Biological activity of agarose-immobilized catecholamines

472

Biochimica et Biophysica Acta, 4 4 4 ( 1 9 7 6 ) 4 7 2 - - 4 8 6 @) E l s e v i e r S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27993

BIOLOGICAL ACTIVITY OF AGAROSE--IMMOBILIZED CATECHOLAMINES

M A R C G. C A R O N a n d R O B E R T

J. L E F K O W I T Z

Departments o f Medicine and Biochemistry, Duke University Medical Center, Durham, N.C. 27710 (U.S.A ) (Received February 3rd, 1976)

Summary Catecholamines substituted to agarose were synthesized in various ways. Norepinephrine and isoproterenol were linked to p-aminobenzamidohexyl agarose by an azo linkage to the catechol ring. Norepinephrine was also coupled to hexyl agarose via the amino group, forming an amino, guanidino or amido bond. Biological activity of the immobilized catecholamines was determined by assessing their abilities to interact with adenylate cyclase in several membrane preparations and intact preparations of erythrocytes. In dog heart membranes, stimulation of adenylate cyclase by the catecholamine-gels could be accounted for by leached hormone which had been released from the gels. In frog erythrocyte membranes, leaching was minimal and no significant stimulation of adenylate cyclase was observed. Agarose-immobilized catecholamines, however, competitively inhibited isoproterenol stimulation of adenylate cyclase in these erythrocyte membranes indicating that catecholamines which are bound to agarose interact with the beta-adrenergic receptors as antagonists rather than agonists. When tested on intact frog erythrocytes, agarose immobilized catecholamines did not increase the intracellular levels of cyclic AMP, although isoproterenol caused as 8--10 fold rise in these levels. Similarly, when tested for antagonist activity in the intact cells the agarose-catecholamines failed to inhibit the stimulation of cyclic AMP caused by isoproterenol. The difference observed in the beta-adrenergic antagonist activity of the agarose-bound catecholamines in membrane preparations and in intact cells can be attributed to steric factors which could have prevented the access of the bead-bound ligands with the surface of the cell or to the possibility that receptors might be buried in the membrane matrix.

Abbreviations: Ad-NE, An-NE, Gu-NE, Azo--NE: norepinephrine linked, respectively, to CHSepharose by an amido bond, to AH-Sephaxosc by an amino bond, to AH-Sepharose by a guanido bond, to agarose by an azo bond; Azo-Iso, isoproterenol linked to agarose by an azo bond.

473 Introduction Hormones and drugs covalently linked to solid supports have proven useful in the study of hormone-receptor interactions, particularly in localizing certain of these receptors to the cell surface [ 1 ]. Several studies have implied that catecholamine bound to various solid supports possess biological activities which are comparable to those of free hormones [2--5]. In some of these studies, however, the possibility that the observed activity was due to free catecholamines which had leached from the solid support was not rigorously excluded. Recently, reports have appeared which suggest that the biological activity of catecholamines immobilized to certain solid supports may be attributable to free ligand which has been released from the solid support [6,7]. The purpose of the studies to be reported here was to determine the biological activity of catecholamines bound to agarose in various ways, using the betaadrenergic receptor-coupled adenylate cyclase as a bioassay system. Methods

Sources of materials Cyclic AMP, ATP, phosphoenolpyruvate, myokinase, (--)isoproterenol, (--)norepinephrine were obtained from Sigma. Triethylamine, methylamine, sulfamic acid, sodium nitrite, sodium tetraborate, sodium dithionite, glutaraldehyde, sodium borohydride and cyanogen bromide were from Eastman. 1-Cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate was from Aldrich. Dowex AG 50W-X2 and AG 50W-X8 were from BioRad Laboratories. Neutral alumina was from ICN. [a-32p]ATP, 3H-labeled cyclic AMP, [3H]norepinephrine and iso[3H]proterenol were from New England Nuclear. AHSepharose 4B, a Sepharose 4B derivative which has a 6 carbon side-arm with a free amino group, and CH-Sepharose 4B, a derivative having a 6 carbon sidearm terminated by a free carboxyl group, were obtained from Pharmacia.

Synthesis of agarose-bound catecholamine gels Five different catecholamine gels were synthesized for this study. Norepinephrine and isoproterenol were used as the two ligands. [ 3H] Norepinephrine and iso[3H]proterenol were used to monitor the extent of coupling of these molecules to the solid support and also to provide a simple and reliable means of assessing the extent to which the bound catecholamines were released from the gels. [3H] Norepinephrine covalently bound to these gels had a specific activity of 62 mCi/mol whereas that of isoproterenol was 125 mCi/mol. For all the gels (except as otherwise noted) the solid support consisted of agarose to which a six carbon aliphatic chain was attached. The chain terminated in either an amine or carboxyl function. These agarose derivatives will be referred to as AH-Sepharose 4B (free terminal NH2) and CH-Sepharose 4B (free terminal COOH). (a) Norepinephrine was linked to CH-Sepharose via the amino group of the molecule to form an amido bond (Ad-NE) by reaction with 1-cyclohexyl-3-(2morpholinoethyl)-carbodiimide me~ho-p-toluene sulfonate (CDI) essentially as described by O'Hara and Lefkowitz [8]. (b) Norepinephrine was substituted to AH-Sepharose to form an amino bond

474 (An-NE) by reaction with glutaraldehyde and subsequent reduction with NaBH4 according to Weinstein et al. [9]. (c) Norepinephrine was attached to agarose via a guanidino linkage (Gu-NE). The free amino group of AH-Sepharose was converted to a reactive cyanamide by activation with BrCN [ 10] , which was then reacted with the amino group of norepinephrine to form a guanidino linkage [8]. (d) Norepinephrine was attached to agarose via the catechol ring. A p-aminob e n z a m i d o h e x y l derivative of agarose was prepared and norepinephrine was coupled to this s uppor t via a diazonium salt intermediate so as to form an azo linkage (Azo-NE) according to the m e t h o d described by Inman and Dintzis [11]. (e) Isoproterenol was attached to agarose via an azo linkage (Azo-lso) to the catechol ring as described in (d). The determination of ligand c o n t e n t of the various gels was carried out exactly as described by O'Hara and Lefkowitz [8]. The efficiency of counting the radioactivity associated with each gel was determined for individual samples by counting a known a m o u n t of [3H] norepinephrine or isoproterenol in the presence of the gel. This procedure was done in order to minimize the potential problem of underestimation of the a m o u n t of material substituted due to quenching by the gels. Complete chemical analyses of the derivatives was n o t p e r f o r m e d in this study. The possible chemical configuration of the various agarose-immobilized catecholamines synthesized is shown in Fig. 1 [8,9,11,12].

Washing of agarose-catecholamine gels After co mp leti on of synthesis each gel (15--30 ml of packed agarose) was washed extensively according to the following procedure. Immediately upon c o m p letio n of the reaction, each gel was collected on a Buchner funnel and washed under suction with 4 1 H 20; 2 1 5 mM HC1; 1 1 2 M NaC1; and 1 1 50 mM sodium acetate buffer pH 5.5. Gels were then placed in dialysis bags and dialyzed at 4°C against 4 1 of 50 mM sodium acetate buffer pH 5.5 with 10--15 changes of buffer over a period of one to two weeks. The e x t e n t of the release of b o u n d catecholamines from the gels was assessed by following radioactivity released into the dialysate. Dialyses were c ont i nued until the radioactivity released declined to a constant value. A small n u m b e r of counts above background could always be d e t e c t e d in the dialysate regardless of the e x t e n t or duration of dialysis. The gels were stored at 4--6°C in the dialysis buffer containing 5 mM EDTA. Immediately prior to an e x p e r i m e n t a 1 ml portion of each gel was further washed with 300- - 400 ml of 200 mM sodium acetate buffer, pH 5.5, 300-400 ml of 2 M NaC1 and 500--600 ml of H 2 0 on a sintered glass funnel and then equilibrated with 50 mM Tris • HC1, pH 7.5. Gels treated in this way were essentially devoid of appreciable radioactive material in solution immediately after washing ( < 1 0 -7 M catecholamine equivalent as determined by released radioactivity). In an a t t e m p t to prepare gels which were devoid of leached material, a gel, Azo-Iso was packed in small columns (0.7 × 4 cm) and washed at elevated t e m p e r a t u r e (37°C) with the following solvents (25 mM Tris • HC1, 10 mM MgC12 pH 7.4 at 37°C, 50% di m e t hyl formamide in the same buffer as well as 50% meth an o l in the same buffer). All buffers contained 2 mM sodium metabisulfate. Columns were washed successively with up to 8 portions of 500 ml of

475

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IS O P R O I " I E ~ Fig. 1. Possible c h e m i c a l s t r u c t u r e s of the v a r i o u s a g a r o s e - i m m o b i l i z e d c a t e c h o l a m i n e s . Each gel was synt h e s i z e d as d e s c r i b e d in M e t h o d s . T h e e x t e n t of s u b s t i t u t i o n of each gel w i t h n o r e p i n e p h r i n c and isoprot e r e n o l , d e t e r m i n e d as d e s c r i b e d in M e t h o d s , was as follows: A d - N E , 4.4 m M ; A n - N E , 6 . 4 m M ; G u - N E , 10.7 raM; A z o - N E , 2.0 raM; A z o - I s o , 1.0 raM.

the various buffers. The Azo-Iso gel which was washed with Tris buffer showed a very low level of leached material after the several washes. Washing of the gel with buffers containing either dimethyl f o rm am i de or m et hanol did n o t produce a gel which was free of leached material.

Preparation o f membrane fractions Myocardial membranes: Washed 10 000 × g membranes derived from canine left ventricular m y o c a r d i u m were prepared essentially as described earlier [13]. Adipose tissue membranes: Membrane preparations from rat paraovarian fat were obtained as described by Czech and L ynn [14] and as described previously [13]. Frog erythrocyte membranes: Blood from grass frogs maintained at r o o m t e m p e r a t u r e was collected and the red cells washed three times with 110 mM NaC1, 10 mM Tris • HC1, pH 7.4. Cells were lysed in 5 mM Tris • HC1, pH 8.1 buffer and the membranes centrifuged at 30 000 × g for 15 min. The lysis process was repeated three times. Membranes were finally suspended in 75 mM Tris • HC1, 25 mM MgC12, pH 8.1, by hom og eni zat i on [15].

476

Adenylate cyclase assays Adenylate cyclase activity was measured using a modification of the method of Krishna et al. [ 16] as described previously [ 13 ]. Incubations were performed for 10 min at 37°C for both myocardial and adipose membrane preparations whereas activity in e r y t h r o c y t e membranes was measured over a 15 min period at 37°C. Adenylate cyclase activity was shown to be linear with time and a m o u n t of protein used in the assays for the various membrane preparations. 32p-Labeled cyclic AMP formed from [a-32p]ATP was isolated by.chromatography either on Dowex AG 50 W-X2 or neutral alumina [17]. Cyclic AMP levels in intact erythrocytes Frog erythrocytes were prepared and washed as for the preparation of erythrocyte membranes. The intact cells were further washed twice with a Ringer's phosphate buffer (84 mM NaC1, 1.9 mM KC1, 1.08 mM CaC12, 10 mM theophylline and 20 mM potassium phosphate pH 7.4) and finally resuspended in the same buffer at a hematocrit of 50%. Incubations were carriedout as follows: 100 gl of 50% hematocrit cells equilibrated at 37°C for 10 min were incubated with the test substances in a final volume of 200 ~1 for 1--5 min at 37°C. Incubations were terminated by addition of a cold solution of 5% trichloroacetic acid containing 4000--5000 cpm of 3H-labeled cyclic AMP to m o n i t o r for analytical losses. Cyclic AMP was purified on Dowex AG 50W-X8 and measured by a competitive protein binding assay [18] as described by Neelon and Birch [191. Pro te in Protein determinations were performed according to the method of Lowry et al. [20]. Results

The ability of agarose-bound catecholamines to stimulate adenylate cyclase activity in various membrane preparations was tested. In dog heart membranes, several of the agarose-bound catecholamines, at concentrations from 50 to 100 pM appeared to stimulate the adenylate cyclase (Fig. 2A). Azo-NE, Ad-NE and Azo-Iso produced a significant stimulation of adenylate cyclase whereas An-NE and Gu-NE gels did not. In the same experiments, in order to m o n i t o r for labeled catecholamines in solution, a Millipore-filtered supernatant of each catecholamine-substituted gel incubation mixture was also tested for its ability to stimulate adenylate cyclase. As shown by the crosshatched bars in Fig. 2A, these supernatants stimulated the adenylate cyclase activity to a comparable or slightly higher level than the gels themselves. Thus, the stimulation of adenylate cyclase observed in the presence of catecholamine substituted agarose gels appears to be accounted for by catecholamines which have apparently leached from the solid support during the incubation. In frog erythrocyte membrane preparations all the gels, except the Azo-Iso, appeared to lack the ability to stimulate the beta-adrenergic receptor-coupled adenylate cyclase activity (Fig. 2B). This lack of activity of the gels in erythrocyte membranes was a reflection of lesser amounts of catecholamines re-

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F i g . 2. A b i l i t y o f a g a r o s e - b o u n d c a t e c h o l a m i n e s t o s t i m u l a t e a d e n y l a t e c y c l a s e i n v a r i o u s m e m b r a n e [)reparations. (A) Dog myocardial membranes: The ability of the various gels to stimulate the adenylate cyclase is s h o w n b y t h e h e i g h t o f t h e o p e n b a r s . T h e f i n a l c o n c e n t r a t i o n s o f t h e a g a r o s e b o u n d c a t e e h o l a m i n e s i n the adenylate cyclase assays were: Ad-NE, 0.2 raM; An-NE, 0.3 mM; Gu-NE, 0.5 mM; Azo-NE, 0.1 raM; and Azo-Iso, 0.05 raM. The ability of Mill[pore-filtered supernatants of gel/membrane incubation mixtures t o s t i m u l a t e t h e c y c l a s e is s h o w n b y t h e c r o s s - h a t c h e d b a r s . T h e b a r s o n t h e r i g h t s h o w t h e e f f e c t s o f i s o proterenol on the adenylate cyclase. In these experiments, each incubation contained 170--240 pg of protein. (B) Frog crythrocyte membranes: T h e b e t a - a d r e n e r g i c a g o n i s t a c t i v i t y o f t h e v a r i o u s g e l s is s h o w n b y t h e b a r s o n t h e l e f t . I n t h e s e e x p e r i m e n t s t h e f i n a l a g a r o s e - b o u n d c a t e c h o l a m i n e c o n c e n t r a t i o n s in t h e adenylate cyclase assays were respectively: Ad-NE, 0.4 mM; An-NE, 0.6 mM; Gu-NE, 1.0 mM; Azo-Ne, 0.2 raM; and Azo-Iso, 0.1 raM. The bars on the right show the effects of isoproterenol alone. Each incubation contained 280--340 pg of erythrocyte membrane protein. (C) Rat adipose membranes: same l e g e n d a s B. T h e c o n c e n t r a t i o n s o f a g a r o s e - b o u n d c a t e c h o l a m i n e s w e r e as d e s c r i b e d u n d e r B. E a c h a d e n y late cyclase incubation assay contained 110--120 pg of protein. The results shown here are the mean of duplicate determinations from (A) two experiments, (B) 7 experiments and (C) 5 experiments and the I-bars represent the standard error.

478 TABLE I RELEASE OF CATECHOLAMINES MEMBRANE PREPARATIONS

FROM

AGAROSE

AFTER

INCUBATION

WITH

VARIOUS

I n t h e s a m e e x p e r i m e n t s , t h a t e f f e c t s o f v a r i o u s gels o n t h e a c t i v i t y o f a d e n y l a t e c y c l a s e i n t h e m e m b r a n e s was e x a m i n e d , d u p l i c a t e i n c u b a t i o n s ( 5 0 0 ttl) w e r e s e t u p u n d e r i d e n t i c a l c o n d i t i o n s t o t h e a d c n y l a t e cyc l a s e a s s a y s ( e x c e p t f o r [ a - 3 2 p ] A T P w h i c h w a s o m i t t e d ) . E a c h gel was t r e a t e d i n t h i s f a s h i o n f o r 1 0 - - 1 5 r a i n . A t t h e e n d o f t h e i n c u b a t i o n , t h e t u b e s w e r e c e n t r i f u g e d a n d t h e s u p e r n a t a n t p a s s e d t h r o u g h Millip o r e f i l t e r s t o r e m o v e t h e a g a r o s e gel a n d t h e m e m b r a n e s f r o m t h e s o l u t i o n . T h e c o n c e n t r a t i o n o f a g a r o s e b o u n d c a t e c h o l a m i n e s u s e d i n t h e s e e x p e r i m e n t s w a s t h e s a m e as d e s c r i b e d i n t h e l e g e n d t o Fig. 2. T h e e x t e n t of l e a c h i n g of the c a t e c h o l a m i n e s in this s o l u t i o n was e s t i m a t e d by m e a s u r i n g thb r a d i o a c t i v i t y present. The concentrations were then calculated from the known specific radioactivities of the catecholamine substituents. The numbers represent the concentration of catecholamines present in a typical aden y l a t e c y c l a s e i n c u b a t i o n a t t h e c o n c l u s i o n o f a 1 0 - - 1 5 r a i n i n c u b a t i o n . T h e r e s u l t s arc r e p r e s e n t a t i v e o f two experiments performed in duplicate.

Azo-NE Ad-NE An-NE Gu-NE Azo-Iso

Dog heart membranes (~M)

Frog erythroeyte (/aM)

220 55 75 130 68

1.1 0.5 0.7 1.0 0.5

membranes

leased f r o m the solid supports. As shown in Table I, the leaching of catecholamines from agarose, as determined by following the radioactivity released in solution, was at least one hundr e d times lower in the presence of e r y t h r o c y t e membranes than in the presence of myocardial membranes. This difference in the e x t e n t of leaching is sufficient to account for the difference in apparent activity of these gels in the two m e m br a ne systems. The concent rat i on of leached radioactivity released from the Azo-Iso gel appeared to represent a sufficient a m o u n t of isoproterenol to elicit the slight stimulation (25%) observed with this gel in the e r y t h r o c y t e membranes. The concentrations of leached norepinephrine achieved with the other four gels were below the " t h r e s h o l d " concentration for stimulation of adenylate cyclase in the frog e r y t h r o c y t e membranes [13]. When the Azo-Iso gel, washed at 37°C with 25 mM Tris • HC1 10 mM MgC12 pH 7.4 2 mM Na2S2Os, was incubated in the presence of e r y t h r o c y t e membranes a considerable release of radioactivity into the supernatant occurred (1 × 10 -7 M isoproterenol). In ad ip o cy te membranes a pattern similar to that of the e r y t h r o c y t e was observed (Fig. 2C). Marginal stimulation was observed only with Azo-Iso and AzoNE gels. In these experiments, no estimates of the concent rat i on of leached ligand was p er f o r m ed. Thus, the data suggest that stimulation of adenylate cyclase by catecholamine gels can be accounted for by leached ligand in solution. When release of leached h o r m o n e is minimal, no activity can be demonstrated. Release of catecholamines covalently coupled to solid supports has been previously r ep o r ted [5--7,21]. The mechanisms which have been suggested to explain the release of small molecules from the solid supports are cleavage of bonds between the ligand and the spacer arm [21,22] and between the spacer arm and the matrix [ 21- - 23] . In order to examine this problem, we have perf o r m e d c h r o m a t o g r a p h y on Sephadex G-10 of material which had leached from an Azo-Iso gel. This c h r o m a t o g r a p h y showed two distinct peaks of radioac-

479 tivity. One of these was in the void volume, indicating that part of the leached material was of higher molecular weight than free isoproterenol Another peak of radioactivity coincided with the elution position of free isoproterenol. These results are consistent with the recent results of Vauquelin et al. [20] which showed that both free isoproterenol and isoproterenol associated with the side arm spacer were both released from Azo-Iso gels of Sepharose 4B during storage and incubation under physiological conditions. Since, in these membrane preparations, agarose-bound catecholamines do not appear to interact with beta-adrenergic receptors as agonists, their ability to act as antagonists was tested. Figs. 3A and B depict the ability of the agarosebound catecholamines to inhibit the isoproterenol stimulation of adenylate cyclase activity in erythrocyte membranes. When dose response curves for isoproterenol stimulation of adenylate cyclase were determined in the presence or absence of fixed concentrations of the various catecholamine gels, a rightward shift of the dose response curve was observed. This shift indicates that the agarose-bound catecholamines act as antagonists of the isoproterenol stimulation [ 2 4 ] Sepharose 4B or Sepharose 4B with the various side-arms showed no inhibitory effect on the isoproterenol activated adenylate cyclase activity. It can be observed from Figs. 3A and B that the antagonism caused by the agarose-bound catecholamines is, in part, competitive. This is shown by the parallelism in the shift of the isoproterenol dose-response curves. Superimposed on this parallel shift, however, is a decrease in the maximal response obtained in the presence of the agarose-bound catecholamines. This indicates a more complex pattern of antagonism [ 24].

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Fig. 3. I n h i b i t i o n o f i s o p r o t e r e n o l - s t i m u l a t e d adenylate cyclase by the various agarose-bound catccholamines. Cateeholaminc-substituted gels w e r e w a s h e d p r i o r t o e a c h e x p e r i m e n t as d e s c r i b e d u n d e r M e t h o d s . T h e final c o n c e n t r a t i o n s o f gel b o u n d c a t e c h o l a m i n e s p r e s e n t in t h e a d e n y l a t e c y c l a s e a s s a y w e r e as follows: Ad-NE, 0.4 raM; An-NE, 0.6 mM;Gu-NE, 1.0mM; Azo-NE, 0.2mMand A z o - l s o , 0.1 m M . E a c h i n c u b a t i o n c o n t a i n e d 2 7 6 - - 3 1 0 # g o f p r o t e i n . T h e r e s u l t s s h o w n are t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s from four experiments.

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Fig. 4. I n h i b i t i n n u f i s o p r o t e r e n o l - s t i m u l a t e d a d e n y l a t e c y c l a s e i n f r o g e r y t h r o c y t c m e m b r a n e s b y G u - N E gel. I n t h e s e e x p e r i m e n t s a d e n y l a t e c y c l a s e w a s a s s a y e d as d e s c r i b e d u n d e r M e t h o d s a n d e a c h i n c u b a t i o n c o n t a i n e d 3 0 0 - - 4 0 0 p g o f p r o t e i n . P a n e l s A, B, a n d C r e p r e s e n t t h e o r e t i c a l c u r v e s f o r t h e v a r i o u s f o r m s o f a n t a g o n i s m [ 2 4 ] . P a n e l D s h o w s i n h i b i t i o n o f t h e G p p ( N H ) p s t i m u l a t e d a d e n y l a t e c y c l a s e b y t h e gel G u - N E . T h e c o n c e n t r a t i o n o f g e l - b o u n d n o r e p i n c p h r i n e i n t h e a s s a y was 1 raM. P a n e l E s h o w s i n h i b i t i o n of isoproterenol-stimulated adenylate cyclase by increasing concentrations of agarose-bound norepincphrine (Gu-NE). Panel F represcnts the isoprotcrenol stimulation of adenylate cyclasein the presence of G u - m c t h y l a m i n e o r G u - N E . B o u n d n o r e p i n e p h r i n e ( G u - N E ) was a t a c o n c e n t r a t i o n o f 1 m M as was G u m c t h y l a m i n e . T h e c o n t r o l gel G u - m e t h y l a m i n e was s y n t h e s i z e d e x a c t l y as d e s c r i b e d u n d e r M e t h o d s f()r t h e G u - N E gel. P a n e l G s h o w s t h e i n h i b i t i o n o f i s o p r o t e r e n o l s t i m u l a t i o n o f a d e n y l a t e c y c l a s e b y t h e c o n t r o l gel G u - m c t h y l a m i n e . E x p e r i m e n t a l c o n d i t i o n s w e r e t h e s a m e as i n F. P a n e l H d e p i c t s t h e i n h i b i t i o n o f i s o p r o t e r e n o l s t i m u l a t e d a d e n y l a t e c y c l a s e b y G u - N E (1 r a M ) . I n t h e s e e x p e r i m e n t s , e a c h i n c u b a t i o n c o n tained 344--363 pg of protein. The results presented in each of the various panels represent the mean of d u p l i c a t e d e t e r m i n a t i o n s f r o m t w o e x p e r i m e n t s e x c e p t f o r p a n e l E w h e r e t h e r e s u l t s p r e s e n t e d arc m e a n s of duplicate determinations.

481

In order to determine, with more precision, the nature of the antagonism observed with the catecholamine substituted gels, a detailed analysis of the antagonism caused by one of the gels (Gu-NE) was performed. The results of this analysis are shown in the various panels of Fig. 4. The results are compared with theoretical curves for various forms of antagonism [24] which are depicted in the center panels of the figure. Panel A represents pure competitive antagonism, characterized by a parallel shift in the dose response curves, with the same m a x i m u m response. Panel B depict noncompetitive antagonism, characterized by a nonparallel shift in the curves and a lower m a x i m u m response. Panel C shows combined competitive and noncompetitive antagonism indicated by a parallel shift in the curves on which is superimposed a decrease in the maximum response. Panels E and H of Fig. 4 demonstrate that the antagonism of isoproterenol activation of adenylate cyclase by Gu-NE has both competitive and noncompatitive components. Furthermore, panel E shows that the antagonism caused by Gu-NE is concentration dependent. As the concentration of bound norepinephrine increases, the isoproterenol dose response curves are shifted to the right in a parallel fashion. The following control experiments were performed. Methylamine was linked to agarose, via a guanidino bond. The resulting gel, (Gu-Methylamine) inhibited the isoproterenol activation of adenylate cyclase in a purely noncompetitive fashion (Panel G). Similarly, Gu-NE inhibited adenylate cyclase activation by the GTP analog Gpp(NH)p in a purely noncompetitive fashion (Panel D). Since stimulation of the adenylate cyclase by Gpp(NH)p is n o t mediated through beta-adrenergic receptors [25], this observation indicates that the noncompetitive portion of the antagonism caused by Gu-NE is completely unrelated to interaction with beta-adrenergic receptors. Finally, if the isoproterenol activation of adenylate cyclase in the presence of Gu-NE is compared with the activation by isoproterenol in the presence of the control gel Gu-methylamine, purely competitive antagonism can be observed (Fig. 4F). To insure that the antagonist activity exhibited by the Gu-NE gel was due only to agarose-bound norepinephrine and n o t to some structurally altered catecholamine released during the incubation, isoproterenol dose-response curves were determined in the presence of Millipore filtered supernatants of the GuNE gel prepared as described in the legend to Table I. In several experiments, these preparations failed to antagonize the isoproterenol stimulation of adenylate cyclase (data not shown). This confirms that the antagonism caused by Gu-NE is in fact, due to the norepinephrine which is bound to agarose. These data indicate that agarose-bound catecholamines are capable of interacting with beta-adrenergic receptors in frog e r y t h r o c y t e membranes, albeit as antagonists rather than as agonists. Accordingly, we next examined the ability of these agarose-bound catecholamines to interact with the beta-adrenergic receptors in intact frog erythrocytes. The intracellular level of cyclic AMP in intact frog erythrocytes rose in response to isoproterenol to about 10 fold the basal level (Figs. 5, 6). A detectable increase could be observed at an isoproterenol concentration of 5 • 10 -~ M and the response reached a m a x i m u m at 10 -s (Figs. 5, 6). As with the membrane systems, the various agarose-bound catecholamine gels were tested for their

482 INTACT FROG

ERYTHROCYTES9001P

3~

L
_J

Azo-hiE Ad-NE An-NE Gu-NEA=0-1=o

-8

-7

-6

-5

F i g . 5. B i o l o g i c a l a c t i v i t y o f v a r i o u s a g a r o s e - i m m n b i l i z e d c a t e c h o l a m i n e s o n i n t a c t f r o g e r y t h r o c y t e s . T h e various agarose-bound catccholamines were present in the incubation mixture at the following concent r a t i o n s : A d - N E , 1.1 m M ; A n - N E , 1 . 6 r a M ; G u - N E , 2 . 7 r a M ; A z o - N E , 0 . 5 r a M ; a n d Azo-lso, 0.25 raM. The right hand side of the figure shows the stimulation of intracellular cyclic AMP levels in intact frog erythrocytes by isoproterenol. In these experiments, the basal level of intracellular cyclic AMP (100%) w a s 2 0 . 7 p m o l / 1 0 0 pl o f P a c k e d e r y t h r o c y t e s . T h e r e s u l t s s h o w n a r e t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s from 4 experiments and the brackets represent the standard error.

ability to interact with beta-adrenergic receptors in the intact cells. All the gels, except Azo-Iso, were inactive as beta-adrenergic agonists (Fig. 5). The small response observed with the Azo-Iso gel could be attributed to isoproterenol which had leached from the gel. Thus, the stimulation caused by Azo-Iso was equivalent to the response elicited by 5 • 10 -7 M native isoproterenol. Although the

ISOPROTERENOL

x plus Gu-NE

.~, / mO~OTER~NOL T 150

,/'~

ISOPROTERENOL ~'d'l~g. Sephorose 4B

/////

Q ~ ioc

Q_

~

50

o

.NNN ~SOPROTIBRENOL-I(M)co~l 0 Fig. 6. E f f e c t s o f agarose b o u n d n o r e p i n e p h r i n e ( G u - N E ) and Sepharose 4 B o n thE' i s o p r o t e r e u o l s t i m u l a t i o n o f i n t r a e e l l u l a r l e v e l s o f c y c l i c A M P in iT,~.act f r o g e r y t h r o c y t e s . A g a r o s e - b o u n d n o r e p i n e p h r i n c w a s p r e s e n t in t h e i n c u b a t i o n m i x t u r e a t 2 . 7 r a M , w h e r : ~ a s S e p h a r o s c 4 B w a s p r e s e n t in a m o u n t s c o m p a r a b / e to the amount of agarose present in incubations with Gu-NE. The crosshatched bars indicate the basal l e v e l s o f c y c l i c A M P a n d t h e l a c k o f e f f e c t o f t h e G u - N E gel a n d S e p h a r o s c 4 B o n t h e l e v e l . T h e r e s u l t s shown here are the means of duplicate determinations from three experiments.

483 T A B L E II EFFECT OF AGAROSE-CATECHOLAMINE GELS ON THE ISOPROTERENOL-STIMULATED CELLULAR LEVELS OF CYCLIC AMP IN INTACT FROG ERYTHROCYTES

INTRA-

T h e e f f e c t o f v a r i o u s a g a r o s e - b o u n d c a t e c h o l a m i n e s on t h e i s o p r o t e r e n o l - s t i m u l a t e d rise in i n t r a c e l l u l a r c y c l i c A M P w a s t e s t e d b y i n c u b a t i o n o f cells in t h e a b s e n c e or t h e p r e s e n c e o f 5, 10 a n d 2 5 nM i s o p r o t e r e n o l . T h e c o n c e n t r a t i o n s of t h e g e l - s u b s t i t u t e d c a t e c h o l a m i n e s w e r e as in t h e e x p e r i m e n t s d e s c r i b e d in Fig. 5. T h e r e s u l t s s h o w n are t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s f r o m t w o e x p e r i m e n t s . Addition

I n t r a c e l l u l a r level of c y c l i c A M P ( p m o l / 1 0 0 pl p a c k e d e r y t b r o c y t e s ) Isoproterenol concn. (nM):

None Ad-NE An-NE Azo-NE Azo-Iso

0

5

10

25

18.8 17.2 20.0 15.2 42.4

27.6 30.4 26.0 20.8 67.2

38.0 36.8 32.0 33.2 62.8

58.6 48.4 59.2 59.2 85.6

actual concentration of leached isoproterenol was n o t determined it is of interest that the response elicited by the Azo-Iso gel appeared to correspond to the concentration (5 • 10 -7 M) which has been found to leach from the Azo-Iso gel in the presence of erythrocyte membranes (Table I). The various gels were also tested as antagonists of the isoproterenol stimulated increase in intracellular cyclic AMP observed in these intact cells. As shown in Fig. 6, there was no effect of either Gu-NE or Sepharose 4B on the cyclic AMP accumulated in these cells in response to isoproterenol. As shown in Table II, the remaining four gels were tested for antagonist properties in intact cells in the presence of only three concentrations of isoproterenol. None of the gels antagonized the isoproterenol mediated rise in cyclic AMP in the cells. As shown in Table II the Azo-Iso gel produced a slight but significant further stimulation of the cyclic AMP (presumably due to the activity of leached isoproterenol released into solution). Discussion In this study the biological activity of catecholamines bound to agarose via various chemical linkages was examined in three different membrane preparations. The data suggest that when the catecholamine-substituted gels stimulate adenylate cyclase, the activity can be explained on the basis of leached hormones present in the medium. In heart membranes, however, two of the gels, An-NE and Gu-NE, did not stimulate adenylate cyclase activity, despite the fact that, as shown in Table I, rather high concentrations of leached material were found in the treated gel supernatants. It is possible that the norepinephrine which was released from these gels may have been chemically altered and hence biologically less active. The activity of Millipore filtered supernatants of treated gels appeared to be slightly higher than that of the gels themselves. This is consistent with the fact that the catecholamine-bound gels can act as weak antagonists of isoproterenol stimulated adenylate cyclase. In frog erythrocyte m e n ,

484 branes considerably less leaching of catecholamines occurred from the various gels. Consequently, in these membranes, only Azo-Iso stimulated the adenylate cyclase. As noted above, the activity of this gel is quite compatible with the levels of leached isoproterenol released during the incubation. The lack of activity of comparable concentrations of leached norepinephrine is a reflection of the f12 character of the beta-adrenergic receptors in these cells which have a much higher (100--1000 times greater) affinity for isoproterenol than norepinephrine [13]. These results suggest that cateeholamines bound covalently to agarose do not interact with the beta-adrenergic receptors as agonists since they fail to activate the adenylate cyelase in membrane preparations. Nonetheless, these immobilized catecholamines are capable of interacting with the beta-adrenergic receptors as antagonists, as shown above. All of the catecholamine substituted gels were capable of antagonizing the isoproterenol stimulated adenylate cyclase in erythrocyte membranes. Gels which presumably had the eatecholamine substituted via the eatechol ring appeared to be more potent than those gels where norepinephrine was attached through the amino nitrogen. The antagonism of isoproterenol stimulation of the cyclase in membrane preparations was of mixed type. One c o m p o n e n t appeared competitive in nature, demonstrated by the parallel shift in the isoproterenol dose response curves. This implies a direct interaction of the immobilized catecholamine with the beta-adrenergic receptors. Presumably, chemical changes introduced by derivatization or steric constraints imposed by the agarose beads alter the nature of this interaction, thus converting the catecholamines from agonists to antagonists. The noncompetitive inhibition which is superimposed on the competitive type appears to be, however, unrelated to beta-adrenergic receptor occupancy. Thus, the Gu-NE gel noncompetitively inhibited both the F- (not shown) and Gpp(NH)p stimulation of adenylate cyclase as well as that caused by catecholamines. The exact cellular location of beta-adrenergic receptors has never been firmly established and has been the subject of some discussion in the literature. They are generally thought to be associated with the cell membranes in proximity to the adenylate cyclase system [26,27]. On the other hand, catecholamines are small molecules which are capable of penetrating the cell surface and reaching a variety of intracellular organelles. Using these gels which are incapable of penetrating inside the cell, we attempted to probe the localization of the beta-adrenergic receptors in the intact frog erythrocyte. An advantage of using these catecholamine gels for studies with intact cells is, as we have demonstrated here, that their biological activity is opposite (antagonists) to that of the cateeholamines from which they were synthesized (agonists). Thus, biological activity of the catecholamine gels as antagonists on intact cells (implying a localization of the receptors at the cell surface) could not be attributed to the presence of free catecholamines which are agonists. However, none of the gels antagonized the isoproterenol stimulated accumulation of cyclic AMP in intact erythrocytes. These results could be interpreted in several ways: First, steric factors introduced by the large Sepharose beads could have prevented access of the bound catecholamines to the receptor sites. Second, the large size of the agarose beads relative to the cells might have p~ecluded a degree of receptor occupancy sufficient to elicit the antagonist effect. Third, it is possible that the receptors, al-

485

though localized in the plasma membrane might not be accessible at the surface of the cell membrane. Rather, they might exist buried within the membrane matrix. Our finding that the apparent agonist properties of agarose-bound catecholamines can most likely be explained by the fact that ligand leached from these gels is in agreement with the results of Yong [6], but at variance with those of Venter et al. [3,4]. It should be noted, however, that the results of Venter et al. were obtained with catecholamines bound to glass beads and hence are not directly comparable to our results. Our findings also shed light on perplexing findings previously published by Weinstein et al. [9]. These authors found that derivatives prepared by linking norepinephrine to Sepharose via rabbit serum albumin bound human leukocytes. The binding could be blocked by propranolol. The implication was that binding was occurring via interaction of the gel-bound catecholamine with beta-adrenergic receptors. Nonetheless, when the ability of these gels to activate adenylate cyclase in leukocytes was tested they were found to be inert. This seeming paradox might be explained by our observation that agarose-catecholamines do interact with beta-adrenergic receptors, but as antagonists rather than agonists. The ability of the derivatives prepared by Weinstein et al. [9] to interact with the sites on intact cells and the inability of ours to do so may have been due to the much longer spacer arm on their gels or to intrinsic differences in the membrane localization of the receptors in the two different types of cells. Acknowledgements The authors wish to thank Dr. F.A. Neelon for supplying the purified preparation of protein kinase for the measurement of cyclic AMP and Dr. R. M. Bell for the adipose membrane preparations. This work was supported by H.E.W. grant No. HL-16037-02 and a grant-in-aid from the American Heart Association with funds contributed in part by the North Carolina Heart Association. Marc. G. Caron is a recipient of a Fellowship from the Medical Research Council of Canada and the government of the province of Quebec. Robert. J. Lefkowitz is an Established Investigator of the American Heart Association. References 1 S c h i m m e r , B.P., U e d a , K. a n d S a t o , G.H. ( 1 9 6 8 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 32, 8 0 6 - - 8 1 1 2 J o h n s o n , C.B., Blecher, M. a n d Giorgio, Jr., N.A. ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 6 , 1035--1041 3 V e n t e r , J.C., D i x o n , J . E . , M a r o k o , P.R. a n d K a p l a n , N.O. ( 1 9 7 2 ) Proc. Natl. A c a d . Sci. U.S. 69, 1141--1145 4 V e n t e r , J.C., Ross, Jr., J., D i x o n , J.E., M a y e r , S.E. a n d K a p l a n , N.O. ( 1 9 7 3 ) Proc. Natl. A c a d . Sci. U.S. 70, 1 2 1 4 - - 1 2 1 7 5 V e n t e r , J.C. a n d K a p l a n , N.O. ( 1 9 7 4 ) Science 1 8 5 , 4 5 9 - - 4 6 0 6 Y o n g , M.S. ( 1 9 7 3 ) Science 1 8 2 , 1 5 7 - - 1 5 8 7 Y o n g , M.S. a n d R i c h a r d s o n , J.B. ( 1 9 7 4 ) Science 1 8 5 , 4 6 0 - - 4 6 1 8 O ' H a r a , D.S. a n d L e f k o w i t z , R.J. ( 1 9 7 4 ) in M e t h o d s of E n z y m o l o g y ( C o l o w i c k , S.P. a n d K a p l a n , N.O., eds.), pp. 6 9 5 - - 7 0 0 , A c a d e m i c Press, N e w Y o r k 9 W e i n s t e i n , Y., M e l m o n , K . L . , B o u r n e , H.R. and Sela, M. ( 1 9 7 3 ) J. Clin. I n v e s t . 52, 1 3 4 9 - - 1 3 6 1 10 A x e n , R., P o r a t h , J. a n d E r n b a c k , S. ( 1 9 6 7 ) N a t u r e ( L o n d o n ) 2 1 4 , 1 3 0 2 - - 1 3 0 4 11 I n m a n , J.K. a n d Dintzis, H.M. ( 1 9 6 9 ) B i o c h e m i s t r y 8, 4 0 7 4 - - 4 0 8 2 12 V e n t e r , J.C., A r n o l d , L.J. a n d K a p l a n , N.O. ( 1 9 7 5 ) Mol. P h a r m a c o l . 11, 1--9

486

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Lefkowitz, R.J. (1975) Biochem. Pharmacol. 24, 1651--1658 C z e c h , M.P. a n d L y n n , W.S. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 5 0 8 1 - - 5 0 8 9 C a r o m M.G. a n d L e f k o w i t z , R . J . ( 1 9 7 4 ) N a t u r e ( L o n d o n ) 2 4 9 , 2 5 8 - - 2 6 0 K r i s h n a , G., Weiss, B. a n d B r o d i e , B.B. ( 1 9 6 8 ) J. P h a r m a c o l . a n d E x p t . T h e r a p . 1 6 3 , 3 7 9 - - 3 8 8 R a m a c h a n d r a n , J. ( 1 9 7 1 ) A n a l . B i o c h e m . 4 3 , 2 2 7 - - 2 3 9 G i l m a n , A.C. ( 1 9 7 0 ) P r o c . N a t l . A c a d . Sci., U.S. 6 7 , 3 0 5 - - 3 1 2 N e e l o n , F . A . a n d B i r c h , B.M. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 8 3 6 1 - - 8 3 6 5 L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 1 V a u q u e l i n , G., L a c o m b e , M . L . , H a n o u n e , J. a n d S t r o s b c r g , A.D. ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res. C o m mun. 64, 1076--1082 C u a t r e c a s a s , P. a n d P a r i k h , I. ( 1 9 7 2 ) B i o c h e m i s t r y 11, 2 2 9 1 - - 2 2 9 9 Tesser, G.I., F i s c h , H . V . a n d S c h w y z e r , R. ( 1 9 7 4 ) Helv. C h e m . A c t a 57, 1 7 1 8 - - 1 7 3 0 A r i e n s , E.J., S i m o n i s , A.M. a n d V a n R o s s u m , J.M. ( 1 9 6 4 ) in M o l e c u l a r P h a r m a c o l o g y , ( A r i e n s , E.J. ed.), p p . 2 8 7 - - 3 9 3 , A c a d e m i c Press, N e w Y o r k L c f k o w i t z , R.J. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 6 1 1 9 - - 6 1 2 4 D a v o r e n , P.R. a n d S u t h e r l a n d , E.W. ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 3 0 1 6 - - 3 0 2 3 (~ye, I. a n d S u t h e r l a n d , E.W. ( 1 9 6 6 ) B i o c h i m . B i o p h y s . A c t a 1 2 7 , 3 4 7 - - 3 5 4