Calcium Influx Measurements

Like other GPCRs, mammalian LPH, Hc110-R, and C. elegans LAT-1 mediate influx of Ca²⁺ ions into the cell upon activation (LAJUS et al., 2006; RAHMAN et al., 1999;

SAEGER, 2000; WILLSON et al., 2004a). COS-7 cells were shown to be an appropriate system for calcium influx experiments with recombinantly expressed mammalian LPH and Hc110-R (LELIANOVA et al., 1997; SAEGER, 2000). Whereas the calcium influx measurements in the work of SAEGER (2000) were performed in single-cell assays, subsequent experiments were performed on cell pools in a cuvette, as was performed in this work. In such experiments, the Hc110-R transfected COS-7 cells react to an α-LTX stimulus with a peak of the intracellular Ca²⁺ concentration, followed by a plateau (personal communication, Hans-Peter Schmitt-Wrede, Heinrich-Heine University Duesseldorf). The observed reaction of the depsiphilin (lat-1) and lat-2 transfected COS-7 cells in this work was similar to these former results, although the results were partially not reproducible.

In the experiments with Lipofectamine^TM transfected cells, the intracellular Ca²⁺ concentration of cells that gave a response started at a lower level than the Ca²⁺ concentration in cell samples that did not react. The importance of this observation is not clear. Furthermore, of seven measurements of depsiphilin (lat-1) transfected cells, four with a stimulus of 500 pM, only two samples did react at all.

The lat-2 transfected cells from the experiments with Lipofectamine^TM transfection reacted only in one out of seven measurements, again four of them with a stimulus of 500 pM. The observed initial peak must be regarded as a nonspecific event, since the peak was also observed in response to the injection of water. It was also observed as a response to the different stimuli in empty-vector transfected cells.

A peak was also observed in the experiments with FuGENE transfected cells, although the peak was broader, lasting for 40 – 60 sec instead of 5 – 10 sec as with Lipofectamine^TM transfected cells. A comparable peak was observed in the pEGFP-N2 transfected cells used as reference. This broader peak must therefore also be regarded as a nonspecific response of the cells to the injection in general. In FuGENE transfected cells, an increased level (plateau) of the intracellular Ca²⁺ concentration was observed in all measurements with depsiphilin (lat-1) and

Discussion 163

lat-2 transfected cells, except for one experiment with a sample of depsiphilin (lat-1) transfected cells that were pooled due to low cell numbers. These cells did react to a stimulus of 100 µM ATP, indicating their viability. The cells might be regarded as an outlying sample, as their impaired growth causing the decreased cell number might indicate negative impacts during their growth phase. Perhaps these cells did not express the receptor as efficiently as the other samples or were impaired in another manner. In contrast, the positive response of one sample of pEGFP-N2 empty-vector transfected cells, used as reference, is not as easily explained. Since the responding sample was a fraction of pooled cells and the other fraction did not respond, it is unlikely that the whole pool of cells was contaminated, e.g. with depsiphilin (lat-1) or lat-2 transfected cells. It is more likely that only the responding sample was contaminated, potentially with ATP, which was used in previous measurements to test the viability of the cells. The shape of the response, however, was comparable to the responses of other samples to α-LTX rather than to ATP, which usually caused a sharp peak without a plateau. The presence of functional endogenous receptors for α-LTX in empty-vector transfected COS-7 cells is also unlikely, since responses of these cells to α-LTX stimuli were neither observed in the second control sample nor in other experiments with pEGFP-N2 empty-vector transfected COS-7 cells (personal communication, Hans-Peter Schmitt-Wrede, Heinrich-Heine University Duesseldorf).

The group of MEE (2004) identified LAT-1, but not LAT-2, as a target for BWSV in C. elegans. In this work, the function of the parasitic LAT-2 as a target for α-LTX could not be clarified. The experiments were performed although it was unknown whether or not LAT-2 acts through a signaling cascade similar to the LAT-1 mediated pathway. The results of calcium influx measurements in the cells upon an α-LTX stimulus were ambiguous, as discussed above. Since the expression of H. contortus LAT-2 in COS-7 cells could not be confirmed by a Western Blot, no statement is possible on whether an intact receptor was expressed in the cells or not.

However, the results do not exclude that parasitic LAT-2 expressed in COS-7 cells might mediate a similar response to α-LTX stimuli as the parasitic LAT-1 orthologs.

The different results of the experiments with Lipofectamine^TM and FuGENE might be due to different transfection efficiencies of these transfection reagents for lat-2.

164 Discussion

Subsequent experiments with larger sample size are planned to further examine the responses of depsiphilin (lat-1) and lat-2 transfected cells and empty-vector transfected cells to α-LTX stimuli.

Accordingly, the results of the calcium influx measurements must be regarded as preliminary results and evaluated carefully. As there were unexpected results which had to be explained, a concluding statement seems not reasonable. The observed responses might be nonspecific events. Recalling the results of the Western Blot of membrane-protein preparations for depsiphilin transfected cells, a possible explanation for the ambiguous results might be the absence of intact receptors due to degradation or impaired translation. Even if depsiphilin was translated correctly and only underwent the potentially natural cleavage at its GPS, the localization of the receptor might not have been in the cell membrane. The receptor fragments detected in the membrane-protein preparations of depsiphilin transfected cells might also be derived from internalized receptors in acidic lysosomes. In previous work, in transiently transfected COS-7 cells expressing an Hc110-R-GFP construct, GFP signals were detected 24 h post transfection in 64 % of the cells in vesicles, in 30 % in vesicles and membrane, and in 6 % solely in the membrane. 48 h post transfection Hc110-R was detected in 83 % of the cells solely in vesicles, in 14 % in cell membrane and vesicles, and in only 3 % in the membrane alone (SAEGER, 2000). Internalization of the receptor may also have occurred in cells expressing O. ostertagi depsiphilin or H. contortus LAT-2. Localization studies were not performed in this study. Therefore, the results of the calcium influx measurements should only be regarded as preliminary, demanding further and more intensive experiments to clarify the potential involvement of parasitic latrophilin-like receptors in mediating the action of α-LTX.

An interesting question that should be considered in future experiments is therefore the localization of the transiently expressed receptors in the cell. If the examined receptors of the present study were also internalized during the measurements, their potential functionality might not have been detectable. Localization could be examined using a GFP-tagged expression construct of the receptor, visualizing the recombinant protein within the cells. A GFP-tag would also allow determination of transfection rate. In this work the receptors were expressed either untagged

Discussion 165

(depsiphilin) or only with a small His-tag (LAT-2) to exclude potential negative impacts on the receptor functionality.

For the design of further experiments, the following factors might be of interest:

In the present experiments as well as in the previous work of SAEGER (2000), the native α-LTX was used, purified from natural BWSV produced by milking spiders.

This toxin was previously described to be involved in pore-formation. VOLYNSKI and coworkers (2000) observed that the pore-forming properties of α-LTX were facilitated by the receptors for α-LTX, although the receptors themselves were not involved in pore-formation. The group proposed that the receptors bind α-LTX and bring it into the vicinity of the membrane. Subsequently the toxin forms pores within the membrane. In single-cell assays for calcium influx measurements in cells expressing LPH constructs, LAJUS and coworkers (2006) observed a biphasic response.

Phase I of the response, consisting of calcium spikes or an increase in frequency and amplitude of calcium spikes in already active cells, was assigned to the GPCR induced cascade, whereas phase II was a plateau-phase, which was regarded as the effect of pore-formation: phase II was observed also in endogenous LPH deficient HIT-T15 β-cells expressing a truncated LPH construct, lacking all but the first TM helix and therefore the C-terminus carrying the structural characteristics of a GPCR. This observation supports the facilitation of pore-formation by the N-terminus independently of the GPCR-characteristic region of the receptor.

Therefore, an influx of calcium into the cells might not be evidence for a signaling cascade mediated by a GPCR.

However, in Hc110-R the influx of calcium ions could be blocked to a large extent with cadmium ions or nifedipine, a blocker for calcium channels of the L-type (SAEGER, 2000). These results indicate a specific influx of calcium via channels rather than through pores. Experiments using calcium channel blockers would clarify whether or not the observed effects of α-LTX on depsiphilin (lat-1) and lat-2 transfected cells were due to a receptor-mediated signaling cascade or to a potential receptor-facilitated formation of membrane pores. Another approach would be the use of the mutant LTX^N4C (VOLYNSKI et al., 2003), which is deficient in pore-forming properties. The use of LTX^N4C would also clarify if the responses are specific to the α-LTX component of the venom. MEE and coworkers (2004)

166 Discussion

suggested that α-LTX might potentially not be the active ligand for nematode LAT-1 but more likely another component of the BWSV, since they observed no effect of highly purified α-LTX on C. elegans, whereas BWSV had a toxic effect on the worms.

The α-LTX-preparations used in the present experiments were bought from ALOMONE Labs. The purity is > 98 % (ALOMONE LABS, 2007). Whether the remaining < 2 % contained the ligand causing the observed responses of Hc110-R transfected cells or whether the response was specific to the α-LTX component, could be clarified using LTX^N4C, as mentioned above, or highly purified α-LTX as described by MEE (2004).

Anyhow, the most interesting task within any future work would be to investigate the reaction of depsiphilin and lat-2 transfected cells to emodepside. The design of these experiments would have to consider the hydrophobic character of the component.

7.5 Real-time PCR

Real-time PCR is a convenient method to examine the quantity of nucleic acids in biological samples. To correct the data for processing variabilities, e.g. different efficiencies in cDNA synthesis in different samples for an analysis of transcription levels, the data can be normalized to reference genes. In the present work developmental stages of H. contortus and O. ostertagi were analyzed for their transcription levels of depsiphilin. Since no reference genes had been previously evaluated, two genes were chosen as putative reference genes: the 18 S rRNA (18 S) gene and the 60 S acidic ribosomal protein (60 S) gene. Both had been previously tested as reference genes in the canine hookworm A. caninum. 60 S was the most stable reference gene in several developmental stages and different strains of this parasitic nematode (TRIVEDI and ARASU, 2005). In the experiments of TRIVEDI and ARASU (2005) 18 S was not rated as an appropriate reference gene.

The cDNA synthesis was performed using an oligo-(dT)-primer. 18 S rRNA does not have a poly-A tail, so the authors relied on sufficient accidental internal priming for cDNA synthesis. In the present work, cDNA-synthesis was performed using gene-specific primers in addition to an oligo-(dT)-primer.

Discussion 167