Reduced corticotropin releasing factor binding sites in the frontal cortex of suicide victims.

Previous studies have provided evidence that corticotropin releasing factor (CRF) is hypersecreted in patients with major depression. This CRF hypersecretion is believed to contribute at least in part to hyperactivity of the hypothalamic-pituitary-adrenal axis in depressed patients. If CRF is chronically hypersecreted in depressed patients, then, due to down-regulation, a reduced number of CRF receptor binding sites should be present in patients with profound depressive disorder. To test this hypothesis, we measured the number and affinity of CRF binding sites in the frontal cortex of 26 suicide victims and 29 controls who died of a variety of causes. There was a marked (23%) reduction in the number of CRF binding sites in the frontal cortex of the suicide victims compared with the controls. These data are consistent with the hypothesis that CRF is hypersecreted in depression.

Adrenergic receptors in aging and Alzheimer's disease: decreased alpha 2-receptors demonstrated by [3H]p-aminoclonidine binding in prefrontal cortex.

Biochemical and pathological studies have described abnormalities in the brainstem locus coeruleus noradrenergic neurones in Alzheimer's disease (AD) and in aging. Loss of cortical noradrenergic fibers originating from the locus coeruleus may cause a decrease in presynaptic receptors or induce an increase in postsynaptic receptors, similar to "denervation supersensitivity" in animal models. Thus far it is unclear whether alpha 2-adrenergic receptors are affected in AD. In this study, we assessed the specific binding of [3H]p-aminoclonidine, an agonist at alpha 2-receptors and at imidazoline-preferring binding sites, to prefrontal cortex and other regions including hippocampus, temporal cortex, putamen and cerebellum from subjects with AD and aging controls. We particularly focused on the prefrontal cortex because of its relatively rich monoaminergic innervation and recent evidence suggesting involvement of noradrenergic mechanisms in cognition in aging nonhuman primates. The other regions, which are also innervated by noradrenergic fibers, were examined for comparison. Ligand binding to prefrontal cortex decreased with age of controls and was also significantly reduced by approximately 50% in AD subjects compared to age-matched controls. This change in AD was related to the maximum binding capacity (Bmax) rather than to an altered affinity of the ligand for the receptor. There were no significant changes in any of the other regions studied. Binding did not change with postmortem delay or with duration of tissue storage. We suggest that presynaptic alpha 2-receptors presumably labeled by [3H]p-aminoclonidine on noradrenergic synapses are those that are selectively decreased in the prefrontal cortex in AD and in aging.

Catecholamines inhibit lipid peroxidation in young, aged, and Alzheimer's disease brain.

Some catecholamines and indolamines inhibit lipid peroxidation. Recent studies indicate that catecholaminergic inhibition of lipid peroxidation may be receptor mediated in vivo and in cell cultures. Because oxidative stress is one of the hypothesized pathogenic mechanisms for neurodegenerative diseases, including Alzheimer's disease (AD), we hypothesized that catecholaminergic and indolaminergic inhibition of lipid peroxidation would be altered in AD as compared to age-matched non-AD. To test this hypothesis we studied the effect of a variety of neurotransmitters and their antagonists on ascorbate-stimulated lipid peroxidation in membrane fragment preparations derived from postmortem human brain. In this in vitro system, the inhibition of lipid peroxidation by dopamine and serotonin did not appear to be receptor mediated. Further, our findings indicate that there is no apparent effect of age or AD on the inhibition of lipid peroxidation by catecholaminergic and indolaminergic agents.

Binding of [3H]-p-aminoclonidine to alpha 2-adrenoceptor states plus a non-adrenergic site on human platelet plasma membranes.

Characterization of the binding of [3H]p-aminoclonidine ([3H]PAC) to purified plasma membranes from human platelets has revealed multiple binding sites. [3H]PAC identified site-1 in the picomolar affinity range (site-1 KD estimates ranged from 13 to 94 pM). Site-1 displayed a rank order of competition by various compounds for [3H]PAC, indicative of an alpha 2-adrenoceptor, and was sensitive to 0.1 mM GTP. [3H]PAC also identified a second site with nanomolar affinity (site-2 KD estimates ranged from 0.7 to 1.7 nM). In the presence of 0.1 mM GTP, site-2 was not diminished significantly. Also in contrast to site-1, site-2 displayed low affinity for yohimbine (YOH), (-)-epinephrine and (-)-norepinephrine (NE). Therefore, site-2 could not be an active alpha 2-adrenoceptor; instead it had properties similar to a previously reported imidazoline-preferring binding site. A third site (site-3) bound [3H]PAC with a KD for site-3 of 26.6 +/- 10.0 nM (SD). Site-3 had a rank order of competition by various compounds for 5 nM [3H]yohimbine ([3H]YOH) binding which was indicative of an alpha 2-adrenoceptor. (-)-NE competed for 5 nM [3H]YOH binding at two sites: site-1 Ki = 32 pM, site-3 Ki = 239 nM. Treatment with 0.1 mM GTP completely removed site-1 and transferred the competitive binding of (-)-NE to low affinity (Ki = 437 nM). Thus, site-3 appears to be a free alpha 2-adrenoceptor. Bmax estimates for untreated membranes, derived from simultaneous multi-experiment curve-fitting analyses, were site-1 = 36 +/- 29 fmol/mg plasma membrane protein, site-2 = 95 +/- 34 fmol/mg and site-3 = 154 +/- 35 fmol/mg. We are the first to report a site for [3H]PAC binding on platelets (site-2) with properties uncharacteristic of an adrenoceptor. This observation appears to be due to our use of purified plasma membrane and low ionic strength buffer. These studies relate to reports of increased binding of [3H]PAC to platelets from depressed patients.

Concurrent neuroborreliosis and Alzheimer's disease: analysis of the evidence.

Several recent reports have claimed a possible association between Borrelia burgdorferi infection and Alzheimer's disease (AD). Herein, we describe our search for additional evidence of neuroborreliosis in AD. Brain tissue from neuropathologically confirmed cases of AD was cultured for B burgdorferi using standard microbiologic methods. Material derived from culture was further examined using electron microscopy, direct immunofluorescence and acridine orange fluorescence. Previous studies have shown high titers of antiborrelia antibodies in CSF in all cases of confirmed neuroborreliosis; therefore, we tested CSF from neuropathologically confirmed cases of AD by indirect immunofluorescence and enzyme-linked immunoassay. In addition, imprint preparations from AD and control brain tissues were studied by direct immunofluorescence using a monoclonal antiborrelia antibody. Finally, a Western blot method was used to analyze protein extracts from cultures and AD brain tissue for the presence of borrelia antigen. Contrary to previous studies, our results do not support an association between infection with B burgdorferi and AD.

3H-spiroperidol binding in human temporal cortex (Brodmann areas 41-42) occurs at multiple high affinity states with serotonergic selectivity.

3H-Spiroperidol labels multiple high affinity states with serotonergic selectivity in human prefrontal cortex and with dopaminergic selectivity in human caudate and putamen. The characteristics of the binding of this ligand in human temporal cortex have not been previously described. Brodmann areas 41-42 in the temporal cortex are associated with primary auditory sensation and, in epileptics, with auditory hallucinatory experiences. We found that in this region of the human brain, antipsychotic ligands bind at multiple high affinity states, the majority of which exhibit serotonergic, rather than dopaminergic, selectivity. Dose-response data is best resolved by a three-site fit. Results of the co-analysis of dose-response data with saturation data indicates that two of the 3H-spiroperidol affinity states may represent the high and low affinity states of the serotonin (5HT)2 receptor, while a third affinity state may represent the 5-HT1A receptor.

3H-clonidine and 3H-yohimbine binding to glass fiber filters: implications for studies with platelet membranes.

3H-Clonidine and 3H-yohimbine were observed to bind to glass fiber filters. The binding was displaced by co-filtration with the corresponding non-radioactive ligand. Phentolamine and (--)-norepinephrine were ineffective in displacing either 3H-clonidine or 3H-yohimbine bound to filters. Failure to correct for filter binding resulted in an over-estimation of specific binding to platelet membranes. Certain published methodologies may have consequently misidentified up to 20% of the specific binding to platelets, that was actually due to displaced filter binding. Experimental conditions are described which eliminate filter binding. These results are significant for the interpretation of data from studies of platelet binding in depressed patients.

The low affinity component of [3H]clonidine binding is absent in the prefrontal cortex of presumptive suicide victims.

Dysfunction in alpha-adrenergic receptor systems has been implicated to be of etiologic significance in depressive illness. The alpha 2-adrenergic ligand [3H]clonidine labels at least two sets of binding sites in normal human prefrontal cortex. We now report that the lower affinity component of [3H]clonidine binding is apparently absent in the prefrontal cortices of presumptive suicide victims.

Guanyl nucleotides decrease antagonist binding at high affinity dopamine receptors.

Guanyl nucleotides induce a loss of high affinity [3H]spiroperidol binding sites in rat striatum that can be demonstrated by rate association, equilibrium dissociation and saturation analyses. [3H]Spiroperidol is an antagonist ligand for rat striatal dopamine receptors. These data therefore demonstrate clear cut effects of guanyl nucleotides on antagonist binding.

[3H]clonidine binds at multiple high affinity states in human prefrontal cortex.

[3H]Clonidine binds at particulate membrane fractions of human prefrontal cortex in a process that demonstrates high affinity, saturability, reversibility, alpha 2-adrenergic selectivity and the existence of multiple affinity states. At 37 degrees C maximal specific [3H]clonidine binding was briefly attained at 10 and lasted only until 15 min, while at 21 degrees C maximal binding was maintained from 20 to 90 min. At 21 degrees C, rate dissociation studies and saturation analyses were at least biphasic, and adrenergic competitors decreased [3H]clonidine binding with Hill coefficients less than 0.70. Analysis of these data showed at least two affinity states with apparent KDs of 0.34 and 6.0 nM, and the order in which ligands decreased [3H]clonidine binding was clonidine greater than (-)-epinephrine greater than (-)-norepinephrine greater than yohimbine greater than (+)-norepinephrine greater than (+/-)-isoproterenol greater than prazosin greater than serotonin.

Do antipsychotic drugs and serotonin down regulate [3H]-spiroperidol binding sites in human cortex?

[3H]-Spiroperidol binds at several specific sites in postmortem human prefrontal cortex at 21 degrees C. The overall selectivity of these sites is serotonergic. At 37 degrees C, there is a marked loss of specific [3H]-spiroperidol binding that occurs between 10 and 60 min. This loss of binding is not apparently due to 3H-ligand degeneration or metabolism, or to non-specific binding site degradation. The loss of binding can also be produced by serotonin (5-HT), haloperidol and chlorpromazine but not by prazosin. The rate of the loss of binding is dependent on 3H-ligand concentration and protein concentration. The loss of binding is correlated with a loss of serotonin selective [3H]-spiroperidol binding sites having a KD of approximately 0.04 nM. Therefore antipsychotic compounds and 5-HT appear to down-regulate a subset of antipsychotic binding sites in human prefrontal cortex.

Specific [3H]UK 14,304 binding in human cortex occurs at multiple high affinity states with alpha 2-adrenergic selectivity and differing affinities for GTP.

[3H]UK 14,034 is a full agonist at alpha 2-adrenergic receptors. Although the characteristics of the binding of the partial alpha 2-adrenergic agonists in postmortem human brain were known, the binding of [3H]UK 14,304 had not been studied in this tissue. Multi-site binding of this radiolabel had been reported in other tissues and guanosine triphosphate (GTP) had been shown to reduce [3H]UK 14,304 binding. We now report that [3H]UK 14,304 labels at least 2 specific binding sites in human brain that both have the characteristics of an alpha 2-adrenergic binding site. GTP decreases agonist binding at both of these sites, but with different potencies at each site.

Specific [3H]-spiroperidol binding sites in human prefrontal cortex: potential site multiplicity and overall serotonin-like selectivity.

[3H]-Spiroperidol specifically binds at sites in human prefrontal cortex. The binding of this ligand is apparently anomalous at 37 degrees C, with a substantial loss of specific binding occurring between 5 and 40 min incubation. However, at 21 degrees C, this loss of binding is not observed even at 60 min. At 21 degrees C, [3H]-spiroperidol binding in human prefrontal cortex is apparently occurring at multiple sites or multiple affinity states of single classes of sites, or at a combination of both. The overall selectivity is predominantly serotonergic, rather than dopaminergic.

Cations decrease specific [3H]-spiroperidol binding in human prefrontal cortex.

Ligand binding at many physiologically relevant receptors is regulated by divalent cations. To determine whether [3H]-spiroperidol binding sites in prefrontal cortex might be physiologically relevant receptors, we examined the effect of ions on the binding of this ligand in postmortem human prefrontal cortex. Our results indicate that several cations decreased [3H]-spiroperidol binding in a dose-dependent fashion. Of these, Cd++ and Zn++ were the most able to decrease [3H]-spiroperidol binding with IC50 of 5.5 +/- 2.4 X 10(-6)M and 5.6 +/- 1.1 X 10(-5)M respectively. These findings indicate that [3H]-spiroperidol may bind at physiologically relevant receptors in human prefrontal cortex.

Pharmacokinetics and central nervous system effects of the novel dopamine D3 receptor antagonist GSK598809 and intravenous alcohol infusion at pseudo-steady state.

GSK598809 is a novel selective dopamine D(3) receptor antagonist, currently in development for the treatment of substance abuse and addiction. In a blinded, randomized, placebo-controlled study, effects of single oral doses of 175 mg GSK598809 were evaluated in healthy volunteers. Pharmacokinetics, central nervous system (CNS) effects and potential for interactions with alcohol were evaluated, using an alcohol infusion paradigm and analysis of eye movements, adaptive tracking, visual analogue scales, body sway, serum prolactin and verbal visual learning test. Adverse effects of GSK598809 included headache, dizziness and somnolence. Plasma concentration of GSK598809 was maximal 2-3 hours postdose and decreased with a half-life of roughly 20 hours. CNS effects were limited to prolactin elevation and decreased adaptive tracking. Co-administration of GSK598809 and alcohol did not affect alcohol pharmacokinetics, but caused a 9% decrease of C (max) and a 15% increase of AUC of GSK598809. CNS effects of co-administration were mainly additive, except a small supra-additive increase in saccadic reaction time and decrease in delayed word recall. In conclusion, GSK598809 causes elevation of serum prolactin and a small decrease in adaptive tracking performance. After co-administration with alcohol, effects of GSK598809 are mainly additive and the combination is well tolerated in healthy volunteers.

Serum protein leakage in aged human brain and inhibition of ligand binding at alpha 2-adrenergic and cholinergic binding sites.

Serum proteins are known to extravasate into the brain parenchyma in senile and presenile dementia (Glenner: Hum. Pathol. 16:433-435, 1986; Wisniewski and Kozlowski: Ann. NY Acad. Sci. 396:119-129, 1982). We have recently demonstrated that human serum Cohn fraction IV (alpha-globulin enriched) inhibits ligand binding at putative dopamine and serotonin2 receptors labeled by [3H]spiroperidol in human brain (Andorn, Pappolla, Fox, Klemens, and Martello: Proc. Natl. Acad. Sci. USA 83:4572-4575, 1986). We now demonstrate that serum proteins can be identified in the neuropil and in neuronal cell bodies in normal aged brain, that alpha-globulin-enriched fractions inhibit ligand binding at alpha 2-adrenergic and muscarinic binding sites in human brain as well, and that serum proteins can be identified within neuronal cytoplasm and axons.

[3H]Spiroperidol binding in rat striatum: two high-affinity sites of differing selectivities.

[3H]Spiroperidol binding to homogenates of rat striatum is saturable and shows either monophasic or biphasic saturation isotherms under specified conditions. In poorly washed membrane fragment preparations, saturation isotherms of [3H]spiroperidol binding are monophasic, revealing an apparently homogeneous set of sites with KD 0.6 +/- 0.3 nM and density 440 +/- 80 fmol/mg protein. However, equilibrium displacement studies of [3H]spiroperidol binding at this site indicate an alpha-adrenergic component in addition to the previously described dopaminergic component. In thoroughly washed membrane fragment preparations, saturation isotherms are clearly biphasic, showing an additional high-affinity site with an approximate KD of 24 +/- 10 pM and an approximate density of 110 +/- 20 fmol/mg protein at a protein concentration of 2.0 mg/ml. Selectivity at this site appears classically dopaminergic, suggesting that the lower affinity site is the primary source of the alpha-adrenergic component of spiroperidol binding.

Alterations in adrenergic receptors of frontal cortex and cerebral microvessels in Alzheimer's disease and aging.

Biochemical and pathological abnormalities are evident in the noradrenergic innervation of the cerebral cortex in Alzheimer's Disease (AD), and there is also a decline in aging, which may lead to changes in adrenergic receptors. To assess this question, we analyzed adrenergic receptor subtypes by ligand binding methods in prefrontal cortex and brain microvessels from subjects with AD and aging controls. Ligand binding to adrenoceptors and their subtypes did not change with postmortem delay in obtaining tissues. alpha 1-adrenergic receptors of the frontal cortex did not correlate with age but there was a small (approximately 25%), though significant, reduction in AD subjects. In cerebral microvessels, there were no changes in these receptors. alpha 2-receptors of the cortex significantly declined with age in controls and were also significantly reduced by approximately 50% in AD subjects. However, in cerebral microvessels alpha 2-adrenergic receptors were significantly increased by approximately 60% in AD. We suggest that presynaptic alpha 2-adrenoceptors on noradrenergic synapses may be those that are selectively decreased in the prefrontal cortex in AD. Total beta-receptors in cortex did not correlate with age, nor were they altered in AD. However, beta 1-receptors were decreased but beta 2-receptors were significantly increased in AD, indicating a change in the relative ratio of beta 1/beta 2-receptors. Similarly, beta 2-receptors of cerebral microvessels were significantly increased in AD. These changes suggest receptor "up-regulation" in response to noradrenergic denervation in AD and may reflect functional changes at the blood-brain barrier.

Ascorbate-stimulated lipid peroxidation in human brain is dependent on iron but not on hydroxyl radical.

Lipid peroxidation has been postulated as a cause of neuronal damage in both aging and neurodegenerative disease. In studies of neurodegenerative disease, iron, iron plus ascorbate, or ascorbate alone has been used to stimulate lipid peroxidation. The mechanism by which ascorbate stimulates lipid peroxidation in human brain is unclear. The studies reported here were performed to determine whether ascorbate-stimulated lipid peroxidation in human brain membrane fragments was dependent on iron, superoxide radical, hydrogen peroxide, or hydroxyl radical. Our findings indicate that although ascorbate-stimulated lipid peroxidation in brain is iron dependent, it apparently does not require hydrogen peroxide or hydroxyl radical, but rather is dependent on superoxide radical formation.