Localization of cytosolic phospholipase A2 messenger RNA mainly in neurons in the rat brain

doi:10.1016/S0306-4522(99)00051-2

Neuroscience

Volume 92, Issue 3, June 1999, Pages 1061-1077

https://doi.org/10.1016/S0306-4522(99)00051-2 Get rights and content

Abstract

Ca²⁺-sensitive 85,000 mol. wt cytosolic phospholipase A2 plays an essential role in the selective and stimulus-dependent release of arachidonic acid from membrane phospholipids. Cytosolic phospholipase A2-catalysed lipid mediators including arachidonic acid and its metabolites have been suggested to be involved in a variety of neuronal functions in the CNS. Since the cellular localization of cytosolic phospholipase A2 is still controversial and obscure, we tried an improved method of rapid processing of each specimens and succeeded in obtaining intense signals of cytosolic phospholipase A2 messenger RNA in the normal rat brain by northern blot analysis and in situ hybridization. Northern blot analysis showed the abundant distribution of cytosolic phospholipase A2 messenger RNA in most regions of the brain, with intense signals observed in the pineal gland and pons. Macroautoradiographs prepared after in situ hybridization with three different antisense riboprobes gave essentially similar patterns of localization; significant signals were widely detected in the gray matter of various regions, i.e. the olfactory bulb, cerebral cortex, hippocampus, amygdala, several thalamic and hypothalamic nuclei and cerebellum. Microautoradiographs showed that most of the intense signals were predominant in neurons, and that faint signals were from glial cells and other non-neuronal cells in the choroid plexus, inner surface cells of veins and the leptomeninges. In addition, the cycloheximide treatment increased the cytosolic phospholipase A2 messenger RNA level in the same cell populations originally possessing messenger RNA signals.

Predominant expression of cytosolic phospholipase A2 messenger RNA in neurons may provide the basis for the contribution of cytosolic phospholipase A2-catalysed lipid mediators to a variety of neurotransmission and synaptic functions in the CNS.

Section snippets

Materials and chemicals

The materials and chemicals used and their sources were as follows: TRIzol^® Reagent, from Life Technologies (Rockville, MD, U.S.A.); cycloheximide, formamide, glutaraldehyde, pancreatic ribonuclease A, sarcosyl and dextran sulfate, from Sigma Chemical Company (St Louis, MO, U.S.A.); paraformaldehyde, from Merck Industries (Darmstadt, Germany); Baker's yeast tRNA, from Boehringer Mannheim Biochemica Corp. (Indianapolis, IN, U.S.A.); T7- and SP6-RNA polymerases, from Promega Corp. (Madison, WI,

Probe specificities for 85,000 mol. wt cytosolic phospholipase A2 messenger RNA

To confirm the specificity for detection of cPLA2 mRNA by radioisotope-labeled probes, we conducted several control experiments. Northern blot analysis with the three different antisense probes revealed the same single 2.9 kbp size of band in various regions of the rat brain (Fig. 1 for antisense probe-286, data for the other probes not shown). In addition, these antisense probes provided quite analogous patterns of localization for cPLA2 mRNA among normal rats (Fig. 4B). For labeled sense

Discussion

The present study demonstrated the precise mapping of cPLA2 mRNA in rat brain by in situ hybridization. cPLA2 mRNA is expressed mainly in neurons in most of brain regions, and also in some glial cells, neuroendocrine cells, ependymal cells, choroid plexus and vascular cells, although the non-neuronal cells are a minor population. To obtain the precise localization of cPLA2 mRNA, we improved the conventional method of in situ hybridization by extensive purification of riboprobes and rapid

Conclusions

In conclusion, cumulative knowledge from neurophysiological studies provided the basis of the possible existence of cPLA2 in neurons, in both pre- and postsynaptic sites.1., 3., 4., 14., 16., 21., 22., 23., 28., 29., 34., 39., 44., 47., 61., 62. The previous reports concerning the cellular localization of this enzyme did not always show the presence of cPLA2 in neurons.15., 42., 48., 53., 59. The present study clearly demonstrated the predominant localization of cPLA2 mRNA signals in neurons,

Acknowledgements

This work was supported in part by Research for the Future Program (RFTF) JSPS-RFTF 98L00201 from the Japan Society for the Promotion of Science (JSPS). The initial phase of this work was supported in part by Subfemtomole Biorecognition Project, ICORP, Japan Science Technology Corporation (JST). The authors thank Dr Larry D. Frye for editorial help with the manuscript.

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Localization of cytosolic phospholipase A2 messenger RNA mainly in neurons in the rat brain

Abstract

Section snippets

Materials and chemicals

Probe specificities for 85,000 mol. wt cytosolic phospholipase A2 messenger RNA

Discussion

Conclusions

Acknowledgements

Cell

Brain Res.

Brain Res.

Brain Res.

Molec. brain Res.

Prostaglandins

Trends biochem. Sci.

Neuron

Neuroscience

J. biol. Chem.

Neuron

J. biol. Chem.

Neuron

Cell

Brain Res.

Prog. Brain Res.

Biochem. biophys. Res. Commun.

Neurosci. Lett.

Prostaglandins

Trends Neurosci.

Molec. Brain Res.

Biochem. biophys. Res. Commun.

Brain Res.

Biochim. biophys. Acta

Cell

Fedn Eur. biochem. Socs Lett.

Brain Res.

Brain Res.

J. biol. Chem.

Brain Res.

Molec. Brain Res.

Neurosci. Lett.