Rapid increase of glial glutamate uptake via blockade of the protein kinase A pathway

Abstract

Glutamate is the main excitatory neurotransmitter in the vertebrate central nervous system. Removal of this transmitter from the synaptic cleft by glial and neuronal transporter systems plays an important role in terminating glutamatergic neurotransmission. The effects of different activators and blockers of PKA and PKC on glutamate uptake were studied in primary glial cells cultivated from the rat cortex using the patch-clamp recording technique and immunocytochemical methods. GF 109203X enhances glutamate-induced membrane currents in a concentration- and time-dependent manner. After pre-application for 40 s the maximal transport capacity was increased by 30-80%. The estimated Km-value of the transport system did not change after drug application and the enhanced glutamate uptake was reversible within a few minutes upon washout. Activators and blockers of the PKC pathway did not affect glutamate uptake, whereas H89, a selective blocker of PKA, mimicked the effects of GF 109203X, indicating involvement of the protein kinase A pathway. The GF 109203X-induced increase in transport capacity is likely to be mediated by GLAST since the GLT-1 selective blocker dihydrokainate was unable to block basal or stimulated glutamate uptake. Furthermore, the increase in transport activity may well be based on an increase in cell surface expression of the transporter protein since preincubation with cytochalasin-B, a protein that blocks actin polymerization, almost completely abolished the effect of GF 109203X and H89. These results indicate that GF 109203X and H89 enhance glial glutamate uptake via blockade of the PKA. The described effect may affect glutamatergic neurotransmission by reducing the glutamate concentration in the synaptic cleft.

Adenosine Monophosphate–activated Protein Kinase Regulates Interleukin-1β Expression and Glial Glutamate Transporter Function in Rodents with Neuropathic Pain

“…The size of GTCs reflects the amount of transported glutamate, which has been widely used as an effective tool to study the function of glial GTs. 14,36,50 We recently reported that mice with pSNL had lower amplitudes of GTCs. 14 After recording baseline GTCs in spinal slices taken from neuropathic GFP-GFAP mice, we perfused the AMPK activator (AICAR, concentration in the bath: 10 μM) into the recording chamber and recorded GTCs again.…”

Adenosine A2A receptors modulate glutamate uptake in cultured astrocytes and gliosomes

“…Fig. Accordingly, different studies have shown that the cAMP/protein kinase A transducing pathway can either enhance (Figiel et al, 2003;Gegelashvili et al, 1997), fail to affect (Danbolt, 2001;Gegelashvili et al, 1997) or decrease the expression or activity of GLT-I and GLAST (Adolph et al, 2007;Lim and Kim, 2003;Nishizaki et al, 2002;Rath et al, 2008) in astrocytic cultures. The presently observed severe decrease of GLT-I and GLAST activity and expression levels (Fig.…”

Increased excitatory amino acid transport into murine prion protein knockout astrocytes cultured in vitro

“…The increased EAAT activity in PrPKO versus WT astrocytes was accompanied by only a modest elevation in EAAT1 expression in PrPKO astrocytes, suggesting the alterations could be due to indirect mechanisms that effect surface localization and/or changes in intrinsic activity. L-Glutamate transporters are subject to interactions with modulating proteins (Jackson et al 2001) and to post-translational modifications that can result in altered transporter function (Adolph et al 2007; Duan et al 1999; Lin et al 2001; Munir et al 2000, Schlag et al 1998). The localization of PrP and EAATs to lipid rafts may provide an environment conducive to direct and/or indirect interactions between these proteins (Butchbach et al 2004; Naslavsky et al 1997).…”