Molecular Brain Research, 20 (1993) 111-117 111 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00 BRESM 70642 Modulation of the phospholipase C activity in rat brain cortical membranes by simultaneous activation of distinct monoaminergic and cholinergic muscarinic receptors Joan Sall6s, Michael A. Wallace and John N. Fain Department of Biochemistry, University of Tennessee, Memphis, TN 38163 (USA) (Accepted 16 March 1993) Key words: Muscarinic M 1 receptor; Muscarinic M 3 receptor; Dopamine D 1 receptor; Phospholipase C; Inositoiphosphate; Rat cortical membrane The activation of phospholipase C (PLC) was examined in membranes of rat cerebral cortex simultaneously exposed to monoaminergic receptor and muscarinic receptor agonists after the treatment of membranes with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (100 /~M EEDQ) and propylbenzilylcholine (10 nM PrBCM). Treatment of membranes with PrBCM results in a selective inactivation of M 3 muscarinic receptors, while treatment with EEDQ results in a relative sparing of M 1 muscarinic receptors 14. Stimulation of PLC by GTPyS alone in rat cortical membranes had an apparent ECs0 of about 0.4/.tM, but in the presence of carbachol (1 mM) was 0.09/zM. Treatment of rat cortical membranes with EEDQ or PrBCM did not modify the concentration-response curves for GTPyS alone, but the ability of carbachol (1 mM) to shift the ECs0 of GTPyS was lost in PrBCM-treated membranes. We have previously shown that dopamine, working through Dl-like dopamine receptors, alters the PLC response to carbachol by preventing this shift in the apparent ECs0 for GTPyS 16. When we reproduced these experiments in EEDQ- and PrBCM-treated membranes, only in EEDQ-treated membranes was dopamine able to inhibit the PLC response to carbachol. The results indicate that the post-receptor mechanisms of PLC activation are distinct for the putative M 1 as opposed to M3 muscarinic receptors in rat cortical membranes. Further, there appears to be a specific interaction between D 1 and M 3 receptors. 5-Methyltryptamine and carbachol were used to study the effects of simultaneous activation of the serotonergic and the putative M 1 and M 3 muscarinic receptor subtypes, respectively, on PLC activity. In EEDQ-treated membranes, the experimental data best fit a model for interdependent interactions. In contrast in PrBCM-treated membranes the data fit a model for independent interaction. INTRODUCTION One major pathway for neurotransmitter signalling involves phosphoinositide-specific phospholipase C (PLC). The enzyme catalyzes the breakdown of the phosphoinositides into two products: diacylglycerol and inositolphosphate(s). The former product is important for the regulation of protein kinase C and as an inter- mediate in overall lipid metabolism. The latter prod- ucts have roles in the regulation of intracellular Ca 2+ levels, most prominently inositol 1,4,5-trisphosphate, which causes release of sequestered calcium from the endoplasmic reticulum. A variety of neurotransmitters have been shown to stimulate PLC activity in brain s. Recently, we have developed an assay for carbachol- activated guanine nucleotide-dependent phospholipase C stimulation using crude membrane preparations from rat brain cortex 4. This system allows direct measure- ment of the activation of phospholipase C by agonists of the muscarinic and serotoninergic receptors 15. Moreover, dopamine inhibits the activation by carba- chol of PLC in rat cortical membranes. Pharmacologi- cal characterization reveals that the effect of dopamine occurs through stimulation of a Dl-like dopaminergic receptor 16. The membrane assays described here al- lowed us to measure both positive and negative modu- lators of PLC interacting at a common postsynaptic membrane site. N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) acts as an irreversible antagonist at various monoaminergic receptors in peripheral tissues and in brain after parenteral administration l'z'l°. Importantly, Correspondence: J. Sall6s. Present address: Departament de Farmacologia i Psiquiatria, Universitat Autbnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Fax: 581 20 04.