51

Autoradiographic Characterization of-Adrenergic Receptor Subtpe in the Canine

Conduction SystemKathryn H. Muntz

It has been hypothesized, based on physiological evidence, that there is a greater proportion of1-adrenergic receptors on the myocytes of the conduction system when compared with the workingmyocardium. The purpose of these studies was to examine f3-adrenergic receptor subtype in theconduction system of the dog by using the technique of coverslip autoradiography. Scintillation studies of['25l]pindolol binding to ventricular sections demonstrated that binding was saturable (dissociationconstant of 116 pM), had the correct order of potency for a 13-receptor, and was stereoselective. Bothbetaxolol (p,-selective) and ICI-118,551 (f2-selective) competition curves fit a two-site model in nonlinearcurve-fitting analyses (78% B13-receptors). Autoradiographic studies determined that the myocytes of thesinoatrial node had approximately twice as many autoradiographic grains as the surrounding atrialmyocytes. The myocytes of the atrioventricular bundle had a number of grains similar to the number insurrounding septal myocytes. Autoradiographic inhibition curves with betaxolol or ICI-118,551 demon-strated that both the sinoatrial node and the atrioventricular bundle had inhibition profiles similar to thesurrounding myocytes (predominantly B13) but unlike the inhibition profiles of arterioles (predominantlyj%). Calculations using the dissociation constants derived from the nonlinear curve-fitting analysis andthe percent specific binding in the presence of 4 10-7 M betaxolol or ICI-118,551 determined that theproportion of 61- to P2-receptors was the same (70-80%o 131) when comparing the sinoatrial node and thesurrounding atrial myocytes. The atrioventricular bundle had a higher percentage of P3-receptors (95%)than the surrounding septal myocytes (79%) with betaxolol, but with ICI-118,551 the difference was notsignificant. Thus, by using techniques that can precisely quantify adrenergic receptors over myocytes ofthe conduction system, it was concluded that, whereas the myocytes of the sinoatrial node haveapproximately twice as many j3-receptors as the surrounding atrial myocytes, the proportion of Pl- tojB2-receptors is the same. (Circulation Research 1992;71:51-57)KEY WoRDS * ,f-adrenergic receptors * subtype * heart * conduction system

P hysiological evidence suggests that heart rate ismore responsive than contractility to 832-adren-ergic receptor agonists.1-3 Thus, it has been

hypothesized that there may be a greater proportion of132-receptors in the conduction system than in the work-ing myocardium. Autoradiographic data in the rat hasfound a greater percentage of P2-receptors on thesinoatrial (SA) node than on the working myocardium.4Additional autoradiographic analyses in the guinea pighave suggested that the atrioventricular (AV) node andbundle have a greater percentage of }32-receptors than isfound in the surrounding myocardium,5,6 although datain the rat has suggested that the AV node has a similarratio of 8f- to f32-receptors as the septal myocardium.7

This manuscript was sent to Harold C. Strauss, ConsultingEditor, for review by expert referees, editorial decision, and finaldisposition.From the Department of Cell Biology and Neuroscience, Uni-

versity of Texas Southwestern Medical Center, Dallas.Supported by US Public Service grant HL-45326.Address for correspondence: Kathryn H. Muntz, PhD, Depart-

ment of Cell Biology and Neuroscience, University of TexasSouthwestern Medical Center, 5323 Harry Hines, Dallas, TX75235-9039.

Received November 13, 1991; accepted March 20, 1992.

The purpose of these studies was to examine the3-receptor subtype in two regions of the conductionsystem in the dog, a species commonly used for cardio-vascular studies, and to compare subtype to workingmyocardium (predominately 81) and arterioles (pre-dominately R2). We used the technique of coverslipautoradiography to precisely quantify grains over con-duction system myocytes, thus excluding blood vessels,nerves, and connective tissue elements that are presentin the conduction system.

Materials and MethodsFive mongrel dogs were anesthetized with pentobar-

bital sodium (30 mg/kg), and the hearts were removed.Serial blocks were removed at the junction of thesuperior vena cava with the right atrial appendage andin the area of the AV bundle. Additional blocks wereremoved from the anterior wall of the left ventricleadjacent to the interventricular septum. The blocks oftissue were mounted on cork and immediately frozen infreon cooled with liquid nitrogen.

Validation of BindingSections (10 ,um) were cut from the left ventricular

blocks and used for validation of [125I]pindolol (Dupont/

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from

52 Circulation Research Vol 71, No 1 July 1992

New England Nuclear, Boston) binding to the sections.For binding, sections were preincubated in 50 mM Tris(pH 7.4), 10 mM MgCl2, 1 mM EDTA, and 10-4 Mascorbic acid for 10 minutes to remove endogenousligand. Sections were then incubated at room tempera-ture in the same buffer with ["Illpindolol. Nonspecificbinding was determined by incubation in [125I]pindololin the presence of 5 ,M (+)-propranolol HCl (SigmaChemical Co., St. Louis, Mo.). Preliminary studiesdetermined that equilibrium was reached at 60 minutes;therefore, incubations were carried out for 70 minutes.Preliminary experiments also determined that optimalremoval of nonspecific binding was achieved by two20-minute ice-cold buffer washes. These washing con-ditions did not remove specific binding. After washing,the slides were quickly dipped in ice-cold distilled waterand dried with a stream of cold air; the sections werescraped off the slide and analyzed by gamma counting.

Several studies were done to determine [1251]pindololbinding characteristics. Sections were incubated in sixto eight concentrations of [1251]pindolol to determine theB.aX and the equilibrium dissociation constant. Agonistorder of potency and stereoselectivity were determinedby incubating sections in 120 pM [1251]pindolol in thepresence of several concentrations of agonists (thebitartrate salts of L-norepinephrine, L-epinephrine,L-isoproterenol, and D-norepinephrine; Sigma).

Sections were incubated in [1251]pindolol in the pres-ence of several concentrations of the f,3-selective ligandbetaxolol (Laboratoires d'Etudes et de Recherches Syn-thelabo, Paris) and the ,82-selective ligand ICI-118,551(Imperial Chemical Industries PLC, Macclesfield, En-gland). Both the saturation curves and the drug dis-placement studies were analyzed using the LIGANDprogram of Munson and Rodbard8 as modified byMcPherson for the personal computer (BIOSOFr).

AutoradiographySections were incubated in 120 pM [1251]pindolol in

the presence of five concentrations (3x10-9 to 2x 10-6M) of either betaxolol or ICI-1 18,551, 5 ,uM propranolol(nonspecific binding), or without drug (total binding).The sections were incubated as described above andprocessed for autoradiography according to previouslydescribed methods.9-11 Coverslips were dipped in NTB2emulsion (Kodak), diluted 1:1 with distilled water(43°C), and the coverslips were allowed to dry for 3hours in the dark. A coverslip was glued (Superglue,Loctite) to one end of each slide that had been incu-bated in [lI]pindolol, and the coverslips were apposedto the tissue section using a No. 20 binder clip and aplastic spacer to protect the coverslip from breakage.The slides were exposed in boxes with Drierite for 5days.

After exposure, the binder clip was removed, thecoverslip was gently lifted, and an alligator clip wasplaced between the coverslip and the slide. The slideswere then developed in D19 (1:1 with distilled water,Kodak), fixed, stained with hematoxylin and eosin, andmounted with Permount.The autoradiographic grains were quantified with a

Nikon Magiscan according to previously describedmethods.11 For features that did not fill the videoscreen, the instrument allowed an outline of the feature

feature, as well as the number of grains over thatfeature. Ten fields were counted from the followingtissues: sinoatrial myocytes, atrial myocytes, atrial arte-rioles, myocytes of the AV bundle, septal myocytes, andseptal arterioles. The fields were selected randomlyusing a low-power objective in which the grains couldnot be visualized. Because there are few arterioles in thesections, arterioles oriented in any way were used. Onlyvessels less than 90 ,um in diameter and in which alumen could be visualized were analyzed.The SA node and the AV bundle were identified in

the serial blocks using a histochemical stain for acetyl-cholinesterase.12 Serial sections were taken from boththese regions, and every fifth section was stained foracetylcholinesterase. The serial sections were consecu-tively numbered, so that the autoradiographs could becompared with the acetylcholinesterase-labeled sec-tions preceding and following the autoradiograph. Thisfacilitated the positive identification of the SA node andthe AV bundle. In addition, the SA node could beidentified by smaller myocytes and the presence of theSA nodal artery present in the center of the node. TheAV bundle was also identified by its position adjacent tothe point where the membranous interventricular sep-tum becomes the muscular septum, before it branchesinto the right and left bundle branches.

After quantitation, the grains were normalized to astandard area (10-2 mm2), and total and nonspecificgrains were calculated. Nonspecific grain densities weresubtracted from grain densities obtained from total,betaxolol, and ICI-118,551 slides; the percent inhibitionof specific binding with each concentration of /3-selec-tive drug was calculated; and inhibition curves wereconstructed.Data collected from one concentration (4 x 10-7 M) of

either betaxolol or ICI-118,551 were analyzed using theequation of Neve et a113 as applied by Murphree andSaffitz14 in autoradiographic studies. These analyses aremore thoroughly described in the "Results" section.

StatisticsTotal and nonspecific grain densities over different

regions of either the atrium or the septum were com-pared using a one-way analysis of variance followed by aTukey's multiple comparison test. Because the atrialsamples and the septal samples were set up at differenttimes, we did not compare grain densities between theatrial and septal sections.The percent specific binding with either betaxolol or

ICI-118,551 was compared among six regions analyzedusing a one-way analysis of variance. Since the percentspecific binding was independent of the variability ingrain density from experiment to experiment, this was avalid analysis. All data are expressed as mean+SEM.The equilibrium dissociation constants are expressed asthe geometric mean+SEM.

ResultsValidation studies of tissue sections determined that

binding was saturable (Figure 1) with an equilibriumdissociation constant of 116 pM (-9.936+0.014 log M,n=3). In addition, binding had the order of potencycharacteristic of a f3-adrenergic receptor and demon-strated stereoselectivity (Figure 2). The results of com-puter competition analyses with betaxolol using scintil-to be analyzed and then determined the area of the

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from

Muntz (3-Receptor Subtype in Canine Conduction System

40-

c c

C 30m t

Xc 20"

cx, O- 10

A

* Total Binding/O Specific BincA Nonspecific

Binding

_~~~~~~~~A

ding

100r

cD 80r0

=

m,o

;. 40

20

100 200 300 400 500[1251]-Pindolol (pM)

FIGURE 1. Representative saturation curve demonstratingtotal, nonspecific, and specific binding of [125I]pindolol tocanine heart sections. With nonlinear curve-fitting techniques,a dissociation constant of 120pM and a Bmax of35.9fmol/mgprotein were obtained.

lation counting of tissue sections are seen in Figure 3A.Nonlinear curve-fitting analyses of individual competi-tion curves obtained from dog ventricular sections de-termined that the binding of betaxolol fit a two-sitemodel (p<0.0001) with an equilibrium dissociationconstant of 1.77x 10-8 M (-7.753+0.072 log M, n=3) forthe high-affinity site (I3P) and 9.51 x 10-7 (-6.022+0.184log M) for the low-affinity site (3,2). In the ventricularsections, there were 78±3% ,l1-adrenergic receptors as

estimated by the LIGAND program. Similar analyses wereapplied to the competition curves of ICI-118,551 per-formed on canine ventricular sections (Figure 3B) andindicated that the binding of ICI-118,551 fit a two-sitemodel (p<0.0001) with an equilibrium dissociation con-stant of 5.08x10-9 (-8.294±0.546 log M, n=3) for thehigh-affinity site ((32) and 4.26x10-7 (-6.371±0.044 logM) for the low-affinity site (13l). In addition, simultane-ous analysis of saturation curves and competition curvesas described by UnnerstallI5 determined that ["'I]pin-dolol did not show significant selectivity for 83-receptors.

Analysis of the autoradiographs determined that themyocytes of the SA node had approximately twice asmany total grains as the atrial myocytes or the atrialarterioles (Figures 4 and 5). In contrast, the myocytes ofthe AV bundle had a similar number of total grains as

100

'c

.5 80m0

n- 60

D~ X

` 400-

.5 20a)0.CLU)

-9 -8 -7 -6 -5 -4log [ Agonist,M ]

FIGURE 2. Graph showing agonist inhibition of [25I1]pin-dolol binding to canine heart sections, demonstrating an

agonist order of potency characteristic of a 3-adrenergicreceptor (L-isoproterenol>L-norepinephrine .L-epinephrine).In addition, stereoselectivity was also demonstrated (L-norepinephrine>D-norepinephrine). The means were deter-mined from two experiments in duplicate.

0

o -9 -8 -7 -6

Log [ICI-1 18,551 ,M]

-5 -4

B100

80

c

60 _0.

0 40

20

00 -9 -8 -7 -6 -5 -4

Log [ICI-1 18,551 ,M]

FIGURE 3. Inhibition of specific ["'5]pindolol binding witheither the 813-selective ligand betaxolol (panel A) or the,2-selective ligand ICI-118,551 (panel B). The data were

plotted as percent maximal binding calculated by the LIGANDprogram versus the concentration of ligand. Analysis of theindividual experiments with LIGAND demonstrated that bothdrugs fit a two-site model in canine heart sections. Values aremean±SEM of three experiments.

the myocytes of the interventricular septum and theseptal arterioles. Nonspecific binding was low, and therewere no significant differences among the three regionsin either the atrium or the septum in nonspecificbinding. Although there was not enough tissue in eitherthe left or the right bundle branch present for adequatequantification, qualitatively these regions appeared tohave a grain density similar to that of the AV bundleand the septal myocytes.Examination of the inhibition curves derived from

autoradiographs determined that the myocytes of theSA node and the atrial myocytes had similar profiles foreither betaxolol or ICI-118,551, whereas atrial arterioleshad a different profile for both drugs (Figure 6). Betax-olol was less potent for arterioles, whereas ICI-118,551was more potent for arterioles. These profiles arecharacteristic of a predominance of 8,2-receptors. Simi-lar profiles were observed in the septum, with themyoc---es of the AV bundle and the septal myocytesdemonstrating P,i profiles and the septal arterioles ex-hibiting 132 profiles (Figure 7).To calculate the percentage of 13l- and 1,2-receptors

from the autoradiographs, the results of the LIGANDanalyses of betaxolol were fit to the followingequation13:

BmaX1 XL BmaX2XLL+Kdlx(1+I/Kil) L+Kd2X(1+I/Ki2)

53

h

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from

54 Circulation Research Vol 71, No 1 July 1992

ATRIUM SEPTUM

FIGURE 4. Bar graphs showing autoradiographicgrain density from three regions in either the atriumor the septum. SA, sinoatrial; AV, atrioventricular.Values are mean±SEM; n5 dogs. *p<0.05 vs.

total grain density in atrial myocytes and arterioles.

Total Nonspecific Total Nonspecific

where B is the amount of radioligand specifically bound,Bmax1 and BmaC2 are the densities of the f3,- and /32-

receptors, respectively, L is the concentration of radio-ligand used in the incubations (120 pM), I is theconcentration of competing ligand (4x 10 ' M), Kdl andKd2 are the equilibrium dissociation constants of theradioligand for the two sites (116 pM), and Ki, and Ki2are the equilibrium dissociation constants of the bindingsites for the competing ligands. It should be noted thatthe dissociation constant of ['251]pindolol used in theseequations was the dissociation constant that was derivedexperimentally and not the dissociation constant frompreviously published calculations.

When no competing ligand was present, the followingequation (where X equals B,axi and Y equals Bmax2) wasobtained based on our data:

B=0.508X+0.508Y (2)

When 4 x 10`' M betaxolol was present in the incuba-tion, the following equation was obtained:

B=0.042X+0.421Y (3)When 4x10` M ICI-118,551 was present in the

incubation, the following equation was obtained:

B=0.348X+0.013Y (4)

ek

el

,

t,

FIGURE 5. Photomicrographs comparing grains over the sinoatrial node with grains over the surrounding atrial myocytes. PanelA: Bright field microscopy of atrial myocytes. Panel B: Dark field microscopy of atrial myocytes, demonstrating totalautoradiographic grains. Panel C: Bright field microscopy of sinoatrial node. Panel D: Dark field microscopy of sinoatrial node,demonstrating a greater grain density than that found in the surrounding atrial myocytes (panel B). Magnification, x344.

E

Ev)

.!

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from

Muntz ,8-Receptor Subtype in Canine Conduction System 55

C 0 Atrial MyocytesA Atrial Arterioles

80 X0

co - -8 -60

40-

20

-9 -8 -7 -6 -9 -8 -7 -6

log [ Betaxolol, M log [ ICI-118,551, M]

FIGURE 6. Graphs demonstrating the inhibition of specific/125Ilpindolol binding by several concentrations of eitherbetaxolol (3I-selective) or ICI-118,551 (1,2-selective) over thesinoatrial (SA) node, atrial myocytes, and atrial arterioles.Betaxolol has an equal affinity for the SA node and the atrialmyocytes, whereas it has a lower affinity for the arterioles.ICI-118,551 has an equal affinity for the SA node and theatrial myocytes but a greater affinity for the arterioles; n=5dogs.

The autoradiographic data (specific binding) in theabsence of betaxolol or ICI-118,551 (100%) and in thepresence of 4x 10` M betaxolol or ICI-118,551 (percentspecific binding) was inserted as B in the appropriateequation. The equation calculated without drug (Equa-tion 2) was solved simultaneously with either the betax-olol equation (Equation 3) or the ICI-118,551 equation(Equation 4) using the methods of Murphree andSaffitz,'4 and the percentage of P1- and 1,2-receptorsubtypes was calculated (Table 1). These data indicatedthat with betaxolol the myocytes of the SA node, atrialmyocytes, and septal myocytes had approximately 80%l1-receptors and that the myocytes of the AV bundlehad a somewhat greater percentage of P1-receptors(95%). Arterioles, on the other hand, had predomi-nately 132-receptors.

Similar results were obtained with ICI-118,551, al-though in most cases the percentage of 1,l-receptors wasslightly lower than that obtained with betaxolol (Table1). The exception was the percentage of 13-receptorsubtypes in the atrial arterioles, and this may be related

cn

.c

CO

cOO n

5 .-

C,

0)

at

100

80

60

40~

20

O AV Bundle0 Septal MyocytesA Septal Arterioles

/

/-A

-9 -8 -7 -6log [ Betaxolol, M ]

5 X ~~~~~~~~~A11/

-9 -8 -7 -6log [ ICI-118,551, M ]

FIGURE 7. Graphs demonstrating the inhibition of specific["151]pindolol binding by several concentrations of eitherbetaxolol (/3,-selective) or ICI-118,551 (,82-selective) over theatrioventricular (AV) bundle, septal myocytes, and septalarterioles. Betaxolol has an equal affinity for the AV bundleand the septal myocytes, whereas it has a lower affinity for thearterioles. ICI-118,551 has an equal affinity for the AVbundle and the septal myocytes but a greater affinity for thearterioles; n=5 dogs.

to the fact that the variation in the specific binding toarterioles was high with betaxolol. With ICI-118,551there was no significant difference in the percent spe-cific binding between septal myocytes and myocytes ofthe AV bundle.

DiscussionPharmacological experiments carried out in vivo have

led to the conclusion that 132-adrenergic receptors arepresent on cardiac myocytes and, in some cases, canmediate the positive chronotropic effects of catechol-amines. In one study, 8,2-agonists potentiated morepronounced chronotropic than inotropic responses,whereas a 13-selective agonist produced the same de-gree of inotropic and chronotropic stimulation.' Theseworkers hypothesized that the SA node had a higherratio of p2- to 1,1-receptors than the working myocar-dium. Our data suggested that in the dog, although,3-receptor number on the SA node was twice that of thesurrounding atrial myocytes, the ratio of f,B- to ,-2receptors was the same. A previous autoradiographicstudy in rats suggested that, in addition to increased,B-receptor number on the SA node, the percentage of132-receptors was greater.4 There could be species dif-ferences, but an additional explanation for the discrep-ancy is the fact that, in the study by Saito et al,4 thetechnique of film autoradiography was used. With thistechnique it is not possible to exclude large nerves,blood vessels, or connective tissue present in the SAnode. The SA node has more connective and nervoustissue than the working myocardium. Both fibroblasts'6and cardiac nerves5 have predominately ,(2-adrenergicreceptors. By using image analysis of coverslip autora-diography, it is possible to exclude connective tissue andlarge nerves from the grain counting so that myocytes ofthe SA node can be analyzed separately.Not only were two 13-receptor blocking agents used,

but five concentrations of each blocking agent were usedin the autoradiographic studies, thus obtaining compe-tition curves that clearly indicated that the inhibitionprofiles of the myocytes of the SA node were similar tothose of the atrial myocytes and not the atrial arterioles.Calculations of the percentage of 1,-receptors usingcoefficients derived from nonlinear curve-fitting analy-ses also supported the conclusion that myocytes of theSA node and the atrial myocytes had similar propor-tions of 1,1-receptors.

Nonlinear curve-fitting analyses of canine ventricularsections yielded a dissociation constant for ["lIlpindololof 116 pM. This is very close to that obtained with["1I]pindolol in canine atrium (120 pM). The rationalefor using ["lIlpindolol instead of ["lI]cyanopindolol wasthat ["lI]pindolol had a lower nonspecific binding (-5%at the dissociation constant) than ["lIlcyanopindolol(20%) in canine heart sections (author's unpublishedobservations).

In doing simultaneous nonlinear LIGAND analyses ofScatchard plots with competition curves of either betax-olol or ICI-118,551, I did not find that ["51]pindolol wasselective for P2-receptors. Neve et al,13 in more exten-sive studies, have reported that ["lIlpindolol was moreselective for 12-receptors. If this is the case, I would beunderestimating the number of 1,-receptors at theconcentrations of ligand used in our studies. However,the present data are in line with other studies using

j; L X. 1so

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from

56 Circulation Research Vol 71, No 1 July 1992

TABLE 1. Percent specific [..'I]Pindolol Binding and Percentage of fl1- and f2-Receptors in Several Regions ofCanine Heart

Specific [l5Ilpindolol 8-Receptorsbinding (%) X Y P3i (%) P2 (%)

BetaxololAtrial myocytes 22.46+4.56 160 37 81.07 18.93Atrial arterioles 73.96+9.03* 24 173 12.22 87.78SA node myocytes 23.24+3.26 158 39 80.03 19.97Septal myocytes 23.64+3.61 156 40 79.49 20.51Septal arterioles 64.57±9.93* 49 148 24.78 75.22AV myocytes 12.05±2.47t 187 10 94.99 5.01

ICI-118,551Atrial myocytes 53.05±3.88 151 46 76.57 23.43Atrial arterioles 23.51+3.43* 62 134 31.78 68.22SA node myocytes 50.69+1.93 144 53 72.99 27.01Septal myocytes 49.93+6.11 141 55 71.84 28.16Septal arterioles 16.32+4.00* 41 156 20.87 79.13AV myocytes 55.25±5.52 157 40 79.91 20.09

SA, sinoatrial; AV, atrioventricular. Values are mean±SEM for specific ['251]pindolol binding; n=5 dogs.X, Y, and percentage of /,l- and /32-receptors were calculated using methods described in the text.*p<O.OOl vs. myocytes.tp<0.05 vs. other myocytes and arterioles.

radioligand binding to membranes and tissue sections inwhich the atrial25,178 and septal6 myocytes have ap-proximately 70-80% 61-receptors. Regardless, a slightselectivity of [1"5I]pindolol for 3,2-receptors would in noway alter the conclusion that the relative proportions of3,l- to /3,-receptors are the same in the myocytes of theSA node and the atrial myocytes. Because ['lI]pindololwas not selective for 3,2-receptors in my system, I electedto use the dissociation constant derived with my meth-ods (116 pM for both 3,B- and 1,2-receptors) rather thantry to estimate constants from the data of Neve et al13 inanother system.

It has been reported that the AV bundle and the AVnode in the guinea pig have a higher proportion of(32-receptors than the myocardium of the interventricu-lar septum, whereas the rat AV node has proportions of(32-receptors similar to those found in interventricularseptal myocytes. In our study, the AV bundle of the dogdid not have a greater proportion of /32-receptors whencompared with the interventricular septum. Indeed,there was evidence for an increased proportion of3,B-receptors in the AV bundle, at least with betaxolol.Variability was greater with ICI-118,551 in the AVbundle and septal myocytes, which may explain why asignificant difference was not detected with this ligand.

Since a higher proportion of 13-receptors was notfound in the SA node when compared with the atrialmyocytes, how does one explain the increased sensitivityof heart rate to 3,2-agonists when compared with con-tractility? It has been shown in canine atrium withradioligand methods that approximately 25% of the,1-receptors are of the 1,2-receptor subtype. However,50% of the shortening of the action potential duration isdue to P2-receptors, suggesting that 1,2-receptors may bemore tightly coupled to physiological responsiveness.17These authors ruled out a role for presynaptic /2-receptors by depleting catecholamines in some animals.Presynaptic 3,2-receptors are thought to facilitate cate-

cholamine release; thus, (32-agonists could indirectlystimulate 13-receptors on cardiac myocytes.Along similar lines, evidence in human atrial prepa-

rations indicates that 1,2-receptors are more tightlycoupled to adenylate cyclase than are 1,1-receptors.18,(2-Receptors generate more cAMP than do 13-recep-tors, although only 30% of the 13-adrenergic receptorpopulation is of the 12 subtype. Thus, in the dog,although the ratio of /,1- to 1,2-receptors is not differentwhen comparing the SA node myocytes with atrialmyocytes, the total number of 132-receptors is increasedin the SA node. If 1,2-receptors were more tightlycoupled to adenylate cyclase in canine myocytes, therewould be a relatively greater effect of 13, stimulation onheart rate.

In conclusion, these studies demonstrate that, al-though the /3-receptor number is twice as high on themyocytes of the SA node than on the surrounding atrialmyocytes, the ratio of 1,X- to ,82-receptors is the same.These observations could have implications regardingthe role of /8-adrenergic receptor subtypes in cardiacfunction.

AcknowledgmentsI acknowledge the excellent technical assistance of Sheffield

Kadane and Rebecca Burkett in preparing the autoradiographsand analyzing them. I would like to thank Laboratoiresd'Etudes et de Recherches Synthelabo, Paris, for kindly provid-ing the betaxolol and Imperial Chemical Industries PLC, Mac-clesfield, England, for generously providing the ICI-118,551.

References1. Carlsson E, Dahloff C-G, Hedberg A, Persson H, Tangstrand B:

Differentiation of cardiac chronotropic and inotropic effects oft3-adrenoceptor agonists. Naunyn Schmiedebergs Arch Pharmacol1977;300:101-105

2. Johansson L-H, Persson H: P2-Adrenoceptors in guinea-pig atria.J Pharm Pharmacol 1983;35:804-807

3. Lemoine H, Kaumann AJ: Regional differences of 3,1 and/,2-adrenoceptor-mediated functions in feline heart. NaunynSchmiedebergs Arch Pharmacol 1991;344:56-69

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from

Muntz f-Receptor Subtype in Canine Conduction System 57

4. Saito K, Torda T, Potter WZ, Saavedra JM: Characterization of,f1- and P2-adrenoceptor subtypes in the rat sinoatrial node andstellate ganglia by quantitative autoradiography. Neurosci Lett1989;96:35-41

5. Molenaar P, Canale E, Summers RJ: Autoradiographic localiza-tion of beta-, and beta-2 adrenoceptors in guinea pig atrium andregions of the conduction system. J Pharmacol Exp Ther 1987;241:1048-1064

6. Molenaar P, Russell FD, Shimada T, Summers RJ: Densitometricanalysis of Pl- and 832-adrenoceptors in guinea pig atrioventricularconduction system. J Mol Cell Cardiol 1990;22:483-495

7. Saito K, Kurihara M, Cruciani R, Potter WZ, Saavedra JM: Char-acterization of Pr- and f2-adrenoceptor subtypes in the rat atrio-ventricular node by quantitative autoradiography. Circ Res 1988;62:173-177

8. Munson PJ, Rodbard D: LIGAND: A versatile computerizedapproach for characterization of ligand-binding systems. Anal Bio-chem 1980;107:220-239

9. Young WS, Kuhar MJ: A new method for receptor autoradiogra-phy: 3H-Opioid receptors in rat brain. Brain Res 1979;179:255-279

10. Muntz KH, Olson EG, Lariviere GR, D'Souza S, Mukherjee A,Willerson JT, Buja LM: Autoradiographic characterization of betaadrenergic receptors in coronary blood vessels and myocytes innormal and ischemic myocardium of the canine heart. J Clin Invest1984;73:349-357

11. Muntz KH, Garcia CA, Hagler HK: a1-Receptor localization in ratheart and kidney using autoradiography. Am J Physiol 1985;249:H512-H519

12. Gomori G: Histochemical demonstration of sites of cholineesterase. Proc Soc Exp Biol Med 1948;68:354-358

13. Neve KA, McGonigle P, Molinoff PB: Quantitative analysis of theselectivity of radioligands for subtypes of beta adrenergic recep-tors. J Pharmacol Exp Ther 1986;238:46-53

14. Murphree SS, Saffitz JE: Delineation of the distribution of ,-ad-renergic receptor subtypes in canine myocardium. Circ Res 1988;63:117-125

15. Unnerstall JR: Computer-assisted analysis of binding data, inYamamura HI, Enna SJ, Kuhar MJ (eds): Methods in Neurotrans-mitter ReceptorAnalysis. New York, Raven Press, Publishers, 1990,pp 37-68

16. Lau YH, Robinson RB, Rosen MR, Bilezikian JP: Subclassifica-tion of P-adrenergic receptors in cultured rat cardiac myoblastsand fibroblasts. Circ Res 1980;47:41-48

17. Liang BT, Frame LH, Molinoff PB: f2-Adrenergic receptors contrib-ute to catecholamine-stimulated shortening of action potential dura-tion in dog atrial muscle. Proc NatlAcad Sci USA 1985;82:4521-4525

18. Brodde O-E: Pl- and f2-adrenoceptors in the human heart: Prop-erties, function and alterations in chronic heart failure. PharmacolRev 1991;43:203-242

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from

K H Muntzconduction system.

Autoradiographic characterization of beta-adrenergic receptor subtype in the canine

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1992 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.71.1.511992;71:51-57Circ Res. 

http://circres.ahajournals.org/content/71/1/51World Wide Web at:

The online version of this article, along with updated information and services, is located on the

  http://circres.ahajournals.org//subscriptions/

is online at: Circulation Research Information about subscribing to Subscriptions: 

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

  document. Permissions and Rights Question and Answer about this process is available in the

located, click Request Permissions in the middle column of the Web page under Services. Further informationEditorial Office. Once the online version of the published article for which permission is being requested is

can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:

by guest on June 22, 2018http://circres.ahajournals.org/

Dow

nloaded from