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Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs.

Dagger F, Valdivieso E, Marcano AK, Ayesta C - Mem. Inst. Oswaldo Cruz (2013)

Bottom Line: When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD).No direct relationship was found between antiproliferative drug treatment and RVD.AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Biología Celular de Parásitos, Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela. chichadagger@gmail.com

ABSTRACT
The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.

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regulatory volume decrease in Leishmania mexicana promas tigotes after anti-microtubule (Mt) drug treatment. Relative changes in cell volume of parasites cultured in the absence or presence of the drugs were followed by monitoring absorbance at 550 nm. Traces fol lowed by control, trifluoperazine (TFP) and taxol treated cells (A) and cells treated with rhizoxin and ansamitocin P3 (AP3) (B). Results are representatives of those obtained from five independent experiments and are expressed in arbitrary absorbance units. OD: optical density.
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f02: regulatory volume decrease in Leishmania mexicana promas tigotes after anti-microtubule (Mt) drug treatment. Relative changes in cell volume of parasites cultured in the absence or presence of the drugs were followed by monitoring absorbance at 550 nm. Traces fol lowed by control, trifluoperazine (TFP) and taxol treated cells (A) and cells treated with rhizoxin and ansamitocin P3 (AP3) (B). Results are representatives of those obtained from five independent experiments and are expressed in arbitrary absorbance units. OD: optical density.

Mentions: Effect of anti-Mt drugs on RVD in L. mexicana promastigotes under hypotonic stress - The drugs used in this study have shown strong antiproliferative effects on Leishmania promastigotes. Based on these results, we next investigated the capacity of these drug-treated promastigotes to carry out RVD under hypotonic stress. When control cells were subjected to a reduction in osmolarity from 300-150 mOsm, they immediately swelled until they became quite rounded, but within a few minutes they began to shrink, reaching the same degree of motility and morphology observed at 300 mOsm. These interesting changes lasted nearly 10 min (results not shown). Light-scattering experiments allowed us to follow the process of volume recovery by monitoring the A550 of the cell suspensions, as cell swelling leads to a decrease in the absorbance reading (Park et al. 1997, Rohloff et al. 2003). All of the experiments were carried out under the same conditions. The promastigotes were cultured for 48 h and the drugs were added at concentrations near the IC50 values. The control cells and the drug-treated cells were centrifuged and resuspended in 300 mOsm buffer. Following a two-fold dilution of the isosmotic cell suspensions with distilled water, the absorbances of the suspensions were monitored for at least 10 min. No differences in the lag time or rate of volume recovery were observed among control, chlorpromazine, taxol or TFP-treated cells (Fig. 2A) (and results not shown). The cells completed RVD in less than 5 min. Our findings were quite different for the AP3 and rhizoxin-treated cells. A decrease in absorbance was also observed in these cells 1 min post-treatment, but this decrease in absorbance was much more dramatic than that in the control cells. Volume recovery was not observed for the AP3-treated cells, whereas the rhizoxin-treated cells slowly returned to their original volume (Fig. 2B).


Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs.

Dagger F, Valdivieso E, Marcano AK, Ayesta C - Mem. Inst. Oswaldo Cruz (2013)

regulatory volume decrease in Leishmania mexicana promas tigotes after anti-microtubule (Mt) drug treatment. Relative changes in cell volume of parasites cultured in the absence or presence of the drugs were followed by monitoring absorbance at 550 nm. Traces fol lowed by control, trifluoperazine (TFP) and taxol treated cells (A) and cells treated with rhizoxin and ansamitocin P3 (AP3) (B). Results are representatives of those obtained from five independent experiments and are expressed in arbitrary absorbance units. OD: optical density.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974315&req=5

f02: regulatory volume decrease in Leishmania mexicana promas tigotes after anti-microtubule (Mt) drug treatment. Relative changes in cell volume of parasites cultured in the absence or presence of the drugs were followed by monitoring absorbance at 550 nm. Traces fol lowed by control, trifluoperazine (TFP) and taxol treated cells (A) and cells treated with rhizoxin and ansamitocin P3 (AP3) (B). Results are representatives of those obtained from five independent experiments and are expressed in arbitrary absorbance units. OD: optical density.
Mentions: Effect of anti-Mt drugs on RVD in L. mexicana promastigotes under hypotonic stress - The drugs used in this study have shown strong antiproliferative effects on Leishmania promastigotes. Based on these results, we next investigated the capacity of these drug-treated promastigotes to carry out RVD under hypotonic stress. When control cells were subjected to a reduction in osmolarity from 300-150 mOsm, they immediately swelled until they became quite rounded, but within a few minutes they began to shrink, reaching the same degree of motility and morphology observed at 300 mOsm. These interesting changes lasted nearly 10 min (results not shown). Light-scattering experiments allowed us to follow the process of volume recovery by monitoring the A550 of the cell suspensions, as cell swelling leads to a decrease in the absorbance reading (Park et al. 1997, Rohloff et al. 2003). All of the experiments were carried out under the same conditions. The promastigotes were cultured for 48 h and the drugs were added at concentrations near the IC50 values. The control cells and the drug-treated cells were centrifuged and resuspended in 300 mOsm buffer. Following a two-fold dilution of the isosmotic cell suspensions with distilled water, the absorbances of the suspensions were monitored for at least 10 min. No differences in the lag time or rate of volume recovery were observed among control, chlorpromazine, taxol or TFP-treated cells (Fig. 2A) (and results not shown). The cells completed RVD in less than 5 min. Our findings were quite different for the AP3 and rhizoxin-treated cells. A decrease in absorbance was also observed in these cells 1 min post-treatment, but this decrease in absorbance was much more dramatic than that in the control cells. Volume recovery was not observed for the AP3-treated cells, whereas the rhizoxin-treated cells slowly returned to their original volume (Fig. 2B).

Bottom Line: When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD).No direct relationship was found between antiproliferative drug treatment and RVD.AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Biología Celular de Parásitos, Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela. chichadagger@gmail.com

ABSTRACT
The trypanosomatid cytoskeleton is responsible for the parasite's shape and it is modulated throughout the different stages of the parasite's life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite's hypotonic stress-induced shape change, as well as its recovery.

Show MeSH
Related in: MedlinePlus