Studies on Nicotinic Acetylcholine Receptors in Mammalian Brain. Characterization of a Microsomal Subfraction Enriched in Receptor Function for Different Neurotransmitters

Abstract

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml−1 appears to be enriched in receptor sites for a number of potential neurotrans‐mitters. These include the cholinergic (nicotinic and muscarinic) and ß‐adrenergic receptors. This microsomal subfraction (P3B2) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients. We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P3B2 by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P3B2, and the postsynaptic membranes in the two fractions appear morphologically distinct since P3B2 does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.

Putative 51,000-Mr protein marker for postsynaptic densities is virtually absent in cerebellum.

“…Actin is widely distributed in the cytoplasm and appears, as well, in other cell organelles (28,31) so that it could not be used with any certainty as a marker of PSD structures in isolated fractions. Other functional components such as Thy-1 antigen, neurotransmitter receptors, phosphodiesterases, and other modulatory proteins have also been postulated to be present in the synapse; however, these components may exist in extrasynaptic regions as well (1,4,12,14,29,34). Recently, an antigenic determinant of 95,000 Mr has been proposed as a rather general marker for synaptic structures (30).…”

The Distribution of Radioligand Binding Activity and 5′‐Nucleotidase Activity in Bovine Caudate Nucleus Subcellular Fractions

“…Although ["Hlspiperone binding activity has been demonstrated in a glial cell fraction (Henn et al, 1980), the fraction used is unlikely to be pure, and a neuronal location of the radioligand binding activities is supported by the loss of [3H]-QNB and [3H]spiperone binding sites upon kainic acid lesioning of rat striatum (Fields et al, 1978). The microsomal preparation has also been fractionated by Mahler and coworkers (De Blas and Mahler, 1978;Near and Mahler. 1981).…”

Dopamine Receptors in Subcellular Fractions from Bovine Caudate: Enrichment of [3H]Spiperone Binding in a Postsynaptic Membrane Fraction

“…The method of Salvaterra and Matthews (1980) was used to obtain fractions containing nuclei (P,), synaptosomes and mitochondria (P2), myelin, synaptic plasma membranes (SPM), and mitochondria. Microsomes (P,) and a microsomal subfraction (P3B2) were prepared as described previously (de Blas and Mahler, 1978). Postsynaptic membranes (PSM,) and related fractions were prepared from SPM following the procedure of Ratner and Mahler (1978), involving extraction with salt and EGTA, sonication, and fractionation on a sucrose gradient.…”