ABSTRACTSWe examined the linkage of cholinergic receptors to the phosphoinositide signaling pathway to elucidate one facet of the autonomic response mechanism in fetal and adult sheep. Cholinergic stimulation with carbachol increases the production of3H-inositol mono-, bis-, and trisphosphates in a time- and concentration-dependent manner in both fetal and adult myocardium. However, the maximal stimulation of inositol polyphosphates above basal activity was much greater in fetal (120 ± 11%) than in adult (20 ± 7%) myocardium (mean ± SEM). Saturation binding analysis of myocardial muscarinic receptors using3H-N-methylscopolamine revealed significantly higher receptor concentration in fetal (240 ± 25 fmol/mg protien) than in adult (78 ± 15 fmol/mg protein) myocardium (mean ± SEM). Binding competition studies revealed a pattern of selectivity—atropine < 4-diphenylacetoxy-N-methylpiperidine methiodide < piren-zepine < (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride < 11–2[[2-[(diethylamino)-methyl]-1-piperdinyl]acetyl]-5,11− dihydro-6H-pyrido[2, 3-b[[1, 4]benzodiazepine-6-one 116—compatible with the presence of muscarinic receptor (MR)2, MR3, and/or MR5 subtypes. Receptor subtype determination by Northern blot analysis revealed mRNA specific for the MR2 subtype in both fetal and adult myocardium, although expression was greater in fetal heart. We conclude that decreases in MR2 subtype protein and mRNA levels parallel the age-related decrease in carbachol-stimulated PLC activity. Our studies demonstrate differences between fetal and adult myocardium in the concentration of muscarinic cholinergic receptors and their linkage to a putative calcium mobilizing signaling pathway and suggest that this pathway may play a different role in the fetus than in the adult. The physiologic significance of this age-dependent change in cholinergic-linked signaling response of the myocardium is not yet known. (Pediatr Res31: 601–605, 1992)