Rats were made hyperammonemic by feeding them
Rats were made hyperammonemic by feeding them and ammonium-containing diet . Membrane expression, phosphorylation of the GluA1 and GluA2 subunits and the mechanisms underlying their alterations in hyperammonemic rats were analyzed in freshly isolated cerebellar slices.
Material & methods
Results Chronic hyperammonemia increases (p<0.01) membrane SC 236 of GluA1 to 184±32% of control rats (Fig. 1A) and reduces (p<0.05) that of GluA2 to 86±6% of control rats (Fig. 1C). Chronic hyperammonemia also increases (p<0.05) the ratio of GluA1 in the membrane/total content to 0.68±0.03 compared to 0.56±0.04 in control rats (Fig. 1B) and reduces (p<0.05) the ratio of GluA2 in the membrane/total content to 0.54±0.02 compared to 0.64±0.03 in control rats (Fig. 1D). Changes in membrane expression are associated with altered phosphorylation. Phosphorylation of GluA1 at Ser831 is increased (p<0.05) in hyperammonemic rats to 183±34% of control rats (Fig. 1E) and phosphorylation of GluA2 at Ser880 is also increased (p<0.01) to 197±33% of control rats (Fig. 1F). In contrast, phosphorylation of Ser845 of GluA1 is reduced (p<0.01) to 79±9% of control rats (Fig. 1G). As Ser880 of GluA2 is phosphorylated by PKC, we assessed whether inhibition of PKC reduces its phosphorylation in hyperammonemic rats. Inhibition of PKC completely normalizes phosphorylation of GluA2 at Ser880 in hyperammonemic rats, reducing it to 112±20% (p<0.05) reaching similar levels to control rats (Fig. 2A). Moreover, this is associated with complete normalization of membrane expression of GluA2, which increases (p<0.01) to 133±11% (Fig. 2B). Increased PKC activity in hyperammonemic rats could be a consequence of enhanced activation by phospholipase C, which, in turn, may be activated by PKA , . To assess this possibility we tested the effects of inhibiting phospholipase C or PKA on phosphorylation of Ser880 and on membrane expression of GluA2. Inhibition of phospholipase C reduces phosphorylation of GluA2 at Ser880 in hyperammonemic rats to 85±5% (p<0.001) (Fig. 3A). However, this is not associated with normalization of membrane expression of GluA2, which decreases to 41±8% (p<0.001) (Fig. 3B). Inhibiton of PKA reduces phosphorylation of GluA2 at Ser880 in hyperammonemic rats to 108±23% (p<0.01) reaching similar levels to control rats (Fig. 3C). Moreover, this is associated with increased membrane expression of GluA2 to 175±43% of basal (p<0.001) as shown in Fig. 3D. These data suggest that increased PKA activity would contribute to the reduced membrane expression of GluA2 in hyperammonemic rats. We then looked for the mechanism by which PKA activity could be increased in hyperammonemic rats. As PKA is activated by cAMP we measured it in cerebellar slices. In control rats cAMP levels were 23±2pmol/mg protein. In hyperammonemic rats cAMP levels were increased significantly to 60±5pmol/mg protein (p<0.001) (Fig. 4A). A main phosphodiesterase degrading cAMP is phosphodiesterase 2 (PDE2), which is activated by cGMP . To test whether increased cAMP levels in hyperammonemic rats could be due to reduced PDE2 activity, we assessed the effects of a PDE2 inhibitor (EHNA) on cAMP levels. As shown in Fig. 4B, inhibition of PDE2 strongly increased (p<0.05) cAMP levels in control rats to 179±39% of basal. In contrast, in hyperammonemic rats, EHNA did not affect cAMP levels, which remained at 66±9% of basal. This supports that PDE2 activity was already inhibited in hyperammonemic rats, thus leading to increased cAMP levels. As PDE2 is activated by cGMP we assessed the effects of adding 8-Br-cGMP, a cell permeable analog of cGMP, on cAMP levels. Enhancing cGMP levels activated PDE2 and reduced cAMP levels similarly in controls, to 51±5% of basal, and in hyperammonemic rats, to 38±8% of basal (Fig. 4B), supporting that increased cAMP levels in hyperammonemic rats are due to reduced PDE2 activity, which may be restored by increasing cGMP.