Sensitive measurement of agonist-stimulated [3H]inositol monophosphate accumulation in rat cortical miniprisms.
Phosphoinositide (PI) breakdown is an important transmembrane signaling mechanism in rat brain and numerous transmitter receptors are linked to this mechanism. Since agonist-stimulated PI breakdown is often changed after drug pretreatment, assessment of changes in PI breakdown represents an important tool in drug development. PI breakdown is commonly monitored by assaying [3H]inositol monophosphate ([3H]IP1) accumulation in the presence of lithium as an inhibitor of inositol monophosphatase. The present protocol presents a lithium-inhibited [3H]IP1 accumulation assay that enhances relatively weak agonist-stimulated [3H]IP1 accumulation signals by thorough oxygenation during agonist stimulation. The protocol is particularly useful in the measurement of [3H]IP1 accumulation after in vitro exposure to relatively weak stimulants, such as serotonin (5-HT), and/or after animal pretreatments that decrease the response to the agonist. In the case of 5-HT stimulation the monoamine oxidase (MAO) inhibitor, tranylcypromine, was added to the incubation medium to inhibit breakdown of exogenous serotonin. We used this protocol to measure 5-HT- and carbachol-stimulated [3H]IP1 accumulation in cortical miniprisms obtained from rats pretreated with D-fenfluramine. D-Fenfluramine is a drug that acutely releases 5-HT into the synaptic cleft and blocks its reuptake.
Erfurth
A
A
Department of Brain and Cognitive Sciences, E25-604 Massachusetts Institute of Technology, Cambridge 02142-1323, USA.
Wurtman
R J
RJ
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002540:Cerebral Cortex / Q000378:metabolism*; D005277:Fenfluramine / Q000494:pharmacology; D005473:Fluoxetine / Q000494:pharmacology; D006652:Histological Techniques; D007295:Inositol Phosphates / Q000378:metabolism*; D008297:Male; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D009462:Neurology / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D012701:Serotonin / Q000494:pharmacology; D018490:Serotonin Agents / Q000494:pharmacology; D014191:Tranylcypromine / Q000494:pharmacology; D014316:Tritium
D007295:Inositol Phosphates; D008996:Monoamine Oxidase Inhibitors; D018490:Serotonin Agents; D005473:Fluoxetine; D014316:Tritium; D005277:Fenfluramine; D012701:Serotonin; D014191:Tranylcypromine; D002217:Carbachol