Rottlerin, an inhibitor of protein kinase Cdelta (PKCdelta), inhibits astrocytic glutamate transport activity and reduces GLAST immunoreactivity by a mechanism that appears to be PKCdelta-independent.
ABSTRACT Protein kinase C (PKC) regulates the activity and/or cell surface expression of several different neurotransmitter transporters, including subtypes of glutamate transporters. In the present study, the effects of pharmacological inhibitors of PKC were studied in primary astrocyte cultures that express the glutamate aspartate transporter (GLAST) subtype of glutamate transporter. We found that general inhibitors of PKC, bisindolylmaleimide I (Bis I), bisindolylmaleimide II (Bis II), staurosporine and an inhibitor of classical PKCs, Gö6976, had no effect on Na+-dependent glutamate transport activity. However, rottlerin, a putative specific inhibitor of PKCdelta, decreased transport activity with an IC50 value (less than 10 micro m) that is comparable to that reported for inhibition of PKCdelta. The effect of rottlerin was very rapid (maximal effect within 5 min) and was due to a decrease in the capacity (Vmax) for transport. Rottlerin also caused a drastic loss of GLAST immunoreactivity within 5 min, suggesting that rottlerin accelerates GLAST degradation/proteolysis. Rottlerin had no effect on cell surface or total expression of the transferrin receptor, providing evidence that the effect on GLAST cannot be attributed to a non-specific internalization/degradation of plasma membrane proteins. Down-regulation of PKCdelta with chronic phorbol ester treatment did not block rottlerin-mediated inhibition of transport activity. These results suggest a novel mechanism for regulation of the GLAST subtype of glutamate transporter and indicate that there is a rottlerin target that is capable of controlling the levels of GLAST by controlling the rate of degradation or limited proteolysis. It appears that the target for rottlerin may not be PKCdelta.
Article: Cardiac glycosides ouabain and digoxin interfere with the regulation of glutamate transporter GLAST in astrocytes cultured from neonatal rat brain.[show abstract] [hide abstract]
ABSTRACT: Glutamate transport (GluT) in brain is mediated chiefly by two transporters GLT and GLAST, both driven by ionic gradients generated by (Na(+), K(+))-dependent ATPase (Na(+)/K(+)-ATPase). GLAST is located in astrocytes and its function is regulated by translocations from cytoplasm to plasma membrane in the presence of GluT substrates. The phenomenon is blocked by a naturally occurring toxin rottlerin. We have recently suggested that rottlerin acts by inhibiting Na(+)/K(+)-ATPase. We now report that Na(+)/K(+)-ATPase inhibitors digoxin and ouabain also blocked the redistribution of GLAST in cultured astrocytes, however, neither of the compounds caused detectable inhibition of ATPase activity in cell-free astrocyte homogenates (rottlerin inhibited app. 80% of Pi production from ATP in the astrocyte homogenates, IC50 = 25 μM). Therefore, while we may not have established a direct link between GLAST regulation and Na(+)/K(+)-ATPase activity we have shown that both ouabain and digoxin can interfere with GluT transport and therefore should be considered potentially neurotoxic.Neurochemical Research 10/2010; 35(12):2062-9. · 2.24 Impact Factor
Bala T S Susarla