EFFECT OF OSMOLALITY ON RB-86(+) UPTAKE AND RELEASE BY LEISHMANIA-DONOVANI

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 Rb-86 such that the final osmolality and ionic composition was as desired. Samples were taken at known times and the amount of intracellular Rb-86 was measured. Similarly, experiments were performed in which Rb-86 was added to the cultures about 18 hr before collection, and the amount of Rb-86 released from the washed cells was measured. Under iso-osmotic conditions only about 1.3% of the intracellular Rb-86 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 Rb-86 release under iso-osmotic conditions and largely inhibits it under hypo-osmotic conditions. The rate of Rb-86 release was not sensitive to K+ concentration and was not altered if chloride was replaced by sulfamate. Ouabain had no effect on either Rb-86 uptake or release, but carbonylcyanide P-trifluoromethoxyphenylhydrazone (FCCP) reduced the rate of Rb-86 release and, after about a 300 sec exposure, completely inhibited Rb-86 uptake. Amiloride partially inhibited Rb-86 release, but had no effect on uptake. A decrease in pH from 7.1-5.9 had little effect on Rb-86 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 Rb-86 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.