ORIGINAL PAPER The Trophic Effect of Ouabain on Retinal Ganglion Cell is Mediated by EGF Receptor and PKC d Activation Gustavo de Rezende Corre ˆa • Karinne Cristinne da Silva Cunha • Aline Araujo dos Santos • Elizabeth Giestal de Araujo Accepted: 11 May 2010 / Published online: 25 May 2010 Ó Springer Science+Business Media, LLC 2010 Abstract It was already shown that ouabain treatment can stimulate PKC isoenzymes leading to the activation of intracellular pathways involved in cell survival, growth and proliferation. We have previously demonstrated that oua- bain or PMA treatment increases retinal ganglion cell survival, an effect mediated by PKC activation. The aim of this work was to investigate the role of EGF receptors in the ouabain effect and also to study which PKC isoform is activated by treatment with ouabain and PMA. Our results show that 2.5 lM tyrphostin, 1.0 lM PP1, 4.0 lM U73122, 1.0 lM JNK inhibitor V and 2.0 lM rottlerin blocked the ouabain effect indicating an involvement of receptors for EGF, Src, PLC, JNK and PKC d respectively. The effect of PMA was only abolished when cultures were treated with rottlerin or with the JNK inhibitor suggesting the involvement of PKC d and JNK. These results indicate that PKC d could be a key regulator of retinal ganglion cell survival. Keywords Retinal ganglion cells Á Ouabain Á Neuronal survival Á PKC d Á Src Á EGF receptor Introduction A well known and important function of Na ? ,K ? -ATPase in mammalian cells is the maintenance of ionic gradients across the plasma membrane. This enzyme presents three subunits (a, b and c) and transports three Na ? outward and two K ? into the cell, using ATP energy [1]. The a subunit contains the binding site for ouabain and ATP [2]. The b subunit is important for the a subunit activity [3]. The c subunit stabilizes the pump conformation [4] and may influence the binding of ouabain to Na ? ,K ? -ATPase [5]. It was already shown that a1 isoform is widely expressed in the nervous system, the a3 isoform is more restricted to neurons, the b1 isoform is found predominantly in neurons while b2 is specific for astrocytes [6–8]. It was demon- strated that Na ? ,K ? -ATPase is also involved in cell sig- naling pathways through protein–protein interactions leading to changes in nuclear responses [1]. The presence of Na ? ,K ? -ATPase pump in caveolae was identified and this specific localization was related to cell growth and proliferation [9, 10] as well as to endocytosis [11]. Ouabain is a glycoside used for many years to treat cardiac dysfunction. This therapeutic application involves an increase in intracellular calcium concentration observed after ouabain treatment. As ouabain binds to the a-subunit of Na ? ,K ? -ATPase, an imbalance of its ionic transport takes place, leading to the accumulation of intracellular Na ? and a consequent increase in intracellular calcium through the Na ? /Ca 2? exchanger [12, 13]. Interestingly, endogenous ouabain has also been identified [14] and its plasmatic concentration ranges from 50 pM to 80 nM G. de Rezende Corre ˆa Á K. C. da Silva Cunha Á A. A. dos Santos Á E. G. de Araujo (&) Departamento de Neurobiologia, Programa de Neurocie ˆncias, Instituto de Biologia, Universidade Federal Fluminense, CP: 100180, Nitero ´i, Rio de Janeiro 24001-970, Brazil e-mail: egiestal@vm.uff.br A. A. dos Santos Instituto de Biofı ´sica Carlos Chagas Filho, Coordenac ¸a ˜o de Ensino de Po ´s-Graduac ¸a ˜o, Centro de Cie ˆncias da Sau ´de, Bloco G, Sala G1-003, Universidade Federal do Rio de Janeiro, Ilha do Funda ˜o, Rio de Janeiro 21949-900, Brazil A. A. dos Santos Departamento de Fisiologia e Farmacologia, Instituto Biome ´dico, Universidade Federal Fluminense, Nitero ´i, Rio de Janeiro 24210-130, Brazil 123 Neurochem Res (2010) 35:1343–1352 DOI 10.1007/s11064-010-0190-7