Trypanosoma brucei: Analysis of cytoplasmic Ca2+ during differentiation of bloodstream stages in vitro

The highly regulated intracellular concentration of calcium (Ca2+) is a well-described regulator of diverse cellular events, including cell cycle control. in the present study we have addressed the regulation of cytosolic Ca2+ in differentiation events in the life cycle of the protozoan parasite Trypanosoma brucei. Bloodstream form (BSF) trypanosomes include the mitotically active long slender forms (LS) which differentiate to two nondividing stages-intermediate (INT) which transform into short stumpy (SS) forms. An axenic in vitro culture system was used to cultivate LS to a density greater than 1.0 x 10(6) cells/ml/day. Populations of the intermediate BSF (INT) and SS were derived from cultured LS by treatment with difluoromethyl ornithine (DFMO, 100 mu M) for 2 and 4 days, respectively. A semiquantitative reverse transcriptase-coupled polymerase chain reaction protocol (SQ-RT-PCR) was developed to objectively distinguish the three BSF by monitoring the relative levels of stage-specific mRNAs-cytochrome oxidase II (COXII), variant surface glycoprotein, and procyclin during the differentiation of LS to SS; showing an increase in COXII and procyclin mRNA expression during this process of differentiation. Basal cytosolic Ca2+ levels [Ca2+](i) of populations of LS, INT, and SS were studied using Indo-1 dual emission fluorometry. [Ca2+](i) was maximal in dividing LS cells and was shown to decrease coincidentally with early events in the process of differentiation to INT and SS. Thapsigargin (1 mu M). reported to cause the release of Ca2+ from the endoplasmic reticulum, elevated [Ca2+](i) by about 30-60 mu M in all BSF; however, the total thapsigargin-releasable stores decreased in parallel with the decrease in basal [Ca2+](i). Control treatments verified that elevations in [Ca2+](i) in response to thapsigargin were intracellular in origin. These results may reflect the cessation of cytosolic Ca2+ transients involved in the regulation of mitosis as the parasite exits from the cell cycle and differentiates from rapidly dividing LS to the nondividing SS. (C) 1996 Academic Press, Inc.