Background

Aiming to develop new drugs against infections caused by different trypanosomatids, efforts have been invested in the elucidation of the physiological mechanism responsible for intracellular ionic homeostasis, in particular Ca²⁺ ions, which are known to play a pivotal role as a second messenger in Trypanosoma cruzi and Leishmania spp.The intracellular Ca²⁺ concentration ([Ca²⁺]_i) is finely regulated (Benaim and García., 2011; Benaim et al, 2020) in these kinetoplastids. Leishmaniases are vector-borne parasitic neglected diseases caused by at least 20 species of the genus Leishmania, and are transmitted between mammalian hosts by female sandflies (Burzaet al., 2018). Chagas disease is another particularly neglected human infection, caused by Trypanosoma cruzi, transmitted by triatomine bugs when infectious parasites in the feces of the vector enter in the skin of the mammalian host at the bite site where scratching is provoked (Santos et al., 2020).

Prior to Fura-2, the most popular method for measuring [Ca²⁺]_i in cells was to monitor the fluorescence of an indicator called Quin-2. Nevertheless this fluorophore has severe limitations due to its short excitation wavelength, non-ratiometric nature and low quantum yield (Grynkiewicz et al., 1985). Also, Quin-2 produces the quenching of large amounts of intracellular Ca²⁺ that could strongly interfere with the minute changes in the [Ca²⁺]_i that are the focus of this protocol. For this reason, Quin-2 has been used instead as an intracellular Ca²⁺ chelator, when information under this extreme cellular condition is required. However, BAPTA is most preferred for this use because even though it has a similar molecular structure and a comparable affinity for Ca²⁺ as does Quin 2, BAPTA has a very weak fluorescence emission, thus having less interference with the [Ca²⁺]_i measurement (Moreno et al, 1994). Fura-2 indeed overcomes all these limitations. The main advantage of using ratiometric dyes like Fura-2, when compared to single wavelength probes, is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length, allowing thereby to accurately determine the concentration of intracellular calcium (Barreto-Chang and Dolmetsch, 2009). In cell suspensions, accurate determinations of [Ca²⁺]_i changes can be made by the use of Fura-2, especially in combination with various available pharmacological agents (Patel et al., 2012).

The simple and relatively fast protocol presented here has been widely used in the search and evaluation of several potential chemotherapeutic anti-parasitic drugs to elucidate their mechanism of action against T. cruzi and several Leishmania spp. For example, one can determine the source of Ca²⁺ (intracellular vs. extracellular) in a particular rise of the [Ca²⁺]_i observed in a particular condition, for example a treatment with a drug, in order to verify if the compound acts at the plasma membrane level (channels, receptors), at intracellular compartments (mitochondria, endoplasmic reticulum, acidocalcisomes) or at both (Benaim et al., 2020). This is simply achieved by performing the same experiment in the presence or absence (using EGTA, a Ca²⁺ chelating agent) of extracellular Ca²⁺.

A typical example of the Fura-2 use in Trypanosoma cruzi is depicted in Figure 1. In A (blue trace) it can be seen that addition of sphingosine (Sph) to epimastigotes loaded with Fura 2 induces an increase in [Ca²⁺]_i, which is specific for this particular sphingolipid, since addition of Sphingosine-1-Phosphate (Sph-1-P), Ceramide-1-Phosphate (Cer-1-P) or Ceramide (Cer) were without any discernible effect. On the other hand, when EGTA was added to study the effect of Sph in the absence of extracellular calcium (red trace), the sphingolipid, instead of inducing an increase in the [Ca²⁺]_i, caused a small decrease.This could represent a release of the low cytoplasmic Ca²⁺ to the extracellular space passing by the channel, when itencountered the chelator EGTA. In Figure1B are depicted the effects of nifedipine, a classical human L Type VGCC antagonist on the parasite channel. The green trace shows that nifedipine fully blocks the action of Sph added (indicated by a green arrow). As it is observed (Black trace) the miltefosine, is also able to open this channel, mimicking its effect in L. mexicana (Benaim et al., 2013). Miltefosine is the only oral drug approved for the treatment of visceral leishmaniasis and is also effective against T. cruzi. But, differently to the effect of the natural sphingolipid, nifedipine was not able to totally block the action of this drug (Blue trace), suggesting that these compounds act via a different mechanism on the Ca²⁺ channel.


Figure 1. Effect of Sphingosine-1-P (Sph-1-P), Ceramide -1-P (Cer-1-P),Ceramide (Cer) and Sphingosine (Sph), Miltefosine and Nifedipine (a specific L-type VGCC channel blocker) on the intracellular Ca²⁺ concentration of Trypanosoma cruzi. A. Sph-1-P, Cer-1-P, Cer and Sph were added sequentially to a final concentration of 10 µM in the presence of 2 mM CaCl₂ (blue trace) and in the absence of extracellular Ca²⁺ (red trace). B. Sphingosine (10 µM, arrow) was added after addition of nifedipine (4 µM, arrow) in the presence of extracellular Ca²⁺ (green trace). Miltefosine (4 µM) was added in the presence of extracellular CaCl₂ (blue trace). Nifedipine (40 µM, arrow) was added before the addition of Miltefosine (4 µM) in the presence of extracellular Ca²⁺ (black trace). (see text for details). Taken from Rodriguez-Duran et al. (2019) FEBS J, 286, 3909-3925. Copyright 2019. FEBS PRESS.

Finally, we used as example the effect of a potent anti-tuberculosis drug known to affect T. cruzi, L. mexicana (Benaim et al., 2020) and L. donovani (Gil et al., 2020) which instead of acting through the plasma membrane Ca²⁺ channels as miltefosine does, it exerts its action on intracellular organelles, like the parasites’ mitochondrion and/or acidocalcisomes. Thus, it can be observed in Figure 2, that the effect of the drug is the same in the presence (A) or in the absence (B) of extracellular Ca²⁺, since the cation is released from the mentioned intracellular organelles.


Figure 2. Effects of the antituberculosis drug SQ109 on Ca²⁺ fluxes in Leishmania mexicana. (A) Intracellular Ca²⁺ concentration in the presence of 2 mM external Ca²⁺ and (B) Same as panel A but in the absence of external Ca²⁺ (2 mM EGTA). Taken from García-García et al. (2016) Antimicrob Agents Chemother. 60: 6386-6389, Copyright 2016. American Chemical Society.