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 phospholipase C (PLC) second messenger system has been demonstrated in central nervous system (CNS) tissue of many animal species. However, little information exists regarding this association in the developing human CNS. Due to the suggested role of acetylcholine in the regulation of development and differentiation of neural cells, the knowledge of these relationships during human fetal development acquires singular importance. Because of this, we examined the cholinergic stimulation of PLC in human fetal CNS organotypic tissue cultures. Agonist treatment of cultures, in the presence of lithium, resulted in a 4-6-fold increase in inositol phosphates formation. This increase was caused principally by the formation of inositol phosphate (IP). However, kinetic studies demonstrated that the levels of IP2, IP3 and IP4 also increased rapidly after stimulation reaching maximum levels before IP. These results support the hypothesis that muscarinic receptor activation results in an increase in the hydrolysis of PIP2. The inositol phosphate formation was dependent on agonist concentration. The obtained EC50 values were approximately 57 +/- 15 microM for carbachol, 8 +/- 2 microM for acetylcholine and 49 +/- 15 microM for oxotremorine. The agonist-dependent formation of inositol phosphates was inhibited by the muscarinic antagonists atropine and pirenzepine. Pirenzepine inhibited carbachol stimulation with high affinity (Ki = 2.90 +/- 1.15 nM), indicating that PLC activation is the result of activation of the m1 subtype of muscarinic receptors. Treatment of cultures with pertussis toxin did not result in inhibition of agonist-dependent activation of PLC. This result suggests that the m1 muscarinic receptor is coupled to PLC through Gq.
Larocca
J N
JN
Department of Neurology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Rodriguez-Gabin
A G
AG
Rashbaum
W K
WK
Weidenheim
K M
KM
Lyman
W D
WD
D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002490:Central Nervous System / Q000187:drug effects / Q000196:embryology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009924:Organ Culture Techniques; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D002217:Carbachol; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium