Effect of osmolality on ⁸⁶Rb⁺ uptake and release by Leishmania donovani

Abstract

Promastigotes from late-log phase cultures of Leishmania donovani were washed and resuspended in Hanks' Balanced Salt Solution without glucose or phenyl red but with 20 mM (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]) (HEPES) (HBSS-, 305 mOsm/kg). They were then added to a solution containing 86Rb such that the final osmolality and ionic composition was as desired. Samples were taken at known times and the amount of intracellular 86Rb was measured. Similarly, experiments were performed in which 86Rb was added to the cultures about 18 hr before collection, and the amount of 86Rb released from the washed cells was measured. Under iso-osmotic conditions only about 1.3% of the intracellular 86Rb was released in 900 sec. This increased about 4-fold if the osmolality was reduced from 305-153 mOsm/kg. This is much slower than the very rapid release of alanine in response to hypo-osmotic stress, indicating that alanine release is not via a non-specific pore. Reducing the temperature from 26 degrees C to 3-4 degrees C completely inhibits 86Rb release under iso-osmotic conditions and largely inhibits it under hypo-osmotic conditions. The rate of 86Rb release was not sensitive to K+ concentration and was not altered if chloride was replaced by sulfamate. Ouabain had no effect on either 86Rb uptake or release, but carbonylcyanide P-trifluoromethoxyphenylhydrazone (FCCP) reduced the rate of 86Rb release and, after about a 300 sec exposure, completely inhibited 86Rb uptake. Amiloride partially inhibited 86Rb release, but had no effect on uptake. A decrease in pH from 7.1-5.9 had little effect on 86Rb release under iso-osmotic conditions and slightly increased the rate of release under hypo-osmotic conditions, but it decreased the rate of uptake under both iso-osmotic and hypo-osmotic conditions. Cells taken from 3-day stationary phase cultures released 86Rb more slowly under iso-osmotic conditions than cells from late log phase cultures, but were more responsive to hypo-osmotic stress than were log phase cells. These data appear to rule out an [Na-K-Cl] transporter or a [K-Cl] cotransporter as the means of K+ release, but are consistent with the possibility that a K+/H+ exchanger is present. The possibility that other carrier systems may be present is also discussed.

Plasma membrane K+/H+‐ATPase From leishmania donovani

“…Taken together these data suggest the involvement of an energy-dependent K'/H+ exchanger, possibly a K+/H+-ATPase, on the L. donovani plasma membrane. Our data corroborate Blum's (1992) report of an amiloride-insensitive K+ uptake and his speculation of the possible existence of a K+/H+ antiporter in L. donovani.…”

A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi

“…Evidence for a regulatory volume decrease mechanism has been observed in various trypanosomatids including Herpetomonas samuelpessoai (Andrade and Andrade, 1988), Leishmania major (Blum, 1992;Darling et al, 1990;LeFurgey et al, 2001;Vieira et al, 1996), L. donovani (Blum, 1992, Blum, 1996, Blum and Balber, 1996 and Crithidia luciliae (Bursell and Kirk, 1996). In L. major a 50% reduction in external osmolarity evoked an extremely rapid efflux of amino acids that apparently completely accounted for the RVD (Vieira et al, 1996).…”

Osmoregulation in the Parasitic Protozoan Tritrichomonas foetus

“…Osmolality was changed by dilution of cell suspensions with appropriate combinations of PBS or culture media and distilled water, 2.5 M NaCl, 2.5 M choline chloride, or 2.5 M NaNO 3 .…”

“…Aliquots of cells resuspended in PBS were added to equal volumes of inulin [ 14 C]carboxylic acid (2 Ci ml Ϫ1 ), 3 H 2 O (10 Ci ml Ϫ1 ), and PBS and incubated at room temperature for 1 min before centrifugation through oil. The supernatant was removed, the pellet was processed, and the radioactivity was counted with a Packard Tri-Carb 1900 liquid scintillation system, as previously described (15).…”

“…The first information provided on K ϩ fluxes in diplomonads revealed that the efflux of K ϩ , together with its counteranion, contributed approximately one-half of the total RVD (2,19). This contrasts with the situation in Leishmania, in which a major efflux of Na ϩ and Cl Ϫ and, to a lesser extent, an efflux of K ϩ occur in response to hypoosmotic stress (3,17). Our findings show that the response of T. foetus to anisomotic challenge is different from the response observed in all other protozoan parasites to date.…”