Protein kinase C-mediated impairment of glutamine outward transport and SN1 transporter distribution by ammonia in mouse cortical astrocytes

Highlights

• Ammonia decreases protein kinase C (PKC) activity in mouse astrocytes.

• PKC activation reverses ammonia-induced decrease of SN1 level in the cell membranes.

• PKC activation reverses ammonia-induced decrease of L-glutamine efflux by system N.

• PKCδ is the isoform involved in SN1 modulation by ammonia in mouse astrocytes.

Abstract

SN1, a system N amino acid transporter specific for astrocytes, is mainly responsible for export of newly synthesized L-glutamine from the cells. Astrocytic retention of L-glutamine which plays a critical role in ammonia-induced astrocytic swelling resulting in brain edema, could be tentatively attributed to the impaired L-glutamine export from astrocytes. The present study demonstrates that treatment of cultured mouse cortical astrocytes for 24 h with 5 mM ammonium chloride (“ammonia”) inhibits the system N-mediated L-glutamine transport out of the cell, and that this inhibition is related to the reduced presence of the SN1 transporter on the cell membrane. Ammonia decreased total protein kinase C (PKC) activity in the absence but not in the presence of PKC activator, phorbol 12-myristate 13-acetate (PMA), and activation of PKC by PMA reversed both the ammonia-induced decrease of system N-mediated L-glutamine release and ammonia-induced SN1 deficit in the membrane fraction. However, while ammonia did not change the protein level of PKCα isoform, it decreased the protein content of PKCδ. Moreover, ammonia treatment increased the cell surface expression of SN1 in cells with silenced PKCα and PKCδ. Silencing of PKCδ abrogated the decrease of system N (SN1)-mediated L-glutamine release by ammonia. The results implicate the involvement of PKCδ in the inhibition of SN1 membrane expression and activity by ammonia.

Introduction

L-glutamine is the most ubiquitous amino acid in all mammalian tissues and body fluids, including the central nervous system (CNS), where its concentration is at least one order of magnitude higher than of any other amino acid (Albrecht et al., 2007; Cynober, 2002; Pithon-Curi et al., 2002). In the brain L-glutamine is formed in astrocytes from L-glutamate and ammonia in an ATP-consuming reaction catalyzed by glutamine synthetase (Albrecht et al., 2010a). A significant proportion of synthesized L-glutamine exits astrocytes and enters neurons, to give rise to the excitatory neurotransmitter amino acid L-glutamate (Albrecht et al., 2010a; Suárez et al., 2002), and the inhibitory neurotransmitter γ-aminobutyric acid (Albrecht et al., 2010a), reflecting the neuronal leg of the glutamate/glutamine cycle (Albrecht et al., 2010a; Waniewski and Martin, 1986). A certain proportion of astrocyte-derived L-glutamine leaves the CNS via the cerebral capillary endothelial cells forming the blood-brain barrier (Albrecht et al., 2010b; Lee et al., 1998).

In CNS diseases associated with hyperammonemia including hepatic encephalopathy, excessive L-glutamine synthesis and its accumulation in astrocytes resulting from detoxification of excess of ammonia is considered to be deleterious to brain function. Astrocytes are the locus of glutamine synthetase in the brain (Suárez et al., 2002), which renders them a primary target of excess ammonia. Specifically, excess of newly synthesized L-glutamine contributes to astrocytic swelling which results from its interference with mitochondrial function and from osmotic action (Kruczek et al., 2011; Sinke et al., 2008). In turn, astrocytic swelling is the primary cause of brain edema, a frequent fatal complication of hepatic encephalopathy (Blei and Larsen, 1999; Häussinger et al., 2000).

Astrocytic L-glutamine transport is mediated by a Na⁺-coupled amino acid transport system N, represented by three carriers SN1, SN2, and SN7 (SLC38A3, SLC38A5 and SLC38A7, respectively) (Pochini et al., 2014). SN1, coded by the Snat3 gene identified and annotated on the chromosome 3p21.31 is the most abundant system N transporter in astrocytes, which is mainly responsible for export of newly synthesized L-glutamine from the cells (Bröer et al., 2004). Recently, silencing of transporters SN1 and SN2 in cultured mouse astrocytes has been reported to cause L-glutamine retention in these cells (Zielińska et al., 2016). Those observations prompted a hypothesis that ammonia may contribute to intra-astrocytic L-glutamine retention by interfering with SN1-mediated glutamine efflux. Having confirmed this hypothesis, we asked about the mechanism by which ammonia alters the expression and activity of SN1.

Phosphorylation by protein kinase C (PKC) is the principal mechanism which controls intracellular distribution and activity of different membrane proteins (Nissen-Meyer and Chaudhry, 2013). SN1 appears to be regulated by this kinase as was documented by other groups (Balkrishna et al., 2010; Nissen-Meyer and Chaudhry, 2013; Nissen-Meyer et al., 2011; Sidoryk-Wegrzynowicz et al., 2011). PKC activation by phorbol esters (PMA) leads to internalization of SN1, a process correlated with its decreased expression and transport activity as shown in X. laevis oocytes and in cultured rat astrocytes in vitro (Balkrishna et al., 2010; Nissen-Meyer et al., 2011; Sidoryk-Wegrzynowicz et al., 2011). In turn, SN1 interactions with different PKC isozymes including α, γ and δ isoforms were shown in cultured rat astrocytes (Nissen-Meyer and Chaudhry, 2013; Nissen-Meyer et al., 2011; Sidoryk-Wegrzynowicz et al., 2011). Basing on the above considerations we hypothesized that the mechanism by which ammonia inhibits L-glutamine efflux from astrocytes during hyperammonemia may be related to altered interaction of PKC with SN1 and the ensuing modulation of its activity. Down this valley, we endeavored the role of PKCδ and α isoforms. To test this hypothesis we analyzed mutual relations between activation of total PKC or silencing the particular PKC isozymes, and the expression and cell membrane distribution of SN1 transporter in cultured mouse cortical astrocytes treated or not with ammonia. Analysis of the effect of activation of PKC by phorbol esters also included the rate of [³H]glutamine release from astrocytes with silenced distinct PKC isoforms.