Phosphoinositide hydrolysis in vivo with group I metabotropic glutamate receptor agonists

doi:10.1016/S0006-8993(99)01065-3

Brain Research

Volume 821, Issue 2, 13 March 1999, Pages 539-545

https://doi.org/10.1016/S0006-8993(99)01065-3 Get rights and content

Abstract

The present report describes the effect of mGluR agonists and antagonists administration on phospholipase C activation by measuring accumulation of [³H] inositol monophosphates (IP) in rats pre-labeled with [³H]myo-inositol (i.c.v. 24 h pre-treatment). The levels of accumulated [³H]IP were then determined from clarified tissue homogenates using ion-exchange chromotography. Following lithium chloride treatment (10 mg/kg, s.c.), (R/S)-3,5-dihydroxyphenylglycine (DHPG), a selective group I mGluR agonist was found to dose-dependently cause a maximal increase in the levels of [³H]IP at 0.3 to 3 μmol/8 μl i.c.v. with lower doses resulting in less efficacious or no responses. This effect was temporal-dependent reaching a plateau at 2 h. The DHPG-induced increases in [³H]IP were most pronounced in the hippocampus where a 3- to 5-fold increase above vehicle was consistently found, but significant approximately 2-fold increases were also seen in the cerebellum, striatum and frontal cortex. The mixed group I and II agonist, (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (1S,3R-t-ACPD), similarly resulted in dose-dependent increases in [³H]IP levels with doses of 1 to 3 μmol i.c.v. Furthermore, this effect was enantiomer specific since the less active 1R,3S-t-ACPD failed to alter phosphoinositol hydrolysis. Administration of the selective mGluR5 agonist (R/S)-2-chloro-5-hydroxyphenyl-glycine (CHPG) resulted in a dose-dependent increase in hippocampal but not cerebellar levels of [³H]IP, consistent with the receptor distribution of the two group I mGluRs. The Group II agonist LY354740 (1S,2S,5R,6S-2-aminobicycl[3.1.0]hexane-2,6-dicarboxylate monohydrate) and the group III agonist L-AP₄ (L-(+)-2-amino-4-phosphonobutyric acid) failed to alter the levels of [³H]IP. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycycloprop-1-yl)-3-(xanth-9-yl)propanoic acid) is a nM potent Group II antagonist. However, LY341495 has also been found to have μM potency in inhibiting mGluR1 and 5. The stimulation of [³H]PI hydrolysis by 1 μmol DHPG was dose-dependently blocked by co-administration of the mGluR antagonists, LY341495 at doses that are constant with an interaction at Group I mGluR's. Taken together these results suggest that stimulation of group I mGluRs results in measurable increases in PI hydrolysis in vivo. This method could be quite useful in determining the doses and routes of administration of agonists and antagonists that are required to interact with group I mGluRs.

Introduction

Glutamate through its many receptors mediates most of the excitatory neurotransmission within the central nervous system. Therefore, it is not surprising to find that modulation of the glutamatergic system through changes in glutamate release or alteration in postsynaptic receptor activation is thought to be involved in a variety of neurological disease states from memory and learning deficits pathways [12], to drug withdrawal and neurodegeneration 11, 20. Glutamate acts via two distinct classes of receptors, the ionotropic receptors that are cation channels, and the metabotropic receptors (mGluRs) that are coupled to G-proteins and modulate intracellular signal transduction pathways [14].

The eight mGluRs are further subdivided based on their amino acid sequence homology determined from isolated clones, their ability to effect certain signal transduction mechanisms, and their known pharmacological properties [14]. For instance, the Group II mGluRs consist of mGluR2 and mGluR3, which have been found to be negatively coupled to adenylate cyclase, and are activated by such compounds as LY354740 (1S,2S,5R,6S-2-aminobicycl[3.1.0]hexane-2,6-dicarboxylate monohydrate) 13, 19. Similarly the Group III mGluRs, including mGluR4, mGluR6, mGluR7 and mGluR8, are negatively coupled to adenylate cyclase and are potently activated by L-AP₄ (L-(+)-2-amino-4-phosphonobutyric acid) [17]. Lastly, the Group I mGluRs, which include the mGluR1 and mGluR5, are known to activate phospholipase C (PLC) thereby resulting in the increased hydrolysis of phosphoinositides and intracellular calcium mobilization. There are several compounds that are reported to activate the Group I mGluRs in in vitro systems including: DHPG (R/S)-3,5-dihydroxyphenylglycine 7, 18which activates both the mGluR1 and mGluR5 receptors, CHPG (R/S)-2-chloro-5-hydroxyphenyl-glycine [5]which is proposed to be selective for the mGluR5, and tADA trans-azetidine-2,4-dicarboxylic acid [9]that may also be selective for the mGluR5 receptor. It should be noted that many of these pharmacological tools are not ideal in that they cross react between groups of receptors. For instance, LY354740 can potently activate the mGluR8 receptors but not the other Group III mGluRs, while other compounds such as 1S,3R-t-ACPD (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid [17]are believed to activate all of the Group I and II mGluRs.

While the in vitro and behavioral effects of the Group I agonists have been investigated, to date there are no reports of the in vivo biochemical effects of these pharmacological tools. Previous work 4, 10, 15, 25has used a pretreatment with [³H]myo-inositol, subsequent injection of lithium and isolation of inositol monophosphates to characterize the in vivo effects on PLC activation with muscarinic agents. Recent work [26]has also successfully used this technique to characterize the in vivo serotonergic and muscarinic PLC-coupled receptor actions of some antipsychotics. The present experiments were undertaken to characterize the effects on PLC activation following administration of selected mGluR agonists and antagonists.

Section snippets

Materials

[³H]Myo-inositol (18 Ci/mmol) was purchased from Amersham Life Sciences (Arlington Heights, IL). DHPG, CHPG, tADA, 1S,3R-t-ACPD, 1R,3S-t-ACPD, L-AP₄ were obtained from Tocris (Ballwin, MO). Pilocarpine was obtained from RBI (Natick, MA). All other compounds were synthesized at Lilly Research Laboratories. All drug solutions were made in purified water with equimolar NaOH added where required to dissolve the compounds.

Animals and treatment

The procedures of Patel and Freedman [15]as modified by Bymaster et al. [4]

Agonist induced [³H]phosphoinositol hydrolysis

Following LiCl treatment, 1 μmol DHPG i.c.v. induced a 2-fold and 5-fold increase in [³H]IP in the cerebellum and hippocampus (Table 1). This is similar to the 3-fold and 6-fold increase seen with the muscarinic agent pilocarpine. A clear dose-dependent response was apparent as 300 nmol to 3 μmol DHPG resulted in maximal increases in [³H]IP within the hippocampus and cerebellum while lower doses resulted in either less efficacious or no significant increases (Fig. 1A). DHPG also demonstrated a

Discussion

Previous work has shown that lithium inhibits inositol-monophosphatase [6]in the phosphatidyl-inositol cycle and that following lithium treatment the brain levels of inositol and inositol monophosphates in rodents are altered 1, 2, 21. By prelabeling a portion of the phospholipid pool with an i.c.v. 24 h [³H]myo-inositol pretreatment, it is possible to isolate [³H]IP formed after lithium treatment with simple ion exchange chromatography methods. In this way, it has been shown that muscarinic

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Although it has been shown that DHPG can stimulate internalization of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and NMDA receptors (Snyder et al., 2001), most likely through GTPase Rab5b facilitated endocytosis (Arnett et al., 2004), key elements of signaling pathways involved in group I mGluR-mediated neuroprotection have not yet been fully identified. Group I mGluRs link to the downstream targets through a PLC-dependent pathway (Rhee, 2001; Llansola et al., 2000; Johnson et al., 1999). At the neuronal level, DHPG reduces both evoked and spontaneous excitatory post-synaptic currents (EPSCs), a phenomenon often referred to as metabotropic glutamate receptor-dependent form of long-term depression or mGluR-LTD (Xiao et al., 2001).
Group I metabotropic glutamate receptor (mGluR) agonist DHPG reduced nerve cell death caused by their exposure to NMDA (“neuroprotective effect”) and attenuated NMDA receptor-mediated currents [Blaabjerg, M., Baskys, A., Zimmer, J., Vawter, M. P., 2003b. Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors. Brain Research, Molecular Brain Research 117, 196–205.]. In the present study, we used organotypic hippocampal culture preparation to examine specific phospholipase C (PLC) inhibitor U73122 effects on DHPG-induced neuroprotection, changes in excitatory synaptic transmission associated with the neuroprotective DHPG treatment and a role of group I mGluR ligands in neurogenesis. Results show that short (10 min) DHPG treatment did not result in neuroprotection but significantly depressed field synaptic potentials (fEPSP) in the Schaffer collateral-CA1 pathway. The fEPSP depression was not affected by the PLC inhibitor U73122. In contrast, prolonged (2-h) treatment of cultures with DHPG induced a significant protective effect that was blocked by a PLC inhibitor U73122 but not by its inactive analog U73343. Voltage-clamp measurements of spontaneous miniature excitatory post-synaptic currents (EPSCs) recorded in CA1 neurons from cultures treated with DHPG (10 μM, 2 h) showed a significant reduction of the EPSC amplitude in DHPG-treated but not control (untreated) cultures. This reduction was completely abolished by U73122, suggesting a PLC involvement. Since activation of PLC is thought to be associated with cell proliferation, we investigated whether group I mGluR agonist DHPG or subtype antagonists LY367385 and MPEP have an effect on dentate granule cells expressing immature neuronal marker TOAD-64. DHPG (100 μM, 72 h) slightly but not significantly increased the number of TOAD-64 positive cells. The mGluR1 antagonists LY367385 (10 μM, 72 h) markedly decreased the number of TOAD-64 positive cells and mGluR5 antagonist MPEP (1 μM, 72 h) had no effect. These data suggest that (1) prolonged activation of group I mGluRs reduces nerve cell susceptibility to excitotoxic injury in a PLC-dependent manner; (2) this reduction is associated with a PLC-dependent depression of excitatory synaptic transmission; and (3) mGluR1 activation may facilitate neurogenesis.
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We have previously shown that the release of central neurotransmitters can be modulated by the activation of Group I and Group II subtypes of G-protein-linked metabotropic glutamate (mGlu) receptors. To date, however, very little is known about the regulation of serotonergic neurotransmission by these receptor subtypes. In the present study, we have utilized in vivo intracerebral microdialysis to elucidate the roles of Group I and Group II mGlu receptors in the regulation of neuronal 5-hydroxytryptamine (5-HT) release in the frontal cortex of conscious, freely moving rats. Dialysate 5-HT was of neuronal origin with basal release showing strong calcium dependency and tetrodotoxin sensitivity and marked elevation following K⁺-induced depolarization. The broad-spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; 1–3 mM] did not significantly modify basal cerebrocortical 5-HT release. Similarly, the Group I mGlu receptor-specific agonist (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG; 1–3 mM] showed no marked effect on cortical dialysate 5-HT levels. To eliminate the possibility that these findings were the result of receptor desensitization, the effects of lower concentrations of (RS)-DHPG (100–300 μM) and shorter ligand exposure time (15 min) were also evaluated. Dialysate 5-HT levels remained unmodified by these manipulations. In comparison, the Group II mGlu receptor agonist, (2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (L-CCG-1; 500 μM), evoked a marked facilitation of release (approximately 150% of basal) which was fully reversed by the Group I/II antagonist, (S)-α-methyl-4-carboxyphenylglycine [(S)-MCPG; 3 mM]. The modulatory action of L-CCG-1 showed a bell-shaped concentration-response relationship. (S)-MCPG (3 mM) and the potent and selective mGlu₅ receptor antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP; 100 μM), when given alone, did not significantly modify 5-HT levels.
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¹: Published on the World Wide Web on 4 February 1999.

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Interactive reportPhosphoinositide hydrolysis in vivo with group I metabotropic glutamate receptor agonists1

Abstract

Introduction

Section snippets

Materials

Animals and treatment

Agonist induced [3H]phosphoinositol hydrolysis

Discussion

Biochem. Biophys. Res. Commun.

Neurosci. Lett.

Brain Res.

Neuropharmacology

J. Biol. Chem.

Neuropharmacology

Neuropharmacology

Trends Pharmacol. Sci.

Prog. Neurobiol.

Eur. J. Pharmacol.

Neurochem. Int.

Neuropharmacology

Neurobio. Aging

Interactive report
Phosphoinositide hydrolysis in vivo with group I metabotropic glutamate receptor agonists¹

Agonist induced [³H]phosphoinositol hydrolysis