Elsevier

Neuropharmacology

Volume 39, Issue 4, 15 March 2000, Pages 643-651
Neuropharmacology

Long-term potentiation in dentate gyrus of the rat is inhibited by the phosphoinositide 3–kinase inhibitor, wortmannin

https://doi.org/10.1016/S0028-3908(99)00169-0Get rights and content

Abstract

The pivotal role of inositol phospholipids in cell signalling has been placed centre-stage again with the recognition that phosphoinositide (PI) 3–kinase is implicated in several cellular processes. Stimulation of PI–3 kinase requires activation of the 85 kD regulatory subunit which relies on tyrosine phosphorylation, one consequence of which is activation of the 110 kD catalytic subunit. In this study, we have investigated the role of PI 3–kinase in the expression of long-term potentiation (LTP) in perforant path-granule cell synapses of the rat. We report that intracerebroventricular injection of wortmannin inhibited expression of LTP, though it did not affect the early change in the synaptic response. Activation of PI 3–kinase was enhanced in tetanized tissue prepared from dentate gyrus, compared with untetanized tissue, but this effect was inhibited in tissue prepared from wortmannin-pretreated rats. LTP was associated with increased glutamate release, as previously described, but this effect was also inhibited in tissue prepared from wortmannin-pretreated rats. The results presented demonstrate that wortmannin also exerted an inhibitory effect on KCl-stimulated glutamate release and calcium influx in hippocampal synaptosomes in vitro. The evidence presented is consistent with the hypothesis that PI 3–kinase activation, possibly by NGF, plays a role in expression of LTP in dentate gyrus.

Introduction

The identification of cell signalling events stimulated by inositol phospholipid products of phosphoinositide 3–kinase (PI 3–kinase) has amplified and consolidated the pivotal role of inositol phospholipids in cell signalling. PI 3–kinase phosphorylates the D–3 position of phosphatidylinositol 4,5–bisphosphate (PtdIns(4,5)P2) to produce phosphatidylinositol 3,4,5–trisphosphate (PtdIns(3,4,5)P3) which on dephosphorylation yields phosphatidyl–inositol 3,4–bisphosphate and phosphatidylinositol 3–phosphate (Cantley et al., 1991, Fry and Waterfield, 1993, Stephens et al., 1993, Kapeller and Cantley, 1994). The immediate product of PI 3–kinase, PtdIns(3,4,5)P3, has been shown to activate calcium-independent isoforms of protein kinase C (Nakanishi et al., 1993, Toker et al., 1994) and protein kinase B (Franke et al., 1995, Franke et al., 1997, Stokoe et al., 1997), while recent evidence indicated that it also activates phospholipase Cγ (Bae et al., 1998), an action shared by arachidonic acid (Hwang et al., 1996, McGahon and Lynch, 1998).

PI 3–kinases, which are activated by cytokines and growth factors (Hawkins et al., 1997), comprise a group of intracellular signalling enzymes which have been implicated in proliferation, differentiation and secretion (Kapeller and Cantley, 1994, Hawkins et al., 1997). PI 3–kinase activity can be stimulated by the βγ subunit of G proteins (Hawkins et al., 1997), though activation as a consequence of receptor-activated tyrosine kinase has also been described (Vanhaesebroeck et al., 1997, Rameh et al., 1995). In this case, the two SH2 domains of the p85 regulatory subunit of PI 3–kinase bind to phosphotyrosine residues on the activated receptor, resulting in translocation and activation of the p110 catalytic subunit. However, cooperation or synergism between G–protein-linked receptors yielding the G–protein βγ subunit and receptors possessing tyrosine kinase activity has been proposed (Kusoru et al., 1997, Tang and Downes, 1997).

There is a good deal of data indicating that tyrosine kinase activation plays a role in expression of LTP. Inhibitors of tyrosine kinase have been shown to inhibit LTP (Abe and Saito, 1993, O'Dell et al., 1991), while tyrosine phosphorylation of protein substrates, PLCγ (McGahon and Lynch, 1998), synaptophysin (Mullany and Lynch, 1998), trkA (Maguire et al., 1998) and the 2B subunit of the NMDA receptor (Rosenblum et al., 1996, Rostas et al., 1996) has been shown to increase following induction of LTP in dentate gyrus. The consequences of increased tyrosine phosphorylation of these substrates remains to be firmly established, though evidence has linked increased phosphorylation of the synaptic vesicle protein, synaptophysin, with increased glutamate release (Mullany and Lynch, 1998). The involvement of tyrosine kinase in LTP has been further supported by the observation that the trk inhibitor, tyrphostin AG879, blocks LTP (Mullany and Lynch, 1998, Maguire et al., 1998), while arachidonic acid or NGF, both of which activate tyrosine kinase, contribute to the LTP-associated increase in glutamate release (McGahon and Lynch, 1996, Kelly et al., 1998). It must be considered that tyrosine phosphorylation of substrates, which have not been identified to date, may also contribute to expression of LTP and the present study was designed to investigate the possibility that one substrate protein might be the p85 regulatory subunit of PI 3–kinase. We therefore investigated the effect of the PI 3–kinase inhibitor, wortmannin, on KCl-stimulated glutamate release in hippocampal synaptosomes in vitro. In addition, since we have observed that the interaction between NGF and ACPD stimulates glutamate release (Kelly et al., 1998), we also assessed the effect of wortmannin on the enhancement of release induced by NGF and ACPD. In parallel with the experiments assessing the effect of wortmannin on glutamate release, its effect on KCl-induced calcium influx was also investigated. This analysis indicated an inhibitory effect of wortmannin on release, therefore we considered that it may affect LTP. To address this question, we analysed the effect of wortmannin on expression of LTP in perforant path-granule cell synapses in parallel with changes which occur in glutamate release and PI 3–kinase activity in control (untetanized) dentate gyrus and dentate gyrus in which LTP was induced in vivo. The data indicate that PI 3–kinase is activated following induction of LTP and are consistent with the possibility that this may contribute to the increase in glutamate release which accompanies LTP in dentate gyrus.

Section snippets

Animals

Male Wistar rats (250–350 g), obtained from the BioResources Unit in Trinity College, Dublin, were housed in groups of 4–6 under a 12-hour light schedule at a temperature of between 22 and 23°C. Rats were killed by cervical dislocation and the hippocampus was rapidly removed and used for analysis of release, calcium influx and tyrosine kinase activity.

Analysis of glutamate release

The synaptosomal preparation P2 was used for analysis of glutamate release as previously described (McGahon and Lynch, 1996). Briefly, samples of

Effect of wortmannin on glutamate release and [45Ca2+] influx in vitro

Addition of 50 mM KCl to saline-pretreated hippocampal synaptosomes increased glutamate release from 0.29 μmol/mg (±0.08) to 0.59 μmol/mg (±0.07) in saline-pretreated synaptosomes and from 0.39 μmol/mg (±0.03) to 0.53 μmol/mg (±0.04) in wortmannin-pretreated synaptosomes; in each case, however, KCl-induced release represented a statistically significant increase over unstimulated release (P<0.05; n=6; ANOVA; Fig. 1(A)). The data indicate that NGF and ACPD significantly enhanced KCl-stimulated

Discussion

The objectives of this study were (a) to establish if PI 3–kinase plays a role in transmitter release and in expression of LTP in perforant path-granule cell synapses and (b) to establish whether NGF contributed to activation of PI 3–kinase and thereby LTP.

In this study we focused on assessing the role of PI 3–kinase in modulating presynaptic, rather than postsynaptic, events therefore as a first step in establishing whether PI 3–kinase plays a role in LTP, we investigated the effect of the PI

Acknowledgements

A.K. is a Trinity Foundation Scholar. We are grateful to Forbairt, Ireland and The Health Research Board for financial support.

References (42)

  • A. Kelly et al.

    Evidence that nerve growth factor plays a role in long-term potentiation in the rat dentate gyrus

    Neuropharmacology

    (1998)
  • D. Leopoldt et al.

    Gbetagamma stimulates phosphoinositide 3–kinase–gamma by direct subunit interaction with two domains of the catalytic p110 subunit

    Journal of Biological Chemistry

    (1998)
  • B. McGahon et al.

    The synergism between metabotropic glutamate receptor activation and arachidonic acid on glutamate release is occluded by induction of long-term potentiation in the dentate gyrus

    Neuroscience

    (1996)
  • H. Nakanishi et al.

    Activation of ζ isozyme of protein kinase C by phosphatidylinositol 3,4,5–trisphosphate

    Journal of Biological Chemistry

    (1993)
  • H. Oda et al.

    Inhibition of protein kinase C–dependent noradrenaline release by wortmannin in PC12 cells

    Archives of Biochemistry and Biophysics

    (1997)
  • P. Ordronneau et al.

    An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates

    Journal of Immunological Methods

    (1991)
  • L.E. Rameh et al.

    Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3–kinase association with tyrosine-phosphorylated proteins

    Cell

    (1995)
  • S.P. Soltoff et al.

    Nerve growth factor promotes the actiation of phosphatidylinositol 3–kinase and its association with trk tyrosine kinase

    Journal of Biological Chemistry

    (1992)
  • L. Stephens et al.

    Agonist-stimulated synthesis of phosphatidylinositol (3,4,5)–trisphosphate: a new intracellular signalling system?

    Biochemical, Biophysical Acta

    (1993)
  • X. Tang et al.

    Purification and characterization of Gβγ–responsive phosphoinositide 3–kinases from pig plateket cytosol

    Journal of Biological Chemistry

    (1997)
  • A. Toker et al.

    Activation of protein kinase C family members by the novel polyphosphoinositides, Ptd–3,4–P2 and Ptd–3,4,5–P3

    Journal of Biological Chemistry

    (1994)
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