Donna D. Flynn

Characterization of l‐[3H]Nicotine Binding in Human Cerebral Cortex: Comparison Between Alzheimer's Disease and the Normal

Abstract: Putative nicotine receptors in the human cerebral cortex were characterized with l‐[3H]nicotine. l‐[3H]Nicotine binding was enhanced by the addition of Ca2+ and abolished in the presence of Na3EDTA. Association and dissociation of the ligand were rapid at 25°C with t1/2 values of 2 and 3 min, respectively. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 5.6 nM and a Hill coefficient of 1.05. There was no effect of postmortem interval on the density of binding sites assayed up to 24 h in rat frontoparietal cortex. Nicotine binding in human cortical samples was also unaltered by increasing sampling delay. In human cortical membranes, binding site density decreased with normal aging. Receptor affinity and concentration in samples of frontal cortex (Brodmann area 10) from patients with Alzheimers disease were comparable to age‐matched control values. Samples of infratemporal cortex (Brodmann area 38) from patients with Alzheimers disease had a 50% reduction in the number of l‐[3H]nicotine sites. Choline acetyltransferase activity was significantly decreased in both cortical areas. Enzyme activities in the temporal pole were reduced to 20% of control values. These data indicate that postsynaptic nicotine receptors are spared in the frontal cortex in Alzheimers disease. In the infratemporal cortex, significant numbers of receptors remain despite the severe reduction in choline acetyltransferase activity. Replacement therapy directed at these sites may be warranted in Alzheimers disease.

Cocaethylene: a unique cocaine metabolite displays high affinity for the dopamine transporter.

Abstract: Concurrent cocaine and alcohol use is common practice in the general population, as indicated by recent prevalence studies. In the presence of ethyl alcohol, cocaine is metabolized to its ethyl homolog, cocaethylene. The transesterification of cocaine and ethanol to cocaethylene takes place in the liver and represents a novel metabolic reaction. Cocaethylene was detected in postmortem blood, liver, and neurological tissues in concentrations equal to and sometimes exceeding those of cocaine. In vitro binding studies demonstrate that cocaethylene has a pharmacological profile similar but not identical to that of cocaine at monoamine transport sites assayed in the human brain. Cocaethylene was equipotent to cocaine at inhibiting [3H]mazindol binding to the dopamine transporter. The blockade of dopamine reuptake in the synaptic cleft by cocaethylene may account for the enhanced euphoria associated with combined alcohol and cocaine abuse.

Differential Regulation of Molecular Subtypes of Muscarinic Receptors in Alzheimer's Disease

Abstract: Molecular subtypes of muscarinic receptors (m1–m5) are novel targets for cholinergic replacement therapies in Alzheimers disease. However, the status of these receptors in human brain and Alzheimers disease is incompletely understood. The m1–m5 receptors in brains from control subjects and Alzheimers disease patients were examined using a panel of specific antisera and radioligand binding. Quantitative immunoprecipitation demonstrated a predominance of the m1, m2, and m4 receptor subtypes in cortical and subcortical regions in control subjects. In Alzheimers disease, normal levels of m1 receptors measured by radioligand binding contrasted with decreased m1 receptor immunoreactivity, suggesting that the m1 receptor is altered in Alzheimers disease. The m2 immunoreactivity was decreased, consistent with the loss of m2 binding sites and the location of this receptor subtype on presynaptic cholinergic terminals. The m4 receptor was up‐regulated significantly and may offer a target for new memory‐enhancing drugs. Differential alterations of molecular subtypes of muscarinic receptors may contribute to the cholinergic component of Alzheimers disease dementia.

Visualizing Dopamine and Serotonin Transporters in the Human Brain with the Potent Cocaine Analogue [125I]RTI-55: In Vitro Binding and Autoradiographic Characterization

Abstract: The cocaine analogue RTI‐55 was evaluated as a probe for in vitro labeling and localization of dopamine and serotonin transporters after death in the human brain. Kinetic, saturation, and competition binding experiments indicated complex interactions of the radioligand with the identification of multiple recognition sites. In membrane binding assays, the association of [125I]RTI‐55 at 25°C to putamen membranes was monophasic. In contrast, dissociation of [125I]RTI‐55 occurred in two phases with t1/2 values of 9.4 and 36.5 min, respectively. Saturation analysis of [125I]RTI‐55 binding demonstrated two binding sites in the human putamen with KD values of 0.10 ± 0.02 and 1.81 ± 0.46 nM. The binding of [125I]RTI‐55 was displaced by a wide range of cocaine analogues and monoamine uptake inhibitors. The rank order of potency demonstrated in competition assays with human putamen membranes indicates that the radioligand labels cocaine recognition sites on the dopamine transporter (mazindol > GBR 12909 > GBR 12935 > paroxetine > nisoxetine > desipramine ≥ fluoxetine > citalopram). In the human occipital cortex, [125I]RTI‐55 recognized multiple binding sites with KD values of 0.02 ± 0.01 and 4.18 ± 0.46 nM. The rank order of potency for inhibition of [125I]RTI‐55 binding to cerebral cortex membranes (paroxetine > citalopram > GBR 12909 ≥ mazindol ≥ nisoxetine > benztropine) suggests that [125I]RTI‐55 labels the serotonin transporter in the human occipital cortex. Autoradiographic mapping of [125I]RTI‐55 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderately elevated densities of [125I]RTI‐55 binding sites were also seen throughout the thalamus, hypothalamus, and substantia nigra. [125I]RTI‐55 binding sites were prevalent throughout the cerebral cortex and amygdala. In autoradiographic studies, the addition of the selective serotonin transport blocker citalopram completely prevented [125I]RTI‐55 labeling in the thalamus, hypothalamus, and throughout most of the cerebral cortex. In the presence of citalopram, [125I]RTI‐55 binding site densities remained elevated over the striatum and substantia nigra, with selective residual labeling also seen in the external segment of the globus pallidus and the lateral nucleus of the amygdala. These results demonstrate that in the human brain, [125I]RTI‐55 labels multiple recognition sites on dopamine and serotonin transporters.

Characterization and Distribution of Transferrin Receptors in the Rat Brain

Abstract: Transferrin receptors were characterized with 125Iferrotransferrin on membrane fractions prepared from the rodent forebrain. The distribution of transferrin receptors in the rat brain was investigated further by in vitro autoradiography. Saturation binding analysis revealed an apparent single class of sites with a dissociation constant of 2 nM and a binding site density of 15 pmol/g. The Hill coefficient derived from these data was 1.05. indicating the absence of cooperativity and that 125I‐ferrotransferrin binds to a single class of sites. Estimates of the kinetically determined Kd for forebrain membranes were within the 2–4 nM range, in agreement with the equilibrium measurements. Apotransferrin and ferrotransferrin competitively displaced the binding of 125I‐ferrotransferrin, while ferritin, albumin, and cytochrome c failed to compete for the binding site. Ceruloplasmin, the copper transport protein, was a weak inhibitor of 125I‐ferrotransferrin binding. Autoradiographic localization studies demonstrate a heterogeneous distribution of transferrin receptors in the rat brain. Transferrin receptor densities were markedly elevated over the cerebral cortex and the hippocampus. Moderate to high 125I‐ferrotransferrin binding was also apparent throughout areas involved in motor functions, including the caudate‐putamen, the nucleus accumbens, the substantia nigra, the red nucleus, and the cerebellum.

The M5 (m5) receptor subtype: Fact or fiction?

By comparison to the other subtypes of muscarinic receptors, very little is known about the binding properties, locations, mechanisms and physiological functions of the M5 (m5)* receptor subtype. Studies of the m5 receptor have been hampered by the lack of m5-selective ligands or antibodies and a source that endogenously expresses predominantly the m5 receptor subtype. We have developed a pharmacological labeling strategy using the non-selective muscarinic antagonist [3H]NMS, in the presence of muscarinic antagonists and toxins in green mamba venom to occlude the m1-m4 receptor subtypes, to selectively label the m5 receptor subtype. This m5-selective labeling approach, along with those developed for the other four receptor subtypes, has permitted for the first time a comparison of the relative expression levels and anatomical localizations of the five muscarinic receptor subtypes in the brain. The distribution profile of the m5 receptor is distinct from the other four subtypes and is enriched in the outer layers of the cortex, specific subfields of the hippocampus, caudate putamen, olfactory tubercle and nucleus accumbens. These studies have also demonstrated that the levels of m5 receptor protein expression are apparently higher and more widespread than anticipated from previous in situ hybridization and immunoprecipitation studies. Taken together, the results suggest a unique and potentially physiologically important role for the m5 receptor subtype in modulating the actions of acetylcholine in the brain.

Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: Implications for cholinergic-based therapies

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimers disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.

Attenuation of muscarinic receptor-G-protein interaction in Alzheimer disease

Cortical M1 muscarinic receptor-G-protein coupling, high-affinity, guanine nucleotide-sensitive agonist binding (Flynn et al., 1991; Warpman et al., 1993) and muscarinic receptor-stimulated [3H]PIP2 hydrolysis (Ferrari-DiLeo and Flynn, 1993) are known to be defective in Alzheimer disease. Whether this defect reflects an alteration in the M1 muscarinic receptor, its respective guanine nucleotide binding (G) protein or both is not known. This study compares the number and both basal and muscarinic receptor-mediated function of G-proteins in synaptosomal membranes from cerebral cortical samples of age-matched control subjects and Alzheimer disease patients. Immunoblotting with anti-G alpha q/11 and anti-G beta antibodies demonstrated no alteration in the number of these G-protein subunits in Alzheimer disease. Basal [35S]GTP gamma S binding and hydrolysis of [gamma-32P]GTP by high-affinity GTPase also were not significantly altered in Alzheimer disease compared to control membrane samples. However, muscarinic agonist-stimulated GTP gamma S binding and GTP hydrolysis were significantly reduced (80-100%) in Alzheimer disease cortical samples. Diminished agonist-stimulated GTP gamma S binding and GTP hydrolysis correlated with the loss of guanine nucleotide-sensitive, high-affinity agonist binding (KL/KH) ratio) to the M1 receptor subtype. These data provide further evidence for the loss of muscarinic receptor-G protein coupling in Alzheimer disease and support the hypothesis that muscarinic receptor-mediated cortical activation may be compromised in Alzheimer disease.

Pharmacological screen for activities of 12=hydroxyibogamine: a primary metabolite of the indole alkaloid ibogaine

The purported efficacy of ibogaine for the treatment of drug dependence may be due in part to an active metabolite. Ibogaine undergoes first pass metabolism and isO-demethylated to 12-hydroxyibogamine (12-OH ibogamine). Radioligand binding assays were conducted to identify the potency and selectivity profiles for ibogaine and 12-OH ibogamine. A comparison of 12-OH ibogamine to the primary molecular targets identified previously for ibogaine demonstrates that the metabolite has a binding profile that is similar, but not identical to the parent drug. Both ibogaine and 12-OH ibogamine demonstrated the highest potency values at the cocaine recognition site on the 5-HT transporter. The same rank order (12-OH ibogamine > ibogaine), but lower potencies were observed for the [3H]paroxetine binding sites on the 5-HT transporter. Ibogaine and 12-OH ibogamine were equipotent at vesicular monoamine and dopamine transporters. The metabolite demonstrated higher affinity at the kappa-1 receptor and lower affinity at the NMDA receptor complex compared to the parent drug. Quantitation of the regional brain levels of ibogaine and 12-OH ibogamine demonstrated micromolar concentrations of both the parent drug and metabolite in rat brain. Drug dependence results from distinct, but inter-related neurochemical adaptations, which underlie tolerance, sensitization and withdrawal. Ibogaine’s ability to alter drug-seeking behavior may be due to combined actions of the parent drug and metabolite at key pharmacological targets that modulate the activity of drug reward circuits.

Diminished muscarinic receptor-stimulated [3H]-PIP2 hydrolysis in Alzheimer's disease.

The functional integrity of the cortical muscarinic receptor (MR)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C signalling pathway was assessed in Alzheimer disease (AD) and age-matched control subjects. There was no difference in the basal hydrolysis of [3H]-PIP2 to [3H]-inositol phosphates between control and AD membrane preparations. However, muscarinic agonist-stimulated PIP2 hydrolysis was significantly diminished in the AD cases. Diminished agonist-stimulated PIP2 hydrolysis correlated with the loss in high affinity agonist binding (KL/KH ratio) to the M1 muscarinic receptor subtype in the disease. These data further support the hypothesis that muscarinic receptor-mediated signal transduction is altered in AD, and that the defect lies at the level of muscarinic receptor-G protein/effector coupling.