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Characterization of muscarinic receptors on cultured microglial cells
Abstracts
Vol. 56, No.s 11112, 1995
1037
71 PHOTOAFFINITY LABELLING OF MUSCARINIC RECEPTORS USING DIAZONIUM SALTS
ACETYLCHOLINE
B. Ilien, F. Autelitnno, and M. Goeldner. Lab. Chimie Bio-Organique, FacultC de Pharmacie - 67401 ILLKIRCH Cedex - FRANCE
U.R.A.
1386 C.N.R.S.,
Structural analysis of muscarinic acetylcholine receptor sites has evolved as a combination of molecular modelling, site-directed mutagenesis and chemical labelling of binding residues. Among these methods, protein modification through affinity reagents certainly provides the strongest information for constitutive analysis of agonist or antagonist binding sites. Up to now, two aziridinium mustard derivatives revealed the same Asp105 residue (ml sequence) as their unique alkylation site. We selected two photoactivatable probes (aryldiazonium salts) for their interesting photochemical labelling properties at muscarinic acetylcholine receptors. Indeed, the diazonium function (Nz,+) mimics the quaternary ammonium head of most cholinergic ligands and generate, upon irradiation, highly reactive species (arylcations) able to react with all kind of residues. Moreover, these compounds display spectral properties allowing Trp-mediated photolabelling through energy transfer. Such a labelling process has proven to be very efficient in alkylating, in a fully atropine-protectable manner, membrane-bound and digitonin-solubilized muscarinic receptors from rat and porcine brain. The two probes were tritiated and allowed to photoreact with affinity-purified muscarinic receptors from porcine striatum. SDS-PAGE analyses of the alkylated species revealed, besides aggregates, a large polypeptide (66-07 kD) band typical of muscarinic receptors and an incorporation of the radioactive label which was prevented by the antagonist atropine. Peptide mapping and sequencin g studies will be undertaken in order to locate and identify the chemically modified residues in the photolabelled muscarinic receptors.
72 CI-L4RACTERIZATION
OF MUSCARINIC
RECEPTORS
ON CULTURED MICROGLIAL
CELLS
G. Ferrari-DiLeo and D.D. Flynn. Dept. of Pharmacology, University of Miami School of Medicine, Miami, FL USA 33 101 Recent studies have suggested that microglia may play a pivotal role in the pathogenesis of Alzheimer’s disease (AD; McGeer & Rogers Neural. 42:447, 1992). In response to neuronal insults, microglia undergo both a morphological and functional transformation from a resting to activated state, and perform macrophage and immune functions including phagocytosis, secretion of superoxide anions and “pseudo-inflammatory” responses. Consistent with these functions are the presence of activated microglia near or within senile plaques of AD and the rationale for the use of non-steroidal anti-inflammatory agents as therapies for AD. However, whether microglia play either a protective or destructive role in the disease process is not clear. Recent evidence suggests that microglial activation may be controlled, in part, by specific neurotransmitters, including acetylcholine (Whittemore et al, Bruin Res. 62159, 1993). In AD and other neurodegenerative diseases characterized by alterations in neurotransmitter levels, the usual homeostatic regulatory mechanisms for microglial activation may be lost. We have characterized muscarinic receptors on cultured microglia isolated from adult and newborn brain tissue and on blood macrophages. Total PHI-NMS receptor binding in brain microglia and blood macrophages was 150-300 and 35 fmol/mg protein, respectively. Quantitative immunoprecipitationusing receptor subtype-specific antisera (provided by Dr. AllanLevey, Emory University) demonstrated that the m5 receptor subtype was predominant in all cell types studied. The recent demonstration that the m5 receptor subtype is coupled to activation of nitric oxide synthase (Wang et al., J.PhurmacoZ.~.Zberup. 268552, 1994) is consistent with the secretion of nitric oxide by activated microglia. Taken together, these studies may provide further insight into muscarinic receptor regulation of microglial function and suggest new approaches for modulating microglial responses to neuronal insults.
Vol. 56, No.s 11112, 1995
1037
71 PHOTOAFFINITY LABELLING OF MUSCARINIC RECEPTORS USING DIAZONIUM SALTS
ACETYLCHOLINE
B. Ilien, F. Autelitnno, and M. Goeldner. Lab. Chimie Bio-Organique, FacultC de Pharmacie - 67401 ILLKIRCH Cedex - FRANCE
U.R.A.
1386 C.N.R.S.,
Structural analysis of muscarinic acetylcholine receptor sites has evolved as a combination of molecular modelling, site-directed mutagenesis and chemical labelling of binding residues. Among these methods, protein modification through affinity reagents certainly provides the strongest information for constitutive analysis of agonist or antagonist binding sites. Up to now, two aziridinium mustard derivatives revealed the same Asp105 residue (ml sequence) as their unique alkylation site. We selected two photoactivatable probes (aryldiazonium salts) for their interesting photochemical labelling properties at muscarinic acetylcholine receptors. Indeed, the diazonium function (Nz,+) mimics the quaternary ammonium head of most cholinergic ligands and generate, upon irradiation, highly reactive species (arylcations) able to react with all kind of residues. Moreover, these compounds display spectral properties allowing Trp-mediated photolabelling through energy transfer. Such a labelling process has proven to be very efficient in alkylating, in a fully atropine-protectable manner, membrane-bound and digitonin-solubilized muscarinic receptors from rat and porcine brain. The two probes were tritiated and allowed to photoreact with affinity-purified muscarinic receptors from porcine striatum. SDS-PAGE analyses of the alkylated species revealed, besides aggregates, a large polypeptide (66-07 kD) band typical of muscarinic receptors and an incorporation of the radioactive label which was prevented by the antagonist atropine. Peptide mapping and sequencin g studies will be undertaken in order to locate and identify the chemically modified residues in the photolabelled muscarinic receptors.
72 CI-L4RACTERIZATION
OF MUSCARINIC
RECEPTORS
ON CULTURED MICROGLIAL
CELLS
G. Ferrari-DiLeo and D.D. Flynn. Dept. of Pharmacology, University of Miami School of Medicine, Miami, FL USA 33 101 Recent studies have suggested that microglia may play a pivotal role in the pathogenesis of Alzheimer’s disease (AD; McGeer & Rogers Neural. 42:447, 1992). In response to neuronal insults, microglia undergo both a morphological and functional transformation from a resting to activated state, and perform macrophage and immune functions including phagocytosis, secretion of superoxide anions and “pseudo-inflammatory” responses. Consistent with these functions are the presence of activated microglia near or within senile plaques of AD and the rationale for the use of non-steroidal anti-inflammatory agents as therapies for AD. However, whether microglia play either a protective or destructive role in the disease process is not clear. Recent evidence suggests that microglial activation may be controlled, in part, by specific neurotransmitters, including acetylcholine (Whittemore et al, Bruin Res. 62159, 1993). In AD and other neurodegenerative diseases characterized by alterations in neurotransmitter levels, the usual homeostatic regulatory mechanisms for microglial activation may be lost. We have characterized muscarinic receptors on cultured microglia isolated from adult and newborn brain tissue and on blood macrophages. Total PHI-NMS receptor binding in brain microglia and blood macrophages was 150-300 and 35 fmol/mg protein, respectively. Quantitative immunoprecipitationusing receptor subtype-specific antisera (provided by Dr. AllanLevey, Emory University) demonstrated that the m5 receptor subtype was predominant in all cell types studied. The recent demonstration that the m5 receptor subtype is coupled to activation of nitric oxide synthase (Wang et al., J.PhurmacoZ.~.Zberup. 268552, 1994) is consistent with the secretion of nitric oxide by activated microglia. Taken together, these studies may provide further insight into muscarinic receptor regulation of microglial function and suggest new approaches for modulating microglial responses to neuronal insults.