Sensitive measurement of agonist-stimulated [ 3H]inositol monophosphate accumulation in rat cortical miniprisms

Abstract

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]IP 1) accumulation in the presence of lithium as an inhibitor of inositol monophosphatase. The present protocol presents a lithium-inhibited [ 3H]IP 1 accumulation assay that enhances relatively weak agonist-stimulated [ 3H]IP 1 accumulation signals by thorough oxygenation during agonist stimulation. The protocol is particularly useful in the measurement of [ 3H]IP 1 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]IP 1 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 [11]and blocks its reuptake [13]. © 1997 Elsevier Science B.V. All rights reserved.