ProtocolSensitive measurement of agonist-stimulated [3H]inositol monophosphate accumulation in rat cortical miniprisms
Section snippets
Types of research
Studies on cellular processes linked to PI breakdown as transmembrane transducing system, in particular:
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studies on neurotransmitter- or agonist-stimulated PI breakdown;
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studies on relatively weak PI stimulants, such as 5-HT, dopamine [19], histamine [7];
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studies on animals with reduced postsynaptic sensitivity, such as aged rats 4, 15;
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studies on PI hydrolysis after animal pretreatment;
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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.
References (20)
- Baird, J.G. and Nahorski, S.R., Potassium depolarisation markedly enhances muscarinic receptor stimulated inositol...
- Berridge, M.J., Downes, C.P. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidylinositol responses in...
- Brown, E., Kendall, D.A. and Nahorski, S.R., Inositol phospholipid hydrolysis in rat cerebral cortical slices. I....
- Burnett, D.M., Bowyer, J.F., Masserano, J.M. and Zahniser, N.R., Effect of aging on alpha-1 adrenergic stimulation of...
- Conn, P.J. and Sanders-Bush, E., Serotonin-stimulated phosphoinositide turnover: mediation by the S2 binding site in...
- Conn, P.J. and Sanders-Bush, E., Regulation of serotonin-stimulated phosphoinositide hydrolysis: relation to the...
- Daum, P.R., Downes, C.P. and Young, J.M., Histamine stimulation of inositol-1-phosphate accumulation in lithium-treated...
- Erfurth, A., Gardier, A.M., Ribeiro, E. and Wurtman, R.J., Effects of subchronic pretreatment with d-fenfluramine or...
- Fowler, C.J., Court, J.A., Tiger, G., Björklund, P.-E. and Candy, J.M., Stimulation of inositol phospholipid breakdown...
- Heathers, G.P., Juehne, T., Rubin, L.J., Corr, P.B. and Evers, A.S., Anion exchange chromatographic separation of...