Platelet H-imipramine binding: A state-dependent marker in depression

1. Reduced density of ³H-imipramine binding sites (B_max) to platelets has been reported in depressed patients during an episode of illness. In the present study we assessed the usefullness of decreased B_max of platelet ³H-imipramine binding as an indicator of the depressed state. We also investigated the effect of long-term treatment with imipramine on platelet ³H-imipramine binding.

2. A comparison of platelet ³H-imipramine binding in 10 drug-free depressed patients and 8 normal volunteers revealed significantly lower mean Bmax values in depressed patients, whereas the affinity (K_d) of ³H-imipramine binding was identical in both groups.

3. A longitudinal study of platelet ³H-imipramine binding in 10 depressed patients during and after imipramine treatment (125–200 mg/day) revealed consistently low B_max values despite clinically meaningful improvement. However, B_max values increased significantly following complete remission and remained elevated even after imipramine had been discontinued for 4 weeks.

4. These findings suggest that decrease in the sensity of platelet ³H-imipramine binding sites in depressed patients is not likely to be a direct drug effect and that normalization of this variable may follow clinical remission.     

H-Clonidine binding to membranes of platelets prepared under physiological conditions

(1) A reproducible binding assay has been established to measure ₂-adrenoreceptors on membranes of human platelets prepared under physiological conditions.

(2) Washed platelet suspensions were obtained from fresh blood by a modified method (Mustard et al., 1972) and membranes were prepared by mechanical homogenization.

(3) ³H-clonidine, a selective ₂-adrenoreceptor agonist was selected as the ligand in a concentration range of 2–64 nM. Specific binding of ³H-clonidine was defined as the difference between total bound radioactivity and that not displaced by excess cold clonidine (6.4 × 10⁻⁵M).

(4) Platelets from 14 healthy young male volunteers were analysed using this technique, and one high affinity binding site, with K_D and B_max values in the reported range was found (K_D: 10.14 ± 1.95 nM; B_max: 428 ± 33 fmol/mg protein (Mean) ± S.E.M.).

(5) This technique will be used in future studies that will examine ₂-adrenoreceptor changes in specific subgroups of depressed patients.     

3H-imipramine binding to freshly prepared platelet membranes in depression

3H-Imipramine binding was measured in freshly prepared platelet membranes from 47 drug-free major depressives and 46 healthy controls. Where possible, platelet binding in depressed subjects was repeated following treatment. A significant negative correlation was found between Bmax and assay protein concentration and Bmax values were corrected for this effect. Adjusted Bmax was significantly lower (by 14%) in female depressed patients than in female control subjects, and the difference was of similar magnitude premenopausally and postmenopausally. No such difference was found in males. Kd did not differ significantly between depressed and control subjects. Multiple regression analysis confirmed significant effects on Bmax of presence of depressive illness, age (positive correlation), and season (higher in summer). Within the depressed sample, Bmax was significantly lower in those subjects with obsessional features. Endogenicity (Research Diagnostic Criteria or Newcastle), dexamethasone suppression test result, drug-free interval, family history of depression, depressive psychosis, suicidal ideation, and past history of suicide attempts were not significantly related to Bmax. Paired comparisons revealed no significant effect on Bmax of 6 weeks' treatment with imipramine, maprotiline, or BRL 14342 or of a course of electroconvulsive therapy.     

Platelet [3H]imipramine binding in affective disorders: trait versus state characteristics

Platelet [3H]imipramine binding (Bmax) was determined in 67 patients with major affective illness (33 euthymic bipolar, 34 depressed unipolar) and 58 normal control subjects. Bipolar patients had significantly lower Bmax values than did control subjects. The mean Bmax in the unipolar patients was lower than in the control subjects, but the difference was not statistically significant. Dissociation constant (Kd) values did not distinguish patients in either category from control subjects. The significantly lower Bmax in euthymic bipolar patients and the apparent state independence of Bmax in some but not all unipolar patients suggest that platelet imipramine binding may be a trait marker in a subset of affective disorders.     

Imipramine binding sites on platelets of patients with major depressive disorder

3H-Imipramine binding to platelets of patients with primary, unipolar major depressive disorder was investigated and compared to that of a normal, healthy control population. No significant differences could be demonstrated between either the Kd or the Bmax values of the two groups. A negative correlation was observed between imipramine Bmax values and the Hamilton anxiety ratings of the depressed patients. Patients who displayed psychomotor retardation tended to have lower platelet imipramine Bmax values than patients with psychomotor agitation. It is suggested that platelet imipramine Bmax values may be a biological marker for subtypes of depression.     

Effects of repeated trazodone administrations on serotonergic neurotransmission: Biochemical studies

1. 1. Repeated administrations of trazodone as well as imipramine or mianserin(10 mg/kg i.p. twice daily for 3 weeks) attenuated the norepinephrine (NE) stimulation of adenylate cyclase studied in brain minces. Therefore trazodone shares with “tricyclic” (imipramine) and “atypic” (mianserin) antidepressants the capability to modulate the beta-adrenergic function.

2. 2. Daily treatments with imipramine or trazodone enhanced the Vmax of neural uptake of serotonin (5HT) in minces prepared from rat frontal cortex; in contrast mianserin failed to modify the [³H]-5HT uptake.

3. 3. Repeated administrations of imipramine but not of trazodone or mianserin reduced the maximum number of [³H]-imipramine recognition sites which are located on serotonergic axon terminals.

4. 4. Differently, only repeated administration of trazodone decreased Bmax values of [³H]-mianserin binding sites which are located on membranes innervated by serotonergic neurons. Moreover trazodone did not change the number or affinity of 5HT₂ receptors either after single or repeated administrations; in contrast even a single administration with mianserin or repeated administrations with imipramine down-regulated [³H]-ketanserin specific binding in membranes prepared from the frontal cortex.

5. 5. Our observations therefore suggest that trazodone, imipramine or mianserin exerts similar effects on the adenylate cyclase system, by acting on a interneuronal loop which links serotonergic and noradrenergic transmission function. However, its exact mechanism of action, in part resembling both tricyclic and atypic antidepressants, requires further exhamination.

Antidepressant binding: Implications for the mode of action and the biology of depression

1. 1. (³H)Imipramine binding sites in the brain are localized mainly on serotonergic nerve terminals. In the hippocampus of rats with a lesion of serotonergic nerve terminals produced by neonatal administration of 5,7-DHT the depletion of serotonin was paralleled by a decrease in (³H)imipramine recognition sites.

2. 2. (³H)Imipramine recognition sites in brain tissue or platelets are associated with serotonin uptake sites. A significant correlation exists between the potency of a series of antidepressants and other compounds to displace high affinity (³H)imipramine binding and to inhibit the neuronal uptake of serotonin but not of norepinephrine.

3. 3. There is a significant correlation between the ability of drugs to displace (³H)desipramine binding and to inhibit norepinephrine but not serotonin reuptake. (³H)Desipramine recognition sites are located at least in part at noradrenergic nerve terminals since destruction of these terminals by 6-OH-DA results in parallel decrease in (³H)desipramine but not in (³H)imipramine Binding.

4. 4. The high affinity recognition sites of (³H)imipramine and (³H)desipramine in the brain could be physiologically And Pharmacologically relevant regulatory sites associated with neuronal uptake of serotonin and norepinephrine respectively. Treatments which clinically lead to improvement of depression (eg. antidepressants ECT REM sleep deprivation) were shown to “down-regulate” (³H)imipramine binding sites in brain of experimental animals.

5. 5. The density of (³H)imipramine binding sites was shown to be lower in platelets from depressive patients and in brains of suicide victims. It appears that decreased binding of (³H)imipramine to platelets of depressed patients is a promising biological marker of depression although there is no conclusive evidence to indicate whether it is a state- or trait-dependent phenomenon.

Differences between sodium-dependent and desipramine-defined [3H]imipramine binding in intact human platelets

Measurements of sodium-dependent [3H]imipramine binding to intact human platelets from 20 human volunteers were made and compared to desipramine-defined binding, a method commonly employed in population studies of platelet [3H]imipramine sites. The density (Bmax) of sodium-dependent [3H]imipramine sides in platelets was significantly lower (449 +/- 36 sites/platelet) and the affinity (Kd) significantly higher (1.15 +/- 0.12 nM) than those obtained when excess desipramine was used to define specific binding (Bmax 654 +/- 33 sites/platelet, p less than 0.001; Kd 1.52 +/- 0.11 nM, p less than 0.001). There was no significant correlation between the density (Bmax) of sodium-dependent and desipramine-defined binding in individual subjects, suggesting that a different proportion of sites are labeled under the two assay conditions. No age-dependent variation was found in either Kd or Bmax values of sodium-dependent or desipramine-defined [3H]imipramine binding. The results suggest determination of sodium-dependent [3H]imipramine binding to intact platelets may be a useful measure for the estimation of [3H]imipramine recognition sites relevant to the serotonin uptake in studies of patients with affective disorders.     

Changes in platelet 3H-imipramine binding with chronic imipramine treatment are not state-dependent

One month of imipramine treatment increased both the Kd and Bmax of platelet 3H-imipramine binding in 11 endogenous unipolar depressed patients. Continued treatment (13 weeks) of 5 patients subsequently lowered the Bmax values of 2 patients who had initially shown the largest increases, so that binding was no longer significantly elevated after 13 weeks. The observed changes in Kd but not in Bmax, could be explained by the carryover of tightly bound drug to the binding assay, although neither of the measures were correlated with plasma imipramine levels. Posttreatment Bmax (4 weeks) values were inversely related to plasma cortisol levels, although a weak but positive correlation was found before treatment. No significant change was found in plasma cortisol with treatment. Clinical responses were not related to cortisol or Bmax changes, although optimal improvement was associated with extreme values (high and low) of pretreatment Bmax. The present results, obtained with imipramine, and similar results obtained after nortriptyline and electroconvulsive shock by others, suggest that at least some antidepressants may induce transient changes in the Bmax of platelet binding that are independent of affective state.     

Characteristics of agonist displacement of [H]ketanserin binding to platelet 5-HT2A receptors: implications for psychiatric research

Brain 5-HT_2A receptors exist in two agonist affinity states as a function of their coupling to G_q protein. This has not yet been shown in platelets. We examined [³H]ketanserin's saturable binding to platelet 5-HT_2A receptors and characteristics of agonist displacement curves of [³H]ketanserin binding in healthy control subjects. [³H]ketanserin saturation curves showed a trend for a two-site model, reflecting two independent binding sites. At low [³H]ketanserin concentrations, agonist displacement curves were flat and best fit a two-site model, indicating the existence of two agonist affinity states. Guanylyl 5′-imidotriphosphate [Gpp(NH)p] induced a significant rightward shift in agonist displacement curves to fit a one-site model. Platelet membrane 5-HT_2A receptors exist in two agonist affinity states that are regulated by G_q protein. Platelet 5-HT_2A receptors provide an accessible model for examining possible dysregulation in the agonist affinity or coupling efficiency to the phosphoinositide system in psychiatric disorders and their modulation by psychotropic medications.     

Opiate peptide receptors on intact NIE-115 neuroblastoma: radioligand binding properties, intracellular response, and effects of increasing membrane cholesterol

1. Binding of [³H]methionine-enkephalin to intact NIE-115 neuroblastoma cells (competing ligand: naloxone) revealed a homogenous population of receptors with a density (B_max) of 79.0 ± 6.5 mol/mg protein (mean SEM, N=3) and an apparent K, of 5.33 ± 1.63 mM.

2. The order of displacement of [³H]met-enkephalin was met-/leu-enkephalin > naloxone > morphine, suggesting that it is of the delta receptor class.

3. Specific binding was heat-labile, stereospecific and sensitive to Na⁺.

4. Adding met-enkephalin to intact neuroblastoma caused reductions of both basal and prostaglandin E₁-stimulated levels of cyclic AMP (41.4 ± 4.0% (N=6) and 45.1 ± 2.4% (N=3) of control levels, respectively). Maximum inhibition (naloxone-reversible) was observed as low as 10⁻⁷ M met-enkephalin.

5. Preliminary results suggest that cells grown in cholesterol-supplemented medium show reduced binding of [³H]met-enkephalin.     

Brain slices in radioligand binding assays: Quantification of opiate, benzodiazepines and beta-adrenergic ([H] CGP-12177) receptors

1. We have characterized and quantified specific binding of [³H]-flunitrazepam (FNZ: (benzodiazepine), [³H]-naloxone (NAL: (opiate) and [³h]cgp-12177(CGP: (beta-adrenergic) to thick slices (230–400 μm) of mouse and rat brain.

2. The binding sites are stereospecific, saturable and of high affinity. In all cases, the binding of the ligands is readily reversible and demonstrates the appropriate drug specificity.

3. In mouse brain [³H]-NAL binding is elevated by chronic treatment with naloxone (via capsules).

4. We have been unsuccessful in quantifying beta adrenoreceptors with the archetypal ligand [³H]-dihydroalprenolol (DHA). However, the use of [³H]-CGP 12177 enabled us to detect high-affinity beta adrenoreceptors in brain slices.

5. [³H]-CGP also permits the demonstration of rapid and reversible agonist-induced down-regulation (internalization) of beta binding sites.

6. We have been successful in quantifying beta adrenergic sites in single pineal glands of rat and hamster.     

Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation

Glutamate is the major excitatory neurotransmitter in the rat visual system. Using quantitative autoradiography the effect of unilateral orbital enucleation on [³H]kainate, [³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) and [³H]glutamate binding to kainate, quisqualate and NMDA receptors respectively has been examined within anatomical components of the visual pathway at 4 time points up to 20 days post-lesion. The time course for the degeneration of retinal projection fibres was assessed in a separate group of animals by quantifying [³H]cyclohexyladenosine ([³H]CHA) binding to presynaptic adenosine A₁ receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A₁ receptor binding in visual structures of the visually deprived hemisphere. However, at 10 days post-lesion [³H]AMPA binding was significantly reduced (30%) in the visually deprived superior colliculus but unaltered in other visual structures. At this time point there was also a significant reduction (50%) in [³H]CHA binding in the visually deprived superior colliculus but not in other retino-recipient nuclei. There were similar changes in [³H]AMPA and [³H]CHA binding at 20 days post-enucleation. [³H]Kainate binding was significantly increased in the visually deprived superior colliculus only at 20 days post-enucleation. Saturation analysis of [³H]kainate and [³H]AMPA binding at this time point indicated a selective increase in the b_max value for the high affinity [³H]kainate binding site and a concomitant decrease in the b_max value for the high affinity [³H]AMPA binding site in the visually deprived superior colliculus. There were, however, no significant alterations in [³H]AMPA or [³H]kainate binding in other primary projection areas or in secondary visual areas (e.g. visual cortex) at any time point. NMDA sensitive [³H]glutamate binding was unaltered in the visually deprived hemisphere up to 20 days post-enucleation. These results suggest an upregulation of kainate receptors in the visually deprived superior colliculus after orbital enucleation and a loss of presynaptic quisqualate receptors on degenerating retinal fibres. The plastic alterations in kainate receptors in the superior colliculus are supportive of electrophysiological data suggesting a physiological role for these sites in mediating excitatory postsynaptic potentials in tectal neurons.     

Regional distribution of [H]imipramine binding in rat brain

[³H]imipramine binding was measured in 23 microdissected areas of the rat brain and compared to published values for the endogenous levels of serotonin, noradrenaline and dopamine in the same areas.

The density of [³H]imipramine binding sites appears to be highly correlated with the distribution of endogenous serotonin especially where the serotonin is located mainly in nerve terminals. A weak but still significant correlation also exists with the distribution of endogenous noradrenaline whereas no such correlation could be detected for endogenous dopamine.     

Inositol 1,4,5-trisphosphate metabolism in the cerebella of Lurcher mutant mice and patients with olivopontocerebellar atrophy

We have investigated inositol 1,4,5-trisphosphate (InsP₃) metabolism in cerebellar membranes of normal humans and patients with dominant ataxia (‘C’ kindred), and also in cerebellar microsomes of Lurcher mutant mouse (a suggested model for cerebellar ataxia). Various [³H]InsP₃ metabolites formed separated by HPLC using 3 successive convex gradients of 1.7 M ammonium formate, pH 3.7. [³H]InsP₃ metabolism was rapid and in 15- and 45-day-old control mice cerebella about 50% of [³H]InsP₃ was metabolized within 20 s. In 15-day-old Lurcher mice the rate of [³H]InsP₃ metabolism was significantly low (40% of normal). [³H]InsP₃ metabolism was almost absent in 45-day-old Lurcher mice cerebellar microsomes. The decreased [³H]InsP₃ metabolism was consistent with decreased recovery of the various inositol polyphosphates formed. Similarly, in cerebellar membranes of human patients with olivopontocerebellar (OPCA) a significant decrease in [³H]InsP₃ metabolism was observed when compared with normal controls. These data suggest that altered phosphoinositide turnover may be associated with the onset of neuronal degeneration in human OPCA.     

5-HT1B receptor binding in degenerative movement disorders

Using [³H]sumatriptan as a radioligand, 5-hydroxytryptamine (5-HT)_1B receptors were examined in posterior striatum and midbrain post-mortem tissue sections of 12 patients who had died from representative degenerative movement disorders as compared to nine controls. In the control human basal ganglia, the highest densities of [³H]sumatriptan binding were observed in the globus pallidus and substantia nigra. No significant change in the density of [³H]sumatriptan binding sites was found in the striatum and substantia nigra of the six Parkinson's disease brains. In the two brains from patients with progressive supranuclear palsy an increase was found in the densities of [³H]sumatriptan binding sites, most marked in the substantia nigra. In contrast, [³H]sumatriptan labelling was almost absent in the striatonigral degeneration brain and was markedly reduced in the three Huntington's disease brains. This study indicates that the status of 5-HT_1B receptors is different in each degenerative movement disorder and suggests that human 5-HT_1B receptors are located somatodendritically on GABAergic and peptidergic caudate-putamen neurons which project to the substantia nigra and globus pallidus, where these receptors are presynaptic.     

High- and low-affinity receptors regulate platelet responses to phorbol diesters and teleocidin

We examined binding of ³H-phorbol dibutyrate (³H-PDBu) to gel filtered human platelets (GFP) and discovered that GFP possess two classes of receptors for phorbol diesters (PDE). High-affinity (HA) receptors, approximately 5000/GFP, bound ³H-PDBu with an apparent dissociation constant (K_D) of approximately 12 nM. Low-affinity receptors were approximately 5 times more numerous (2.4 × 10⁴/GFP) and had a 10-fold lower affinity for ³H-PDBu (apparent K_D = 115 nM). The potencies of phorbol myristate acetate (PMA) and PDBu paralleled their binding affinities to the PDE receptors. Teleocidin (Tel), although structurally distinct from PDE, competed with ³H-PDBu for its HA-receptors (K_I Tel = 1.9 nM). Binding of PDE to HA- or LA- receptors was rapid, reversible, saturable and stereospecific. The HA- and LA-receptors modulated different platelet responses. HA-receptors regulated the secretion of β-thromboglobulin from -granules and the release of N-acetyl-β-D-hexosaminidases from lysosomes. LA-receptors mediated both platelet aggregation and the release of serotonin from dense granules. This is the first demonstration of two physiologically active classes of PDE/Tel receptors in human platelets, and demonstrates that particular platelet responses may be directed by distinct classes of receptors for specific agonists.     

Comparative autoradiographic distribution of calcium channel antagonist binding sites for 1,4-dihydropyridine and phenylalkylamine in rat, guinea pig and human brain

1. The autoradiographic distribution of calcium channel antagonist binding sites for 1,4-dihydropyridine and phenylalkylamine has been investigated in rat, guinea pig and human brain.

2. 1,4-dihydropyridine([³H] (+) PN200-110) and phenylalkylamine ([³H] (−) D-888) binding sites are identically distributed in the brain of the three mammalian species studied.

3. High densities of calcium antagonist binding sites are present in brain areas enriched in synaptic contacts such as the hippocampus, cortex and striatum. Low to moderate densities of sites are found in other regions such as the thalamus, hypothalamus and brain stem.

4. These data demonstrate the existence of specific calcium antagonist binding sites in mammalian brain including man. These sites are discretely distributed with highest concentrations present in the hippocampus and cortex. Moreover, the similar distribution of binding sites for [³H](+)PN200-110 and [³H](−) D-188 suggests that 1,4-dihydropyridine and phenylalkylamine bind to the same receptor site complex in mammalian brain.     

Biological markers in juvenile depression

3H-p-Aminoclonidine binding to platelets of children and adolescents with major depressive disorder was compared to that of a healthy control population. Significantly higher alpha 2-adrenoceptor Kd and Bmax values were observed in the patient population. 3H-Dihydroalprenolol binding to lymphocyte membranes of the same patient population showed significantly higher beta-adrenoceptor Bmax values than controls. Control females had significantly higher beta-adrenoceptor Kd values than control males, and the female patients had significantly lower beta-adrenoceptor Kd values than control females. 3H-Imipramine binding to platelets of these patients showed significantly higher imipramine Kd values in patients with a suicide attempt, whereas the imipramine Bmax values were significantly increased in patients with major depressive disorder with or without a suicide attempt. We propose that increased platelet alpha 2-adrenoceptor Kd and Bmax values, together with increased platelet imipramine Kd values, may serve as possible biological markers for children and adolescents with major depressive disorder and a tendency toward suicide. Elevated platelet imipramine and lymphocyte beta-adrenoceptor Bmax values may be biological markers for juvenile depression, and decreased beta-adrenoceptor Kd values may be a biological marker for depression in young females.     

Functional and biochemical basis for multiple muscarinic acetylcholine receptors

1. The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity.

2. [³H]PZ has been used extensively to identify and characterize the putative M₁ (high affinity for PZ) mAChR subtype, which predominates in central nervous system (CNS) and ganglia.

3. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists.

4. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M₁ and M₂ subtypes.

5. Therapeutic and untoward effects of muscarinic drugs may be mediated by independent mAChR subpopulations which may be pharmacologically exploited to produce more highly selective as well as efficacious new drugs.