Elsevier

Brain Research

Volume 1195, 21 February 2008, Pages 1-11
Brain Research

Research Report
Regulation of AQP4 protein expression in rat brain astrocytes: Role of P2X7 receptor activation

https://doi.org/10.1016/j.brainres.2007.12.023Get rights and content

Abstract

ATP has been recognized as an important extracellular signaling molecule and P2X receptors are membrane ion channels activated by the binding of extracellular ATP. Since both AQP4 and P2X7 receptor (P2X7R) are known to be present in astrocytes, we examined whether P2X7R activation plays a role in the regulation of AQP4 expression in astrocytes. Immunoblotting and immunocytochemistry confirmed the expression of both P2X7R and AQP4 in primary cultured rat astrocytes. Co-immunoprecipitation assays of the HEK293 cells expressing both proteins revealed no protein–protein interaction. An activation of P2X7R in primary cultured astrocytes by a P2X7R agonist significantly decreased the AQP4 protein expression, which was abolished by the pre-treatment of a P2X7R antagonist. In addition, AQP4 expression was not affected by high extracellular copper, zinc, or iron concentrations. In a rat model with anoxia-induced brain injury where extracellular ATP levels could be increased, whole brain AQP4 expression was significantly decreased, whereas P2X7R expression was unchanged. Importantly, pre-treatment of P2X7R antagonist in rats significantly inhibited the AQP4 down-regulation in anoxic brain injury, consistent with the in vitro results observed in astrocytes. In conclusion, P2X7R activation in astrocytes was associated with down-regulation of AQP4 in rat brain astrocytes in vitro and in vivo, and this was prevented by P2X7 receptor blockade. Thus, an activation of P2X7R in astrocytes in response to brain injury is likely to play a role in the protective down-regulation of AQP4, which might inhibit water influx to the cells and attenuate the acute cytotoxic brain edema after acute brain injury.

Introduction

Brain edema is associated with many intracranial neuropathological states and metabolic diseases (Gunnarson et al., 2004, Kimelberg, 2004, Unterberg et al., 2004). Although a number of studies have addressed the underlying molecular mechanisms of brain edema, it is still unclear how regulation of water transport takes place across the blood–brain barrier, the cerebrospinal fluid–brain interface, and between extracellular and intracellular compartments in brain parenchyma. The AQP family of water channel proteins is the major pathway by which water rapidly crosses cell membranes (Agre et al., 2002, Nielsen et al., 2002). It is known that AQP4 is the predominant water channel protein in brain and it has a highly polarized distribution in ependymal cells and astroglial cells, where AQP4 could facilitate water flux (Frigeri et al., 1995, Hasegawa et al., 1994, Jung et al., 1994, Nielsen et al., 1997, Rash et al., 1998). Recently, it has been reported that AQP4 contributes to the development of acute brain edema (Bloch et al., 2005, Manley et al., 2000, Vajda et al., 2002).

ATP has been recognized as an important extracellular signaling molecule. Neuropathological conditions are associated with increased extracellular ATP levels originated from damaged cells (Bodin and Burnstock, 1998, Dubyak and el Moatassim, 1993). Recent studies on primary cultures of astrocytes revealed the presence of different kinds of P2 purinoreceptors, i.e., P2X and P2Y receptor subtypes (Fumagalli et al., 2003, Jacques-Silva et al., 2004, Weisman et al., 2005). In particular, P2X receptors are membrane ion channels activated by the binding of extracellular ATP (Khakh and North, 2006). Currently, seven cloned P2X receptor subtypes (P2X1-7) have been identified, and P2X7 receptor subtypes (Surprenant et al., 1996) are particularly present in the microglial, neuron, and astroglial cells, in contrast to the localization of P2X1-6 receptor subtypes mainly on neurons (North, 2002). Moreover, P2X7 receptor density is known to be up-regulated in response to brain ischemia (Cavaliere et al., 2004, Collo et al., 1997, Franke et al., 2004) and an activation of P2X7 receptors in rat astrocyte cultures induces an influx of extracellular Ca2+ (Ballerini et al., 1996, North, 2002) and exerts its effects through PKC/MAPK signaling pathway(Wang et al., 2003). Importantly, previous studies revealed that AQP4 function is, at least partly, dependent on the intracellular [Ca2+] and/or Ca2+-dependent PKC activity (Han et al., 1998, Zelenina et al., 2002). Specifically, Han et al. (1998) demonstrated that PKC activation significantly reduced the rate of AQP4-expressing oocyte swelling. Furthermore, Yamamoto et al. (2001) revealed the PKC-mediated decrease of AQP4 mRNA and protein levels in rat astrocyte culture.

Therefore, in the astroglial cells where both AQP4 and P2X7 receptors are present, it is hypothesized that increased extracellular ATP levels in response to brain injury, and hence an activation of P2X7 receptor may change the protein expression of AQP4 which could result in the altered osmotic water permeability of astrocytes. The purposes of the present study were 1) to study whether P2X7 receptor activation regulates AQP4 protein expression in primary cultured astroglial cells in vitro; 2) to examine whether an anoxia-induced brain I/R injury in rats, which might be associated with increased extracellular ATP levels, is associated with decreased AQP4 expression in vivo; and 3) to examine whether P2X7 receptor blockade in rats with anoxia-induced brain I/R injury attenuates the down-regulation of AQP4 in rat brain.

Section snippets

Both P2X7 receptor and AQP4 proteins were present in primary cultured rat brain astroglial cells

As demonstrated in Fig. 1, immunoblotting revealed that P2X7 receptor (Fig. 1A) and AQP4 (Fig. 1B) proteins were present in rat brain astroglial cells as well as in HEK293 cells transiently transfected by cDNA encoding P2X7 receptor (pcDNA3.1-P2X7-EE, Fig. 1A) or AQP4 (pEGFP N1-AQP4, Fig. 1B). Immunocytochemistry demonstrated that both P2X7 receptor (Fig. 1C) and AQP4 (Fig. 1D) immunolabeling was associated with primary cultured astroglial cells. Co-immunoprecipitation assays of the HEK293

Discussion

We demonstrated that P2X7 receptor activation in astroglial cells by high extracellular ATP levels could be implicated in the down-regulation of AQP4 protein expression in vitro. Thus, an activation of P2X7 receptors of the astroglial cells in vivo by extracellular ATP released from damaged or dying cells in response to brain injury is likely to play a role in the protective down-regulation of AQP4, which might inhibit water influx to the cells and attenuate the acute cytotoxic brain edema

Primary culture of rat brain astroglial cells and preparation of HEK293 cells

Rat brain astroglial cells were prepared from 2–3 day-old Sprague–Dawley rats (Orient Bio, Pusan, Korea) by the method of McCarthy and De Vellis (1980) with some modifications (Suk et al., 2002). The animal protocols have been approved by the Animal care and Use Committee of the Kyungpook National University, Korea. Briefly, whole brains were dissociated in the modified eagle's medium (Sigma, St. Louis, MO) supplemented with 31.3 mM NaHCO3, 24.4 mM d-glucose, 10% fetal bovine serum (Gibco BRL,

Acknowledgments

The authors thank Jung-Suk Lim and Ji-Hyun Earm for their expert technical assistance. This study was supported by Korea Science and Engineering Foundation Grant funded by the MOST (R01-2007-000-20441-0), Brain Korea 21 Project in 2007, Danish National Research Foundation (Danmarks Grundforskningsfond), WIRED program (Nordic Council and the Nordic Centre of Excellence Program in Molecular Medicine), and Dongguk University.

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