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Modulation of signal transduction in rat synaptoneurosomes by platelet activating factor

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Molecular and Chemical Neuropathology

Abstract

The potential involvement of platelet activating factor (PAF, 1-O-alkyl 2-O-acetyl-sn-glycero-3-phosphocholine) in aggravation of ischemic brain injury has been recently postulated. Reported evidences in support of this thesis include increases of brain PAF concentration during ischemia and the neuroprotective effect exerted by PAF antagonists. In this article, we demonstrate that several PAF-mediated biochemical responses in synaptoneurosomes in vitro resemble these observed previously in schemic brain and are widely acknowledged as the potentially causal factors in this pathology. In synaptoneurosomes prepared from rat hippocampus, 10 nM PAF caused an observable elevation of intracellular calcium as measured by fluorescence Fura-2A probe. A similar elevation of synaptoneurosomal [Ca2+]i was evoked by 1 mM glutamate treatment. As an effect of calcium entry after PAF application, a translocation of protein kinase C (PKC) toward plasma membranes was demonstrated by3H-labeled phorbol-binding method. It was followed by an increase of 50 kDa proteolytic fragment of the enzyme (PKM) recognized on Western blots with anti-PKC antibody. Incubation of synaptoneurosomes in the presence of calcium chelators abolished these effects of PAF and significantly decreased the content of PKC in the membranes. Furthermore, PAF treatment markedly attenuated the receptor- and postreceptor-activated cAMP accumulation in synaptoneurosomes. The decrease of cAMP level seems to be secondary to the PAF-induced calcium entry with subsequent activation of cAMP-specific phosphodiesterase, since it was completely blocked by IBMX, a potent inhibitor of this enzyme. Our observations indicate that PAF in a concentration found in ischemic brain can elevate [Ca2+]i and potentiate calcium-dependent intracellular signalling in synaptoneurosomes in vitro, including PKC translocation/activation and proteolysis, followed by IBMX-sensitive inhibition of cAMP production. The relative contribution of these events to ischemic brain injury is currently under extensive investigation.

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Abbreviations

IBMX:

3-isobutyl-1-methylxanthine

Fura 2AM:

fura-2-acetoxy-methyl-ester

DMSO:

dimethyl-sulfoxide

[Ca2+]i :

intracellular free calcium concentration

PBDu:

4-β-phorbol-12, 13-dibutyrate

PAF:

platelet-activating-factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine)

Lyso-PAF:

1-O-hexadecyl-sn-glycero-3-phosphocholine

PKC:

protein kinase C

NE:

norepinephrine

2CA:

2-chloroadenosine

BN52021:

specific PAF receptor antagonist belonging to ginkgolides

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  1. Laboratory of Molecular Neuropathology, Department of Medical Research Centre, Polish Academy of Sciences, Dworkowa 3, 00-784, Warsaw, Poland

    Krystyna Domańska-Janik & Barbara Zabłocka

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  1. Krystyna Domańska-Janik
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  2. Barbara Zabłocka
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Domańska-Janik, K., Zabłocka, B. Modulation of signal transduction in rat synaptoneurosomes by platelet activating factor. Molecular and Chemical Neuropathology 25, 51–67 (1995). https://doi.org/10.1007/BF02815086

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