doi:10.1152/japplphysiol.01071.2005 100:1709-1718, 2006. J Appl PhysiolD. L. Kellogg, Jrchallengesvasoconstriction in humans during thermoregulatory In vivo mechanisms of cutaneous vasodilation andYou might find this additional info useful...89 articles, 57 of which can be accessed free at: This article cites /content/100/5/1709.full.html#ref-list-135 other HighWire hosted articles, the first 5 are: This article has been cited by [PDF] [Full Text] [Abstract], November 19, 2013; . J R Army Med CorpsElise M Hindle and J D HenningCritical care at extremes of temperature: effects on patients, staff and equipment[PDF] [Full Text] [Abstract], March , 2014; 38 (1): 87-92. Advan in Physiol EduE. A. Tansey, S. M. Roe and C. D. Johnsonof blood flowThe sympathetic release test: a test used to assess thermoregulation and autonomic control[PDF] [Full Text] [Abstract], October 28, 2014; . 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J Diabetes Sci Technoland Thomas HaakAndreas Pftzner, Itamar Raz, Gabriel Bitton, David Klonoff, Ron Nagar, Norbert HermannsDatain a Safer and More Efficient Prandial Insulin TreatmentA Review of the Existing Clinical Improved Insulin Absorption by Means of Standardized Injection Site Modulation Resultsincluding high resolution figures, can be found at: Updated information and services /content/100/5/1709.full.html can be found at: Journal of Applied Physiology aboutAdditional material and information http://www.the-aps.org/publications/japplThis information is current as of January 8, 2015.ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2006 by the American Physiological Society.those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American publishes original papers that deal with diverse areas of research in applied physiology, especially Journal of Applied Physiologyon January 8, 2015Downloaded from on January 8, 2015Downloaded from Invited ReviewHIGHLIGHTEDTOPIC A Physiological Systems Approach to Human andMammalian ThermoregulationIn vivo mechanisms of cutaneous vasodilation and vasoconstriction in humansduring thermoregulatory challengesD. L. Kellogg, Jr.Geriatric Research, Education, and Clinical Center, Department of Veterans Affairs, South Texas Veterans HealthCare System, Audie L. Murphy Memorial Veterans Hospital Division; and Division of Geriatrics and Gerontology,Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TexasKellogg, D. L., Jr. In vivo mechanisms of cutaneous vasodilation and vasoconstrictioninhumansduringthermoregulatorychallenges.JApplPhysiol100:17091718,2006;doi:10.1152/japplphysiol.01071.2005.This reviewfocuses onthe neural andlocal mechanisms that have been demonstrated to effect cutaneous vasodilationand vasoconstriction in response to heat and cold stress in vivo in humans. First,our present understanding of the mechanisms by which sympathetic cholinergicnervesmediatecutaneousactivevasodilationduringreexresponsestowholebody heating is discussed. These mechanisms include roles for cotransmissionas well as nitric oxide (NO). Next, the mechanisms bywhichsympatheticnoradrenergicnervesmediatecutaneousactivevasoconstrictionduringwholebody cooling are reviewed, including cotransmission by neuropeptide Y (NPY)acting through NPYY1 receptors. Subsequently, current concepts for themechanisms that effect local cutaneous vascular responses to direct skinwarmingare examined. These mechanisms include the roles of temperature-sensitive afferent neurons as well as NOincausingvasodilationduringlocalheating of skin. This section is followed by a review of the mechanisms that causelocal cutaneous vasoconstriction in response to direct cooling of the skin, includingthe dependence of these responses on intact sensory and sympathetic, noradrenergicinnervation as well as roles for nonneural mechanisms. Finally, unresolved issuesthat warrant further research on mechanisms that control cutaneous vascularresponses to heating and cooling are discussed.thermoregulation; heat stress; cold stress; skin blood ow; cotransmissionMECHANISMSOFCUTANEOUSVASCULARCONTROLDURINGHEATSTRESSANDCOLDSTRESSINHUMANSHUMANTHERMOREGULATIONOCCURS in response to changes ininternaltemperature[orcoretemperature(Tc)]andskintem-perature (Tsk). The cutaneous circulation is a major effector ofhumanthermoregulation. Duringheatstress, elevatedTcandTskleadtocutaneous vasodilationthroughcombinations ofneural mechanisms and the local effects of higher temperatureson the skin vessels themselves. Conversely, during cold stress,reduced temperatures lead to cutaneous vasoconstrictionthroughcombinedneural andlocal mechanisms. Undernor-mothermic conditions, skin blood ow (SkBF) averages 5%of cardiac output; however, the absolute amount of blood in theskincanvaryfromnearlyzeroduringperiods of maximalvasoconstrictionasincoldstresstoasmuch60%ofcardiacoutput distributedoverthebodysurfaceduringmaximal va-sodilation in heat stress (65).Inglabrous, or nonhairy, regions of skin(palms, plantaraspect of feet, andlips), cutaneousarteriolesareinnervatedsolely by noradrenergic sympathetic vasoconstrictor nerves(18, 3537, 67). All thermoregulatory reexes in these regionsare mediated by changes in noradrenergic vasoconstrictor toneandtheeffectsoflocaltemperaturesonskin(3537, 67). Innonglabrous,orhairy,areasofskin(limbs,head,andtrunk),reexchangesinSkBFaremediatedbytwobranchesofthesympathetic nervous system(noradrenergic vasoconstrictornerves andcholinergicactivevasodilator nerves, whichareunique to humans) in addition to the local effects of tempera-ture (10, 37, 67). These dual sympathetic neural control mech-anisms effect the major aspects of thermoregulatory responsesover most of the human bodys surface.Underresting, normothermicconditions, cutaneousarte-rioles in supine humans receive little neural stimulation;hencethesmoothmusclecellsof thecutaneousarteriolesare near basal tone. Withcoldstress, fallingTskandTcinitiate a thermoregulatory reex to conserve body heat.This reex is mediated by increased noradrenergic vasocon-strictor tone that causes an arteriolar vasoconstriction and adecrease in SkBF. Conversely, during heat stress, rising TskAddressforreprintrequestsandothercorrespondence:D.L.Kellogg,Jr.,Div. of Geriatrics and Gerontology, Dept. of Medicine, Univ. of Texas HealthScience Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229(e-mail: kelloggd@uthscsa.edu).J Appl Physiol 100: 17091718, 2006;doi:10.1152/japplphysiol.01071.2005.8750-7587/06 $8.00 Copyright 2006 the American Physiological Society http://www. jap.org 1709on January 8, 2015Downloaded from andTcinitiateathermoregulatoryreextofacilitatebodycooling(seeFig. 1).Withthe inductionof mildheat stress, as Tskincreases,SkBF is controlled by small variations in both cutaneousvasoconstrictor andvasodilator nerveactivities(39, 68). Asheat stress progresses and Tc begins to rise with Tsk, there is theabolition of any extant vasoconstrictor tone. As Tc continues torise, it reaches a threshold value at which the cutaneous activevasodilator systembecomes fully activated. Sweating alsobegins at the same Tcthreshold under resting conditions;however, thevasomotorandsweatingthresholdscanbedis-similar during exercise or baroreceptor challenges (55, 73). Aswhole body heat load increases, active vasodilator tone to thecutaneous arterioles is increased. This increase in neural activ-itymediatesadecreaseinsmoothmuscletone, anarteriolarvasodilation, and effects an increase in SkBF. High SkBFdelivers heat to the body surface where it is dissipated to theenvironment inconjunctionwiththe evaporationof sweat.Overall, the active vasodilator system is responsible for 8095%of the elevation in SkBF that accompanies heat stress (37, 66).Neural Control Mechanisms of the Cutaneous VasculatureDualvasoconstrictornervesandvasodilatornervesinskinwererst suggestedin1931byLewis andPickering(51);however, the rst denitive evidence came from work by Grantand Holling (24). They measured Tsk as an index of blood owin the human forearm and found large increases in response toheat stress that could be abolished by sympathectomy or nerveblockade. Theynotedthat whereassympathectomyornerveblockadecausedonlyaslight cutaneousvasodilationduringnormothermia, heatstresselicitedamuchgreaterincreaseinSkBF. Inaddition, nerveblockadeduringestablishedhyper-thermia and vasodilation abolished the increase in SkBF. Theseresultssuggestedthat cutaneousvesselsinnonglabrousskinreceived sympathetic active vasodilator as well as sympatheticvasoconstrictor innervation.Inthe1950s, thendingsofGrantandHolling(24)wereconrmedbyEdholmet al. (14) andbyRoddieet al. (64).More recently, studies found that bretylium tosylate (a prejunc-tional noradrenergic neuronal blockingagent) abolishes thecutaneous vasoconstriction induced by cold stress, but it doesnot alter the vasodilator responses induced by heat stress (41).Thisconrmedthat dual efferent neural systemscontrol thecutaneous arterioles: anoradrenergicvasoconstrictor systemand a nonadrenergic active vasodilator system.Cutaneousactivevasodilatormechanisms. Despite the factthat the cutaneous active vasodilator system is the most studiedof the mechanisms that control SkBF, the precise mechanismsbywhichthecutaneous activevasodilator systemfunctionsremainsomewhatenigmatic(24,51,63).Severalhypothesesfor how this system works have been proposed; however, nonehas been clearly proven.SUDOMOTOR ACTIVITY AND ACTIVE VASODILATION. In the initialdescriptions of cutaneous active vasodilation, it was noted thatsweatingandactivevasodilationbeganat approximatelythesame time in resting, heat-stressed persons (24). This observa-tion led to a proposal that the mechanism of cutaneous activevasodilation involved cholinergic sudomotor nerve activity (9,19, 21, 24). Additional evidence favoring the possible associ-ation of active vasodilation with sweating included the obser-vationthatpersonswithanhidroticectodermaldysplasia, thecongenital absence of sweat glands, also lack a cutaneousactive vasodilator response to heat stress (9).Along-heldtheoryoftherelationshipbetweensudomotoractivityandactivevasodilationwasdrawnfromworkdonewithsalivaryglands(19). FoxandHilton(19)hypothesizedthat during heat stress the activation of sweat glands bycholinergic sudomotor nerves caused release of an enzyme intotheinterstitial spacethat cleavedbradykininfrominterstitialglobulins near cutaneous resistance vessels, leading to vasodi-lation. Such an indirect relationship could explain how cholin-ergic-receptorblockadewithatropinecouldabolishsweatingbut onlydelayandslightlyreduceincreasesinSkBFduringhyperthermia (63, 64). This hypothesized mechanismwasrefuted by the nding that complete blockade of bradykinin B2receptorsinskinwiththespecicreceptorantagonist HOE-140, had no effect on the extent of cutaneous active vasodila-tionduringheatstress.GiventhesendingsandthefactthatonlybradykininB2receptorsarepresent inhumanskin, thebradykinin Hypothesis cannot be correct (44).The precise relationshipbetweensudomotor activityandactivevasodilationremainsuncertain. Indeed, althoughit isclear that cholinergic sudomotor nerves innervate sweatglands, whether the sudomotor and vasodilator nerves are oneandthe same or separate nerves has not beendetermined;however, the observation that patients with anhidrotic ectoder-mal dysplasia, wholacksweat glands, alsofail toactivelydilate skin vessels during heat stress and the close relationshipbetweensweat productionandvasodilator skinsympatheticnerve activity (SSNA) suggests that the sudomotor nerves andvasodilator nerves could well be one and the same (9, 39, 80, 81).COTRANSMISSIONANDCUTANEOUSACTIVEVASODILATION. Asillustrated in Fig. 2, an alternative to the bradykinin hypothesisFig. 1. Skin blood ow responses to cold stress and heat stress. During periodsofnormothermia, whenskinbloodowrepresents5%ofcardiacoutput,cutaneousvesselsreceivevariable, yet relativelyminor neural inputsfromactive vasoconstrictor and vasodilator nerves. During periods of hypothermia,falling core and skin temperatures lead to reex increases in sympathetic activevasoconstrictornerveactivitytoreduceskinbloodowandconservebodyheat. During periods of heat stress, increasing core and skin temperatures leadto reex increases in sympathetic active vasodilator nerve activity to increaseskin blood ow. Increases in skin blood ow can reach 60% of cardiac outputduringsevere heat stress. Highskinbloodowacts inconjunctionwithevaporationofsweat toreducebodyheat. VC, increasedactivevasocon-strictor nerve activity;VC, decreased active vasoconstrictor nerve activity;VD, increasedactivevasodilatoractivity;Threshold, coincidentcoretem-peratureat whichcutaneous activevasodilationandsweatingbeginunderresting conditions.Invited Review1710 CUTANEOUS VASCULAR CONTROL MECHANISMSJ Appl Physiol VOL100 MAY2006 www.jap.orgon January 8, 2015Downloaded from was proposed by Hokfelt (32) that cutaneous active vasodila-tion relies on a cotransmitter system and was based on studiesofatropine-resistant, cholinergiccotransmittersystemsinthecat pawthat reliedoncoreleaseofacetylcholine(ACh)andvasoactive intestinal peptide (VIP). According to this hypoth-esis, a single set of neurons could control both active vasodi-lationofcutaneousarteriolesandsweatingbyreleasingbothACh and the neuropeptide cotransmitter VIP. ACh wouldcause sweating, and VIP would effect active vasodilation (32).This atropine-resistant cotransmitter mechanism could explainwhyatropineabolishessweatingbut not activevasodilation(46, 64).TheHokfelthypothesishasbeenattractiveforseveralrea-sons: 1) VIPisavasodilator (viacAMP), 2) it isfoundinhumannerveendingsassociatedwithsweat glands(88)andblood vessels (28), and 3) it is colocalized with ACh (88). VIPhas also been implicated in the control of sweat glands (83, 96).Forexample,exogenousVIPappearstoincreasemuscarinic-receptor afnity for methacholine (a muscarinic-receptor ago-nist) and may thus promote sweat production as well as activevasodilator (83, 96).Theinitial test oftheHokfelt hypothesisinhumanactivecutaneous vasodilationwas reportedbySavage et al. (69).Nerves from patients with cystic brosis (CF) were known tohavelittleornoVIP(31).Savageetal.reasonedthatifVIPwere the cotransmitter for active vasodilator, CF patientswould have reduced cutaneous vasodilation in response to heatstress; however, they found that patients with CF had normalactive vasodilator responses tobodyheating. Skinbiopsiesrevealed very sparse levels of VIPin their patients. Theyconcludedthat VIPprobablywasnot theelusivetransmitterthat effected cutaneous active vasodilator (69). This work leftthe issue of cotransmission in human skin unresolved.Subsequent studies showed conclusively that the cutaneousactivevasodilatorsystemdidinvolvecotransmission:speci-cally, acholinergiccotransmittersystem(46). Therst ofaseries of studies conrmed the classic results of Roddie et al.(64) that local application of atropine to skin abolished sweat-ing completely, but it did not abolish active vasodilation duringheat stress. In addition, iontophoretic pretreatment of skin withatropineblockedallvasomotorresponsestoexogenouslyap-plied ACh, demonstrating that all cutaneous vascular responsesto ACh are mediated by muscarinic receptors. These twostudies ruled out ACh as the sole neurotransmitter of the activevasodilator system (46). A third nding was that an intradermaldoseofbotulinumtoxin,takenupspecicallybycholinergicnerve terminals and interrupting the release of all neurotrans-mitters from those terminals, completely abolished both cuta-neousactivevasodilationandsweatinginthetreatedareaofskin. This series of threeobservations ledtothefollowingconclusions: 1) the only functionally important cutaneous vas-cular cholinergic receptors are muscarinic, 2) active vasodila-tioniseffectedbycholinergicnerves, and3)thesubstancescausing vasodilation must include at least one neurotransmittercoreleased with ACh from cholinergic nerves (46).Incontrast tothe ndings of Savage et al. (69), inCFpatients, recent workbyBennett et al. (5, 6) supports theinvolvement of VIPasacotransmitter inactivevasodilator.This worktestedwhether activevasodilator is aredundantsystem in which ACh and VIP are coreleased from cholinergicnerves. The neuropeptide fragment VIP10O28was used toblock the effects of VIP at VIP type 1 and VIP type 2 receptors.VIP10O28was chosenfor thesestudies becauseit not onlyblocked the two major receptors for VIP but it also blocked theeffects of peptide histidine-methionine (PHM). PHM and VIParestructurallyrelatedneuropeptidesformedfromthesameprepropeptide, andbotharereportedtobepresent inhumanskin (33). VIP10O28 was given alone and in combination withatropine. VIP10O28, alone or incombinationwithatropine,attenuated (but did not abolish) the rate of rise of SkBF duringheat stress. This ndingsupportedarolefor VIPinactivevasodilation(5, 6);however, recentworkwithVIP10O28hasfailed to replicate the antagonist effect of the agent during heatstress, andthus decisiveconclusions about theroleof VIPremain problematic (94).NITRICOXIDEANDACTIVEVASODILATION. In the 1990s, workby Taylor et al. (84, 85) showed a role for nitric oxide (NO) inthermoregulatory reex-mediated active vasodilation of therabbit ear. This work provided the rationale to study and clarifyrolesfor theNOsystemincutaneousactivevasodilationinhumans (13, 40, 72). Althoughinitial workbasedonintra-arterial infusions of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) was unable to establish such a role (13),subsequentstudies(40, 42)basedonintradermaladministra-tion of NOsynthase (NOS) inhibitors [NG-nitro-L-argininemethyl ester (L-NAME and L-NMMA)] via microdialysisprobesduringhyperthermiaprovedsucharoletobeextant.Thesestudiesfoundthat theincreaseinSkBFmediatedbyactive vasodilation during heat stress was signicantly attenu-ated by NOSinhibition (40, 42). Shastry et al. (72) alsoinvestigated this issue with laser-Doppler owmetry from fore-armskin in combination with intra-arterial infusions of L-NMMAafter a markedcutaneous vasodilationeffectedbywholebodyheating.TheyfoundthatNOSblockadereducedSkBF modestly. The results of the studies by Kellogg et al. (40)and Shastry et al. (72) show that an active vasodilator requiresfunctional NOS to achieve full expression.Theforegoingstudies might leadonetoassumethat thefunctional NOS required for an active vasodilator meant thatNO levels in skin increased during active cutaneous vasodila-tion; however, a novel alternative was proposed by Farrell andBishop (15). Their proposal was based on their studies of howNOfunctionsasavasodilatorintherabbitearduringhyper-thermia(15). Theynotedthatnitroprussidecouldrestorethevasodilationinhibitedbyprior NOSblockadeduringhyper-thermia, but the same dose of nitroprusside infused into the earFig. 2. Sympatheticcholinergicnervecotransmissionandcutaneousactivevasodilation. Initially based on work done in the cat paw, it has been found thatcutaneous active vasodilation is mediated by cholinergic nerve cotransmission(46). Acetylcholineandothercotransmittersreleasedfromcholinergicsym-patheticnerves dilatecutaneous vessels andactivatesweatingduringheatstress. The exact roles and interactions of acetylcholine and the cotransmittersthat effect active vasodilation and sweating during thermoregulatory reexesremainincompletelydened. At present, evidencesuggeststhat vasoactiveintestinal peptide may be a cotransmitter in this system.Invited Review1711 CUTANEOUS VASCULAR CONTROL MECHANISMSJ Appl Physiol VOL100 MAY2006 www.jap.orgon January 8, 2015Downloaded from circulation in normothermia did not raise ear blood ow. Theimplicationwas that anactivevasodilator intherabbit earrequiredbothNOproductioninadditiontoactivationofthevasodilator nervesandthat thesetwoelementswerenot ar-ranged in series (vasodilator activity did not increase NOproduction) ashadbeensupposed. Instead, their studiesledthemtothenovelconclusionthatNOservedapermissiverole in the active vasodilation in the rabbit ear. They proposedthatNOhadtobepresentforvasodilationtobeeffectedbyanother neurotransmitter but that the absolute level of NO didnot increase in heat stress.AninitialtestoftheFarrellandBishop(15)hypothesisinhuman skin examined whether increased levels of NO break-down products could be found in skin during hyperthermia, butno such increases were found. This suggested that NO acted asapermissivefactorratherthanasaneffectorofcutaneousactivevasodilation. Kelloggetal. (47)repeatedthisstudytoexamine NO levels in hyperthermia by measuring bioavailableNOinvivoasdetectedbyNO-selectiveamperometricelec-trodes (47). In contrast to the initial study based on NObreakdownproducts, thisstudyfoundthat, duringhyperther-mia, both SkBF and bioavailable NO concentrations began toincrease at the same Tc. In addition, both SkBF and bioavail-ableNOconcentrationsincreasedduringheatstress, demon-strating that bioavailable NO does increase in skin during heatstressinhumans,attendanttoactivevasodilation.Thisresultsuggests that NO has a role beyond that of a permissive factorintheprocess;rather, NOcouldwellbeaneffectorofcuta-neous vasodilation during heat stress (43, 45, 47, 48).Additional evidence in favor of NO as an active effector ofcutaneous active vasodilation came fromrecent work byWilkinset al. (93). Theseauthorsusedintradermal microdi-alysis to deliver the NOS inhibitorL-NAME during heat stressandlowdosesoftheNOdonornitroprussidetoskinduringbothnormothermiaandheat stress. Theyfoundthat, duringheat stress, NOS inhibition alone attenuated cutaneous vasodi-lation and that administration of low doses of nitroprusside torestoreNOlevelsduringcontinuedNOSinhibitionfailedtoovercome the attenuation. Theyconcludedthat duringheatstress, NOdid not act permissively but rather directlycauses a portion of vasodilation during heat stress. In addition,theyfoundthat lowdosesof nitroprussidecausedagreatercutaneousvasodilationduringheat stressthaninnormother-mia. This suggests that NO may have a synergistic vasodilatoryrelationshipwiththeneurotransmittersthat effect cutaneousactive vasodilation.Another question about the role(s) of NO in active vasodi-lator involves the factor(s) that mediate the NOincreaseoccurring in heat stress. This issue was recently addressed bytwo studies. Shastry et al. (71) investigated the hypothesis thatAChfromcholinergicnervesincreasedNOviamuscarinic-receptor stimulation. They combined muscarinic-receptorblockade by atropine with NOS blockade byL-NAME. Theseagents weregivenafter activationof theactivevasodilatorsystem in prolonged heat stress, when SkBF had already risensignicantly. These authors found that atropine had little effectonestablishedactivevasodilator but that L-NAMEreducedSkBFduringestablishedactivevasodilatorinprolongedheatstress. Theyconcludedthat, althoughproductionofNOwasrequired, neither sweatingnor muscarinic-receptor-mediatedNO production was needed to sustain active vasodilator duringthe late, established phase of heat stress (71).Subsequently, Shibasaki et al. (74) published work address-ingwhethermuscarinicreceptoractivationleadstoNOpro-duction early in heat stress. They postulated that ACh contrib-utedtoactivevasodilatorbecauseAChisreleasedfromcho-linergic nerves during heat stress and ACh vasodilates throughmuscarinic-receptor-mediated NO production. To test this hy-pothesis, theycombinedacetylcholinesteraseinhibitionwithneostigminetomagnifytheagonisteffectsofAChandNOSblockadewithL-NAMEtoabolishNOeffects.IncontrasttoShastry et al. (71), Shibasaki et al. (74) gave the drugs beforeinitiationofbodyheating, whenactivevasodilationwasnotactivated, and continued throughout a period of hyperthermia.Shibasaki et al. found that early in body heating (when Tsk wasincreased but Tc was not), SkBF increased at sites treated withneostigmine (with presumably augmented ACh levels) beforeSkBF increased at untreated sites. They found that augmentingeffects of acetylcholinesterase inhibition was abolished byNOSinhibition.Lateinheatstress,whenactivevasodilationwas well established, SkBF at neostigmine-treated sites did notdiffer fromSkBFat untreatedsites, but SkBFat L-NAMEtreatedsites was lower that at untreatedsites. Their resultssuggested that ACh-mediated NOproduction was possibleearly in heat stress but not after substantial cutaneous vasodi-lationhadoccurred(74). Shibasaki et al. foundthat at theneostigmine treated sites sweating and cutaneous vasodilationoccurredwiththe elevationinskintemperature but beforemeasurable changes in internal temperature. This ndinglimitedtheiroverallconclusionsaboutmechanismsofactivevasodilator inlate heat stress (74); however, the importantnding by Kamijo et al. (39) that active vasodilator tone can beincreased by elevation of Tsk alone during whole body heatinglends strength to the argument that ACh mediates NO produc-tion early in heat stress.Recently Wong et al. (95) reported evidence that H1 hista-mine receptors may play a role in the generation of NO duringcutaneous active vasodilation in hyperthermia. They found thatthe rst-generation antihistamine pyrilamine attenuated the riseof SkBF during heat stress. They also found that part of the NOgeneratedduringactivevasodilationwasmediatedbyhista-mine type 1 receptors. The source of histamine remains to beidentied; however, recent work by Wilkins et al. (92) suggeststhat 1) VIP-induced release of histamine from mast cells couldbe involved, and2) VIP and histamine both have NO-depen-dent components to their effects. Thus there appear to beseveral pathways that maygenerateNOintheskinduringhyperthermia (39, 74, 92, 95).Cutaneous active vasoconstrictor mechanisms. Earlyevi-dence for the control of SkBF by sympathetic active vasocon-strictornervescamefromstudiesoftheeffectsofperipheralsympathectomies in human patients (1, 18, 67). These studiesrevealedthat interruptionof sympathetic neural activitybysurgicalorpharmacologicalmeansleadtoincreasesinSkBFwhendoneinacool environment, anobservationconsistentwith the release of active vasoconstrictor tone.Unliketheratherenigmaticactivevasodilatorsystem, thecutaneous sympathetic vasoconstrictor systemhas beenas-sumedtobeacomparativelywell-understood,simplesystemthat effects vasoconstrictionthroughclassical noradrenergicmechanisms involving the action of norepinephrine on 1- andInvited Review1712 CUTANEOUS VASCULAR CONTROL MECHANISMSJ Appl Physiol VOL100 MAY2006 www.jap.orgon January 8, 2015Downloaded from 2-receptors (7, 14, 18, 24, 26, 34, 49, 50, 67). This has beenclearlydemonstratedbystudies usingprejunctional sympa-thetic nerve blockade with bretylium and postjunctional block-ade with -receptor antagonists (7, 27, 41, 52); however, recentworkhasshownthatthecutaneousvasoconstrictorsystemisnot so simple.Cutaneous vasoconstrictor nerves have been known to con-tain several neurotransmitters colocalized with norepinephrine,includingneuropeptideY(NPY)andATP, whichhavebeenshown to participate in noradrenergic vasoconstriction in ani-mal models (8, 29, 30, 57, 58, 90). Similar results have beenreported from in vitro studies with isolated human tissues andvascular smooth muscle cells (61, 62). In vivo work by Taddeiet al. (82) showedthat suchsystemswereextant inhumanforearm circulation and mediated the vasoconstriction inducedbybaroreexunloading.Thesestudiesprovidedtherationalefor examining the role of cotransmission in the control of thehuman cutaneous circulation.In 2001, Stephens et al. (76) demonstrated that humancutaneous active vasoconstrictor nerves, like human activevasodilator nerves, represent a cotransmitter system. Theseauthors compared the effects of differing combinations of 1-,2-, and -receptor antagonists on the SkBFresponses tohypothermia. Their results conrmed a role for postjunctionalexcitationof 1- and2-receptorsinreducingSkBFduringhypothermiaasinducedbywholebodycooling. Inaddition,theyfoundthat thepretreatment ofskinwiththe-receptorantagonist propranolol producedamoreconsistent vasocon-striction during hyperthermia. This suggests that a -receptor-mediated vasodilation may modulate-receptor-mediated va-soconstriction during hypothermia. The most signicant resultof their work was the nding that simultaneous and completeblockade of 1-, 2-, and-receptors failedtoabolishthecutaneous vasoconstriction induced by hypothermia. Com-binedwiththeir conrmationthat bretyliumblockadeof allneurotransmitter release from cutaneous noradrenergic nervestotally abolished reductions in SkBFduring body cooling,Stephens et al. concluded that nonnoradrenergic, cotransmittermechanisms effect reductions of SkBF during activation of thecutaneous active vasoconstrictor system in hypothermia.Subsequent work by Thompson and Kenney (87) conrmedthe foregoing results and further characterized the importanceof cotransmissioninactive vasoconstriction, These authorsfound that combined blockade of - and -receptors in forearmskinattenuatedthemaximalvasoconstrictionofforearmskineffectedbywholebodycoolingby40%inyoungsubjects.Combined blockade completely abolished the vasoconstrictionto cold stress in subjects 61 yr and older. Prejunctional block-ade of all sympathetic neurotransmitter release with bretyliumtosylate completely abolished vasoconstriction in both groups.These ndings revealed the great contribution that cotransmis-sion makes to cutaneous active vasoconstriction in youth andhow this contribution is lost with advancing age.A recent study by Stephens et al. (78) has further exploredthe role of cotransmission in active cutaneous vasoconstriction.TheytestedthehypothesisthatNPYactedasacotransmitteralong with norepinephrine to mediate reductions in SkBFduring hypothermic periods in humans. They further hypothe-sizedthat NPYY1receptors mediatedtheeffects of NPY.Theyfoundthat theNPYY1antagonist BIBP-3226signi-cantly attenuated reductions in SkBF during whole body coldstress. Furthermore, the combination of NPYY1 receptorantagonismwithcomplete-and-receptorblockadeabol-ished the cutaneous vasoconstriction attendant to hypothermia.As illustratedinFig. 3, theseresults clearlyshowthat thecutaneous active vasoconstrictor systemis a cotransmittersystem that effects reductions in SkBF via the release of NPYandnorepinephrineandthepostjunctionalactivationofNPYY1, 1-, 1-, and perhaps -receptors (78).Local Temperature Control Mechanisms of theCutaneous VasculatureLocal warming of the skin and vasodilation. In response toincreases in local Tsk as occurs with hyperthermia, cutaneousbloodvessels dilatebylocal temperature-dependent mecha-nismsinadditiontothepreviouslydiscussedneural mecha-nisms. Withlocal warmingofskin, local SkBFincreasesindirect proportiontothetemperatureachieved, withmaximallocal SkBF reached when local Tsk is held at 42C for 3555min (86). The local vasodilation is biphasic with an initial briskvasodilation followed by a prolonged plateau phase (see Fig. 4).Themechanismsthat effect thelocal, temperature-depen-dent cutaneous vasodilation involve both local neural mecha-nismsaswell aslocal generationofNO. Thesetwomecha-nisms appear to be independent of each other (56). The previ-ouslydiscussedneural cutaneous active vasodilator systemdoes not appear to be involved in the cutaneous vascularresponse to local skin warming because neither botulinumtoxin-induced abolition of active vasodilation nor muscarinic-receptor blockade alters the vasodilator response (23, 46).Prostanoids do not appear to be involved either, becausecyclooxygenaseinhibitionfailstoalterlocal temperature-de-pendent vasodilation (23).Theinitial phaseof thelocal warmingresponsehasbeenfound to be mediated by local activation of afferent cutaneoussensory nerves. This portion of the vasodilation can be greatlyattenuated by topical anesthesia directly at the locally warmedsite but not by cutaneous nerve blockade at points distant fromthe heated site (56, 60).Recent work by Stephens et al. (77) has further characterizedthe role of sensory afferents in the vasodilation caused by localskin warming. On the basis of studies done with topicalapplication of the vanilloid type 1-receptor activator capsaicin,theyproposedthatlocalincreasesinskintemperaturestimu-lates heat-sensitive vanilloid type 1 receptors on afferentnerves. Thisactivatesalocal axonreextocausetheanti-dromicreleaseof avasodilatoryneurotransmitter (or neuro-transmitters)that effect local skinbloodowincreases(77).Fig. 3. Sympathetic noradrenergic nerve cotransmission and cutaneous activevasoconstriction. Noradrenergiccutaneous activevasoconstrictionhas longbeen known to be mediated by norepinephrine (NE) release acting on 1- and2-receptors on the vascular smooth muscle of cutaneous arterioles. Stephenset al. (76, 78) have recently demonstrated that the neuropeptide cotransmitterneuropeptideY(NPY)isalsoinvolvedinactivevasoconstrictionandworksthrough postjunctional NPY Y1 receptors.Invited Review1713 CUTANEOUS VASCULAR CONTROL MECHANISMSJ Appl Physiol VOL100 MAY2006 www.jap.orgon January 8, 2015Downloaded from Thenatureofthevasodilatoryneurotransmitteroftheinitialphase of local temperature-dependent vasodilation remainsuncertain.The prolonged plateau phase of local temperature-dependentvasodilationhasbeenshowntobemediatedbylocalgenera-tion of NO. This phase can be greatly attenuated by pretreat-ment of the locally warmed area of skin with the NOS inhibitorL-NAME(43, 56). Shastryet al. (70) usedthe heat shockprotein 90 (HSP90) inhibitor geldanamycin to further examinethe mechanisms involved. HSP90has beenshowntobindendothelial NOS(eNOS; typeIII NOS) andenhanceeNOSactivationandNOgeneration(22).Theyfoundthatgeldana-mycin attenuated increases in SkBF caused by local skinwarmingby20%andthussuggest that eNOSmaybetheNOS isoform that mediates the prolonged plateau phase of thecutaneousvascular responsetolocal hyperthermia(70) (seeFig. 5).Local cooling of the skin and vasoconstriction. As with localincreases in Tsk, decreases in local Tsk with local cooling of theskin causes a local, temperature-dependent vasoconstriction. Incontrast tothe local warmingvasodilatoryresponse that isindependent of cholinergic cutaneous active vasodilator mech-anisms, the local cooling vasoconstrictor response is dependenton intact noradrenergic cutaneous active vasoconstrictor nerves(38, 59, 60). The requirement for intact noradrenergic neuronswasmadeevident fromstudiesthat usedbretyliumtoblockneurotransmitter release. With intact noradrenergic nerves,local coolingcauses a progressive reductioninSkBFwithfalling local temperature. Pretreatment of skin with bretylium,whichblocksprejunctional releaseofneurotransmittersfromsympathetic vasoconstrictor nerves, reverses the initial portionof the local cooling-induced vasoconstriction into a local cool-ing-induced vasodilation. It is only with continued cooling thatbretylium-treated sites show a reduction in SkBF (27, 59, 60).Recent work by Johnson et al. (38) has further dened howthe cutaneous active vasoconstrictor system participates in thevasoconstrictor response to local skin cooling in the absence ofTc changes by examining the roles of noradrenergic receptors,NPYY1receptors, andafferent sensorynerves. Theyfoundthat blockade of - and - receptors altered the response as didbretylium; i.e., combined - and -receptor blockade reversedthe initial phase of vasoconstriction was reversed to a vasodi-lation followed by vasoconstriction as cooling continued. NPYY1-receptor blockadewithBIBP-3226didnot alter there-sponseat all. Finally, topical anesthesiawithEMLAcreamalso reversed the initial vasoconstriction to a dilation followedbyconstrictionwithprolongedcooling. Johnsonet al. con-cluded that local cooling of skin occurs in two phases: an initialFig. 4. Local warming responses and vasodi-lator mechanisms in human skin. Increases inthe local temperature of cutaneous blood ves-sels increaseskinbloodowinabiphasicresponse: aninitial rapidincreasetoapeakfollowedbyaprolongedplateauphase. Theinitialphaseismediatedbyantidromicneu-rotransmitter releasefromsensoryafferentsmediatedbytemperature-sensitivevanilloidtype1receptors(VR-1). Sympatheticactivevasodilator nerves are not involved. The pla-teauphasethat is observedwithprolongedlocal warming of the skin is mediated bynitricoxide(NO)generation. Thesourceofthis NO appears to be at least partiallythrough heat shock protein 90 (HSP90) acti-vationof endothelial NOsynthase(eNOS).None of the responses to increased local skintemperature depend on the presence of intactcutaneous active vasodilator nerves (augmented effects due to increased tempera-ture; ? unknown neurotransmitter).Invited Review1714 CUTANEOUS VASCULAR CONTROL MECHANISMSJ Appl Physiol VOL100 MAY2006 www.jap.orgon January 8, 2015Downloaded from phase lasting a few minutes, and a prolonged phase. The initialphase is mediated by activation of cold-sensitive afferentneurons that effect the release of norepinephrine from sympa-theticcutaneousvasoconstrictornerves. Norepinephrinethenvasoconstricts skin vessels through postjunctional -receptors.NPY and NPY Y1 receptors appear not to be involved in theeither the initial or prolonged phases. In addition, the mecha-nisms for theprolongedphaseappear tobenonneurogenicbecause no manipulations altered the prolonged cutaneousvasoconstrictionduringlocalcooling;however,thesemecha-nisms have not been dened further (38).Work done over the last several decades has suggested a rolefor 2-adrenoreceptors in causing the cutaneous vasoconstric-tion induced by local skin cooling (2, 3, 11, 16, 17, 89). Thisworkwas basedontheinvivoobservationthat local skincoolingaugmented2-adrenergicvasoconstriction, butatten-uated 1-vasoconstriction in humans (20). In particular, Flava-han and colleagues have extensively explored the role of-adrenergic receptors. Their studies have used the mouse tailas well as isolated human tissues and cells. With these models,theyhaveshownthatlocalcoolingcausesaugmentedadren-ergic vasoconstriction through 2C-adrenoreceptors (11).These receptors have been found not to be directly thermore-active; rather they are translocated from the trans-Golgi appa-ratus of vascular smooth muscle cells to the plasma membranethroughthecold-inducedactivationofRhoAandRhokinase(2, 3). RhoA and Rho kinase activation also appears to enhancethe calcium sensitivity of the vascular smooth muscle contrac-tile apparatus (2).Recently, Flavahan (17) published work that the generationofreactiveoxygenspecies(ROS)frommitochondriainvas-cular smooth muscle may mediate the vasoconstriction inducedby local cooling. Baily et al. (3) found that cooling of mousetail arteries ledtoanincrease inROSinvascular smoothmuscle cells. Manipulationof ROSlevel byapplicationofrotenone, N-acetylcysteine, or a superoxide dismutase mimeticabolishedthevasoconstrictorresponseto2C-adrenoreceptoractivation. These manipulations of ROS levels also abolishedtheactivationof RhoAinculturedhumanvascular smoothmusclecells. Theseresultssuggest that thevasoconstrictioncaused by local cooling is effected by increased ROS genera-tion in mitochondria in vascular smooth muscle cells. In-creasedROSactivatesRhoA/Rhokinaseactivationandcon-sequent mobilization of 2C-adrenoreceptors to the cell mem-branewheretheycanbeactivatedbycatecholamines fromsympatheticneurons(3). Whetherthiselegantmodelpartici-Fig. 5. Local cooling responses and vasocon-strictor mechanisms in human skin. De-creases in the local temperature of cutaneousblood vessels reduces skin blood owthrough mechanisms that require intact sym-pathetic noradrenergic innervation. Effects oflocal temperaturedecreasesareindependentof any change in core temperature. Localtemperaturereductions aresensedbycold-sensitive afferent nerves. These nerves effectthe release of norepinephrine fromsympa-theticactivevasoconstrictornerves. Norepi-nephrineactsthrough-and-receptorstoproduce an initial vasoconstriction. Localcoolingalsomediatesaninitial vasodilationthat competeswiththeinitial noradrenergi-cally mediated vasoconstriction. This dilationis mediatedthroughnonneural mechanismsandis observedwhenrelease or postjunc-tionaleffectsofnorepinephrineareblocked.Finally, continued local cooling effects a pro-longed vasoconstriction through nonneuralmechanisms. The nature of the nonneuralmechanisms is unknown in humans.Invited Review1715 CUTANEOUS VASCULAR CONTROL MECHANISMSJ Appl Physiol VOL100 MAY2006 www.jap.orgon January 8, 2015Downloaded from pates in the in vivo human cutaneous response to local coolingis unproven.FUTURERESEARCHDIRECTIONSCutaneous Active Vasodilator MechanismsAnalternativeexplanationforthendingbySavageetal.(69) that the vasodilation induced by hyperthermia is preservedinCFdespitesparsecutaneouslevelsofVIPisbasedonthepossibility of redundancy between ACh and VIP in cutaneousactive vasodilator control, as occurs in other cotransmittersystems (4, 53). According to this explanation, in CF patients,an apparently normal increase in SkBF during heat stress couldbe caused by ACh release. The active vasodilator system in CFpatients would be highly dependent on ACh-mediated vasodi-lation and hence highly sensitive to muscarinic-receptor block-ade. Similarly, inhealthy, non-CFpersons, coreleasedAChand VIP each contribute to cutaneous active vasodilator duringheat stress. Blockadeof postjunctional muscarinicreceptorswith atropine in non-CF persons would leave the VIP portionofactivevasodilatorintactandgivetheimpressionthatAChwas not involved. Furthermore, blockade of prejunctional mus-carinic receptors with atropine would reduce prejunctionalinhibitionof neuropeptiderelease, produceanenhancedre-lease of VIP, and further mask the role of ACh (4, 53).Conversely, innon-CFpersons, blockade of VIPreceptorswouldleavetheACh-mediatedportionunaltered, suggestingthat VIP was not important to active vasodilator. Cotransmittersystems are known to have redundancies whereby lack of oneneurotransmittercanbecompensatedforbyanother(4, 53).Whether this occurs in the cutaneous active vasodilator systemis unknown.An additional complexity of cotransmitter systems is that thereleaseofthedifferentneurotransmittersdependsonthefre-quency of nerve ring (4, 54). In nerves with colocalized AChand VIP, ACh is preferentially released at low ring frequen-cies and VIP at high ring frequencies (4, 54). Such differentialreleaseof classicalneurotransmitterssuchasAChat lowfrequencies andneuropeptide release at higher nerve ringfrequenciesarewell documented(4, 54). Giventhat SSNAincreases progressively with Tcduring heat stress (79), itwould not be surprising to nd that ACh plays a role early inheat stress(whenSSNAislow) andVIPand/or other neu-ropeptides play roles late in heat stress (when SSNA is high).The studies by Shibasaki et al. (74) and Shastry et al. (71)are consistent with the differential release of neurotransmittersmentioned in the foregoing section and suggest the possibilityofdifferent mechanismsofNOgenerationwithintheactivevasodilator system. Given that bioavailable NO increases in theinterstitial space duringheat stress (47) andthe results ofShibasaki et al. (74) andShastryet al. (71), it maybethatdifferent mechanisms are involved in the early and late phasesof active vasodilationingeneral andinNOproductioninparticular.A related and unanswered question about the role of NO intheactivevasodilator systempertains towhichisoforms ofNOS participate in active vasodilator. Both neuronal NOS andeNOShave beendetectedinnormal humanskin(75, 91).Although it is clear that NOS generation of NO mediates partof active vasodilator system, it is unclear which NOS isoform(s)produce the increased NO required for active vasodilator.Cutaneous Active Vasoconstrictor MechanismsNPYcaneffect vasoconstrictioneitherdirectlyorthroughthe potentiation of noradrenergic -receptor activation. BasedontheworkbyStephenset al. (76, 78), it isclearthat bothNPYandnorepinephrineacttogethertoreduceSkBFduringhypothermia, but whether NPYacteddirectlyoncutaneousvascular smooth muscle or through ostentation of noradrener-gicmechanismswasunclear.Theydidnotethat,duringnor-mothermia, exogenous norepinephrine caused small and tran-sient vasoconstriction at sites with complete - and -receptorblockade. Furthermore, the addition of NPY Y1-receptorblockade abolished this vasoconstriction. However, because ofthe transient nature of the response and because of the require-ment for tonic NPY release under normothermic conditions fortheir observation to be true, the authors were reluctant toconclude whether NPY acts as a direct cutaneous vasoconstric-tor or whether it potentates the effects of noradrenergic recep-tor activation. This remains an unresolved issue.Local Warming of the Skin and VasodilationThe major area of uncertainty regarding the mechanisms bywhich local hyperemia effects vasodilation is what neurotrans-mitterorneurotransmittersarereleasedbyafferentnervestocause local SkBF increases. The C-ber afferents in skin thatarelikelyinvolvedintheprocessareknowntocontainsub-stance P and calcitonin gene-related peptide (CGRP). Whethersubstance Pand/or CGRPare the actual neurotransmittersinvolved remains to be proven in humans.Another unansweredquestionrelatestothegenerationofNO that mediates the prolonged plateau phase of the cutaneousvascular responsetolocal skinhyperemia. It hasbeensug-gested that HSP90 causes calmodulin to displace eNOS fromcaveolin-1. This leads to eNOS activation and potentiallyincreased NO generation (25). The observation that HSP90 isnecessary for full expression of the NO-dependent, prolongedplateauphase of the local warmingresponse suggests thateNOS is involved in the process and that calcium and calcium-dependent proteins are involved. These mechanisms remain tobe studied.Local Cooling of the Skin and VasoconstrictionThe role of sympathetic vasoconstrictor nerves in the initialphase of vasoconstriction effected by local cooling of the skinis well characterized; however, the nonneural mechanisms thatmediate the prolonged response to local cooling are unknown.The intricate model of cold-induced ROS generation leading toRhoA/Rhokinaseactivationand2C-adrenoreceptortranslo-cation proposed by Flavahan and colleagues (2, 3, 11) must beveriedinvivoinhumans. Other mechanisms that maybeinvolved include alterations of endothelial function, bloodviscosity,receptorafnity,and/orcutaneousvascularsmoothmuscle function. None of these mechanisms have been inves-tigated at present in humans.GRANTSD. L. Kellogg, Jr., is supported by National Heart, Lung, and Blood InstituteGrant HL-65599.Invited Review1716 CUTANEOUS VASCULAR CONTROL MECHANISMSJ Appl Physiol VOL100 MAY2006 www.jap.orgon January 8, 2015Downloaded from REFERENCES1. 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