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

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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]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 [11]and blocks its reuptake [13]. © 1997 Elsevier Science B.V. All rights reserved.

Section snippets

Types of research

Studies on cellular processes linked to PI breakdown as transmembrane transducing system, in particular:

  • studies on neurotransmitter- or agonist-stimulated PI breakdown;

  • studies on relatively weak PI stimulants, such as 5-HT, dopamine [19], histamine [7];

  • studies on animals with reduced postsynaptic sensitivity, such as aged rats 4, 15;

  • studies on PI hydrolysis after animal pretreatment;

  • studies in tissues with relatively low PI response to agonist stimulation (in rat hippocampus the 5-HT-stimulated

Time required

Steps A to G of Section 4require approximately 6 h.

Animals

Male Sprague–Dawley rats (Charles River Breeding Laboratories, Wilmington, MA; 250–350 g) were housed in pairs under diurnal lighting conditions (light on between 08:00 and 20:00 h). They were provided with rat chow and tap water ad libitum. Environmental temperature was maintained at 22°C.

Special equipment

Brinkmann/McIlwain tissue chopper (Mickle Laboratory Engineering Co., Gomshall, Surrey, UK), Precision Scientific Dubnoff metabolic shaker (Thomas Scientific, Swedesboro, NJ), Beckman TJ-CR centrifuge

Detailed procedure

We used this procedure to monitor 5-HT- and carbachol-stimulated ([3H]IP1) accumulation in cortical miniprisms obtained from rats pretreated with d-fenfluramine. Each single experiment was performed with one vehicle-pretreated and one substance-pretreated animal. The rats were injected intraperitoneally (i.p.) for 4 consecutive days (between 10:00 a.m. and 11:00 a.m.) with vehicle (2 ml/kg) or d-fenfluramine (10 mg/2 ml/kg). The animals were decapitated 72 h after the last injection.

Effects of tranylcypromine in vitro

Monoamine oxidase inhibitors are known to prevent the breakdown of exogenous serotonin during the agonist stimulation period [5]. Fig. 1 shows that the serotonin-induced signal is enhanced in the presence of 100 μM of the monoamine oxidase inhibitor, tranylcypromine.

Pretreatment experiments

We examined the effects of subchronic pretreatment with d-fenfluramine on rat cortical miniprisms.

No significant difference was found between basal [3H]phospholipid accumulation in vehicle (43 100±3700 d.p.m./20 μl gravity-packed

Discussion

Our data show that subchronic pretreatment with d-fenfluramine leads to a down-regulation of 5-HT-stimulated PI breakdown in rat cortical miniprisms (for further discussion of this result, see Erfurth et al. [8]). 5-HT-stimulated PI breakdown was monitored by a lithium-inhibited [3H]IP1 accumulation assay (modified from Berridge et al. [2]and Brown et al. [3]) which is particularly sensitive to 5-HT stimulation because of the addition of the MAO inhibitor, tranylcypromine, and of 95% O2/5% CO2

Quick procedure

  • A. Prepare buffers. Oxygenate for 30 min before use.

  • B. Prepare miniprisms. Equilibrate miniprisms for 60 min in lithium-free buffer.

  • C. Label tissue with myo-[3H]inositol for 60 min.

  • D. Incubate with lithium-containing buffer (10 min).

  • E. Incubate with agonist for 60 min, oxygenate all vials thoroughly, add monoamine oxidase inhibitor when stimulating with 5-HT.

  • F. Separate inositol phosphates.

  • G. Extract [3H]IP1 over columns containing anion exchange resin.

  • H. Count radioactivity.

  • I. Express data and

Essential literature references

References [2], [3], [5], [9], [14].

Acknowledgements

These studies were supported by a fellowship from the Deutsche Forschungsgemeinschaft to A.E. and by grants from the Center for Brain Sciences and Metabolism Charitable Trust.

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