Muscarinic Cholinergic Receptors in Human Foetal Brain: Characterization and Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Frontal Cortex

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Abstract
A total of 22 frontal cortices from normal human fetal brains of gestational ages ranging from 16-40 wk and 5 postnatal brains ranging from 5-50 yr were analyzed for the ontogeny of muscarinic receptors using [3H]quinuclidinylbenzilate (QNB) as the ligand. QNB binding sites were stable up to 4.5 mo. of storage at -70.degree. C. QNB binding was characterized in frontal cortices of 28-wk-old fetal brains as muscarinic receptors by the following criteria: it was localized mainly in particulate fractions; binding was saturable at a concentration of 1.5 nM; the cholinergic antagonists atropine and scopolamine competed for the binding, with IC50 [median inhibitory concentration] values of 1 and 0.8 nM, respectively. The agonists oxotremorine, carbachol and pilocarpine gave IC50 values of 1, 15 and 18 .mu.M, respectively. Nicotinic receptor ligands and noncholinergic drugs could not compete for the binding. Bimolecular association and dissociation rate constants for the reversible binding are 6.23 .cntdot. 108 M-1 .cntdot. min-1 and 2.0 .cntdot. 10-2 .cntdot. min-1, respectively. The equilibrium dissociation constant is 33 pM. The Kd obtained by saturation binding data is 103 pM. Ontogeny of muscarinic receptors showed 3 distinct phases: phase I, they appear between 16 and 18 wk [average concentration 109 fmol/ml protein of total particulate fraction (TPF)] and slowly increase up to 20 wk (average concentration 147 fmol/mg protein TPF). Phase II is a lag period between 20 and 24 wk at which time receptor concentration does not change perceptibly (average concentration 167 fmol/mg protein TPF). Phase III is a rapid increase of receptors occurring in the 3rd trimester, reaching a maximum at birth (average concentration 450 fmol/mg TPF). After birth the receptor content declines and the values in adult frontal cortices are 50% of that at birth, indicating synapse elimination postnatally. Affinity of the receptor to QNB did not change during fetal development. NaCl and GTP decrease the agonist affinity, as observed by carbachol competition of QNB binding. This effect was observed even at 16-18 wk. The magnitude of Na effect remained 2- to 3-fold at all ages but the GTP effect increased from about 1.4-fold at 16-18 wk to 2- to 3-fold at birth. Evidently, the modulatory elements appear simultaneously with the receptors.