Differential modulation of the glutamate transporters GLT1, GLAST and EAAC1 by docosahexaenoic acid.

At present, the ability of polyunsaturated fatty acids (PUFAs) to regulate individual glutamate transporter subtypes is poorly understood and very little information exists on the mechanism(s) by which PUFAs achieve their effects on the transport process. Here we investigate the effect of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the activity of the mammalian glutamate transporter subtypes, GLT1, GLAST and EAAC1 individually expressed in human embryonic kidney (HEK) cells. Exposure of cells to 100 muM DHA increased the rate of d-[(3)H]aspartate uptake by over 72% of control in HEK(GLT1) cells, and by 45% of control in HEK(EAAC1) cells. In contrast, exposure of HEK(GLAST) cells to 200 muM DHA resulted in almost 40% inhibition of d-[(3)H]aspartate transport. Removal of extracellular calcium increased the inhibitory potential of DHA in HEK(GLAST) cells. In contrast, in the absence of extracellular calcium, the stimulatory effect of DHA on d-[(3)H]aspartate uptake in HEK(GLT1) and HEK(EAAC1) cells was abolished, and significant inhibition of the transport process by DHA was observed. Inhibition of CaM kinase II or PKC had no effect on the ability of DHA to inhibit transport into HEK(GLAST) cells but abolished the stimulatory effect of DHA on d-[(3)H]aspartate transport into HEK(GLT1) and HEK(EAAC1) cells. Inhibition of PKA had no effect on the modulation of d-[(3)H]aspartate transport by DHA in any of the cell lines. We conclude that DHA differentially modulates the GLT1, GLAST and EAAC1 glutamate transporter subtypes via different mechanisms. In the case of GLT1 and EAAC1, DHA appears to stimulate d-[(3)H]aspartate uptake via a mechanism requiring extracellular calcium and involving CaM kinase II and PKC, but not PKA. In contrast, the inhibitory effect of DHA on GLAST does not require extracellular calcium and does not involve CaM kinase II, PKC or PKA.

An investigation into the role of calcium in the modulation of rat synaptosomal D-[3H]aspartate transport by docosahexaenoic acid.

The effect of the polyunsaturated fatty acid cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the high-affinity, sodium-dependent uptake of D-[3H]aspartate into purified rat brain synaptosomes was examined. Incubation of the synaptosomes with 20 microM DHA caused over 50% inhibition of the maximum velocity (V(max)) of D-[3H]aspartate transport. This inhibition was significantly potentiated by pre-exposure of the synaptosomes to the fatty acid for 10 min prior to the start of the transport assay. Less highly unsaturated fatty acids such as arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid), linolenic acid (cis-9,12,15-octadecatrienoic acid) and oleic acid (cis-9-octadecenoic acid) were significantly less potent than DHA. Removal of extracellular calcium, or reduction of the intracellular calcium concentration using the intracellular calcium chelator BAPTA/AM (10 microM), did not reduce the inhibition caused by DHA. On the other hand, an increase in the concentration of intracellular calcium mediated by thapsigargin (25 microM) or the calcium ionophore A23187 (10 or 100 nM) led to a reduction in the rate of D-[3H]aspartate transport in the absence of DHA. The CaM kinase II inhibitor, KN-93, reduced D-[3H]aspartate uptake independently of whether DHA was also present, but had no effect on the inhibition of D-[3H]aspartate uptake by either A23187 or thapsigargin. We conclude that whereas DHA inhibits synaptosomal D-[3H]aspartate uptake in a calcium-independent manner, a calcium-based mechanism exists that can also modulate glutamate transporter activity.