Glutamate Exocytosis and MARCKS Phosphorylation Are Enhanced by a Metabotropic Glutamate Receptor Coupled to a Protein Kinase C Synergistically Activated by Diacylglycerol and Arachidonic Acid

Abstract

4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K+ channel responsible for stabilizing the membrane potential . We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4,ß-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGIuR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] mimics the action of 48phorbol dibutyrate, but only in the presence of 2 btM arachidonic acid (AA) . In this article, we investigate the role of AA . AA plus (1S,3R)-ACPD is without effect on KCIinduced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca 2+ channel or Ca 2 '-secretion coupling . Diacylglycerol concentrations are greatly enhanced by (1 S,3R)-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C . Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC . mGIuR activation by (1S,3R)-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGIuR, consistent with the known behaviour of certain purified PKC isoforms . The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity . Key Words: Glutamate exocytosis-MARCKS phosphorylation-Protein kinase C-Metabotropic glutamate receptor-Diacylglycerol-Arachidonic acid .

Noncompetitive Metabotropic Glutamate5Receptor Antagonist (E)-2-Methyl-6-styryl-pyridine (SIB1893) Depresses Glutamate Release through Inhibition of Voltage-Dependent Ca2+Entry in Rat Cerebrocortical Nerve Terminals (Synaptosomes)

“…The activation of group I mGlu receptors has been shown to be facilitatory at nerve terminals, an effect likely to be the result of DAG production and PKC activation (Herrero et al, 1992;Coffey et al, 1994;Lu et al, 1997;Reid et al, 1999;Thomas et al, 2000). In the present study, we found that SIB1893-mediated inhibition of glutamate was reduced from 19.8 Ϯ 1.5 to 3.0 Ϯ 1.8% and 4.7 Ϯ 1.5% in the presence of PDBu and Ro 32-0432, respectively.…”

“…To confirm that an mGluR5/PLC/PKC cascade is being suppressed by SIB1893 in its inhibition of glutamate, we next examined whether the SIB1893-mediated effect was sensitive to PKC inhibition. Control 4AP (3 mM)-evoked release of 19.4 Ϯ 0.5 nmol/mg/4 min was attenuated by the PKC inhibitor Ro 32-0432 (1 M) to 10.8 Ϯ 0.4 nmol/mg/4 min (n ϭ 5; P Ͻ 0.01), reflecting an inhibition of the reported basal PKC activity present in nerve terminals (Coffey et al, 1994). Crucially, however, in the presence of Ro 32-0432, SIB1893 addition had no further effect on 4AP-evoked glutamate release (10.3 Ϯ 0.7 nmol/ Fig.…”

“…Whether the inhibition of glutamate release from nerve terminals underlies this neuroprotective action of mGlu 5 R blockade specifically is subject of debate given that the relative contributions of the two group I mGluRs (mGlu 1 R and mGlu 5 R) to the facilitation of glutamate remain contentious (Sistiaga et al, 1998;Reid et al, 1999;Fazal et al, 2003). Another connected issue arises from studies with isolated cerebrocortical nerve terminals (synaptosomes) reporting remarkably high PKC activity even in unstimulated conditions and in the effective absence of ligand (Coffey et al, 1994). This latter observation begs the question as to whether the high basal PKC activity reflects a basal, "constitutive" activity of mGluRs reported in the absence of ligand (Gasparini et al, 2002).…”

Sevoflurane depolarizes pre‐synaptic mitochondria in the central nervous system

“…The synaptosomal plasma membrane can be depolarized with K + producing a clamped depolarisation, which does not, however, mimic physiological conditions (60). A more physiological membrane depolarisation is evoked by the K + channel inhibitor 4-AP (13).…”

Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages

“…A direct role for PKC in protein secretion per se is supported by its established roles in mediating the secretion of various cargoes, such as glutamate and noradrenaline from neuronal cell lines (32)(33)(34)(35), mucin from colonic tumor cell lines (36), histamine from rat basophilic leukemia mast cells (37), and insulin and glucagon from pancreatic cells (38 -41). Furthermore, PKC has been reported to interact with a number of proteins associated with intracellular transport (e.g.…”