Elsevier

Brain Research

Volume 864, Issue 1, 5 May 2000, Pages 52-59
Brain Research

Research report
Immunohistochemical localization of phospholipase D1 in rat central nervous system

https://doi.org/10.1016/S0006-8993(00)02134-XGet rights and content

Abstract

Phospholipase D (PLD) is one of the intracellular signal transduction enzymes and plays an important role in a variety of cellular functions. We investigated the distribution of PLD isozyme, PLD1 in the rat brain and spinal cord using an immunological approach. Western blot analysis showed the presence of PLD1 protein in all tissues studied, with significantly higher levels in the brainstem and spinal cord, which was correlated with the results obtained from PLD activity assay. Prominent and specific signals of PLD1 were observed in many functionally diverse brain areas, including the olfactory bulb, medial septum–diagonal band complex, cerebral cortex, brainstem, cerebellum, and spinal cord. In the brainstem, the red nucleus, substantia nigra, interpeduncular nucleus, cranial motor nuclei (trigeminal motor, abducent, facial, and hypoglossal), sensory cranial nerve nuclei (spinal trigeminal, vestibular, and cochlear), as well as nuclei of the reticular formation, all showed intense immunoreactivity. Purkinje cells and deep cerebellar nuclei of the cerebellum were also labeled intensely. However, no significant labeling was found in the thalamus, epithalamus, and basal ganglia. Although many of the PLD1 immunoreactive cells were neurons, PLD1 was also expressed in glial cells such as presumed astrocytes and tanycytes. These findings suggest that PLD1 may play an important role in the central nervous system of the adult rat.

Introduction

Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline, has been suggested to play an important role in receptor-mediated signal pathways leading to cell proliferation [1], [18], apoptosis [11], [19], [24], cell differentiation [17], [23], cytoskeletal reorganization [4], [15], and membrane trafficking and secretory events [3], [14], [21], possibly including neurotransmitter release [2]. PA has been shown to act directly as a signaling molecule and can be further converted to other messenger molecules, such as 1,2-diacylglycerol and lysophosphatidic acid [6], [9].

Recently, two mammalian isoforms of phospholipase D, PLD1 and PLD2, have been characterized at the molecular level [7], [8]. However, the functional role of PLD in cells is still poorly defined. Likewise, there are only a few published studies concerning the regulation of PLD expression in cell culture systems.

The identification of PLD in a novel signal transduction pathway engenders a special interest in the function of PLD in the nervous system. Although brain PLD has been studied in various systems from isolated subcellular organelles to cell cultures, brain slices, and tissues [5], [12], only limited data are currently available concerning the regional distribution and cellular localization of PLD within the nervous system. Information concerning cell-specific expression of PLD can provide potential clues to the functional significance of the PLD isozymes. In this study, we therefore examined the localization of PLD1-immunoreactive profiles within the brain and spinal cord of the rat by Western blot analysis, an assay for PLD activity, and immunohistochemistry.

Section snippets

Experimental animals and tissue processing

Ten male Sprague–Dawley rats (3–6 months old) were deeply anesthetized with 4% chloral hydrate (1 ml/100 mg). Five rats were used for Western blot analysis and PLD activity assay, and five rats for immunohistochemistry. Animals were sacrificed either by decapitation followed by dissection of brains and spinal cords for Western blot analysis and an assay for PLD activity, or by transcardial perfusion with the fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 for

PLD1 immunoreactivity: specificity of staining

Anti-PLD1 antibody recognition of the protein was specifically blocked when the blots or sections were incubated with anti-PLD1 antibody that had been preadsorbed with antigen (Fig. 1A,B), indicating the specificity of PLD1 immunoreactivity on Western blot and immunohistochemical staining.

Western blot analysis and phospholipase D activity assay

As shown in Fig. 2A, anti-PLD1 antibody specifically recognized approximately 120 kDa of the PLD1 protein band in various regions of rat brain tissues. The expression of PLD1 protein was observed in all

Discussion

The precise function of PLD in mammalian cell regulation is unknown. In an initial attempt to gain some insight into the functional role of PLD, we investigated the distribution of PLD in the central nervous system using an anti-PLD1 antibody. The specificity of the affinity-purified anti-PLD1 antibody used in the present study was underscored by the absence of detectable immunoreactivity under staining conditions in which the primary PLD1 antibody was deleted, or more importantly, preadsorbed

Acknowledgements

The authors thank Hee-Duck Rho and Byoung-Ouk Hong for their excellent technical assistance. This work was supported by The Catholic Medical Center Research Fund for Special Projects (98-3 for M.-Y. Lee) and by a grant (HMP-98-N-1-0012) from the Good Health R&D Project and the Ministry of Health and Welfare, South Korea.

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