J Appl Physiol 109: 95–100, 2010.
First published April 15, 2010; doi:10.1152/japplphysiol.01187.2009.
VIP/PACAP receptor mediation of cutaneous active vasodilation during heat
stress in humans
Dean L. Kellogg, Jr.,1,2,3 Joan L. Zhao,1,2 Yubo Wu,1,2 and John M. Johnson3
1
Geriatric Research, Education, and Clinical Center, Department of Veterans Affairs, South Texas Veterans Health Care
System, Audie L. Murphy Memorial Veterans Hospital Division; and 2Division of Geriatrics and Gerontology, Department
of Medicine and 3Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas
Submitted 19 October 2009; accepted in final form 13 April 2010
laser-Doppler flowmetry; VPAC2; PAC1; thermoregulation; microdialysis
is a major effector of human
thermoregulatory reflex responses. During periods of cold
stress, constriction of skin blood vessels with consequent
reductions in skin perfusion facilitates maintenance of thermal
homeostasis by reducing loss of heat to the environment. In
periods of heat stress, thermal homeostasis is maintained by
dilation of the cutaneous vasculature to increase heat loss to the
environment by delivering excess heat to the body surface
where it can be removed in conjunction with evaporative
cooling through sweat production.
THE CUTANEOUS VASCULATURE
Address for reprint requests and other correspondence: D. L. Kellogg, Jr.,
Div. of Geriatrics and Gerontology, Dept. of Medicine, Univ. of Texas Health
Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229
(e-mail: [email protected]).
http://www.jap.org
In nonglabrous (hairy) skin, blood flow (SkBF) changes
during thermoregulatory reflexes are effected by two branches
of the sympathetic nervous system. During cold stress, cutaneous vasoconstriction is mediated by increased tone in vasoconstrictor nerves that release norepinephrine and NPY to
effect reductions in SkBF (17, 41). During heat stress, cutaneous vasodilation is mediated by increased activity in the cutaneous active vasodilator system. This system involves release
of acetylcholine (ACh) as well as one or more cotransmitters
from cutaneous cholinergic sympathetic nerves (24). According to present understanding, the release of ACh and one or
more cotransmitters (probably neuropeptides, but at present of
uncertain identity) from cholinergic nerves increases SkBF
during heat stress to cause cutaneous vasodilation to facilitate
heat loss in combination with sweat production (23). This
cutaneous active vasodilator system is critical to human tolerance of endogenous and endogenous heat stress as heat-related
illnesses including heat exhaustion and heat stroke occur when
it fails (21, 23, 33). This system can cause SkBF to increase to
as much as 8 l/min or 60% of cardiac output at the peak of heat
stress (34). While the vasodilator potential and overall physiological import of this system is well understood, the nature of
the neurotransmitters involved remains unclear.
Neuropeptides of the secretin/glucagon/VIP/pituitary adenylate cyclase activating peptide (PACAP) family have been
implicated in cotransmission in the cutaneous active vasodilator system (2, 18) albeit not without controversy (49, 51). The
members of this neuropeptide superfamily that have been
identified in human skin are the cotranslated and coexpressed
peptides, VIP and peptide histidine methionine (PHM), along
with the related peptide, PACAP. VIP and PHM are structurally related peptides that are encoded by the same gene and are
cosynthesized from the same precursor and therefore co-occur
in essentially equal amounts in neurons (9, 45). PACAP has a
68% homology with VIP (9, 19, 29, 32, 37, 40). PACAP occurs
in two forms: PACAP38 and a truncated derivative, PACAP27.
PACAP38 has been found in cutaneous nerves in association
with both sweat glands and blood vessels while PACAP27
appears to be absent from skin (29, 40). VIP/PHM and PACAP
can be colocalized with ACh and all cause vasodilation with
PACAP being the most potent and PHM the least potent (32);
thus any or all of these peptides may be considered as candidate neurotransmitters for the cholinergic cutaneous active
vasodilator system (30, 47, 48).
Members of the secretin/glucagon/VIP/PACAP peptide
family mediate their effects through G protein-coupled receptors. Since initially identified, several nomenclatures for these
receptors have been used, i.e., VIP1, VIP2, PACAP1, etc.;
however, a specific nomenclature has now been established
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Kellogg DL Jr, Zhao JL, Wu Y, Johnson JM. VIP/PACAP
receptor mediation of cutaneous active vasodilation during heat stress
in humans. J Appl Physiol 109: 95–100, 2010. First published April
15, 2010; doi:10.1152/japplphysiol.01187.2009.—Vasoactive intestinal peptide (VIP) is implicated in cutaneous active vasodilation in
humans. VIP and the closely related pituitary adenylate cyclase
activating peptide (PACAP) act through several receptor types: VIP
through VPAC1 and VPAC2 receptors and PACAP through VPAC1,
VPAC2, and PAC1 receptors. We examined participation of VPAC2
and/or PAC1 receptors in cutaneous vasodilation during heat stress by
testing the effects of their specific blockade with PACAP6 –38.
PACAP6 –38 dissolved in Ringer’s was administered by intradermal
microdialysis at one forearm site while a control site received Ringer’s
solution. Skin blood flow was monitored by laser-Doppler flowmetry
(LDF). Blood pressure was monitored noninvasively and cutaneous
vascular conductance (CVC) calculated. A 5- to 10-min baseline
period was followed by ⬃70 min of PACAP6 –38 (100 ␮M) perfusion
at one site in normothermia and a 3-min period of body cooling.
Whole body heating was then performed to engage cutaneous active
vasodilation and was maintained until CVC had plateaued at an
elevated level at all sites for 5–10 min. Finally, 58 mM sodium
nitroprusside was perfused through both microdialysis sites to effect
maximal vasodilation. No CVC differences were found between
control and PACAP6 –38-treated sites during normothermia (19 ⫾
3%max untreated vs. 20 ⫾ 3%max, PACAP6 –38 treated; P ⬎ 0.05
between sites) or cold stress (11 ⫾ 2%max untreated vs. 10 ⫾
2%max, PACAP6 –38 treated, P ⬎ 0.05 between sites). PACAP6 –38
attenuated the increase in CVC during whole body heating when
compared with untreated sites (59 ⫾ 3%max untreated vs. 46 ⫾
3%max, PACAP6 –38 treated, P ⬍ 0.05). We conclude that VPAC2
and/or PAC1 receptor activation is involved in cutaneous active
vasodilation in humans.
96
VIP/PACAP RECEPTORS AND CUTANEOUS ACTIVE VASODILATION
Glossary
CVC
LDF
MAP
PACAP
PACAP6 –38
PAC1
PHM
PR
Cutaneous vascular conductance
Laser-Doppler flowmetry
Mean arterial pressure
Pituitary adenylate cyclase activating peptide
VPAC2 and PAC1 receptor antagonist
PACAP Receptor
Peptide histidine methionine
Pulse rate
J Appl Physiol • VOL
SkBF
SNP
Tloc
Tor
Tsk
VPAC1
VPAC2
VIP
VIP10 –28
Skin blood flow
Sodium nitroprusside
Local skin temperature
Oral temperature
Skin temperature
VIP and PACAP receptor, type 1
VIP and PACAP receptor, type 2
Vasoactive intestinal peptide
VPAC1 and VPAC2 receptor antagonist
METHODS
Antagonism of VPAC2 and PAC1 receptors was achieved by
administration of PACAP6 –38, a VPAC2 and PAC1 receptor selective antagonist that is without activity at VPAC1 receptors (27). Drug
delivery was achieved by intradermal microdialysis to permit direct,
local administration of PACAP6 –38 into the interstitial space of a
small area of skin. This approach permitted simultaneous local monitoring of SkBF from an untreated control site and an adjacent locally
treated site within each subject without confounding systemic effects.
SkBF was monitored by laser-Doppler flowmetry (LDF, MoorLab,
Moor Instruments, Devon, UK) from the same small volume of skin
(⬃1 mm3) over each microdialysis probe. LDF measurements are
specific to skin and are uninfluenced by blood flow in the underlying
skeletal muscle (36).
Preliminary experiments were performed to determine the appropriate PACAP6 –38 concentration to achieve an optimal VPAC2/
PAC1 receptor blockade that would consistently antagonize the receptors without the potentially confounding effects of altering basal
SkBF (51). In the first of these studies, we used intradermal microdialysis and tested the effects of 50, 100, and 200 ␮M PACAP6 –38
concentrations on basal SkBF. These four studies showed that 50 ␮M
and 100 ␮M PACAP6 –38 did not alter basal SkBF but that 200 ␮M
PACAP6 –38 increased SkBF to 32 ⫾ 3% of maximal CVC. Maximal
absolute CVC levels as achieved with 58 mM sodium nitroprusside
(SNP) were not altered by 50, 100, or 200 ␮M PACAP6 –38. Based
on these preliminary studies, we chose to further evaluate the antagonist properties of 100 ␮M PACAP6 –38 for use in our whole body
heat stress protocol.
As there is no specific VPAC2/PAC1 agonist available (25–27), we
chose to examine the ability of PACAP6 –38 to antagonize the
vasodilation produced by 30 ␮M PACAP38, an agonist at VPAC1,
VPAC2, and PAC1 receptors. This concentration of PACAP38 was
found to increase cutaneous vascular conductance (CVC) to 89 ⫾ 7%
of maximal levels. VIP was not chosen because this peptide is only a
weak agonist at PAC1 receptors compared with PACAP38 (25–27).
As illustrated in Fig. 1, 100 ␮M PACAP6 –38 consistently attenuated
the vasodilation induced by 30 ␮M PACAP38 (P ⬍ 0.05). Based on
our preliminary studies, we chose 100 ␮M PACAP6 –38 for use in our
whole body heat stress protocol.
Nine healthy subjects (4 men and 5 women) participated in the main
portion of this study. The subjects’ average age ( ⫾ SE) was 34 ⫾ 4 yr,
average weight was 62 ⫾ 2 kg, and average height was 169 ⫾ 2 cm. All
subjects were in good health as documented by medical history and
physical examination and were taking no medications, and all were
nonsmokers. All subjects gave their informed consent to participate in
this institutionally approved study. There was no caffeine intake on the
day of the study. The menstrual phase of the female subjects at the time
of the study was not controlled for.
Microdialysis probes were made in our laboratory from polyimide
tubing and a 1-cm length of capillary microdialysis membrane
(200-␮m diameter, molecular mass cutoff 20 kDa) reinforced by a
51-␮m-diameter coated stainless steel wire placed in the lumen of the
membrane and tubing. Upon arrival in the laboratory, each subject had
two probes placed at separate sites on the ventral aspect of one
forearm. Ice was used at each skin site to achieve temporary local
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with VPAC1, VPAC2, and PAC1 as accepted receptor terminology (15). VPAC1 and VPAC2 receptors have essentially
equivalent high affinities for both VIP and PACAP38. PAC1
receptors have high affinity for PACAP38 and bind VIP with
much lower affinity (0.01%). PHM has little if any affinity for
VPAC1 receptors and binds to VPAC2 receptors with a much
lower affinity than does either VIP or PACAP (25–27, 32, 45).
VPAC1, VPAC2, and PAC1 receptors all have been found in
human nonglabrous skin: VPAC2 and PAC1 receptors appear
to be the predominant types while the number of VPAC1
receptors was reported to be sparse (28).
Evidence implicating the secretin/glucagon/VIP/PACAP
peptide family in cutaneous active vasodilation in humans is
mixed. Bennett et al. (2) found that VIP10 –28, a truncated
derivative of VIP produced by mast cells with VPAC1 and
inconsistent VPAC2 receptor antagonist properties (5, 42),
significantly attenuated the increase in cutaneous vascular
conductance (CVC) relative to internal temperature during
whole body heating (2). These results suggested an important
mechanistic role for VPAC1 and/or VPAC2 receptors and VIP
in cutaneous active vasodilation; however, contrary evidence
also exists. For example, patients with cystic fibrosis have
sparse but not zero levels of VIP in nonglabrous skin, yet these
patients appear to have normal cutaneous active vasodilator
responses to heat stress (22, 37, 50). In addition, Wilkins et al.
(49, 51) found that a higher concentration of VIP10 –28 than
used by Bennett at al (2) augmented cutaneous active vasodilation in heat stress. To add further uncertainty, the antagonist
properties of VIP10 –28 on VPAC2 receptors appear to be
variable (13, 46). The realization that PACAP coexists with
VIP in skin neurons compounds the complexity of defining
roles for these neuropeptides and their receptors in cutaneous
active vasodilation (10).
The foregoing summary of in vivo evidence for and against
a mechanistic role for neuropeptides of the secretin/glucagon/
VIP/PACAP peptide family in cutaneous active vasodilation
outlines why such a role remains under debate. We sought to
clarify this issue by examining the effects of an alternative to
the VPAC receptor antagonist, VIP10 –28, on the vasodilation
induced by whole body heating to test whether members of this
superfamily of peptides participate in cutaneous active vasodilation. We hypothesized that the two more common receptors
for these peptides in skin, specifically VPAC2 and PAC1
receptors rather than VPAC1 type that is relatively sparse in
skin (28), are activated to increase SkBF during heat stress in
humans. We tested our hypothesis by examining the effect of
PACAP6 –38, a truncated version of PACAP38 with recognized antagonist properties for VPAC2 and PAC1 receptors, on
responses in SkBF during thermoregulatory reflexes in humans.
VIP/PACAP RECEPTORS AND CUTANEOUS ACTIVE VASODILATION
anesthesia before microdialysis probe placement (16). For probe
placement, a 25-gauge needle was inserted through the dermis using
sterile technique. Entry and exit points were approximately 2.5–3 cm
apart. A microdialysis probe was subsequently threaded through the
lumen of the needle, which was withdrawn, leaving the probe in place
with the microdialysis membrane entirely within the dermis. After
probe insertion and before additional instrumentation, subjects waited
2 h or longer for resolution of insertion trauma (1).
To induce thermoregulatory reflexes, subjects wore a tube-lined
suit to control skin temperature (Tsk) by perfusion with water to
control Tsk of different temperatures (35). For normothermia, the suit
was perfused with water at approximately 33–34°C. The suit was
perfused with 16 –17°C water to decrease Tsk and induce cold stress
and 48°C water to raise Tsk to 38 –39°C during heating periods. Over
the suit, subjects wore a water-impermeable plastic garment to insulate them from the environment and prevent sweat evaporation. The
suit and garment covered the entire body with the exception of the
head, hands, feet, and the forearm from which measurements were
made.
Internal temperature was monitored with a thermocouple held in the
sublingual sulcus (Tor). Tsk was recorded as the weighted electrical
average from six thermocouples taped on the skin surface (20, 44). Pulse
rate (PR) and mean arterial pressure (MAP) were recorded continuously
from a finger (Finapres BP Monitor, Ohmeda, Madison, WI).
For the study, subjects were placed in the supine position and
instrumented. Tsk was maintained at 34°C. Data collection began with
a 5- to 10-min baseline control period during which the microdialysis
probes were perfused with Ringer’s solution only. Perfusion was done
at a rate of 3 ␮l/min using a microinfusion pump. After this control
period, the perfusate at one site was changed to 100 ␮M PACAP6 –38
in Ringer’s. Perfusion was maintained with Ringer’s only at the
second site. Perfusion was maintained with these solutions for 40 – 45
min under normothermic conditions to allow PACAP6 –38 to reach a
steady state in the intradermal space. Following this normothermic
period, Tsk was decreased to induce cold stress for 3 min. Tsk was
J Appl Physiol • VOL
then raised to 38 –39°C and maintained at that level for 35–50 min to
induce heat stress and thereby stimulate the active vasodilator system.
After heat stress, subjects were cooled and returned to a normothermic
Tsk of 34°C. All microdialysis sites were then perfused with 58 mM
SNP to effect maximal vasodilation at each site. CVC values were
normalized to these maximal levels for data analysis. The protocol is
illustrated in Fig. 2.
Data are presented as means ⫾ SE. CVC values were indexed as
LDF/MAP and were normalized to their respective maxima as elicited
by 58 mM SNP to facilitate comparisons among sites both within and
among subjects after verifying that there were no statistically significant differences among the absolute LDF values achieved with SNP
perfusion (P ⬎ 0.05 between sites). The CVC responses at the two
microdialysis sites were analyzed by comparing the Tor thresholds at
which CVC began to increase during whole body heating. The internal
temperature threshold for the onset of vasodilation for each site was
defined as the Tor that prevailed when a sustained increase in CVC
began during whole body heating. Tor thresholds were chosen from
graphs of CVC vs. Tor by an investigator who was blinded as to the
conditions, subjects, and antagonist treatment. These thresholds were
compared by ANOVA for repeated measures. Normothermic baseline
CVC values, CVC levels during the final minute of drug infusion in
normothermia, CVC during the final minute of cold stress, and CVC
from the final 3 min of heat stress were also compared among sites by
ANOVA, followed by Student-Newman-Keuls test. MAP, pulse rate
(PR), and Tor changes from normothermia to the peak of heat stress
were compared by paired t-tests. The level of statistical significance
was defined as P ⬍ 0.05.
RESULTS
An example of the responses in skin blood flow to the body
heating protocol is illustrated in Fig. 3. In normothermia,
during perfusion with Ringer’s at both microdialysis sites,
CVC values did not differ between sites (18 ⫾ 2 vs. 17 ⫾
2%max, P ⬎ 0.05). Changing the microdialysis perfusate to
100 ␮M PACAP6 –38 at one site did not alter CVC (P ⬎ 0.05
between sites). In the last 3 min of normothermia, CVC at sites
perfused with 100 ␮M PACAP6 –38 averaged 19 ⫾ 3%max
while at control sites CVC averaged 20 ⫾ 3%max (P ⬎ 0.05
between sites).
During cold stress, CVC was reduced at both sites (P ⬍ 0.05
cold stress vs. normothermia). CVC values fell to 11 ⫾ 2%max
CVC at sites treated with PACAP6 –38 and to 10 ⫾ 2%max
Fig. 2. Whole body heat stress protocol. This protocol was designed to examine
the effects of antagonism of VPAC2 and PAC2 receptors for VIP and pituitary
adenylate cyclase activating peptide (PACAP) on the cutaneous vasodilation
induced by whole body heat stress. Two intradermal microdialysis sites were
used to perfuse intradermal microdialysis fibers with Ringer’s solution alone at
an untreated control site and with 100 ␮M PACAP6 –38, a VPAC2/PAC1
receptor antagonist. Maximal cutaneous vasodilation was achieved at the end
of the study by perfusion with 58 mM sodium nitroprusside (SNP) at both sites.
The perfusion rate at all microdialysis sites was 3 ␮l/min. Tsk, skin temperature.
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Fig. 1. Summary of attenuating effect of 100 ␮M PACAP6 –38 on the
vasodilation induced by 30 ␮M PACAP38 in 4 subjects. PACAP6 –38 did not
alter basal cutaneous vascular conductance (CVC) levels (P ⬎ 0.05).
PACAP38 increased CVC significantly from basal levels in the absence and
presence of PACAP6 –38 (P ⬍ 0.05); however, the vasodilation induced by
PACAP38 was significantly reduced by PACAP6 –38 (P ⬍ 0.05).
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VIP/PACAP RECEPTORS AND CUTANEOUS ACTIVE VASODILATION
Fig. 3. CVC response to the body heating protocol in 1 subject. Perfusion with
100 ␮M PACAP6 –38 Ringer’s began 10 min into the study. Periods of whole
body cooling (CS) and heating are indicated. Perfusion with 58 mM SNP
began at 120 min. The site receiving PACAP6 –38 showed less vasodilation
during heat stress than did the control site.
DISCUSSION
The main finding of this study is that antagonism of VPAC2
and PAC1 receptors in forearm skin significantly attenuates the
increases in SkBF during thermoregulatory reflex responses to
whole body heat stress in humans. This finding demonstrates
that activation of one or both of these receptors by members of
the secretin/glucagon/VIP superfamily of peptides mediates a
significant part of thermoregulatory reflex cutaneous active
vasodilation in nonglabrous skin.
In contrast to attenuating the vasodilation caused by whole
body heat stress, we found that VPAC2/PAC1 receptor antagonism did not alter reflex vasoconstriction caused by whole
body cooling. This result shows that these receptors and hence
members of the VIP/PACAP peptide family are not involved in
effecting cutaneous vasoconstriction during cold stress. In
addition, that vasoconstriction at untreated control sites did not
differ from that observed at VPAC2/PAC1 blocked sites indicates that PACAP6 –38 did not alter the responsiveness of the
J Appl Physiol • VOL
Fig. 4. Summary of CVC responses. Under normothermic conditions during
perfusion of both microdialysis sites with Ringer’s solution, CVC values,
expressed as a percentage of their respective maxima, did not differ significantly (P ⬎ 0.05 between sites). In normothermia, changing the perfusion of
one site to 100 ␮M PACAP6 –38 in Ringer’s did not produce any significant
difference with CVC at sites perfused with Ringer’s alone (P ⬎ 0.05). Cold
stress significantly reduced CVC at both sites compared with normothermia
(Normo) (P ⬍ 0.05). These responses did not differ between sites (P ⬎ 0.05).
During heat stress, CVC increased at both microdialysis sites compared with
normothermia (P ⬍ 0.05); however, this increase was significantly attenuated
at sites perfused with 100 ␮M PACAP6 –38 (P ⬍ 0.05, PACAP6 –38 vs.
Ringer’s).
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CVC at control sites. These responses did not differ between
sites (P ⬎ 0.05).
In normothermia, MAP averaged 77 ⫾ 3 mmHg and averaged 71 ⫾ 4 mmHg at the peak of heat stress (P ⬎ 0.05,
normothermia vs. heat stress). PR averaged 62 ⫾ 2 beats/min
in normothermia and increased to 84 ⫾ 5 beats/min at the peak
of heat stress (P ⬍ 0.01). Tor averaged 36.34 ⫾ 0.09°C in
normothermia and increased to 37.11 ⫾ 0.10°C at peak heat
stress (P ⬍ 0.01).
During whole body heating, CVC at the sites perfused with
Ringer’s began to rise when Tor reached values of 36.62 ⫾
0.09°C and 36.59 ⫾ 0.19°C for sites perfused with PACAP6 –
38. There was no statistically significant difference between
the threshold values at the two sites (P ⬎ 0.05).
At the peak of heat stress, CVC at sites perfused with
Ringer’s increased to 59 ⫾ 3%max CVC (P ⬍ 0.05 vs.
normothermia). CVC at sites perfused with PACAP6 –38 increased to 46 ⫾ 3%max CVC (P ⬍ 0.05 vs. normothermia), a
response that was significantly attenuated compared with that
at the Ringer’s sites (P ⬍ 0.05). Averaged CVC responses for
the protocol are summarized in Fig. 4.
Following heat stress, maximal CVC in response to 58 mM
SNP infusion did not differ between sites that were untreated
and those that received PACAP6 –38 (P ⬎ 0.05).
vascular smooth muscle itself, precluding a nonspecific attenuated vascular responsiveness as an explanation of the attenuating effects of PACAP6 –38 on SkBF increases during heat
stress.
The foregoing conclusions are based on our demonstration
that PACAP6 –38 attenuated the vasodilator response evoked
by administration of the agonist PACAP38 significantly. We
were, however, unable to block completely the vasodilation
induced by exogenous PACAP38 with PACAP6 –38. While we
may have been able to achieve greater blockade with higher
concentrations of PACAP6 –38 we found higher concentrations induced a vasodilation, perhaps due to activating effects
of higher concentrations or inflammatory mechanisms such as
mast cell destabilization (14, 32). The failure to achieve complete blockade could be a consequence of limiting the concentration of PACAP6 –38 to 100 ␮M to avoid any increase in
basal SkBF. We limited our concentration of PACAP6 –38 to
100 ␮M to avoid potentially confounding effects of pharmacological increases in baseline as identified by Wilkins et al.
(51). This allowed us to be certain that cutaneous active
vasodilation was actually attenuated by PACAP6 –38 rather
than an artifact of baseline elevation. We therefore chose a
PACAP6 –38 concentration of 100 ␮M as an optimal one that
would allow us to define whether VPAC2/PAC1 receptor
activation was involved in the mechanism of cutaneous active
vasodilation. We reasoned that although this choice may have
precluded estimation of the absolute VPAC2/PAC1 contribution to cutaneous active vasodilation because of uncertainty
about the extent of receptor blockade, an attenuation of cutaneous active vasodilation without potentially confounding
baseline effects would best prove or refute our hypothesis of
VPAC2/PAC1 receptor involvement.
Another factor that could have contributed to our apparent
inability to abolish the vasodilation induced by PACAP38 may
be because it is an agonist for VPAC1 as well as VPAC2 and
VIP/PACAP RECEPTORS AND CUTANEOUS ACTIVE VASODILATION
J Appl Physiol • VOL
nonglabrous skin; however, their precise location has not been
elucidated (40). These findings show that the receptors for both
VIP and PACAP38 are also found in microanatomic locations,
consistent with a mechanistic role in cutaneous active vasodilation.
How VIP and/or PACAP38 activation of the VPAC/PAC1
receptors effects cutaneous vasodilation is unknown, however,
given that activation of these receptors by physiological concentrations of VIP and/or PACAP activates adenylyl cyclase in
general. On sweat glands in particular, cAMP seems likely to
be involved as a second messenger (6, 7, 43). Less likely is the
release of histamine. Although exogenous VIP and PACAP38
have been found to induce histamine release from mast cells,
such release occurs only at high (micromolar) concentrations
of agonist by non-receptor-mediated, direct interaction with G
proteins (14, 29, 49). Nitric oxide production may also be
involved (49).
Finally, the antagonism by 100 ␮M PACAP6 –38 accounted
for ⬃30% of the increase in CVC from normothermia to the
peak of heating. The source of the remaining 70% of the
response is not clear. As previously discussed, the remainder
could be due to incomplete blockade of the VPAC2/PAC1
receptors or to activation by the native agonist of unblocked
VPAC1 receptors. It might also be ascribed to other transmitter-receptor groups, for example ACh (24, 31, 38, 39), or
neurokinins (52, 53). Further research with combinations of
more specific antagonists will be required to distinguish among
these possibilities.
In summary, antagonism of VPAC2/PAC1 receptors for
VIP, PACAP38, and related members of their neuropeptide
family attenuates SkBF increases induced by whole body heat
stress. This shows that one or more of these neuropeptides and
their cognate receptors are involved in increasing SkBF during
whole body heat stress. Overall, these data strongly support a
role for VPAC2/PAC1 receptor activation in cutaneous active
vasodilation and therefore suggest that members of the secretin/glucagon/VIP/PACAP superfamily of neuropeptides are
also involved.
GRANTS
This work was supported in part by National Heart, Lung, and Blood
Institute Grant HL-065599.
DISCLOSURES
No conflicts of interest (financial or otherwise) are declared by the authors.
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PAC1 receptors. PACAP6 –38 antagonizes VPAC2 and PAC1
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cannot exclude a role for VPAC1 receptors in active vasodilation during whole body heat stress.
A prior study from our laboratory showed that VIP10 –28, an
antagonist of VPAC1, and inconsistently of VPAC2 receptors
(13, 46), attenuated the rise of SkBF during heat stress (2).
While this study supported a role for activation of these
receptors in the mechanism of cutaneous active vasodilation,
subsequent work by Wilkins et al. (49, 51) suggested that
VIP10 –28 had some characteristics of a receptor agonist.
VIP10 –28 is a truncated version of VIP that is produced
in vivo by lymphocytes and has a relatively low affinity for
VPAC1 and VPAC2 receptors (42). These attributes are problematic in interpreting in vivo effects of this agent; however,
the findings of the present study are consistent with the work
by Bennett et al. (2).
The present demonstration that VPAC2/PAC2 receptor activation is involved in the mechanism of cutaneous active
vasodilation indicates that release of neuropeptides belonging
to the VIP/PACAP peptide family is involved in the process.
Within this peptide family, both VIP and PACAP38 are found
in skin, while the truncated version of PACAP38, PACAP27,
is not (40, 47, 48). Both VIP and PACAP38 are potent
vasodilators in skin (10, 47, 48). PHM, another member of the
VIP/PACAP peptide family, has also been found in human
skin colocalized in equal amounts with VIP (9, 19). No specific
receptor has been found for PHM in mammals (45). PHM has
a much lower binding affinity than VIP and PACAP for
VPAC1 and VPAC2 receptors, and especially PAC1 receptors
(26) and has a potency several orders of magnitude lower than
those of VIP or PACAP (10). Since VIP and PHM co-occur
and are coreleased, any contribution of PHM to cutaneous
vasodilation would be relatively smaller than that of VIP (10,
25–27).
Cutaneous immunohistochemical studies of VIP-containing
neurons have revealed abundant VIP-containing fibers that
innervate cutaneous arterioles and sweat glands (8, 11, 12).
PACAP38 has also been found in skin nerves innervating
arterioles and sweat glands (40). Both VIP and PACAP38 are
found colocalized with ACh in cutaneous cholinergic nerves, a
type known to be intimately involved in cutaneous active
vasodilation (10, 24, 47). They are also found in cutaneous
afferent nerves that may be involved in the process (10, 32, 52,
53). These findings show that both VIP and PACAP38 are
found in microanatomic locations consistent with a mechanistic role in cutaneous active vasodilation as demonstrated by the
attenuating effects of PACAP6 –38 on the process.
As previously mentioned, VPAC1, VPAC2, and PAC1 receptors are all found in human nonglabrous skin. Of these,
VPAC1 receptors appear to be quite limited in number in skin
in general and in cutaneous arterioles in particular (28). In
contrast, VPAC2 receptors have been found to be abundant in
skin, although mRNA for VPAC2 receptors appears to be
absent from dermal vascular smooth muscle cells (11, 12).
VPAC2 receptors are also abundantly expressed in sweat
glands (11, 12). PAC1 receptors are also found in human
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VIP/PACAP RECEPTORS AND CUTANEOUS ACTIVE VASODILATION