Abstract
The involvement of the astrocytic SN1 (SNAT3) transporter in ammonia-induced l-glutamine retention was recently documented in mouse-cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its cellular localization were determined using real-time qPCR, Western blot, and confocal microscopy. Sp1 binding to Snat3 promoter was analyzed by chromatin immunoprecipitation. The role of Sp1 in SN1 expression and SN1-mediated [3H]glutamine uptake in ammonia-treated astrocytes was verified using siRNA and mithramycin A. The involvement of protein kinase C (PKC) isoforms in Sp1 level/phosphorylation status was verified using siRNA technology. Sp1 translocation to the nuclei and its enhanced binding to the Snat3 promoter, along with Sp1 dependence of system N-mediated [3H]glutamine uptake, were observed in astrocytes upon ammonia exposure. Ammonia decreased the level of phosphorylated Sp1, and the effect was reinforced by long-term incubation with PKC modulator, phorbol 12-myristate 13-acetate, which is a treatment likely to dephosphorylate Sp1. Furthermore, silencing of the PKCδ isoform appears to enhance the ammonia effect on the Sp1 level. Collectively, the results demonstrate the regulatory role of Sp1 in regulation of SN1 expression and activity in ammonia-treated astrocytes and implicate altered Sp1 phosphorylation status in this capacity.
Highlights
L-glutamine (Gln) is the most abundant amino acid in the central nervous system (CNS) where its concentration is at least one order of magnitude higher than any other amino acid [1,2,3]
The glutamate/glutamine cycle is known as a metabolite shuttle in which Gln transported from astrocytes to adjacent neurons is converted to Glu or gamma-aminobutyric acid (GABA) in an enzymatic reaction catalyzed by glutaminase or glutamate decarboxylase [4,5,6,7]
In vitro experiments documented that in astrocytes treated with pathologically relevant concentrations of ammonia, as well as in cells exposed to a high-concentrations of Gln [14,15], create the induction of a mitochondrial permeability transition accompanied the astrocytic swelling [16,17,18]
Summary
L-glutamine (Gln) is the most abundant amino acid in the central nervous system (CNS) where its concentration is at least one order of magnitude higher than any other amino acid [1,2,3]. Gln is synthesized in astrocytes from L-glutamate (Glu) and ammonia in the reaction catalyzed by glutamine synthetase [4]. The glutamate/glutamine cycle is known as a metabolite shuttle in which Gln transported from astrocytes to adjacent neurons is converted to Glu or gamma-aminobutyric acid (GABA) in an enzymatic reaction catalyzed by glutaminase or glutamate decarboxylase [4,5,6,7]. Gln is considered to play an important role in the pathogenesis of neurological diseases associated with hyperammonemia, including hepatic encephalopathy (HE), due to its excessive synthesis and accumulation in astroglial cells [8,9,10,11]. It has been shown that induction of mitogen-activated protein kinases (MAPK) occurs in the astroglial cells exposed to the excess of Gln [21]. An efficient transport via specific amino acid carriers guaranteeing proper Gln inter-cellular distribution in hyperammonemic astrogliopathy is of importance
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