[Drug nM

FIGURE 1. Saturation of fHfMDA and [3H]MDMA binding in rat brain synaptosomes

NOTE: lncreasing amounts of unlabeled MDA or MDMA were added to 1.0 mL of incubation buffer containing 2 nM [ H]MDA or 2 nM [ H]MDMA, respectively. Experiments were performed on tissue in the presence of 0.27 M sucrose. Data are expressed as percent of [3H]MDA or [3H]MDMA bound to tissue in the absence of added nonradioactive drug. Nonspecific binding was assessed by measuring the amount of [ H]ligand bound to boiled synaptosomes incubated in the presence of 0.27 M sucrose.

The high capacities (i.e., Bmax value) of [3H]MDA and [3H]MDMA binding sites, as well as those that have been reported for [3H] amphetamine binding sites (60 pmol/mg protein) (Hauger et al. 1984) and [ H]fenfluramine binding sites (63 pmol/g tissue) (Garattini et al. 1987), argue against bimolecular interactions of these drugs with monovalent protein-binding sites. Although the mechanism by which sucrose acts to preserve low affinity [3H]MDA binding is yet to be determined, a similar phenomenon has been observed for [ H] amphetamine binding (Hauger et al. 1984). In the latter study. a wash of tissue in isotonic sucrose prior to incubation was reported to increase nearly threefold the specific binding over a wash with 50 mM Tris-HCl alone (Hauger et al. 1984).

Pharmacology of Specific [3H]MDA Binding in Rat Brain

The pharmacology of [3H]MDA binding was determined by examining the effects of other monoamine reuptake blockers and related amphetamine analogs on the inhibition of [3H]MDA binding. The pattern of paroxetine, desipramine (DMI), dimethoxymethamphetamine (DOM), and MDA inhibition of specific [3H]MDA binding is shown in figure 3. Experiments were performed in the presence of 0.27 M sucrose using 2 nM [3H]MDA, conditions under which both high- and low-affinity [3H]MDA binding sites are labeled. Paroxetine was the most potent inhibitor (IC50= 1.6 ^M) followed by DMI (IC50= 5.9 ^M), DOM (IC50 = 17 ^M), and MDA (IC50 = 43 ^M). Analysis of paroxetine and DMI inhibition curves revealed Hill coefficient values (nH) close to 1.00 (paroxetine, % = 0.85; DMI, % = 0.91). MDA and DOM gave rise to much shallower inhibition curves (MDA, nH = 0.56; DOM, nH = 0.53). providing additional evidence for the existence of multiple apparent [3H]MDA binding sites.

Eadee-Scatchard transformation of saturation data of [3H]MDA binding in rat brain synaptosomes

NOTE: Increasing amounts of unlabeled MDA were added to 1.0 mL of incubation buffer containing 2 nM [ H]MDA. Experiments were performed on tissue in the presence (open circles) and absence (closed circles) of 0.27 M sucrose. Nonspecific binding was assesed by measuring the amouut of [ H]MDA bound to boiled synaptosomes incubated in the presence of 0.27 M

Eadee-Scatchard transformation of saturation data of [3H]MDA binding in rat brain synaptosomes

NOTE: Increasing amounts of unlabeled MDA were added to 1.0 mL of incubation buffer containing 2 nM [ H]MDA. Experiments were performed on tissue in the presence (open circles) and absence (closed circles) of 0.27 M sucrose. Nonspecific binding was assesed by measuring the amouut of [ H]MDA bound to boiled synaptosomes incubated in the presence of 0.27 M

FIGURE 3. Inhibition of [3H]MDA incorporation into synaptosomes

NOTE: [3H]MDA binding assays were performed in 50 mM Tris-HCl (ph 7.1) containiag 0.27 M sucrose and 2.0 nM [3H]MDA as described in the text. Results are expressed as percent inhibition of specific [ H]MDA incorporation in the absence of inhibitors. Boiled tissue blanks were performed at each concentration of drug.

The inhibition of [3H]MDA binding by several other related compounds is seen in table 1. All compounds were tested at a concentration of 10 ^M under conditions that favored the labeling of the high-affinity [3H]MDA binding site (zero sucrose, 2 nM [3H]MDA) and at 100 ^M concentration under conditions designed to favor the study of the low-affmity [3H]MDA binding site (0.27 M sucrose, 3 ^M [3H]MDA). A significant positive correlation (r2=0.80, p<0.01) between the relative inhibition potencies of the test drugs at the high- and low-affinity [3H]MDA binding sites was observed upon linear regression analysis (figure 4A).

EFFECTS OF OSMOLARITY AND DETERGENTS

A possible explanation for the large capacity (i.e., high Bmax values) of [3H]MDA binding sites and stimulation of [ H]MDA binding by isotonic sucrose is intrasynaptosomal internalization and sequestration of [3H]MDA. Studies of apparent chloride-dependent [3H] glutamic acid binding (Pin et al. 1984; Zaczek et al. 1987) have demonstrated this type of phenomenon for labeled glutamate. This possibility was examined by measuring [3H]MDA binding in the presence of varying concentrations of sucrose and estimating the relative synaptosomal volume by measuring [3H]H2O incorporation into synaptosomes in parallel experiments. As shown in figure 5, decreasing the concentration of sucrose in the incubation medium led to a decrease in the level of [3H]MDA binding. This contrasted with an increase in intrasynaptic volume, as indicated by an increase in the capacity of the synaptosomes to retain [3H]H2O. A significant negative correlation (r2=0.84; p<0.02) was obtained when [ H]H2O incorporation was correlated with [3H]MDA incorporation by linear regression analysis. These data argue against a sequestration phenomenon, since the amount of [3H]MDA binding should increase with increasing vesicular volume, if intrasynaptosomal internalization and sequestration was occurring.

TABLE 1. Pharmacology of inhibition of [H]MDA binding

High-Affinity

Low-Affinity

Drug

Binding

Binding

Amphetamine

47 ± 21

31 ± 13

Mescaline

36 ±4

12 ± 6

MDMA

48 ± 12

32 ± 3

N,N-DMT

5 9± 11

56 ± 20

PCA

84 ± 7

56 ± 8

DOM

70 ± 3

35 ± 8

Fenfluramine

63 ± 6

34 ± 9

Paroxetine

100± 1

91 ± 8

Desipramine

96 ± 5

83 ± 9

Imipramine

96 ± 3

61 ± 15

NOTE: Inhibition of high-affinity [3H]MDA binding was performed in 50 mM Tris-HCl (pH 7.1) in the presence of 2 nM [ H]MDA to preferentially label the high-affinity site. Drugs were tested at 10 ^M concentrations for inhibition of high-affinity binding. Low-affinity [3H]MDA binding inhibition was performed in 50 mM Tris-HCl (pH 7.1) containing 0.27 M sucrose in the presence of 3.0 ^M [ H]MDA to preferentially label the low-affinity site. Inhibition was peformed using 100 ^M concentrations of the drugs tested. Values represent percent inhibition of specific [ H]MDA binding (mean ± SEM) performed in the absence of inhibiting drugs. Boiled tissue was no in simultaneous assays to assess nonspecific binding.

NOTE: Inhibition of high-affinity [3H]MDA binding was performed in 50 mM Tris-HCl (pH 7.1) in the presence of 2 nM [ H]MDA to preferentially label the high-affinity site. Drugs were tested at 10 ^M concentrations for inhibition of high-affinity binding. Low-affinity [3H]MDA binding inhibition was performed in 50 mM Tris-HCl (pH 7.1) containing 0.27 M sucrose in the presence of 3.0 ^M [ H]MDA to preferentially label the low-affinity site. Inhibition was peformed using 100 ^M concentrations of the drugs tested. Values represent percent inhibition of specific [ H]MDA binding (mean ± SEM) performed in the absence of inhibiting drugs. Boiled tissue was no in simultaneous assays to assess nonspecific binding.

Another approach used to examine the possible existence of [3H]MDA sequestration into synaptosomes was to investigate the effects of the detergents Triton X-100 and digitonin on the level of [3H]MDA incorporation into rat brain synaptosomes (table 2). Concentrations of detergents lower than 0.01 percent did not affect specific [3H]MDA binding. Digitonin. at a concentration of 0.01 percent, caused a 30 percent decrease (p<0.05) in the level of apparent [3H]MDA binding as compared to control, and 0.01 percent Triton caused a 71 percent decrease (p<0.01). These data provide additional evidence against intrasynaptosomal internalization and sequestration of [3H]MDA since relatively high concentrations (0.01 percent)

Binding Affinity
FIGURE 4. Correlation between the relative inhibitory potencies of various drugs at high- and low-affinity [3H]MDA binding and between drug lipophilicities and inhibition potencies of [3H]MDA binding

NOTE: Panel A represents the relationship between the relative inhibitory potencies of various drugs a3 high- and low-affinity [ H]MDA binding. Percent inhibition by test drugs of low-affinity [3H]MDA binding is plotted vs. the inhibition of high-affinity binding. Panels B and C represent the relation between the lipophilicity of test drugs and their ability to inhibit high and low [ H]MDA binding, respectively. In both cases, the retention times of test drugs on reverse-phase HPLC are plotted vs. percent inhibition of [3H]MDA binding. Pearson's r values and levels of significance are derived from linear regression analysis of the data.

of the detergents were required to cause significant decreases in [3H]MDA incorporation into the tissue. Furthermore, these decreases were only partial, which is in contrast to what is generally observed when labeled substances am released from a membrane-intemalized pool by pore-forming detergents, which abruptly release the total contents of membrane-sequestered compounds.

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