RESUMO
We report the identification of an invertebrate Na(+)/K(+)/2Cl(-) cotransporter, NKCC. As a model system, we used the immune cells (coelomocytes) of the Mediterranean sea urchin Paracentrotus lividus. These cells are particularly interesting because they can be activated to undergo a rapid and dynamic change in cell shape. We demonstrate that forskolin, a cyclic AMP agonist known to regulate NKCC, induced coelomocyte transformation at doses of 10 micromol l(-)(1) and greater. Using two distinct monoclonal antibodies (T4 and T9) raised against the human intestinal epithelial NKCC, we have identified a high-molecular-mass (195 kDa) protein in coelomocyte extracts. We propose a novel method for the isolation of NKCC in one step by using bumetanide-Sepharose affinity chromatography under low-[Cl(-)] conditions. This method was successful in isolating coelomocyte 195 kDa NKCC. The T4 monoclonal antibody was used in immunocytochemical experiments to localize NKCC in resting and activated coelomocytes. In petalloid coelomocytes, a punctate, cytoplasmic distribution was observed in close proximity to actin filament bundles; in transformed coelomocytes, the immunofluorescence was distributed along the length of the filopodia and uniformly throughout the perinuclear region. The change in subcellular distribution of NKCC between the resting and the activated state was further investigated by using cell surface biotinylation followed by immunoprecipitation. These studies revealed an upregulation of NKCC at the plasma membrane upon activation, a process that was blocked by the F-actin-stabilizing drug phalloidin. These studies identify a novel model system in which to investigate a newly identified invertebrate Na(+)/K(+)/2Cl(-) cotransporter.