Journal of Neurochernisiry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites Peter C. G. Nijssen, Tammy Sexton, and Steven R. Childers Department of Physiology and Pharmacology, Bowman Gray School oj’hfedicine, Wake Forest University, Winston-Salem, North Carolina, U.S.A.

Abstract: Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat stnatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone),irreversible antagonists [naloxonazine, j3-funaltrexamine, and j3chlornaltrexamine (8-CNA)], and phospholipase A,. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled ~alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (p), ’H-labeled 2-~serine-5-~-leucine-6-~-threonine enkephalin (6), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity p, 6, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the ICm and the maximal inhibition of adeny-

lyl cyclase by opioid agonists were identical to values in untreated membranes. Only j3-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the ICsoof opioid agonists. This effect of j3-CNA was not due to nonspecific interactions with Gi, G,, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by 8-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity p, 6, or EKC sites identified in brain by traditional binding studies. Key Words: Opioid receptor-Adenylyl cyclase-GTP-binding proteins-Cyclic AMP. Nijssen P. C. G. et al. Opioid-inhibited adenylyl cyclase in rat brain membranes: Lack ofcorrelation with high-affinity opioid receptor binding sites. J. Neurochem. 59,225 1-2262 (1992).

Although opioid receptor binding sites have been well characterized in brain since 1973, the effector systems coupled to these receptors to produce their responses are less well understood. The best-characterized effector system is opioid-inhibited adenylyl cyclase (Collier and Roy, 1974; Sharma et al., 1975; Traber et al., 1975). This reaction is representative of the inhibitory class of adenylyl cyclase-linked receptors (Neer and Clapham, 1988; Birnbaumer et al., 1990): The reaction requires both sodium and GTP (Blume et al., 1979), and it is attenuated by pertussis toxin (Hsia et al., 1984; Abood et al., 1987). The three

major pharmacological types of opioid receptors inhibit adenylyl cyclase; these are 6 (Sharma et al., 1975; Traber et al., 1975), p (Frey and Kebabian, 1984; Yu et al., 1986; 1990), and K (Attali et al., 1989; Konkoy and Childers, 1989). Although the initial discovery of opioid-inhibited adenylyl cyclase occurred in brain membranes (Collier and Roy, 1974), much of the information about this second-messenger system has come from transformed cell lines, most frequently 6 receptors in NG108-15 neuroblastoma X glioma hybrid cells (Sharma et al., 1975; Traber et al., 1975; Blume et al.,

Received November 26, 199 1 ; revised manuscript received May 6, 1992; accepted May 30, 1992. Address correspondence and reprint requests to Dr. S. R. Childers at Department of Physiology and Pharmacology, Bowman Grey School of Medicine, Wake Forest University, 300 South Hawthorne Road, Winston-Salem, NC 27 103, U.S.A. Abbreviufionswed: CAMP, cyclic A M P p-CNA, P-chlornaltrex-

arnine; D-Ala enk, 2-~-alanine-rnethionine enkephalinarnide; DAMGO, ~alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin; DSLET, 2-~-senne-5-~-leucine-6-~-threonine enkephalin; EKC, ethylketocyclazocine; (3-FNA, (3-funaltrexarnine; G, and Gi, stirnulatory and inhibitory GTP binding regulatory proteins of aden ylyl cyclase, respectively; GTP-y-S, guanosine 5’-0-(3-thiotriphosphate); OPNH, oxyrnorphone-pnitrophen ylhydrazone.

225 I

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P. C. G. NIJSSEN ET AL.

1979) and p receptors in SK-N-SH neuroblastoma cells (Yu et al., 1986, 1990) and 7315c tumor cells (Frey and Kebabian, 1984; Puttfarcken and Cox, 1989). However, even these model systems provide complex relationships between receptor binding and opioid-inhibited adenylyl cyclase (Law et al., 1985). One study (Costa et al., 1985) demonstrated the existence of several receptor states in NG108-15 cells, only one of which was coupled to adenylyl cyclase, whereas another study (Fantozzi et al., 198 1) showed that irreversible blockade of 95% of receptor binding sites in NG108-15 cells did not affect 6 opioid-inhibited adenylyl cyclase. One important question that cannot be answered in transformed cell lines is the identity of the opioid receptor types coupled to adenylyl cyclase in normal, nontransformed brain tissue. Several studies demonstrated opioid-inhibited adenylyl cyclase in brain membranes (Collier and Roy, 1974; Law et al., 198 1; Cooper et al., 1982; Beitner et al., 1989) but the pharmacological characteristics of this response were difficult to quantitate because of the low (10-20%) inhibition evident in brain membranes. In our laboratory, studies of opioid-inhibited adenylyl cyclase have focused on procedures which modify the interaction of both Gila and G, proteins with receptors and effectors. One such technique involves preincubation of brain membranes at pH 4.5 before assay of receptor binding and adenylyl cyclase at pH 7.4 (Childers and LaRiviere, 1984). The effect of low-pH pretreatment was to change the function of guanine nucleotide-binding proteins in brain membranes by (1) increasing GTP and NaCl regulation of opioid agonist binding without affecting binding in the absence of GTP or NaCl (Lambert and Childers, 1984) and (2) eliminating G,stimulated adenylyl cyclase, while simultaneously increasing opioid-inhibited adenylyl cyclase (Childers and LaRiviere, 1984; Rasenick and Childers, 1989). In low-pH-pretreated brain membranes, opioid agonists inhibited adenylyl cyclase by a maximum of 3040% of basal activity (Childers, 1988; Konkoy and Childers, 1989). The present study employs this increased signal of receptor-inhibited adenylyl cyclase to explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase. This study utilizes several agents to block specific opioid receptor binding sites, including (1) naloxonazine, which at low concentrations irreversibly blocks p , sites (Wolozin and Pasternak, 1981); (2) P-funaltrexamine (P-FNA), which blocks traditional p sites (Takemon et al., 1981; Ward et al., 1982); and (3) 8-chlornaltrexamine (P-CNA), which blocks several subtypes of opioid receptors (Ward et al., 1982). Other agents include the irreversible agonist oxymorphone-p-nitrophenylhydrazone(OPNH) (Hahn et al., 1985) and phospholipase A,, which inhibits agonist binding at low concentrations (Pasternak and Snyder, 1974). These experiments explore the effect of specific

J. Neurochem.. Vol. 59, No.6, 1992

receptor blockade on opioid-inhibited adenylyl cyclase in brain membranes. MATERIALS AND METHODS Materials Labeled ligands for receptor binding studies, 3H-labeled ~-serine-5-~-leucine-6-~-threonine enkephalin (['HIDSLET; 34.5 Ci/mmol), [3H]ethylketocyclazocine (EKC; 15.4 Ci/mmol), and 'H-labeled ~alanine-4-N-methylphenylalanine-5-glycine-01-enkephalin ( ['HIDAMGO; 32.3 Ci/ mmol) were purchased from New England Nuclear Corp. (Boston, MA, U.S.A.). ['HJATP (31 Ci/mmol) was o b tained from ICN Radiochemicals(Irvine, CA, U.S.A.). Unlabeled peptides,including2-~-alanine-methionineenkephalinamide (D-Ala enk), DSLET, and DAMGO were purchased from Peninsula Laboratories (Belmont, CA, U.S.A.).0-CNA and P-FNA were purchased from Research Biochemicals, Inc. (Wayland, MA, U.S.A.). Opioid hydrazone derivatives, including naloxonazine and OPNH, were kind gifts from Dr. Gavril Pastemak. Unlabeled nucleotides, creatine phosphate, creatine phosphokinase, and adenosine deaminase were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Sodium acetate and methanol were HPLC-grade reagents from Fisher (Orlando, FL, U.S.A.). All other chemicals were reagent-grade materials. Adenylyl cyclase assay Opioid-inhibited adenylyl cyclase was assayed by the lowpH pretreatment procedure as described previously (Childersand LaRiviere, 1984;Childers, 1988).Striata were removed from male Sprague-Dawley rats (150-200 g) and homogenized in Tris/MgZfbuffer (50 mMTris-HCl, 3 mM MgCI,, pH 7.4) with a Polytron homogenizer.After centrifugation at 48,000 g for 10 min, the pellet was resuspended in 1 ml of pH 4.5 buffer (50 mM sodium acetate, 5 mM MgCI,, 1 mM dithiothreitol, 1 mM EGTA, pH 4.5)and incubated with occasional mixing at 0°C for 20 min. The membranes were then diluted with a fivefold excess of Tris/ MgZf buffer, centrifuged, and resuspended in fresh Tris/ Mg2+buffer. Membranes (20- 100 pg of protein) were added to tubes containing 20 mM creatine phosphate, 10 U of creatine phosphokinase, 10 mMtheophylline, 30 p A 4 cyclic AMP(cAMP),100 pMATP, and 1 pCi of [3H]ATPtogether with various drug additions to a final volume of 100 pl. When opioid-inhibited adenylyl cyclase was assayed, the tubes contained 100 mMNaCl and 50 p M GTP in addition to various concentrations of opioid agonists and/or antagonists. Reactions were initiated by the addition of ATP, and tubes were incubated at 30°C for 10 min and then immersed in boiling water for 2 min. Enzyme blanks consisted of the same materials incubated in the presence of brain membranes that had been boiled for 2 min before the addition of ATP. All tubes were cooled on ice for 5 min, then 0.75 U of adenosine deaminase was added, and the tubes were incubated at 30°C for 5 min and placed on ice. The remaining ATP was precipitated by the sequential addition of 150 pl of 1 MBa(OH), followed by 150 pl of 1 MZnSO,, with 5 min between each addition. The supernatant was prepared by centrifugation of assay tubes at 1,000 g for 15 min. [3H]CyclicAMP formed by adenylyl cyclase was assayed by an HPLC procedure (Childers, 1986)consisting of a 3-pn Microsorb C- 18 column (Rainin Instruments),with

OPIOID RECEPTORS AND BRAIN ADENYLYL CYCLASE 0.8 Msodium acetate, pH 5.0, containing 10%methanol as the mobile phase. Samples (250-pl volume) were injected by an automatic injector 6 min apart, and the ['Hlcyclic AMP peak eluting approximately 4 min after injection was detected and collected by ultraviolet absorbance of the unlabeled cyclic AMP in the sample, which triggered a fraction collector through an automatic peak detector (1x0, Lincoln, NE, U.S.A.). The [3H]cyclic AMP (volume, approx. 0.6 ml) was collected in a scintillation vial, and radioactivity was determined by liquid scintillation spectrophotometry at 45% efficiency after the addition of 5 ml of Liquiscint scintillation fluid (National Diagnostics, S o m e d e , NJ, U.S.A.).

Opioid receptor binding In experiments comparing adenylyl cyclase with receptor binding, both assays were conducted in the same membrane preparation. After treatment with the various agents described below, striatal membrane preparations were split in half and centrifuged at 48,000 g for 10 min. One tube of membranes was resuspended in Tris/MgZ*buffer for assay of adenylyl cyclase as described above. The other tube was resuspended in 50 mMTris-Ha, pH 7.7, for receptor binding assays (Childers and Snyder, 1980). Membranes (1.9 ml, containing approximately 0.9 mg of protein) were added to tubes containing 1 nM labeled ligands. Ligands included [3H]DSLET for direct assay of 6 sites, ['HIDAMGO for direct assay ofp sites, and ['HIEKC in the presence of 100 nM unlabeled DSLET and DAMGO for assay Of K sites. Because it is not yet established that ['HIEKC sites in the presence of unlabeled DAMGO and DSLET bind to genuine K sites, these sites are termed EKC sites. Nonspecific binding was defined as the difference between total binding and binding in the presence of 1 pMlevallorphan. Tubes were incubated at 25°C for 40 min, and reactions were terminated by rapid filtration through Whatman GF/B glass-fiber filters. Bound radioactivity was determined after overnight extraction of the filters in 8 ml of Liquiscint scintillation fluid.

Membrane treatments For assay of phospholipase A, effects on brain adenylyl cyclase and opioid receptor binding, a modification of the procedure of Pasternak and Snyder (1974) was utilized. Membranes were prepared from rat striatum through the low-pH-pretreatment step as described above. After centrifugation of membranes, membranes were resuspended in 1 ml of Tris/Mg2+buffer containing 1 mMCaCl,, and phospholipase A, (100 ng/ml) was added. The tubes were incubated at 25°C for 20 min, and the reaction was terminated by the addition of 5 mM EGTA. Following centrifugation, membranes were resuspended in either Tris buffer for receptor binding assays or Tris/Mg2+ buffer for adenylyl cyclase assays. In experiments using irreversible opioids, the method of Childers and Pasternak (1 982) was employed. Striatal membranes, prepared up to the low-pH-pretreatment step, were resuspended in 1 ml of fresh Tris/Mg2+ buffer and incubated at 25°C for 20 min with various concentrations (0.1 to 10 p M ) of irreversible opioids, including naloxonazine, OPNH, P-FNA, and P-CNA. Control tubes contained either buffer alone or equal concentrations of naloxone to control for washing out reversible components of the drugs. After incubation, 15 ml of fresh buffer was added to the tubes, and after centrifugation, membranes were resuspended in pH 4.5 buffer for low-pH pretreatment as described above.

2253

After one further centrifugation, membranes were resuspended in either Tris buffer or Tris/Mg2+ buffer for assay of receptor binding or adenylyl cyclase as described above.

Data analysis Unless indicated otherwise, results are data from triplicate determinations in representative experiments that were repeated at least three times. The standard deviation oftriplicate determinations was less than 6%. To analyze IC, values of agonist inhibition curves in adenylyl cyclase assays, maximum efficacy was calculated by determination of the agonist concentration required to reach maximal inhibition. ICso values were calculated by computer analysis of log-logit data; all curves exhibited Hill slopes not significantly different from unity.

RESULTS Agonist/antagonist properties of irreversible opioids on brain adenylyl cyclase Opioid-inhibited adenylyl cyclase can be quantitated in striatal membranes when the membranes are pretreated at pH 4.5 before assay of activity at pH 7.4. Under these conditions, opioid agonists produce 3040% maximal inhibition at micromolar concentrations (Childers and LaRiviere, 1984; Childers, 1988; Konkoy and Childers, 1989). Because one of the major goals of these studies was to block opioid receptor binding sites by incubation of striatal membranes with irreversible opioids, experiments were first designed to test whether these compounds had ago&/ antagonist properties in the brain opioid-inhibited adenylyl cyclase system. One compound, OPNH, has been utilized in other studies as an irreversible agonist, with long-acting agonist properties in vivo and persistent blockade of receptor binding in brain membranes after multiple wash steps in vitro (Hahn et al., 1985). Figure 1 compares the inhibition of striatal adenylyl cyclase by two peptide opioid agonists,

1

0

Concentration Agonist (pM)

FIG. 1. Inhibition of adenylyl cyclase in low-pH-pretreated rat striatal membranes by DSLET, DAMGO. and OPNH. Compounds, at 0.1-50 IJ\n concentrations, were incubated with striatal membranes during the adenylyl cyclase assay as described in Materials and Methods. Data are expressed as percentage of basal activity, which was 83 pmol of cAMP/min/rng protein.

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DSLET (binding to 6 sites) and DAMGO (binding to sites), with inhibition produced by OPNH. All of these compounds produced significant inhibition of adenylyl cyclase in low-pH-pretreated membranes, with maximal inhibition between 30 and 35% ofbasal activity. Of the three compounds tested, DSLET was most potent, with an IC,, value of -0.7 pM. The other opioids were less potent, both showing IC,, values of -3 p M . This inhibition was mediated through opioid receptors in all cases, as 1 pM naloxone blocked the inhibition of 5 pM DSLET and 20 pM DAMGO and OPNH (data not shown). Several other compounds were tested for antagonist properties. Figure 2 compares the effect of various concentrations of naloxone and naloxonazine on adenylyl cyclase inhibited by 10 pMD-Ala enk (a concentration of agonist that produces maximal inhibition of adenylyl cyclase). Both compounds blocked agonist inhibition, with full blockade occumng at 1 p M concentrations, although naloxonazine was slightly less potent than naloxone. Neither compound alone had any effects on adenylyl cyclase when assayed at concentrations up to 10 p M (data not shown). Another method of examining antagonist properties of different compounds is to calculate effects of antagonists on agonist IC,, values. The results in Table 1 show that all of the compounds tested were effective antagonists, with 0.5 pM concentrations of every compound providing at least sevenfold shifts in agonist dose-response curves. The K, values of these compounds were all within the same order of magnitude, ranging from 40 to 80 nM (Table 1). p

-

Irreversible actions on adenylyl cyclase and opioid receptor binding To compare the effects of irreversible opioids on both opioid receptor binding sites and opioid-inhib-

.01

.I

1

Concentratlon Antagonist

10

100

(pM)

FIG. 2. Blockade of opioid-inhibited adenylyl cyclase in striatal membranes by opioid antagonists. Adenylyl cyclase was assayed in low-pH-pretreatedmembranes in the presence of 10 p M D-Ala enk with various concentrations (0.1-10 f l )of naloxone and naloxonazine. Data are expressed as percentage of basal activity, which was 72 pmol of cAMP/min/mg of protein.

J. Neurochem.. Vol. 59. No. 6, 1992

TABLE 1. Efects of opioid antagonists on 0-Ala enkinhibited adenylyl cyclase Drug

None Naloxone Naloxonazine 0-CNA 8-FNA

DAIa enk IC, 0.7 -t 0.2 8.1 1.6 6.3 f 1.4 9.9 2.0 5.2 k 0.9

* *

(pM)

Shift ratio 11 9

14 7

K. (nM) 50 62 38 83

Adenylyl cyclase was assayed in low-pH-pretreated membranes from rat striatum as described in Materials and Methods, and agonist dose-response curves for inhibition of activity were obtained using 0.05 to 50 pMD-Ala enk in the presence of 0.5pMantagonist. ICso values (mean t SEM of three determinations) were calculated as the concentration of DAIa enk that produced half of the maximal inhibition level of adenylyl cyclase. Maximal inhibition levels were 36 6% inhibition of basal activity (75 pmol of cAMP/min/ mg of protein) and were not significantlychanged by the addition of antagonists.

*

ited adenylyl cyclase, striatal membranes were incubated with various concentrations of different compounds, washed and preincubated at pH 4.5 to remove reversibly acting components of the drugs, and split in half so that the same preparation of membranes was assayed for opioid receptor binding and opioid-inhibited adenylyl cyclase. To control for washing effectiveness, equal concentrations of naloxone were added to some membranes and incubated and washed under the same conditions as membranes treated with irreversible agents. In each experiment, naloxone-incubated membranes displayed the same receptor binding as buffer-treated controls (data not shown). Therefore, the wash steps were sufficient to eliminate reversibly acting opioids. However, these steps, which were necessary to remove reversibly acting drugs, did eliminate some of the opioid-inhibited adenylyl cyclase. In untreated membranes, assayed after washing and preincubations, maximal inhibition levels by D-Ala enk were -25%, compared with the 35% maximal inhibition observed in unwashed membranes. Table 2 shows the results with the irreversible agonist OPNH. Incubation of membranes with 10 pM OPNH resulted in a profound loss of receptor binding sites, with 99% loss of p sites, 80% loss of 6 sites, and 85% loss of EKC sites. Interestingly, although 10 p A 4 OPNH produced significant inhibition of adenylyl cyclase as an agonist (Fig. 1) after OPNH-treated membranes were washed, no persistent inhibition of adenylyl cyclase was observed, as basal adenylyl cyclase activity was unchanged by pretreatment with OPNH (Table 2). Moreover, adenylyl cyclase continued to be inhibited by 10 p M D-Ala enk to a level (25% inhibition) similar to the maximal inhibition seen in untreated membranes, and the inhibition produced by D-Ala enk was blocked by 1 pA4 naloxone (Table 2). Therefore, OPNH did not act as an irreversible ago-

OPIOID RECEPTORS AND BRAIN ADENYLYL CYCLASE

2255

TABLE 2. Effect of OPNH on opioid receptor binding and opioid-inhibited adenvivi cvclase in rat striatal membranes A. Receptor binding Control

Treated

Ligand

cpm bound

% control

cpm bound

% control

['HIDAMGO [3H]DSLET ['HJEKC

3,168 f 221 3,169 f 155 872 f 66

100 100

27 f 58 619 f 79 131 f 52

20 15

I00

1

B. Adenylyl cyclase (% basal activity) Additions

Control

Treated

None ~ - A l aenk ~ A l enk, a naloxone

100 r 5 74 f 7 94 f 8

101 f 8 76 f 4 96 f 5

Striatal membranes were incubated with 10 p M OPNH, washed, pretreated at pH 4.5, and split into two parts for assay of receptor binding and opioid-inhibited adenylyl cyclase as described in Materials and Methods. Receptor assays using ['HIEKC included 100 pM DAMGO and DSLET to block p and b sites. Opioid-inhibited adenylyl cyclase was measured with 10 pMDAla enk in the presence and absence of 1 pMnaloxone; data are precentage of basal activity (62 pmol of cAMP/min/mg of protein). Values are the means 2 SEM of three experiments.

nist with regard to opioid inhibition of adenylyl cyclase, despite the profound loss ofp, 6,and EKC binding sites. Table 3 shows the results of incubation of striatal membranes with 10 pM naloxonazine. As described in the previous experiment, the same membranes were assayed for both receptor binding and opioid-in-

hibited adenylyl cyclase. Incubation of membranes with naloxonazine produced 98% loss of p sites, 75% loss of 6 sites, and 80% loss of EKC sites. Although 10 pA4 naloxonazine is more than sufficient to block the inhibition of adenylyl cyclase caused by 10 pMDAla enk (see Fig. 2), 10 pA4 DAla enk inhibited adenylyl cyclase to the same maximal inhibition level (approx.

TABLE 3. Effect of naloxonazine on opioid receptor binding and opioid-inhibited adenylyl cyclase in rat striatal membranes A. Receptor binding Control

Treated

Ligand

cpm bound

% control

cpm bound

% control

['HIDAMGO ['HIDSLET [3H]EKC

3,479 f 201 2,993 f 132 546 f 63

100 100 I00

82 f 61 742 f 98 99 '' 57

2 25 18

B. Adenylyl cyclase (% basal activity) Additions

Control

Treated

None D-Ala enk D-Ala enk. naloxone

100f7 76 k 6 95 f 8

96 + 4 74 4 92 ? 7

*

Striatal membranes were incubated with 10 p M naloxonazine, washed, pretreated at pH 4.5, and split into two parts for assay ofreceptor binding and opioid-inhibited adenylyl cyclase as described in Materials and Methods. Receptor assays using ['HIEKC included 100 nM DAMGO and DSLET to block p and b sites. Opioid-inhibited adenylyl cyclase was measured with 10 pM D-Ala enk in the presence and absence of 1 p M naloxone. Data represent mean values k SEM of three experiments.

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TABLE 4. Efect ofp-CNA on opioid receptor binding and opioid-inhibited adenylyl cyclase in rat striatal membranes A. Receptor binding cpm bound at j3-CNA concentration (& ofI) Ligand

0

0.1

0.5

1.o

['HIDAMGO

2,676 f 154 1,860 f 98 592 f 69 2,358 134

1,955 f 177 (73%) 1,655 f 122 (89%) 503 f 78 (85%) 1,784 f 101 (76%)

1,151 f 72 (56%) 1,525 ? 56 (82%) 415 f 44 (71%) 1,303 f 99 (55%)

905 f 81 (33%) 1,321 f 72 (71%) 366 f 33 (62%) 998 f 88 (41%)

[ 'HIDSLET

['HIEKC ['HINaloxone

*

B. Adenylyl cyclase (% basal activity) % at j3-CNA concentration (&

of

~

0

0. I

0.5

I .o

D-Ma enk

100 f 4 74 f 3

GTP-7-S Forskolin NaF

80 7 224 f 18 256 f 29

108 f 9 79 1- 8 82 f 4 233 f 31 287 f 15

97 f 6 81 1-4 83 f 5 201 f 22 263 f 21

I04 t 5 89 f 3 81 f 6 241 f 19 241 f 12

Additions ~

None

*

Striatal membranes were incubated with jf-CNA, washed, pretreated at pH 4.5, and split into two parts for assay of receptor binding and adenylyl cyclase as described in Materials and Methods. Concentrations of drugs were 10 pMDala enk, 1 N G T P - r - S , and 10 p M forskolin. In a separate experiment, fluoridestimulated adenylyl cyclase (10 mM NaF) was assayed in membranes before low-pH pretreatment, and results are expressed as percentage of basal activity (69 pmol of cAMP/min/mg of protein) in those membranes. Binding data are expressed as specific counts per minute bound, with percentage of control (no PCNA) in parentheses. Adenylyl cyclase data are percentage of basal activity (8 I pmol of cAMP/min/mg of protein). Values are the means f SEM of three experiments.

25%) in treated membranes as in untreated controls. The same findings were observed for 10 p A 4 P-FNA (data not shown). Different results were obtained, however, using another irreversible antagonist, @-CNA.Incubation of membranes with 1 pA4 P-CNA produced a time-dependent decrease in receptor binding that reached maximal levels by 15 min of preincubation (data not shown). This concentration of P-CNA significantly inhibited high-affinity binding for all three ligands, with 67% loss of p sites, 29% loss of 6 sites, and 38% loss of EKC sites (Table 4). Lower concentrations of 8-CNA produced lesser effects on receptor binding (data not shown). Scatchard analysis of high-affinity p and 6 binding was performed after pretreatment of brain membranes with 100 nM P-CNA (Fig. 3). For ['HIDAMGO binding, the K , was 0.78 f 0.16 nMin naltrexone-pretreated membranes and 0.94 f 0.15 nM in 8-CNA-pretreated membranes, whereas the B,,, of ['HIDAMGO decreased from 0.22 k 0.09 to 0.073 f 0.006 pmol/mg of protein in P-CNA-pretreated membranes (a 67% decrease; mean values t SEM from three experiments). For ['HIDSLET binding, the K , was 2.0 -+ 0.2 nM in naltrexone-pretreated membranes and 2.1 f 0.4 nM in P-CNA-pretreated membranes, whereas the B,,, of ['HIDSLET decreased from 0.20 k 0.03 to 0.1 1 f 0.02 pmol/mg of protein in P-CNA-pretreated membranes (a 44% decrease; mean values SEM from three experiments). These data confirmed that the effect of P-CNA was to

. I Neurochem.. . Vol. 59. No. 6, 1992

inactivate receptor binding sites, not reduce the affinities of existing sites. This inactivation was again demonstrated by showing that the addition of I pA4 DAMGO or DSLET to membranes after 0-CNA pretreatment, followed by the standard low-pH pretreatment and washing steps, had no effect on the inactivation of binding by P-CNA (data not shown). Despite the fact that P-CNA produced smaller effects on receptor binding than the other agents de0.05

0.04

I

J

I

\

't

0 DSLET-CNA

O DSLET+CNA

A DAMGO-CNA A DAMGO+-

%

Bound, prnoleshng protein

FIG. 3. Scatchard analysis of high-affinity p and 6 opioid receptor binding sites after treatment with 0-CNA. Rat brain membranes were preincubated with 100 nM j3-CNA for 15 min at 25°C then washed and assayed for [3H]DAMG0 and r3H]DSLET binding as described in Materials and Methods. Saturation experiments were performed using 0.1-6 nM concentrations of each labeled lgand.

OPIOID RECEPTORS AND BRAIN ADENYL YL CYCLASE scribed above, it blocked at least a portion of opioidinhibited adenylyl cyclase after washing of membranes (Table 4). Although 0.1 pM P-CNA did not significantly reduce maximal inhibition by 10 pM D Ala enk, higher concentrations of P-CNA (0.5 and 1 p M ) blocked approximately one-half of the DAla enk-induced inhibition. Interestingly, this blockade of agonist inhibition occurred with P-CNA at a concentration (1 p M ) that produced less receptor binding blockade than seen earlier with 10 p M naloxonazine, OPNH, and P-FNA. It is important to note that 8CNA had no effect on either basal or forskolin-stimulated adenylyl cyclase in low-pH-pretreated membranes or fluoride-stimulated adenylyl cyclase in pH 7.4-pretreated membranes (Table 4). Thus, the actions of P-CNA on opioid-inhibited adenylyl cyclase were not caused by nonspecific effects of this compound either on the catalytic unit of adenylyl cyclase or on G,-stimulated activity. To determine whether 0-CNA had any nonspecific effects on Gi function, adenylyl cyclase activity was assayed in low pH-pretreated membranes with 1 p M guanosine 5'-0-(3-thiotnphosphate)(GTP-7-S). Previous results (Hatta et al., 1986; Rasenick and Childers, 1989) showed that low concentrations of nonhydrolyzable guanine nucleotides inhibit, and high concentrations stimulate, adenylyl cyclase. Our studies (not shown) revealed similar results, with up to I pLM GTP-7-S inhibiting approximately 20% of activity and higher ( 10-50 p M ) concentrations stimulating activity up to threefold in untreated mem-

225 7

branes. In low-pH-pretreated membranes, maximal inhibition by GTP-7-S was still seen at 1 p M , but higher concentrations failed to stimulate activity over basal levels. This is consistent with the loss of G,-stimulated adenylyl cyclase in low-pH-pretreated membranes (Childers and LaRiviere, 1984; Rasenick and Childers, 1989). The addition of j3-CNA to low-pHpretreated membranes (Table 4) had no effect on the 18-20% inhibition of adenylyl cyclase by 1 pMGTP73,suggesting that the loss in opioid-inhibited adenylyl cyclase by P-CNA was due to loss of receptors, and not Gi proteins. Effect of phospholipase A, on opioid receptor binding and opioid-inhibited adenylyl cyclase Phospholipase A, inhibits opioid agonist binding (Pasternak and Snyder, 1974) but does not inhibit basal adenylyl cyclase activity in brain (Lad et al., 1979). To compare the effects of phospholipase A, on opioid receptor binding and opioid-inhibited adenylyl cyclase, membranes were preincubated with 100 ngml of phospholipase A, before assay of receptor binding and adenylyl cyclase in the same membrane preparations. The results (Table 5) showed that phospholipase A, produced 97% loss of p sites, 100% loss of 6 sites, and 93% loss of EKC sites. Despite this profound loss of opioid receptor binding, there was no significant effect of phospholipase A, on opioid-inhib ited adenylyl cyclase. Although the enzyme did stimulate basal adenylyl cyclase activity to a small degree (1 8%), similar to effects reported previously (Lad et

TABLE 5 . Eflect of phospholipase A , on opioid receptor binding and opioid-inhibitedadenylyl cyclase in rat striatal membranes A. Receptor binding

Control

Treated

Ligand

cpm bound

% control

[ 'HIDAMGO ['HIDSLET

3,478 f 103 3,319 f 141 612 f 83

100 100 100

['HIEKC

cpm bound 99 12

% control

* 71 * 59

3 0

I

42 f 66

B. Adenylyl cyclase (% basal activity) Additions

Control

Treated

None D-Ala enk DAla enk, naloxone

10026 71 _ + 4 97 4

118+9(100) 88 f 5 (74) 117 6 (99)

*

*

Striatal membranes were incubated with phospholipase A, (100 ng/ml), washed, pretreated at pH 4.5, and split into two parts for assay of redeptor binding and opioid-inhibited adenylyl cyclase as described in Materials and Methods. Opioid-inhibited adenylyl D-Ala enk in the presence and absence of I p M cyclase was measured with 10 ~ L M naloxone. For adenylyl cyclase data, numbers in parentheses are data corrected for the 18% stimulation of basal activity by phospholipase A,; data are percentage of basal activity (69 pmol of cAMP/rnin/mg of protein). Values are the means f SEM of three experiments.

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al., 1979), 10 pM DAla enk produced the same degree of inhibition (26%)as in untreated membranes. Moreover, the inhibition by DAla enk was blocked by l pA4 naloxone; thus, the inhibition occurred through opioid receptors. Previous studies (Lin and Simon, 1978) showed that the effects of phospholipase A, were reversed by the addition of defatted proteins (e.g., bovine serum albumin) to absorb lysolecithins generated by the enzymatic reaction. Thus, one explanation of these results is that high concentrations of protein in the adenylyl cyclase assay (namely, the creatine phosphokinase regeneration system) would reverse the actions of the enzyme in that assay. To control this possibility, the experiment above was repeated using 100 ng/ ml phospholipase A,, then adding creatine phosphate/creatine phosphokinase to the receptor binding assay at concentrations equivalent to those used in the adenylyl cyclase assay. Results (not shown) revealed that phospholipase A, decreased [3H]DSLETbinding by 95% in normal membranes and by 92% in membranes with creatine phosphate/creatine phosphokinase. In the same membranes, 10 p M D-Ala enk continued to inhibit adenylyl cyclase by 23%, as in untreated membranes. Therefore, the lack of effect of phospholipase A, on opioid-inhibited adenylyl cyclase is not an artifact of the assay. Effect of treatments on opioid agonist inhibition curves One explanation for the lack of correlation between opioid receptor binding and opioid-inhibited adenylyl cyclase is the phenomenon of spare receptors. If there were a large population of spare receptors in brain membranes, then blockade of even 99.9% of receptor binding sites might not affect maximal inhibi-

0 Control

0 P-llpse A

.1

1

10

100

Concentratlon D-ala enk (pM)

FIG. 4. Effect of preincubation of striatal membranes with phospholipase A2, 0-CNA, or buffer control on D-Ala enk-inhibited adenylyl cyclase. Membranes were preincubated with 1 p M 0CNA or 100 ng/ml phospholipaseA2, washed, treated at pH 4.5, and assayed for adenylyl cyclase activity in the presence of 0.150 CJM o-Ala enk. Data are expressed as percentage of basal activity, which was 69 pmol of cAMP/min/mg of protein.

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TABLE 6 . Efect of P-CNA on opioid-inhibited adenylyl cyclase in rat striatal membranes 8-CNA concentration (PM)

0 0.1 0.5

I .o

~ A l enk a ICSO (04

Maximal inhibition (W)

0.82 ? 0.46 4.9 -+ 2.3 6.1 t 3.4 4.8 ? 1.9

26 a 5 21 + 4 19?7 1 1 a5

~

Membranes were incubated with P-CNA and pretreated at pH 4.5 as described in the footnote to Table 4. Adenylyl cyclase was assayed in the presence of 0.05-50 pA4 BAla enk; the IC, values were calculated as the concentration of DAla enk that produced half-maximal inhibition. Maximal inhibition data are the maximal percentage inhibition of basal activity (70 pmol of cAMP/min/mg of protein) produced by 10 p M DAla enk. Data represent mean values ? SEM of three experiments.

tion of adenylyl cyclase by opioid agonists. However, if spare receptors did exist, then blockade of receptor binding sites would cause a rightward shift in the agonist concentration-effect curves. Maximal inhibition levels would not be changed until the population of spare receptors was completely blocked. To test this hypothesis, membranes were treated with either 100 ng/ml phospholipase A, or 1 pM P-CNA, and adenylyl cyclase was assayed in the presence of 0.1-50 p M D-Ala enk to generate agonist inhibition curves. The results (Fig. 4)show different effects of the two treatments. Treatment with phospholipase A, produced no significant change in the agonist inhibition curve, with the same IC,, value and maximal inhibition caused by DAla enk. However, incubation with PCNA produced significant effects on the inhibition curve, increasing the IC,, value for DAla enk as well as decreasing the maximal inhibition produced by DAla enk. These results are supported by examining the effect of several concentrations of @-CNAon DAla enk inhibition curves (Table 6). A low (0.1 p M ) concentration of @-CNAhad no significant effect on the maximal inhibition produced by DAla enk but did increase the IC,, value for DAla enk by approximately sixfold. Increasing the concentration of PCNA had no further effect on the IC,, values but did decrease the maximal inhibition, with only 1 1% inhibition by DAla enk occurring after incubation with 1 p M @-CNA.These results are consistent with the spare receptor hypothesis when membranes are incubated with P-CNA but not with phospholipase A,. Pretreatment in the presence of sodium and GTP When comparing opioid receptor binding sites with opioid-inhibited adenylyl cyclase, it is important to note that receptors exist in two states under the two assay conditions. In Tris buffer, without sodium and guanine nucleotides, high-affinityagonist binding predominates, whereas the sodium and GTP that are both required for opioid-inhibited adenylyl cyclase

OPIOID RECEPTORS AND BRAIN ADENYLYL CYCLASE ffl

.I

Basal

None

I

T

PLA

T

PLA+NaCI+GTP

Addition

FIG. 5. Effect of NaCl and GTP preincubation with phospholipase A, on D-Ala enk-inhibited adenylyl cyclase in rat striatum. Membranes were preincubated with 100 ng/ml phospholipase A, at 25°C for 20 min in the presence and absence of 100 mM NaCl and 100 f l GTP. The membraneswere then washed, treated at pH 4.5, and assayed for adenylyl cyclase in the presence and absence of 10 phl D-Ala enk. Results are expressed as picornoles of CAMPper minute per milligramof protein; data represent mean values SEM of five separate experiments.

*

shift high-affinity receptors into low-affinity states (Childers and Snyder, 1980; Remmers and Medzihradsky, 1991). To determine whether blockade of low-affinity receptors would block a higher level of opioid-inhibited adenylyl cyclase. membranes were preincubated with phospholipase A, in the presence and absence of the same concentrations of NaCl and GTP used in the adenylyl cyclase assay. Results (Fig. 5) showed that the addition of NaCl and GTP increased the effect of phospholipase A, on opioid-inhibited adenylyl cyclase. Preincubation with phospholipase A, alone had no significant effect on the maximum inhibition by DAla enk, similar to the results shown in Fig. 3. However, when membranes were preincubated with phospholipase A, together with NaCl and GTP, the maximum inhibition by DAla enk was significantly decreased, from 27% in membranes treated with phospholipase A, alone to 8% in membranes pretreated with NaC1, GTP, and phospholipase A,. Pretreatment with NaCl and GTP alone had no effect on D-Ala enk-inhibited adenylyl cyclase (data not shown).

DISCUSSION The present study examined the effect of irreversible opioid receptor blockade on opioid-inhibited adenylyl cyclase in brain membranes. Several of the drugs used in these experiments have specific receptor-blocking properties if used at concentrations low enough. Thus, naloxonazine blocks p , sites (Wolozin and Pasternak, 1981), whereas P-FNA blocks traditional p sites (Ward et al., 1982). At higher concentrations, the specificity of these compounds is lost. In

225 9

these experiments, 1-10 pA4 concentrations of these compounds did not provide specificity in differential blockade of receptor subtypes. This conclusion is supported by the effects of these agents on receptor binding, where p, 6, and EKC sites were all inhibited, although to somewhat different degrees. Clearly, the absolute specificity of these agents and, therefore, the need to use low concentrations of these compounds became irrelevant when none of them (except PCNA) had any effect on opioid-inhibited adenylyl cyclase. The surprising conclusion of these studies is that blockade of greater than 90% of receptor binding sites did not block inhibition of adenylyl cyclase by opioid agonists. Although similar results were obtained with naloxonazine, P-FNA, and OPNH, the most dramatic result was obtained with phospholipase A,, which after blocking 93-100% of p, 6, and EKC sites not only failed to reduce the maximal inhibition produced by D-Ala enk, but also failed to shift the agonist concentration-effect curve. Except for phospholipase AZ, all agents had more effect on reducing high-affinity p binding than on 6 binding, thus implying that high-affinity 6 sites might play a role in inhibition of adenylyl cyclase. However, a comparison of the results obtained with P-CNA and phospholipase A, effectively demonstrates that high-affinity 6 sites cannot be correlated with opioid-inhibited adenylyl cyclase. P-CNA inhibited 6 binding sites by only 30%at the same concentration that markedly reduced both the maximal inhibition and the affinity of DAla enk in inhibiting adenylyl cyclase. On the other hand, blockade of 100% of 6 sites by phospholipase A, had no effect on D-Ala enk-inhibited adenylyl cyclase. The actions of P-CNA were explored in more detail than the other irreversible agents because, unlike OPNH and naloxonazine, 0-CNA decreased both high-affinity opioid receptor binding and opioid-inhibited adenylyl cyclase. In some respects, this is not surprising because P-CNA contains the highly reactive aminochloroethyl alkylating group and reacts nonselectively with all opioid receptor types (Ward et al., 1982). Its effects in the present study were consistent with those of an irreversible antagonist, as it decreased the B,, and not the K , of high-affinity receptor binding. Moreover, the addition of 1 p M DAMGO or DSLET to membranes after P-CNA pretreatment had no effect on the ability of P-CNA to decrease high-affinity binding. These two experiments suggest that the higher (micromolar) concentrations of agonists that are required for opioid-inhibited adenylyl cyclase did not reverse the actions of P-CNA in these membranes. On the other hand, in one respect, the actions of P-CNA do not correspond with those of traditional irreversible antagonists. Unlike reversible antagonists, whose binding kinetics are first-order, irreversible compounds should display zero-order kinetics, and loss of opioid receptor binding should be linear with time. Indeed, at any given

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P. C. G. NIJSSEN ET AL.

P-CNA concentration greater than the B,,, of recep tors, loss of opioid receptor binding should be complete if the reaction is given sufficient time. Interestingly, this effect is never observed: The time course of P-CNA inactivation of opioid receptor binding is not linear; instead, a maximal loss in binding occurs at each concentration of 0-CNA. This effect has been observed not only in the present study, but also in previous reports with P-CNA (e.g., Fantozzi et al., 1981).It is also not unique to the opioid receptor system: A highly reactive irreversible muscarinic cholinergic antagonist displayed the same effect in blocking receptor binding in membranes (Bolden and Baker, 1990). This phenomenon has never been adequately explained but is probably related to the fact that these compounds contain highly reactive alkylating groups that react with nonspecific sites in membranes. Thus, over time, a certain amount of P-CNA is lost for specific reaction with opioid receptors. In the present study, a 15-min preincubation with 50-400 nM PCNA provided maximal loss in high-affinity opioid receptor binding. Increasing the time of preincubation did not provide a more significant effect on opioid-inhibited adenylyl cyclase. When analyzing the effects of alkylating agents on receptor binding and function, it is important to distinguish between reversible and irreversible actions of these compounds. Even with agents containing highly reactive alkylating groups, such as 0-CNA, only a small portion of the drug may be covalently bound to the receptor. Instead, most of these compounds produce reversible agonist or antagonist effects on receptors (Takemori et al., 1981). The data in Figs. 1 and 2 demonstrate that K, values can be obtained for antagonist actions and IC,, values can be obtained for agonist actions. However, such values are not necessarily relevant to the potencies of compounds such as irreversible ligands. Indeed, for true irreversible ligands, the concepts of IC,, and K, values have no meaning. Instead, concentrations of P-CNA were empirically chosen to produce a desired effect on receptor binding over a given time period; such concentrations are meaningful because of the phenomenon (asdiscussed above) that maximal effects on binding occur with each concentration of P-CNA. The principal goal of the present study was to compare opioid-inhibited adenylyl cyclase in brain membranes with high-affinity opioid receptor binding sites. When opioid-inhibited adenylyl cyclase was quantitated in low-pH-pretreated brain membranes (Childers and LaRiviere, 1984; Childers, 1988; Konkoy and Childers, 1989), several differences between opioid receptor binding and opioid-inhibited adenylyl cyclase became clear. The most obvious was the affinities of agonists in the two systems. Because agonist receptor binding is assayed in Tris buffer, in the absence of sodium and guanine nucleotides, affinities are high, in the nanomolar range. However, both so-

J. Neurochem.. Vol. 59. No.6, 1992

dium and GTP are required for opioid-inhibited adenylyl cyclase, and therefore agonist affinities in inhibiting adenylyl cyclase are in the micromolar range. This difference can be explained by the different buffers used in the two assays: When receptor binding is conducted in adenylyl cyclase buffer (i.e., Tris/ Mgz+buffer containing NaCl and GTP), 3H-labeled agonist binding is markedly reduced, and agonist potencies in displacing [3H]naloxonebinding are micromolar, identical to their potencies in inhibiting adenylyl cyclase (Werling et al., 1985; Puttfarcken and Cox, 1989). The differences between high- and lowaffinity sites may also explain the lack of correlation between receptor binding and adenylyl cyclase. This explanation is supported by the data in Fig. 5, where preincubation of membranes with sodium and GTP increased the effect of phospholipase A, on opioid-inhibited adenylyl cyclase. These data suggest that whereas phospholipase A, blocks high-affinity receptor binding sites in the absence of sodium and GTP, this enzyme blocks opioid-inhibited adenylyl cyclase only when it reacts with low-affinity receptor sites. Therefore, the lack of correlation between opioid-inhibited adenylyl cyclase and high-affinity opioid receptor binding sites is most likely due to the fact that opioid inhibition of adenylyl cyclase occurs only under conditions where high-affinity sites exist in low proportions compared to low-affinity sites. The pharmacological properties of low-affinity opioid receptors in brain have not been fully explored, although other results have demonstrated that opioid agonists have micromolar affinities at receptor binding sites under these conditions (Werling et al., 1985; Puttfarcken and Cox, 1989). The physiological relevance of these findings is supported by electrophysiological experiments, which also require micromolar concentrations for opioid agonists in changing cell firing rates (Wen and Macdonald, 1983; North et al., 1987;Gross et al., 1990).Another important point is that even in NG108-15 cells, which possess only 6 receptor sites (Chang et al., 1978), the correlation between receptor binding and opioid-inhibited adenylyl cyclase is not simple. Although detailed pharmacological studies support the concept that opioid-inhibited adenylyl cyclase in NGlO8-15 cells is coupled to 6 receptors (Law et al., I985), other studies have demonstrated that multiple receptor states exist in these cells and that adenylyl cyclase may not be coupled to the high-affinity form traditionally measured in binding studies (Costa et al., 1985). Moreover, blockade of 95% of receptor binding sites by 0-CNA in NG108- 15 cells did not reduce opioid-inhibited adenylyl cyclase; in fact, the dose-response curve for agonists was shifted to the left by this receptor blockade (Fantozzi et al., 1981). The finding that P-CNA decreased agonist potency by sixfold (Table 6) does not agree with the finding of Fantozzi et al. (198 1) that P-CNA increased agonist potency in inhibiting adenylyl cyclase

OPIOID RECEPTORS AND BRAIN ADENYLYL CYCLASE in NG108-15 cell membranes. The most likely explanation for this disparity is that different populations of spare receptors exist in the two tissues. The decrease in potency observed in the present study is consistent with spare receptors, or “receptor reserve” as defined by the method of Furchgott and Burstyn (1967). The explanation for increased potency in NG108-15 cells following receptor blockade is not clear; however, in preliminary studies in brain membranes, we (S. Childers and T. Sexton, unpublished observations) have observed an increased potency of DAla enk-inhibited adenylyl cyclase in membranes pretreated with phospholipase A,. The mechanism of this effect is currently under investigation. There are several alternative explanations for the apparent lack of correlation between high-affinity binding sites and opioid-inhibited adenylyl cyclase in brain membranes. One possibility, that there is a large excess of receptor reserve in which only a fraction is responsible for inhibition of adenylyl cyclase, is unlikely because of the lack of evidence for a large number of spare receptors in both rat (as reported in this study) and guinea pig brain membranes for the K receptor system (C. Konkoy and S. Childers, submitted for publication). Another possibility is that the receptors responsible for opioid-inhibited adenylyl cyclase represent a unique subset of receptors, different from those identified in binding assays. Indeed, it is conceivable that the sites responsible for inhibition of adenylyl cyclase are not being detected in classical binding assays. Such questions can be addressed in binding assays under conditions (in the presence of sodium and GTP) that allow agonist inhibition of adenylyl cyclase. Unfortunately, the lack of selective labeled opioid antagonists makes such studies difficult, although we are currently conducting studies with [3H]naloxone binding to address this issue directly. The most likely explanation is that the low-affinity sites that are responsible for inhibition of adenylyl cyclase are not as sensitive to protein-modifying reagents as are high-affinity sites. This explanation is supported by the funding in the current study (Fig. 5) that the addition of sodium and GTP increased the ability of phospholipase A2to inactivate opioid-inhibited adenylyl cyclase. This explanation is also supported by two other findings: (1) [3H]naloxone binding sites, which are increased in brain membranes after incubation with NaCl, are not blocked by N-ethylmaleimide or phospholipase A, (Nijssen and Childers, 1987); and (2) the effect of P-CNA on blocking both high-affinity agonist binding and opioid-inhibited adenylyl cyclase is increased when membranes are preincubated in the presence of NaCl and GTP (S. Childers and T. Sexton, in preparation). It is important to note that, with the potential of constant exchange of GTP and GDP occurring on G proteins, both high- and low-affinity states exist in equilibrium and that, even in the presence of sodium and GTP, it is likely that transient

2261

high-affinity sites exist. The lack of correlation between high-affinity binding and opioid-inhibited adenylyl cyclase suggeststhat such sitesare not responsible for adenylyl cyclase inhibition. Instead, the lowaffinity sites that are responsible for this second-messenger system may exist in a confonnational state that renders them less susceptible to irreversible blockade from a variety of agents. Further studies with ‘opioid-inhibitedadenylyl cyclase, opioid-stimulated GTPase, and receptor binding are in progress to help differentiate between these possibilities. Acknowledgment: This study was supported by PHS grant DA-02904 from the National Institute on Drug Abuse.

REFERENCES Abood M. E., Lee N. M., and Loh H. H. (1987) Modification of opioid agonist binding by pertussis toxin. Brain Res. 417,7074. Attali B., Saya D., and Vogel Z. (1989) K-Opiate agonists inhibit adenylate cyclase and produce heterologous desensitization in rat spinal cord. J. Neurochem. 52, 360-369. Beitner D. B., Duman R. S., and Nestler E. J. (1989) A novel action of morphine in the rat locus coeruleus: persistent decrease in adenylate cyclase. Mol. Pharmacol. 35, 559-564. Birnbaumer L., Abramowitz J., and Brown A. M. (1 990) Receptoreffector coupling by G-proteins. Biochim. Biophys. Acfa 1031, 163-224. Blume A. J., Lichtshtein L., and Boone G. (1979) Coupling of opiate receptors to adenylate cyclase: requirement for sodium and GTP. Proc. Natl. Acad. Sci. USA 76,5626-5630. Bolden C. P. and Baker S. P. ( I 990) Effect of acetylethylcholine mustard on muscarinic receptor-coupled attenuation ofcAMP formation in intact GH3 cells. J. Pharmacol. Exp. Ther. 254, 136-141. Chang K. J., Miller R. J., and Cuatrecasas P. (1 978) Interaction of enkephalin with opiate receptors in intact cultured cells. Mol. Pharmacol. 14,961-970. Childers S . R. (1986) A high-performance liquid chromatography assay of brain adenylate cyclase using [’HIATP as substrate. Neurochem. Res. 11, 161- 17 I. Childers S. R. ( I 988) Opiate-inhibited adenylate cyclase in rat brain membranes depleted of G,-stimulated adenylate cyclase. J. Neurochem. 50,543-553. Childers S . R. (1991) Opioid receptorcoupled second messenger systems. Life Sci. 48, 1991-2003. Childers S. R. and LaRiviere G. (1984) Modification of guanine nucleotide regulatory components in brain membranes. 11. Relationship between guanosine-5’-triphosphateeffects on opiate receptor binding and coupling with adenylate cyclase. J. Neurosci. 4, 2764-277 1. Childers S. R. and Pasternak G. W. (1982) Naloxazone, a novel opiate antagonist: irreversible blockade of rat brain opiate receptors in vifro.Cell. Mol. Neurobiol. 2, 93-103. Childers S. R. and Snyder S. H. (1980) Differential regulation by guanine nucleotides of opiate agonist and antagonist receptor interactions. J. Neurochem. 34,583-593. Collier H. 0.J. and Roy A. C. (1974) Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenates. Nafure 248, 24-27. Cooper D. M. F., Londos C., Gill D. L., and Rodbell M. (1982) Opiate receptor-mediated inhibition ofadenylate cyclase in rat stnatal plasma membranes. J. Neurochem. 38, 1 164-1 167. Costa T., Wuster M., Gramsch C., and Herz A. (1985) Multiple states of opioid receptors may modulate adenylate cyclase in

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Opioid-inhibited adenylyl cyclase in rat brain membranes: lack of correlation with high-affinity opioid receptor binding sites.

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor bin...
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