Dopamine activates the extracellular signal-regulated kinase 1/2 (ERK 1/2) through a mechanism mediated by reactive oxygen species

ACQUIER, A.; MORI SEQUEIROS GARCIA, M.; BRION, L.; GOROSTIZAGA, A.; GOMEZ, N.; PAZ, C.; MENDEZ, C.F.

Milano

Congreso; World Congress of Nephrology 2009; 2009

European Renal Association, European Dialysis and Transplant Association, International Society of Nephrology

INTRODUCTION AND AIMS: Dopamine (DA) produced intrarenaly exerts a natriuretic effect by inhibiting Na+,K+-ATPase activity. In the proximal tubule, DA regulation of Na+,K+-ATPase involves the activation of D1- and D2-like receptors, stimulation of protein kinase C and generation of hydroxylated metabolites of arachidonic acid (AA). Phosphorylation of cytosolic phospholipase A2 by the extracellular signal-regulated kinase 1/2 (ERK 1/2, members of the MAP kinase family) increases its intrinsic enzymatic activity and augments Ca2+-induced AA release. The metabolism of DA generates reactive oxygen species (ROS) that could act as novel intracellular second messengers under subtoxic conditions, inducing the activation of various signal transduction processes, including the MAP kinase pathway. Therefore, the aim of this work was to determine if ERK 1/2 participates in the pathway activated by DA and the receptor involved in this process. We also aimed to study the participation of ROS in the signal pathway activated by DA. METHODS: We used opossum kidney (OK) cells, a known model of proximal tubule function. OK cells were stimulated with DA, or the D1 or D2 receptor specific agonists SKF-38393 (300 nM) or PPHT (500 nM) respectively, alone or in the presence of the antioxidants N-acetylcysteine (1 mM NAC) or Trolox (100 µM), or the monoamino oxidase (MAO) inhibitor pargyline (1 µM). Activation of ERK 1/2 was determined through the detection of the phosphorylated form of ERK 1/2 (p-ERK) by Western blot and immunocytochemistry using specific antibodies. RESULTS: DA promoted a dose- and time-dependent activation of ERK 1/2. Maximal activation occurred rapidly (5 min) and was evident with low concentrations (1 µM) of DA. Stimulation of either D1 or D2 receptors led to activation of ERK 1/2. Immunocytochemistry showed that DA treatment of the cells resulted in increased cytosolic and nuclear specific fluorescence, confirming ERK 1/2 activation and translocation. The effect of DA was partially reduced by NAC, indicating the participation of ROS on DA-induced ERK 1/2 activation, whereas the antioxidant Trolox had no effect. Pretreatment of the cells with pargyline also prevented, in part, ERK 1/2 activation by DA. Finally, the stimuli that results in ERK 1/2 activation (1 µM, 5 min) produced no effect on cell proliferation measured 24, 48 and 72 hours after incubation. CONCLUSIONS: Our results suggest that DA promotes ERK 1/2 activation and translocation through D1 and D2 receptors via a mechanism that involves, at least in part, MAO-dependent ROS production.