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Notes: Abstract: To investigate a possible function of the nervous tissuespecific protein kinase C substrate B-50/GAP-43 in regulati of the dynamics of the submembranous cytoskeleton. we studii the interaction between purified 6–50 and actin. Both the phosphorylated and dephosphorylated forms of 8–50 cosedi-mented with filamentous actin (F-actin) in a Ca2+-independent manner. Neither 6–50 nor phospho-6–50 had any effect on the kinetics of actin polymerization and on the critical concentration at steady state, as measured using pyrenylated actin. tight scattering of F-actin samples was not increased in the presence of 550, suggesting that 550 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 550 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 6–50, We conclude that 6–50 is an actin-binding protein that does not directly affect actin dynamics.

Notes: Abstract: To study the involvement of the protein kinase C (PKC) substrate B-50 [also known as growth-associated protein-43 (GAP-43), neuromodulin, and F1] in presynaptic cholecystokinin-8 (CCK-8) release, highly purified synaptosomes from rat cerebral cortex were permeated with the bacterial toxin streptolysin O (SL-O). CCK-8 release from permeated synaptosomes, determined quantitatively by radioimmunoassay, could be induced by Ca2+ in a concentration-dependent manner (EC50 of ∼10-5M). Ca2+-induced CCK-8 release was maximal at 104M Ca2+, amounting to ∼10% of the initial 6,000 ± 550 fmol of CCK-8 content/mg of synaptosomal protein. Only 30% of the Caa+-induced CCK-8 release was dependent on the presence of exogenously added ATP. Two different monoclonal anti-B-50 antibodies were introduced into permeated synaptosomes to study their effect on Ca2+-induced CCK-8 release. The N-terminally directed antibodies (NM2), which inhibited PKC-mediated B-50 phosphorylation, inhibited Ca2+-induced CCK-8 release in a dose-dependent manner, whereas the C-terminally directed antibodies (NM6) affected neither B-50 phosphorylation nor CCK-8 release. The PKC inhibitors PKC19–36 and 1 −(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), which inhibited B-50 phosphorylation in permeated synaptosomes, had no effect on Ca2+-induced CCK-8 release. Our data strongly indicate that B-50 is involved in the mechanism of presynaptic CCK-8 release, at a step downstream of the Ca2+ trigger. As CCK-8 is stored in large densecored vesicles, we conclude that B-50 is an essential factor in the exocytosis from this type of neuropeptide-containing vesicle. The differential effects of the monoclonal antibodies indicate that this B-50 property is localized in the N-terminal region of the B-50 molecule, which contains the PKC phosphorylation site and calmodulin-binding domain.

Notes: Abstract: 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.

Notes: Abstract: 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.

Notes: Abstract: We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B-50 (GAP-43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration-dependent manner by elevating the free Ca2+ concentration from 10−8 to 10−5M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2+-induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2+-induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2+- and phorbol ester-induced release were not additive at higher phorbol ester concentrations (〉10−7M). We compared the sensitivities of Ca2+- and phorbol ester-induced release of noradrenaline to the protein kinase inhibitors H-7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B-50. Ca2+-induced release was inhibited by B-50 antibodies and polymyxin B, but not by H-7; phorbol ester-induced release was inhibited by polymyxin B and by H-7, but only marginally by antibodies to B-50. We suggest that phorbol esters and Ca2+ stimulate noradrenaline release through different mechanisms and that the essential role of B-50 in Ca2+-induced noradrenaline release may involve other properties of B-50 besides protein kinase C–mediated phosphorylation.

Notes: Abstract: 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.

Notes: Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca2+-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 10−5M Ca2+ The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca2+-induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca2+-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 Ca2+-induced NA release. PKC pseudosubstrate PKC19-36, which inhibited B-50 phosphorylation (IC50 value, 10−5M), failed to inhibit Ca2+-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 Ca2+-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 Ca2+-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca2+- 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 Ca2+-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.

Notes: Abstract: The neuron-specific protein B-50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B-50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B-50/CaM binding, we used the cross-linker disuccimidyl suberate (DSS) to form a covalent B-50/CaM complex, which is stable on SDS-PAGE. Upon addition of DSS, purified B-50 and calmodulin form a 70-kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti-B-50 and anti-CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70-kDa B-50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B-50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B-50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.

Notes: Abstract: A phosphatidylinositol 4-phosphate (PIP) kinase (EC 2.7.1.68) was purified from bovine brain membranes in a six-step procedure involving solubilization of the enzyme with 170 mM NaCl followed by chromatography on diethylaminoethyl-cellulose, phosphocellulose, Ultrogel AcA44, hydroxylapatite, and ATP-agarose. The enzyme preparation was nearly homogeneous and was purified 5,600-fold with a final specific activity of 85 nmol/min/mg of protein and a yield of 20%. Its molecular mass was 110 kilodaltons, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific for PIP; phosphorylation of phosphatidylinositol and diacylglycerol was not observed.

Notes: Abstract: In the in vitro hippocampal slice preparation a short tetanus induces long-term potentiation (LTP) and an increase in the post hoc phosphorylation of a 52-kDa protein in synaptosomal plasma membranes (SPM) prepared from these slices. This 52-kDa SPM phosphoprotein closely resembles the predominant phosphoprotein in coated vesicles, pp50, with respect to the insensitivity of its phosphorylation to Ca2+/calmodulin and cyclic AMP. This resemblance prompted us to compare in rat brain the 52-kDa SPM protein with pp50 in isolated coated vesicles. Both proteins appear to be very similar on basis of the following criteria: (1) relative molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) peptide mapping, (3) phospho-amino acid content, and (4) isoelectric point. Since coated vesicles are thought to be involved in receptor-mediated endocytosis and membrane recycling, our data suggest that LTP-correlated changes in 52-kDa phosphorylation may reflect increased coated vesicle activity.