A calmodulin-activated (Ca2+-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi

Abstract

High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.

Ceramide and sphingosine have an antagonistic effect on the plasma-membrane Ca2+-ATPase from human erythrocytes

“…For example, ceramide induces apoptosis, whereas sphingosine and sphingosine 1-phosphate stimulate mitogenic pathways [21,23]. The results presented in this work demonstrate that this sphingolipid inhibits the Ca# + -ATPase in a dose-dependent manner, affecting also its affinity for Ca# + .…”

“…Sphingosine is derived directly from ceramide and in many systems acts antagonistically with ceramide [21][22][23]. For example, ceramide induces apoptosis, whereas sphingosine and sphingosine 1-phosphate stimulate mitogenic pathways [21,23].…”

“…Ca# + transport was determined by the use of arsenazo III as a calcium indicator using the wavelength pair 675-685 nm, following the methodology described in [23]. To determine Ca# + transport, aliquots of IOVs was diluted in a final volume of 1 ml of a buffer containing 160 mM KCl, 50 µM arsenazo III, 0.5 mM MgCl # , 25 µM CaCl # and 10 mM Tris\HCl (pH 7.4) at 37 mC.…”

“…Other reagents were from Sigma. CaM was purified from bovine brain according to the method of Guerini et al [22], with the modifications introduced in [23].…”

The plasma-membrane Ca2+-ATPase of Leishmania donovani is an extrusion pump for Ca2+

“…These vesicles of defined polarity will be invaluable tools in elucidating ion movements, metabolite transport, drug accumulation and efflux in the parasite. Sealed vesicles were prepared from other trypanosoma [32,33] including L. dono ani [31], but, in the absence of associated microtubular structures, their polarities could not be ascertained. Using these vesicles of undefined polarity and by measuring medium Ca# + by the arsenazo method, ATP-dependent Ca# + entry was demonstrated in Trypanosoma cruzi [32] and Trypanosoma brucei [33].…”