Esam E. El-Fakahany - US grants

brain cell; aging;

The process of aging is known to be accompanied by loss of memory. Due to the importance of central muscarinic receptors in memory, the present proposal aims at investigating the possible changes in brain muscarinic receptors that take place during aging. Dissociated rat brain cell aggregates and brain slices will be used to study the distribution and subtypes of muscarinic receptors in animals of different ages. In preliminary studies, the binding properties of muscarinic receptors in these preparations will be characterized, including tissue linearity, regional distribution and the kinetics of association and dissociation. Moreover, saturation and displacement experiments will be conducted using different radioactive muscarinic receptor ligands and displacers to assess the contribution of receptor subtypes and affinity states to total binding. These results will be compared with those obtained in brain homogenates. Subsequently, these experiments will be repeated in intact brain tissue prepared from rats of different ages. Consequently, the effect of age on the rates of synthesis and degradation of the receptors, the rate of agonist-induced receptor down-regulation, and antagonist-induced up-regulation, will be studied. Intact brain cells and slices will also be used to study muscarinic receptor-mediated responses, such as increases in cyclic GMP formation and phosphoinositide hydrolysis, in addition to decreases in cyclic AMP synthesis, since the first two responses can be elicited only in intact cells. Moreover, different agonist-mediated biochemical responses will be measured in brain cells and slices obtained from young and aged animals. Furthermore, the ability of high agonist concentrations to desensitize these responses will be assessed in animals of the different age groups. The proposed work is expected to increase our knowledge regarding the changes in the properties of brain muscarinic receptors which accompany aging.

Pirenzepine is a novel antagonist of muscarinic acetylcholine receptors, since it demonstrates considerable tissue selectivity. Although there is a substantial volume of literature related to the pharmacological and biochemical effects of pirenzepine, significantly less efforts have been devoted in order to characterize the nature of interaction of this drug with the muscarinic receptors and the relationship of its binding sites on the receptor to those of classical receptor antagonists. The present proposal is designed to provide better understanding of the features of binding of pirenzepine to brain muscarinic receptors. The relationship between pirenzepine binding sites in the brain and those of [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine will be investigated in detail. Several criteria will be used in order to define whether pirenzepine interacts with the binding sites of these ligands competitively or noncompetitively. These criteria will be assessed by studying: 1) the effect of pirenzepine on the saturation isotherms, in addition to the rates of association and dissociation of the ligands in rat brain, 2) displacement of increasing ligand concentrations by pirenzepine, 3) effects of preincubation with pirenzepine on subsequent ligand binding, and 4) the ability of pirenzepine to protect the binding sites of these ligands against irreversible alkylation. A second part of the project deals with investigating whether the hydrophilic nature of pirenzepine might explain its unique receptor binding features and pharmacological selectivity. To provide a satisfactory answer for this speculation, the binding profile of both pirenzepine and N-methylscopolamine, a quaternary muscarinic receptor ligand, will be compared. The proposed experiments include studying the regional distribution of the binding sites of both ligands and its relationship to [3H]quinuclidinyl benzilate binding sites. In addition, the functional selectively of N-methylscopolamine, if any, will be compared to that of pirenzepine in terms of their ability to block agonist-induced responses in mouse neuroblastoma cells. Taken together, the experiments suggested in the present proposal are expected to provide useful information concerning the mode of interaction of pirenzepine with brain muscarinic cholinergic receptors.

muscarinic receptor; receptor binding; neuropharmacologic agent; biological signal transduction; allosteric site; nitric oxide; inositol phosphates; ligands; mutant; drug design /synthesis /production; hydrolysis; antineoplastics; biological response modifiers; cell differentiation; chemical association; drug screening /evaluation; enzyme inhibitors; muscle relaxants; receptor coupling; receptor expression; tissue /cell culture; site directed mutagenesis;

muscarinic receptor; receptor expression; muscle contraction; second messengers; receptor binding; gallbladder; nitric oxide synthase; enzyme activity; receptor coupling; phosphatidylinositols; messenger RNA; alkylating agents; chemical kinetics; chemical association; hydrolysis; enzyme inhibitors; smooth muscle; adenylate cyclase; cyclic AMP; nitric oxide; solution hybridization; radiotracer; guinea pigs; transfection; tissue /cell culture;

DESCRIPTION (provided by applicant): This amended competing renewal for years 15-18 aims at investigating the mechanisms of the unconventional mode of activation of muscarinic acetylcholine receptors by the muscarinic receptor agonist, xanometine, in cultured cerebral cortical neurons. Xanomeline demonstrates unique functional selectivity in activating M1 muscarinic receptors that are important for learning ad memory. Thus, xanomeline-like muscarinic agonists are considered promising candidates for drug development for cholinergic replacement therapy in Alzeimer's disease. During the current funding period, we serendipitousiy discovered that xanomeline activates M1 muscarinic receptors in a long-lasting and wash-resistant manner. Moreover, we obtained strong evidence that this novel property of xanomeline is not due to an experimental artifact or nonspecific interaction with the cell membrane, but is rather the result of its avid binding to a specific and saturable site located on the M1 muscarinic receptor. We have also obtained preliminary evidence using pharmacology and computer modeling of receptor structure that this persistent interaction of xanometine with the M1 muscarinic receptor takes place at an "exosite" that is distinct from where conventional agonists bind. The proposed project aims at applying a combination of pharmacologic tools, computer modeling, sitedirected mutagenesis and structure-activity relationship studies in an iterative manner to delineate the molecular nature of the muscarinic receptor exosite and the interaction of xanomeline at that site. These studies are expected to have significant impact on the development of additional muscarinic receptor agonists that exhibit long-lasting receptor activation for therapy of Alzheimer's dementia. They may also illustrate novel mechanisms of activation of muscarinic receptors that might be applicable to other members of the G protein coupled receptor family.