Brain Receptor Methodologies: Amino Acids. Peptides. Psychoactive Drugs

90024.

General Preface

Neurobiology or neuroscience is a multidisciplinary subject that has grown out of a common interest in nervous tissue by biochemists, physiologists, and pharmacologists. Researchers in this field therefore require an expertise within their own specialty as well as knowledge of other related areas. The degree of cross-fertilization between the various subdisciplines within neurobiology is extensive, and in most cases is required for the conduct of relevant research in the field.

The Neurobiological Research series provides a comprehensive and current view of various subdisciplines within neurobiology. Each volume will cover a specific area and will present in great detail the methods involved, so that the reader can grasp the general scope of the subdiscipline as well as have sufficient information to actually perform a given methodology. Each subdiscipline will be covered in an extensive manner in order to maximize the probability of finding a given methodology within each volume. This series, therefore, will differ from most existing works in this area, which generally present a few long reviews of selected areas within the realm of neurobiology and do not provide comprehensive coverage of any subdiscipline or the details of methodology.

An additional major emphasis throughout the series will concern how each methodology can be used to address various basic and clinical problems. Critical evaluations of each technique and the meanings of the data obtained from it are intended from each contributor. It is a major goal of the series to facilitate the flow of basic research strategies toward clinical application, and authors have been encouraged to review and to evaluate both past and potential future clinical studies. In this regard the editors are keenly aware of the need for a more rational and critical approach toward clinical neuroscience research.

The Neurobiological Research series should be a unique and valuable addition to the libraries of all neuroscientists. It is hoped that the series will be of equal value for both basic as well as clinical scientists. The first volume (Parts A and B) of the series deal with the area of neurotransmitter and neuromodulator receptors in brain, and future volumes will cover the subdisciplines of neuroanatomy, neurophysiology, brain-specific macromolecules, neurochemistry, and behavioral neurobiology.

Preface to Part B

Part B continues from Part A with the rest of Section II, specific receptor binding methodologies. Subsection II,B deals with receptors for amino acids and neuropeptides and covers areas including GABA (Chapter 1 by DeFeudis), glycine (Chapter 2 by Young), carnosine (Chapter 3 by Hirsch and Margolis), opiates (Chapter 4 by R. S. Zukin), bombesin (Chapter 5 by Moody), CCK (Chapter 6 by Innis et al.), TRH (Chapter 7 by Burt), and substance P (Chapter 8 by Too and Hanley). The major omissions are glutamate and aspartate receptors. Amino acids probably represent the majority of brain neurotransmitter substances, at least relative to the amines and acetylcholine, although with the exception of GABA, the amino acids remain relatively uncharacterized in brain. Their further study should receive high priority.

The neuropeptides also represent an area of rapidly expanding knowledge. Whether these agents represent neurotransmitters or neuromodulators is currently debated, and the chapters in this section should be reviewed with regard to Chapter 4 by Polak and Bloom in Part A. It is likely that much of the current explosion in neuropeptide research will soon settle, and that clearer concepts of their role in neurotransmission will emerge.

Subsection II,C deals with receptors and binding sites for psychoactive drugs. This represents an intriguing area of receptor research in that systems have been defined for which the endogenous ligands have not been identified, and includes reviews on the benzodiazepines (Chapters 9 and 10 by Marangos and Patel and by Leeb-Lundberg and Olsen, respectively), picrotoxin (Chapter 11 by Ticku and Olsen), PCP (Chapter 12 by S. R. Zukin), neuroleptics (Chapter 13 by Leff and Creese), tricyclic antidepressants (Chapter 14 by Rehavi et al.), and adenosine (Chapter 15 by Patel et al.). The characterization of these sites has added enormously to our understanding of the mechanism of action of these drugs and holds the potential of defining new neurotransmitter and neuromodulator systems if and when endogenous ligands for some of the sites are identified. The potential for such studies is best illustrated by the developments that have occurred concerning the opiate peptides during the past 10 years. The adenosine receptor has been included in this subsection because it was not appropriate in any other section, although it is obviously not considered a psychoactive drug receptor at this time.

Brain Receptor Methodologies, Parts A and B, provide a treatment of brain receptors that is of broader scope than has previously been attempted. The information contained here can well constitute the cornerstone of one’s library relating to receptor psychopharmacology.

Contents of Part A

1. Receptors: A Historical Perspective

Iain C. Campbell

2. Preparation of Labeled Receptor Ligands

Yieh-Ping Wan and Stephen D. Hurt

3. Heterogeneous Receptors and Binding Curve Analysis in Neurobiology

Peter J. Munson

4. The Distribution of Peripheral Regulatory Peptides: A Dual Immunochemical (Immunocytochemistry and Radioimmunossay) Approach

Julia M. Polak and Stephen R. Bloom

5. The Solubilization of Membrane Proteins

Andrew C. Newby

6. Solubilization and Characterization of Brain Benzodiazepine Binding Sites

John W. Thomas and John F. Tollman

7. Solubilization of the Dopamine Receptor

Pierre M. Laduron

8. Autoradiographic Demonstration of Receptor Distributions

Miles Herkenham

9. Afffinity and Photoaffinity Labeling of Receptors

Mordechai Sokolovsky

10. Cyclic Nucleotide and Adenylate Cyclase in Brain: Electrophysiological Studies

Trevor W. Stone

11. Calmodulin in the Nervous System

Robert J. DeLorenzo and James R. Goldenring

12. Preparation of the Catalytic Subunit of cAMP-Dependent Protein Kinase

David A. Flockhart and Jackie D. Corbin

13. Phospholipid Methylation in Brain and Other Tissues

Fulton T Crews

14. β-Adrenergic Receptors

Ernst Bürgisser and Robert J. Lefkowitz

15. α-Adrenergic Receptors in Neural Tissues: Methods and Applications of Radioligand Binding Assays

Bruce D. Perry and David C. U’Prichard

16. Dopamine Receptors in Brain

Philip Seeman

17. Radioactive Ligand Binding Studies: Identification of Central

Serotonin Receptors

M. Ramon

18. Muscarinic Receptor [³H]Ligand Binding Methods

Frederick J. Ehlert, William R. Roeske, and Henry I. Yamamura

Index

Section II

Specific Receptor-Binding Methodologies

Outline

Chapter 1: GABA RECEPTORS IN THE VERTEBRATE CNS

Chapter 2: GLYCINE RECEPTORS IN THE NERVOUS SYSTEM

Chapter 3: IDENTIFICATION AND CHARACTERIZATION OF A CARNOSINE BINDING SITE

Chapter 4: OPIATE RECEPTORS: CURRENT ISSUES AND METHODOLOGIES

Chapter 5: RECEPTORS FOR BOMBESIN-LIKE PEPTIDES

Chapter 6: CENTRAL AND PERIPHERAL CCK RECEPTORS

Chapter 7: PITUITARY AND CNS TRH RECEPTORS

Chapter 8: PERIPHERAL AND CENTRAL SUBSTANCE P BINDING SITES

Chapter 9: THE BENZODIAZEPINE RECEPTOR

Chapter 10: BENZODIAZEPINE-GABA RECEPTOR INTERACTIONS

Chapter 11: PICROTOXININ BINDING SITES IN BRAIN

Chapter 12: PHENCYCLIDINE RECEPTORS IN BRAIN: CURRENT METHODOLOGICAL ISSUES

Chapter 13: NEUROLEPTIC BINDING SITES IN BRAIN

Chapter 14: HIGH-AFFINITY BINDING SITES FOR TRICYCLIC ANTIDEPRESSANTS IN BRAIN AND PLATELETS

Chapter 15: ADENOSINE: ITS ACTION AND SITES OF ACTION IN THE CNS

B

Amino Acids and Peptides

Outline

Chapter 1: GABA RECEPTORS IN THE VERTEBRATE CNS

Chapter 2: GLYCINE RECEPTORS IN THE NERVOUS SYSTEM

Chapter 3: IDENTIFICATION AND CHARACTERIZATION OF A CARNOSINE BINDING SITE

Chapter 4: OPIATE RECEPTORS: CURRENT ISSUES AND METHODOLOGIES

Chapter 5: RECEPTORS FOR BOMBESIN-LIKE PEPTIDES

Chapter 6: CENTRAL AND PERIPHERAL CCK RECEPTORS

Chapter 7: PITUITARY AND CNS TRH RECEPTORS

Chapter 8: PERIPHERAL AND CENTRAL SUBSTANCE P BINDING SITES

Chapter 1

GABA RECEPTORS IN THE VERTEBRATE CNS

F.V. DeFEUDIS¹,     Institut Henri Beaufour Laboratories, Le Plessis-Robinson, France

Publisher Summary

As γ-aminobutyric acid (GABA) is involved in physiological mechanisms that govern various normal or altered behaviors in vertebrates, it becomes essential to learn more about the membrane receptors that are associated with these actions of GABA. The ligand binding technique has been widely employed to study such GABA receptors in central nervous system (CNS) sub-cellular particles, a major aim of these studies being the separation of GABA binding into components related to its uptake (inactivation) and receptor interaction. This chapter discusses representative protocols for studying GABA binding, either in the presence or absence of added Na+. It has been found that GABA receptors might be considered to consist of several macromolecules: a GABA recognition site, a Cl--ionophore, and a benzodiazepine receptor; also, certain proteins, gangliosides, phospholipids, and perhaps other membrane constituents might be involved in modulating the interaction of GABA and its recognition site. It is evident that the binding of radio-labeled GABA agonists to CNS membrane sites involves neuronal GABA receptors and that the ligand binding method should be useful for further characterizing and isolating these receptors.

I. Introduction

II. Methods for Studying GABA Binding

A. Preparation of Tissue Particles

GABA Binding Assay in the Presence of Na+ and Other

B. Inorganic Ions

C. GABA Binding Assay Using a Crude Membrane Fraction in the Absence of Added Na+

D. GABA-Binding Assay in Tissue Culture Particles in the Absence of Added Na+

III. Kinetic Analysis of the Data

A. Lineweaver-Burk Analysis

B. Scatchard Analysis

C. Eadie-Hofstee Analysis

Pellet-Supernatant Distribution Ratios; Correction of Data

D. Using Sucrose Spaces

E. Hill Plots

F. Determination of IC50 Values

IV. Representative Experimental Results

A. [³H]GABA Binding to a P2 Fraction in a Bicarbonate-Buffered Physiological Medium

B. [³H]Muscimol and [³H]GABA Binding to Cerebral Subcellular Particles in the Absence of Added Na+

V. CNS Regional Distribution of GABA Binding

VI. Subcellular Distribution of GABA Binding

VII. Effects of Various Factors on GABA Binding

A. Kinetic Constants for GABA Binding to Various Subcellular Preparations in the Presence or Absence of Added Na+

B. Analog Specificity of GABA Binding in the Presence or Absence of Added Na+

C. Effects of Some Psychoactive Agents on GABA Binding

D. Effects of Inorganic Ions on GABA Binding

E. Comments on the Use of Tris Buffers

F. Effects of Freezing-Thawing, Washing, and Detergents on GABA Binding; Endogenous Inhibitors

G. Effects of Enzymes on GABA Binding

H. Effects of Phospholipids and Gangliosides on GABA Binding

VIII. Populations of GABA Receptors

IX. Basic Findings Relating GABA Receptors to Physiology and Behavior

A. Convulsions

B. Extrapyramidal Functions

C. Maturation and Aging

D. Other Functions and Behaviors

X. GABA Receptors in the Human CNS; Therapeutic Implications

A. Epilepsy

B. Extrapyramidal Disorders

C. Other Disorders

XI. Practical Limitations and Pitfalls of the Ligand Binding Method

XII. Concluding Remarks

References

I INTRODUCTION

Since γ-aminobutyric acid (GABA) appears to be involved in physiological mechanisms that govern various normal or altered behaviors in vertebrates, it seems essential to learn more about the membrane receptors that are associated with these actions of GABA. The ligand binding technique has been widely employed to study such GABA receptors in CNS subcellular particles, a major aim of these studies being the separation of GABA binding into components related to its uptake (inactivation) and receptor interaction.

Representative protocols for studying GABA binding, either in the presence or absence of added Na+, will be discussed herein, and the literature on this subject will be briefly reviewed.

II METHODS FOR STUDYING GABA BINDING

A Preparation of Tissue Particles

1 Whole Brain or CNS Regions

Male Sprague-Dawley or Wistar rats (200–250 g) are decapitated and their brains or CNS regions are excised, weighed, and homogenized in 10 volumes of ice-cold 0.32 M sucrose solution using glass-Teflon tissue grinders (0.1–0.15 mm radial clearance; 7 strokes/sample). All further operations are performed at 0 to 4°C. Synaptosomal–mitochondrial (P2) fractions are prepared using a Beckman Model J-21 centrifuge or a Sorvall Model RC-2B centrifuge. Four-ml portions of homogenates are centrifuged at 1000 g for 10 min, and 3.0-ml portions of resultant supernatant fractions are recentrifuged at 17,000 g for 30 min. P2 pellets are resuspended in 3.0 ml of 0.32 M sucrose solution [or in Na+-free or Na+-containing medium (see following sections)] and recentrifuged at 17,000 g, 30 min, to decrease ribosomal–microsomal contamination and endogenous GABA content of the particles. Washed P2 fractions are weighed after removing adherent fluid from the centrifuge tubes (see DeFeudis et al., 1979a). GABA binding can be studied using either these washed P2 fractions, more thoroughly washed P2 fractions, or frozen-thawed hypoosmotically shocked membrane particles prepared from these fractions (Zukin et al., 1974; Enna and Snyder, 1975).

To prepare a crude membrane fraction, the procedure of Greenberg and colleagues (1976) can be followed. Rats are decapitated and their brains (or CNS regions) are removed, weighed, and homogenized in 10 volumes of ice-cold Na+-free, Tris–citrate buffer (50 mM, pH 7.1). Homogenates are pooled, sonicated for 30 sec with a Brinkman Polytron P-10 (setting No. 6) and then centrifuged in a Beckman L5–65 ultracentrifuge at 50,000 g for 10 min (Type-30 rotor). Supernatants are discarded, and pellets are resuspended in 20 ml of Tris–citrate buffer, pooled, and sonicated again (as above). Then, samples are recentrifuged (50,000 g for 10 min), the resultant supernatants are discarded, and pellets are weighed. Pellets can be frozen at −25°C for 2 weeks to 3 months with negligible loss of their binding activities for [³H]GABA or [³H]muscimol.

2 Tissue Cultures

Cultured cells are collected, pooled in large centrifuge tubes (four or five Petri dishes/tube), and packed by centrifugation at 1000 g for 10 min. All further operations are conducted at 0 to 4°C using Tris–citrate medium (50 mM, pH 7.1). After decanting supernatants, cells are washed into homogenizer tubes using 15 ml of medium, homogenized, and then pooled in an Erlenmeyer flask. The combined suspension is sonicated for 30 sec with a Brinkman Polytron PT-10 (setting No. 6) and then distributed into tared tubes and centrifuged at 50,000 g for 10 min (Type-30 rotor), using a Beckman Model L5–65 ultracentrifuge. Supernatants are discarded, pellets are resuspended in 20 ml of medium, pooled, and sonicated again (as above). After recentrifugation at 50,000 g for 10 min, the resulting pellets may be stored at −25°C for 1 to 4 weeks with negligible loss of binding activity (Ossola et al., 1980; DeFeudis et al., 1980a, b).

B GABA Binding Assay in the Presence of Na+ and Other Inorganic Ions

A procedure that has been used to study GABA binding in the presence of Na+ and other inorganic ions will be described. This procedure is based on the displacement of labeled GABA from its highest-affinity binding sites by excess unlabeled bicuculline methiodide (BMI) (DeFeudis et al., 1979a). A newer procedure, developed by Kurioka and colleagues (1981a, b), which involves treating cerebral membranes with Triton X-100 to destroy Na+-dependent GABA uptake sites and then examining [³H]GABA binding in the presence of Na+, will not be described here but should also be considered for such studies.

Washed P2 fractions are weighed, resuspended in 3.0 ml of medium, and kept on ice for 15 min. Aliquots (0.25 ml) of these suspensions are pipetted into weighed centrifuge tubes. Then 0.2 ml of medium, either free of added substance, or containing enough freshly prepared BMI (Pierce Chem. Corp.) or unlabeled GABA (Calbiochem Corp.) to provide final concentrations of 10−3M, is added to each tube. These high concentrations of BMI and GABA are used to estimate BMI-sensitive and GABA-sensitive [³H]GABA binding sites. Samples are mixed for 4 to 5 sec and kept on ice for 15 min. Then 0.5 ml of medium, providing final concentrations of 1.5 × 10−9 to 5.3 × 10−4M of [³H]GABA (γ-[2,3-³H(N)aminobutyric acid; New England Nuclear Corp.; 34.5 Ci/mmole, supplemented with unlabeled GABA when necessary) and 3.4 × 10−8−4.2 × 10−7M of [¹⁴C]sucrose ([U-¹⁴C]sucrose; Radiochemical Centre, Amersham, U.K.: 381 mCi/mmole), is added. Suspensions are mixed and kept on ice for 15 min before final centrifugation at 17,000 g for 30 min (JA-14 rotor). It should be noted that this method permits determination mainly of slowly reversible [³H]GABA binding.

GABA binding is examined in glucose-free bicarbonate-buffered medium (pH 7.4), the osmolarities of the ions in this medium being: Na+, 147.3; K+, 3.5; Ca²+, 1.3; Mg²+, 1.2; Cl−, 128.5; HCO3−, 24.55; PO4³−, 0.45; and SO4²−, 1.2 mOsm/liter (DeFeudis et al., 1975).

Radioactivity due to ³H and ¹⁴C and the protein contents of resuspended pellet fractions are determined as previously described (Lowry et al., 1951; DeFeudis, 1974).

C GABA Binding Assay Using a Crude Membrane Fraction in the Absence of Added Na+

Frozen pellets are resuspended in 20 ml of deionized water, kept at 22°C for 20 min, and then centrifuged at 50,000 g for 20 min. This step is repeated twice more, and then pellets are weighed and resuspended in Na+-free Tris–citrate buffer (4.0 ml buffer for pellets representing about 2 g original fresh weight of tissue). All further operations are conducted at 0 to 4°C using Na+-free Tris–citrate medium.

Aliquots (100 μl) of tissue suspension (representing about 22 mg original fresh weight of tissue, or about 0.8 to 1.0 mg protein) plus 100 μl of Tris–citrate medium, either free of added substance or containing sufficient unlabeled GABA (Calbiochem Corp.) to provide a final concentration of 10−5 or 10−3M, are mixed in small (1.2 ml) centrifuge tubes and kept on ice for 10 min. Then, 250 μl of buffer, containing either [³H]GABA (γ-[2,3-³H(N)] aminobutyric acid; 36.12 Ci/mmole) or [³H]muscimol (3-[methylene-³H(N)]hydroxy-5-aminomethylisoxazole; 13.68 Ci/mmole) in a final concentration range of 3.08 × 10−9 to 3.08 × 10−8M plus [¹⁴C]sucrose ([U-¹⁴C]sucrose; 673 Ci/mmole) in a final concentration range of 6.2 × 10−8 to 6.2 × 10−7M, are added. Samples are mixed, kept on ice for 5 min, and then centrifuged at 57,000 g for 5 min (Type-25 rotor). The binding of both [³H]GABA and [³H]muscimol are maximal under these conditions, and both radioligands are maximally displaced by 10−5 M unlabeled GABA.

After final centrifugation, radioactivity due to ³H and ¹⁴C is determined in 100 μl aliquots of supernatants and in 250 μl aliquots of resuspended pellet fractions (DeFeudis, 1974). Protein is determined by the method of Lowry et al. (1951).

D GABA-Binding Assay in Tissue Culture Particles in the Absence of Added Na+

Frozen pellets of cultures are homogenized in 20 ml of deionized water, kept at 23°C for 20 min, and then centrifuged at 50,000 g for 20 min. Resuspension and centrifugation steps are repeated twice more. The resultant pellets (weighing about 0.4 to 0.7 gm) are resuspended in 3.4 to 4.0 ml of ice-cold Na+-free Tris–citrate medium (50 mM; pH 7.1); all subsequent operations are performed at 0 to 4°C using Na+-free Tris–citrate medium. Aliquots (0.1 ml) of suspension, representing about 0.4 mg protein, plus 0.1 ml of medium, either free of added substance or containing various concentrations of unlabeled muscimol, GABA, other GABA analogs, or drugs, are mixed and kept on ice for 10 min. Then, 0.25 ml of medium containing (as final concentrations) [³H]muscimol or [³H]GABA at 3.1 × 10−9 to 6.2 × 10−8 M plus [¹⁴C] sucrose at 6.3 × 10−8 to 1.25 × 10−6 M is added. (Excess unlabeled GABA (10−3 or 10−5 M) is used to estimate specific binding of [³H]muscimol or [³H]GABA.) Selected concentrations of various GABA analogs or drugs are used to determine their IC50 values. Samples are kept on ice for 20 min and then centrifuged at 57,000 g for 5 min. ([³H]Muscimol and [³H]GABA are maximally bound under these conditions and are maximally displaced by 10−5 M GABA). [¹⁴C]Sucrose provides estimates of the amount of supernatant fluid trapped in the pellets (DeFeudis, 1974; see Section III,D). Radioactivity in pellets and supernatants and the protein contents of pellets are determined as described in Section II, C.

Unlabeled γ-aminobutyric acid, δ-aminovaleric acid, β-alanine, (–)-2,4-diaminobutyric acid, picrotoxinin, and strychnine-SO4 may be purchased from Sigma Chemical Corp.; isoguvacine-HBr, muscimol, (±)nipecotic acid, guvacine-HBr, bicuculline-methobromide, and 3-aminopropanesulfonic acid may be obtained from Prof. C. G. Wermuth, Dr. E. Costa, Prof. P. Krogsgaard-Larsen, Dr. J. F. Collins, and Prof. G. A. R. Johnston.

III KINETIC ANALYSIS OF THE DATA

Data obtained in receptor binding assays often fit a rectangular hyperbolic curve that passes through the origin. The dependent variable is the amount of ligand that is bound to a given particulate site(s) after correction for nonspecific binding (B); the independent variable is the free ligand concentration (F). Maximal binding capacity (Bmax) and the binding, or dissociation, constant (KB) are determined. The general equation may be represented as

(1)

Methods that are useful for graphing such data are described below.

A Lineweaver-Burk Analysis

Using the method of Lineweaver and Burk (1934), or the double-reciprocal plot, 1/B is plotted as a function of 1/F. This plot, which has no point corresponding to B = 0, F = 0 because division by zero is not defined, may be represented by the following linear equation:

(2)

in which 1/Bmax is the y-intercept, KB/Bmax is the slope, and −1/KB is the x-intercept.

B Scatchard Analysis

The method of Scatchard (1949), in which B/F is plotted against B, is represented by the following linear equation:

(3)

KB can be derived from the slope of the line, which is −1/KB, and Bmax is the x-intercept.

C Eadie-Hofstee Analysis

When using the method of Eadie and Hofstee (see Hofstee, 1952), B is plotted against B/F, in accordance with the following equation:

(4)

With this method, Bmax is the y-intercept, –KB is the slope, and Bmax/KB is the x-intercept.

D Pellet–Supernatant Distribution Ratios; Correction of Data Using Sucrose Spaces

Binding data may be presented as pellet-supernatant distribution ratios (DR), as mole of ligand bound/mg protein, as mole of ligand bound/g pellet (corrected for sucrose space), or as mole of ligand bound/g original wet weight of tissue (DeFeudis, 1974). Distribution ratios are calculated as follows:

(5)

The density of pellets may be assumed to be 1.043 g/ml (density of 0.32 M sucrose), and that of supernatants may be assumed to be 1.008 g/ml (approximate density of the incubation medium employed). Corrected distribution ratios [DR (coir.)] are calculated as follows:

(6)

in which DRG = uncorrected ligand distribution ratio and DRS = distribution ratio of sucrose, which is used to estimate the amount of supernatant fluid that is trapped within the pellets. The equation used to calculate the amount of ligand bound/g pellet (corrected for sucrose space) and detailed descriptions of its application have been published (DeFeudis et al., 1975, 1977; Somoza and DeFeudis, 1978).

E Hill Plots

The graph of log (B/Bmax – B) versus log of free ligand concentration (log F) is a straight line [Eq. (7)] and the slope is designated nH (Hill number or Hill coefficient; Hill, 1910). In binding studies Hill plots are used to determine whether positive or negative cooperativity is involved in the interactions.

F Determination of IC50 Values

The IC50 value, that is, the concentration of a substance (inhibitor) that produces 50% inhibition of specific (displaceable) binding of the ligand can be estimated by plotting the log of specifically bound ligand (log B) on the ordinate versus –log of the inhibitor concentration on the abscissa. The point of intersection between the inhibition curve and the line representing 50% specific binding gives –log IC50. These values are generally determined using log-probit plots, assuming competitive inhibition. IC50 values can be converted to Ki values (inhibition constants) using the equation:

(8)

in which C = concentration of labeled ligand and KB = binding (dissociation) constant of the ligand.

IV REPRESENTATIVE EXPERIMENTAL RESULTS

A [³H]GABA Binding to a P2 Fraction in a Bicarbonate-Buffered Physiological Medium

Using a balanced bicarbonate-buffered medium and P2 fractions of rat cerebral cortex, calculation of DR (corr.) using Eq. (6) revealed a significant displacement of bound [³H]GABA by both excess unlabeled GABA and BMI in the presence of a physiological concentration of Na+ (Fig. 1) (DeFeudis et al., 1979a). With this method, both low- and high-affinity BMI-sensitive binding processes were detected. A Scatchard analysis [Eq. (3)] revealed that the Bmax for [³H]GABA binding to its lower-affinity site was decreased by about 500 pmol/mg protein by BMI (10−3 M), whereas its affinity was not appreciably altered, indicating noncompetitive inhibition by BMI at this site (Fig. 2). Using lower concentrations of [³H]GABA (1.5–15.3 nM) a higher-affinity binding component was detected, which appeared to be competitively inhibited by BMI (Fig. 3). Hill coefficients [Eq. (7)] for the BMI- and GABA-sensitive components of these binding processes were about unity, indicating that neither negative nor positive cooperativity was involved in these interactions (Fig. 4) (see also Enna and Snyder, 1975; DeFeudis and Somoza, 1977). On a protein basis, the highest affinity BMI-sensitive [³H]GABA-binding site detected in the presence of Na+-containing medium had K4 × 10−8 M and B2 pmol/mg protein or 140 pmol/gm original wet weight of cerebral cortex.

Fig. 1 Corrected distribution ratios [DR (corr.); , Control; •, 10−3 M GABA; Δ, 10−3 M BMI. Reproduced with permission from DeFeudis et al. (1979a).

Fig. 2 Scatchard plots [Eq. (3)] of the binding of [³H]GABA to a synaptosome-enriched fraction of rat cerebral cortex in a balanced bicarbonate-buffered medium containing physiological concentrations of Na+ and other inorganic ions. Total binding (•) and that which occured in the presence of 10−3M ) or 10−3M ) are indicated on a protein basis; 3–5 separate samples per point. Note that BMI decreased the capacity of [³H]GABA binding by about 500 pmol/mg protein without altering appreciably its affinity. Binding that occurred in the presence of excess unlabeled GABA represents an estimate of the nonspecific component. ——, Bmax ≅ 2490 pmol/mg; KB ≅ 4.7 × 10−6M. - - - -, Bmax ≅ 1990 pmol/mg; KB ≅ 4.3 × 10−6M. Reproduced with permission from DeFeudis et al. (1979a).

Fig. 3 Lineweaver-Burk plots [) and the binding that occurred in the presence of 10−3M BMI (Δ) are shown. Values were corrected for trapped ligand using [¹⁴C] sucrose distribution ratios [Eq. (6)]. ——, Bmax ≅ 2.8 nmol/g; KB ≅ 3.86 × 10−8M. - - - -, Bmax ≅ 3.2 nmol/g; KB ≅ 5.29 × 10−8M. Reproduced with permission from DeFeudis et al. (1979a).

Fig. 4 ), binding that occurred in the presence of 10−3M unlabeled GABA (•) or 10−3M ) are shown. Hill numbers (nH) are indicated below; 3–14 separate samples per point. B , nH = 1.01; Δ——Δ, nH = 0.98; - - - -, nH = 1.01; …, nH = 0.98. Reproduced with permission from DeFeudis et al. (1979a).

B [³H]Muscimol and [³H]GABA Binding to Cerebral Subcellular Particles in the Absence of Added Na+

A comparison of the binding of [³H]GABA and [³H]muscimol that occurred in a particulate fraction of rat brain in Na+-free Tris–buffered medium is shown in Fig. 5 (DeFeudis et al., 1979c). These data, which are presented as pellet-supernatant distribution ratios [see Eq. (5)], revealed that excess unlabeled GABA displaced a greater amount of [³H]muscimol than [³H]GABA from high-affinity binding sites. Apparent Bmax values were 1.9 pmol/mg protein for [³H]muscimol and 1.0 pmol/mg protein for [³H]GABA under the conditions employed [calculated using Eq. (2)] (see also DeFeudis et al., (1979b).

Fig. 5 Distribution ratios [, control; •- - -•, 10−3M , 10−5M GABA. Reproduced with permission from DeFeudis et al.