• GLAST/EAAT1 regulation in cultured Bergmann glia cells: role of the NO/cGMP signaling pathway.

GLAST/EAAT1 regulation in cultured Bergmann glia cells: role of the NO/cGMP signaling pathway.

Neurochemistry international (2013-11-12)
Alberto Balderas, Alain M Guillem, Zila Martínez-Lozada, Luisa C Hernández-Kelly, José Aguilera, Arturo Ortega

Glutamate, the major excitatory amino acid, activates a wide variety of signal transduction cascades. Ionotropic and metabotropic glutamate receptors are critically involved in long-term synaptic changes, although recent findings suggest that the electrogenic Na(+)-dependent glutamate transporters, responsible for its removal from the synaptic cleft participate in the signaling transactions triggered by this amino acid. Glutamate transporters are profusely expressed in glia therefore most of its uptake occurs in this cellular compartment. In the cerebellar cortex, Bergmann glial cells enwrap glutamatergic synapses and participate in the recycling of its neurotransmitter through the glutamate/glutamine shuttle. It has long been acknowledged that glutamatergic transmission in the cerebellar molecular layer results in cGMP accumulation within Bergmann glia cells. In this context, we decided to investigate a plausible role of the nitric oxide/cGMP-signaling pathway in the regulation of Bergmann glia glutamate transporters. To this end, the well-established model of primary cultures of chick cerebellar Bergmann glial cells was used. Confluent monolayers were exposed to the nitric oxide donor, sodium nitroprusside, or to the non-hydrolysable cGMP analog dbcGMP and the [(3)H] D-aspartate uptake activity measured. An increase in uptake activity, related to an augmentation in VMax, was detected with both treatments. The signaling cascade includes NO/cGMP/PKG and Ca(2+) influx through the Na(+)/Ca(2+) exchanger and might be related to the plasma membrane glutamate transporters turnover. Interestingly enough, an inhibitor of the cGMP dependent protein kinase was capable to abolish the sodium nitroprusside induced Ca(2+) influx. These results provide an insight into the physiological role of cGMP in the cerebellum.

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Methylene Blue solution, 0.05 wt. % in H2O
Methylene Blue solution, for microscopy, concentrate according to Ehrlich, concentrated, aqueous solution
Rp-8-Bromo-β-phenyl-1,N2-ethenoguanosine 3′,5′-cyclic monophosphorothioate sodium salt, ≥98% (HPLC), powder
Sodium thiophosphate tribasic hydrate, ≥90%
Methylene blue, certified by the Biological Stain Commission
Methylene Blue Solution 1.4%(w/v)95%ethanol | 7220-79-3, 1.4 % (w/v) in 95% ethanol