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Muscarinic Cholinergic Receptors in Human Foetal Brain: Characterization and Ontogeny of [ 3 H]Quinuclidinyl Benzilate Binding Sites in Frontal Cortex
Author(s) -
Ravikumar B. V.,
Sastry P. S.
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb07136.x
Subject(s) - muscarinic acetylcholine receptor , oxotremorine , quinuclidinyl benzilate , endocrinology , medicine , carbachol , dissociation constant , pilocarpine , chemistry , cholinergic , acetylcholine receptor , receptor , ontogeny , atropine , muscarinic antagonist , biology , biochemistry , neuroscience , epilepsy
Twenty‐two frontal cortices from normal human foetal brains of gestational ages ranging from 16 to 40 weeks and five postnatal brains ranging from 5 to 50 years were analysed for the ontogeny of muscarinic receptors using [ 3 H]quinuclidinyl benzilate (QNB) as the ligand. QNB binding sites were shown to be stable up to 41/2 months of storage at – 70°C. QNB binding was characterized in frontal cortices of 28‐week‐old foetal brains as muscarinic receptors by the following criteria: (1) it was localised mainly in particulate fraction; (2) binding was saturable at a concentration of 1.5 n M ; (3) the cholinergic antagonists atropine and scopolamine competed for the binding, with IC 50 values of 1 and 0.8 n M , respectively. The agonists oxotremorine, carbachol, and pilocarpine gave IC 50 values of 1, 15 and 18 μ 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 ± 10 8 M −1 ± min −1 and 2.0 ± 10 −2 ± min −1 , respectively. The equilibrium dissociation constant is 33 p M. The K D obtained by saturation binding data is 103 p M. Ontogeny of muscarinic receptors showed three distinct phases: In phase I, they appear between 16 and 18 weeks [average concentration 109 fmol/mg protein of total particulate fraction (TPF)] and slowly increase up to 20 weeks (average concentration 147 fmol/mg protein TPF). Phase II is a lag period between 20 and 24 weeks 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 third 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 foetal development. Sodium chloride and GTP decrease the agonist affinity, as observed by carbachol competition of QNB binding. This effect was observed even at 16–18 weeks. The magnitude of sodium effect remained two‐ to threefold at all ages but the GTP effect increased from about 1.4‐fold at 16–18 weeks to two‐ to threefold at birth. These results suggest that the modulatory elements appear simultaneously with the receptors.