Heterogeneity of cholinergic muscarinic receptors coupled to phosphoinositide metabolism in immature rat brain

The effects of muscarinic agonists and antagonists on phosphoinositide (PtdIns) metabolism were examined in the cerebral cortex and brainstem of 7-day-old rats, in order to evaluate the role of muscarinic receptor subtypes in this process. Additionally, comparative experiments were performed in cortices from adult animals. Accumulation of [³H]inositol phosphates ([³H]InsPs) in [³H]inositol pre-labeled brain slices was taken as an index of PtdIns hydrolysis. In neonatal cortex, maximal stimulation induced by the full agonists acetylcholine, carbachol and methacholine was 8-10 fold over basal [³H]InsPs accumulation. The effect of the partial agonists bethanechol, pilocarpine and oxotremorine varied from 3 to 4 fold over basal. Smaller responses to cholinergic stimulation were found in the brainstem and in the adult cortex. In neonatal cortex, muscarinic antagonists inhibited the stimulatory responses with the following order of potency: 4-DAMP > pirenzepine > AF-DX 116 ≅ p-F-HHSiD. Pirenzepine inhibition of full agonist-induced [³H]InsPs accumulation showed biphasic curves, with two thirds of the response being inhibited with high affinity. When partial agonists were used, the resulting pirenzepine curves were better described by interaction at one high affinity site. No differences were found between immature and adult rats in the effect of pirenzepine on [³H]InsPs accumulation induced by carbachol, methacholine, or bethanechol. Inhibition by pirenzepine of PtdIns hydrolysis induced by carbachol or methacholine showed biphasic curves also in the brainstem. In this area, only one third of the response was inhibited with high affinity, and p-F-HHSiD was more potent as an antagonist. Inhibition of the agonist responses by 4-DAMP, AF-DX 116, or p-F-HHSiD resulted in monophasic curves in both cortex and brainstem. Based on pirenzepine antagonism, it is likely that more than one muscarinic receptor subtype is involved in the activation of PtdIns metabolism in the immature rat brain. M₁ receptors appear to mediate the high affinity component of the response. When full agonists are employed, the remaining component is probably activated by the M₃ receptor subtype. Partial agonists appear to stimulate PtdIns metabolism by activating M₁ receptors only.