OSMOTIC MODULATION OF THE OUABAIN-SENSITIVE (NA-PROLIXUS(+K+)ATPASE FROM MALPIGHIAN TUBULES OF RHODNIUS)

The presence and regulation by hyperosmotic medium of the ouabain-sens itive (Na++K+)ATPase of the Malpighian tubule cells of Rhodnius prolix us was investigated. The ouabain-sensitive (Na++K+)ATPase activity was 5.4 +/- 0.5 nmol Pi x mg(-1) x min(-1). Vanadate 100 mu M completely abolished this ATPase activity. In hyperosmotic medium, obtained by ad dition of 180 mar 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 mediu m, 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. Furthermore, phorbol ester (TPA), an activator of protein kinase C, mimicked the effect of hyperosmotic shock on (Na++K+)ATPase activity. The increase in Ca2+ co ncentration decreased the (Na++K+)ATPase activity by 60% in isosmotic medium, with maximal effect obtained in 10(-6) M Ca2+. No effect was o bserved 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 Malpighlan t ubule is regulated by both increasing Ca2+ concentration and by the os molality of the medium by different and integrative ways.