active vasodilation in the TEA-only site,
which was not observed.
A more likely explanation is that blockade of KCa channels
in addition to NOS inhibition augments active vasodilation by
necessitating vasodilation via other pathways. EDHFs have
often been implicated as a ‘back-up’: mechanism when other
routes of vasodilation are impaired, as the effects of EDHFs are
commonly not observed until NOS is blocked (1, 26, 38).
Furthermore, cross-talk is known to exist between EDHFs and
other vasodilatory pathways (3, 43). Two candidates for vasodilatory pathways that may be upregulated during active vasodilation when NOS and KCa channels are simultaneously
blocked are KIR and KATP channels.
Protocol 2: cross-talk between KCa and KIR channels. Protocol 2 was designed to test the hypothesis that when NOS and
KCa channels are blocked, KIR and/or KATP channels are
activated, thus allowing dilation to still occur, as shown in
protocol 1. Our results from protocol 2 confirm this hypothesis
as the augmentation of active vasodilation observed with combined blockade of NOS and KCa channels compared with NOS
block alone was removed by the addition of BaCl2. Furthermore, BaCl2 alone had no effect on dilation, indicating that the
involvement of KIR and/or KATP channels is triggered by
combined blockade of NOS and KCa channels.
These data suggest cross-talk between NO, KCa channels,
and KIR and/or KATP channels. Currently, there is little known
about the potential mechanisms responsible for cross-talk between KCa and KIR channels, mostly owing to the fact that KIR
channels are still relatively poorly understood. EDHFs activate
KCa channels by eliciting calcium sparks from the sarcoplasmic
reticulum (10); however, calcium has been shown to inhibit
both KIR (36) and KATP channels (32). KIR channels have been
shown to be activated by protein kinases (46). It is possible the
change in calcium conductance within the cell, combined with
other changes that occur during passive heating, leads to a
protein kinase signaling cascade which activates KIR channels.
KATP channels can be opened by EETs (50), which may occur
to a greater extent when KCa channels are also blocked.
Additionally, hyperpolarizing pathways may have been triggered due to changes in membrane potential. TEA significantly
reduced baseline CVC, indicating depolarization at rest. Although depolarization likely would not directly open KIR or
KATP channels during whole body heating (35), it may have
played some role in their involvement.
Limitations: pharmacological agents. The present study and
all studies conducted in intact humans in vivo are limited in
two ways. First, we are limited to using only pharmacological
agents that can safely be administered in humans, and thus we
are unable to more specifically pharmaco-dissect the mechanisms at play. While more specific blockers exist than the ones
used in the present study, they are unsuitable for use in
humans. Second, we are faced with the challenge of not being
able to determine exactly which pathways are being affected by
our interventions. Many pharmacological agents can have
multiple effects, which may or may not be dependent on the
dose of the drug. The technique of microdialysis, while pro-
•
KCa Channels and Cutaneous Active Vasodilation
AUTHOR CONTRIBUTIONS
Author contributions: V.E.B., N.F., and C.T.M. conception and design of
research; V.E.B. and N.F. performed experiments; V.E.B. analyzed data;
V.E.B. and C.T.M. interpreted results of experiments; V.E.B. prepared figures;
V.E.B. drafted manuscript; V.E.B., N.F., and C.T.M. edited and revised
manuscript; V.E.B., N.F., and C.T.M. approved final version of manuscript.
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