Life Sciences, Vol. 31, pp. 2019-2023 Pergamon Press Printed in the U.S.A.

[3H]PIRENZEPINE SELECTIVELY IDENTIFIES A HIGH AFFINITY POPULATION OF MUSCARINIC CHOLINERGIC RECEPTORS IN THE RAT CEREBRAL CORTEX 1

Mark Watson, William R. Roeske 2 and Henry I. Yamamura

Departments of Pharmacology, Biochemistry, Psychiatry, Internal Medicine and the Arizona Research Laboratories, University of Arizona

Health Sciences Center, Tucson, Arizona 85724

(Received in final form August 2, 1982)

SUMMARY

The specific binding of [3H]pirenzepine was investigated in homogenates of rat cerebral cortex, cerebellar cortex, and heart. Specific binding of [3H]pirenzepine in the cerebral cortex as de- fined by displacement with atropine sulfate (I~M) was of high affin- i ty (K d = 4-10 nM, receptor density = 1.06 pmoles/mg protein), stereoselective, and competitive with drugs specific for the mus- carinic receptor. In contrast, few [3H]pirenzepine binding sites were demonstrated in cerebellar and heart homogenates.

The specific binding of [3H]agonists and [3H]antagonists to musca- rinic cholinergic receptors has been extensively studied in several tissues (1-6). Although similar aff inity constants for classical muscarinic antag- onists in various tissues (2,4,6,7) suggest homogeneity among these binding sites, there is also evidence which implies the existence of independent subclasses of muscarinic receptors {8). Multiple agonist aff inity states could reflect modulation of a single recognition site (4,6,9). However, the existence of antagonist heterogeneity has been more di f f icul t to rationalize within a unitary concept (4,5,8,9).

Pirenzepine, the tricyclic compound 5,11-dihydro-11-[4-methyl-1-piper- azinyl)acetyl]-6H-pyride [2,3-I][1,4] benzodiazepine-6-one-dihydro-chloride, is clinically useful in the treatment of peptic ulcer disease (10-12). This novel muscarinic antagonist has been proposed to differentiate subpopula- tions of muscarinic receptors (8,12-14), although its selectivity has been disputed recently (15). In the present study, we provide evidence that [3H]pirenzepine ([3H]pz) binds wi th high affinity and specificity to muscarinic receptors in homogenates of the rat cerebral cortex.

METHODS

The cerebral cortices of male Sprague-Dawley rats (350-450 g) were ra- pidly dissected over ice immediately following their sacrifice. A 5.0 per- cent homogenate of the cortical sample was prepared by homogenization

IA preliminary presentation of these data was given at the CNS Receptors: Molecular Pharmacology to Behavior meeting at Mount Saint Odile, France, September 2, 1982. 2Send correspondence to Dr. William R. Roeske, Department of Internal Med- icine, University of Arizona Health Sciences Center, Tucson, Arizona 85724.

0024-3205/82/182019-05503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

2020 High Affinity [3H]pz Binding Vol. 31, No. 18, 1982

with a Brinkmann polytron (2x, 15 sec, setting 5.5) in ice cold 50mM sodium- potassium phosphate buffer, pH 7.4.

The specific binding of [3H]pz (75.0 Ci/mmole, obtained from New England Nuclear) was determined using a centrifugation assay. Aliquots (100~I) of tissue nomogenate were incubated at 0°-4°C with various concen- trations of [~H]Pz in a final assay volume of 2 ml containing the 50mM sodium-potassium phosphate buffer. The time of incubation was 120 minutes. All binding measurements were determined in duplicate or t r ip l icate in at least 3 separate experiments, except where noted. Specific binding was de- fined as the difference between measurements made in the presence and ab- sence of atropine sulfate (I~M). Follow~ngoincubation, the tubes were cen- trifuged at 27,000xg for 20 minutes at 0 -4 C. After rapidly decanting the supernatants, the pellets were surface washed with three 2 ml aliquots of ice cold 50mM sodium-potassium phosphate buffer. After drying, the pellets were solubilized with 300 ~I of tissue soluDilizer (NCS, Amersham) and vor- texed. Following a 30 min incubation period in a 37°C water bath, they were again vortexed. The radioactivity of each sample was quantitated by liquid sc int i l la t ion spectroscopy with an efficiency of 43 percent after the addi- tion of 4 ml of l iquid sc int i l la t ion f lu id (Omnifluor/toluene/acetic acid). TEpically, about 90 percent of the counts were specific for the binding of [JH]Pz (see Figure 1), and assays without tissue showed binding to the vial was mimimal (<5 percent of total counts).

800-

~ 600-

v

4 0 0 l

200'

1

, ~ 5 I 0

FREE ['H] PIRENZEPINE (nM)

Figure I . A representative experiment of specif ic (o) and nonspecific (0) [3H]pirenze~ine binding to homogenates of rat ce- rebral cortex. Specific LJH]Pz binding was experimentally deter- mined as the difference between tota l binding and nonspecific bin- ding using paral le l assays performed in t r i p l i ca te in the absence and presence of atropine sulfate (I~M). The K d was 9.5 nM and the Bma x was 1540 fmol/mg protein.

A tota l concentration of 0.6 nM [3H]pz was used for measurements of the competitive inhibit ion of [3H]pz binding by various nonlabelled cho- l inergic drugs under these conditions. Nonlinear least squares regression analyses were employed using a computerized i terat ive procedure on these da- ta. Protein content was determined according to the method of Lowry et al. (16) using bovine serum albumin as the standard.

¢ -

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RESULTS

Determinations of specific [3H]pz binding at 0°-4°C demonstrated that [3H]pz, over a concentration range of 0.02 nM to 10.0 nM binds in a satur- able manner with a high a f f i n i t y site in homogenates of rat cerebral cor- tex. Measurements carried out at 30, 90, and 120 minute intervals insured a valid analysis of data obtained at 120 minutes, since equilibrium is reached at this time.

To further insure that proper conditions for [3H]pz binding were achieved, tissue l inear i ty studies were performed. Specific binding was linear in cortical homogenates of 2.5, 5.0, 7.5 and 10.0 percent when [3H]pz concentrations were varied between 0.02 nM and 10.0 nM (data not shown). Specific and nonspecific [3H]pz binding in a tyRical rat cortical homogenate is i l lustrated in Figure 1. Nonspecifi~ [~H]Pz binding in- creased l inearly with increasing concentrations of [JH]Pz and was not sa- turable, whereas specific [3H]pz binding showed a downward concavity al- though

Table 1

The effects of muscarinic cholinergic drugs on the inhibit ion of [3H]pz in rat cerebral cortical homogenates, a

Drugs ICso(nM) Hi l l Slope

Pirenzepine 11.8 0.93 (7.6 - 17) (0.71 - 1.2)

Atropine 1.7 0.88 (0.5 - 4.4) (0.57 - 1.3)

Dexetimide i . I 1.13 (0.6 - 1.5) (0.87 - 1.5)

Levetimide Z970 0.76 (2230 - 4770) (0.60 - 1.1)

Oxotremorine 285 0.60 (74 - 428) (0.50 - 0.87)

Pilocarpine 4,140 0.80 (2,670 - 6,840) (0.63 - 0.99)

Acetylcholine (physostigmine, I~M)

16,440 ; 16,550 0.61 ; 0.61

Acetyl-B-methylcholine 25,900 0.49 (14,700 - 33,200) (0.42 - 0.57)

Carbamylcholine 34,700 0.48 (19,500 - 53,800) (0.30 - 0.70)

a See Methods for experimental details. Experiments were per- formed 2 to 7 times, each point in duplicate or t r ip l i ca te . Data are presented as the mean with the range in parenthesis. Diazepam and propranolol were ineffective at IO0~M concentrations.

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saturation was not achieved at a concentration of 10 nM. Scatchard plots of the data showed linearity over the concentration range examined, which is consistent with mass action behavior. Further, nonlinear least squares re- gression analysis yielded an average high aff inity dissociation constant (K d) of 6.2 nM (range 3.8 to 9.5, n=5) and an accompanying receptor den- sity of 1.06 pmoles/mg protein (range 0.70 to 1.54, n=5) or 90 fmol/mg t is- sue (range 54 to 135). The average Hil l slope for these saturation iso- therms was 0.98 (range 0.8 to 1.1). Results of [3H]pz binding in the rat cerebellum and heart using homogenates prepared by our previous methods (17,18) indicate that l i t t l e i f any specific [JH]Pz binding occurs under the present assay conditions.

Pharmacological specificity of [3H]pz binding in the cerebral cortex was demonstrated by testing several muscarinic agonists and antagonists for their abil i ty to inhibit [JH]Pz binding. The IC50 values of these var- ious nonlabelled cholinergic ligands, which were measured by competitive in- hibition of [3H]pz binding, are shown in Table 1. Levetimide and dexeti- mide, the stereoisomers of benzetimide, were employed to demonstrate the stereo-specificity of [3H]pz binding. Dexetimide was more efficacious in displacing the [JH]Pz binding with an IC50 value of 1.1 nM.

DISCUSSION

The results of the present study clearly demonstrate the presence of a high affinity [3H]pz binding site in the rat cerebral cortex and suggest that [3H]pz will become a useful probe in the investigation of muscarinic receptor-effector mechanisms. The existence of both pharmacological speci- f ic i ty and stereospecificity strongly suggest that [3H]pz binding rep- resents a specific interaction with muscarinic cholinergic receptors.

The criteria for specific binding of [3H]pz to a receptor have been satisfied (19). Interestingly, the number of sites labelled by [3H]pz in the cerebral cortex is somewhat less than in our recent studies using C3H](-)QNB in similar buffers (3,17). The existence of high and low af- f ini ty pirenzepine binding sites has been postulated by others using compe- t i t ion curves and physiological studies (8,13,14,20,21). Since only a low affinity pirenzepine site has been seen in the heart using physiological techniques (20,21), i t is not surprising that we did not detect many [JH]Pz sites in the heart under our conditions. Similarly, only a low af- f in i ty pirenzepine binding site has been predicted for the rat cerebellum using indirect methods (8) and we found almost no high affinity [3H]Pz binding in this tissue. These data suggest that pirenzepine is a selective antagonist for muscarinic receptors in different tissues.

While high affinity pirenzepine sites have been previously predicted (8) using indirect methods, we have used a direct method to demonstrate the spe- cif ic binding using radiolabelled pirenzepine. Although an unpublished stu- dy (cited in 8) reports a 2OhM site for [JH]Pz in the cerebral cortex, i t was unclear from this preliminary data whether the criteria for specific binding to the muscarinic receptor were met and whether specific [JH]Pz binding was fou,~d i~l uther tissues.

The inhibition of [3H]Pz binding by muscarinic drugs clearly demon- strates that muscarinic receptors are being specifically labelled. While the antagonist pirenzepine is potent in inhibiting [3H]pz, muscarinic ago- nists have shallow Hill slopes and are less potent than antagonists, thus supporting the notion that [3H]Pz labels an antagonist site (3,4). Fur- thermore, dexetimide is about 2000 times more potent than levetimide, demon- strating stereoselectivity (3,18). The selective properties of [3H]Pz

Vol. 31, No. 18, 1982 High Affinity [3H]Pz Binding 2023

are clearly evident under the conditions of our present assay and further studies using this unique compound are currently being done in an effort to determine i f the concept of dist inct multiple antagonist muscarinic receptors can be ful ly accepted (22).

ACKNOWLEDGEMENTS

Portions of this study was supported in part by USPHS grants MH-30626, MH-27257, and Program Project Grant HL 20984, a Research Scientist Develop- ment Award Type II (MH-O0095) from the National Institute of Mental Health to H.I. Yamamura, and a Research Scientist Development Award (HL-00776) from the National Heart, Lung, and Blood Institute to W.R. Roeske. We thank Ms. Terry Austin for her secretarial assistance.

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