Long-term potentiation (LTP) is a well known experimental model for studying the activity-dependent enhancement of synaptic plasticity, and because of its long duration and its associative properties, it has been proposed as a system to investigate the molecular mechanisms of memory formation. At present, there are several lines of evidence that indicate that pre-and postsynaptic kinases and their specific substrates are involved in molecular mechanisms underlying LTP. Many studies focus on the involvement of protein kinase C (PKC). One way to investigate the role of PKC in long-term potentiation is to determine the degree of phosphorylation of its substrates after in situ phosphorylation in hippocampal slices. Two possible targets are the presynaptic membrane-associated protein B-50 (a.k.a. GAP 43, neuromodulin and F1), which has been implicated in different forms of synaptical plasticity in the brain such as neurite outgrowth, hippocampal LTP and neurotransmitter release, and the postsynaptic protein neurogranin (a.k.a. RC3, BICKS and p17) which function remains to be determined. This review will focus on the protein kinase C activity in pre-and postsynaptic compartment during the early phase of LTP and the possible involvement of its substrates B-50 and neurogranin.

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We studied the molecular events underlying K(+)-induced phosphorylation of the neuron-specific protein kinase C substrate B-50. Rat cortical synaptosomes were prelabelled with 32P-labelled orthophosphate. B-50 phosphorylation was measured by an immunoprecipitation assay. In this system, various phorbol esters, as well as a synthetic diacylglycerol derivative, enhance B-50 phosphorylation. K+ depolarization induces a transient enhancement of B-50 phosphorylation, which is totally dependent on extracellular Ca2+. Also, the application of the Ca2+ ionophore A23187 induces B-50 phosphorylation, but the magnitude and kinetics of A23187-induced B-50 phosphorylation differ from those induced by depolarization. The protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and staurosporine antagonize K(+)- as well as PDB-induced B-50 phosphorylation, whereas trifluoperazine and calmidazolium are ineffective under both conditions. We suggest that elevation of the intracellular Ca2+ level after depolarization is a trigger for activation of protein kinase C, which subsequently phosphorylates its substrate B-50. This sequence of events could be of importance for the mechanism of depolarization-induced transmitter release.

To investigate a possible function of the nervous tissue-specific protein kinase C substrate B-50/GAP-43 in regulation of the dynamics of the submembranous cytoskeleton, we studied the interaction between purified B-50 and actin. Both the phosphorylated and dephosphorylated forms of B-50 cosedimented with filamentous actin (F-actin) in a Ca(2+)-independent manner. Neither B-50 nor phospho-B-50 had any effect on the kinetics of actin polymerization and on the critical concentration at steady state, as measured using pyrenylated actin. Light scattering of F-actin samples was not increased in the presence of B-50, suggesting that B-50 does not bundle actin filaments. The number of actin filaments, determined by [3H]cytochalasin B binding, was not affected by either phospho- or dephospho-B-50, indicating that B-50 has neither a severing nor a capping effect. These observations were confirmed by electron microscopic evaluation of negatively stained F-actin samples, which did not reveal any structural changes in the actin meshwork on addition of B-50. We conclude that B-50 is an actin-binding protein that does not directly affect actin dynamics.

The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca(2+)-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (> 60%) ATP-dependent manner as a result of an elevation of the free Ca2+ concentration from 10(-8) to 10(-5) M Ca2+. The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca(2+)-induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca(2+)-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca(2+)-induced NA release. PKC pseudosubstrate PKC19-36, which inhibited B-50 phosphorylation (IC50 value, 10(-5) M), failed to inhibit Ca(2+)-induced NA release, even when added before the Ca2+ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca(2+)-induced NA release. Concerning the Ca2+ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca2+ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca(2+)-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca(2+)- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca(2+)-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca2+ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca2+ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca2+ influx resulting in exocytosis of NA.

B‐50 (GAP‐43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin‐binding domain of B‐50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B‐50 into streptolysin O‐permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N‐terminal residues 39–43 of rat B‐50 dose‐dependently inhibited Ca2+‐induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C‐terminal‐directed (residues 132–213) anti‐B‐50 antibodies were without effect in the same dose range. NM2 inhibited PKC‐mediated B‐50 phosphorylation at Ser41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32P‐B‐50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B‐50 in the absence of Ca2+. Similar concentrations of NM6 did not affect B‐50 phosphorylation or dephosphorylation or B‐50/calmodulin binding. We conclude that the N‐terminal residues 39–43 of the rat B‐50 protein play an important role in the process of Ca2+‐induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca2+‐ and phosphorylation‐dependent fashion.

Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca2-regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munci 8, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca2 /calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca2~-induced release of [3H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca2~/calmodulindependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles. Key Words: Rat-ExocytosisPhosphatase-Dynamin I.

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. AbstractExocytosis from nerve terminals is triggered by depolarization-evoked Ca*+ entry, which also activates calmodulin and stimulates protein phosphorylation. Ba2+ is believed to replace Ca2+ m triggering exocytosis without activation of calmodulin and can therefore be '+ used to unravel aspects of presynaptic function. We have analysed the cellular actions of Ba in relation to its effect on transmitter release from isolated nerve terminals. Barium evoked specific release of amino acid transmitters, catecholamines and neuropeptides (EC,, 0.2-0.5 mM), similar to K'/Ca" -evoked release both in extent and kinetics. Ba2 +-and Ca2+ -evoked release were not additive. In contrast to Ca'+, Ba2+ triggered release which was insensitive to trifluoperizine and hardly stimulated protein phosphorylation. These observations are in accordance with the ability of Ba2+ to replace CaZf m exocytosis without activating calmodulin. Nevertheless, calmodulin appears to be essential for regular (Cazf -triggered) exocytosis, given its sensitivity to trifluoperizine.Both Ba2+-and Ca'+-evoked release were blocked by okadaic acid. Furthermore, anti-calcineurin antibodies decreased Ba'+-evoked release. In conclusion, BaZf replaces Ca'+/calmodulin in the release of the same transmitter pool. Calmodulin-dependent phosphorylation appears not to be essential for transmitter release. Instead, our data implicate both CaZf -dependent and -independent dephosphorylation in the events prior to neurotransmitter exocytosis.Keywords: Exocytosis; Ba*+; Ca'+-dependent release; Calmodulin; Okadaic acid, Phosphatase IntroductionThe regulated exocytosis of neurotransmitters and neuropeptides is triggered by depolarization evoked Ca*+ entry. Although this process is reasonably specific for Ca2+, several divalent cations with similar physico-chemical properties, such as Ba'+, Sr2+ and Pb2+, were found to evoke transmitter release as well (Zengel and Magleby, 1980;Augustine and Eckert, 1984;Heldman et al., 1989;Shao and Suszkiw, 1991;McMahon and Nicholls, 1993;Sihra et al., 1993). Among these, Ba2+ is the best documented and probably the most effective. However, Ba2+ was found to be ineffective in binding and activating the