In H. contortus a receptor for emodepside and α-LTX was previously identified (SAEGER et al., 2001). Hc110-R transfected HEK-293 cells responded with calcium influx to an α-LTX stimulus. The influx was shown to occur via voltage-dependent calcium channels of the L-type (SAEGER, 2000). In the free-living nematode C. elegans the putative signaling cascade for emodepside was described to be mediated by the receptor LAT-1 (WILLSON et al., 2004a). The mechanism of action engages a characteristic GPCR-mediated pathway, including the G-protein Gαq (egl-30), phospholipase Cβ (PLC-β, egl-8), and a presynaptic receptor for diacylglycerol (DAG), UNC-13 (unc-13). The involvement of the BK-type potassium channel SLO-1 is proposed (personal communication Lindy Holden-Dye, School of Biological Sciences, Southampton). Previously, SLO-1 was described to be important for precise regulation of neurotransmitter release, most likely activated by the influx of calcium via voltage-gated calcium channels (WANG et al., 2001).
In mammals, the action of α-LTX is also mediated by GPCRs, the LPH. The binding of α-LTX to mammalian LPH leads to calcium influx, also occuring through calcium channels of the L-type. Outward potassium currents, which are provoked by repeated depolarizing voltage stimuli, are inhibited by α-LTX in endogenous LPH-expressing and therefore α-LTX-susceptible MIN 6 β-cells. This effect does not occur when BK-type potassium channels are blocked by the inhibitor iberiotoxin, indicating that the potassium currents occur through BK-type potassium channels.
The phospholipase C inhibitor U73122 also blocked the effect of α-LTX on the potassium currents in MIN6 β-cells (LAJUS et al., 2006). LIU and coworkers (2005) showed the involvement of protein kinase C in mediating the effects of α-LTX.
These results indicate that mammalian LPH utilize for mediating the effects of α-LTX a pathway similar to that previously described for C. elegans LAT-1 (WILLSON et al., 2004a). Therefore, it seems reasonable to propose, that the pathway might be conserved, and that depsiphilins could potentially engage a similar signaling cascade. The endogenous ligands of C. elegans LAT-1 and of mammalian LPH are unknown. About 7 % of all genes in the genome of C. elegans, namely approx.
1300 genes and approx. 400 pseudogenes, code for GPCRs. GPCRs in C. elegans
174 Discussion
are involved in developmental and behavioural functions as well as in chemoreception (BERGAMASCO and BAZZICALUPO, 2006). As described above (3.6), many GPCRs are receptors for neurotransmitters. The involvement of the LAT-1 mediated pathway in transmitter release indicates a potential physiological role as a receptor for neurotransmitters as well. The utilization of components of the nervous system of nematodes as targets is also known from other anthelmintic drugs such as the macrocyclic lactones, nicotinergic agonists or piperazine. The endogenous ligand of LAT-1, however, remains to be identified.
In this work, LAT-1 orthologs were identified in C. oncophora and O. ostertagi and were called depsiphilins. They show high identities with their ortholog Hc110-R in H. contortus and moderate identities with LAT-1 in C. elegans and C. briggsae.
Structural similarities with these receptors and with mammalian LPH indicate a function as a GPCR. Polyclonal antibodies against three epitopes in Hc110-R were developed in rabbits and were tested on the prokaryotically expressed N-termini of the receptors. A specific binding of α-LTX to the isolated N-termini could not be confirmed. This result, however, does not exclude potential binding affinities of the native receptors to α-LTX. Furthermore, orthologs of LAT-2, a receptor previously discussed as an alternative target for emodepside, were identified in H. contortus and C. oncophora. O. ostertagi depsiphilin and H. contortus LAT-2 were also expressed in eukaryotic cells. The transiently transfected COS-7 cells were examined for their responses to α-LTX by means of calcium influx. The results were ambiguous, but did not exclude binding of α-LTX to the receptors. Speculating, depsiphilin and LAT-2 might be targets for similar endogenous ligands, or for the same ligand that activates different pathways.
An interesting experiment would be the stimulation of depsiphilin or lat-2 expressing cells with emodepside, to examine the properties of the receptors as targets for this anthelmintically active compound. The setup of respective experiments, however, would have to consider the hydrophobic nature of emodepside.
The experiments planned for the near future include more intensive examination of the receptor-mediated responses of depsiphilin or lat-2 expressing cells to α-LTX. For this purpose, expression plasmids for GFP-tagged receptors will be prepared and a
Discussion 175
larger sample size for receptor expressing and empty-vector transfected cells will be examined.
The real-time PCR based investigation of transcription levels of depsiphilins in developmental stages of H. contortus and O. ostertagi showed that depsiphilin was transcribed in all motile stages, and at even higher levels in eggs. In previous studies hatching of nematode eggs was unaffected by emodepside. This observation therefore does not indicate a reduced or absent transcription of the putative receptor depsiphilin. The comparison of 18 S rRNA and the 60 S acidic ribosomal protein gene as reference genes in real-time PCR indicated 18 S to be the more stable reference gene in the present experiments. The small sample size and the different results for H. contortus and O. ostertagi L3, however, demand more experiments with larger sample size. These experiments are also planned for the near future.
The contribution of a calcium-gated potassium channel to the action of emodepside was previously suggested by WILLSON and coworkers (2003) and recently emphasized by HOLDEN-DYE (personal communication), who observed knockout mutants for the potassium channel SLO-1 to be highly resistant to emodepside. The observations are partially consistent with the involvement of BK-type potassium channels in the effect of α-LTX on mammalian cells, as described by LAJUS (2006).
Orthologs of BK-type potassium channels had not yet been identified in parasitic nematodes. In the present work, orthologs of SLO-1 were identified in H. contortus and C. oncophora, and as a preliminary sequence in O. ostertagi. The sequences showed relatively high identities with potassium channels of the BK-type in several other organisms. To date, no functional investigations of the role of the parasitic nematode SLO-1 in mediating the actions of emodepside have been undertaken.
Nevertheless, their identification is the first step toward further experiments.
The extent to which all newly identified proteins contribute to the anthelmintic action of cyclooctadepsipeptides needs to be determined. The experiments planned for the near future are further calcium influx measurements in eukaryotic cells expressing the receptors as discussed above and the examination of transcription levels in larger sample size in real-time PCR.
176 Discussion
Most promising experimental setups include the expression of the parasitic genes in C. elegans knockout mutants. A plasmid for the expression of O. ostertagi depsiphilin driven by the C. elegans lat-1 promotor was already prepared. Such experiments could clarify, whether the parasitic genes were able to fulfill the physiological and pharmacological tasks of the respective endogenous genes. Another interesting project will be the functional comparison of mammalian LPH with parasitic depsiphilins and parasitic LAT-2, and of mammalian BK-type potassium channels with parasitic SLO-1. The results could be of value for the optimal adaptation of the anthelmintic components to their targets in parasites.
Appendix 177