Inhibitory adenosine receptors in the heart: Characterization by ligand binding studies and effects on β-adrenergic receptor stimulated adenylate cyclase and membrane protein phosphorylation

j Mol Cell Cardiol 16, 931-942 (1984)

Inhibitory Adenosine Receptors in the Heart: Characterization by Ligand Binding Studies and Effects on ~-adrenergic Receptor Stimulated Adenylate Cyclase and Membrane Protein Phosphorylation M. M a r l e n e H o s e y *l, K a t h r y n K. M c M a h o n I a n d R i c h a r d D. G r e e n 2

1Department o/Biological Chemistry and Structure, University of Health Sciences/The Chicago Medical School, North Chicago, IL 60064; and 2 Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA (Received 1 August 1983, accepted in revisedform 17January 1984) M. M. HosEY, K. K. McMA~IONAND R. D. GREEN. Inhibitory Adenosine Receptors in the Heart : Characterization by Ligand Binding Studies and Effects on fl-adrenergic Receptor Stimulated Adenylate Cyclase and Membrane Protein Phosphorylation, Journal of Molecularand CellularCardiology(1984) 16, 931-942. Adenosine causes negative chronotropic and inotropic effects on cardiac tissue. We have investigated the nature of the cardiac adenosine receptor and its effector mechanisms in preparations of newborn chick heart. The adenosine analog [3H]N6(L,phenylisopropyl)adenosine (L-PIA), an agonist at R-type adenosine receptors, bound with high affinity to receptors in crude and highly-purified membrane preparations. The K D was 3-5 nM. The receptor density was low in crude membranes (10 fmol/mg protein) but significantly enriched in purified sarcolemma (164 fmol/mg protein). Competition studies showed that N-ethylcarboxamide adenosine and N6(D-phenylisopropyt)adenosine were less potent than N6(L-phenylisopropyl)adenosine at the chick heart adenosine receptor, as expected for an Ri-type adenosine receptor. Gpp(NH)p decreased the binding of [3H]N6~L-phenylisopropyl)adenosine to chick heart membranes, suggesting that the guanine nucleotide converted the receptor to a lower affinity state. N6(L-phenylisopropyl)adenosine inhibited fl-adrenergic receptor stimulated adenylate cyclase activity. The ICsofor cyclase attenuation by N6(L-phenylisopropyl)adenosine was 1 #~. N6(L-phenylisopropyl)adenosine reversed the effect of the fl-receptor agonist isoproterenol on phospholamban phosphorytation in 32p-labelled slices of newborn chick hearts. This effect of N6(L phenylisopropyl)adenosine was evident by 2 rain, had an ICsoof200 nM, and was prevented by the adenosine receptor antagonist 8-phenyltheophylline. Taken together, the results suggest that the antiadrenergic effects of adenosine on cardiac tissue are mediated by a decrease in membrane protein phosphorylation signalled by activation of Ri-adenosine receptors. The coupling mechanism between receptor activation and protein phosphorylation may be an attenuation of adenylate cyclase. K~'r WORDS: Adenosine receptors; [3H]N6(L,phenylisopropyl)adenosine binding; Adenylate cyclase; Membrane protein phosphorylation; Phospholamban.

Introduction A d e n o s i n e p r o d u c e s negative' i n o t r o p i c effects in c a r d i a c tissue. T h i s effect m a y be o f physiological significance b e c a u s e it is k n o w n t h a t a d e n o s i n e is c o n t i n u a l l y released i n t o the circ u l a t i o n [1, 9, 28, 36J a n d t h a t this release is i n c r e a s e d d u r i n g periods o f stress, such as d u r i n g exercise or a n o x i a [1, 9, 28, 36]. I n m a m m a l i a n or chick ventricles, a d e n o s i n e , like a c e t y l c h o l i n e , o n l y p r o d u c e s n e g a t i v e ino-

tropic effects in tissue p r e v i o u s l y s t i m u l a t e d by a f l - a d r e n e r g i c r e c e p t o r a g o n i s t [3, 35]. T h e s e effects h a v e b e e n t e r m e d ' a n t i - a d r e n e r g i c ' or ' i n d i r e c t ' effects. T h e a n t i - a d r e n e r g i c effect o f a d e n o s i n e on c o n t r a c t i l e force is t h o u g h t to result f r o m the a b i l i t y o f a d e n o s i n e to reverse the effect o f f l - a d r e n e r g i c agonists on the C a 2 + c u r r e n t [3]. T h e a d e n o s i n e r e c e p t o r s i n v o l v e d in the a n t i a d r e n e r g i c effect a p p e a r to be the R i - t y p e

* To whom.requests for offprints should be addressed. 0022-2828/84/100931 + 12 $03.00/0

9 1984 Academic Press Inc. (London) Limited

932

M.M. Hosey et aL

[2, 3, &lO, 35]. By definition, Ri-adenosine receptors attenuate adenylate cyclase activity [22]. Unlike P-type adenosine sites, which also attenuate cyclase, Ri-sites are blocked by methylxanthines. Adenosine decreases cAMP levels in cardiac tissue and this effect is blocked by methylxanthines [-3, 8-10, 35]. The rank potency order of adenosine analogs at Ri-type receptors is usually N 6 (Lphenylisopropyl)adenosine (L-PIA) x > Nethylcarboxamide adenosine (NECA) >_ N 6 (D-phenylisopropyl) adenosine (D-PIA) [6-8]. Indeed, L-PIA is more potent than NECA in attenuating cardiac adenylate cyclase [19] and L-PIA is a potent inhibitor of contractile force [-2, 11]. One should be able to directly detect cardiac Ri-receptors using the radiolabelled agonist [3H]L-PIA. However, no direct characterization of cardiac adenosine receptors by ligand binding studies have been reported. It also should be possible to gain further information about the effector mechanisms utilized by the adenosine receptor system. It is widely held that agonists that increase cAMP formation in cardiac tissue ultimately increase contractile force by a cAMP-mediated phosphorylation of the Ca2+-channel or a component thereof [32]. While the identity of these phosphorylated membrane component(s) is unknown, it is known that fladrenergic agonists dramatically increase the phosphorylation of a cardiac membrane protein in perfused hearts and in cardiac slices [14, 20, 45]. If the adenosine receptor produces its physiological effects on cardiac tissue via attenuation of cAMP generation and consequent inhibition of cAMP-dependent protein kinase activity [10], then the antiadrenergic effects of adenosine on Ca 2+ channel activity could be caused by decreased membrane protein phosphorylation. The present study demonstrates for the first time: (1) the presence of Ri-adenosine receptors in newborn chick heart by direct agonist binding studies using [3H]L-PIA, and (2) that activation of cardiac adenosine receptors dramatically affects the phosphorylation of cardiac membrane proteins in cardiac slices.

Experimental Procedures

Materials Fertilized White Leghorn eggs were obtained from SPAFAS. Newborn chicks were used 7 to 10 days post-hatching. N 6(Lphenylisopropyl)- adenosine (L-PIA) was purchased from Boehringer-Mannheim. N 6 (D-phenylisopropyl) adenosine (D-PIA) was a gift from Dr John Daly, National Institutes of Health, Bethesda, MD. Nethylcarboxamide adenosine (NECA) was a gift from Dr H. Stein, Abbot Labs, North Chicago, IL. 8-phenyltheophylline was from Calbioehem. [3H]N6 (L-phenylisopropyl) adenosine (methyl-2- phenylethyladenosine, L-N6-1-[adenine-2,8-3H, ethyl-2-3H]) was purchased from New England Nuclear at a specific activity of 49.9 Ci/mmol. Adenosine dearninase (type 3), ATP, dATP, deoxycyclic AMP, creatine phosphate (disodium salt), creatine phosphokinase and isoproterenol were obtained from Sigma. Cyclic AMP, G T P and guanylylimidodiphosphate [Gpp(NH)p] were from P-L Biochemicals. The non-xanthine phosphodiesterase inhibitor R O 20-1724 was a gift from Dr H. Sheppard, Hoffman-LaRoche, Nutley, NJ. [0~-32p]ATP and [-~-32p]dATP were purchased from ICN. [3H]cAMP and inorganic 32p were from Amersham. [3H]deoxy-cAMP was from New England Nuclear. All other reagents were fi-om sources as previously described [14].

Preparation of membranesfor ligand binding and adenylate cyclase studies Crude membrane preparations of whole newborn (5 to 10 days old) chick hearts were prepared by homogenizing minced tissue in 4 vols of 10 mM histidine, pH 7.5, 1 mM EDTA (buffer A) with a Polytron (PT-10) homogenizer for two 5-s periods at setting 7. The homogenate was incubated for 10 rain at 37~ with 5 units/ml of adenosine deaminase [-49]. The solution was then diluted 10 to 15 fold with buffer A and centrifuged at 25,300 x gay for 40 min. The resulting pellet was resuspended in 40 vol of i mM NaHCO3, 1

1 Abbreviationsused : L-PIA, N6(L-phenylisopropyl)adenosine; D-PIA, N 6 (D-phenylisopropyl)adenosine; NECA, N-ethylcarboxamideadenosine;Gpp(NH)p, guanylylimidodiphosphate;NaDodSO4,sodiumdodecylsulfate.

Action of Adenosine on Cardiac Tissue

mM E D T A (pH 7.5), incubated for 30 min at 0~ and centrifuged as above. The pellet was resuspended in 4 vol of buffer A and used immediately or stored briefly in liquid N 2. Purified membranes were prepared as previously described [12, 13]. Ligand binding studies were performed with membranes designated as T1 and T z [12, 13]. Adenylate cyclase studies were performed with membranes designated 15/36 (method 1, [12]). We previously documented that the Ta, Tz and 15/36 membranes are enriched in sarcolemma by several criteria [12, 13]. For example, T I is enriched 15-fold in muscarinic receptors [12], 15-fold in Na +, K+-ATPase, 30-fold in 5'nucleotidase, and 50 60-fold in sialic acid [13]. The data at hand do not allow a distinction to be made as to the relative contributions of myocardial v. non-myocardial cell membranes to the membrane preparations.

Ligand binding assay The binding of [3H]N 6(L-phenylisopropyl)adenosine to cardiac membranes was performed essentially according to the methods described by Yeung and Green for the Ri-receptor agonist [3H]cyclohexyladenosine [49]. The reaction mixture contained: 10 m~ histidine, p H 7.5, 10 mM MgClz, 5 units/ml of adenosine deaminase, varying concentrations of [ 3 H ] N 6(L-phenylisopropyl)adenosine and either 0.3 0.4 mg of crude membranes or 15 to 30 pg of purified membranes in a total volume of 0.2 ml (for crude membranes) or 0.1 ml (for purified membranes). The reactions were carried out in triplicate at 37~ for 15 rain and were processed as previously described [49]. Non-specific binding was defined as that obtained in the presence of 0.1 mM non-radioactive N 6 (L-phenylisopropyl) adenosine [49].

Adenylate cyclaseassay Purified t5/36 membranes [12] were pretreated with 5 units/ml of adenosine deaminase for 5 or 10 min at room temperature and then assayed for adenylate cyclase activity in the presence and absence of isoproterenol and varying concentrations of N 6 (Lphenylisopropyl)adenosine. Two different assays were utilized. One utilized [e-32p]ATP

933

as substrate [34] while the other used [~3zp]dATP [7]. The reaction mixtures contained: 10 mM histidine, pH 7.5, 9 m~ MgC12, 0.1 mM [~32-p]ATP or [e-32p]dATP (100 ct/ min/pmol), 2 mM cAMP or 0.1 mM deoxycyclic A M P (in accordance with the substrate used), 5 mM phosphocreatine, 0.4 mg/ml phosphocreatine kinase, 50 mM NaC1 or NH4C1, 10 #M GTP, 0.1% bovine serum albumin, 0.5 units/ml adenosine deaminase, _+0.5 mM R O 20-1724 (a phosphodiesterase inhibitor), and 10 to 15 ~ug membrane protein in a total volume of 0.1 ml. The reactions were carried out in duplicate at 37~ for 10 min. The samples which contained ATP as substrate were processed according to Salomon et al. [34] while those which contained d A T P as substrate were processed according to Cooper and Londos [7]. Recovery of [3Zp]cAMP or [3ap]deoxy-cyclic A M P was measured by calculating recovery of [3H]cAMP or [3H]deoxy-cyclic A M P (15,000 ct/min) added to the samples.

Protein phospho~ylation studies Tissue slices from 7 to 9-day-old chick hearts were prepared with a Stadie-Riggs tissue slicer and incubated in 32pi-containing Tyrode's solution as previously described [14]. All tissue groups were incubated with 2 U/ml of adenosine deaminase for 10 min prior to and during the control or drug treatment to metabolize exogenous adenosine. Isoproterenol was used at 10 .6 M. When 8phenyltheophylline was used, it was added to the tissue samples in dimethylsulfoxide at a final concentration of solvent of 0.2%. In those experiments all controls also received 0.2% dimethylsulfoxide. Tissue slices for control or drug-treated groups were freezeclamped and processed as previously described [14]. Membrane fractions designated T1 and T z [12, 13], containing sarcolemma and sarcolemma plus sarcoplasmic reticulum, respectively, were prepared and stored briefly in liquid N 2 prior to analysis by polyacrylamide gel electrophoresis and autoradiography [14].

NaDodSO 4opolyacrylamidegel electrophoresis NaDodSO 4 polyacrylamide gel electrophoresis was performed according to Laemmli

934

M.M. Hosey et al.

[16] but gradient gels were used as specified. Duplicate samples were dissolved in the N a D o d S O 4 sample buffer [13], one of which was heated at 37~ for 10 min while the other was boiled for 1 min prior to electrophoresis [14]. Autoradiograms were p r e p a r e d from the dried gels using K o d a k No-Screen X - r a y film. Molecular weight standards were phosphorylase a (94,000), bovine serum albumin (68,000), ovalbumin (43,000), carbonic anhydrase (29,000), trypsin inhibitor (21,000), fllactoglobulin (i8,400), myoglobin (16,700), cytochrome c (12,500), C-terminal fragment (Ser 237--Ala 335, designated EP -2 in [26]) of fructose 1,6-bisphosphatase (11,000), and the N-terminal fragment (N-acetyl T h r 1--Ata 60, [19]) of fructose 1,6bisphosphatase (6,500). T h e fructosebisphosphatase peptides were kindly supplied by Drs Frank Marcus and T a p a t i Chatterjee (Chicago Medical SchooI) and their exact size determined by a m i n o acid sequence analysis

[267. Miscellaneous methods A T P concentration [40] and specific activity [6"] determinations were performed as previously described [14]. T h e specific activity of the endogenous [y-32p]ATP formed in the a2P-labelled cardiac slices in the present studies varied between groups from 0,05 to 0.15 ct/min/nrnol, and was unaffected by drug treatment. Protein determinations were performed according to Bradford [4-]. Results

L(gand binding studies [ 3 H I N6 (L-phenylisopropyl) adenosine bound with high affinity in a specific and saturable m a n n e r to crude (Fig. 1 (a)) and highly purified (Fig. l(b)) chick heart membranes. T h e K D was 4.3 __ 1.5 ma (n = 3) for crude m e m branes and 4.7 ___ 0.4 (n = 2) nM f o r the purified membranes. T h e high affinity binding of [3H]N6 (L-phenylisopropyl) adenosine to cardiac m e m b r a n e s was similar to that previously described for the adenosine agonist [3H]cyclohexyladenosine binding to the high affinity state of the Ri-adenosine receptor in h i p p o c a m p u s [49]. T h e receptor density was very low in the crude m e m b r a n e s (Bmax = 9.2

(o)

4

t

9

3L

3

e

9

.e ~

B

/

X

~

I

"I 0

L..g.T-x'~

'l ~

I

v, % .

!

.

.

.

.

.

.

'~ ,rF>-L.j

't 2

4

6

8

I0

12

14

FIGURE l. Binding of [3H]N6 (L-phenylisopropyl)adenosine to crude membrane (a) and purified membrane (b) preparations of newborn chick heart. Saturation isotherms are shown in terms of cpm bound/ sample. (a) 385 g protein/sample; (b) 12,5 g protein/ sample. 0 - - -D, Total ct/min bound ; x-x, non-specific ct/min bound; O- - - 9 specific ct/min bound. Scatchard plots are shown in the insets, where B/F = fmol/mg protein x nM, and B = fmol bound/mg protein. The values Obtained were: A, KD= 3.3 riM; Bmax=9.9 fmol/mg protein; B, K o = 5.1 n~, Bma~ = 164 fmol/mg protein: Data Shown are from representative experiments. Data from a series of similar experiments are summarized in the text. _+ 1,2 fmol/mg protein, n = 3), but was enriched greater than 15-fold in the purified m e m b r a n e s (Bmax = 164 fmol/mg 3 protein). T h e fold-enrichment of the [ H ] N (Lphenylisopropyl)adenosine binding sites in the purified m e m b r a n e s was similar to that observed for sarcolemma markers [12, 13]. We previously showed these m e m b r a n e s were enriched 10 to 20 fold in muscarinic cholinergic receptors [12], N a +, K + - A T P a s e and 5'nucleotidase, while being essentially free of mitochondrial markers [13].

Action of Adenosine on Cardiac Tissue

The stereoisomer of N 6 (L-phenylisopropyl)adenosine (L-PIA), N 6(D-phenylisOpropyl)adenosine ( D - H A ) , was less potent than L-PIA in binding to the cardiac adenosine receptor ( T a b l e 1). A Hill plot of the inhibition of [3H]N6 (L-phenylisopropyl)adenosine binding by D-PIA was characterized by a pseudo-Hill coefficient of 0.77 _+ 0.12 (n = 3), which was not significantly different from one. This indicated that, under the conditions used, the agonists were mainly recognizing a single high affinity state of the receptor [46], and the ICs0 value for D - P I A (Table 1) binding to this affinity state could be transformed to an approximate Ki value [46] of 20 nM. By this criteria, D - P I A was 4 to 5-fold less potent than L-PIA in binding to the cardiac receptors. Similar conclusions can be made from experiments in which we compared L-PIA and D - P I A to compete for [ 3 H ] L - P I A binding (data not shown). Another adenosine analog, Nethylcarboxamide adenosine, was similarly less potent than L-PIA at the cardiac binding sites as it had an ICs0value of 54 nM (Table 1) and an approximate Ki value of 16 nM. The binding of [3H]N6(L,phenylisopropyl)adenosine to cardiac adenosine receptors was decreased 70% by 100 #M of the guanine nucleotide G p p ( N H ) p . The ICsofor G p p ( N H ) p was 20 #M (Table 1). This observation was consistent with the expected conversion of a high affinity state of the cardiac adenosine receptor to a lower affinity state as previously described for adenosine receptors in the hippocarnpus by Yeung and Green

[491.

935

Adenylate cyclasestudies The effects of varying concentrations of L - P I A on adenylate cyclase in purified chick heart membranes are summarized in Figure 2. L-PIA inhibited isoproterenol-stimulated adenylate cyclase by 60%. The approximate ICs0for this effect was 1 #M assuming maximal inhibition was at 0.1 mM. Higher concentrations of L - H A than 0.1 mM were not tested because of the limited solubility of the compound. The results shown summarize experiments performed with either A T P or d A T P as substrate. The degree of stimulation by isoproterenol and inhibition by L-PIA or the muscarinic receptor agonist oxotremorine (data not shown) were identical with either A T P or dATP. The only difference observed was that basal activity was higher with d A T P (389.7 + 3 9 . 9 pmol deoxycyclic A M P / m g protein/rain, n = 6) than with ATP (264.5-4- 11.6 pmol cAMP/mg protein/rain, n = 10). L-PIA was considerably less effective than the musearinic receptor agonist oxotermorine in attenuating isoproterenol-stimulated cyclase (Table 2). At equal concentrations, L-PIA reduced isoproterenol-stimulated activity less than 25%, while oxotremorine reduced activity by 80%. Furthermore the maximal effect elicited by L-PIA was a 50% to 60% inhibition (at 100 #M) compared to the near total inhibition caused by oxotremorine 2. In addition, L-PIA had no significant effect on basal adenytate cyclase activity, while oxotremorine consistently attenuated basal activity by 20% to 25%

TABLE 1. Inhibition by D-PIA, N-ethylcarboxamide adenosine and Gpp(NH)p on the specific binding of [aH]N6(L-phenylisopropyl) adenosine to purified membrane preparations of newborn chick heart Addition

ICs0

n

N6(D-phenylisopropyl)adenosine N-ethylcarboxamide adenosine Gpp(NH)p

68.7 __. 5.8 nM

3

54.0 -t- 0.1 nM 20.3 _ 8.7/~M

3 3

The concentrationsof [3H]N6(L-phenylisopropyl}adenosineused were 9.8 to I0.3 nM. n refers to number of determinations, iC~0 values given are mean -I-s.~.M.

936

M . M . H o s e y et aL

ioo .~_

~

eo

3 .

6o

I 8

t 7

[ 6

I 5

I 4

-,o~ [L-PlAI, M

FIGURE 2. Inhibition of isoproterenol-stimulated adenylate cyclase by varying concentrations of N6(Lphenylisopropyl)adenosine.Adenylate cyclasewas assayed as described in Experimental procedures. Isoproterenolwas 1 #M. Numbers in parentheses are the number of experiments performed in duplicate, and the values are the mean ___S.E.M. (Table 2). All attempts to amplify the L - P I A response on basal or isoproterenol-stimulated cyclase activity were without success. These included determinations of optimal concentrations of G T P a n d m o n o v a l e n t cations a n d v a r y i n g the concentrations of adenosine deaminase.

Membrane protein phosphorylation studies T h e fl-adrenergic receptor agonist isoproterenol increased the phosphorylation in cardiac slices of a cardiac m e m b r a n e protein (Fig. 3) that possesses electrophoretic proper-

ties characteristic of p h o s p h o l a m b a n [13-15, 17, 18, 24]. W e previously d o c u m e n t e d the t i m e - d e p e n d e n t increase in the phosphorylation of this protein in slices by isoproterenol [14]. T h e molecular weight (Mr) of the protein is 27,000 daltons in samples heated at 37~ for 10 m i n prior to electrophoresis or 11,000 in samples boiled for 1 m i n (Fig. 3(b)). L-PIA, at 10 - 6 M, produced a time dependent i n h i b i t i o n of the isoproterenol induced phosphorylation of the 2 7 K / 1 1 K protein (Fig. 3(c) (e)). These experiments with L - P I A

TABLE 2. Comparison of the effects of N6(L phenylisopropyl)adenosine and the muscarinic receptor agonist oxotremorine on basal and isoproterenol stimulated cardiac adenylate cyclase Addition Isoproterenol, 1 /2u Oxotremorine, 1 pM N6(L-phenylisopropyl)adenosine, 1 /~M N6(L-phenylisopropyl) adenosine, 100 #M

Basal activity

(%)

129.6 _+ 2.6 (12) 81.8 _ 3.9 (9) 99.5 _ 2.4 (5) N.D.

Isoproterenolstimulated activity

(%) --

19.7 + 8.9 (7) 77.4 ___6.9 (8) 47.2 + 11.5 (6)

Values given are mean + S.E.M.Number in parentheses is number of determinations. N.D.means not determined. Each determination was performed in duplicate.

Action of Adenosine

on Cardiac

Tissue

937

2 and 6 m i n exposures to isoproterenol alone (data not shown) a n d found, in agreement 43, with our previous study [14], that the 27/11K 34,000* *27, protein is m a x i m a l l y phosphorylated b y 2 24,000* min. However, a moderate phosphorylation of 21, 20,000" the peptides of 34,000, 24,000 a n d 20,000 18, became a p p a r e n t in the samples exposed to *11, isoproterenol for 6 min. T h e effect of L - P I A to decrease the phosphorylation of the 2 7 K / 1 1 K protein was doset__J L~J t___J t_._J t.___l d e p e n d e n t (Fig. 4). For these studies, (a) (b) (c) (d) (e) increasing concentrations of L - P I A were Isoproterenol + + + + added 2 m i n after isoproterenol. T h e total N 6 (L-phenylisopropyl) I' 2' 5' exposure time to L - P I A was 4 rain, and to adenosine FIGURE 3. Autoradiogram depicting the time- isoproterenol 6 min. T h e results shown are of dependent antagonism of isoproterenol-stimulated radioautograms of T 1 a n d T 2 fractions cardiac membrane phosphorylation by NS(L- analyzed by densitometry. T h e effects of 3/~M phenylisopropyl)adenosine. Membrane fraction T l was L - P I A were m a x i m a l (data not shown) a n d prepared as previously described [10] from 32p-labelled a n ICs0 value of approximately 200 nM for heart slices treated as follows: control (a); isoproterenol, 1 #M, 2 min (b) ; isoproterenol, 2 min, then L-PIA, 1 pM L - P I A was calculated for both fractions. It was i m p o r t a n t to determine if the L - P I A for 1 min (c), 2 rain (d), or 5 min (e) in the continued presence of isoproterenol. Duplicate samples (20 #g response could be antagonized by a n adenprotein each) are shown. In each case, the sample on the osine receptor antagonist. We chose to use 8left was treated at 37~ for 10 min and the sample on the right was treated at 100~ for 1 rain prior to electro- p h e n y l t h e o p h y l l i n e as the antagonist, since it phoresis. NaDodSO4 gel electrophoresis was performed has been reported to be a potent antagonist at on a 14% gel. Phosphoproteins affected by the drugs are the adenosine receptor b u t a less effective indicated by asterisks next to their Mr values. Other values indicate the positionsof the M, markers.

67,

6O

were performed similarly to those used to d e m o n s t r a t e the antiadrenergic effects of adenosine on contractile force [2, 3, 9, 10, 35]. Isoproterenol was added first for 2 min, a n d then L - P I A was added for 1, 2 or 5 m i n (in the c o n t i n u e d presence of isoproterenol). T h e r e did not a p p e a r to be an L - P I A response at 1 min, b u t at 2 a n d 5 m i n the phosphorylation of the 2 7 K protein was decreased by 25 a n d 80%, respectively (Fig. 3(c)-(e)), as determ i n e d by densitometric analysis of the radioautogram. The phosphorylation of three phosphopeptides with M r of 34,000, 24,000 a n d 20,000 (Fig. 3(e)) was moderately e n h a n c e d in the samples c o n t a i n i n g L - P I A for 5 min. T h e phosphorylation of these peptides might be due to the exposure to L - P I A itself a n d / o r to the longer exposure to isoproterenol (total = 7 min). I n separate experiments we tested the effects of L - P I A alone, i.e. in the absence of isoproterenol, a n d did not observe the phosphorylation of these c o m p o n e n t s (e.g. see Fig. 5(e)). We also compared the effects of

Tt

o

~ ~;

40

~8

2o I

o~

6o

9==

20

o r,-- o N ~ 40

I

I

AAA T~

I

I

I

3 30 500 Ns (L-phenylisopropyl) adenosine, nM 0

FIGURE 4. Concentration dependency of NS(Lphenylisopropyl)adenosine effect on isoproterenolstimulated membrane phosphorylation. Membrane fractions T 1 and T 2 were prepared from a2p-labelled heart slices treated with isoproterenol (1 /~M)for 2 rain, and then with N6(L-phenylisopropyl)adenosineat 0, 3, 30, or 300 nMfor an additional 4 min. Duplicate samples were treated as described in the legend to Fig. 3 and electrophoresed on 10 to 16% gradient gels. The results are shown as densitometric tracings of the 27,000 dalton protein on the resulting autoradiograms.

938

M.M. Hosey et aL

phosphodiesterase inhibitor [39]. For these studies we modified the experimental design slightly. Instead of adding L-PIA 2 min after isoproterenol, both agonists were added simultaneously for a total of 5 rain. 8phenyhheophylline (3 /~M) was added 5 min prior to the agonists. After isoproterenol (10- 6 M) plus L-PIA (10 -7 M) treatment, the phosphorylation of the 27K/11K protein in the T2 fraction treatment was 65% of that observed in the presence of isoproterenol alone (Fig. 5(c)). The L-PIA response was totally prevented by 8-phenyhheophylline (Fig. 5(d)). These results show that L-PIA attenuates membrane phosphorylation at an Ri-type rather than a P-type site. 8phenyhheophylline did not enhance the isoproterenol response (Fig. 5(b)). Similar results were observed with the T 1 fraction (data not shown), The phosphorylation of a peptide with a M, of 20,000 (not labeled) was slightly enhanced by the isoproterenol plus L-PIA treatment (Fig. 5(c)) and prevented by the presence of 8-phenyhheophylline (Fig. 5(d)).

67,000 -43,000" 2 7 , 0 0 0 --

2,,ooo . 18,400

*11,000 6,500

.

.

......

-- -

I5oproferenol 8 - phenyltheophylline N6 ( L- pheny]isopropyl) odenosine

.

--

~.__1L__j ~__~ t__~ L---J L--J (a) (b) (c) (d) (e) ( f ) + + + + -

+

-

+

-

+

+

+

+

+

F I G U R E 5. Autoradiogram depicting inhibition of N6(L-phenylisopropyl)adenosine effect on membrane phosphorylation by 8-phenyhheophylline. Membrane fraction T 2 was prepared fi'om s2p-labelled heart slices treated as illustrated: (a), 1 #M isoproterenol alone; (b) isoproterenol plus 8-phenyltheophylline (3 #M); (c) isoproterenol plus L-PIA (0~1 ,uM); (d) isoproterenol plus 8-phenyltheophylline plus L-PIA; (e) L-PIA alone; (f) L-PIA plus 8-phenyhheophylline. 8-phenyltheophylline or vehicle was added 5 min prior to the agonists. Total agonist exposure time was 5 min. T h e gel was a 10% to 18% gradient gel. Other conditions are as in Fig. 3 or are given in the text.

Discussion

The results show that the adenosine analog L-PIA adenosine binds with high affinity to cardiac membranes, attenuates isoproterenolstimulated adenylate cyclase and antagonizes the effect of isoproterenol on cardiac membrane protein phosphorylation in heart slices. Taken together the results strongly argue for the presence of Ri-adenosin e receptors in t h e chick heart and provide information concerning the functional mechanisms of receptor : effector coupling. The data are consistent with previous studies which suggested the presence of Ri-adenosine receptors in cardiac tissue on the basis of adenosine induced decreases in cAMP in intact hearts or heart slices [2, 3, 8-10, 35] and in membrane preparations [19-]. T h a t L-PIA was acting to inhibit adenylate cyclase and protein phosphorylation via activation of Ri rather than P sites [21, 22] is illustrated by both the potency of the L-PIA effect (Figs. 2 and 4) and the antagonism of the effect by 8phenyhheophylline (Fig. 5). L-PIA is not effective at P-sites even at the highest concentration tested (0.8 mM) [36], and actions of adenosine analogs at P-sites are not prevented by methylxanthines [21]. The ligand binding studies of the Ri-adenosine receptor in the chick heart are technically difficult because of the low receptor density. This condition severely limits the studies that can be performed with a [SH]ligand for these receptors. It also explains why others [30] were previously unable to demonstrate the existence of adenosine receptors in cardiac tissue using [3H]cyclohexyladenosine with a specific activity 3 to 4 times lower than the L-PIA we used in this study. However, the limited data that was obtained was highly reproducible and consistent. We feel it is sufficient to argue for the presence of high-affinity Ri-adenosine receptors in cardiac tissue. A more suitable ligand for future characterization of this receptor system would be a ligand labelled with 125I [99, 38]. Most receptors coupled to adenylate cyclase usually exhibit multiple affinities for agonists. The high affinity binding site for L-PIA that we were able to demonstrate in the chick heart compares favorably with the

Action of Adenosine on Cardiac Tissue

high aitinity state of the Ri-adenosine receptor previously documented in rat hippocampus by Yeung and Green (KD = 2 nM, [49]) and with [ 3 H ] L - P I A binding to membranes from whole rat brain [37] and rat fat cell membranes [43]. The decrease in [ 3 H ] L - P I A binding caused by G p p ( N H ) p (Table 1) probably represents the expected conversion of the receptor to a lower affinity state. We were unable to directly quantitate this lower affinity state of the cardiac receptor because of the low density of receptors and low signal : noise ratio (Fig. 1). The affinity of the lower affinity state of the cardiac Ri-adenosine receptor may be similar to that of the G p p ( N H ) p induced affinity state of the hippocampal Ri-receptor (K D = ~ 3 0 nM) or even lower. In this regard, a low affinity state (K D = 400 mM) was indirectly demonstratable in hippocampus [49]. This value is similar to the potency of L - P I A to attenuate membrane protein phosphorylation (IC5o= 200 nM, Fig. 4). With many cyclase-linked receptors the affinity of ligands for the low affinity state correlates well with the potency of ligands to produce physiological effects. The lower potency of D - P I A [5, 37, 43, 47] and N-ethylcarboxamide adenosine [22] compared to L-PIA for the cardiac adenosine receptor are expected for an Ri-adenosine receptor. While the potency difference we observed is not as great as in some other tissues [22], others have recently demonstrated that N-ethylcarboxamide adenosine was only 4 to 5-fold less potent in inhibiting adenylate cyclase in guinea-pig myocardial membranes [19]. This potency ratio agrees very well with our results on the potencies of these two agonists in binding to the receptor (Fig. 1, Table 1). A similar situation was observed by Londos et al. for cerebral cortex [23] in which L-PIA was only < 10-fold more potent in inhibiting cyclase than Nethylcarboxamide adenosine. The present results extend our understanding of the antiadrenergic effect of adenosine by showing that Ri-receptor activation antagonizes the effect of isoproterenol on cardiac membrane protein phosphorylation. This effect may occur as a result of the attenuation of the adenylate cyclase system mediated by the Ri-adenosine receptors. Accordingly, the time course for the L-PIA

939

induced decrease in isoproterenol-stimulated phospholamban ph0sphorylation agrees with the time course for adenosine induced decrease in isoproterenol-stimulated cAMP formation in slices of rat ventricular muscle [8]. An additional action of L-PIA, such as activation of a phosphatase or inhibition of a protein kinase, may also be possible. These possibilities are suggested by the apparent differences in affinities of L - P I A to attenuate isoproterenol-stimulated adenylate cyclase and protein phosphorylation. However, direct comparisons are difficult, since the cyclase assays were performed in isolated membranes and the protein phosphorylation in tissue slices, and all assays were performed under different conditions. The IC50 concentration of L - P I A required to inhibit protein phosphorylation in our studies is in the range of that required to produce negative inotropic effects in rat atria (ICso = 40 nM in the presence of 10 .8 M isoproterenol, [11]) and in perfused guinea pig hearts (ICs0 ~ 2 0 0 nM after a bolus injection of 4 x 10 -11 mole isoproterenol, [2]). No data is available on the inotropic effects of L-PIA on ventricular strips. The 2 7 K / l l K protein whose phosphorylation is stimulated by isoproterenol and inhibited by L-PIA (and muscarinic agonists, [14]) is found in sarcoplasmic reticulum [20, 25, 41, 42, 45] as well as in many types of preparations of sarcolemma [13, 25, 33]. The sarcoplasmic reticulnm peptide was originally termed phospholamban by Katz and coworkers [42]. This peptide possesses a characteristic electrophoretic mobility in that it is converted from a larger (24 to 27,000 daltons) to a smaller form (11,000 daltons) after it is heated at 100~ [13--15, 17, 18, 24]. This may be due to a tetramer to dimer conversion, with the monomer being a peptide of 5500 daltons [15], or to the dissociation of a trimer of nonidentical subunits, one of which is an 11,000 dalton phosphoprotein [24]. In one previous communication we assigned a M r of 14,000 to the small form of the phosphopeptide that is observed in boiled preparations [14], while in another we referred to it as 11,000 [13]. These differences are due to the difficulty in accurately determining the M r of small peptides. In the present studies, we used as a M r marker a peptide from the C-terminus of fructose 1,6-

940

M . M . Hosey et al.

bisphosphatase (Ser 23NAla 335) which has a Mr of 10,820 as determined by amino acid sequence [26]. The relative mobilities of this peptide and the small phosphopeptide were essentially identical in 10% to 18% gradient gels. In addition, the small phosphopeptide migrated considerably slower than another fructose 1,6-bisphosphatase peptide (Nterminal fragment, N-acetyl-Thr 1 Ala 60) that has a M r of 6,500 [26]. Based on these observations a Mr of 11,000 is probably a reasonable estimate for the smaller form of the isoproterenol-stimulated phosphopeptide. In sarcoplasmic reticulum, phospholamban phosphorylation enhances Ca 2 § uptake and is believed to account for the increased rate of relaxation caused by catcholamines [20, 41, 45]. Whether a phospholamban-type protein is a true and/or functional constituent of sarcolemma is controversial [25]. In any event, it is clear that its phosphorylation is sensitively controlled in slices and in perfused hearts by agents that produce positive and negative inotropic effects on cardiac tissue [14, 20, 45, this paper]. The phosphorylation of several peptides (M r = 34,000, 24,000 and 20,000) was slightly enhanced under several conditions. These included a longer treatment with isoproterenol in the absence (data not shown) or presence (Fig. 3(e)) of L-PIA. Phosphorylation of one of these peptides (20,000) also appeared to be enhanced in the presence of L - P I A plus isoproterenol (Fig. 5(c)) compared to isoproterenol alone (Fig. 5(a)). Furthermore, this later effect was blocked by 8-phenyltheophylline (Fig. 5(d)). We do not yet know the nature of these reactions, nor whether they are of physiological significance. It is possible that they do not occur in myocardial cells, but rather in epithelial or vascular tissue. The time course is rather slow and the effect is modest. Thus they have not been further characterized. It is interesting to compare the actions of adenosine and acetylcholine since they both have direct and indirect (or antiadrenergic) effects on cardiac tissue. (That the inotropic and cAMP-lowering effects of adenosine on cardiac tissue are not due to release of acetyl-

choline has been demonstrated by Dobson [8] who showed that these effects of adenosine cannot be prevented by the muscarinic receptor antagonist atropine). The number of muscarinic cholinergic receptors in newborn chick heart is at least 50-fold greater [12] than the number of Ri-adenosine receptors (Fig. 1). Whether or not all of these muscarinic receptors are coupled to effectors is not known. In other tissues it has been shown that there are a significant number of spare muscarinic receptors. It is interesting, however, that muscarinic agonists appear more effective than adenosine receptor agonists in inhibiting flreceptor stimulated adenylate cyclase (Table 2) and membrane protein phosphorylation [14] in the chick heart. The muscarinic agonist oxotremorine inhibits the isoproterenol-stimulated adenylate cyclase by 80% to 100% 2, while L-PIA only inhibits by 50% to 60% (Fig. 2). Similarly, comparisons of the effects of oxotremorine and L-PIA on isoproterenol-stimulated membrane protein phosphorylation also showed that, in a given time, oxotremorine inhibited a greater percentage of phosphorylation than L-PIA (Hosey, unpublished observations). Whether these effects reflect a greater number of muscarinic receptors coupled to effectors or a higher efficiency of muscarinic receptor:effector coupling remains to be determined. The data from this and other studies suggest that the antiadrenergic effects of acetycholine and adenosine both appear to be due, at least in part, to attenuation of the cAMP-generating and cAMP-dependent protein phosphorylating systems. The direct effects of both agonists are most likely due to other effector mechanisms.

Acknowledgements The authors wish to thank Mrs J u d y Ptasienski for expert and dedicated technical assistance. This work was supported by grant # H L 23306 from the N I H and by the Chicago Heart Association. M. M. Hosey is an Established Investigator of the American Heart Association.

2 McMahon, K. K. and Hosey,M., unpublished observations.

Action of Adenosine on Cardiac Tissue

941

References 1 BACCHUS,A. N., ELY, S. W., KNABB, R. M., RUBIO, R., BERNE, R. M~ Adenosine and coronary blood flow in conscious dogs during normal physiological stimuli. A m J Physio1243, H628-H633 (1982). 2 BAUMANN,G., SCHRADER,J., GERLACn, E. Inhibitory action of adenosine on histimine- and dopamine-stimulated cardiac contractility and adenylate cyclase in guinea pigs. Circ Res 48, 259-266 (1981). 3 BELARDINELLI,L., VOGEL, S., LINDEN, J., BERNE, R. M. Antiadrenergic action of adenosine on ventricular myocardium in embryonic chick hearts.J Mol Cell Cardiol 14, 291 294 (1982). 4 BRADFORD,M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248 254 (1976). 5 BRUNS, R. F., DALY, J. W., SNVDER, S. H. Adenosine receptors in brain membranes: Binding of N 6cyclohexyl[SH]adenosine and 1,3-diethyl-8-[3H]phenylxanthine. Proc Natl Acad Sci USA 77, 5547 5551 (1980). 6 COGOLI,J. M., DoBson, J. G. An easy and rapid method for the measurement of the [732-p]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi . Anal Biochem 110, 331 337 (1981). 7 COOPER,D. M. F., LoNI)OS, C. Evaluation of the effects of adenosine on hepatic and adipocyte adenylate cyclase under conditions where adenosine is not generated endogenously. J Cyclic Nucleotide Res 5, 289-302 (1979). 8 DOBSON, J. G. Reduction by adenosine of the isoproterenol induced increase in cyclic adenosine 3',5'monophosphate formation and glycogen phosphorylase activity in rat heart muscle. Circ Res 43, 785-792 (1978). 9 DOBSON,J. G., FENTON, R. A. Anti-adrenergic effects of adenosine in the heart. In Regulatory Function of Adenosine, R. M. Berne, T. W. Rail and R. Rubio Eds. pp. 363-376, Martinus Nijhoff, Hague, Netherlands (1983). 10 DOBSON,J. G. Mechanism of adenosine inhibition of catecholamine-induced responses in heart. Circ Res 52, 151-160 (1983). 11 ENOOH,M., MARUYAMA,M., TAIRA, N. Modification by islet-activating protein of direct and indirect inhibitory actions of adenosine on rat atrial contraction in relation to cyclic nucleotide metabolism. J Cardiovasc Pharmacol 5, 131-142 (1983). 12 HosE'/, M. M., FIELDS, J. Z. Quantitative and qualitative differences in muscarinic eholinergic receptors in embryonic and newborn chick hearts.J Biol Chem 256, 6395 6399 (1981). 13 HosEY, M. M. Chick heart plasma membranes. Isolation and analysis of autophosphorylation. Biochim Biophys Acta 690, 106-116 (1982). 14 IWASA,Y., HosEY, M. M. Cholinergic antagonism of fl-adrenergic stimulation of cardiac membrane protein phosphorylation in situ. J Biol Chem 258, 4571 4575 (1983). 15 KIRCHBER6ER, M. A., ANTONETZ, T. Phospholamban: Dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 1 t,000 and 5,500 molecular weight forms. Biochem Biophys Res Commun 105, 152-156 (1982). 16 LAEMMLI,U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature [Lond] 227,680-685 (1970). 17 LAMERS,J. M.J., STINIS,J. T. Phosphorylation of low molecular weight proteins in purified preparations of rat heart sarcolemma and sarcoplasmic reticulum. Biochim Biophys Acta 624, 443459 (1980). 18 LASERS,J. M.J., STINIS, H. T., DEJoNGE, H. R. On the role of cyclic AMP and Ca2+-calmodulin-dependent phosphorylation in the control of (Ca 2+ + Mg2+)-ATPase of cardiac sarcolemma. FEBS Lett 127, 139-143 (1981). 19 LEUNG,E., JOHNSTON, C. I., WOODCOCK,E. A. Demonstration ofadenylate cyclase coupled adenosine receptors in guinea pig ventricular membranes. Biochem Biophys Res Commun 110, 208-215 (1983). 20 LINDEMANN,J. P., JONES, L. R., HATHAWAY,D. R., HENRY, B. G., WATANABE,A. M. ~-adrenergic stimulation of phospholamban phosphorylation and Ca 2 +-ATPase activity in guinea pig ventricles. J Biol Chem 2511, 464-471 (1983). 21 LONDOS,C., WOLFF,J. Two distinct adenosine-sensitive sites on adenylate cyclase. Proc Natl Acad Sci USA 74, 5482-5486 (1977). 22 LoNnos, C., COOPER, D. M. F., WOLFF,J. Subclasses of external adenosine receptors. Proc Natl Acad Sci USA 77, 2551-2554 (1980). 23 LONDOS,C., WOLFF,J., COOPER, D. M. F. Adenosine receptors and adenylate cyclase interactions. In Regulatory Function of Adenosine, R. M. Berne, T. W. Rall, R. Rubio Eds. pp. 17 30, The Hague: Martinus Nijhoff (1983). 24 Louis, C. F., MAFnTT, M.,JARVlS, B. Factors that modify the molecular size ofphospholamban, the 23,000 dalton cardiac sarcoplasmic reticulum phosphoprotein. J Biol Chem 257, 15182-15186 (1982). 25 MANALAN,A. S., JONES, L. R. Characterization of the intrinsic cAMP-dependent protein kinase activity and endogenous substrates in highly purified cardiac sarcolemmal vesicles. J Biol Chem 257, 10052-10062 (1982). 26 MARCUS, F., EDELSTEIN,I., REARDON, I.~ HEINRIKSON,R. L. Complete amino acid sequence of pig kidney fructose- 1,6-bisphos phatase. Proc Natl Aead Sci USA 79, 7161-7165 ( 1982). 27 MCMAHON, K. K., HOSEY, M. M. Potentiation of monovalent cation effects on ligand binding to cardiac muscarinic receptors in N-ethylmaleimide treated membranes. Biochem Biophys Res Commun 111, 41-46 (1983). 28 McKENzlE, J. E., STEFFEN, R. P., HADDY, F. J. Relationships between adenosine and coronary resistance in conscious exercising dogs. Am J Physio1242, H21 H29 (1982). 29 MUNSHI,R., BAER, H. P. Radioiodination of p-hydroxyphenyl isopropyladenosine: development of a new ligand for adenosine receptors. CanJ Physiol Pharmaco160, 1320-1322 (1982).

942

M . M . H o s e y et aL

30 MURPHY,K. M. M., SNYDER,S. H. Adenosine receptors in rat testes: labeling with 3H-cyclohexyladenosine. Life Sci 28, 917-920 ( 1981). 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

46 47 48 49

OSTERRmDER,W., BRUNN, G., HESCHELER,J., TRAUTWEtN, W., FLOCKERZI, V., HOt,ANN, F. Injection ofsubunits of cyclic AMP-dependent protein kinase into cardiac myocytes modulates Ca 2+ current. Nature 298, 576-578 (1982). REUTER,H. A. Properties of two inward membrane currents in the heart. Annu Rev Physio141,413-424 (1979). RINALDI,M. L., LEP~ucrI, C. J., DE~L~ILLE,J. G. The epinephrine-induced activation of the cardiac slow Ca z+ channels is mediated by the cAMP-dependent phosphorylation of calciductin, a 23,000 M, sareolemma protein. FEBS Lett 129, 277-281 (1981). SALO~ON,Y., LONOOS, C., ROVBELL, M. A highly sensitive adenylate cyclase assay. Anal Biochem 58, 541-548 (1974). SCHRAOER,J., BAUMANN,G., GERLACn, E. Adenosine as an inhibitor of myocardial effects of catecholamines. Pfluegers Arch 372, 29 35 (1977). SCH/iTZ, W., SCHRAnER,J,, GERLACH, E. Different sites of adenosine formation in the heart. Am J Physiol 240, H963-H970 (1981). SCnWABE, U., TROST, T. Characterization of adenosine receptors in rat brain by (-)[SH]N6phenylisopropyladenosine. Naunyn-Schmiedeberg's Arch Pharmaco1313, 179-187 (1980). SCHWABE, U., LENSCnOW, V., UKENA, D., FERRY, D. R., GLOSSMAN, H. [12Sl]N6-pHydroxyphenylisopropyladenosine, a new ligand for R i adenosine receptors. Naunyn-Schmiedeberg's Arch Pharmaco1321, 84 87 (1982). SMELUE, F. W., DAws, C. W., DALY, J: W., WELLS, J. N. Alkylxanthines: Inhibition of adenosine-elicited accumulation of cyclic AMP in brain slices and of brain phosphodiesterase activity. Life Sci 2'1, 2475-2482 (1979). STANLE'Z,P. E., WILLIAMS, S. G. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem 29,381-392 / 1969). TADA,M., KIRCHBERGER,M. A., REPKE, D: I., KATZ, A. M. The stimulation of calcium transport in cardiac sarcoplasmic reticulum by adenosine 3!-5'-monophosphate-dependent protein kinase. J Biol Chem 249, 6174-6180 (1979). TADA, M., KmCrmEROER, M. A., KArZ, A. M. Phosphorylation of a 22,000 dalton component of the cardiac sarcoplasmic reticulum by adenosine 3'-5'-monopnosphate-dependent protein kinase. J Biol Chem 250, 2640-2647 (1975). TROST,T., SCnWABE, U. Adenosine receptors in fat cells. Identification by (-)-N6-[3H]phenylisopropyladenosine binding. Mol Pharmaco119, 228235 (1981). VOOEL, S., SPERELAKIS, N. Induction of slow action potentials by microiontophoresis of cyclic AMP into heart cells.J Mol Cell Cardiol 13, 5t-64 (1981). WATANASE,A. M., LXNDEMAN,J.P.,JoNES, L. R., Bmcn, H. R., BAtLEY,J. C. Biochemical mechanisms mediating neural control of the heart. In Disturbances in Neurogenic Control of the Circulation, F. M. Abboud, H. A. Fozzard,J. P. Gilmore, D.J. Reis, pp. 189 203. Bethesda.. WEILAND,G. A., MOUNOEV,P. B: Quantitative analysis of drug-receptor interactions: 1. Determination of kinetic and equilibrium properties. Life Sci 29, 313-330 ( 1981 ). WmHAMS, M., RISLEV, E. A. Biochemical characterization of putative central purinergic receptors by using 2-chloro[3H] adenosine, a stable analog of adenosine. Proc Natl Acad Sci USA 77, 6892-6896 (1980). WOLFf, J., LoNDos, C., CooK, G. H. Adenosine interactions with thyroid adenylate cyclase. Arch Biochem Biophys 191,161-168 (1978). YEUNG,S.-M. H., GREEN, R. D. Agonist and antagonist affinities for inhibitory adenosine receptors are reciprocally affected by 5'-guanylylimidodiphosphate or N-ethylmaleimide. J Biol Chem 258, 2334-2339 (1983).