[3H]WIN 35,428 binding in the human brain.

The binding of [3H]WIN 35,428 was studied in post-mortem human brain, including extrastriatal regions. In the putamen, dopamine almost completely inhibited the [3H]WIN 35,428 binding. Paroxetine inhibited the binding with similar affinity as cocaine, in the range 200-300 nM. In the frontal cortex, [3H]WIN 35,428 labelled cocaine- and alaproclate sensitive binding sites, of which a major fraction was of protein nature. The elucidation of the cocaine sensitive sites in the frontal cortex should be the subject of further research.

Dopamine uptake sites in Parkinson's disease and in dementia of the Alzheimer type.

The binding of [3H]GBR-12935 to dopamine (DA) uptake sites was studied in post-mortem putamen from a control group and from patients with Parkinson's disease (PD) or dementia of the Alzheimer type (DAT). The specific binding (Bmax) was almost completely abolished in the PD group and reduced by 65% in the DAT group. There were no significant differences in apparent binding affinity (Kd) between the DAT group and controls. The decreases in [3H]GBR-12935 binding to DA uptake sites in this study indicate a marked degeneration of DA neurites in the putamen in PD and also in DAT.

Adenylyl cyclase activity and G-protein subunit levels in postmortem frontal cortex of suicide victims.

Basal and stimulated adenylyl cyclase activities and Gs and Gi protein alpha-subunit levels (Gs alpha and Gi alpha) were compared in postmortem frontal cortex from 18 suicide cases and 22 matched controls. Basal, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated and forskolin stimulated enzyme activities were significantly lower in the suicide cases, compared to controls. These effects were most apparent in those suicides that had died from violent means or that had had a history of depression and appeared to reflect the lowered basal activity rather than a reduced ability of either GTP gamma S or forskolin to activate the enzyme. No significant correlations were found between adenylyl cyclase activity and either subject age or postmortem delay. Western blotting revealed no significant differences in Gs alpha and Gi alpha levels between control and suicide cases. However, levels of the smaller Gs alpha isoform (Gs alpha-S) showed a tendency to be increased in the violent death suicide and depressed suicide subgroups, compared to controls. Levels of the larger Gs alpha isoform (Gs alpha-L) showed a significant positive correlation with subject age. Gi alpha levels showed a significant negative correlation with subject age and a positive correlation with postmortem delay. These results support the hypothesis that suicidal behaviour and depressive illness may be associated with an altered regulation of adenylyl cyclase.

[3H]GBR-12935 binding to human cerebral cortex is not to dopamine uptake sites.

The binding of the dopamine uptake inhibitor [3H]GBR-12935 to 16 regions of the human brain was investigated in competition experiments with increasing concentrations of GBR-12909, mazindol, and dopamine. The methodology used included a relatively high tissue concentration (8 mg/ml) and addition of 5 mM KCl in the assay buffer. GBR-12909 inhibited 80-90% of the binding in most regions, whereas dopamine only inhibited the binding in the striatum. Mazindol inhibited only part of the cortical binding at concentrations of > 1 microM, whereas the inhibition in the caudate and the putamen also contained a high-affinity component representing the dopamine uptake site. It is concluded that the [3H]GBR-12935 binding sensitive to GBR-12909 cannot be regarded as specific binding to the dopamine uptake site because the displaceable binding most likely is not related to the dopamine uptake site.

[3H]GBR-12935 binding to cytochrome P450 in the human brain.

The presence of multiple [3H]GBR-12935 binding sites in the human brain has been revealed in several recent studies. One site represents the dopamine uptake site. In rat brain it was demonstrated that [3H]GBR-12935 also binds to nondopaminergic "piperazine acceptor sites." One of these sites has been identified as cytochrome P450IID1 in canine brain. [3H]GBR-12935 binding to the piperazine acceptor sites in the human brain was investigated in the present study. A pharmacological definition of the piperazine acceptor sites is presented: the [3H]GBR-12935 binding fraction that could be discriminated by 10 microM GBR-12909 in the presence of 0.3 microM mazindol. This binding fraction was saturable, with binding affinity in the range of 3-8 nM. It was also demonstrated that the piperazine acceptor or cytochrome P450-sensitive drugs cis-flupentixol and proadifen (SKF 525 A) compete for the same binding sites, suggesting the cytochrome P450 nature of the binding. The findings presented support the proposal that at least part of this fraction represents cytochrome P450IID6, the human form of P450IID1. The distribution of [3H]GBR-12935 binding to the suggested P450IID6-site in 12 brain regions was examined, without significant differences in binding densities between the regions. The significance of the present findings on the cytochrome P450 system in brain is discussed.

Inhibition of [3H]paroxetine binding by various serotonin uptake inhibitors: structure-activity relationships.

Fifty-four compounds structurally related to zimeldine or alaproclate and eight reference substances were examined as inhibitors of the high affinity binding of [3H]paroxetine to rat cerebral cortical membranes as a measure of the affinity of the 5-hydroxytryptamine (5-HT) transporter. None of the compounds had an affinity as high as paroxetine (KD = 0.026 nM). The most potent compound, 3-(4-methoxyphenyl)-1-methyl-3-phenylpropylamine (2) had a 5 times lower affinity than paroxetine. Some other diphenyl-1-methyl-propylamines displayed high affinity, e.g. the 4-bromo (4) and 2-bromo (7) derivatives. The primary amine analogue of zimeldine substituted with an alpha-methyl group (19) had an affinity only slightly less than that of norzimeldine (11) but an almost 100 times higher affinity than that of the unsubstituted primary zimeldine analogue (57). These observations indicate that a methyl group on the alpha-carbon and on the nitrogen both increase the affinity for the [3H]paroxetine binding site. The structure activity relationship for the compounds to inhibit [3H]paroxetine binding was highly significantly correlated to the inhibition of 5-HT uptake in mouse brain slices (P less than 0.01) and to the inhibition of noradrenaline uptake in the same slices (P less than 0.05). QSAR analysis of the zimeldine series of compounds indicates that substitution of halogens of the 2-position of the phenyl ring is unfavourable. The cis configuration promotes higher activity than the trans configuration.

Loss of dopamine uptake sites labeled with [3H]GBR-12935 in Alzheimer's disease.

The binding of the dopamine uptake inhibitor [3H]GBR-12935 to postmortem putamen from a control group and patients with Alzheimer's disease/senile dementia of Alzheimer type (AD/SDAT) or vascular dementia (VD) was studied. The binding density (Bmax) in AD/SDAT was significantly reduced to 50% of control. A reduction of Bmax in VD was also noted, but it did not reach statistical significance. No differences in apparent binding affinity (Kd) between controls and dementia groups were obtained. The concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid were also determined. The concentrations of DA and DOPAC were reduced by 30-40% in AD/SDAT and VD, but the reductions did not reach statistical significance. The concentration of 3-MT was reduced by 40% in AD/SDAT and by 30% in VD. The [3H]GBR-12935-binding densities correlated significantly with corresponding concentrations of DA in control brains. It is suggested that the loss of [3H]GBR-12935-binding sites in human putamen in AD/SDAT reflects a degeneration of dopamine neurites.

[3H]GBR-12935 binding to dopamine uptake sites in rat striatum.

The binding of the selective dopamine uptake inhibitor [3H]GBR-12935 to rat striatum was studied. Competition by mazindol and dopamine against [3H]GBR-12935 binding revealed monophasic binding curves. The addition of 100 microM dopamine to the mazindol competition inhibited only 80% of the binding, indicating more than one [3H]GBR-12935 binding site in rat striatum. When a binding fraction that could be discriminated by 1 microM mazindol or 1 mM dopamine was defined as specific binding, a single site binding model was obtained. The [3H]GBR-12935 binding was of protein nature, since it was abolished after protease treatment. Drug inhibition studies with the addition of low concentrations of mazindol and dopamine resulted in alterations in apparent Kd values only, suggesting competitive inhibition by these compounds against [3H]GBR-12935 binding. It is concluded that the [3H]GBR-12935 binding to rat striatum discriminated by 1 microM mazindol reflects binding to the substrate recognition site for the dopamine uptake.

Binding of some antidepressants to the 5-hydroxytryptamine transporter in brain and platelets.

Antidepressant agents with properties to inhibit 5-hydroxytryptamine (5-HT, serotonin) uptake in brain tissue and platelets bind with high affinities to neuronal and platelet membranes. [3H]Imipramine, [3H]paroxetine and [3H]citalopram label specific binding sites related to the 5-HT transporter. [3H]Paroxetine and [3H]citalopram appear to be better ligands than [3H]imipramine. The former label a homogenous population of binding sites, whereas the displaceable binding of [3H]imipramine is heterogenous. Recent observations in several laboratories, which have taken the heterogeneity of [3H]imipramine binding into account, indicate that the binding of antidepressants to the 5-HT transporter probably occurs to the same site that binds 5-HT for transport and not to a separate site as previously suggested. Additional bonds to subsites in close vicinity to the 5-HT recognition site may contribute to the binding. No convincing evidence has been presented of the existence of an endogenous ligand other than 5-HT itself that binds to the [3H]imipramine binding site. Recent studies also suggest that repeated treatment of rats with antidepressant agents does not produce any alterations of the binding of [3H]imipramine or [3H]paroxetine to membranes of cerebral cortex. It is also doubtful whether the density of the 5-HT uptake site in platelets measured with these ligands is decreased in affective disorders as first reported.

High- and low-affinity [3H]desipramine-binding sites in human postmortem brain tissue.

The binding of [3H]desipramine to human brain tissue was characterized. Competition studies in the frontal cortex and hypothalamus revealed a single-site binding model for noradrenaline (Ki 120-190 microM). The noradrenaline uptake inhibitors nisoxetine, nortriptyline and desipramine fitted two-site binding models and these compounds exhibited 10-80 times lower Ki values than the serotonin uptake inhibitor citalopram. The high-affinity component of the nisoxetine-sensitive [3H]desipramine binding (Ki 50-110 nM) approximated the binding sensitive to noradrenaline. This binding fraction was defined as that sensitive to 1 microM nisoxetine and showed a maximum binding capacity (Bmax) of 380 +/- 80 fmol/mg protein and an apparent Kd of 5.1 (4.5-5.7) nM in the hypothalamus. The binding was also investigated in 25 additional brain regions without finding detectable amounts of binding. However, when the specific binding was defined as that sensitive to 100 microM nisoxetine, low-affinity binding where Bmax and Kd were not possible to determine was obtained in all brain regions investigated. It is concluded that [3H]desipramine binding to human brain tissue represents multiple binding sites. Only when regarding binding sensitive to noradrenaline and to the high-affinity component of noradrenaline uptake inhibitors is the binding saturable and of high affinity. It is possible that this site represents the uptake site for noradrenaline.

Unaltered [3H]GBR-12935 binding after chronic treatment with dopamine active drugs.

Rats were injected intraperitoneally with haloperidol 0.5 mg/kg, raclopride 1 mg/kg, bromocriptine 2.5 mg/kg, d-amphetamine 2.5 mg/kg, or cocaine 10 mg/kg twice daily for 21 days. The animals were sacrificed 72 h after last injection. Control rats were injected with saline, following the same schedule. The radioligand [3H]GBR-12935 was used as a presynaptic marker for dopamine neurites. There were no significant differences in [3H]GBR-12935 binding to striatum between drug-treated rats and controls.

Age-correlated loss of dopamine uptake sites labeled with [3H]GBR-12935 in human putamen.

The effects of age (19-100 years) upon dopamine uptake sites labeled with [3H]GBR-12935 in human postmortem putamen from 20 individuals were studied. There was a 70% decrease in binding density (Bmax) over the adult age range. No significant changes in binding affinity (Kd) were detected, the mean Kd being 1.0 +/- 0.2 nM (mean +/- S.E.M.). Nor were there any changes in binding related to the postmortem delay. Based on the findings that [3H]GBR-12935 labels the uptake site for dopamine, it is suggested that the age-related loss of [3H]GBR-12935 binding in human putamen reflects a degeneration of dopamine neurites.

[3H]desipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurons.

The pharmacological and biochemical characteristics of [3H]desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one [3H]desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The [3H]desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 microM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of approximately 1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). In contrast, when 10 microM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with KD values of 0.5 and greater than 100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (Bmax) for binding defined by 0.1 microM nisoxetine varied between brain regions, with very low density in the striatum (Bmax not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of [3H]noradrenaline uptake but not with 5-[3H]hydroxytryptamine uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug inhibition indicates a single-site model of the 5-HT uptake site/antidepressant binding site in rat and human brain.

Drug inhibition against [3H]paroxetine binding to rat cortex and human putamen was investigated in saturation experiments. The addition of 5-HT, imipramine, citalopram and clomipramine all produced changes in apparent binding affinity (Kd) without changes in the number of binding sites (Bmax). These data suggest that there is no heterogeneity of specific [3H]paroxetine binding, supporting a single site model of the 5-HT uptake site and antidepressant binding site.

Characterization of [3H]paroxetine binding in rat brain.

The binding of the 5-hydroxytryptamine (5-HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 microliter, the binding was optimized with an apparent dissociation constant (KD) of 0.03-0.05 nM. Competition experiments with 5-HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single-site binding model. Fluoxetine and imipramine revealed, in addition to a high-affinity (nanomolar) site, also a low-affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5-HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5-HT- and fluoxetine-sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5-HT (100 microM) sensitive [3H]paroxetine binding were also consistent with a single-site binding model, and the binding was competitively inhibited by 5-HT and imipramine. The number of binding sites (Bmax) for 5-HT-sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p-chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a greater than 60% reduction in the striatum and hypothalamus, indicating a selective localization on 5-HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5-HT uptake in rat brain.

Adrenergic, serotoninergic, histaminergic, and imipramine binding sites in post-mortal human cerebral microvessel preparations.

Cerebral microvessels were prepared from fresh and frozen human brain samples obtained from autopsy cases. Structural integrity and purity of the microvessels were confirmed by light and electron microscopy, and by measurement of the enzymatic marker gamma-glutamyltranspeptidase. Similar morphological and enzymatic characteristics were found for the microvessels prepared from fresh and frozen brain samples. Radioligand binding experiments indicated the presence both in the "fresh" and "frozen" microvessel preparations of specific alpha 1-, alpha 2-, and beta-adrenergic, histamine H1, serotonin S1 and imipramine binding sites, although the density of beta-adrenergic and histamine H1 specific binding sites were lower in the frozen samples than in the fresh samples. Low levels of specific binding to muscarinic, GABAergic and serotonin S2 sites (with respect to the specific binding densities in the crude homogenates) were found in the microvessel preparations.

5-Hydroxytryptamine-sensitive [3H]imipramine binding of protein nature in the human brain. II. Effect of normal aging and dementia disorders.

Recently, a high-affinity [3H]imipramine binding site of protein nature that appeared related to the 5-hydroxytryptamine (5-HT, serotonin) uptake mechanism was demonstrated in the rat brain. In a preceding paper a similar [3H]imipramine binding site of protein nature and displaceable by 5-HT was demonstrated in the human brain. Most previous [3H]imipramine binding studies of the human brain have used desipramine-sensitive binding, which appears to contain a significant amount of additional binding not related to 5-HT neurons. Therefore this study of the human brain in the normal aging, in Alzheimer's disease/senile dementia of Alzheimer type (AD/SDAT) and in multiinfarction dementia (MID) presents data on 5-HT-sensitive [3H]imipramine binding. The influence of normal aging (17-100 years) was studied in the frontal and cingulate cortices, in the putamen, caudate nucleus, amygdala and in the hippocampus. An age-related change in 5-HT-sensitive [3H]imipramine binding was only noted in the cingulate cortex with a 50% loss in Bmax over the adult age range. In contrast, desipramine-sensitive [3H]imipramine binding studied in the frontal cortex and in the putamen showed marked increases in Bmax with age which correlated with increases in Kd. It is suggested that these increases are related to an increased binding to lipophilic membrane components not related to 5-HT neurons. The 5-HT-sensitive [3H]imipramine binding (Bmax) was reduced to 60% of control in the cingulate cortex and to 50% in the putamen in AD/SDAT. In MID there was a 50% loss of [3H]imipramine binding sites (Bmax) in the putamen, but a 30% loss in the cingulate cortex did not reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptamine-sensitive [3H]imipramine binding of protein nature in the human brain. I. Characteristics.

[3H]Imipramine binding sites were characterized in the human brain by investigating the sensitivity to protease treatment, dependency on NaCl and the effects of drug inhibition. The binding was found to consist of a protease sensitive and a protease resistant fraction. These two fractions could be discriminated by 5-hydroxytryptamine (5-HT) but not desipramine. The [3H]imipramine binding discriminated by 5-HT was found to be sodium dependent. The 5-HT-sensitive [3H]imipramine binding displayed a regional variability with Bmax values ranging from 50 to 100 fmol/mg protein in neocortical areas to 400-500 fmol/mg protein in the substantia nigra and hypothalamus. The Kd values for 5-HT-sensitive [3H]imipramine binding were 1-2 nM throughout the brain. Additional [3H]imipramine binding insensitive to 5-HT, but displaceable by desipramine showed little regional variation, with the binding capacity in the hypothalamus approximating that found in cortical areas. This binding fraction was of low affinity, was not dependent on the presence of NaCl and was insensitive to protease treatment. Drug inhibition studies revealed that the addition of low concentrations of 5-HT or norzimeldine to 5-HT-sensitive [3H]imipramine binding sites produced changes in affinity, consistent with a competitive interaction. It is suggested that the 5-HT-sensitive [3H]imipramine binding may represent the substrate recognition site for 5-HT uptake in the human brain.

Divergent changes in D-1 and D-2 dopamine binding sites in human brain during aging.

The density of D-1 and D-2 dopamine receptors in human caudate nucleus and putamen, obtained postmortem, were studied throughout the adult lifespan using [3H]fluphenazine as the dopamine receptor ligand. The D-1 subtype increased progressively with age in both regions, while the D-2 subtype declined in caudate nucleus. The ratio of D-1/D-2 Bmax in both regions increased from approximately 1 at age 20 to 2 by age 75. The dopamine content in putamen declined with age and was inversely correlated with D-1 receptor density. We suggest that D-1 receptor density is up-regulated by loss of dopamine during aging. The D-2 receptor density in caudate nucleus was positively correlated with choline acetyltransferase activity, suggesting that loss of intrastriatal neurons with age may contribute to the decrease in D-2 sites. These divergent changes in dopamine receptor subtypes with age result in an altered complement of dopamine receptors in older humans and may provide a basis for selective pharmacotherapy in disorders of the basal ganglia.

Single-site model of the neuronal 5-hydroxytryptamine uptake and imipramine-binding site.

Recently, a high affinity [3H]imipramine-binding site of protein nature that appeared to be related to the 5-hydroxytryptamine (5-HT, serotonin) uptake mechanism was demonstrated. This binding site was only part of desipramine-displaceable [3H]imipramine binding, which contained a significant amount of additional binding not related to 5-HT uptake. The present study further investigates the [3H]imipramine-binding site of protein nature in the rat brain. Displacement by 5-HT and 6-methoxytetrahydro-beta-carboline (6-MeO-TH beta C) revealed monophasic displacement patterns with 60% displaceable binding. This binding fraction was abolished by protease treatment of the brain tissue prior to binding assay. Saturation studies of [3H]imipramine binding (1-30 nM) in rat cortex showed that the binding displaced by 30 microM 5-HT [Bmax 322 +/- 16 fmol/mg of protein, Kd 4.17 +/- 1.07 nM (means +/- SE)] was not different from the binding displaced by 1.0 microM norzimeldine (Bmax 349 +/- 15 fmol/mg of protein, Kd 4.47 +/- 1.07 nM) or 30 microM 6-MeO-TH beta C (Bmax 439 +/- 28 fmol/mg of protein, Kd 5.49 +/- 1.09 nM). When 100 microM desipramine was used in saturation studies, the binding was different from that displaced by 5-HT with Bmax 608 +/- 42 fmol/mg of protein and Kd 6.68 +/- 1.09 nM. Both displacement and saturation studies in which two displacing agents were combined indicated that most of the binding competed by 5-HT (30 microM) and norzimeldine (1.0 microM) is identical. Similarly, the binding displaced by 5-HT or norzimeldine is subsumed within 6-MeO-TH beta C (30 microM)-displaceable binding. Lesion studies with parachloroamphetamine, a selective toxin for 5-HT terminals, which resulted in a 83% reduction of [3H] 5-HT uptake ( [3H]noradrenaline uptake unaffected), abolished cortical [3H]imipramine binding displaced by 30 microM 5-HT or 1.0 microM norzimeldine. (greater than 80% reduction). However, with 100 microM desipramine as displacer, 40% of the binding remained in lesioned animals. The [3H]imipramine binding displaced by 30 microM 5-HT or 1.0 microM norzimeldine was sodium dependent, and an increase in NaCl concentration from 0 to 120 mM resulted in a 10-fold increase in affinity without effect on Bmax, whereas no change in binding was observed with increasing concentrations of LiCl.(ABSTRACT TRUNCATED AT 400 WORDS)