Na+/K(+)ATPase activity inhibition and isoform-specific translocation of protein kinase C following angiotensin II administration in isolated eel enterocytes

In the eel, angiotensin II (Ang II) has a role at the level of both gill ch loride and kidney tubular cells, regulating sodium balance and therefore os moregulation. The present study extends these findings to another important osmoregulatory organ-the intestine. Enterocytes were obtained from sea-wat er (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K(+)ATPase activity, because in SW-acclimated animals the intestine re presents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimula ted with increasing Ang II concentrations (0.01-100 nM) showed a dose-depen dent inhibition of the NA(+)/K(+)ATPase activity. The threshold decrease wa s at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and d id not decrease further with the use of higher hormone doses. These hormona l effects were blocked by a specific competitive antagonist of the AT1 rece ptor subtype, DuP-753 (100% inhibition at 10 muM), indicating that these ef fects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([C a2+](i)), followed by a lower sustained phase. Removal of extracellular Ca2 + did not reduce the initial transient response and completely abolished th e plateau phase. The inhibition of the Na+/ K(+)ATPase activity was depende nt on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Wes tern blot analysis with antibodies to PKC alpha, betaI, beta II, gamma, del ta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expres sed the conventional isoforms (alpha and betaI), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the t ranslocation from the cytosol to the plasma membrane of PKC alpha, PKC delt a and PKC eta isoforms. In conclusion, our results suggest that Ang II modu lates intestinal Na+/K(+)ATPase in SW-acclimated eels via calcium mobilizat ion and PKC activation.