Role of adenosine in the sympathetic activation produced by isometric exercise in humans

Role of adenosine in the sympathetic activation

produced by isometric exercise in humans.

F Costa, I Biaggioni

J Clin Invest. 1994;93(4):1654-1660. https://doi.org/10.1172/JCI117147.

Isometric exercise increases sympathetic nerve activity and blood pressure. This exercise pressor reflex is partly mediated by metabolic products activating muscle afferents

(metaboreceptors). Whereas adenosine is a known inhibitory neuromodulator, there is increasing evidence that it activates afferent nerves. We, therefore, examined the hypothesis that adenosine stimulates muscle afferents and participates in the exercise pressor reflex in healthy volunteers. Intraarterial administration of adenosine into the forearm, during venous occlusion to prevent systemic effects, mimicked the response to exercise, increasing muscle sympathetic nerve activity (MSNA, lower limb

microneurography) and mean arterial blood pressure (MABP) at all doses studied (2, 3, and 4 mg). Heart rate increased only with the highest dose. Intrabrachial adenosine (4 mg) increased MSNA by 96 +/- 25% (n = 6, P < 0.01) and MABP by 12 +/- 3 mmHg (P < 0.01). Adenosine produced forearm discomfort, but equivalent painful stimuli (forearm ischemia and cold exposure) increased MSNA significantly less than adenosine. Furthermore,

adenosine receptor antagonism with intrabrachial theophylline (1 microgram/ml forearm per min) blocked the increase in MSNA (92 +/- 15% vs. 28 +/- 6%, n = 7, P < 0.01) and MABP (38 +/- 6 vs. 27 +/- 4 mmHg, P = 0.01) produced by isometric handgrip (30% of maximal voluntary contraction) in the infused arm, but not the contralateral arm. Theophylline did not prevent […]

Research Article

Find the latest version:

Role of Adenosine

in the

Sympathetic

Activation

Produced by Isometric

Exercise

in Humans

Fernando Costa and Italo Biaggioni

DivisionofClinicalPharmacology,DepartmentsofMedicineandPharmacology, VanderbiltUniversity,Nashville,Tennessee37232-2195

Abstract

Isometric exercise increases sympathetic nerve activity and bloodpressure. This exercise pressor reflex is partly mediated bymetabolic products activating muscle afferents (metabore-ceptors). Whereas adenosine is a known inhibitory neuromodu-lator, there is increasing evidence that it activates afferent nerves.We,therefore, examined the hypothesis that adenosine stimulates muscle afferents and participates in the exercise pressorreflexinhealthy volunteers. Intraarterial administra-tionof adenosine into theforearm, duringvenousocclusion to prevent systemic effects, mimicked the response to exercise, increasing muscle sympathetic nerve activity (MSNA, lower limb microneurography) and mean arterial blood pressure

(MABP)atall dosesstudied(2, 3, and 4 mg).Heartrate in-creased only with thehighest dose. Intrabrachial adenosine (4 mg) increased MSNA by96±25%(n=6,P <0.01) and MABP by 12±3mmHg(P<0.01).Adenosineproducedforearm

dis-comfort, butequivalent painfulstimuli(forearmischemia and coldexposure)increased MSNAsignificantlyless than adeno-sine.Furthermore, adenosine receptorantagonismwith intra-brachialtheophylline (1 ytg/mlforearm permin)blocked the increase in MSNA(92±15%vs.28±6%,n= 7,P <0.01)and MABP(38±6vs.27±4 mmHg,P=0.01)produced by isomet-richandgrip (30%ofmaximal voluntarycontraction)inthe in-fused arm, butnotthe contralateralarm.Theophyllinedidnot prevent the increase in heart rate produced by handgrip, a response mediated moreby central command than muscle af-ferent activation. We propose that endogenous adenosine contributes to the activation of muscle afferents involved in the exercise pressor reflex in humans. (J. Clin. Invest.

1994.93:1654-1660.) Keywords:adenosine *

theophylline

-

iso-metric exercise * muscle

sympathetic

nerve

activity

Introduction

Isometricexercisesetsinmotionaseriesofeventsthat work in tandem to activate the

sympathetic

nervous system and in-creaseblood pressure. Thecortical

impulses

thatinitiate volun-tarymuscle contractions

(the

so-called central

command)

con-tributetothe increase in

sympathetic

tone.Localmechanisms alsoplayapivotalrole in the

triggering

of thisreflex. Muscle contraction activates afferent nerves sensitive tomechanical

Dr.ItaloBiaggioni,Clinical ResearchCenter,AA3228MCN, Vander-biltUniversity, Nashville,TN37232-2195.

Receivedfor publication12July1993andinrevisedform17

De-cember1993.

deformation ("mechanoreceptors"). The increase in muscle metabolism and the relative ischemia that results from com-pression of blood vessels by the contracting muscle generate metabolic products that then activate chemo-sensitiveafferent

nerves ("chemoreceptors" or "metaboreceptors"). These che-moreceptors constitute the afferent limb of a reflex that results in sympathetic activation and increased blood pressure. Our understanding of this reflex in humans has been improved by the ability to measure muscle sympathetic nerve activity

(MSNA)'

directly usingmicroneurographic techniques (1). Us-ingthis approach it has been shown that voluntary isometric handgrip increases sympathetic nerve activity and blood pres-sure inhumans, in large part due to stimulation of these fore-armafferents(1).

Adenosineis known to be produced during increased meta-bolic demands and, in particular, during muscle contraction andischemia (2). It could be proposed, therefore, that adeno-sine accumulates during isometric exercise and activates mus-cleafferents. Inagreement with thishypothesis, there is grow-ingevidence that adenosine excites a variety of afferent nerves leading to sympathetic activation (3). Adenosine activates renalafferents(4),myocardial afferents (5), and carotid body andarterial(6)chemoreceptors. Activation of afferentnerves byadenosineappearstobeparticularlyprominentin humans, and may explain most ofthecardiovascular and respiratory

effects of intravenousadenosinein thisspecies.

Given theexcitatoryeffect ofadenosineonother afferent

fibers,the purpose of the presentstudieswasto testthe hypoth-esis thatadenosine activatesthe muscleafferents involved in theexercisepressor reflex. Towardsthisaim,wehave usedtwo complementary approaches.First,wedeterminedif theeffects ofexogenousadenosine, acting locallyintheforearm,mimic thoseproduced bytheexercisepressorreflex.Forthesestudies weadministeredincreasingdosesof adenosine via the brachial artery while thevenouscirculation ofthearm wasoccluded,to avoidspillover of adenosineinto thesystemiccirculation. Sec-ond, we determined if blockade ofendogenous adenosine

would alter the exercise pressorreflex. Forthesestudies, the

adenosine receptor

antagonist theophylline

wasadministered

viathebrachialarterytominimize

systemic

effects.Isometric exercisewasperformedduringintrabrachial administration of saline or theophylline while

monitoring

the heart rate and blood pressure responses. Concomitantmeasurementsof

sym-patheticnerveactivityweremade in thelowerlimb, throughan

electrodeplacedintheperonealnerve,as an assessmentofthe systemicsympatheticactivationproducedbyexercise.

Methods

Westudiedatotalof 19normal,healthyvolunteers of bothgenders,

19-44 yr old (mean, 25±2 yr). Subjects were nonsmokers, whowere 1.Abbreviation usedinthispaper: MSNA,musclesympatheticnerve

activity.

1654 F. Costa andI.Biaggioni

J. Clin. Invest.

© The AmericanSocietyforClinicalInvestigation,Inc.

0021-9738/94/04/1654/07 $2.00

free of medications and had abstained from methylxanthines for at least 72 h before the study day. Theprotocolwas_{approved by}the VanderbiltUniversityInstitutional Review Board. Volunteerswere in-formedof thecharacteristics ofthestudyand gave writtenconsent.

Instrumentation. Subjects werestudied fasted and in thesupine position.Heartratewasmonitoredbysurfaceelectrocardiography

cou-pledto a ratecomputer. Arterial blood pressurewasmeasuredthrough

anindwellingcatheterplacedin the left brachial artery and connected

throughtwothree-wayvalvestoapressure transducer. The second and third portswereused for intraarterialdrugadministration. Cardiovas-cularsignalsweremodulatedonsignalconditioners anddisplayedona

thermal array recorder(TA2000;GouldInc., Cleveland, OH).To

mea-sureexpiratory volume,thesubjectsbreathedthrougha_mouthpiece,

andinspirationandexpirationwereisolated with one-way valves.

Ex-piratoryflowwasmeasured withaheatedpneumotachograph (Fleisch pneumotach;GouldInc.)connectedto adifferential transducer (Vali-dyne Engineering Corp., Northridge, CA). Expiratory volume was

measuredbyintegratingtheexpiratoryflowsignalovertime(integrator signal conditioner,GouldInc.).

Sympatheticnerveactivitywasmeasuredaspreviouslydescribed

(7).Theapproximatelocationoftherightperonealnerveatthe levelof thefibularheadwasdeterminedbytransdermalelectricalstimulation (10-60V,0.01-msduration),which producedpainlessmuscle

contrac-tions of the foot.Atungsten needleelectrode,withashaft diameterof 200 gm andanuninsulatedtipdiameter of1-5Aim,wasinserted into thenerve.Asimilar electrode withalargeruninsulatedtipwasinserted subcutaneously near therecordingelectrodeto serve as areference electrode. Therecordingelectrodewaspositionedwithin thenerve to

obtain multiunitrecordingsofsympatheticefferentactivity.Placement of the electrodewas_guidedbyelectrical stimulation (1-4 V, 0.01-ms

duration),which_producedmuscletwitchesofthe foot butnot paresthe-sias.Electricalstimulationwasdonewithastimulatorconnectedto an

isolation unit(S88andSIU8TB;GrassInstruments, Quincy,MA). The electrodewasthenswitchedto a_recordingmode andfine adjustments of itspositionweremadetoobtainasatisfactoryrecording siteas

de-scribed below. Recorded signals were fed to a preamplifier (gain,

X1,000)andwerefiltered usingabandwidth between 700 and 2,000 Hz.Thefilteredsignalwasrectified, amplified (gain,X100),and

inte-gratedina_{resistance-capacitance}networkusingatimeconstantof0.1 (NerveTrafficAnalysisSystem 662C-3;Universityof Iowa Bioengi-neering,IowaCity,IA).Thefinalsignalwasmonitoredusingastorage

oscilloscope (SIllA;Tektronics, Beaverton,OR) andrecordedafter

fourfoldamplification(TA-2000recorder;GouldInc.)

Criteria foranadequatemusclesympatheticnerve_activity

record-ing were as follows: (a) electrical stimulation produced muscle

twitches, but no paresthesias; (b) stretch ofthe tendons in the foot evokedproprioceptiveafferentsignals,whereascutaneousstimulation

by slight stroking ofthe skin did not; (c)held expirationincreased neuralactivityat asitewhere arousalstimuli did not; and(d)nerve

activityincreasedduringthehypotensive phaseof the valsalva maneu-verandwassuppressedafter the release of valsalvaduringblood

pres-sureovershoot. Musclesympatheticnerveactivitywasalsomonitored

throughoutthestudyusingaloudspeaker.Thishelpedin the identifica-tionofpotential artifacts,suchaselectrostaticdischarges,whichwere

alsoidentified in theintegrated neurogrambytheirrapid onsetand shorterduration.

Study protocols. Subjectswereallowedto restinaquietroomfor 20-30 min after instrumentationtoensurethat allcardiovascularand

microneurographic parameters had returned to restingvalues. The

subjectswerewarned aheadof time of allprocedures done,and that

theymayproducediscomfort,in ordertoavoidanticipatoryreactions. Intraarterial bolusinjectionsofeither salineorincreasingdoses ofaden-osine_{(2, 3,}and4mg)werethenadministered inasingle-blinded fash-ion whilemonitoringthesubject'sheart rate, blood pressure, and mus-cle sympathetic nerve _activity. Intraarterial adenosinewas

adminis-tered during simultaneous occlusion of the venous circulation to

prevent itsspilloverinto the_systemiccirculation. For thispurposea

pneumaticcuffwasplacedproximaltotheinjectionsiteandinflatedto

50mmHg immediately before injection, andwasmaintained inflated for the 2min after the adenosine injection. The highest dose of adeno-sine(4 mg)wasalso given intravenously and intraarterially without occluding thevenouscirculation inordertoexamine thesystemic ef-fects of adenosine, andtodetermine the effectiveness ofvenous occlu-sion in avoiding these systemic effects. Sufficient time wasallowed between injections to allow parameters to return toward baseline values.

Intraarterial administration of adenosine has been reported to pro-duce forearmpain (8). It is possible, therefore, that activation of

sen-sory afferents may contributetothechanges in the MSNA produced by intrabrachial adenosine. To determine this potential contributionwe

compared the effects oftwopainful stimuli, ischemia and cold expo-sure,tothoseproducedby intrabrachial adenosine. For this purpose, saline andincreasing doses of adenosine (0.5-4 mg)wereadministered intrabrachially in a single-blinded fashion duringvenous occlusion. Subjectswereaskedtorank theintensity of painon ascale from0 (no pain)to 10 (excruciating pain) after each dose. Forearm ischemia of increasing duration (1, 2, 3, and 5 min) was induced in the same sub-jects by inflatingacuff placed around the arm to 50 mmHg above systolic blood pressure. The effects of cold exposureweredetermined by sequentially applyingone pack of ice (140 x 130 mm) overthe forearm, followed bytwopacks of iceovertheforearm, and finally by placing the hand in icewaterfor 1 min. The subjectswereaskedtorank theintensity of the painatthe end of each period of ischemiaorcold exposure,asdescribed above.Wepaired the dose of adenosine in each subject with the duration of ischemiaortheextentof cold exposure thatproduced similar intensities of pain. We then compared the effect of thosepaired stimuli on MSNA.

Asecond groupof volunteerswasstudied to determine the role of endogenous adenosine in the exercise pressor reflex. We used isometric handgrip to elicit an exercise pressor reflex, and theophylline as an adenosine receptor antagonist. Subjects were instrumented as de-scribed above and allowedto restinaquiet room for 20-30 min. Saline wastheninfused into the left brachial artery at the same rate ofinfusion calculatedfortheophylline(see below). After 5 min of saline infusion, weperformed three experimental protocols in the following sequence: (a)Handgripfollowed by ischemia. Volunteerswereaskedtoperforma

sustained handgrip for 3 minat30% of maximal voluntary contraction,

usingahandgripdynamometer(LafayetteInstrumentCo.,Lafayette, IN). A pneumatic cuff on the upper arm was inflated to 50 mmHg above thesystolic blood pressure immediately after release ofthe hand-grip andwasmaintained for 2 min (posthandgrip ischemia). (b) Con-trolhandgrip.Static isometric exercise was performed at 30% of maxi-malvoluntary contractionfor3 min in the right arm. (c) Simultaneous

handgripandischemia. Circulatory arrest was induced by inflating a pneumatic cuff, placed in the upper left arm, to 50 mmHg above the

systolicblood pressure. Thesubjectwassimultaneouslyasked to per-formasustained handgrip ofthe left hand at 30% of maximal voluntary contraction. Both exercise and ischemia were maintained for 2 min.

After this experimental protocol, the infusion of saline was replaced byaninfusion ofaminophylline(2.5 mg/ml) into the left brachial ar-teryat a rateof 1 Ag/mlofforearm volume/min. This dose has been

previouslyshowntoproducenegligiblesystemic effects (9). 5 min after

startingtheaminophyllineinfusion the exercise protocols described abovewererepeatedin thesamesequence. Heart rate, blood pressure, and MSNAweremonitoredthroughoutthestudy. Blood pressurewas

monitored continuously either through the left brachial catheter or with an automated device placed in the right arm (Finapres 2300; Oh-meda, Englewood, CO)during the periods of circulatoryarrestof the leftarm.Blood samplesweredrawnfrom the antecubital vein of both

arms atthe endof thestudyformeasurementof plasmatheophylline levels.

Drugs,analytical methods,and statisticalanalysis.Adenosine was

mg/ml, 85%anhydrous theophylline)was purchased from American RegentLaboratories,Inc.Shirley, NY.

Meanarterial bloodpressure was calculated as two-thirds of the

diastolicpressure plusone-thirdofthe systolic pressure. Measurements

of muscle sympatheticnerveactivitywere made from the original trac-ings of the mean voltageneurogramsusing a digitizer tablet coupled to

SigmaScanSoftware(JandelScientific,Corte Madera, CA) in a micro-computer. The amplitude of each "burst" was measured in millimeters and totalactivitywasdefinedas the sum of "burst" amplitude over a 60-speriodandexpressed in arbitraryunits. The effect of a given inter-vention (e.g., drugadministration)on muscle sympathetic nerve activ-ity was expressed as percent change from the resting period preceding theintervention.

Theophyllinewasmeasuredbyhigh performanceliquid chromotog-raphy asdescribed previously (10).Data were analyzed using the Num-berCruncher Statistical System software (NCSS, Kaysville,Utah)ina

microcomputer. Statistical analysiswasperformedusing t test for sin-glecomparisons, and analysis of variance (ANOVA) for multiple

com-parisons.Posthoc tests(Duncan's)were used todetermine individual differences only if significantgroupdifferenceswerefound with

AN-OVA.ValuesofP.0.05 wereconsideredsignificant.Resultsare ex-pressed asmean±SEM.

Results

Effects of intrabrachial administration ofadenosine. The

ef-fectsof bolusinjectionsof adenosineinto the antecubitalvein and into the brachialarteryduringsimultaneous venous occlu-sionarecomparedinFig.1. Intravenous adenosineproduceda

biphasicbloodpressure responseasreported previously (1

1),

withaninitialincrease followed byadecrease in blood pres-sure. The pressor phase was accompanied by anincrease in minute ventilation andaninitialincrease in MSNA followed by atemporary suppression in MSNA. Thedepressor phase

25

E

20-0.

K

10

15-cc15

-15

a

E E

10-0.

.4 5

_ie-/o

125

-._.100

-b

75-Z ₅₀

-0,

O-wY

SALINE

*

p<0.01

I 1

2 3

ADENOSINE (mg)

4

Figure 2.Cardiovascularandsympatheticeffects ofintrabrachial adenosine. Linegraphs show the effects of saline and increasing

in-traarterialbolusinjections ofadenosine on heart rate (HR, top), mean

arterialbloodpressure

(MABP,

middle), and muscle sympathetic nerveactivity (MSNA, bottom). Data are expressed as changes (A) or

percentchanges(%A)fromtheprecedingresting period. Measure-ments wereperformed duringthe period from 30 to 90 s after

injec-tions. Drugswereadministered intothebrachialartery during simul-taneous venousocclusion oftheforearm.n=6; P values are those

from ANOVA; asterisks indicate significant differencescompared withsaline, usingpost hoc tests(Duncan's).

mmHg

0 ... ...

TV ILt

100

...

HR

bpm

O{

BP 101 _fllifl

mmHg

TV _YI\~

ILt __

100.-.

HR 50 ---n I,,

bpm

0

44t,4^,\:~~~~~~~~~~~~~~\V'Y y.yl

. ...

tAdo4mgW.v

AA4AAA(1s/k/\/\/.A(/ N_,IA/n~~~~~~~~~~-i NA

_._. ...

.~~~~~~~~~~....I

...AdAdo4mg I.a...

Figure1.Tracing showsrecordingsofintegratedneurograms of

mus-clesympatheticnerveactivity (MSNA),blood pressure(BP),tidal volume(TV),andheart rate(HR) atbaseline,and after bolus

injec-tionof4mg adenosine whenadministeredeitherintravenously(top),

orintraarterially duringsimultaneousvenousocclusion(bottom).

wasaccompaniedbyanincrease in heartrateand MSNA. All oftheseeffectswereoverin 60s.Identical effectswereobserved

afterbolusinjections ofadenosine intothebrachialartery(data notshown),implying that,atthese doses,intrabrachial adeno-sine reaches the systemic circulation. A different pattern of

effectswasobserved whenadenosinewas

injected

intothe

bra-chialarteryduring simultaneousvenous

occlusion;

blood pres-sure and MSNA increasedgraduallyand in

parallel,

whereas

minute ventilation didnotchangesignificantly.

Thedose-responsecurveofthese changesareshown inFig. 2. Intraarterial bolusinjectionsof adenosine during simulta-neous venousocclusion producedadose-dependentincrease in MSNA (n=_6,P<0.01by ANOVA) and in bloodpressure(P

<0.01). Heart ratealso increased(P=_{0.03) but only}atthe highest dose of adenosine. Adenosine (4 mg) increased MSNA by 96±25% and raisedmean arterial bloodpressureby 12±3 mmHg. ThedatapresentedinFig.2 wereobtained by averag-ing heart rateand blood pressure values atthe time ofpeak effects ofeach bolusof salineoradenosine,andby

integrating

MSNAover 1 min,starting30safterdrug administration.

Adenosineproducedagreaterincrease in MSNA than cold exposure or ischemia,when both stimuliwere

paired

to pro-duce thesameintensityof forearmdiscomfort

(Fig.

3).

I II

i

USNA II A_NM

B

-uJ 0

o

4-z

2-

0-6

-CC

4,-0

co Z ₂

0

A

ADO COLD

PAIN

- 100

-80

-60 Z

-20

ADO COLD

MSNA

B

ADO ISCH ADO ISCH

PAIN MSNA

-40

30

K

*20 z

10 M

-0

Figure 3.Comparison oftheeffects of intrabrachial adenosineto

otherpainful stimuli. (A) Comparisonof the effects of intrabrachial

adenosine (ADO,2.7±0.6mg, openbars,n = 6)and coldexposure (COLD, hatchedbars)onpainscore(leftbars,maximalpainscore

= 10) and musclesympatheticnerveactivity (MSNA,rightbars,

ex-pressed asthe percent change form theprecedingbaselineperiod).

(B)Comparison of the effect of intrabrachial adenosine (ADO,

1.8±0.5 mg,openbars, n=6)andforearmischemia (ISCH, hatched bars,duration ofischemia=280±20s).Inbothcasesstimuliwere

titratedtoproduceasimilarlevelofforearm painin thesame indi-vidual.Other methods are similar to those ofFig.2.

Effect of adenosinereceptorblockade withtheophyllineon

theexercise pressorreflex. Intrabrachialinfusionof theophyl-line produced small changes in heartrate,bloodpressure, and MSNA thatwere notdifferent from those producedbysaline (TableI). Venous forearmsamplestakenatthe end oftheophyl-line infusion revealed theophylline plasma levels of5.1±1.1

,gg/ml

(28±6

,gM)

in theinfusedarm.Plasmalevels in the con-tralateral arm, reflecting the degree ofsystemic exposure to theophylline,were 1.0±0.2

ktg/ml

(5±1 AiM).

Static isometric exerciseproducedasignificantincreasein MSNA that progressed throughoutthe duration of the hand-grip and reached amaximal increase of 92±15% during the thirdminute. MSNA remained abovebaseline duringthe pe-riod ofpost-handgrip ischemia (Fig. 4). Similar time-course changes inbloodpressure and heart rate were observed. The increasein MSNA produced byisometric exercisewas signifi-cantlyinhibitedduring intrabrachial administration oftheoph-ylline (92±15% vs. 28±6% duringthethird minute of hand-grip),and wascompletelyabolished duringtheperiod of post-handgrip ischemia (Fig. 4; n = 7, P < 0.01). The increasein bloodpressure produced byhandgripwasalsoblunted by in-trabrachial theophylline (P=_{0.01, 38±6}vs. 27±4mmHg dur-ingthethird minute ofhandgrip). Onthe other hand,

theophyl-TableI.Effect ofIntrabrachialInfusion

ofSalineorTheophyllineonBaseline Parameters

Saline Theophylline

A HR(bpm) -1±1.4 -2±1

A SBP(mm Hg) +6±3 +3±2

A DBP(mmHg) +9±2 -4±3

AMSNA(%) +47±6 +33±10

line hadnosignificanteffectonthechangesin heartrate

pro-ducedbyisometricexercise.

Isometricexercisewasrepeatedduringsimultaneous circu-latoryarrestoftheexercisingmuscleinanattempttoprovidea

morepotentmetabolic stimulus.Isometric exerciseduring

cir-culatory arrestproducedagreaterincreasein muscle

sympa-theticnerveactivitythanisometricexercise alone.MSNA in-creasedby 77±16% duringthe second minute of isometric

ex-ercise andby173±39% duringthe second minute ofisometric exerciseduring circulatoryarrest.The increase inMSNA

pro-duced by isometric exercise during circulatory arrest was

blunted byintrabrachial theophylline to 44±19% during the secondminute(Fig. 5;n=7,P<0.01 by ANOVA). Theophyl-line alsoinhibitedtheincreaseinmeanarterial bloodpressure

produced by isometric handgrip during circulatory arrest

(45±7vs.27±1 mmHgduringthe second minute ofhandgrip), althoughthisdifferencedidnotreachstatistical significance(P

* *SALINE

30 O-- THEO

20-10i p-NS

0j

-40

-z E

E 30

-< ₂₀

-10

-100

-75

50

z

25- ....0p0.01

HG ' HG 2' HG3' PHIl' PHI 2'

TIME COURSE

Figure 4. Effect of intrabrachial theophyllineonthecardiovascular and sympatheticresponsetoisometric exercise. Linegraphsshowthe effects of 3min of sustained handgrip (HG, 30% of maximal

volun-tarycontraction) followed by 2 min of posthandgrip ischemia (PHI, circulatoryarrestof the forearm during thereleaseof handgrip).

Pro-cedureswererepeated during infusion of saline (K)ortheophylline (o, 1 qg/ml forearm/min).n=6;Pvaluesarefrom analysis of variance

for theoverallcomparisonbetween saline and theophylline. Other

methods andabbreviationsaresimilartothoseofFig.2.

p-0.01

= 0.06byANOVA). The increase in heart rate produced by isometric handgripduring circulatory arrest was unaffected by theophylline.

We thought it important to use each subject as his own control becauseoftheinterindividual variability in the magni-tudeofsympathetic activation produced by the exercise pres-sor reflex. For this reason, the salineinfusionperiod preceded the theophylline infusion period inall subjects. The order of infusionscould not be reversed because of the half-life of the-ophylline. Responses to isometric exercise of the right hand wereused as a control to account for possibletime-related ef-fects andfor potential systemic effects ofintrabrachial theoph-ylline. Intrabrachial infusionoftheophyllineintothe left arm didnotalter the increase inMSNA produced by isometric exer-cise of the rightarm(Fig.6).

Isometrichandgrip of the leftarm produced a greater in-crease inMSNAthan isometric handgrip oftheright arm. It haspreviouslybeen shown thatexerciseof the dominant, and therefore, more conditioned arm elicits a lesser increase in MSNA than exercise ofthenondominantarm(12).Ourresults during saline administrationcanbeexplained by this mecha-nism because all the volunteers participating in this protocol studywereright handed.

Discussion

Thespecificmetaboliceventsinvolved intheexercisepressor reflexare notcompletely understood.It has beenproposedthat

- ₃₀

-a 20

-10

-,, 40

-z

E E

30-I.n

<

20-10

-150

-m-100

-z

O

--e@SALINE

o---oTHEO

p-NS

p-0.0S

T- p<0.01

ISHG 1' ISHG 2' TIME COURSE

Figure5. Effect of intrabrachialtheophyllineonthecardiovascular

andsympatheticresponsetoisometric exerciseduringsimultaneous forearm ischemia. Linegraphsshowtheeffects of 2minofsustained

handgrip (30%of maximalvoluntary contraction) during

simulta-neouscirculatoryarrestof theforearm(ischemic handgrip, ISHG).

Procedureswererepeatedduring infusion of saline(-)ortheophylline

(o, 1,ug/ml forearm).n=7;Pvaluesarefromanalysisofvariance fortheoverallcomparisonbetweensalineandtheophylline. Other

methods andabbreviationsaresimilartothoseof Fig.2.

100*

80

-60

z

E 40

20

0

T

SALINE THEO

LEFT ARM

SALINE THEO

RIGHT ARM

Figure6. Bar graph shows the increase in muscle sympathetic nerve activity(MSNA,expressed as the percent change from the preceding resting period) produced during the third minute ofisometric hand-grip (30% of maximal voluntary contraction) of the left or right arm

duringinfusion of salineortheophylline(THEO) into the left brachial artery.Asteriskdenotessignificant differencecomparedtosaline

us-ingttest; n=5.

hydrogen ions are involved in muscle afferent activation. In favor of this proposal, itwasfoundthat a decrease in muscle intracellular pH parallels the increase in muscle sympathetic activity inresponse toisometric exercise (13,14).Furthermore, muscle contraction in patients withmusclephosphorylase defi-ciency (McArdle's disease) isnotaccompaniedbyglycogen deg-radation or a decrease in muscle pH, and MSNA does not increase inresponse toisometric exercisein thesepatients(15). It has been argued, however, that other metabolic products may alsoplayarole inthis reflex. Forexample, exercise ofa nonconditionedmuscle producesagreaterincrease inMSNA than exercise ofaconditioned muscle, eventhoughasimilar decrease in musclepH is produced in bothcases(12), suggest-ing that the decrease in muscle pH isnotthesolemediator of thisreflex. However, otherpotential metabolic mediators re-sponsiblefor the triggeringof the exericsepressorreflex have notbeenpreviouslyidentified.

Adenosinecanbeproposedasalikelycandidate because it isknowntobeproducedinskeletal muscles

during

increased

metabolicdemands and ischemia (2).There is extensive evi-dence, however, thatadenosineacts as an

inhibitory

neuromod-ulator.Adenosine

hyperpolarizes

neurons,decreasesnerve fir-ing, prevents seizures, and inhibits neurotransmitter release (16, 17). Theseinhibitoryeffectsareparticularly

prominent

in efferentnervesand in the centralnervoussystem. Even inthe

central nervoussystem, however, adenosine may have excit-atoryactions,

enhancing

the releaseofglutamateinthe nucleus ofthesolitariitract(18)and inhippocampalneurons

(19).

On theotherhand,adenosinehas

prominent excitatory

actions in afferentnerves.To thebest ofourknowledge,adenosine acti-vates allafferentssofarexamined, includingrenal and myocar-dialafferents,andarterialchemoreceptors. We,

therefore,

hy-pothesizedthat adenosine would also activate the muscle affer-entsinvolved in the exercisepressorreflex.

Insupportof thishypothesis,wepreviouslyfoundthat in-trabrachialinjectionsof adenosineproducedamodestbut

sig-nificant increase in MSNA (20).Anincrease in bloodpressure was also observed, but this effect was not significant atthe relativelylow dosesused(0.5-1.5mg).Inthepresentstudywe

were able toadminister

higher

dosesbyoccludingthevenous 1658 F.Costaand I. Biaggioni

-circulation ofthe forearmtoavoidsignificant spilloverof intra-arterial adenosine into the systemic circulation. This approach ispossible because of the extremely short half-life of adenosine inblood,reportedly < I s.(21). The effectiveness of this strat-egyis evidencedby the lack ofventilatorystimulation, adeno-sine's most consistent and dramatic systemic effect, when it wasadministeredintraarterially duringvenousocclusion.

Using this approach,wefound that intrabrachial adenosine producedgradual andparallel increases in MSNA and blood pressure at dosesthat hadnoeffectonheartrate(2 and 3mg). Isometricexercise, by comparison, also increases heartratein additiontoincreasingbloodpressureand MSNA.Theincrease in heart rate produced by voluntary exercise, however, is largely due to thevoluntary central command initiating the

exercise,rather thantoactivation of muscle afferents(22).The effects of intrabrachial adenosine, therefore, mimic those ex-pectedto result from theselective activation of muscle affer-entsby isometric exercise.

Before we can conclude that adenosine indeed activates muscleafferents in theforearm, alternative explanationsto our findingsneed tobeconsidered. Adenosine isapotent vasodila-tor in the forearm. It could be proposed, therefore, that the systemic sympathetic activation produced by intrabrachial adenosine is dueto local vasodilatation. Thisseemsunlikely because we havepreviously shownthatintrabrachial

nitroprus-side, administeredat adosetitratedtoproduce thesame mag-nitude of forearm vasodilationasadenosine, hasnoeffecton MSNA (20). Intrabrachial adenosine also produces forearm

discomfort (8),possiblythrough activation ofsensoryafferents (23) that share the morphological and electrophysiological characteristics of musclesympatheticafferents (slow-conduct-inggroup IVC-fibers) (24). The increase in MSNA produced by intrabrachialadenosine, however,was greaterthan that pro-duced by coldorischemia when these stimuliweretitratedto produce acomparableintensity of pain. Therefore, activation ofsensoryafferentsmaycontributeto,but doesnotcompletely explain,theincrease in MSNA produced by intrabrachial aden-osine.

Two limitations of this first set of experimentsshouldbe considered. First, it remains possible that adenosine activates otherasyetundeterminedstructuresin the forearm,leading to sympatheticactivation. From theresultsobtained using exoge-nousadenosine,therefore,wecannotdefinitelyconclude that adenosineactivates muscle afferents. Second, itcan be argued thatweusedsupraphysiologicaldosesof adenosine. Itis likely,

however, thatmostof theintraarteriallyinjectedadenosine is trapped by the endothelium because of thehighlyeffective nu-cleosideuptakesystem, and verylittlereaches theinterstitium (25),ourputative site of action. Itis, therefore,impossibleto estimate how much of the adenosine injected intraarterially

actually reaches the muscle interstitium, and whether such

concentrationsconstitute"supraphysiological" levels. Because of these limitationsand asa complementary ap-proach tothisproblem, we also determined ifblocking endoge-nous adenosine may alter the cardiovascular and neural re-sponses totheexercisepressorreflex. For this purpose we ad-ministered the adenosine receptor antagonist theophylline intrabrachially(26,27) at doses that lacked significant systemic effectsbut weresufficienttoantagonize the increase in MSNA producedby exogenous adenosine, while the exercise pressor reflexwasevoked

by

isometric

handgrip.

Intrabrachial

theoph-ylline

bluntedtheincreaseinMSNA andmeanarterialblood

pressureproduced by isometric exercise,but had no effect on the increase in heart rate. Similar effects were observed if a greater metabolic stimuliwasinducedbyperformingexercise during circulatoryarrestof theforearm. The fact that intrabra-chial theophylline selectively blocksthesympatheticactivation andthe increase in bloodpressure produced by exercise,but not the increase inheart rate, supportsthehypothesis that en-dogenousadenosine contributes to muscle afferent activation. As mentionedpreviously,theincrease in heart rateduring iso-metric exercise isto a large extentduetothevoluntarycentral commandinitiatingthe exercise rather than to muscle afferent activation.

Wealsomust considerpotentiallimitations of this second set ofexperiments. First, theophylline seemed to producea greater inhibition of theincrease in MSNA than the increase in blood pressure produced byisometric exercise. Several reasons may accountfor thisapparentdifference. Theophylline didnot blunt theexercise-induced tachycardia, whichmaycontribute tothe pressoreffect byincreasing cardiacoutput. Likewise, if our hypothesisis correct,intrabrachial theophylline would se-lectively blockadenosine-induced muscleafferent activation, but willhavenoeffectoncentral commandor muscle mecha-noreceptors, which may increase blood pressurethrough mech-anisms that are notreflected in MSNA. Second, it could be argued that theophylline may have other nonspecific effects, such asinhibitionofphosphodiesterase. Thepotency oftheoph-ylline as a phosphodiesterase inhibitor (IC50 of 0.1-0.2 mM [28,29]) is muchlowerthanas anadenosinereceptor antago-nist

(IC50

of2-50

_,M

[30]). Bycomparison, the plasma con-centration oftheophylline obtained in the infused arm was 28±6,uM.Third,it could be argued that theeffect of theophyl-lineon theexercise pressorreflexwasdue toasystemiceffect rather than local inhibition of adenosine in the exercising fore-arm. Systemic plasma levels of theophylline, however, were significantlylower (5±1 jiM)thanthoseconsidered therapeuti-cally effective (20-50 jiM). More importantly, theophylline had noeffecton theexercisepressor response toexercise ofthe contralateral arm, implying that theophylline had no signifi-cant systemiceffects. These resultsalsoimplythat the differ-ences in response to exercise of thetheophylline-infused arm were not due totime-related effects. This is an important con-sideration given the sequential design ofour study(saline fol-lowed bytheophylline).

prominent in other animalspecies. It is possible,therefore, that theapparent contradictionbetweenourfindingsand those re-ported in catsareexplainedbyspecies differences. Important

speciesdifferences have also been reported forotheractions of

adenosine(34).

It has beenproposedthat the primary roleofadenosineisto

maintainlocal homeostasis by counteractingexcessive

meta-bolic demandson the cell. It is generally consideredthat adeno-sine fulfills thisrolethroughits inhibitory actions(e.g.,

vasodi-lation, inhibition ofnorepinephrine release). The excitatory action of adenosine reported here wouldappear to be atodds with this role. However, by activatingmuscleafferents,

adeno-sinewillproduce systemic sympathetic activationandincrease blood pressure,which will result in improved perfusion pres-sure to the exercising muscle. At the same timeadenosinewill act locally to inhibit norepinephrine release and to produce

vasodilatation,thusprotectingthe exercising muscle from the

increased sympathetic activity.

Inconclusion, wepostulate adenosineas one of the

meta-bolic productsthatcontributeto the activation of muscle affer-entsinvolvedintheexercisepressor reflex in humans. Further-more, wespeculate that these excitatory effects ofadenosine

works in tandem with its inhibitory actions toprotect the

exer-cisingmuscle.

Acknowledgments

The authorswould liketothankDr.DavidRobertsonfor hisinputin theformulation ofthehypotheses underlying these studies, andMrs. JaneEstradaand Dorothea Boemer foreditorial assistance.

Thisworkwassupportedin part by grantsHL-14192(Specialized

CenterofResearch inHypertension)andRR-0095(Clinical Research

Center)from theNational Institutes of Health, andagrantfromthe Life Sciences Institute.

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