Thomas W. Vickroy

Effects of bilateral ibotenate-induced lesions of the nucleus basalis magnocellularis upon selective cholinergic biochemical markers in the rat anterior cerebral cortex

The relationship of choline acetyltransferase (ChAT) activity and high affinity binding of the potent and selective sodium-dependent choline uptake inhibitor [3H]hemicholinium-3 ([3H]HC-3) to high-affinity binding of the muscarinic agonist [3H](+)-cis-methyldioxolane ([3H](+)CD), the putative M1 selective antagonist [3H]pirenzepine ([3H]PZ) and the classical antagonist [3H](-)-quinuclidinyl benzilate ([3H](-)QNB) in homogenates of the rat neocortex was studied. ChAT activity was 42% lower in rats with ibotenate-induced lesions of the nucleus basalis magnocellularis (nbm) when compared to controls, and [3H]HC-3 binding was similarly reduced by 44%. However, equilibrium dissociation constants (Kd values) for [3H]HC-3 (0.8-1.0 nM), [3H](-)QNB (11-24 pM), [3H]PZ (4.0-4.3 nM) and [3H](+)CD (2.1-2.9 nM) were each unchanged. Mean Bmax values (total binding site densities) for [3H](+)CD were significantly altered in both hemispheres of the anterior cerebral cortex, showing a 25% reduction in the number of sites which display the highest affinity conformation for this potent muscarinic agonist. The decreased ChAT activity and [3H]HC-3 binding after nbm lesions were associated with only slight reductions in putative M1 muscarinic site density (14%) and [3H](-)QNB binding site density (13%). Thus, it appears that while [3H]PZ and [3H](-)QNB label predominantly postsynaptic muscarinic binding sites, a significant number of sites labeled by [3H](+)CD may be associated with presynaptic cholinergic nerve terminals. These data suggest that cholinergic input differentially regulates the drug binding sites of anterior cerebral cortical muscarinic receptors, exerting a substantial effect upon the highest affinity conformational state for agonists.

Quantitative light microscopic autoradiography of [3H]hemicholinium-3 binding sites in the rat central nervous system: a novel biochemical marker for mapping the distribution of cholinergic nerve terminals

The distribution of specific [3H]hemicholinium-3 ([3H]HC-3) binding sites throughout the rat forebrain was studied by means of quantitative light microscopic autoradiography. Tissue sections were labeled with 2.5 nM[3H]HC-3, apposed to tritium-sensitive film for 2 months and analyzed by computer-assisted densitometry. Regions of intense [3H]HC-3 labeling include the caudate-putamen, nucleus accumbens, olfactory tubercle, amygdala, habenula and the granule cell layer of the dentate gyrus. Little or no specific binding was detected in the corpus callosum, a white matter region. This distribution of specific [3H]HC-3 binding sites is compatible with a selective labeling of central cholinergic nerve terminals.

Differential regulation of high-affinity agonist binding to muscarinic sites in the rat heart, cerebellum, and cerebral cortex

The muscarinic agonist [3H]cismethyldioxolane ([3H]CD) was used to characterize the effects of regulators upon high-affinity agonist binding sites of the rat heart, cerebral cortex and cerebellum. Comparative studies with sodium ions (Na+), magnesium ions (Mg++), N-ethylmaleimide (NEM) and the guanine nucleotide Gpp(NH)p revealed tissue-specific effects. Mg++ preferentially enhanced while Gpp(NH)p and NEM reduced high-affinity [3H]CD binding in the heart and cerebellum. By comparison NEM enhanced high-affinity agonist binding in the cerebral cortex while Gpp(NH)p and Mg++ had little or no effect. Kinetic studies support an allosteric mechanism for these effects and provide further evidence for muscarinic receptor subtypes in mammalian tissues.

Functional and biochemical basis for multiple muscarinic acetylcholine receptors

The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity. [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. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M1 and M2 subtypes. 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.

Autoradiographic localization of muscarinic agonist binding sites in the rat central nervous system with (+)-cis-[3H]methyldioxolane

(+)-cis-[3H]Methyldioxolane ((+)-[3H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[3H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[3H]CD states and binding sites for the muscarinic antagonists [3H]pirenzepine (r = −0.79) and (-)-[3H]quinuclidinyl benzilate (r = −0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.

Differential Regulation of Putative M1/M2 Muscarinic Receptors: Implications for Different Receptor-Effector Coupling Mechanisms

This chapter focuses upon results from our recent studies concerning the drug specificities and regulatory profiles of high-affinity muscarinic agonist binding site subtypes. The data are presented in conjunction with other evidence for muscarinic receptor subtypes and a significant portion of the discussion emphasizes the potential involvement of distinct receptor-effector coupling mechanisms for these subtypes. Other reviews relevant to this topic are currently available (1–4).

Identification of Putative M1 Muscarinic Receptors Using [3H]Pirenzepine: Characterization of Binding and Autoradiographic Localization in Human Stellate Ganglia

The advent of selective antimuscarinic drugs such as pirenzepine has led to increased acceptance of the concept of distinct subclasses of muscarinic recegtors. This chapter focuses upon our studies of [3H]pirenzepine ([3H]PZ), which we suggested is an effective ligand for the investigation of putative M1 receptor- effector mechanisms (26–31). Data indicating that [3H]PZ labels putative M1 muscarinic receptors with high affinity is discussed in relation to the growing body of evidence which supports the M1/M2 hypothesis. Emphasis is placed upon our studies of human stellate ganglia. Several relevant reviews have recently appeared in the literature (3, 22, 23, 29).