Characterization of [3H]hemicholinium-3 binding associated with neuronal choline uptake sites in rat brain membranes

Brain Research, 348 (1985) 321-330

321

BRE 11166

Characterization of [3H]Hemicholinium-3 Binding Associated with Neuronal Choline Uptake Sites in Rat Brain Membranes KATHRYN SANDBERG and JOSEPH T. COYLE

Departrnents of Neuroscience, Pharmacology and Experimental Therapeutics, Psychiatry and Behavioral Sciences, and Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD (U.S.A.) (Accepted February 12th, 1985)

Key words: choline uptake - - choline carrier - - acetylcholine - - hemicholinium-3- - receptor

Hemicholinium-3 (HCh-3) is a potent and specific inhibitor of the high-affinity choline transport process (HAChT) localized on cholinergic neurons. In this study, the specific binding of [3H]HCh-3 (120 Ci/mmol) was characterized in crude synaptic membranes prepared from rat brain. The binding of [3H]HCh-3 to forebrain membranes was saturable, reversible and specific with an apparent K~l under optimal conditions of 35 nM and a Bmax of 56 fmol/mg protein. The potency of various HAChT inhibitors correlated with their apparent affinities for the specific [3H]HCh-3 binding site. The specific binding of [3H]HCh-3 exhibited an uneven regional distribution in the adult rat brain that corresponded to the activity of the HAChT in these regions. Transsection of the fornix, which causes a degeneration of the septal hippocampal cholinergic pathway, resulted in comparable reductions of the specific [3H]HCh-3 binding and the specific activity of choline acetyltransferase, a presynaptic marker for cholinergic terminals in the hippocampal formation; the lesion did not affect the specific activity of glutamic acid decarboxylase, a presynaptic marker for GABAergic neurons within the hippocampus. Maximal binding occurred in the presence of 200 mM NaCl: potassium, lithium, rubidium and calcium substituted poorly for sodium; and bromide, fluoride, iodide, sulfate and phosphate were less effective anions than chloride. Increasing concentrations of NaC1 increased the affinity of the site for [3H]HCh-3 with no significant effect on the maximal number of sites; the enhancement of affinity was due to a selective slowing of the rate of dissociation of the ligand from its binding site. These findings indicate that [3H]HCb-3 binds to the carrier site mediating the HAChT on cholinergic neurons; thus, this radioligand may be a useful probe for investigating this presynaptic component (HAChT) of cholinergic neurons.

INTRODUCTION

process that has a broad distribution on ceils 35. A variety of lesion studies have shown that degeneration

The synaptic inactivation of several biogenic amine neurotransmitters including dopamine ( D A ) , norepinephrine and serotonin (5-HT) occurs primarily through their reuptake by a s o d i u m - d e p e n d e n t high-affinity carrier system into the nerve terminals which released the neurotransmitter. Although the action of acetylcholine (ACh) is t e r m i n a t e d at the synapse through hydrolysis via acetylcholinesterase (ACHE), the existence of a s o d i u m - d e p e n d e n t highaffinity choline transport process ( H A C h T ) has been demonstrated on cholinergic nerve terminals 16. The kinetics and pharmacologic characteristics of this H A C h T on cholinergic nerve terminals are distinguishable from a lower affinity choline transport

of cholinergic nerve terminals results in a selective loss of the H A C h T both in braint4,15.20,25 and in the periphery21,30, 31.

Considerable

evidence

suggests

that the rate-limiting step in the synthesis of A C h in cholinergic n e u r o n s is d e p e n d e n t upon the intra-terminal availability of choline which is determined, in part, by the H A C h T 2,3,13. F u r t h e r m o r e , the activity of the H A C h T appears to be regulated by the antecedent activity of the cholinergic neurons with increased velocity of uptake observed in association with conditions that increase the turnover of endogenous ACh2. Thus, H A C h T appears to be a relatively specific presynaptic marker for cholinergic neurons that is intimately involved with the synthesis of ACh.

Correspondence: J.T. Coyle, Division of Child Psychiatry, Meyer 4-163, The Johns Hopkins University School of Medicine. 600 North Wolfe Street, Baltimore, MD 21205, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

322 Recently, a number of laboratories have exploited ligand-binding techniques to characterize the carriers mediating the sodium-dependent high-affinity uptake processes for biogenic amines. Thus, tricyclic antidepressants and related compounds are potent competitive inhibitors of these transport processes and have proved to be useful ligands for labeling specific carriers. For example, [3H]desipramine binds with a high specificity and affinity to the norepinephrine carrier
Materials. [3H]Hemicholinium-3 (120 Ci/mmol), [14C]acetylcoenzyme A (50 mCi/mmol), L-[14C]glu tamic acid (50 mCi/mmol) and liquid scintillation fluor (Formula 947) were obtained from New England Nuclear (Boston, MA). Glass fiber filters (No. 32) were obtained from Schleichert and Schuell (Baltimore, MD). Boric acid, butanol, methanol, sodium bromide, sodium fluoride, sodium hydroxide, sodium iodide and sodium phosphate were purchased from J.T, Baker (Phillipsburg, N J). Dowex 1X8 200400 mesh, chloride form, was purchased from BioRad Labs. (Richmond, CA). HEPES (N-2-hydroxyethylpiperazine-N-2-ethane formic acid) and pyri-

doxal phosphate (grade A) were obtained from Calbioehem-Behring (La Jolla~ CA). Equithesin was purchased from Jensen Salsbury Labs. (Kansas City, MO). Copper sulfate and phenol reagent solution 2N (Folin-Ciocalteau) were purchased from Fisher Scientific (Fairlawn, N J). BRIJ-35 (Formula T21-0110) was obtained from Technicon (Tarrytown, NY). Bovine serum albumin (BSA), CaCI 2, choline chloride, ethylenediamine tetraacetate, eserine ~u[fate, glycylglycine, hemicholinium-3, LiCI, magnesium chloride, mercaptoethanol, polyethylenimme, KC1, potassium tartrate, RbC1, sodium carbonate, NaCI, sodium sulfate, sucrose, Tris-[hydroxymethylaminomethane]-citrate, Tris-hydrochloride, Tris-maleate and trichloroacetic acid were purchased from Sigma (St. Louis, MO). Choline analogues were kindly donated by Dr. Michael Kuhar. All other reagents were of the highest available purity from standard sources. ffH]Hemicholinium-3 binding. Brain tissue from male Sprague-Dawley rats (150-250 g) was homogenized in 10 vols. of ice-cold 0.32 M sucrose with a glass homogenizer fitted with a Teflon pestle, and the homogenate was centrifuged at 1000 g for 10 rain. The pellet (Pl) was discarded; the supernatant was spun at 20,000g for 20 rain. This crude mitochondrial pellet was resuspended in 20 vols. of ice-cold distilled water and dispersed with a Brinkmann PT-10 Polytron at a setting of 5 for 10 s, and the homogenate centrifuged at 8000 g run for 20 rain. The supernatant and buffy coat were collected 7 and pelleted at 48,000 g for 20 rain. This pellet was washed 4 times in 20 vols. of buffer by resuspending via Polytron and centrifuging at 48,000 g for 15 rain. After the final wash, the pellet was resuspended. Unless otherwise stated, 100 #1 of the membrane preparation (approximately 800 gg protein) were incubated in triplicate at 25 °C for 30 min in a 500M volume. The final concentration of [3H]HCh-3 was 10 nM (5.4 × 105 cpm) in 50 mM glycylglycine buffer, pH 7.8, containing 200 mM NaCI. The incubation was terminated by the addition of 5 ml of ice-cold assay buffer to each tube followed by immediate filtration using a Brandell Cell Harvester through glass fiber filters which had been presoaked in 0.1% (v/v) polyethylenimine solution ~ and 0.5% BSA to reduce non-specific binding. Filters were washed with 15 ml of ice-cold 50 mM Tris-HCl containing 200 mM NaCI: and radioactivity measured by liquid scintillation spectrometry. Non-specif~

323 ic binding was defined as binding in the presence of 10 #M unlabeled HCh-3. Specific binding was calculated by subtracting the respective non-specific binding from total binding and usually expressed as femtomoles [3H]HCh-3 bound per milligram protein. Protein was determined according to the method of Lowry et al. 19with BSA as standard. Fornix transections. Sprague-Dawley rats (190-250 g) were anesthetized with Equithesin (1.0 ml, i.p.) and placed in a David Kopf small animal stereotaxic apparatus, The fornix was transected bilaterally by making cuts 1 mm posterior to bregma, 2 mm lateral to the midline and 4.5 mm ventral to the pia surface 36. After 7 days, the rats were decapitated, their hippocampi were rapidly dissected at 5 °C and homogenized in 0.32 M sucrose. Aliquots were taken for measurement of neurotransmitter synthesizing enzymes and for [3H]HCh-3 binding. Enzyme assays. For measurement of neurotransmitter synthesizing enzymes, aliquots of the 0.32 M sucrose homogenates were homogenized in 50 mM Tris-HCI (pH 7.4) containing 0.2% Triton X-100, and the homogenates were centrifuged at 10,000 g for 10 min to remove protein. Aliquots of the supernatant fluid were assayed for the activity of choline acetyltransferase (CHAT) by the method of Bull and Oderfeld-Nowak et al. 5. Glutamic acid decarboxylase (GAD) was measured by the method of Wilson et al.34. RESUL~IS

Characteristics of the specific binding of [3H]HCh-3. Optimal conditions for specific binding of [3H]HCh-3 were observed with freshly prepared, washed synaptic membranes from the forebrain, incubated at 25 °C with 50 mM glycylglycine buffer, pH 7.8, containing 200 mM NaC1. The incubation was terminated by rapid filtration through glass-fiber filters. The filters were washed with ice-cold buffer containing 200 mM NaC1. If NaC1 was absent from the wash buffer, greater than 25% of the radioligand bound to the filters in the absence of tissue. Prior treatment of the filters with 0.1% polyethylenimine and 0.2% BSA resulted in non-specific binding of [~H]HCh-3 to the filter that was several-fold lower than to the untreated filters. Under these conditions, 800 ug of fresh forebrain synaptic membranes incu-

1, t ..~

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ze

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t

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Fig. 1. pH dependency of specific [~H]HCh-3 binding to rat forebrain synaptosomal membranes. Specific binding of [3H]HCh-3 was measured under standard conditions (see Materials and Methods). Values are the result of triplicate determinations and are expressed as specific I3H]HCh-3 bound (fmol/mg protein _+S.E.M.).

bated for 30 rain with 10 nM [3H]HCh-3 bound a total of 1925 + 85 cpm, whereas non-specific binding (measured in the presence of 10/~M unlabeled HCh3) was 985 +_ 85 cpm (n = 20; + S.E.M.). Specific binding exhibited tissue linearity between 200-800 #g protein and was reduced (> 85%) by prior heat denaturation of the membrane (100 °C x 15 min). Specific binding exhibited a sharp pH optimum of 7.8. Binding decreased rather abruptly at more acid and alkaline pH values so that total binding at pH 6 is only 19% of levels at 7.8, while binding at pH 8.5 falls to 62% of levels observed at pH 7.8. Non-specific binding does not markedly change over the pH range 5 to 8.5 (Fig. 1). Prior freezing of the membranes resulted in nearly a 50% loss in specific binding. The temperature optimum appeared to be 25 °C as specific binding at 37 °C or at 0 °C was reduced by approximately 15-20%. Glycylglycine buffer was superior to Tris, borate, phosphate and HEPES buffers when controlled for molarity and pH (Table I).

Saturability of [3H]HCh-3 binding to rat forebrain membranes. The saturability of HCh-3 was assessed by examining the reduction of [3H]HCh-3 bound in the presence of increasing amounts of non-radioactive HCh-3 (Fig. 2). Bound [)H]HCh-3 is reduced progressively with increasing concentrations of unlabeled HCh-3. Maximal reduction of specific binding is apparent at concentrations greater than 100 nM. A Scatchard analysis of these data indicates a single population of binding sites with a dissociation con-

324

Kinetic,; of the specific binding of [-¢H]HCh-3. The

TABLE I

Effects of buffer, temperature and tissue preparation on specific [3H]HCh-3 binding to rat forebrain synaptosomes Binding data are expressed as femtomoles (3H]HCh-3 specifically bound per milligram tissue protein +_ S.E.M. and as mean percent of control. Rat forebrain synaptosomal membranes was performed as described under Materials and Methods. NaCI was added to the buffer at a final concentration of 200 raM. The pH was adjusted to 7.8 for all above conditions.

Buffer (50 mM)

Temperature Tissue (°C) preparation

Specific [~H]HCh-3 bound ~tool~rag % prot, control

Glycylglycine Glycytglycine Glycylglycine Glycylglycine Tris-HC1 Tris-maleate Tris-citrate Borate Sodium phosphate HEPES

25 25 37 0 25 25 25 25

12.1 -+0.3 6.7+0.8 10.0_+1.0 l 0.2__+_1.3 7.5_+0.7 7.4_+0.3 7.1 _+0,5 9.2-+0,9

fresh frozen fresh fresh fresh fresh fresh fresh

100 55 83 84 62 60 59 76

initial time-course ot: [3H]HCh-3 binding to rat forebrain membranes at 25 °C is linear for approximately 10 min (Fig, 3A). The rate of binding then slows and reaches an apparent steady-state at abou~ 25 minl By contrast, non-specific binding assayed in the presence of 10/zM HCh-3 is maximal at the earliest time points examined. The bimolecular rate constant for association was estimated from the linear portion of the association curve. Assuming pseudo first-order

~.

lO

~

8 //

1 m

25 25

fresh fresh

6.5+0,8 8.2__+0,9

54 68

4

o

stant (Ka) of 35 +_ 8 nM and a Hill coefficient of 0.87 (Fig. 2), The maximum number of sites is about 56 _+ 12 fmol/mg tissue protein.

2b

S

16

12

m~.utes

80 ~- . . . . . . .

20 go TIME (minutes)

100

12

10

64

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(-~ -r-

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60 i

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minutes

.........

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80 0

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1.5

2.0

.] HCh 3) 40

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i

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-Log

i 8

i 7

i 6

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[ UNLABELED H C h - 3 ]

Fig, 2. Saturation isotherm for the specific binding of [3H]HCh-3 to rat forebrain synaptosomal membranes. Values represent the results of triplicate determinations and are representative of 4 experiments. Inset: Scatchard plot of saturation isotherm. Values are expressed as mean femtomoles of [3H]HCh-3 specifically bound per milligram tissue protein.

2

4

6 B TrME ( m i n u t e s )

10

12

14

Fig. 3. A: kinetic analysis of the association of [3H]HCh-3 to rat forebrain membranes. Total and non-specific binding were determined under standard conditions as described in Materials and Methods at the indicated time intervals to yield the specific binding values. The data were plotted according to a pseudo first-order reaction. The second-order rate constant was determined from the initial portions of the association curves as described under Results. B: kinetic analysis of the dissociation of specifically bound pH]HCh-3 to rat forebrain membranes. Dissociation experiments were performed by incubating forebrain membranes to achieve equilibrium (25 °C for 30 rain) under standard conditions as described under Materials and Methods. Dissociation was then initiated by adding 10/~M cold HCh-3 and the residual specifically bound [3H]HCb-3 was measured at indicated time points. Specific and non-specific binding at time zero was defined as the equilibrium level, The first-order dissociation rate constant was estimated from the half-life of the dissociation rates for specific binding. Data are the means of 3 separate determinations performed in duplicate,

325 conditions for both [3H]HCh-3 concentration (10~M) and binding site concentration (56 fmol/mg), the association rate constant is 8.5 x 105 M - b s -1 at 25 °C. Dissociation of bound [3H]HCh-3 from forebrain membranes was assessed by adding 10 ~M HCh-3 to membranes previously incubated for 30 rain at 25 °C with 10 nM [3H]HCh-3 and estimating the amount of radioactivity remaining bound at various time intervals (Fig. 3B). When plotted on a semilogarithm scale, bound [3H]HCh-3 decreased linearly, indicating a first-order process. The half-life for dissociation is 74 s at 25 °C. The first order rate constants for dissociation are calculated to be 9.4 x 10 .3 at 25 °C. The kinetics of association and dissociation of [3H]HCh-3 in forebrain membranes yielded a calculated K d value from the ratio of K_I/K 1 of 11 + 8 nM, which is within the range obtained from Scatchard analysis. Pharmacology of [3H]HCh-3 binding sites. Displacement of [3H]HCh-3 with choline, the presumed endogenous ligand for the recognition site, revealed a Ki of 40 g M calculated by log-probit analysis with a Hill coefficient of 0.85, which suggests that they are interacting at a c o m m o n site. Of the choline analogues examined, ethylcholine proved to be t h e most potent and N-butylcholine the least potent inhibitor. The HCh-3 analogue, terphenyl-hemicholinium-3, was the most potent analogue examined with a K i of 15 nM (Table II). Ethylcholine aziridinium ion, an alkylating choline analogue that irreversibly inhibits (ref. 23) the H A C h T - b l o c k e d [3H]HCh-3 binding by > 86% after incubating for 15 min at 25 °C at concentrations as low as 5 gM. There was a highly significant correlation (r = 0.86; P < 0.01) between the affinity of the active 12 analogues for the [3H]HCh-3 specific binding site and their reported potencies in inhibiting the sodium-dependent synaptosomal highaffinity uptake process for [3H]choline3,13~27. Further evidence for the specificity of this labeling procedure was provided by the finding that several pharmacologic agents known not to directly interact with synaptic H A C h T sites were also inactive in displacing [3H]HCh-3 binding. Regional distribution of [3H]HCh-3 binding sites. The specific binding of [3H]HCh-3 exhibited an uneven regional distribution (Table III) that correlated with the specific activity of the H A C h T ~3. The highest specific binding was observed in the striatum.

TABLE II

Drug inhibitory potencies on [3H]HCh-3 binding and [3H]choline uptake in ratforebrain synaptosomes ICs0 values for inhibition of [3H]HCh-3 binding by various drugs were determined from linear regression of log-logit plots. The values are the means of two separate experiments performed in triplicate. ICs0 values for choline uptake inhibitors were obtained from Barker and Mittag 3 and Jope 13. The following drugs exhibited ICs0 values > 0.1 mM: monoethylcholine, diethylcholine, triethylcholine, scopolamine, physostigmine, arecholine, atropine.

Analogues

[3H]HCh-3 binding 1C5o(~M)

[3H]choline uptake IC5o (~M)

N-ethylcholine N-butylcholine a-Methylcholine Monocholine N-methylquinudidin-3-ol Betaine Pyrrolcholine Terphenyl HCh-3 Fluoroethylcholine Ethylpyrrolidinocholine Carbamylcholine Acetylcholine

1.4 16.0 13 2.2 1.4 450 6.0 0.08 10.3 44 35.8 > 100

5.8 62.8 53.4 12.8 6.9 950 27.6 0.065 45.3 89.2 74.6 550

Binding was much lower in all other brain regions examined with the lowest found in the cerebellum (15% of striatal values). Kinetic analysis (n = 4) of the specific binding in striatum and hippocampal formation indicated that both regions had comparable Kds (28 + 5 and 33 + 8 nM, respectively), whereas the BmaxS differed by approximately 4-fold. The striatum has a Bmax 312 + 45 fmol/mg protein, and the hippocampus TABLE III

Regional distribution of specific [.~H]HCh-3 binding to rat brain synaptosomal membranes Binding data are expressed as femtomoles specific [3H]HCh-3 bound per milligram tissue protein +_S.E.M. (n = 5). Synaptosomal membranes from 6 rat brain regions and [3H]HCh-3 binding were performed as described under Materials and Methods.

Brain regions

Specific [~H]HCh-3 bound (fmol/mg protein)

Striatum Hippocampus Hypothalamus-thalamus Medulla pons Cortex Cerebellum

81.9 + 4.2 20.5 + 3.0 9.3 + 1.0 6.3 _+0.8 2.5 _+5.1 1.0 + 0.6

326 has a Bmax of 79 + 12 fmol/mg protein. Thus, regional binding differences appeared to reflect differences in binding site density and not in the apparent affinity of the recognition site for the ligand. Subcellular distribution. To ascertain the subcellular localization of [3H]HCh-3, rat brain homogenates were subjected to differential centrifugation (see Materials and Methods). After homogenizing in 10 vols. of 0.32 M sucrose, the crude nuclear pellet (P1) was obtained by centrifuging the homogenate at 1000 g for 10 min. This PI contained about 12% of the total recovered binding. The crude mitochondrial pellet (P2) (20,000 g for 10 min) contained 78% of total recovered binding while the mitochondrial-myelin pellet (remains of the disrupted P2) pellet after removal of the buffy coat) contained only 10% of total recovered counts. Thus, specific binding is most enriched in the synaptic membrane fraction of rat brain (Table IV) which contains primarily pinched-off nerve-ending particles and also contains the greatest levels of uptake activity 3s. Fornix transections. The medial septat nucleus provides the major source of cholinergic innervation to the hippocampal formation 14As,>. In order to determine whether the specific binding sites for [3H]HCh-3 are localized to cholinergic axons, we examined the effect of fornix transection (Table V), which causes a degeneration of this cholinergic proTABLE IV Subcellular distribution of [3H]HCh-3 binding to rat forebrain synaptosomes

Whole homogenate particulate was the pellet which resulted from a 20,000 g centrifugation of a 15 vols, 0.32 M sucrose whole brain homogenate. The crude nuclear pellet (Pt) resulted from the initial 1000 g centrifugation of the whole brain homogenate. The mitochondria-myelin pellet was the fraction that remained from the disrupted P2 pellet after removal of the buffy coat. All fractions were washed 3 times in 10 vols. of buffer. Values are the results of two separate experiments performed in triplicate. Fraction

Specific/SH]HCh-3 bound J~nol/mg protein

Whole homogenate particulate 3.8 _+0.7 Crude nuclear pellet (P0 4.3 ± 3.6 Osmotically shocked P2 subfraction mitochondria-myelin pellet 5.1 ± 6.2 crude synaptic membranes 12.4 _+3.1

Relative specific activity

100 113 134 326

TABLE V Effect of fornix transections on neurotransmitter cnzvme,s and on specific {~H]HCh-3 binding in the rat htppocamlms

Rat hippocampal synaptosome preparations, ncurotransnainer enzyme assays and [3H]HCh-3 binding were performed as described under Materials and Methods. The ~csults are mean (± S.E.M.) of 6 preparations. Parameter

Control

Forni_~

~;,

[r6lnsec[~?d

( ontroi

Neurotransmitter enzyme., ChAT(nmol/mgprotein/h) 58.4±5.1 GAD (nmol/mg protein/h) 12.5±1.8

12.8±3.0" 11.6~L9

22 89

Specific PHI binding Kd(nM ) Bmax (fmol/mg protein)

26.7±8.0 21.4+5.4"

81 27

33.8+0.8 79.3 +10

* P < 0.01 as compared to control by t-test.

jection to hippocampus. Sevens days after the lesion, C h A T activity, a presynaptic marker for the ch01inergic terminals, was reduced by 78%, whereas the specific activity of G A D , a presynaptic marker for G A B A e r g i c neurons intrinsic to the hippocampal formation 29 was not significantly altered by the fornix lesion. Saturation isotherms for the specific binding of [3H]HCh-3 to membranes derived from the same preparations revealed that the lesion had no significant effect on the apparent Ka; however, the Bmax was reduced by 73%. Thus, specific binding sites for [3H]HCh-3 appear to be localized to cholinergic axons and terminals. Ionic dependence o f the specific binding o f [SH]HCh-3. The activity of the H A C h T process is highly dependent upon the concentration of NaCV, 28,35 With glycylglycine buffer free of NaCI, no specific binding of [3H]HCh-3 could be detected in forebrain membranes. Increasing concentrations of NaCI were associated with increased specific binding of [3H]HCh-3 with half maximal binding occurring at 85 mM NaCI and maximal at 200 mM NaCI. Equimolar substitution of KCI, LiC1, RbCl or CaCI, for 200 mM NaC1 resulted in an 8 0 - 9 0 % reduction in the specific binding of [3H]HCh-3 (Table VI). The mechanism of sodium stimulation of [:~H]HCh-3 binding was examined in greater detail. Saturation isotherms with [3H]HCh-3 conducted at NaCI concentrations of 40, 85 and 200 mM revealed that the enhancement in binding was due to a progressive decrease in the K,t with no significant effect on the Bm.x

327 TABLE VI

TABLE VII

Sodium dependency of specific [3H]HCh-3 binding to rat forebrain synaptosomes

Effects of sodium chloride on [3H]HCh-3 binding to rat forebrain synaptosomes

The specific binding of [3H]HCh-3 to forebrain synaptosomal membranes was performed as described in Materials and Methods. The results are the mean (+ S.E.M.) of triplicate determinations from two assays.

Scatchard analysis was performed on HCh-3 saturation isotherms at 3 NaC1 concentrations. K a and Bmax values were determined by linear regression of Scatchard plots (Fig. 4). The results are the means of two separate experiments performed in triplicate. K 1 was determined by linear regression analysis of dissociation experiments. K 1 was determined by pseudo firstorder reaction analysis of association experiments. Values for kinetic experiments are the means of two separate experiments each performed in triplicate. See the legend to Fig. 3 for details.

200 mM

Specific [3H]HCh-3 binding

NaCI KCI LiCI RbCI CaCI 2

fmol/mg protein

%control

12.9 + 2.3 2.8 +_.0.5 2.1 _+ 0.4 1.4 + 0.7 1.5 + 0.8

100 22 16 11 13

(Fig. 4). S o d i u m slowed the dissociation o f [3H]HCh-3 f r o m its binding site in a c o n c e n t r a t i o n - d e p e n d e n t

NaCI (mM)

Ka (nM)

Bma~ (fmol/mg protein)

K_ 1 (s 1)

KI (nM-1.s -1)

K I/K~ (nM)

40 85 200

134 71 35

44 58 56

3.8 x 10-~ 2.0 x 10-2 9.4 × 10-3

7.2 x 105 7.0 x 105 8.5 x 105

53 28 11

m a n n e r . T h u s , the h a l f - t i m e for dissociation in t h e p r e s e n c e of 200 m M NaC1 was a b o u t 3 t i m e s g r e a t e r

tions in the specific binding of [3H]HCh-3. H o w e v e r ,

t h a n the h a l f - t i m e with 85 m M NaC1. O n t h e o t h e r

b r o m i d e c o u l d partially substitute for c h l o r i d e as the

h a n d , no significant a l t e r a t i o n in the association rate

c o u n t e r a n i o n for s o d i u m ,

(K1) was o b s e r v e d with the d i f f e r e n t NaC1 c o n c e n t r a tions

(Table

VII).

Thus,

the

enhancement

of

DISCUSSION

[3H]HCh-3 b i n d i n g by s o d i u m can b e a t t r i b u t e d to a slowing of the dissociation of t h e ligand f r o m the reco g n i t i o n site.

T h e m a j o r finding of the p r e s e n t study is that highaffinity binding of [3H]HCh-3 can be d e m o n s t r a t e d in

T h e specific binding of [3H]HCh-3 was also aff e c t e d by a n i o n c o n c e n t r a t i o n

brain

membranes

which

is a s s o c i a t e d

with

the

and characteristics

H A C h T of c h o l i n e r g i c n e u r o n e s . S e v e r a l lines of evi-

( T a b l e V I I I ) . S o d i u m salts of f l u o r i d e , i o d i n e , sulfate

d e n c e s u p p o r t this c o n c l u s i o n . [ 3 H ] H C h - 3 b i n d i n g is

and p h o s p h a t e w e r e a s s o c i a t e d with 7 0 - 8 5 % r e d u c -

r e v e r s i b l e , s a t u r a b l e with high affinity ( K , = 1 1 35 n M ) and with substrate specificity suggestive of physiologically r e l e v a n t interactionsS.X0. T h e strucTABLE VIII

Chloride dependency of specific [3H]HCh-3 binding to rat forebrain synaptosomes

c_

The specific binding of [3H]HCh-3 to forebrain synaptosomal membranes was performed as described in Materials and Methods. NaC1 was replaced by an equal concentration of the salts indicated. The results are the mean of triplicate determinations performed in two separate experiments.

O ca

0

20

40

60

200 mM

BOUND (frnol/mg protein)

Fig. 4. Scatchard plots of [3H]HCh-3 binding to rat forebrain synaptosomal membranes in the presence of 40 mM (a), 85 mM (b) and 2(10 mM (c) NaCI. Values are expressed as mean femtomoles specific [3H]HCh-3 bound per milligram tissue protein (BOUND) and as the fraction of specific [3H]HCh-3 bound over free [3H]HCh-3 (BOUND/FREE). Each point is the mean of triplicate determinations.

Specific [3H]HCh-3 binding fmol/mg protein

NaCI NaBr NaF Nal NaSO 4 NaPO 4

12.9 _+ 2.3 9.2 + 0.5 2.1 _+ 0.3 3.7 _+ 0.2 1.9 _+_0.4 3.2 __+0.1

control

100 71 16 29 15 25

328 ture-activity relationship studies of the affinities of various analogues of choline and other agents that inhibit HAChT reveal a highly significant correlation (r = 0.86) between their ICs0 values at the specific binding site for [3H]HCh-3 and their potencies at inhibiting HAChT in synaptosomal preparations. The ICs0 values for inhibiting HCh-3 binding are roughly an order of magnitude higher than for inhibiting HAChT; however, the conditions for measuring choline uptake are different from those for HCh-3 binding (e.g. pH, temperature, tissue preparation and buffer). There is a reasonably close correspondence between the apparent density of the specific binding sites for [3H]HCh-3 and the specific activity of the synaptosomal HAChT in rat brain 13. Prior lesion of a well-defined cholinergic projection to the hippocampal formation which is associated with a marked reduction in HAChT and in the specific activity of CHAT, a specific presynaptic marker for chotinergic terminals 14, resulted in a comparable reduction in the density of the specific binding sites for [3H]HCh-3, consistent with a recent report 33. It is important to emphasize that under the conditions used, [3H]HCh-3 appears to label a single class of non-interacting recognition sites and thus, the low-affinity choline carrier, which is relatively insensitive to HCh-3 (ref. 3), does not appear to be a factor. Considerable evidence from in vitro studies of HAChT in synaptosomal preparations have demonstrated that both cations and anions affect the velocity of transport of [3H]choline. The present study reveals that the specific binding of [3H]HCh-3 closely parallels the ion requirements for choline uptake 28. [3H]HCh-3 specific binding is markedly sensitive to the alkali metal composition of the buffer. Virtually no specific binding could be demonstrated in the glycylglycine buffer in the absence of alkali metal cation, and sodium was the most efficacious of the ions examined. Maximal binding occurred in the presence of 200 mM sodium with a half maximal binding observed at 85 mM (Fig. 4). Kinetic analysis revealed that the increase in binding at a single, subsaturating concentration of ligand resulted from an increase in the affinity of the recognition site for [3H]HCh-3, whereas the number of binding sites was not significantly affected. Analysis of the kinetic factors that might account for this decrease in Kd with increasing

concentrations of sodium in the incubation buffer revealed that decreased rates of dissociation of the ligand from the recognition site without alterations in the rate of association accounted for the effect (Table VII). Anions have also been noted to influence the velocity of synaptosomal accumulation of [3H]choline by the HAChT. Similar to the findings with the synaptosomal HAChT studies 28, chloride proved to be the optimal counter anion for sodium with bromide somewhat less efficacious. Phosphate. sulfate, iodine and fluoride were much less effective substitutes for chloride (Table VIII). These studies on the specific binding of [-~H]HCh-3 further extend ligand binding approaches for characterizing presynaptic carrier sites on aminergic neuronal systems. Despite the fact that the carriers for norepinephrine, DA and 5-HT play a primary role in the inactivation of their neurotransmi.tters, whereas the HAChT is primarily involved in ACh synthesis, there are striking parallels in the kinetic characteristics of these carriersl~,16,32. Thus, the cation and anion dependence of [3H]HCh-3 for HAChT has reportedly been seen for [3H]desipramine, [3H]mazindol and [3H]imipramine for the norepinephrine, DA and 5-HT carriers, respectively. The ionic dependent characteristics of this transport system are very similar to those of other sodium-dependent transport systems for a variety of organic solutes in a number of biological systems 26. The predominant effect of sodium appears to be in increasing the affinity of these carriers for their respective ligands by reducing the rate of dissociation. The allosteric regulation of carrier affinity for their ligands would seem consistent with enhanced binding of carrier substrates in the extracellular space, which contain physiologic concentrations of sodium. These mechanistic similarities point to more fundamental molecular similarities among these carrier proteins. That is, a sodium recognition site, as well as a chloride recognition site, may form part of the macromolecular complex involved in these transport process. The development of a method for labeling the HAChT by ligand binding in broken cell preparations offers a number of advantages for studying cholinergic neuronal function. First, the conditions allow for quantitative in vitro autoradiographic localization of the choline carrier site in frozen sections obtained from brain::. Secondly, the requirements of

329 [3H]HCh-3 b i n d i n g by s o d i u m s h o u l d facilitate analy-

such as A l z h e i m e r ' s d e m e n t i a 6. S y n a p t o s o m a l accu-

sis of h o w c h o l i n e u p t a k e is r e g u l a t e d by this ion.

m u l a t i o n of c h o l i n e c a n n o t a c c u r a t e l y be s t u d i e d in

T h i r d l y , this p r o v i d e s a strategy for p u r i f i c a t i o n and

f r o z e n p o s t m o r t e m brain s p e c i m e n s . T h u s , the stabil-

c h a r a c t e r i z a t i o n of the c a r r i e r site that is n o t d e p e n d -

ity of [3H]HCh-3 binding sites in f r o z e n m e m b r a n e s

ent u p o n the level of tissue integrity r e q u i r e d to

should permit additional biochemical characteriza-

m e a s u r e H A C h T . Finally, c o n s i d e r a b l e e v i d e n c e has

tion of c h o l i n e r g i c t e r m i n a l integrity in p o s t m o r t e m

d e v e l o p e d for i m p a i r m e n t s in the s t r u c t u r e and func-

brain.

tion of cortical c h o l i n e r g i c p r o j e c t i o n s in d i s o r d e r s

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