Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta

In this work, we describe the ability of living hemocytes from an insect (M anduca sexta, Lepidoptera) to hydrolyze extracellular ATP, In these intact cells, there was a low level of ATP hydrolysis in the absence of any divale nt metal (8.24 +/- 0.94 nmol of P-i/h x 10(6) cells). The ATP hydrolysis wa s stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of P-i/h x 10(6) cells. Both activities were linear with cel l density and with time for at least 90 min. The addition of MgCl2 to extra cellular medium increased the ecto-ATPase activity in a dose-dependent mann er. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained wi th 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ repl aced Mg2+. The apparent K-m values for ATP(-4) and Mg-ATP(2-) were 0.059 an d 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffect ed by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other AT Pase and phosphatase activities, such as oligomycin, sodium azide, bafilomy cin A(1), ouabain, furosemide, vanadate, sodium fluoride, tartrate, and lev amizole. To confirm the observed hydrolytic activities as those of an ecto- ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene- 2,2'-disulfonic acid), as well as suramin, an antagonist of P-2-purinorecep tors and inhibitor of some ecto-ATPases, These two reagents inhibited the M g2+-independent and the Mg2+-dependent ATPase activities to different exten ts. Interestingly, lipopolysaccharide, a component of cell walls of gram-ne gative bacteria that increase hemocyte aggregation and phagocytosis, increa sed the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity. (C) 2000 Academic Press.