MUSCARINIC RECEPTOR-DEPENDENT ACTIVATION OF PHOSPHOLIPASE-C IN HUMAN FETAL CENTRAL-NERVOUS-SYSTEM ORGANOTYPIC TISSUE-CULTURE

The coupling of muscarinic-cholinergic receptors (mAchR) with the phos pholipase C (PLC) second messenger system has been demonstrated in cen tral nervous system (CNS) tissue of many animal species. However, litt le information exists regarding this association in the developing hum an CNS. Due to the suggested role of acetylcholine in the regulation o f development and differentiation of neural cells, the knowledge of th ese relationships during human fetal development acquires singular imp ortance. Because of this, we examined the cholinergic stimulation of P LC in human fetal CNS organotypic tissue cultures. Agonist treatment o f cultures, in the presence of lithium, resulted in a 4-6-fold increas e in inositol phosphates formation. This increase was caused principal ly by the formation of inositol phosphate (IF). However, kinetic studi es demonstrated that the levels of IP2, IP3 and IP4 also increased rap idly after stimulation reaching maximum levels before IP. These result s support the hypothesis that muscarinic receptor activation results i n an increase in the hydrolysis of PIP2. The inositol phosphate format ion was dependent on agonist concentration. The obtained EC(50) values were approximately 57 +/- 15 mu M for carbachol, 8 +/- 2 mu M for ace tylcholine and 49 +/- 15 mu M for oxotremorine. The agonist-dependent formation of inositol phosphates was inhibited by the muscarinic antag onists atropine and pirenzepine. Pirenzepine inhibited carbachol stimu lation with high affinity (K-i = 2.90 +/- 1.15 nM), indicating that PL C activation is the result of activation of the mi subtype of muscarin ic receptors. Treatment of cultures with pertussis toxin did not resul t in inhibition of agonist-dependent activation of PLC. This result su ggests that the m1 muscarinic receptor is coupled to PLC through G(q).