The Inhibition of Glutamate Release by Metabotropic Glutamate Receptor 7 Affects Both [Ca2+] and cAMP

Abstract

Metabotropic glutamate receptors (mGluRs) from group III reduce glutamate release. Because these receptors reduce cAMP levels, we explored whether this signaling pathway contributes to release inhibition caused by mGluRs with low affinity for L-2-amino-4-phosphonobutyrate (L-AP4). In biochemical experiments with the population of cerebrocortical nerve terminals we find that L-AP4 (1 mM) inhibited the Ca 2؉ -dependent-evoked release of glutamate by 25%. This inhibitory effect was largely prevented by the pertussis toxin but was insensitive to inhibitors of protein kinase C bisindolylmaleimide and protein kinase A H-89. Furthermore, this inhibition was associated with reduction in N-type Ca 2؉ channel activity in the absence of any detectable change in cAMP levels. In the presence of forskolin, however, L-AP4 decreased the levels of cAMP. The activation of this additional signaling pathway was very efficient in counteracting the facilitation of glutamate release induced either by forskolin or the ␤-adrenergic receptor agonist isoproterenol. Imaging experiments to measure Ca 2؉ dynamics in single nerve terminals showed that L-AP4 strongly reduced the Ca 2؉ response in 28% of the nerve terminals. Moreover, immunochemical experiments showed that 25-35% of the nerve terminals that were immunopositive to synaptophysin were also immunoreactive to the low affinity L-AP4-sensitive mGluR7. Then, mGluR7 mediates the inhibition of glutamate release caused by 1 mM L-AP4, primarily by a strong inhibition of Ca 2؉ channels, although high cAMP uncovers the receptor ability to decrease cAMP. Metabotropic glutamate receptors (mGluR)1 from group III consist of four different subtypes (mGluR4, -6, -7, and -8) and are activated by the selective agonist L(ϩ)-2-amino-4-phosphonobutyrate (L-AP4) (Refs. 1-5; for review, see Refs. 6 and 7). The localization of these receptors within presynaptic active zones (8, 9) is consistent with their role as autoreceptors mediating the feedback inhibition of glutamate release (10 -13). In neuronal preparations the inhibition of glutamate release by these receptors has been considered to be mediated by the reduction of voltage-dependent Ca 2ϩ channel activity (11,14,15). However, these receptors also decrease cAMP levels both in heterologous expression systems (1-5) and in neuronal preparations (13, 16). Nevertheless, it remains unclear what influence this signaling pathway has on the inhibition of glutamate release.The activity of L-AP4-sensitive mGluRs is developmentally regulated in different brain areas (13,(17)(18)(19). In the cerebral cortex mGluRs with high affinity for L-AP4 (EC 50 2.3 M) potently reduce glutamate release in nerve terminals from young (13) but not from adult rats (18). It is therefore possible that in the cerebral cortex of adult rats, mGluRs with low affinity for L-AP4 act as presynaptic receptors that mediate synaptic inhibition. In preparations of this tissue, we have examined whether a mGluR with low affinity for L-AP4 reduces glutamate release in cerebrocortical nerve ter...

Co-expression of Metabotropic Glutamate Receptor 7 and N-type Ca2+ Channels in Single Cerebrocortical Nerve Terminals of Adult Rats

“…The video images were obtained using a slow scan CCD camera (Hamamatsu C4880) operating at 12-bit digitalization (4096 levels), and the output from the camera was stored by the computerized imaging system Lucida 3.0 (Kinetic Imaging, Ltd.). Ca 2ϩ images were analyzed as described previously (11,12). For [Ca 2ϩ ] cyt measurements, synaptosomes were stimulated by 10-s pulses (indicated in each graph by a bar) of 30 mM KCl or 1 mM 4AP in the absence and presence of pharmacological agonists or Ca 2ϩ channel toxins.…”

“…In the adult rat preparation of cerebrocortical nerve terminals, this release was inhibited by high concentrations of the group III mGluR agonist L-AP4 through the activation of mGluR7 (11). Because this inhibition of release is the result of a reduced influx of Ca 2ϩ into the nerve terminals (11), we sought to determine which type of Ca 2ϩ channel was being inhibited by the receptor.…”

“…We demonstrated previously that the cerebrocortical nerve terminals from adult rats express mGluR7 (11). To establish whether other L-AP4-sensitive mGluRs are also present in this preparation, nerve terminals were double-labeled with antisera against synaptophysin and the L-AP4 sensitive mGluRs (subtypes 4, 7, and 8) expressed in the brain.…”

“…In cerebrocortical nerve terminals, the release of glutamate is predominantly controlled by P/Q-type Ca 2ϩ channels, although a significant but less important contribution comes from N-type Ca 2ϩ channels. However, we have recently found that the activation of mGluR7 only inhibits the N-type Ca 2ϩ channel coupled release (11).…”

“…Through their coupling to G i/o proteins, activation of these receptors inhibits adenylyl cyclase (6). However, the inhibition of evoked glutamate release has primarily been associated with a reduction in the activity of the Ca 2ϩ channels that govern the exocytotic process (7)(8)(9)(10)(11)(12). In cerebrocortical nerve terminals, the release of glutamate is predominantly controlled by P/Q-type Ca 2ϩ channels, although a significant but less important contribution comes from N-type Ca 2ϩ channels.…”