Plasma catecholamine modulation of alpha 2 adrenoreceptor agonist affinity and sensitivity in normotensive and hypertensive human platelets

Plasma catecholamine modulation of alpha 2

adrenoreceptor agonist affinity and sensitivity

in normotensive and hypertensive human

platelets.

A S Hollister, … , J H Nadeau, D Robertson

J Clin Invest.

1986;

77(5)

:1416-1421.

https://doi.org/10.1172/JCI112452

.

We measured alpha 2-adrenoreceptor density as well as affinity for and sensitivity to

agonist on intact platelets of normotensive and hypertensive subjects before and after

physiological increases in plasma catecholamines. In normotensives, posture-induced rises

in plasma catecholamines correlated with reduced alpha 2-adrenoreceptor agonist affinity

and fewer high affinity state receptors. Platelet aggregation and inhibition of adenylate

cyclase by L-epinephrine also was reduced. Hypertensive subjects had similar rises in

plasma catecholamines with upright posture, but showed no change in receptor affinity or

sensitivity. No change in platelet alpha 2-adrenoreceptor number occurred in these studies.

In vitro incubation with L-epinephrine revealed that platelets from hypertensives had slower

desensitization than those from normotensives. Binding studies at different temperatures

and with varying sodium concentrations found no thermodynamic or sodium-dependent

differences between normotensive and hypertensive groups. These studies demonstrate

that platelets from hypertensive subjects exhibit a defect in the ability of physiological

concentrations of agonist to desensitize the alpha 2-adrenoreceptor.

Research Article

Find the latest version:

Plasma Catecholamine

Modulation

of Alpha2 Adrenoreceptor Agonist

Affinity

and Sensitivity

in

Normotensive and

Hypertensive

Human

Platelets

Alan S. Hollister, Jack Onrot, Suzanna Lonce, John H. J. Nadeau, and David Robertson

Departments ofMedicine and Pharmacology, Division of Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee37232

Abstract

We measured a2-adrenoreceptor densityaswellasaffinityfor

andsensitivitytoagonistonintactplateletsof normotensive and hypertensive subjects before and after physiological increases

in plasma catecholamines. In normotensives, posture-induced rises in plasma catecholamines correlated with reduced

C2-ad-renoreceptoragonistaffinity and fewer high affinitystate

recep-tors.Platelet aggregation andinhibition ofadenylate cyclase by L-epinephrinealsowasreduced.Hypertensive subjectshad

sim-ilar rises in plasma catecholamines with upright posture, but

showednochange inreceptoraffinityorsensitivity.Nochange

inplatelet a2-adrenoreceptornumber occurred in-these studies. In vitro incubation with L-epinephrine revealed thatplatelets

fromhypertensives hadslower desensitization thanthosefrom

normotensives. Binding studiesatdifferenttemperaturesand with varying sodium concentrations foundnothermodynamic or

so-dium-dependent differences between normotensive and

hyper-tensivegroups.

These studies demonstrate that platelets from hypertensive subjects exhibitadefectinthe ability ofphysiological

concen-trations of agonisttodesensitize thea2-adrenoreceptor. Introduction

The influence of the sympatheticnervoussystemonblood

pres-sureismediatedpredominantly bythe catecholamines

norepi-nephrine and epinorepi-nephrine acting atalphaand beta

adrenore-ceptors. Many prior investigationsofthesympatheticnervous

systeminhumanhypertension haveconcentratedonattempts todetect elevatedplasma catecholamines inhypertensive

sub-jects. Althoughthese studies variedgreatlyindesign, the data donot supportthehypothesis that catecholamineexcessisthe

solecauseofhypertension (1, 2). However,recentstudies have shown that hypertensive subjects have enhanced pressor

re-sponsivenesstocatecholamine andsympathomimeticamine in-fusions(3-7). These findingsare consistent with the proposal

thatvasoconstrictor alpha adrenoreceptors of hypertensive

sub-jectsare moresensitivetocatecholamines thanarethose of

nor-motensives.

Parts ofthisworkwerepresentedtotheAmerican Federation for Clinical Researchmeetings, May 1983 and 1984, and abstracted in Clin. Res.

31:331A (1983) and 32:333A (1984).

Address all correspondence to Dr. Hollister, Division of Clinical

Pharmacology,Department of Medicine, VanderbiltUniversity,

Nash-ville,Tennessee 37232.

Receivedfor publication19April 1985 andinrevisedform9 January

1986.

Increased responsiveness ofadrenergicreceptors may be

sec-ondary to an increase in the number of receptors and/or an

increase in receptor-effector coupling efficiency.An increasein platelet a2-adrenoreceptor density combinedwithpressor,

car-dioacceleratory and proaggregatory hypersensitivity to

cate-cholamines has been reported in orthostatic hypotensivesubjects with chronically low circulating catecholamines (8-10). How-ever,several reports have found normal numbers of platelet

2-adrenoreceptors in hypertensive subjects (11-13), suggesting that the enhanced sensitivity of hypertensives tocatecholaminesis

not associated with an increase in adrenoreceptor number. Wehave demonstrated recently that plasmacatecholamines inversely regulate platelet a2-adrenoreceptor and lymphocyte #2-adrenoreceptor affinity for and sensitivitytoagonistsover short

time periods in normotensive subjects (14-16).Since enhanced

pressorsensitivity of hypertensives cannot be attributedto

in-creaseda-adrenoreceptor density,westudied agonist regulation ofa2-adrenoreceptor agonist affinityandsensitivityin order to

determine whether abnormal affinitystateregulationisassociated

with enhanced receptor sensitivity found in hypertension. We report here thathypertensives exhibit a defect in agonist-me-diated desensitizationofplateleta2-adrenoreceptorsinvivo. Methods

Materials.([3H]methyl)yohimbine(sp.act.75-90Ci/mol,NewEngland Nuclear, Boston, MA, or Amersham Corp.,ArlingtonHeights, IL)was

stored in ethanolunder N2at-20'C untiluse.L-Epinephrine bitartrate, adenosine-3',5'-cyclicmonophosphate (cAMP),adenosine-5'-diphosphate

(ADP),adenosine-5'-triphosphate(ATP), creatinephosphateand phen-ylmethylsulfonyl fluoride(PMSF)'werepurchased fromSigma Chemical Co.,Milwaukee, WI; Hepes fromU.S. Biochemical Corp., Cleveland, OH; creatine kinase fromBoehringerMannheim Gmbh, Federal Re-public of Germany,a-[32P]adenosine-5'-triphosphatefromNewEngland Nuclear, andprostaglandinEl (PgEl)fromUpjohn Diagnostics, Kala-mazoo,MI.Timolol wasagift from Merck, SharpeandDohme,West

Point,PA.

Platelet isolation. Plateletswereisolated fromfreshlydrawn blood forradioligand bindingstudiesaspreviouslydescribed (14).Inbinding

studiesontheeffect of varying extracellular sodiumconcentrations,

N-methyl-D-glucaminewassubstituted for thesodium in the wash solutions andincubation buffer.Foraggregation studies, platelet-rich plasmawas

diluted with platelet-poor plasmatoapproximately 300,000

platelets/,l

and aggregatory responsetoI X 10- to IX 10-8ML-epinephrineand 3 x10-sto 3X 10-7MADP wasdetermined in a Payton dual channel

aggregometerbetween 45 and 75min after bloodwasdrawn.Sensitivity

1.Abbreviations used in thispaper:ECso, concentration of epinephrine producing half-maximalaggregation; ECos,concentration of epinephrine producing half-maximal inhibition of prostaglandinE,-stimulated ade-nylate cyclase; AG', Gibbs free energy change; GTP, guanosine

tri-phosphate;AH ,enthalpy change;K., equilibriumassociation constant;

Kd,equilibrium dissociation constant; ONS, overnight bedrest;PgE1,

prostaglandin El; PMSF, phenylmethylsulfonylfluoride;R, gas constant; AS, entropy change;T,temperature; 3 h Up, 3 hours of upright posture.

1416 A.S.Hollister,J. Onrot, S.Lonce,J. H. J.Nadeau, andD.Robertson J.Clin.Invest.

© The AmericanSocietyfor ClinicalInvestigation,Inc. 0021-9738/86/05/1416/06 $1.00

toADP-inducedaggregationwasexpressedastheconcentration of ADP necessaryto cause biphasic plateletaggregation (17). Sensitivity to

L-epinephrine-induced aggregationwasassessedby plottingtheslopeof

thefirstphaseofaggregation againstthelogof theL-epinephrine

con-centration from which the concon-centration ofepinephrine producing half-maximal aggregation

(EC5o)

wasdeterminedgraphically. In vitro epi-nephrinedesensitization experiments involved incubation of

platelet-rich plasma fromhypertensivesandnormotensives withvehicle, I X I0O

MandI X 10-7 ML-epinephrine at250Cwithoutagitation. At30, 60,

and 120min,aliquotsweretestedfor aggregatory responsiveness to I X10-1ML-epinephrineand theslope of the firstphaseofaggregation wasexpressed as apercentage of the simultaneous vehicle-incubated

sample.

Bindingassay. Competition bindingusing 19 concentrations of

1-epinephrine against6-10nM

[3H]yohimbine

was_performedasdescribed

previously (14). Datawereanalyzed bytheweighted,nonlinear, curve-fittingprocedure basedonthe lawofmassactiondescribedbyDeLean

etal.(18)for averageagonist affinity,high and low agonist affinities (one

andtwobindingsitecurve-fitting), theproportion of receptors in each

affinity state, and the total numberof receptors. Since thecalculated

affinityconstants arelog-normally distributed, meanaffinities expressed in thetext aretheantilog of the mean log affinity constants. Statistical comparisons ofaffinityconstants weremadebetween means and standard deviations of thelogarithmictransformations.

Plateletadenylatecyclase assay. Adenylate cyclase activity was mea-sured inlysedplateletpreparationsby a modification of the method of Johnson(19).Washed plateletswerepelleted ona50% albumin cushion and resuspended in ice-cold 50mMHepes, pH 7.5,4mMdithiothreitol, 0.4mMEGTAand0.04mM PMSF. Samples were frozen, allowed to

thaw,thenhomogenizedtwice inaBrinkmann Polytron at setting 5 for 20sat

40C.

Adenylate cyclase activity was determined in 10-min in-cubationsat370Ccontaining50mMHepes, pH 7.5, 2mMMgCI2, 0.1 mM cAMP, 12.5MMATP,5mMcreatine phosphate, 40mUcreatine

kinase, I x 10- M timolol, 1 x

l04

M prostaglandin El, and0-1

X 10- ML-epinephrine, containing 300,000 cpm

a-[2PJATP

in 200Ml

final volume. Reactionswerestopped by the addition of 600Ml120mM

Znacetateplus 500 Ml144mMNa2CO3, centrifuged, andl-mlaliquots

wereplacedover l-ml alumina columns.

[32P]cAMP

was eluted with

4 ml0.1 MTris, pH7.5, and counted by Cherenkov radiation.

PgE,-stimulatedadenylatecyclaseactivitywaslinear with respect to amount ofplatelet lysateadded andtotime foratleast 15 min and was inhibited

40-80% byL-epinephrineinadose-relatedmanner.Values arereported

astheconcentration ofL-epinephrinenecessarytohalf-maximally inhibit the

PgEl-stimulated

cyclase

(ECO.5)-Plasma catecholamine concentrations were determined by a

ra-dioenzymaticassayaspreviously described(20).

Subjects.Subjectsfor this study were normotensive and hypertensive

males,20-41 yr of age, who abstained from all medications for at least 2wkbefore the study.Allprocedures in this study were reviewed and

approved bytheVanderbilt University Committee for the Protection of HumanSubjects.Subjectswereadmitted to the Elliot V. Newman Clinical Research Centerandafter 8hof overnight

bedrest

(ONS) a supine blood

samplewasdrawnforplasma catecholamineconcentrations and platelet studies. Thesubjectsthenambulated about the ward for 3 h prior to a secondbloodsample. Platelets isolated from each blood sample were dividedinto three parts for simultaneous determination of radioligand

binding,platelet aggregationandadenylate cyclase assay. Mean age, blood pressure,andheartrateforthesubjects are shown in TableI. Results

Effect of

ONSand 3h ofupright posture (3 h Up) on plasma catecholamine concentrations andaverageplatelet

a2adreno-receptoragonistaffinity. Incomparison with ONS, 3 h Up pro-duceda135%increaseinplasma norepinephrine(P<0.01) and a70%risein

plasma

epinephrine

in normal subjects (Table II).

Similarly,

hypertensives

showed increases of 113% in

plasma

Table I. Age, Resting Blood Pressure and Heart RateofNormotensive andHypertensiveSubjects

Blood

Subjects (n) Age pressure Heart rate

Normotensives (10) 29±3

112±4

69±4

73±2

Hypertensives (16) 31±3

136±3*

75±3

97±2*

Mean±SEM.*P <0.001 vs. normotensives.

norepinephrine (P<0.01)and 37% in plasma epinephrine after 3 h Up. There werenosignificant differences between

normo-tensives and hypernormo-tensivesinplasma catecholamine

concentra-tionsineitherposture.

Competition radioligand binding of L-epinephrine for

[3H]yohimbine-labeled

a2-adrenoreceptors on intact platelets

after ONS and 3 h Up ambulation isillustrated fora

normo-tensive (Fig. 1 A)and a hypertensive (Fig. 1 B) subject. After ONS, the platelets of both the normotensive andhypertensive subjects exhibited shallow competition bindingcurves forthe agonist epinephrine. After 3 h Up however, the epinephrine competition binding curve for the normotensive subject was

shifted to the right andwas steeper. The hypertensivesubject showednochangeinagonist competition binding after 3hUp.

Inthegroup of normotensive subjects, the mean affinity of platelet a2-adrenoreceptors for L-epinephrine fell from 0.93±0.15 AMatONSto1.84±0.38

MM

(P

<

_0.01)

after 3 h

Up.

Incontrast, thehypertensive subjects as a group showed no overall change

(ONS,

1.11+0.21 M,vs.3hUp, 0.94±0.13

,M;

notsignificant). At3hUp,platelet a2-adrenoreceptors from hypertensive subjects exhibitedasignificantly higher affinity for agonist than those of normotensive subjects (P<0.05). In normotensives, the change incirculating norepinephrine wassignificantly correlatedwith thefall in platelet a2-adrenoreceptor affinity for the agonist,

L-epinephrine(n = 10,r =0.86,P <0.001). Fig. 2Adepicts the relationship between plasma norepinephrine and the affinity of platelet a2-adrenoreceptors for agonist in each subject after ONS and 3 h Up. In contrast, despite similar rises in plasma cate-cholamines in the hypertensive subjects, therewas noconsistent

reduction in platelet a2-adrenoreceptor affinity for agonist (n = 16,r= -0.0040,P > 0.2, Fig. 2 B).

Analysis ofreceptorhigh andlowaffinitystate.Computerized weighted, curvefitting analysis of agonist competition binding revealed twoagonist affinity states for the platelet

a2-adrenore-ceptors in the ONS samples from normotensive and hypertensive

TableILPlasmaCatecholamines

Subjects (n) Norepinephrine Epinephrine ng/liter ng/liter

Normotensives (10)

ONS 233±37 40±10 3hUp 548±57* 68±11

Hypertensives(16)

ONS 292±25 52±10

3 hUp 622±79* 71±10

I00

0

-80 80

aQ 60 60

40 40

~20

20--8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 Log Epinephrine Log Epinephrine

Figure 1.Competitionradioligand binding ofL-epinephrinefor

[3H]yohimbine-labeleda2-adrenoreceptorsonintactplateletsafter ONS(solidlines) and 3hUp(dashedlines). (A)Normotensive sub-ject. (B) Hypertensivesubject. Linesareconstructed fromthe

weighted,nonlinearcurve-fittingparameterscalculatedfrom each data

set.

subjects. The mean affinityconstantfor thehighaffinitystate

was notsignificantlydifferent in hypertensives vs. normotensives (0.37±0.09

,uM

vs.0.28±0.08

,M,

respectively). Similarly,there

was nosignificantdifference in the lowaffinitystateequilibrium dissociationconstant

(Kd)

(9.1±4.9 MMvs.8.9±4.1 uM, respec-tively).Therewere nodifferencesin the number of receptor sites perplatelet between groups(hypertensives, 210±17;

normoten-sives, 183±11).

Composite competition bindingcurves generated by

com-puter from thehigh and low affinity Kd's (Fig. 3 A)demonstrate ashifttotherightin thebindingdisplacementbyagonistwhen the 3 h Up platelet samplesof normotensivesubjectsare

com-pared withONS. Plateletsof hypertensive subjectsexhibiteda

nonsignificant shift to the leftover thesame time period. In

normotensive subjects, themeanproportion ofreceptorsin the highaffinity state for agonist fellfrom 71 to38% after3 h Up (P<0.001)(Fig. 3 B).Incontrast,hypertensivesubjectsexhibited nochange intheproportionofhighaffinitystatereceptorsdespite similar increases in circulating plasma catecholamines. After 3 h Up, plateletsfromhypertensives had a significantlyhigher proportion of their receptors in the highaffinitystatethandid normotensives (P<0.001).

B

Normotensives

*1WA/

200 400 600 800 1000

Plasma NE ng/l

Hypertensives

200 400 600 800 1000

Plasma NE ng/l

Figure 2. Plateleta2-adrenoreceptoraffinity foragonist plotted against

plasma norepinephrine concentration.(A) Normotensives.(B)

Hyper-tensives.Affinity for agonist andplasmanorepinephrinewere

mea-suredafter8 h ONS and again after3 h Up. Each lineconnectsthe twodeterminationsinasingle subject.

X 80 .8 6 8

co4

a]

.E 60

E

-E₀

-c 40

ID

20

A

lb

l~~~sk~

:I

WI

B

80a

40

la

60 2

>.E

40 =a

IE 20 _,z,

X

8 7 6 5 4 3 -Log [Epinephrine]

Figure3.(A)Composite, computer-generated competition binding

curvesforL-epinephrine displacement of [3H]yohimbine in normoten-sivesubjects (open circles)andhypertensive subjects (closed circle) af-terONS (solid lines) and 3 h Up (dashed lines).(B)Percentageof

plateleta2-adrenoreceptorsexhibiting high affinity binding of agonist after ONS(solidbars) and after 3 h Up(striped bars).Normotensive subjects,n = 10;hypertensives, n = 16.*P<0.001 vs.ONS; tP

<0.001 vs.normotensives 3 h Up.

Aggregatorysensitivity toL-epinephrineand ADP. In order to determine whether the failure of hypertensives' platelet

a2-adrenoreceptors to reduce affinity after agonist exposure was

reflected insensitivity changes, the same platelet samples ob-tained after ONS and 3hUp were tested for in vitro aggregatory sensitivitytoL-epinephrine andADP. The EC50's from L-epi-nephrinedose-responsecurves ateachtime pointarefound in TableIII. Aggregatory sensitivity of platelets toL-epinephrine decreasedsignificantly in normotensives after 3 h Up (P<0.05), whilehypertensives did not. After 3 h Up, hypertensives' platelets weresignificantly more sensitive toL-epinephrine-induced-

ag-gregation thanwere normotensives (P<0.02). Thesefindings

agree well by regression analysis with the changes in agonist affinity foundin simultaneous binding experiments (r=0.70,

P < 0.01).

Thethreshold for biphasic aggregation stimulated by ADP was2.2±0.3

_jaM

for normotensives and 2.8±0.4

.M

for

hyper-tensives. These did notchange significantlyin eithergroup after 3 h Up (2.3±0.3

1M

and2.9±0.6 MM, respectively). Thus,the

change in aggregatorysensitivitytoepinephrinein normal

sub-jects didnotgeneralizetoanother typeofreceptor.

Inhibition ofPgE,-stimulated platelet membrane adenylate cyclase activity by L-epinephrine. Another measure ofplatelet

a2-adrenoreceptor sensitivity is the ability of L-epinephrine to

inhibit adenylate cyclase. Theoretically, adecrease inreceptor

affinity for agonist should result in a shift to the right of the dose-responsecurvefora2-adrenoreceptor-mediated inhibition

of the cyclase. Using thesameplatelet samples obtained after

TableIII.ECs0 for Epinephrine-inducedAggregation

Subjects(n) ONS 3 hUp

AM AM

Normotensives (10) 1.2±0.2 2.1±0.4* Hypertensives (10) 1.1±0.2 1.0±0. It * P<0.05vs.

ONS.

tP<0.02vs.normotensives 3 h Up. Hollister,

A

4.Or

2.0k

I.0 3.0

Table IV.PlateletAdenylate Cyclase Inhibition:

L-EpinephrineConcentration for Half-Maximal Inhibition

ONS 3 hUp

,M gM

Normotensives 0.54±0.10 1.03±0.17* Hypertensives 0.61±0.08 0.52±0.09t

*_{P <0.02 vs. ONS.}

tP<0.02vs.normotensives 3hUp.

ONS and3hUpfromnormotensive and hypertensive subjects, wedetermined the

EC0.5

(TableIV).

After 3 h Up, platelets from normotensive subjects showed

a significant increase in the

ECO.5.

In contrast, platelets from hypertensives exhibited no change in the ability of L-epinephrine toinhibit

adeny!ate

cyclase and, at the3hUpsampling time, were significantly more sensitive to L-epinephrine than nor-motensives' platelets. The magnitude and direction of changes in L-epinephrine-inhibited adenylate cyclase are consistent with the changes measured by the mean receptoraffinity for L-epi-nephrine and by the

_EC5o

forL-epinephrine-induced aggregation.

Invitrodesensitizationofnormotensive'sand hypertensive's platelet

a2-adrenoreceptors.

In order toexamine more closely thedifferences inagonistdesensitization ofplatelet a2-adreno-receptors between normotensives and hypertensives, we mea-sured theeffect of in vitro exposure of platelets to L-epinephrine

onL-epinephrine-stimulatedaggregation (Table V). Platelet-rich plasma from normotensive and hypertensive subjects was in-cubatedat25°Cwithout agitation with 1 X 10-6Mor1 X 10-7

ML-epinephrine or vehicle, then tested for aggregatory response

to

10-'

ML-epinephrine. Whenexpressed asapercentage of the vehicle-incubated response, in vitro incubation with L-epineph-rine resulted in asignificanttime- andconcentration-dependent loss in aggregatory response to 10-5ML-epinephrine in platelet samples obtainedfrom normotensive and hypertensive subjects (pooled

X2

test;time, x2 =63.9, d.f.=4,P <0.001;

concentra-tion, x2 = 116.8,d.f. =4,P <0.001). However, platelets from hypertensive subjects showed significantly less desensitization after L-epinephrine exposure than did normotensives (pooled

x2

= 14.0, d.f. = 4, P<0.01). Athigh

L-epinephrine

concen-trations,thisdifferencewasmost apparentatthe 30-min time point,whereasafter10-7ML-epinephrine incubationsthe dif-ferences persisted throughout the2 h ofincubation. Thus, in vitro incubation with supraphysiological concentrations of a2-adrenergic agonistalso demonstratesadeficiencyin agonist-me-diated receptordesensitizationin platelets fromhypertensives.

Thermodynamic analysis ofagonist and antagonistbinding

toplatelet

aradrenoreceptors

ofnormotensiveandhypertensive subjects. If the differences between normotensives and hyper-tensivesinagonist-mediated a2-adrenoreceptor desensitization

Table V EffectofL-Epinephrine Incubation

onAggregatory ResponsetoL-Epinephrine

Percentage of vehicleresponse

incubation time (min) Epinephrine

Subjects concentration 30 60 120

Normotensives 10-6M 55±2 52±2 28±2

Hypertensives 10-6M 77±7 59±2 30±1 Normotensives I0- M 86±2 76±3 59±2 Hypertensives 10-7M 96±6 89±4 67±4

Table VI. Equilibrium Thermodynamic Parameters

forAgonist and AntagonistBindingtoIntact PlateletsfromNormotensives andHypertensives

AGo AH' ASo

kcal/mol kcal/mol cal/mol-deg

Agonist(epinephrine)

Normotensives -8.45 -17.5 -30.4

Hypertensives -8.49 -17.9 -31.7

Antagonist (yohimbine)

Normotensives -11.3 +.994 +4 1.3

Hypertensives -11.1 +.568 +39.1 areduetoeither altered recognition of agonistor anabnormality in the receptor-G-protein complex, analysis ofagonist and

an-tagonist bindingatdifferenttemperatures maydifferentiate

be-tweensubjectgroups. Wedeterminedtheplatelet a2-adrenore-ceptoraffinity constants at 15°C, 25°C and 370C for L-epi-nephrine and yohimbine of five hypertensive and five

normo-tensivesubjects. The Gibbs freeenergychange (AG0)was cal-culatedfrom the equation, AG0 =-RTIn

Ka,

andaVan't Hoff plot of l/Tvs.lnKayieldedaslope of(AH0)/R. Theentropic change was then calculated from the equation; AG0 = AH0

-TAS0.TableVIshowsthethermodynamicparametersderived from normotensive and hypertensive subjects' platelet

a2-ad-renoreceptoragonist and antagonist binding.

These dataindicate that the thermodynamic driving forces for agonist bindingarethe AH0, which is opposed by thermo-dynamically unfavorable decreases inentropy. Incontrast,

an-tagonist binding is driven byentropy. The absence ofa large difference in thermodynamic driving forces provides evidence againstthe existence of marked differences in receptor-ligand and/or receptor-G-protein interaction between platelet a2-ad-renoreceptors from normotensive and hypertensive subjects.

Influence of sodiumconcentrationonthe agonist

affinity

of

platelet

_{a_-adrenoreceptors.}

Platelet a2-adrenoreceptor affinity for agonist has been showntobeinversely proportional tothe sodiumconcentration (21, 22). Since the effect of sodium appears

to be exerted on the internal side of the membrane (22) and hypertensive subjectshave beenshowntoexhibit altered

mem-brane sodium fluxes (23, 24), we examined the influence of varying sodium concentrationonhypertensives' and

normoten-sives' platelet a2-adrenoreceptor affinity for agonist (Fig. 4).

Lowerassay sodium concentrations produced highermean

affinity constants for intact platelet a2-adrenoreceptor agonist binding without changing the proportionofreceptorsin thehigh affinity state (analysisofvariance:

_F3,21

= 539.0,P < 0.0005). The platelets fromfive hypertensive subjects exhibited no sig-nificant differenceinagonist binding affinity at any assay sodium

concentration whencomparedwithfive normotensivesubjects (analysisof variance:F1.8 =0.756,P >0.5).Theproportion of receptorswith highaffinity for agonist did notdiffer between

groupswith mean±SEM of 75±6%at 1 mMNa+, 79±4%at 10

mMNa+, 78±5%at 30mMNa+and76±6%at 100mMNa+. These data reduce the possibility that an altered sodium "site"

on the a2-adrenoreceptor characterizes essential hypertension

and isresponsiblefor the defect inagonist-mediated desensiti-zation.

Discussion

KDPM

.8_-,

.

10 30 100

[No+]mM

Figure 4. Affinity of intact plateleta2-adrenoreceptorsfor

L-epineph-rineinisotonicassay mediacontainingvarying sodium concentra-tions. Mean±SEM forfivenormotensive(solid line) and five

hyper-tensive(dashed line) subjects.

These findings confirm our prior work ( 14) and extend it to the identification of a loss of high affinity state receptors as the source of the meanaffinitychange. Since no change in the total number ofreceptors occurred during the affinity change, we conclude thatthe loss of high affinity state receptors is most probably a result of the conversion of high affinity state receptors to low affinity state receptors. Although these data were collected in intact platelet binding studies, the results are consistent with the modulation of a2-adrenoreceptor agonist affinity by guanine nucleotide regulatory protein and guanosine triphosphate (GTP),

amodel derived from platelet membrane studies (21, 22). The affinity change wasaccompanied by a loss of aggregatory sen-sitivitytoagonist as well as the ability of agonist to inhibit ad-enylate cyclase. These data provide further evidence for the con-cept of the high affinity state of the a2-adrenorecon-ceptor being the "coupled" or sensitive state. In addition, since long-term ex-posure to adrenergic agonists results in a loss of adrenergic re-ceptors in vivo (8, 25, 26), whereas short-term exposure results

inmean affinity changes, our results support the principle of long-term regulation of sensitivity via receptor number changes and short-termregulationviaaffinitychanges. Whether the

mo-lecularmechanisms for affinity regulation are identical to the model derived from membrane studies or are unique to the intact platelet was not determined by our studies. Nonetheless, both binding andsensitivity measurements indicate desensitization ofintact platelet a2-adrenoreceptors after in vivo or in vitro exposuretoagonist.

Incontrast tonormotensives,hypertensivesubjectsdid not exhibit changesinmeanaffinity,proportionofhighaffinitystate

receptors, or sensitivity to either epinephrine-induced aggregation

orinhibition of adenylate cyclase afterphysiological increases in plasmacatecholamines.Thisfailuretodesensitizeafter agonist exposure resulted in platelet a2-adrenoreceptorsofhypertensive subjects being significantly more sensitive than those of

nor-motensive subjects after 3hUp.

Similarfindingsofadefect in adrenoreceptordesensitization in hypertension have been observed in the

/32-adrenoreceptor

systemofadipocytes and lymphocytes (27, 28). Lymphocyte 2-adrenoreceptors of supine hypertensives were less sensitive to

agonistthanthose of normotensives and showed no alteration

inagonist affinity after 3 h Up. In contrast, normotensives' p2-adrenoreceptorsdesensitized with uprightposture, ultimatelyto

thesamelevel ofsensitivityashypertensives. If thedynamicsof adrenergic receptor sensitivity regulation of circulating blood

elements canbegeneralizedtovascular adrenoreceptors, these studiessuggestacombined defectof reduced

,32-adrenoreceptor

vasodilatation and enhanced a2-adrenoreceptor vasoconstriction in hypertension. This defect would be secondary to impaired agonist-mediatedadrenoreceptor desensitization in hypertensive subjects. Direct studies of vascular receptor sensitivity are nec-essary to confirm these hypotheses.

Severalmembrane-limited mechanisms for altering agonist affinity of platelet a2-adrenoreceptors have been demonstrated (21, 22, 24). In rabbitplateletmembrane preparations, elevated GTP and sodium concentrations lower the affinity of platelet 02-adrenoreceptors foragonists (29); GTP, by shifting high ag-onistaffinity site binding to low affinitysites, and sodium by inducing a parallel rightward shift ofthe agonist binding without alterations in the proportion of receptors in the high affinity state. Using intact human platelets, ourstudies confirmed the shift in agonist binding affinity caused by increasing sodium concentrations and found no change in theproportion of

re-ceptors in the high affinity state in eithernormotensivesor hy-pertensives. Becauseoftheparallel shiftinaffinityand the lack of differences between normotensives and hypertensives, the possibilitythatanalteration in thea2-adrenoreceptor "sodium site" occurs in hypertension is lessened.

Another potential cause for the defect inagonist-mediated a2-adrenoreceptordesensitization in

hypertensives

isan altera-tion in the receptor and/orreceptor-guanine nucleotide regu-latory protein interaction. Changes in structure ofthese mole-cules oralterations intheirbinding toeach othermighthave resulted inthermodynamicdifferences inagonistandantagonist binding between hypertensives and normotensives. However, similar to the results found in the

fl-adrenergic

system(30,31), wedetermined that agonist binding by the a2-adrenoreceptorof intact platelets was temperature sensitiveand driven by large decreases in enthalpy, whereasantagonist bindingshowed little change with temperature and was driven by positive changes in entropy. We found no difference in the thermodynamic driving forces for agonist and antagonist binding between platelet a2-adrenoreceptors from these subjects when the platelet samples weredrawn after ONS. These results are consistent with, but do notprove, the hypothesis that no gross structural change in a2-adrenoreceptors and/or their associated guanine nucleotide reg-ulatoryproteindifferentiates hypertensives from normotensives. These studies have decreased the possibilitythat either

so-dium concentration differences or

receptor-G-protein

changes underliethedefectin

agonist-mediated

platelet

a2-adrenorecep-tordesensitization foundinhypertensivesubjects. Another po-tential site for this defectis an alteration in membrane "mi-croviscosity"whichmay secondarilyaffect

receptor-modulating

mechanisms. An increased platelet membrane microviscosity has been described inspontaneously hypertensiveratsand

as-cribed eithertoprotein (32)or tofattyacid(33) constituentsof the membrane. Thefattyacidcontentsof plateletand red cell membranes appear to be geneticallydeterminedand associated with eitherafamily historyorthe development ofhypertension in rats and men (34, 35). Exactly how thesemembranealterations interact withagonist-mediated adrenergicreceptor desensitiza-tion and whetherthey may be thesourceof thedefect identified in human essentialhypertensionremainstobe elucidated.

In summary, in normotensive human subjects we have

identified an agonist-induced reduction in platelet

a2-adreno-receptor

affinityforagonist whichischaracterizedbyareduction in

the

proportion of receptors in thehigh affinity state and is correlated withafall in receptorsensitivity. Incontrast, hyper-tensive

subjects

failed toexhibit

changes

in

a2-adrenoreceptor

agonist affinity

or

sensitivity

when

exposed

tothesameinvivo stimuli. Therewere no

significant

differencesbetween

tensives and hypertensivesinthe number ofa2-adrenoreceptors

perplatelet,the rise inplasmacatecholamines after 3 hUp,the

thermodynamic driving forces for agonistandantagonist binding andthe influenceof sodium onplatelet a2-adrenoreceptor agonist affinity. The precise molecular basis forthe defect in agonist-mediated a2-adrenoreceptor desensitization in hypertension

re-mainstobeidentified. We conclude that essential hypertension is characterized by adefect in agonist-mediated platelet

a2-ad-renoreceptordesensitization and postulate that this defectmay

be relatedtothehyperresponsiveness of hypertensivesto adren-ergicpressor agents.

Acknowledgments

The authors expresstheirappreciation for the excellent technical

assis-tanceofLorettaSpeier,Natalie Johnson and OscarSafeek,toDr.Roger Johnson for the adenylate cyclaseassaymethodology, andtoDr.Joel

Hardmanfor advice and review ofthemanuscript.

Dr.Hollister is therecipient ofaBurroughs-WellcomePostdoctoral Fellowship and is the Clinical AssociatePhysicianofthe Eliot V. Newman ClinicalResearchCenter,RF-00095,andadditionally supported byHL 31419.Dr.Robertson is therecipient ofaResearch Career Development Award,GM-00494, and is supported by HL-14192 andGM-31304. References

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