Evidence for a rol of calmodulin in calcium-induced noradrenaline release from permeated synaptosomes: effects of calmodulin antibodies and antagonists

The nervous tissue-specific protein B-50 (GAP-43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin-binding proteins. To investigate to what extent calmodulin and the interaction between B-50 and calmodulin are involved in the mechanism of Ca2+-induced noradrenaline release, we introduced polyclonal anti-calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W-7, calmidazolium, and polymyxin B into streptolysin-O-permeated synaptosomes prepared from rat cerebral cortex. Anti-calmodulin antibodies, which inhibited Ca2+/calmodulin-dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca2+-induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W-7, and calmidazolium inhibited Ca2+-induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca2+-induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B-50 and calmodulin, thus lending further support to the hypothesis that B-50 serves as a local Ca2+-sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca2+/calmodulin-dependent enzymes involved in the regulation of one or more steps in the release mechanism

Monoclonal antibody NM2 recognizes the protein kinase C phosphorylation site in B-50 (GAP-43) and in neurogranin (BICKS)

Mouse monoclonal B-50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B-50 (GAP-43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted with peptide fragments of rat B-50 containing the unique protein kinase C (PKC) phosphorylation site at serine-41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B-50 (34-52). To narrow down the epitope domain synthetic B-50 peptides were tested in ELISAs. In contrast to NM6, NM2 immunoreacted with B-50 (39-51) peptide, but not with B-50 (43-51) peptide or a C-terminal B-50 peptide. Preabsorption by B-50 (39-51) peptide of NM2 inhibited the binding of NM2 to rat B-50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B-50 during endogenous phosphorylation of synaptosomal plasma membrane proteins. Preabsorption of NM2 by B-50 (39-51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B-50 and neurogranin. Therefore, NM2 may be a useful tool in physiological studies of the role of PKC-mediated phosphorylation and calmodulin binding of B-50 and neurogranin

Glutamate and γ-aminobutyric acid content and release of synaptosomes from temporal lobe epilepsy patients

During surgical intervention in medically refractory temporal lobe epilepsy (TLE) patients, diagnosed with either mesial temporal lobe sclerosis (MTS)- or tumor (T)-associated TLE, biopsies were taken from the anterior temporal neocortex and the hippocampal region. Synaptosomes, isolated from these biopsies were used to study intrasynaptosomal Ca2+ levels ([Ca2+]i), and glutamate and -aminobutyric acid (GABA) contents and release. All synaptosomal preparations demonstrated a basal [Ca2+]i of about 200 nM, except neocortical synaptosomes from MTS-associated TLE patients (420 nM). K+-induced depolarization resulted in a robust increase of the basal [Ca2+]i in all preparations. Neocortical synaptosomes from TLE patients contained 22.9 ± 3.0 nmol glutamate and 4.6 ± 0.5 nmol GABA per milligram synaptosomal protein, whereas rat cortical synaptosomes contained twice as much glutamate and four times as much GABA. Hippocampal synaptosomes from MTS-associated TLE patients, unlike those from T-associated TLE patients, contained about 70% less glutamate and 55% less GABA than neocortical synaptosomes. Expressed as percentage of total synaptosomal content, synaptosomes from MTS-associated TLE patients exhibited an increased basal and a reduced K+-induced glutamate and GABA release compared to rat cortical synaptosomes. In MTS-associated TLE patients, only GABA release from neocortical synaptosomes was partially Ca2+-dependent. Control experiments in rat synaptosomes demonstrated that at least part of the reduction in K+-induced release can be ascribed to resection-induced hypoxia in biopsies. Thus, synaptosomes from MTS-associated TLE patients exhibit a significant K+-induced increase in [Ca2+]i, but the consequent release of glutamate and GABA is severely impaired. Our data show that at least part of the differences in glutamate and GABA content and release between human biopsy material and fresh rat tissue is due to the resection time