Biomedical subjects

J Michael McIntosh

Publications and source records attributed to J Michael McIntosh.

58 records · Page 4Linked to original sources

PubMed2002

Characterization of nicotinic agonist-induced [(3)H]dopamine release from synaptosomes prepared from four mouse brain regions.

The inhibition of uptake of [(3)H]dopamine into synaptosomes prepared from four mouse brain regions was investigated. The inhibition curves demonstrated that in three regions, striatum, nucleus accumbens, and olfactory tubercle, [(3)H]dopamine was taken up exclusively by dopaminergic terminals. In frontal cortex, however, only a portion of the uptake was into dopaminergic terminals, with a larger amount taken up by noradrenergic terminals, and another small portion by serotonergic terminals. Release studies in frontal cortex indicated that in this region only dopaminergic and noradrenergic terminals are capable of packaging [(3)H]dopamine in a form allowing vesicle-mediated release; additionally, only the dopaminergic terminals have functional presynaptic nAChRs that, when stimulated by nicotinic agonists, evoke [(3)H]dopamine release. Agonist-stimulated [(3)H]dopamine release was characterized from synaptosomes prepared from four mouse brain regions. alpha-Conotoxin MII was a partial inhibitor of dopamine release in all of the brain regions, which suggests that a minimum of two nicotinic cholinergic receptors (nAChRs) are expressed in the nerve terminals of all four brain regions. No nicotine-induced [(3)H]dopamine release was detected in any brain region when the synaptosomes were prepared from beta2 null mutant mice, which indicates that the beta2 subunit is required for all nAChRs mediating this release. Dose-response curves were constructed for seven agonists in each of the brain regions. The pharmacological properties of synaptosomal [(3)H]dopamine release appear similar across the four brain regions. The results suggest that all four regions express the same nAChRs, although subtle regional differences may exist.

Animals↗

PubMed2002

Methyllycaconitine is a potent antagonist of alpha-conotoxin-MII-sensitive presynaptic nicotinic acetylcholine receptors in rat striatum.

The plant alkaloid methyllycaconitine (MLA) is considered to be a selective antagonist of the alpha7 subtype of neuronal nicotinic acetylcholine receptor (nAChR). However, 50 nM MLA partially inhibited (by 16%) [(3)H]dopamine release from rat striatal synaptosomes stimulated with 10 microM nicotine. Other alpha7-selective antagonists had no effect. Similarly, MLA (50 nM) inhibited [(3)H]dopamine release evoked by the partial agonist (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene (UB-165) (0.2 microM) by 37%. In both cases, inhibition by MLA was surmountable with higher agonist concentrations, indicative of a competitive interaction. At least two subtypes of presynaptic nAChR can modulate dopamine release in the striatum, and these nAChR are distinguished by their differential sensitivity to alpha-conotoxin-MII (alpha-CTx-MII). MLA was not additive with a maximally effective concentration of alpha-CTx-MII (100 nM) in inhibiting [(3)H]dopamine release elicited by 10 microM nicotine or 0.2 microM UB-165, suggesting that both toxins act at the same site. This was confirmed in quantitative binding assays with (125)I-alpha-CTx-MII, which displayed saturable specific binding to rat striatum and nucleus accumbens with B(max) values of 9.8 and 16.5 fmol/mg of protein, and K(d) values of 0.63 and 0.83 nM, respectively. MLA fully inhibited (125)I-alpha-CTx-MII binding to striatum and nucleus accumbens with a K(i) value of 33 nM, consistent with the potency observed in the functional assays. We speculate that MLA and alpha-CTx-MII interact with a presynaptic nAChR of subunit composition alpha3/alpha6beta2beta3* on dopamine neurons. The use of MLA as an alpha7-selective antagonist should be exercised with caution, especially in studies of nAChR in basal ganglia.

Aconitine↗

PubMed2002

Loss of nicotinic receptors in monkey striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment is due to a decline in alpha-conotoxin MII sites.

Nicotinic acetylcholine receptors (nAChRs) in the basal ganglia are a potential target for new therapeutics for Parkinson's disease. As an approach to detect expression of nAChRs in monkeys, we used 125I-epibatidine, an agonist at nAChRs containing alpha2 to alpha6 subunits. 125I-Epibatidine binding sites are expressed throughout the control monkey brain, including the basal ganglia. The alpha3/alpha6-selective antagonist alpha-conotoxin MII maximally inhibited 50% of binding in the caudate-putamen and had no effect on 125I-epibatidine binding in the frontal cortex or thalamus. In contrast, inhibition experiments with nicotine, cytisine, and 3-(2(S)-azetidinylmethoxy)pyridine-2HCl (A85380) showed a complete block of 125I-epibatidine binding in all regions investigated and did not discriminate between the alpha-conotoxin MII-sensitive and -insensitive populations in the striatum. To assess the effects of nigrostriatal damage, monkeys were rendered parkinsonian with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Animals with moderate striatal damage (dopamine transporter levels approximately 30% of control) had a 40 to 50% decrease in 125I-epibatidine binding. Inhibition studies showed that the decrease in epibatidine binding was due to loss of alpha-conotoxin MII-sensitive nAChRs. Monkeys with severe nigrostriatal damage (dopamine transporter levels < or = 5% of control) exhibited a 55 to 60% decrease in 125I-epibatidine binding, which seemed to be due to a complete loss of alpha-conotoxin MII nAChRs and a partial loss of other nAChR subtypes. These results show that nAChRs expressed in the primate striatum have similar affinities for nicotine, cytisine, and A85380, that alpha-conotoxin MII discriminates between nAChR populations in the caudate and putamen, and that alpha-conotoxin MII-sensitive nAChRs are selectively decreased after MPTP-induced nigrostriatal damage.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMedDate not supplied

Synthesis of fluorescent analogs of alpha-conotoxin MII.

Alpha-conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. Alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3beta2 and rat chimeric alpha6/alpha3beta2beta3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.

Animals↗