Date Published: April 18, 2018
Publisher: Public Library of Science
Author(s): Gihan S. Gunaratne, Nawal A. Yahya, Peter I. Dosa, Jonathan S. Marchant, Timothy G. Geary. http://doi.org/10.1371/journal.pntd.0006420
Abstract: The anthelmintic praziquantel (±PZQ) serves as a highly effective antischistosomal therapy. ±PZQ causes a rapid paralysis of adult schistosome worms and deleterious effects on the worm tegument. In addition to these activities against the parasite, ±PZQ also modulates host vascular tone in blood vessels where the adult worms reside. In resting mesenteric arteries ±PZQ causes a constriction of basal tone, an effect mediated by (R)-PZQ activation of endogenous serotoninergic G protein coupled receptors (GPCRs). Here, we demonstrate a novel vasodilatory action of ±PZQ in mesenteric vessels that are precontracted by high potassium-evoked depolarization, an effect previously reported to be associated with agonists of the transient receptor potential melastatin 8 channel (TRPM8). Pharmacological profiling a panel of 17 human TRPs demonstrated ±PZQ activity against a subset of human TRP channels. Several host TRP channels (hTRPA1, hTRPC3, hTRPC7) were activated by both (R)-PZQ and (S)-PZQ over a micromolar range whereas hTRPM8 showed stereoselective activation by (S)-PZQ. The relaxant effect of ±PZQ in mesenteric arteries was caused by (S)-PZQ, and mimicked by TRPM8 agonists. However, persistence of both (S)-PZQ and TRPM8 agonist evoked vessel relaxation in TRPM8 knockout tissue suggested that canonical TRPM8 does not mediate this (S)-PZQ effect. We conclude that (S)-PZQ is vasoactive over the micromolar range in mesenteric arteries although the molecular mediators of this effect remain to be identified. These data expand our knowledge of the polypharmacology and host vascular efficacy of this clinically important anthelmintic.
Partial Text: Schistosomiasis is a socioeconomically devastating helminth infection afflicting over 200 million people worldwide . The resulting disease burden of chronic schistosomiasis is estimated to encumber third world economies with an annual loss of 70 million disability-adjusted life years [2, 3]. In infected individuals, the prolific egg laying capacity of paired adult worms (>1000 eggs/day deposited in tissues, ) triggers localized inflammatory responses around eggs trapped within host tissues. Chronic infections progress toward fibrosis and obstructive disease in gastrointestinal tissues and liver (S. mansoni, S. japonicum), genitourinary disease (S. haematobium), anemia, undernutrition and a heightened risk for other comorbidities. Effective drug therapy for schistosomiasis is therefore a healthcare priority [1–3].
The contractile tone of vessel strips isolated from mouse mesenteric arteries was evaluated using wire myography. A typical experiment trace is shown in Fig 1, where mounted vessel strips exhibited a sustained contraction to high K+ media (KPSS) that rapidly reversed upon solution exchange (Fig 1A). At resting tone, addition of ±PZQ caused a marked contraction, consistent with recent data showing vasoconstriction mediated by (R)-PZQ activation of host 5-HT2B receptors (Fig 1A, ). However, an additional action of ±PZQ was observed in vessels precontracted by KPSS exposure. Addition of ±PZQ to vessels contracted with KPSS caused a marked relaxation (Fig 1A). This vasodilatory effect of ±PZQ was dose-dependent, and sufficient to relax the contracted vessel by 61±9% at high concentrations of ±PZQ (100μM, Fig 1B). Relaxation evoked by ±PZQ was phasic, with successive additions of ±PZQ (10μM) resulting in a dose-dependent relaxation of vessel tone toward precontracted levels (Fig 1C).
Here we demonstrate functional interactions between the resolved enantiomers of ±PZQ and a subset of human TRP channels over the micromolar range (Fig 3). These interactions may have significance for understanding the mechanism of action of ±PZQ in both host and parasite.
In terms of host biology, this concentration range is compatible with (R)-PZQ and (S)-PZQ concentrations attained within the splanchnic vasculature during ±PZQ treatment [13, 28, 29]. While the majority of human TRP channels were unaffected by (R)-PZQ and (S)-PZQ (Fig 2), the subset of TRP channels engaged by PZQ enantiomers (hTRPA1, hTRPC3, hTRPC7, hTRPM8) are all expressed in host blood vessels inhabited by adult worm pairs, where their activation causes vasorelaxation. Activation of TRPC3 in mesenteric endothelium mediates agonist-evoked vasodilation [30–32], via various signaling mechanisms (nitric oxide (NO)-dependent signaling, hyperpolarization). TRPC7, which complexes with TRPC3 , mediates store-operated Ca2+ entry in portal vein myocytes . TRPA1 activation also causes vasodilation: in mesenteric beds, this is mediated via TRPA1 activation releasing calcitonin gene related peptide (CGRP) from perivascular nerves. [35, 36]. Finally, TRPM8 is highly expressed in mesenteric artery and pharmacological activation of TRPM8 channels relaxes contracted vessels [20, 24, 25], effects attenuated in TRPM8 knockout mice . These data suggest vasodilation of contracted blood vessels as a possible physiological outcome of host TRP channel engagement by ±PZQ. We note TRPM5, a transducer of bitter taste signaling was not activated by (S)-PZQ (Fig 3). While taste is a side effect associated with (S)-PZQ , another target in the bitter tasting pathway must explain this association.
Discovery of TRPs as human targets of ±PZQ is also informative for efforts to define the parasitic target(s) of ±PZQ, as precedent has now been established for ±PZQ action as both a GPCR ligand and TRP channel modulator. Despite the molecular divergence between human and flatworm proteins and ligand binding pockets [19, 47], it is not unreasonable to anticipate (R)-PZQ or (S)-PZQ affinities for flatworm target(s) within both the GPCR or TRP channel families. Both 5-HT2BR (Gq coupled) and the individual TRP channel targets (hTRPA1, hTRPC3, hTRPC7, hTRPM8) elevate cytoplasmic Ca2+, and the ability of ±PZQ to dysregulate Ca2+ homeostasis in both parasitic schistosomes and free-living flatworms is well appreciated [6, 48–50]. Moreover, the activity of serotonergic GPCRs and TRP channels can be coupled through amplifying interactions–GPCR mediated Ca2+ store depletion activates TRP mediated Ca2+ entry, which can itself stimulate serotoninergic pathways [51–53]. Perhaps the unique host-parasite polypharmacology of ±PZQ to engage reinforcing parasite targets deleterious to worm viability together with host pathways that mediate beneficial responses combating infection underpins the unique clinical efficacy of ±PZQ that has proved difficult to replicate over 35 years of clinical usage.