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 to be selectively enriched with the mRNA for the m5 subtype, suggesting that muscarinic modulation of DA neurons may have a distinct pharmacology. In the present study we have used dissociated cell cultures of fetal rat ventral mesencephalon to characterize muscarinic modulation of DA neurons. [H-3]DA release stimulated by activation of N-methyl-D-aspartate (NMDA) receptors was potentiated by carbachol, a mixed muscarinic-nicotinic agonist, and by oxotremorine-M, a muscarinic agonist. Neither carbachol nor oxotremorine-M had an effect on [H-3]DA release evoked by the non-NMDA agonists, kainate or quisqualate. A nicotinic agonist, DMPP, had no effect on NMDA-stimulated release. Potentiation of NMDA-stimulated [H-3]DA release by oxotremorine-M was inhibited by the broad spectrum muscarinic antagonist, QNB, and by low concentrations of a putative Ml antagonist, pirenzepine, while much higher concentrations of a purported M2 antagonist, AF-DX 384, were required to reverse the oxotremorine-M effect. The muscarinic antagonist, 4-DAMP, was active in a concentration range between that required for pirenzepine and AF-DX 384. Further experiments examined intracellular messenger mechanisms 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 turnover, pilocarpine and arecoline, had no effect on NMDA-stimulated [H-3]DA release. Potentiation of NMDA-stimulated [H-3]DA release by oxotremorine-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 indicate that the muscarinic receptors responsible for potentiation of NMDA-stimulated [H-3]DA release in mesencephalic cultures are linked to phosphoinositide hydrolysis and protein kinase C activation via pertussis toxin insensitive G proteins. The data are also consistent with the identification of these receptors as the m5 subtype of cloned receptor, on the basis of the relative potency of muscarinic agonists and antagonists in this system, although involvement of ml receptors cannot be excluded solely on this basis. Taken together with previous reports showing selective localization of m5 receptor mRNA in regions containing DA cell bodies, these studies suggest that use of dissociated cell cultures may thus be an appropriate model for studying direct modulation of dopaminergic neurons by cholinergic agents interacting with putative m5 receptors. (C) 1993 Wiley-Liss, Inc.