The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 +/- 0.35 nmol/mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.

Sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is critical for initiating differentiation of the PC12 cell to a sympathetic-like neurone. The neuropeptide, pituitary adenylyl cyclase-activating peptide (PACAP), has been demonstrated to cause cells to adopt a neuronal phenotype, although the mechanism of this activity is unclear. PACAP through its type I receptor stimulates a biphasic activation of ERK1/2; a >10-fold increase within 5 min, followed by a >5-fold increase that is sustained for 60 min. An equivalent stimulation is seen in PC12 cells expressing a dominant negative Ras mutant. However, the mitogen-activated kinase/ERK kinase 1/2 (MEK1/2) inhibitor PD98059 blocked both PACAP-induced stimulation of ERK1/2 activity and neurite outgrowth. Thus, the activation signal from the PACAP type I receptor on the ERK1/2 cascade pathway is received downstream of Ras, either at Raf or MEK. Down-regulation of protein kinase C or its inhibition by calphostin C blocked the ability of PACAP to stimulate ERK1/2. We conclude that activation of PACAP type I receptor activates protein kinase C, which then activates the ERK1/2 cascade in a Ras-independent manner at either Raf or MEK1/2.The two forms of pituitary adenylyl cyclase-activating polypeptide 1 are neuropeptides of the secretin/glucagon/vasoactive intestinal peptide/growth hormonereleasing hormone family. They share the same 27 aminoterminal amino acids and arise from a precursor peptide by post-translational processing (1). Two receptor subtypes have been identified for PACAP; both are G protein-coupled receptors. The PACAP type I receptor (found in hypothalamus, brain stem, pituitary, adrenal gland, and testes) is specific for PACAP, having a K d of 0.5-2.0 nM. There are several splice variants that demonstrate different abilities to activate adenylyl cyclase and phospholipase C (2). The type II receptor does not discriminate between PACAP and VIP and is only positively coupled to adenylyl cyclase.PACAP38 potently stimulates neuritogenesis of neuroblastoma cells (3), neonatal chromaffin cells (4), corticotrope cells (5), and PC12 cells (6 -8).The MAP kinases ERK1/2 are thought to be key players in the control of gene transcription events that lead to proliferation or differentiation in PC12 cells in response to epidermal growth factor or NGF, respectively. It has been suggested that the determinant of the nature of the response of PC12 cells (proliferation or differentiation) correlates with the duration of ERK1/2 activation and its translocation to the nucleus (9). ERK1/2 activation following agonist stimulation of G protein-coupled receptors has been reported (reviewed in Ref. 10), although various effectors are employed to couple these receptors to the MAP kinase cascade. In light of the neurite-stimulatory role of PACAP38 in pituitary neural cells and neuroblastoma cell lines, we have investigated the relationship of PACAP38 stimulation to ERK1/2 activation and compared this to that for NGF. In this study, we have demonstrated that ...

The adenosine modulation of glutamate exocytosis from guinea pig cerebrocortical synaptosomes is investigated. Endogenously leaked adenosine is sufficient to cause a partial tonic inhibition of 4-aminopyridine-evoked glutamate release, which can be relieved by adenosine deaminase. The adenosine A1 receptor is equally effective in mediating inhibition of glutamate exocytosis evoked by 4-aminopyridine (where K(+)-channel activation would inhibit release) and by elevated KCl (where K(+)-channel activation would have no effect), arguing for a central role of Ca(2+)-channel modulation. In support of this, the plateau phase of depolarization-evoked free Ca2+ elevation is decreased by adenosine with both depolarization protocols. No effect of adenosine agonists is seen on membrane potential in polarized or KCl- or 4-aminopyridine-stimulated synaptosomes. The interaction of protein kinase C with the A1 receptor-mediated inhibition is examined. Activation of protein kinase C by 4 beta-phorbol dibutyrate has been shown previously by this laboratory to modulate glutamate release via K(+)-channel inhibition, and is shown here to have an additional action of decoupling the adenosine inhibition of glutamate exocytosis.

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.