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Metabotropic glutamate receptor analogues inhibit p[NH]ppG-stimulated phospholipase C activity in bovine brain coated vesicles: involvement of a pertussis toxin-sensitive G-protein.

ABSTRACT: Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG)-stimulated phospholipase C (PLC) activity in bovine brain coated vesicles is inhibited by glutamate agonists. In the present study we show that quisqualic acid (QA), (+/-)-trans-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD), glutamic acid and ibotenic acid inhibited p[NH]ppG-stimulated PLC by 44, 41, 36 and 25% respectively. Carbachol also produced an inhibition of p[NH]ppG-stimulated PLC by 45%. The inhibition caused by trans-ACPD and QA was dose-dependent. DL-2-Amino-3-phosphonopropionic acid and (RS)-alpha-methyl-4-carboxyphenylglycine, specific antagonists of metabotropic glutamate receptors (mGluRs), abolished these inhibitory effects. trans-ACPD inhibition of p[NH]ppG-stimulated PLC was also observed in the presence of ionotropic glutamate receptor antagonists. When carbachol and QA or trans-ACPD were combined, additive inhibitory effects were observed. Preincubation of bovine brain coated vesicles with pertussis toxin abolished the inhibitory effects of mGluR analogues and carbachol on p[NH]ppG-stimulated PLC activity. The presence of Gs alpha and pertussis toxin substrates, Gi alpha and Go alpha subunits as well as PLC beta 1 in bovine brain coated vesicles has been confirmed by immunoblot. These results support the coupling of mGluRs to a PLC in an inhibitory manner through a pertussis toxin-sensitive G-protein in bovine brain coated vesicles.

SUBMITTER: Martin M

PROVIDER: S-EPMC1136726 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

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Project description:Phospholipase C (PLC) beta4, one of the four isoforms of PLCbetas, is the sole isoform expressed in the mouse ventral posterolateral thalamic nucleus (VPL), a key station in pain processing. The mouse thalamus also has been shown to express a high level of metabotropic glutamate receptor type 1 (mGluR1), which stimulates PLCbetas through activation of Galphaq/11 protein. It is therefore expected that the thalamic mGluR1-PLCbeta4 cascade may play a functional role in nociceptive transmission. To test this hypothesis, we first studied behavioral responses to various nociceptive stimuli in PLCbeta4 knock-out mice. We performed the formalin test and found no difference in the pain behavior in the first phase of the formalin test, which is attributed to acute nociception, between PLCbeta4 knock-out and wild-type mice. Consistent with this result, acute pain responses in the hot plate and tail flick tests were also unaffected in the PLCbeta4 knock-out mice. However, the nociceptive behavior in the second phase of the formalin test, resulting from the tissue inflammation, was attenuated in PLCbeta4 knock-out mice. In the dorsal horn of the spinal cord where PLCbeta1 and PLCbeta4 mRNAs are expressed, no difference was found between the wild-type and knock-out mice in the number of Fos-like immunoreactive neurons, which represent neuronal activity in the second phase in the formalin test. Thus, it is unlikely that spinal PLCbeta4 is involved in the formalin-induced inflammatory pain. Next, we found that pretreatment with PLC inhibitors, mGluR1 antagonists, or both, by either intracerebroventricular or intrathalamic injection, attenuated the formalin-induced pain behavior in the second phase in wild-type mice. Furthermore, activation of mGluR1 at the VPL enhanced pain behavior in the second phase in the wild-type mice. In contrast, PLCbeta4 knock-out mice did not show such enhancement, indicating that mGluR1 is connected to PLCbeta4 in the VPL. Finally, in parallel with the behavioral results, we showed in an electrophysiological study that the time course of firing discharges in VPL corresponds well to that of pain behavior in the formalin test in both wild-type and PLCbeta4 knock-out mice. These findings indicate that the thalamic mGluR1-PLCbeta4 cascade is indispensable for the formalin-induced inflammatory pain by regulating the response of VPL neurons.

Project description:1. Phospholipase D (PLD) is the key enzyme in a signal transduction pathway leading to the formation of the second messengers phosphatidic acid and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR agonists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2. The mGluR-selective agonist (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced a concentration-dependent (10-300 microM) activation of PLD in the hippocampus, neocortex, and striatum, but not in the cerebellum. The effect was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3. The rank order of potencies for agonists stimulating the PLD response was: quisqualate > ibotenate > (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine > (1S,3R)-ACPD > L-cysteine sulphinic acid > L-aspartate > L-glutamate. L-(+)-2-Amino-4-phosphonobutyric acid and the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyphenylglycine (100300 microM), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 microM (1S,3R)-ACPD. 4. (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a competitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 microM (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5. 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13-dibutyrate (PDBu), activators of protein kinase C, at 1 microM had a stimulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-induced phosphoinositide hydrolysis. The protein kinase C inhibitor, staurosporine (1 and 10 microM) reduced PLD activation induced by 1 microM PDBu but not by 100 microM (1S,3R)-ACPD. 6. Our results suggest that PLD-linked mGluRs in rat hippocampus may be distinct from any known mGluR subtype coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.

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Metabotropic glutamate receptor analogues inhibit p[NH]ppG-stimulated phospholipase C activity in bovine brain coated vesicles: involvement of a pertussis toxin-sensitive G-protein.

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