Article

Modulation of synaptosomal protein phosphorylation/dephosphorylation by calcium is antagonised by inhibition of protein phosphatases with okadaic acid.

Neuroscience Group, Faculty of Medicine, University of Newcastle, N.S.W., Australia.
Neuroscience Letters (Impact Factor: 2.03). 06/1991; 126(2):203-6. DOI: 10.1016/0304-3940(91)90554-7
Source: PubMed

ABSTRACT The protein phosphatase inhibitor okadaic acid was used to investigate the protein phosphatases involved in the endogenous dephosphorylation of proteins in intact synaptosomes. Despite the fact that the calcium-dependent protein phosphatase (calcineurin) is most concentrated in synaptosomes and accounts for approximately 0.3% of synaptoplasmic protein, the majority of the dephosphorylation activity under both basal and depolarisation conditions is due to protein phosphatase type 1 (PP1) and/or protein phosphatase type 2A (PP2A). Nevertheless our results do suggest that calcineurin is active in synaptosomes and has 2 effects: a rapid, direct dephosphorylation of a limited range of substrates and an indirect activation of PP1 presumably by dephosphorylation of protein phosphatase 1 inhibitor-1.

0 Bookmarks
 · 
35 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: This study was performed to determine the effect of prolonged status epilepticus on the activity and subcellular location of a neuronally enriched, calcium-regulated enzyme, calcineurin. Brain fractions isolated from control animals and rats subjected to pilocarpine-induced status epilepticus were subjected to differential centrifugation. Specific subcellular fractions were tested for both calcineurin activity and enzyme content. Significant, status epilepticus-induced increases in calcineurin activity were found in homogenates, nuclear fractions, and crude synaptic membrane-enriched fractions isolated from both cortex and hippocampus. Additionally, significant increases in enzyme levels were observed in crude synaptic fractions as measured by Western analysis. Immunohistochemical studies revealed a status epilepticus-induced increase in calcineurin immunoreactivity in dendritic structures of pyramidal neurons of the hippocampus. The data demonstrate a status epilepticus-induced increase in calcineurin activity and concentration in the postsynaptic region of forebrain pyramidal neurons.
    Neurobiology of Disease 01/2004; 14(3):483-93. · 5.62 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The functions of crucial proteins in the nervous system are modulated by kinases and phosphatases which catalyze opposing reactions of phosphorylation and dephosphorylation. During spinal cord central sensitization, serine/threonine protein phosphatase 2A (PP2A) may play an important role in determining the excitability of nociceptive neurons in the spinal cord by modulating the phosphorylation state of some critical proteins. The effects of a general inhibitor of PP2A, okadaic acid (OA), and a specific inhibitor, fostriecin, on the behavioral responses of rats following capsaicin injection were investigated in this study. Hyperalgesia was initiated by injection of capsaicin into the plantar surface of the hindpaw of rats. An intrathecal catheter was previously implanted into the subarachnoid space of the spinal cord for the administration of a variety of drugs. Rats were tested for responses to mechanical stimuli using von Frey filaments of different bending forces applied at a site outside the area of injection. Responses to heat stimuli were detected from a site near the injection area. The responses were recorded before and after injection of capsaicin with the perfusion of ACSF, OA negative control, OA or fostriecin at different time points. The results demonstrated that secondary mechanical hyperalgesia and allodynia can be induced by the intradermal injection of capsaicin. Compared to administration of ACSF or the OA negative control, infusion of the phosphatase inhibitor OA or of fostriecin into the subarachnoid space enhanced the secondary mechanical hyperalgesia and allodynia by making the intradermal capsaicin-induced hyperalgesia and allodynia last longer.
    Pain 01/2004; 106(3):443-51. · 5.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The inhibitors okadaic acid (OA), fostriecin (FOS) and cyclosporin A (CsA), were used to investigate the roles of protein phosphatases in regulating exocytosis in rat brain synaptosomes by measuring glutamate release and the release of the styryl dye FM 2-10. Depolarization was induced by 30 mM KCl, or 0.3 mM or 1 mM 4-aminopyridine (4AP). OA and FOS produced a similar partial inhibition of KCl- and 0.3 mM 4AP- evoked exocytosis in both assays, but had little effect upon exocytosis evoked by 1 mM 4AP. In contrast, CsA had no effect upon KCl- and 0.3 mM 4AP-evoked exocytosis, but significantly enhanced glutamate release but not FM 2-10 dye release evoked by 1 mM 4AP. None of the phosphatase inhibitors changed calcium signals from FURA-2-loaded synaptosomes either before or after depolarization. Pretreatment with 100 nM phorbol 12-myristate 13-acetate abolished the inhibitory effect of OA on exocytosis induced by 0.3 mM 4AP. Taken together, these results show that exocytosis from synaptosomes has a phosphatase-sensitive and phosphatase-insensitive component, and that there are two modes of phosphatase-sensitive exocytosis that can be elicited by different depolarization conditions. Moreover, these two modes are differentially sensitive to phosphatase 2A and 2B.
    Journal of Neurochemistry 07/2003; 85(5):1190-9. · 3.97 Impact Factor