Anne C. Andorn

Adrenergic Receptors in Aging and Alzheimer's Disease: Increased β2-Receptors in Prefrontal Cortex and Hippocampus

Abstract: Loss of pigmented noradrenergic locus ceruleus neurons occurs in Alzheimers disease (AD) and, to a lesser extent, in aging. We studied β‐adrenergic receptors and their subtypes, β1 and β2, by the specific binding of 125I‐pindolol to particulate membrane preparations from prefrontal cortex, hippocampus, putamen, and cerebellum and to sections from frontal cortex by in vitro autoradiography. In prefrontal cortex from controls, numbers of total β‐ and β‐adrenoceptors did not significantly correlate with age, but number of β1‐adrenoceptors showed a weak but significant negative correlation. Binding in tissue particulate preparations to total β‐receptors did not reveal significant differences in samples from prefrontal cortex between AD subjects and age‐matched controls. However, β1‐adrenoceptors were decreased and β2‐adrenoceptors were increased in number by ∼30‐50% in AD subjects. Thus, the relative ratio of β/β2‐receptors was decreased in AD. Binding by in vitro receptor autoradiography performed in a subset of samples of frontal cortex also showed β2‐adrenoceptors, and less consistently total β‐and β1recep‐tors, to be increased significantly in number in cortical laminae II, III, IV, and V of tissue sections from AD subjects. In these subjects, number of locus ceruleus cells and norepinephrine concentrations in putamen and frontal cortex were markedly reduced compared with values in controls. In the hippocampus, total β‐ and both β2‐ and β1‐adrenoceptors were increased in number in AD. In contrast, in the putamen. where β‐receptors predominate, total β‐ and β1‐receptors were significantly decreased in number with no consistent change in content of β2‐receptors in AD. There were no significant changes in the cerebellum. Specific pindolol binding was not affected by interval between death and sampling of tissue at autopsy. Our results indicate selective changes in number of β‐receptors in AD. These changes in the cortex and hippocampus suggest receptor upregulation in response to noradrenergic deafferentation from the locus ceruleus or may simply reflect glial proliferation in AD.

Binding of [3H]-p-aminoclonidine to α2adrenoceptor 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.

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 Kp 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 a‐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 α‐adrenergic component of spiroperidol binding.

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

Abstract: 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.

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.

[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.

Specific [3H] UK14, 304 binding in human cortex occurs at multiple high affinity states with alpha2-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.

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.

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

Abstract3H-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.

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.