Characterization of α1-adrenergic receptors in perfused rat heart

J Mol

Cell

Cardiol20,

1025-1034

Characterization Stephen

(1988)

of a,-Adrenergic

J. Edwards,

Stephen

Receptors

Rattigan, Eric Q Colquboun, and Michael G. Clark?

in Perfused

Rat

Elizabeth

Heart

A. Woodcock”

Departmentof Biochemistry,Universityof Tasmania,GPO Box 25i’CHobart, Tasmania7001,Australia (Received 1 March 1988,accepted in revisedfol-m SJub 1988) S. J. EDWARDS, S. RATTIGAN, E. Q COLQUHOUN, E. A. WOODCOCK AND M. G. CLARK. Characterization of ai-Adrenergic Receptors in Perfused Rat Heart. Journal ofMolecular and Cellular Card&Q (1988) 20, 1025-1034. The characteristics of a,-adrenergic receptors were investigated in perfused rat hearts at 37°C. [aH]Prazosin was bound in a time-dependent manner and reached equilibrium at 15 min. Scatchard analysis of the specific binding isotherm for [3H]prazosin indicated a population of high affinity sites (Kd = 0.41 no, B,,,,, = 13.2 pmol/g wet wt). Prazosin binding was displaced by epinephrine as well as by the adrenergic antagonists prazosin > phentolamine > yohimbine > propranolol. Specific prazosin binding was defined as that portion of the binding inhibited by 10 PM phentolamine; phentolamine and epinephrine displaced ‘H-prazosin to the same level. [3H]Prazosin was not metabolized by the heart. When pre-labelled hearts were perfused at 37°C with prazosin-free medium non-specific binding of [3H]prazosin decreased more rapidly (t,,, = 4 min) than specific binding (I,,, = 38 min). Perfusion of the heart at lower temperatures (< 10%) decreased the rate ofloss of nonspecific binding and prevented the loss of specific binding. Fractionation of J3H]prazosin perfused hearts at 0°C when dissociation was minimal, led to a loss of binding so that sarcolemma-enriched fractions contained approximately 2”/0 of the binding sites present in the perfused heart. The binding characteristics ofsarcolemmaenriched fractions (Kd 0.10 no, B,, 300 fmol/mg protein) differed significantly from those of the perfused heart. Exposure of the heart to 10 min of ischaemia prior to binding studies did not alter the characteristics of the [sH]prazosin binding sites. It is concluded that the perfused rat heart contains a population ofa,-adrenoceptors which differ from those of isolated sarcolemma preparations perhaps because of alterations that occur during sarcolemma isolation. The perfused heart should be an appropriate model system in which to study the relationship between receptor occupancy and biological response as well as the direct effects of perturbations such as ischaemia. KEY

WORDS:

a,-Adrenoceptors;

Perfused

heart;

Binding

Introduction The synthesis of radioactive ligands of high specific activity has made it possible to characterize receptors that mediate specific biochemical responses. However, while binding studies are usually conducted with membrane preparations, most biochemical responses are studied in intact tissues or cells. For the rat heart, a,-adrenergic receptor mechanisms appear to mediate inotropy [13] and may predominate in the control of glucose metabolism (for a review see [fl). a,-Adrenoceptors are present on sarcolemma-enriched membrane

studies

in perfusion;

Ischaemia.

preparations of heart [22, 15, 173. Methods d preparation of sarcolemma-enriched fractions may produce an altered sarcolemma fraction in response to an altered distribution (of binding sites caused by the agents under study. This is especially possible when one considers the low yields and qualitative nature of many enriched membrane preparations. Such problems arising from tissue manipulation, which may alter the properties of the receptors or of the receptor fraction under study, are reduced to a minimum with the perfused heart. It the present study prazosin

This work was supported in part by grants from the National Heart Foundation, the Ramaciotti the National Health and Medical Research Council ofAustralia. * Present address: Monash University Department of Medicine and Medical Research Centre, pital, St Kilda Road, Melbourne, Victoria 3004, Australia. t Please address all correspondence to: M. G. Clark, Department of Biochemistry, University Box 252C, Hobart, TAS. 7001, AUSTRALIA. 0022-2828/88/l

11025

+ 10 $03.00/O

0

1988

Foundations Prince

Henry’s

of Tasmania,

Academic

Press

and HosGPO Limited

1026

S. J. Edwards

(al) binding sites in perfused rat heart have been characterized and compared with sites on sarcolemma-enriched membrane fractions.

Materials

Sarcolemma-enriched

Male rats (180 to 230 g body wt) of the Hooded Wistar strain, maintained ad libitum on a standard laboratory chow diet [a were used for these experiments. Hearts were perfused in the Langendorff manner using a system based on that of Williamson [Zq. The perfusion medium was Krebs-Henseleit bicarbonate buffer containing 1.27 mM CaCl,, 0.05 mM EDTA and 5 mM glucose, and the perfusion temperature was 37°C. The hearts were pre-perfused at a constant pressure (80 cm H,O) to give a flow rate of 5 to 8 ml/min for 10 min in a non-recirculating manner prior to commencing the experiment. of [3H&razosin

Samples of perfusate at various times during perfusion with C3H]p razosin and chloroform/ methanol (2 part: 1 part by vol) extracts of frozen heart powders were analyzed using reverse-phase HPLC. Samples were applied to a C-18 PBondpak column (Waters Associates) and eluted isocratically using 70% methanol and 1% triethylamine in water at a flow rate of 2 ml/min. The radioactivity content of the eluted solution was monitored and the retention time for eluted material with a standard profile of compared C3Hlp razosin. Fractionation

with buffer containing 0.6 M KC1 and 20 mM imidazole at pH 6.8 by resuspension and centrifugation (44000 g, 2”C, 10 min). The final pellet was collected for radioactivity measurement.

and Methods

Heart perfusions

Metabolism

et al.

of [3tjlprazosin

binding sites

Sarcolemma enriched cardiac membranes were prepared [S] from frozen powdered C3H]prazosin perfused hearts. Hearts were homogenized in 10 mM NaHCO, containing 5 mM NaNs using an Ultra-Turrax homogenizer (full speed 2 x 15 s). The homogenate was centrifuged (13 000 g, 2”C, 10 min) and the pellet collected for radioactivity measurement. The supernatant was recentrifuged (13 000 g, 2”C, 10 min) and the pellet collected for radioactivity measurement. The supernatant was centrifuged (44000 g, 2”C, 10 min) and the resultant pellet washed twice

membrane preparation

This was also isolated using the fractionation method outlined above and based on the procedure described by Hancock et al. Cl?], a modification of the method of Besch et al. [A. Non-prazosin perfused hearts were used and the final pellet was resuspended in a small volume of 0.2 M sucrose containing 30 mM Lhistidine-HCl at pH 7.0 and stored at -85°C until used in binding assays. Binding assays were conducted as described elsewhere [Ia. Protein was determined [9] using bovine serum albumin as standard. Perfused heart binding studies After 10 min of pre-perfusion the perfusion was changed to a recirculating mode with buffer containing various concentrations of C3H]prazosin and 10 PM phentolamine where indicated. [U-‘4C]Sucrose was included as a marker for the extracellular space and the the amount added was varied with [3H]prazosin to set the perfusate 3H/‘4C at approximately 3 at the commencement of the experiment. The perfusate was sampled at the beginning and end of the perfusion and counted to determine the 3H/14C. After a defined time the hearts were blotted dry, weighed, powdered in liquid N, and 100 mg digested in 1 ml of tissue solubilizer for 3 h at 50°C. The solution was decolourized by the addition of 0.3 ml benzoyl peroxide followed by heating at 50°C for 30 min. Six ml of scintillant was added for counting. Specific binding was defined as the difference in bound radioactivity in the absence compared to the presence of 10 pM phentolamine. Competition binding studies were conducted as described above except that varying concentrations of competing antagonist were included in the recirculating perfusion medium together with an initial concentration of 0.2 to 0.4 nM C3H]prazosin. For binding studies in which epinephrine was the competing ligand against [3H]prazosin, rats were injected with pargyline (100 mg/kg body

a,-Adrenoceptors

in Perfused

20 Time

1027

Heart

30

(mln)

FIGURE 1. Time-dependent binding of [3H]prazosin to perfused rat hearts. Hearts were prep&used for 10 min in a non-recirculating manner as described in Methods and then switched to medium containing 0.4 nM [3H]prazosin and 0.22 rnM [U-“%]sucrose and perfused for the times indicated. After correction for entrapped, extracellular medium the specific prazosin binding was calculated and defined as the difference between total binding (0) and binding in the presence of 10 p~ phentolamine (a). Mean + S.E. are shown for 1z = 4 hearts. The inset shows the change in perfusate concentration of [3H]prazosin (ordinate) expressed as percentage of original amount of labelled ligand added as a function of perfusion time.

wt i.p.) 3 h prior to perfusion and the recirculating perfusion medium included 30 PM cocaine, 30 PM corticosterone and 0.28 mM ascorbate. The perfusions were conducted for a period of 15 min. For dissociation studies hearts were perfused with 0.4 nM r3H]prazosin (-tphentolamine) and [U-r4C]sucrose for 15 min as described above and then the perfusion was changed to prazosin-free medium run in a non-recirculating manner. Hearts were removed at various times to assess total (no phentolamine) or non-specific ( + phentoiamine) binding. To investigate the effects of ischaemia, hearts were pre-perfused for 10 min (nonrecirculating) then medium entering the heart was stopped for 10 min. The hearts were maintained at 37°C. C3H]Prazosin binding was then determined as described above

during a 15 min recirculating taining various concentrations osin ) 10 PM phentolamine.

perfusion conof C3H]praz-

Calculations The following relationships were used to calculate binding : Free [3H]prazosin = total C3H]prazosin added at the commencement of the perfusion - bound C3H]prazosin as determined at the end of the perfusion. Bound r3H]prazosin (pmol/g heart) = {3H(dpm/g) - [r4C(dpm/g heart) x 3H(dpm)/ 14C(dpm) ratioinperfusateJ)/{Specific radioactivity of C3H]p razosin (dpm/pmol) >. Saturation experiments with Increasing concentrations of radioligand were analysed according to Scatchard [Kj. The apparent affinity of C3H]prazosin (X,) was determined

et al.

S. J. Edwards

1028

-lo

-9

A’

log [Inhib,tmg

FIGURE 2. Inhibition of specific ergic blockers (a) and epinephrine initial concentration of C3H]prazosin phentolamine (0)) yohimbine (0) peting ligand rats were pre-injected 0.28 rnM ascorbate. Values represent

-17 hgand]

-‘6

-8

14

CM)

[‘Hlprazosin binding to the perfused heart by increasing concentrations of adren(b). Perfusions were conducted for 15 min at 37°C as described in Methods. The was between 0.2 and 0.4 nM. Additions were: epinephrine (e), prazosin (A), and L-propranolol (a). For binding studies in which epinephrine was the comwith pargyline and perfusions contained 30 /L(M cocaine, 30 PM corticosterone and means f S.E. of four perfusions.

the slope of the line and the maximal number of binding sites (B,,), expressed as pmol/g wet wt of heart was calculated from the intercept with the abscissa. from

Adrenergic antagonists Prazosin hydrochloride (Pfizer) and phentolamine methane sulphonate (CIBA-Geigy Ltd) were generous gifts. C3H]Prazosin and [U“C]sucrose were obtained from the Radiochemical Centre. DL-propranolol hydrochloride, epinephrine bitartrate, L-phenylephrine hydrochloride and yohimbine hydrochloride were obtained from Sigma Chemical Co. (St Louis, MO). Results Metabolism of C31i3prazosin by theperfwed heart Samples of perfusate as well as chloroform/ methanol extracts of heart were analysed for

the formation of 3H-labelled products by reverse-phase HPLC. Commercially supplied C3H]prazosin contained a contaminant of 4.2% which was slightly less lipophillic in character than prazosin. However no evidence for metabolism of [3H]prazosin or of the contaminant was obtained; there was no formation of new 3H-labelled compounds in the heart or perfusate (data not shown). Time course of C3h?praZosin binding to perfused rat heart The isolated perfused rat heart bound increasing amounts of [3H]prazosin up to a maximum at 15 min (Fig. 1). In the following 15 min period a slight, though not statistically significant, decrease in both non-specific (C3H]prazosin not displaceable by 10 /.iM phentolamine) and total binding occurred. The concentration of C3H]prazosin in perfusion (inset Fig. 1) reflected the trend reach-

a,-Adrenoceptors

in Perfused

6’,=13.2 ,Y,=0.41

Prozos~n

concentrotlon

Heart

1029

pmollg

“M

(nh4)

FIGURE 3. Binding of [‘Hlprazosin to the isolated perfused rat heart as a function of increasing concentrations of radioligand. Hearts were preperfused for 10 min in a non-recirculating manner as described in Methods and then switched to medium containing the concentrations of [sH]p razosin shown, [U-14C]sucrose and 10 /LCLM phentolamine as indicated. The perfusions for binding were carried out for 15 min at 37°C. After correction for entrapped extracellular medium the specific prazosin binding (A) was calculated and defined as the difference between total binding (0) and binding in the presence of 10 PM phentolamine (0). Mean f S.E. are shown for n = 4 hearts; error bars are within the symbols. The inset shows the Scatchard analysis of the specific binding data. Ordinate: bound (B)/free concentration (F) radioligand expressed as pmol/g wet wt ofheart per IN. Abscissa: bound radioligand/g wet wt ofheart.

ing a minimum at maximum [3H]prazosin was obtained.

the point at which binding to the heart

Analysis of the nature of [3H@razosin

binding

Efficiency of displacement of C3H]prazosin by adrenoceptor antagonists was in the order propranolol < yohimbine < phentolamine < prazosin. This is consistent with an crl-adrenoceptor subtype [Fig. 2(a)]. In a manner similar to [13H]prazosin displacement from intact BC3H-I muscle cells [I] the adrenoceptor agonist phenylephrine was also shown to displace [3H]prazosin at relatively high concentrations (approximately 50-fold that of phentolamine; data not shown). To ensure that phentolamine displacement of [3H]prazosin does not measure lipophillic or other binding sites distinct from the adrenergic receptor it was necessary to show

[Fig. 2 (b)] that epinephrine inhibited C3H]prazosin binding to achieve the same level of residual non-specific binding as phentolamine. Experiments with epinephrine showed a reduced level of specific C3H]prazosin binding as indicated by the difference between the zero points for the phentolamine and epinephrine dose curves. This difference was due to the presence of pargyline, cocaine and corticosterone necessary to inhibit uptake and metabolism of epinephrine in the perfused rate heart. Specijic binding of [3H_]prarosin Figure 3 shows a nonspecific phentolamine independent binding of C3H]prazosin which is linearly dependent on concentration an’d non-saturable in the range tested. Phentolamine displaceable (specific) binding of C3HJprazosin upon analysis [l6J suggests a

et al.

S. J. Edwards

1030

single population of a limited number of high affinity binding sites. The apparent dissociation constant (K,) was greater than the value for isolated myocytes [Z/I] and was assessed at 0.41 (pK, = 9.39) from 4 experiments. However, the calculated maximal number of binding sites from the present experiments was 13.2 pmol/g wet wt of heart. This corresponds to 7.9 x 101’ binding sites/g wet wt of heart and 2.8 x lo5 binding sites/ myocyte (assuming 2.8 x lo7 cells/g wet wt of heart [4]) and agreed very well with the value calculated from the data of Skomedal et al. [ZO] of 2.4 x lo5 phentolamine-displaceable prazosin binding sitesjmyocyte (assuming 150 mg protein/g wet wt of heart). Dissociation of bound [3h7prazosin Figure 4(a) shows the dissociation of bound C3H]prazosin from the rate heart when perfused with medium at 37°C. Nonspecifically bound C3H]prazosin decreased markedly to less than 10% after 10 min then progressively to less than 1% after 60 min. The loss of spe-

(0)

Fractionation

of bound [3Ejlprazosin

Since loss of specifically bound C3H]prazosin was minimal at low temperatures (< 10°C) labelled hearts were fractionated at 5°C to follow the distribution of bound ligand. Table 1 shows that approximately 2% of the total specifically bound C3H]prazosin was recovered in the sarcolemma-enriched fraction. Thus partial purification of the plasma membrane according to the method of Hancock et al. [S] led to the recovery of a relatively small proportion of the original binding sites present in the intact heart. These experiments (Table 1) also indicated that some loss of specifically bound C3H]prazosin occurred as a

(b) I 10

I 20

1 30

I 40

I 50

I

I IO

6( Time

FIGURE a non-recirculating and 0.22 rn~ prazosin run extracellular binding (0)

cifically bound C3H]prazosin followed a logarithmic function with a half life of 38 min. Reduction of the perfusate temperature to less than 10°C reduced the loss of both nonspecifically and specifically bound [3H]prazosin [Fig. 4(b)]. Specifically bound [3H]prazosin did not decrease significantly even after 2 h at < 10°C.

I 20

I 30

I 40

I 50

I 6C

(min)

4. Dissociation of bound [‘Hlprazosin from the perfused rat manner as described in Methods and then switched [U-“%]sucrose and perfused for 15 min at 37°C. Medium in a non-recirculating manner, and maintained at 37°C (a) medium the specific prazosin binding (A) was calculated and binding in the presence of 10 PM phentolamine (0).

heart. Hearts were preperfused for 10 min in to medium containing 0.4 nM [sH]prazosin was then changed to one containing no or < 10°C (b). After correction for entrapped and defined as the difference between total

a,-Adrenoceptors

TABLE

1031

Heart

1. Recovery of specifically bound ligand from C3H]prazosin perfused hearts isolation of sarcolemmaduring enriched membrane fractions % Specific prazosin bound

Isolation step Homogenate 13000 g, 10 13000 g, 10 44000 g, 30 44000 g, 30 44000 g, 30 The

in Perfused

method

min min min min min used

100 99 1.3

pellet 1 pellet 2

2.8 2.0

2.1 was essentially

that

of Hancock

J

et al.

[8] and is described in the text. All fractions were maintained at &5”C. Homogenization

result of homogenization. Specific binding for the whole heart perfused at 0.25 nM prazosin was 3.5 pmol/g wet wt of heart. If the same heart was homogenized in buffer at pH 7.65 and the membrane material collected by centrifugation the equivalent amount of specifically bound C3H]prazosin was 3.3 pmol/g wet wt. Successive homogenization/centrifugation steps progressively decreased the amount of specifically bound ligand (Fig. 5). Mechanical disruption of the ligand-receptor complex appeared to be the likely cause of this loss as no evidence for solubilized hormone-receptor complexes was obtained by gel-exclusion chromatography of the filtrates. Homogenate binding sites Whole heart homogenates bound C3H]prazosin. Scatchard analysis for specific binding from two experiments indicated K, was 1.1 f 0.1 nM and B,, was 6.4 & 0.4 pmol/g wet wt of heart ( f range). Efect of ischaemia on [3H@razosin binding a-Adrenoceptor mechanisms have been claimed to become enhanced in ischaemia [19], and may be due to altered a-receptor binding characteristics. To investigate this possibility C3H]prazosin binding to the perfused heart was reassessed following 10 min of ischaemia at 37°C. Comparison of the data of Figure 6 with Figure 3 show that neither K, or B,.as a result of ischaemia. . ..a.% was altered

number

Effect of homogenization on specific and nonspecific binding of C3H]prazosin by heart membranes. Hearts were preperfused for 10 min in a nonrecirculating manner as described in Methods and then switched to medium containing 0.25 nM [3H]prazosin and 0.22 rnM [U-‘%]sucrose f 10 pM phentolamine and perfused for 15 min at 37°C. Frozen heart powders (510 mg in triplicate) were homogenized in 5 ml 75 rnM TrisHCI pH 7.65 containing 25 mM MgCI, at 0°C. The homogenates were centrifuged (100 000 g, 5”C, 30 min), supernatants were discarded and the pellet dissolved in tissue solubilizer for counting. In other experiments the pellets were rehomogenized and centrifuged two or three times before the addition of tissue solubilizer. Specifically bound C3H]prazosin (A) is defined as the difference between total binding and binding in the presence of 1.0 FIGURE

PM phentolamine

5.

(0).

Discussion

Prazosin binding characteristics of the perfused heart This study reports our attempt to investigate the characteristics of aI-adrenoceptors in the perfused heart. In this system tissue manipulation, normally associated with in uitro radioligand binding studies, and which may alter receptor properties, has been reduced to a minimum without the complications of true zJ1 vivo studies. It appears likely that binding to nonmyocytes in this preparation represents only a very small proportion of the total specific C3H]prazosin binding. The number of binding sites per myocyte, at 2.8 x IO’, calculated from the Scatchard analysis of our data is in close agreement with values calculated

1032

S. J. Edwards

et al.

8,=12.8 Kd ~0.44

I I

pmol/g “M

I 2 Prazosin

(nM)

FIGURE 6. Effect of ischaemia on the binding of [3H]prazosin to the isolated perfused rat heart as a function of increasing concentrations of radioligand. Hearts were preperfused for 10 min in a non-recirculating manner as described in Methods and then made ischaemic for 10 min at 37°C. Reperfusion was commenced with medium containing the concentrations of [3H]prazosin shown, [U-‘%]sucrose and 10 pM phentolamine as indicated. The perfusions for binding were carried out for 15 min at 37°C. After correction for entrapped extracellular medium the specific prazosin’binding (A) was calculated and defined as the difference between total binding (0) and binding in the presence of 10 PM phentolamine (e). M ean + S.E. are shown for n = 4 hearts; error bars are within the symbols. The inset shows the Scatchard analysis of the specific binding data. Ordinate: bound (B)/f ree concentration (F) radioligand expressed as pmol/g wet wt of heart per nM. Abscissa: bound radioligand/g wet wt of heart.

for isolated myocytes from the data of other workers [ZO]. Therefore C3H]prazosin binding to vascular smooth muscle, endothelial and mast cells a pears insignificant when to [ PHlprazosin binding to compared myocytes of the ventricle. In addition to allowing the characterization of C3H]prazosin binding sites in perfused heart, data from the present study has permitted a comparison of the binding characteristics between the various preparations. Whereas isolated myocytes and the isolated perfused heart showed a similar number of total specific prazosin binding sites, the disso-

ciation constants differed significantly. For the intact heart (this study) and heart tissue slices [12] X, was approx. 0.4 nM and for isolated myocytes Kd was 0.16 nM [ZU]. This difference is not readily explained although it may be possible that collagenase/hyaluronidase perfusion alters receptor characteristics. In addition the redistribution of the receptors originally located on perhaps one or two faces of the myocyte to all sides may also alter the binding characteristics for prazosin. It is noteworthy that sarcolemma-enriched fractions represented only 2% of the total specific binding sites of the perfused heart and the

u,-Adrenoceptors K, was significantly less. These data may therefore suggest that (1) isolation of an enriched sarcolemma preparation selectively concentrates a minor population of high affinity binding sites or that (2) isolation of the sarcolemma may alter the binding characteristics of the cr,-adrenoceptors. However since it is usual to conduct binding studies on material sedimenting between 500 and 45000 g without further purification (e.g. see [27-j), this may not constitute a problem. Whole homogenates which theoretically should contain all of the specific binding sites for C3H]prazosin differed significantly in binding characteristics from both the perfused heart and the sarcolemma-enriched fraction. The apparent dissociation constant, K, was 1.1 nM and the maximal number of binding sites, B,, was 6.4 pmol/g wet wt of heart. This was similar to values reported by Wei and Sulakhe [21] for a-receptor binding by rat ventricular homogenates of 4.8 to 6.6 pmol/g wet wt using [3H]dihydroergocriptine. Thus crude homogenates showed impaired binding, with a decreased number of specific binding sites and a decreased affinity for prazosin. The use of the isolated perfused heart may be a preferable method to in vivo binding studies [Z] where characterization of the binding sites is difficult. The perfused heart affords many of the advantages claimed for the in vivo studies [Z] and distribution of [3H]prazosin binding sites into various structures (e.g. ventricle, atrium, papilliary muscle) could be determined. Nonspecific binding by the isolated perfused heart was considerable (Fig. 3) and represented 40% of the total [3H]prazosin bound at equilibrium. However this could be significantly separated, if required, by homogenization of the hearts in Tris-HC1/MgClz buffer, pH 7.4 at 0°C and collection of the membrane fraction for counting on glass fibre filters (data not shown). Possible disadvantages of using the isolated perfused rat heart for studies on [3H]prazosin binding include metabolism of the ligand, the of diffusion presence barriers and the occurrence of phentolamine-inhibitable (specific) uptake. No evidence was obtained for either three of these possibilities. Although commercially supplied C3H]prazosin con-

in Perfused

Heart

1033

tained a small contaminant there was no indication that this or other related compounds were formed from [3H]prazosin during the perfusion labelling period. Disruption of cell structure did not reveal additional (unoccupied) binding sites (data not shown) and all specifically bound [3H]prazosin was associated with particulate material. Indeed loss of specific binding at 0°C occurred only as a result of mechanical disruption (Fig. 5) and could be induced to occur progressively by repeated homogenization. In addition no evidence was obtained for soluble [3H]prazosinprotein complexes.

Applicationof perfusionbindingstudies a-Adrenoceptors have been identified in heart by both physiological responses [7, 10, 141and by direct radioligand binding experiments using isolated sarcolemma-enriched fractions [231. In addition, it has been suggested that cardiac a-adrenergic responses may assume greater physiological significance under conditions of /I-adrenergic blockade [Ill, hypothyroidism [18l, and myocardial ischaemia [19] and decrease with age [Uj. Since it is difficult to discount the possibility that either /I-adrenergic blockade, hypothyroidism, ischaemia or aging do not alter the composition of the isolated membrane fraction, perfusion binding studies could be used istead of isolated membranes to confirm these effects. In ischaemia, enhanced a-adrenergic responsivene:ss occurs and, although the role has been que:stioned, this could be the primary mediator of the electrophysiological derangements resulting from catecholamine-mediated malignant dysrhythmias [7j. In the present study exposure of the heart to 10 min of ischaemia prior to binding studies did not alter the binding characteristics for prazosin. This may imply that ischaemia does not per se alter a,-adrenergic receptors and that previously reported changes arise from an altered composition of the isolated membrane fraction. Alternatively the change due to ischaemia, exteriorization of internal (e.g. involving receptors) may reverse during the nonischaemic binding period in perfusion. In summary, the present study indicates that the perfused rat heart contains a population of ccl-adrenoceptors which differ in character from those of isolated sarcolemma

S. J. Edwards

1034

preparations but which may do so because of alterations that occur during sarcolemma isolation. The absence of a detectable diffusion barrier to specific binding and of specific uptake suggest that the perfused heart should

zt al.

be an appropriate model in which to study the relationship between receptor occupancy and biological response as well as the direct effects of perturbations which are believed to alter a-adrenoceptor properties.

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