MUSCARINIC POTENTIATION OF EXCITATORY AMINO ACID-EVOKED DOPAMINE RELEASE IN MESENCEPHALIC CELLS - SPECIFICITY FOR THE NMDA RESPONSE AND ROLE OF INTRACELLULAR MESSENGERS

Of the five cloned muscarinic receptor subtypes, dopamine (DA) neurons in the substantia nigra and ventral tegmental areas have been shown t o be selectively enriched with the mRNA for the m5 subtype, suggesting that muscarinic modulation of DA neurons may have a distinct pharmaco logy. In the present study we have used dissociated cell cultures of f etal rat ventral mesencephalon to characterize muscarinic modulation o f DA neurons. [H-3]DA release stimulated by activation of N-methyl-D-a spartate (NMDA) receptors was potentiated by carbachol, a mixed muscar inic-nicotinic agonist, and by oxotremorine-M, a muscarinic agonist. N either carbachol nor oxotremorine-M had an effect on [H-3]DA release e voked by the non-NMDA agonists, kainate or quisqualate. A nicotinic ag onist, DMPP, had no effect on NMDA-stimulated release. Potentiation of NMDA-stimulated [H-3]DA release by oxotremorine-M was inhibited by th e broad spectrum muscarinic antagonist, QNB, and by low concentrations of a putative Ml antagonist, pirenzepine, while much higher concentra tions of a purported M2 antagonist, AF-DX 384, were required to revers e the oxotremorine-M effect. The muscarinic antagonist, 4-DAMP, was ac tive in a concentration range between that required for pirenzepine an d AF-DX 384. Further experiments examined intracellular messenger mech anisms coupled to the muscarinic receptors modulating NMDA-stimulated [H-3]DA release. In contrast to oxotremorine-M, two muscarinic agents with only weak partial agonism with respect to phosphoinositide turnov er, pilocarpine and arecoline, had no effect on NMDA-stimulated [H-3]D A release. Potentiation of NMDA-stimulated [H-3]DA release by oxotremo rine-M was inhibited by staurosporine, an inhibitor of protein kinase C, but was unaffected by H8, an inhibitor of cAMP- and cGMP-dependent protein kinases. Pretreatment of cultures with pertussis toxin did not alter oxotremorine-M potentiation of the NMDA response. Our results i ndicate that the muscarinic receptors responsible for potentiation of NMDA-stimulated [H-3]DA release in mesencephalic cultures are linked t o phosphoinositide hydrolysis and protein kinase C activation via pert ussis toxin insensitive G proteins. The data are also consistent with the identification of these receptors as the m5 subtype of cloned rece ptor, on the basis of the relative potency of muscarinic agonists and antagonists in this system, although involvement of ml receptors canno t be excluded solely on this basis. Taken together with previous repor ts showing selective localization of m5 receptor mRNA in regions conta ining DA cell bodies, these studies suggest that use of dissociated ce ll cultures may thus be an appropriate model for studying direct modul ation of dopaminergic neurons by cholinergic agents interacting with p utative m5 receptors. (C) 1993 Wiley-Liss, Inc.