[Show abstract][Hide abstract] ABSTRACT: Protein kinase A and protein kinase C are involved in processes that enhance glutamate release at glutamatergic nerve terminals. However, it is not known whether these two kinases co-exist within the same nerve terminal, nor is it clear what impact their simultaneous activation may have on neurotransmitter release. In cerebrocortical nerve terminals, co-application of forskolin, which increases cAMP levels and activates protein kinase A, and 4beta-phorbol dibutyrate, a direct activator of protein kinase C, synergistically enhanced the spontaneous release of glutamate. This enhancement exhibited both tetrodotoxin-sensitive and tetrodotoxin-resistant components. Interestingly, the tetrodotoxin-resistant component of release was not observed when cyclic AMP-dependent protein kinase (PKA) and calcium- and phospholipid-dependent protein kinase (PKC) were activated separately, but developed slowly after the co-activation of the two kinases, accounting for 50% of the facilitated release. This release component was dependent on voltage-dependent Ca2+ channels that opened spontaneously after PKA and PKC activation and occurred in the absence of Na+ channel firing. These data provide functional evidence for the co-existence of PKA- and PKC-signalling pathways in a subpopulation of glutamatergic nerve terminals.
Journal of Neurochemistry 01/2004; 87(5):1101-11. DOI:10.1046/j.1471-4159.2003.02065.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in cerebrocortical nerve terminals of adult rats, the inhibition of glutamate release is mediated by mGluR7. In this preparation, the major component of glutamate release is supported by P/Q-type Ca2+ channels (72.7%). However, mGluR7 selectively reduced the release component that is associated with N-type Ca2+ channels (29.9%). Inhibition of P/Q channels by mGluR7 is not masked by the higher efficiency of these channels in driving glutamate release when compared with N-type channels. Thus, activation of mGluR7 failed to reduce the release associated with P/Q channels when the extracellular calcium concentration, ([Ca2+]o), was reduced from 1.3 to 0.5 mm. Through Ca2+ imaging, we show that Ca2+ channels are distributed in a heterogeneous manner in individual nerve terminals. Indeed, in this preparation, nerve terminals were observed that contain N-type (31.1%; conotoxin GVIA-sensitive) or P/Q-type (64.3%; agatoxin IVA-sensitive) channels or that were insensitive to these two toxins (4.6%). Interestingly, the great majority of the responses to l-AP4 (95.4%) were observed in nerve terminals containing N-type channels. This specific co-localization of mGluR7 and N-type Ca2+-channels could explain the failure of the receptor to inhibit the P/Q channel-associated release component and also reveal the existence of specific targeting mechanisms to localize the two proteins in the same nerve terminal subset.
[Show abstract][Hide abstract] ABSTRACT: The release properties of glutamatergic nerve terminals are influenced by a number of factors, including the subtype of voltage-dependent calcium channel and the presence of presynaptic autoreceptors. Group III metabotropic glutamate receptors (mGluRs) mediate feedback inhibition of glutamate release by inhibiting Ca(2+) channel activity. By imaging Ca(2+) in preparations of cerebrocortical nerve terminals, we show that voltage-dependent Ca(2+) channels are distributed in a heterogeneous manner in individual nerve terminals. Presynaptic terminals contained only N-type (47.5%; conotoxin GVIA-sensitive), P/Q-type (3.9%; agatoxin IVA-sensitive), or both N- and P/Q-type (42.6%) Ca(2+) channels, although the remainder of the terminals (6.1%) were insensitive to these two toxins. In this preparation, two mGluRs with high and low affinity for l(+)-2-amino-4-phosphonobutyrate were identified by immunocytochemistry as mGluR4 and mGluR7, respectively. These receptors were responsible for 22.2 and 24.1% reduction of glutamate release, and they reduced the Ca(2+) response in 24.4 and 30.3% of the nerve terminals, respectively. Interestingly, mGluR4 was largely (73.7%) located in nerve terminals expressing both N- and P/Q-type Ca(2+) channels, whereas mGluR7 was predominantly (69.9%) located in N-type Ca(2+) channel-expressing terminals. This specific coexpression of different group III mGluRs and Ca(2+) channels may endow synaptic terminals with distinct release properties and reveals the existence of a high degree of presynaptic heterogeneity.
[Show abstract][Hide abstract] ABSTRACT: In nerve terminals, the activation of Ca(2+) channels of either the P/Q or N type triggers glutamate release. In turn, presynaptic metabotropic glutamate receptors control this process through the modulation of these Ca(2+) channels. By measuring glutamate release from cerebrocortical nerve terminals we show that at physiological concentrations of extracellular Ca(2+), the agonist L(+)-2-amino-4-phosphonobutyrate only reduced the N-type channel coupled release. However, at lower concentrations of extracellular calcium the contribution of N-type channels was abolished and P/Q-type channels were entirely responsible for the remaining release of glutamate. Under these conditions, L(+)-2-amino-4-phosphonobutyrate was capable of reducing the P/Q-type channel mediated release. These results indicate that N-type Ca(2+) channels are less efficiently coupled to glutamate release than P/Q-type channels and that this differential coupling may have important implications for presynaptic modulation of synaptic transmission.
[Show abstract][Hide abstract] 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 forl-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 Ca2+-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 Ca2+ 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 β-adrenergic receptor agonist isoproterenol. Imaging
experiments to measure Ca2+ dynamics in single nerve terminals showed thatl-AP4 strongly reduced the Ca2+ 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 affinityl-AP4-sensitive mGluR7. Then, mGluR7 mediates the inhibition of glutamate release caused by 1 mm
l-AP4, primarily by a strong inhibition of Ca2+channels, although high cAMP uncovers the receptor ability to decrease cAMP.