Assessment of nicotinic acetylcholine receptor-mediated release of [(3)H]-norepinephrine from rat brain slices using a new 96-well format assay.
The study of the modulatory effects of nicotinic acetylcholine receptor (nAChR) agonists on neurotransmitter release from tissue slices has been hampered by laborious and limiting superfusion techniques. A new methodology was developed utilizing 96-well filter plates. This new method produced comparable results to previously published data, yet expanded throughput to permit more complete pharmacological characterization. Rat brain slices, preloaded with [(3)H]-norepinephrine ([(3)H]-NE), were distributed onto 96-well filter plates. Following a 5 min preincubation, the slices were incubated for 5 min with nicotinic agonists or antagonists. (-)-Nicotine (NIC) and 1,1-dimethyl-4-phenylpiperazine (DMPP) evoked release of [(3)H]-NE from a number of brain regions and spinal cord, with the highest response seen in the hippocampus. Concentration-response curves revealed a rank order of potency of (+/-)-epibatidine>>anatoxin-a>A-85380>DMPP=NIC=(-)-cytisine in the hippocampus, thalamus, and frontal cortex. EC(50) values were approximately 0.005, 0.2, 1, 5, 5 and 5 microM, respectively. Concentration-inhibition curves of nicotine evoked [(3)H]-NE release from hippocampal and thalamic slices resulted in a rank order of potency of mecamylamine>hexamethonium>d-tubocurare>DHbetaE. Schild analysis revealed apparent noncompetitive antagonism of [(3)H]-NE release from hippocampus by mecamylamine, hexamethonium, and DHbetaE. In contrast, DHbetaE antagonism of [(3)H]-dopamine release from striatal slices using a similar methodology was competitive.
Anderson
D J
DJ
Neurological and Urological Diseases Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064-6125, USA. david.j.anderson@abbott.com
Puttfarcken
P S
PS
Jacobs
I
I
Faltynek
C
C
D000818:Animals; D001923:Brain Chemistry / Q000502:physiology*; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D017397:Prefrontal Cortex / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism; D013788:Thalamus / Q000187:drug effects / Q000378:metabolism
D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D004246:Dimethylphenylpiperazinium Iodide; D009538:Nicotine; D009638:Norepinephrine