Ecto-nucleotidase activities in the fat body of Rhodnius prolixus

In this study, we describe the ability of intact fat body of an insect, Rhodnius prolixus, to hydrolyze extracellular ATP. In these fat bodies, the ATP hydrolysis was low in the absence of any divalent metal, and was stimulated by MgCl2. Both activities (in the absence or presence of MgCl2) were linear with time for at least 30 min. In order to confirm the observed nucleotidase activities as ecto-nucleotidases, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid). This reagent inhibited both nucleotidase activities and its inhibitory effect was suppressed by ATP. Both ecto-nucleotidase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin, ouabain, vanadate, molybdate, sodium fluoride, levamizole, tartrate, p-NPP, sodium phosphate, and suramin. Concanavalin A, activator of some ecto-ATPases, was able to stimulate the Mg2+-independent nucleotidase activity, but not the Mg2+-dependent one. The Mg2+-independent nucleotidase activity was enhanced with increases in the pH in the range between 6.4-8.0, but the Mg2+-dependent nucleotidase activity was not affected. Besides MgCl2 , the ecto-ATPase activity was also stimulated by CaCl2, MnCl2, and SrCl2, but not by ZnCl2. ATP, ADP, and AMP were the best substrates for the Mg2+-dependent ecto-nucleotidase activity, and CTP, GTP, and UTP produced very low reaction rates. However, the Mg2+-independent nucleotidase activity recognized all these nucleotides producing similar reaction rates, but GTP was a less efficient substrate. The possible role of the two ecto-nucleotidase activities present on the cell surface of fat body of Rhodnius prolixus, which are distinguished by their substrate specificity and their response to Mg2+, is discussed.