Osmotic Modulation of the Ouabain-Sensitive (Na++K+)ATPase from Malpighian Tubules of Rhodnius prolixus

Abstract

The presence and regulation by hyperosmotic medium of the ouabain-sensitive (Na++K+)ATPase of the Malpighian tubule cells of Rhodnius prolixus was investigated. The ouabain-sensitive (Na++K+)ATPase activity was 5.4 ± 0.5 nmol Pi x mg-1 x min-1. Vanadate 100 μM completely abolished this ATPase activity. In hyperosmotic medium, obtained by addition of 180 mᴍ mannitol, the (Na++K+)ATPase activity was inhibited by 60%. When the cell lysates were preincubated in hyperosmotic medium for 30 minutes and the ATPase activity was assayed in isosmotic medium, the (Na++K+)ATPase activity was not modified. Addition of 50 ng/ml sphingosine, a protein kinase C inhibitor, abolished the inhibition of (Na++K+)ATPase activity in hyperosmotic medium. Furtherm ore, phorbol ester (TPA), an activator of protein kinase C, mimicked the effect of hyperosmotic shock on (Na++K+)ATPase activity. The increase in Ca2+ concentration decreased the (Na++K+)ATPase activity by 60% in isosmotic medium, with maximal effect obtained in 10-6 m Ca2+. No effect was observed in hyperosmotic medium. The inhibitory effect of Ca2+ on the (Na++K+)ATPase was not reversed by sphingosine. These results indicate that the ouabain-sensitive (Na++K+) ATPase activity of the Malpighian tubule is regulated by both increasing Ca2+ concentration and by the osmolality of the medium by different and integrative ways.