Chapter 93: Thermoregulation 121

Chapter 93
Thermoregulation
Dean L. Kellogg, Jr.

CUTANEOUS RESPONSES TO
HEAT
Cutaneous Active Vasodilator
Mechanisms
Despite the fact that the cutaneous active vasodilator system has been studied for many decades,
the specific mechanisms by which the cutaneous
active vasodilator system functions are only partly
understood.19,20,24 Several hypotheses have been
proposed for how active vasodilation in skin works,
but none has been completely proven.
SUDOMOTOR ACTIVITY AND ACTIVE
VASODILATION.
In the initial descriptions of cutaneous active
vasodilation, it was noted that sweating and active
vasodilation began at about the same time in a resting person subjected to heat stress.20 This observation led to the hypothesis that the mechanism of
cutaneous active vasodilation involved cholinergic
sudomotor nerve activity.20,2527 Additional evidence
that favored a close mechanistic relationship between active vasodilation and sweating comes from
observations of persons with anhidrotic ectodermal
dysplasia and Ross Syndrome. Persons with this
congenital anhidrotic ectodermal dysplasia lack
sweat glands (see Chapter 83), do not sweat nor do
they have cutaneous active vasodilator responses
to heat stress.27 Persons with Ross syndrome have
an acquired loss of cutaneous cholinergic neurons
and, hence, do not sweat or vasodilate skin during heat stress.28 Patients with either of these rare
conditions are extremely heat intolerant. Despite
observations such as this, the precise relationship between sweat glands and active vasodilation remains poorly defined. While it is clear that
cholinergic sudomotor nerves control sweat glands,
whether sudomotor and vasodilator nerves are one
and the same or separate systems is unknown.29,30
COTRANSMISSION AND CUTANEOUS
ACTIVE VASODILATION.
An alternative hypothesis with strong support
proposes that cutaneous active vasodilation is
mediated by a complex cotransmitter system (eFig.

93-2.1). This proposal is based on studies of an

atropine-resistant, cholinergic cotransmitter system
in the cat paw that relies on corelease of acetylcholine (Ach) and vasoactive intestinal peptide (VIP) to
accomplish vasodilation.31 Based on these observations, it was proposed that a single set of neurons in
human skin could control both active vasodilation
of skin arterioles and sweating by releasing both
Ach and the neuropeptide cotransmitter VIP. Ach
would cause sweating and VIP would cause active
vasodilation.31 This atropine-resistant cotransmitter
mechanism could explain why atropine abolishes
sweating, but not active vasodilation during heat
stress in humans.4,32
The cotransmission hypothesis is attractive for
several reasons: (1) VIP is a vasodilator (via cyclic adenosine monophosphate [cAMP]); (2) VIP is found
in human nerve endings associated with sweat
glands33 and blood vessels34; and, (3) VIP is colocalized with Ach in cholinergic nerve terminals.33 VIP
has also been implicated in the control of sweat
glands; exogenous VIP increases muscarinic receptor affinity for methacholine (a muscarinic receptor
agonist) and may thus promote sweat production
as well as active vasodilation.35,36
The cotransmission hypothesis was tested in
humans in a series of three studies designed to
examine the involvement of cotransmission in the
cutaneous active vasodilator system.37 The first of
these studies confirmed the classic results of Roddie et al22 that local application of atropine to skin
abolished sweating completely, but only slightly
attenuated and delayed active vasodilation during
heat stress. A second study showed that iontophoretic pretreatment of skin with atropine blocked all
vasodilatory responses to exogenously applied Ach,
demonstrating that all cutaneous vascular responses to Ach are mediated by muscarinic receptors.
These two studies demonstrated that Ach could not
be the only neurotransmitter that mediated active
vasodilation.37 In a third study, botulinum toxin was
injected into discrete areas of skin and the effect
on subsequent skin blood flow responses to heat
stress was evaluated. Botulinum toxin is taken up
specifically by cholinergic nerve terminals and
interrupts the release of all neurotransmitters from
those terminals. Treatment of skin with this agent
completely abolished both cutaneous active va-

sodilation and sweating in the treated area of skin

during heat stress. This series of three studies led
to the following conclusions: (1) the only functionally important cholinergic receptors on skin blood
vessels are muscarinic; (2) active vasodilation and
sweating are mediated by cholinergic nerves; (3)
the substances causing vasodilation must include
at least one neurotransmitter coreleased with Ach
from cholinergic nerves.37

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122 Chapter 93: Thermoregulation

One of the current areas of interest in thermoregulatory research is the nature of the cotransmitter
(or cotransmitters) that affect active vasodilation.
Recent work supports the involvement of VIP as a
cotransmitter in active vasodilation.38,39 This work
tested whether active vasodilation is mediated, in
part, by VIP coreleased from cholinergic nerves with
Ach. The neuropeptide fragment, VIP10-28, was
administered by microdialysis to block the effects of
VIP at VPAC1 and VPAC2 receptors. This agent was
chosen because it not only blocks the two major
receptors for VIP, it also blocks the effects of peptide
histidine methionine (PHM). These two neuropeptides share a close structural relationship, are
formed from the same prepropeptide, and are both
reported to be present in human skin.40 VIP10-28
attenuated (but did not abolish) the increase in skin
blood flow during heat stress. The combination of
VIP10-28 with atropine did not enhance the degree
of attenuation achieved with VIP10-28 alone. This
finding showed a role for VIP in active vasodilation; however, since the combination of muscarinic
receptor with VIP receptor blockade did not differ
from VIP receptor blockade only, it was postulated
that additional cotransmitters may well be involved
in the process.38,39
Recent evidence suggests a role for the neurokinin Substance P in cutaneous active vasodilation in
heat stress.41 Neurokinin receptors can be desensitized by repeated exposures to Substance P so
that less vasodilation in response to subsequent
exposures to Substance P.42 It has been shown that
repeated exposure to Substance P reduced the
subsequent vasodilator response to heat stress, in
support of a role for neurokinin receptors and perhaps Substance P as a mediator of cutaneous active
vasodilation.
NITRIC OXIDE AND ACTIVE VASODILATION.
In the 1990s, a series of studies were done that investigated the control of skin blood flow in the rabbit ear. Rabbits (and other lagomorphs) thermoregulate by actively dilating and constricting the blood
vessels in their ears. Rabbits thus provided a possible animal model for study of the dual vasomotor
controls of human nonglabrous skin. The studies in
the rabbit ear showed a role for nitric oxide (NO) in
thermoregulatory reflex-mediated active vasodilation.43,44 This work provided the rationale to study
and clarify roles for the NO system in cutaneous
active vasodilation in humans.4547 Although initial
work based on intra-arterial infusions of the nitric

oxide synthase (NOS) inhibitor NG-monomethyl-larginine (l-NMMA) was unable to establish such a
role,47 later work45,48 used intradermal microdialysis
to deliver NOS inhibitors NG-nitro-l-argininemethyl ester (l-NAME) and l-NMMA into small areas
of skin. These studies found that increases in skin
blood flow caused by active vasodilation during
heat stress were significantly attenuated but not
abolished by NOS inhibition.45,46,48 The results of
these studies show that active vasodilation in skin
requires functional NOS to achieve full expression.
The foregoing discussion of NO might lead one to
believe that since functional NOS is required for
active vasodilation, NO levels in skin must increase
during heat stress to cause active cutaneous
vasodilation; however, based on studies of how NO
functions as a vasodilator in the rabbit ear during
heat stress a novel theory was proposed.49 It had
been noted that NOS antagonists abolished heat
stress-induced active vasodilation in skin of the rabbit ear. Administration of low doses of the NO donor,
nitroprusside, could restore ear skin vasodilation
during heat stress despite NOS blockade; however,
the same dose of nitroprusside infused into the ear
circulation in normothermia did not raise ear blood
flow at all. The implication was that active vasodilation in the rabbit ear required the presence of NO
and activation of vasodilator nerves, but that these
two elements were not arranged in series. This lead
to the idea that vasodilator nerve activity did not
increase NO production, but rather that NO needed
to be present to permit another neurotransmitter
to effect increases in ear skin blood flow. It was thus
proposed that NO served a permissive role in the
active vasodilation rather than as an actual effector
of the process; i.e., NO had to be present for vasodilation to be effected by another neurotransmitter,
but that the absolute level of NO did not increase in
heat stress.
An initial test of this proposal in human skin examined whether increased levels of NO breakdown
products could be found in skin during hyperthermia, but no such increases were found.50 This suggested that NO acted as a permissive factor rather
than as an effector of cutaneous active vasodilation;
however, the study of NO breakdown products is
fraught with problems. Subsequent examinations
of NO changes in heat stress were done by measuring bioavailable NO by NO-selective amperometric electrodes in vivo.38 This technique measures
bioavailable NO directly from the tissue of interest.
In contrast to the initial study based on NO break-

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Chapter 93: Thermoregulation 123

down products, it was found that during heat stress

both skin blood flow and bioavailable NO concentrations increased during heat stress. This proved
that NO does increase in skin during heat stress
in humans, attendant to active vasodilation. This
result suggests that NO has a role beyond that of a
permissive factor in cutaneous active vasodilation;
rather, since NO increases in heat stress, it could
well be an effector of cutaneous vasodilation during heat stress.38,48,51
Additional evidence in favor of NO as an active effector of cutaneous active vasodilation came from
recent work using an NO-clamp technique.52 Intradermal microdialysis was used to deliver a combination of NOS inhibition by l-NAME with the NO donor
nitroprusside into skin. The effect of this combination was to clamp NO at a constant level, i.e., NO
was present but did not increase because of NOS
blockade. When NO levels were clamped and not
allowed to increase, active vasodilation during heat
stress was attenuated. This showed that NO did not
act permissively, but rather was an active effector
of vasodilation. It is thus clear that NO production
increases during heat stress and that NO acts as an
actual effector of cutaneous active vasodilation.
It should be noted that all of the foregoing work
on NO in human skin was done in nonglabrous skin.
This type of skin has a paucity of AVAs. In contrast,
the skin of the rabbit ear is rich in AVAs. It may well
be that NO plays a permissive role in AVA dilatation, but not in the papillary loops of nonglabrous
skin.
Another question about the role of NO in active
vasodilation involves the factors that mediate
increased production of NO during heat stress. One
hypothesis was that Ach from cholinergic nerves
increased NO via muscarinic receptor stimulation.53
This was investigated by examining the effects
of combined muscarinic receptor blockade by
atropine with NOS blockade by l-NAME on vasodilation during heat stress. These agents were given
after activation of the active vasodilator system in
prolonged heat stress, when skin blood flow had
already risen significantly. While atropine was found
to have little effect on skin blood flow, l-NAME was
found to significantly reduce skin blood flow during
established active vasodilation in prolonged heat
stress. This showed that although production of
NO was required for active vasodilation, muscarinic
receptor-mediated NO production was not needed
to sustain active vasodilation during the late, established phase of heat stress.53

Subsequent work has examined whether muscarinic receptor activation leads to NO production early in heat stress.54 Based on earlier studies
that showed atropine delayed the onset of active
vasodilation during heat stress, it was postulated
that Ach contributed to active vasodilation through
muscarinic receptor mediated NO production early
in the process. To test this hypothesis, the effect of
a combination of acetylcholinesterase inhibition
with neostigmine (to magnify the agonist effects of
Ach) and NOS blockade with l-NAME (to abolish NO
effects) on active vasodilation was examined. Neostigmine and l-NAME were delivered prior to initiation of body heating, when the active vasodilator
system was quiescent, and continued throughout
the early and late periods of heat stress. The results
showed that early in body heating (when skin temperature was increased but internal temperature
was not), skin blood flow increased sooner at sites
treated with neostigmine (with presumably augmented Ach levels). The augmenting effects of acetylcholinesterase inhibition were abolished by NOS
inhibition. Late in heat stress, when active vasodilation was well established, the effect of neostigmine
was lost, but skin blood flow at l-NAME treated
sites was attenuated. These results suggested that
Ach mediated the increase in NO production early
in heat stress, but not after substantial cutaneous
vasodilation had occurred.54
H1 receptors for histamine may play a role in the
generation of NO during cutaneous active vasodilation in heat stress.55 Administration of the first-generation antihistamine pyrilamine attenuated, but
did not abolish, the rise of skin blood flow during
heat stress. In addition, it was found that the NO
generated during active vasodilation was mediated
by H1 receptor activation. Thus, there appear to be
several pathways that may generate NO in the skin
during heat stress.
The recent development of antagonists for the
different NOS isozymes has allowed investigations
into the roles of these isoforms in active cutaneous vasodilation. These investigations found that
inhibition of NOS I (neuronal NOS, nNOS) reduced
the cutaneous vasodilator response to heat stress.56
In contrast, inhibition of NOS III (endothelial NOS,
eNOS) had no significant effect on the vasodilator
response to body heating.57 These results are also
consistent with NOS I having an important role in
thermoregulatory reflex vasodilation.58

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124 Chapter 93: Thermoregulation

Local Waming of the Skin

and Vasodilation
The foregoing section outlined thermoregulatory
reflex responses to heat that are mediated primarily by the sympathetic nervous system. Skin vessels
themselves also respond directly to changes in local
temperature. For example, in response to increases
in local skin temperature, cutaneous blood vessels
dilate. This dilation is accomplished by purely local
temperature-dependent mechanisms, the effects
of which are in addition to the previously discussed
neural effects.
With local warming of skin, local skin blood flow
increases in direct proportion to the temperature
achieved. If heating is continued at a maximally
tolerable level of 42C44C for 3555 minutes,
maximal local skin blood flow can be achieved.59
The local vasodilation is biphasic with an initial brisk
vasodilation. This initial dilation rapidly reaches a
peak and is followed by a slight fall in flow. With
continued heat application, a second dilation occurs that can reach maximal levels in a prolonged
plateau phase. For unknown reasons, the plateau
phase declines after approximately 1 hour despite
continued heating60 (eFig. 93-2.2).
The mechanisms that mediate the local, temperature-dependent cutaneous vasodilation involves
local sensory nerve, axon reflex mechanisms and
the local generation of NO. These two mechanisms
appear to be independent of each other.61 The previously discussed neural cutaneous active vasodilator system does not appear to be involved in this
local response as neither botulinum toxin induced
abolition of active vasodilation nor muscarinic receptor blockade with atropine alter the skin blood
flow response to local skin heating.37 Prostanoids
do not appear to be involved either, as cyclooxygenase inhibition has no effect on the vasodilation
induced by local skin heating.62
The initial phase of the local warming response
produces a brief peak value and is mediated by local activation of afferent cutaneous sensory nerves.
This initial phase of the vasodilation can be greatly
attenuated by topical anesthesia directly applied
to the heated site. Cutaneous nerve blockade at
points distant from the heated site that interrupt
sensory nerve function at the heated site have no
effect.61,63 This demonstrates that the initial effects
of local skin heating are mediated by a neural axonreflex mechanism. The specific neurotransmitters

involved in local heating responses are uncertain,

however, based on studies done with topical application of the vanilloid receptor (VR-1) activator
capsaicin. It has been proposed that local increases
in skin temperature stimulate heat-sensitive VR-1
receptors on afferent nerves. This activates the local
axon reflex to cause the antidromic release of a vasodilatory neurotransmitter (or neurotransmitters)
that mediates local vasodilation.64
The prolonged plateau phase of vasodilation in
response to local skin heating is mediated by local
generation of NO. The plateau phase can be greatly
attenuated by pretreatment of the locally heated
area of skin with the NOS inhibitors48,61; thus, NO
generation is clearly necessary for this phase. Recent findings with specific isoforms for endothelial
NOS (eNOS, type III NOS) and neuronal NOS (nNOS,
type I NOS) suggests that endothelial NOS may
mediate increased NO production in forearm skin
during local skin heating, but that neuronal NOS
may mediate increased NO production in the skin
of the leg.5658,65 In addition, HSP90 has been shown
to bind eNOS, enhance eNOS activation, and thus
increase NO generation.66 Treatment of skin with
the heat shock protein 90 (HSP90) inhibitor, geldanamycin, attenuates the plateau phase of local skin
heating by approximately 20%. This suggests that
eNOS may be the NOS isoform that mediates the
prolonged plateau phase of the cutaneous vascular
response to local skin heating67 (see eFig. 93-2.2).

CUTANEOUS RESPONSES
TO COLD
Cutaneous Active
Vasoconstrictor Mechanisms
Initial evidence for the control of skin blood vessels
by sympathetic active vasoconstrictor nerves came
from observations of the effects of peripheral sympathectomies in humans.68,69 In these studies, the
interruption of sympathetic nerves by sympathectomy led to increases in skin blood flow when the
intervention was done in a cool environment. This
observation was consistent with the interruption of
vasoconstrictor activity leading to subsequent relaxation of cutaneous and, hence, passive increases
in blood flow.
The sympathetic vasoconstrictor system in skin
is well understood and causes vasoconstriction
through noradrenergic stimulation of 1- and 2adrenergic receptors.4,20,21,6972 This has been clearly

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demonstrated by studies using prejunctional

sympathetic nerve blockade and postjunctional
blockade with -receptor antagonists.23,71,73,74
Sympathetic nerves in skin contain several different neurotransmitters colocalized with norepinephrine, including neuropeptide Y (NPY) and
adenosine triphosphate (ATP). This suggests that
the sympathetic vasoconstrictor system could be
a cotransmitter system. In studies that compared
the effects of differing combinations of 1 and 2 on
the skin vasoconstriction during cold stress it was
noted that the vasoconstriction was attenuated.
These results confirmed a role for postjunctional
excitation of 1-and 2-adrenergic receptors. It
was also noted that pretreatment of skin with the
-receptor antagonist propranolol led to a more
profound vasoconstriction during cold stress. This
suggested that a -receptor-mediated vasodilation
modulated an -receptor-mediated vasoconstriction during whole body cooling. It is interesting to
observe, however, that simultaneous and complete
blockade of 1-, 2-, and -receptors fails to abolish
the cutaneous vasoconstriction induced by cold
stress although blockade of all neurotransmitter
release from cutaneous noradrenergic nerves with
bretylium tosylate does abolish the response. These
studies suggest that nonnoradrenergic, cotransmitter mechanisms mediate the cutaneous vasoconstriction during cold stress.64
The observation that cold stress responses in skin
are mediated by a cotransmitter system has led
to studies designed to identify the cotransmitters
of that system. Recently, it was proposed that NPY
acted as a cotransmitter along with norepinephrine
to mediate reductions in skin blood flow during
cold stress. It was hypothesized that NPY worked
through activation of NPY Y1 receptors on cutaneous arterioles. Administration of the NPY Y1 antagonist BIBP-3226 was found to significantly attenuate
reductions in skin blood flow during whole body
cold stress. Further, the combination of NPY Y1
receptor antagonism with complete 1-, 2-, and
-receptor blockade abolished reductions in skin
blood flow during cold stress. These results clearly
demonstrate that the cutaneous active vasoconstrictor system is a cotransmitter system that works
via the release of NPY and norepinephrine. These
neurotransmitters cause the postjunctional activation of NPY Y1, 1-, 2-, and perhaps -receptors,
which in turn constrict cutaneous arterioles, reduce
skin blood flow, and, thus, conserve body heat during cold stress75 (eFig. 93-2.3).

Local Cooling of the Skin and

Vsoconstriction
Decreases in skin temperature with local cooling
of the skin cause a local, temperature-dependent
vasoconstriction. While the vasodilatory response
to local heating of the skin is independent of the
cholinergic cutaneous active vasodilator system,
the vasoconstriction mediated by local skin cooling is dependent on intact noradrenergic active
vasoconstrictor nerves.63,76,77 Confirmatory studies
used bretylium tosylate to block neurotransmitter
release from prejunctional noradrenergic nerve
terminals in skin. When noradrenergic nerves are
intact, local cooling causes a prompt and progressive reduction of skin blood flow as local temperature falls. Interruption of neurotransmitter release
with bretylium changes the initial portion of the
local cooling response from a prompt vasoconstriction into a prompt vasodilation. The vasodilation
induced by local cooling is transient and continued
local skin cooling leads to the progressive diminution of the vasodilation and eventual replacement
by a progressive vasoconstriction.4
Recent work has defined how the cutaneous
active vasoconstrictor system participates in the
vasoconstriction caused by local skin cooling, and
established roles for noradrenergic receptors and
afferent sensory nerves76 (eFig. 93-2.4).
It was observed that blockade of -receptors in
skin altered the local cooling vasoconstriction in
the same way that bretylium did, i.e., combined and -receptor blockade reversed the initial phase
of vasoconstriction into a vasodilation followed
eventually by vasoconstriction with continued
local cooling. Topical anesthesia with EMLA (a
eutectic mixture of lidocaine anesthetic) cream also
reversed the initial vasoconstriction to a dilation followed again by constriction with prolonged cooling.
NPY Y1 receptor blockade with BIBP-3226 had no
effect on the response to local cooling. Based on
this work, it is clear that local cooling of skin occurs
in two phases: (1) an initial rapid phase lasting a few
minutes, and (2) a prolonged late phase. The initial
phase is caused by activation of cold-sensitive afferent neurons that mediate release of norepinephrine from sympathetic cutaneous vasoconstrictor
nerves. The released norepinephrine vasoconstricts cooled skin vessels through postjunctional
-receptors. NPY Y1 receptors do not appear to
be involved in either phase of the local cooling

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126 Chapter 93: Thermoregulation

response. It was been speculated that local cooling

stimulated norepinephrine release from adrenergic
nerves through cold receptors acting through axon
reflexes; however, this was recently shown to be
incorrect leaving the exact mechanism whereby
afferent sensory nerves contribute to the vasoconstriction induced by local cooling unclear.78 The
mechanisms for the prolonged, late vasoconstrictor
phase of cooling appear to be nonneurogenic as no
manipulations alter this portion of the response.76

The NO system also plays a role in causing vasoconstriction during local skin cooling. Antagonism
of NO production during local cooling attenuates
the skin vasoconstrictor response.85 Combined
antagonism of both the NO system and adrenergic
mechanisms eliminates the vasoconstrictor response to local cooling.86 These findings show that
direct local cooling of skin causes vasoconstriction
through both the NO-dependent and adrenergic
mechanisms.

A significant role for 2-receptors in causing the

cutaneous vasoconstriction by local skin cooling
has been proposed.7983 For example, local skin
cooling increases 2-adrenergic receptor-mediated
vasoconstriction. In contrast, local cooling does not
augment constriction mediated by 1-adrenergic
receptors.84 In mouse skin, it has been shown that
local cooling causes augmented noradrenergic
constriction through 2C-adrenergic receptors.82
While these receptors are not directly thermosensitive themselves, they have been found to be translocated from the trans-Golgi apparatus of vascular
smooth muscle cells to the plasma membrane
through the cold-induced activation of RhoA and
Rho kinase.80,81 In this way, more 2C-receptors appear on the surface of vascular smooth muscle. In
addition, activation of these kinases enhances the
calcium sensitivity of the vascular smooth muscle
contractile apparatus. These factors explain how
local cooling causes vasoconstriction.80
The generation of reactive oxygen species (ROS)
from mitochondria in vascular smooth muscle appears have a role in the vasoconstriction induced
by local cooling. Cooling of mouse tail skin arteries leads to increased ROS generation in vascular
smooth muscle cells. Alteration of ROS levels by
application free radical scavengers abolishes the
vasoconstrictor response to 2C-adrenergic receptor stimulation. Manipulation of ROS levels also
abolishes the activation of RhoA in cultured human
vascular smooth muscle cells. Taken together, these
results suggest that the vasoconstriction caused by
local cooling is caused by enhanced ROS generation in mitochondria of vascular smooth muscle. Increased ROS then activates RhoA/Rho kinase, which
causes a consequent movement of 2C-adrenergic
receptors to the cell membrane. Upon reaching the
cell surface, 2C-adrenergic receptors can be activated by norepinephrine released by sympathetic
neurons and cause vasoconstriction.81

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