Joint Meeting 

of the International Society for Neurochemistry (ISN)
and the European Society for Neurochemistry (ESN),
to be held in Berlin, Germany from August 8 - 14, 1999

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ESN Award Lectures Berlin '99

Award Lecture 1:
JAN JAKUBIK
"ASPECTS OF THE REGULATION AND MOLECULAR ASSEMBLY OF MUSCARINIC ACETYLCHOLINE RECEPTORS"
J. Jakubík, National Institutes of Health, Bethesda, MD 20892, U.S.A., and Institute of Physiology, Academy of  Science, 14220 Prague, Czech Republic

Several neuromuscular blockers are negative allosteric modulators of muscarinic acetylcholine receptors and diminish their affinity for agonists and antagonists. The neuromuscular blocker alcuronium has been found to enhance the affinity muscarinic receptors for certain antagonists (1), but the question remained open as to whether it is also possible to allosterically enhance the affinity of muscarinic receptors for their agonists. We investigated interactions between five allosteric
modulators and twelve muscarinic agonists on the M1 - M4 muscarinic receptor subtypes and discovered that each of the modulators (alcuronium, brucine,
eburnamonine, strychnine and vincamine) enhanced the affinity of at least one subtype of muscarinic receptors for at least one agonist (2). The possibility to enhance the affinity for acetylcholine appears important for the development of pharmacological approaches to muscarinic receptors. We have also found that muscarinic receptors can be activated from their allosteric binding sites, as shown by changes in the production of cyclic AMP or inositol phosphates (3). The nature of receptor activation from the allosteric site is different from that evoked from the classical binding site (4). It seems clear, however, that the binding of allosteric ligands affects the conformation of the whole receptor, not just the conformation of the allosteric and classical binding sites. In the most recent work, we developed a sandwich-ELISA strategy to study the association of the N- and C-terminal fragments of muscarinic receptors into functional receptor complexes (5). This method is likely to become useful also  in the study of the allosteric properties of muscarinic receptors.

(1) Tucek S. et al.: Mol. Pharmacol. 38, 674-680, 1990.
(2) Jakubík J. et al.: Mol. Pharmacol. 52, 172-179, 1997.
(3) Jakubík J. et al.: Proc. Natl. Acad. Sci. USA 93, 8705-8709, 1996.
(4) Jakubík J. et al.: Mol. Pharmacol. 54, 899-906, 1998.
(5) Jakubík J.,  Wess J.:  J. Biol. Chem. 274, 1349-1358, 1999.

Oxidative stress and deficiency of the antioxidant glutathione in brain appear to be connected with several diseases characterized by neuronal loss. In order to study peroxide detoxification and the glutathione metabolism of brain cells, cultured neurons and astroglial cells were used. Neuron-rich cultures cleared H₂O₂ more slowly from the incubation buffer than confluent astroglial cultures. However, if the differences in protein content were taken into consideration the ability to dispose of H₂O₂ of the cells in the two culture types was identical. The clearance rate by neurons for H₂O₂ was strongly reduced by inhibition of catalase, a situation contrasting with that in astroglial cultures. This indicates that for the rapid clearance of H₂O₂ by neurons the glutathione system cannot functionally compensate for the loss of the catalase reaction. Compared to astroglial cultures, lower specific activities of glutathione peroxidase and of enzymes regenerating NADPH as well as a lower content of glutathione contribute to the lower efficiency of the neuronal glutathione system. The content of glutathione in neurons was doubled within 24 h, if neurons were co-incubated with astroglial cells. This astroglia-mediated increase in neuronal glutathione was suppressed by inhibition of the astroglial ectoenzyme g-glutamyl transpeptidase (gGT), which generates CysGly from glutathione. This dipeptide served in micromolar concentrations as precursor of neuronal glutathione, indicating the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme gGT uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly as precursor for neuronal glutathione.
 

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