Heterogeneous binding of [³H]4‐DAMP to muscarinic cholinergic sites in the rat brain: Evidence from membrane binding and autoradiographic studies

Abstract

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.

Comparative ontogenic profile of cholinergic markers, including nicotinic and muscarinic receptors, in the rat brain

“…For all muscarinic radioligands that were used (["HIQNB,["HJPZ,, a similar protocol was followed Regenold et al, 1989;Araujo et al, 1991;Aubert et al, 1992Aubert et al, , 1994. Experiments were performed at room temperature for L3H]QNB, L"H14-DAMP, and L3HH]PZ and at 4°C for ["HJAF-DX 116 and…”

“…Slides were then transferred sequentially through three rinses (4°C; 4 minutes each) in Tris (50 mM)-HCI buffer, pH 7.4, followed by a rapid dip in cold distilled water. Sections were finally dried under a stream of cold air and tightly juxtaposed to films for 8 days for ["HIQNB and ["H] 95,95,85,70, and 85% of bound ligand, respectively, in the adult rat brain Regenold et al, 1989;Araujo et al, 1991;Aubert et al, 1992Aubert et al, , 1994.…”

“…The pharmacologically defined muscarinic MI receptor class was identified by using [3H]pirenzepine (PZ; Spencer et al, 19861, whereas muscarinic Mz receptors were investigated by using both L3H]AF-DX 116 [ll1 [2-[(diethyl-amino) methyl]-1-piperidinyl] acetyll-5, ll-dihydro-6H-pyrido[2,3-bJ 1 1,4J benzodiazepine-6-one; Regenold et al, 19891 and [3H]AF-DX 384 [5, ll-dihydro-l1-[[(2-(2-[(dipropylamino) methyl]-1-piperidinyl) ethyl) amino] carbonylJ-6H-pyrido [2, 3-b] [1,4J-benzodiazepine-6-one; Aubert et al, 19921. L3HJ4-DAMP (4-diphenylacetoxy-Nmethylpiperidine methiodide) was used to provide information on the ontogenic profile of the muscarinic M1/MS receptor subtypes (Michel et al, 1989;Araujo et al, 1991). This comparative study aimed to provide evidence on the temporal establishment of several components of the cholinergic systems in different areas of the developing rat brain.…”

Pharmacological Characterization of Muscarinic Cholinergic Receptors in Cat Pons and Cortex

“…Atropine is known to bind with equal and high affinity to all muscarinic receptor subtypes [27], Pirenzepine binds with high affinity to M 1 receptors [28], AF-DX 116 binds with high affinity to M2 receptors [29], and 4-DAMP binds with high affinity to M3 and M 1 recep tors [30,31]. Fifteen to 23 competitor concentrations, ranging from 10"12 to 10"3 mol/1, were used.…”

M3 Muscarinic Receptor‐Mediated Enhancement of NMDA‐Evoked Adenosine Release in Rat Cortical Slices In Vitro

“…The predominant role of M3 receptors is consistent with anatomical findings indicating that the rat cerebra! cortex contains high concentrations of [ 3H] 4-DAMP binding sites (Araujo et al, 1991;Zubieta and Frey, 1993), mRNA for m3 muscarinic receptors (Buckley et a!., 1988;Wei et a!., 1994), and immunoreactivity for m3 receptor protein (Levey et a!., 1994). By contrast, the levels of mRNA for m2 and m4 receptors are low in the cortex (Buckley et al, 1988;Wei et a!., 1994).…”