Cholinergic receptors on intestine cells of Ascaris suum and activation of nAChRs by levamisole

Abstract

Cholinergic agonists, like levamisole, are a major class of anthelmintic drugs that are known to act selectively on nicotinic acetylcholine receptors (nAChRs) on the somatic muscle and nerves of nematode parasites to produce their contraction and spastic paralysis. Previous studies have suggested that in addition to the nAChRs found on muscle and nerves, there are nAChRs on non-excitable tissues of nematode parasites. We looked for evidence of nAChRs expression in the cells of the intestine of the large pig nematode, Ascaris suum, using RT-PCR and RNAscope in situ hybridization and detected mRNA of nAChR subunits in the cells. These subunits include components of the putative levamisole receptor in A. suum muscle: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8. Relative expression of these mRNAs in A. suum intestine was quantified by qPCR. We also looked for and found expression of G protein-linked acetylcholine receptors (Asu-gar-1). We used Fluo-3 AM to detect intracellular calcium changes in response to receptor activation by acetylcholine (as a non-selective agonist) and levamisole (as an L-type nAChR agonist) to look for evidence of functioning nAChRs in the intestine. We found that both acetylcholine and levamisole elicited increases in intracellular calcium but their signal profiles in isolated intestinal tissues were different, suggesting activation of different receptor sets. The levamisole responses were blocked by mecamylamine, a nicotinic receptor antagonist in A. suum, indicating the activation of intestinal nAChRs rather than G protein-linked acetylcholine receptors (GARs) by levamisole. The detection of nAChRs in cells of the intestine, in addition to those on muscles and nerves, reveals another site of action of the cholinergic anthelmintics and a site that may contribute to the synergistic interactions of cholinergic anthelmintics with other anthelmintics that affect the intestine (Cry5B).

Keywords: Calcium signaling; Intestine; Levamisole; Nicotinic acetylcholine receptors.

Graphical abstract

Fig. 1

Hematoxylin and Eosin stained transverse section through Ascaris suum showing various regions: the cuticle (C); hypodermis (H); lateral lines (L); contractile spindles (S); arms (A) dorsal nerve cord (DN); ventral nerve cord (VN); muscle bags (B); Canals containing perienteric fluid (Cn) and intestine (I).

Fig. 2

Localization of nAChR subunits in the muscle bag region and intestine of A. suum. RT-PCR analysis of muscle bag (1b, 2b, 3b, 4b, 5b) and intestine (1i, 2i, 3i, 4i, 5i) of five separate adult female A. suum worms. Each lane represents an individual worm that muscle bags and intestinal tissue was taken for analysis. M = FastRuler Middle Range DNA Ladder (Thermo Fisher Scientific) and n.c = negative control. (A) Asu-unc-29 (B) Asu-unc-63 (C) Asu-unc-38 (D) Asu-acr-8. Note the reduced intensity of the bands (encircled white) with Asu-unc-29 (A) from the intestine.

Fig. 3

Differential expression of nAChR subunits in muscle and intestine of Ascaris suum. Bar charts (expressed as mean ± SEM) demonstrating qPCR experiments of the relative mRNA levels of Asu-unc-38 (black bar), Asu-acr-8 (blue bar), Asu-unc-29 (red bar) and Asu-unc-63 (green bar) in: (A) muscle bag region (n = 3 individual worms) and (B) intestinal tissue (n = 3 individual worms) of A. suum. qPCR experiments were repeated three times for each gene: 3 biological replicates each with 3 technical replicates. For muscle bags, analysis revealed that Asu-unc-38 (32.83 ± 0.62) had the highest relative mRNA levels than Asu-acr-8 (1.23 ± 0.41), Asu-unc-29 (1.63 ± 0.22, n = 3) and Asu-unc-63 (5.77 ± 1.19 ); *** P < 0.001. Asu-unc-63 had higher mRNA levels of expression than Asu-acr-8 and Asu-unc-29; * P < 0.05. The means for the mRNA expression of the intestinal were lower but showed similar trends with Asu-unc-38 (4.97 ± 1.49) being the highest, followed by Asu-acr-8 (3.37 ± 1.34), Asu-unc-29 (0.17 ± 0.17) and Asu-unc-63 (2.03 ± 1.53) but the differences did not reach statistical significance (P = 0.1250; n=3 for all subunit genes). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Subcellular localization of nAChR subunits by RNAscope in situ hybridization in A. suum intestine. Representative images of A. suum intestine. (A) Transverse section of the intestine showing the Basolateral, Nuclear, Central and Apical regions that were observed for nAChR subunit expression. (B) Negative control probe (Bacillus DapB) where there are no pink punctate dots. (C) Asu-unc-29 probe (D) Asu-unc-63 probe (E) Asu-unc-38 probe (F) Asu-acr-8 probe. Pink punctate dots (mRNA transcript) indicate positive signal. Both Asu-unc-29 and Asu-unc-63 (C and D) have pink punctate dots within the basolateral region only, while Asu-unc-38 and Asu-acr-8 (E and F) have higher abundance of punctate dots distributed throughout all regions of the intestine, specifically the central region. Arrows indicate individual mRNA transcripts (n=3 worms). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Quantitative subcellular distribution of nAChR subunit mRNA transcripts in A. suum intestine. Bar charts (mean ± SEM) showing the mean density of mRNA transcripts (punctate dots)/μm² for Asu-unc-38, Asu-acr-8, Asu-unc-29, and Asu-unc-63 in various regions of the intestine. A) Mean density of mRNA transcripts for each nAChR subunit in all intestinal tissue visible in the cross section. * Significantly different to Asu-unc-38 (P < 0.05). B) Mean density of mRNA transcripts for each nAChR subunit in basolateral region. * Significantly different to Asu-unc-38 (P < 0.05); **Significantly different to Asu-unc-38 (P < 0.01) and ***Significantly different to Asu-unc-38 (P < 0.001). C) Mean density of mRNA transcripts for each nAChR subunits in the Central region of A. suum intestine. *** Significantly different to Asu-unc-38 or Asu-acr-8 (P < 0.001). Asu-unc-38 (n = 36 image frames from 3 worms); Asu-acr-8 (n = 34 image frames from 3 worms); Asu-unc-29 (n = 39 image frames from 3 worms) and Asu-unc-63 (n = 39 image frames from 3 worms).

Fig. 6

Acetylcholine and levamisole generate Ca²⁺ signals in Fluo-3AM treated Ascaris intestines. A) Micrographs of Ascaris suum intestinal section under white light (left), untreated fluorescing under blue light (center) and 5 μM Fluo-3AM treated under blue light (right), after 60-min incubation at 35 °C with 10% Pluronic F-127. Key structures, tower/column and cells are highlighted. B) Representative trace of a 10 mM CaCl₂ stimulated signal. Grey box indicates application of the stimulus. C) Amplitudes of Ca²⁺ signals in intestines exposed to APF containing 500 μM CaCl₂ (untreated) and 10 mM CaCl₂ (Black bar). **** Significantly different from 500 μM CaCl₂ APF (P < 0.0001, t = 18.67, df = 925, unpaired t-test). 500 μM CaCl₂ APF n = 238 recordings from 8 intestines from 8 individuals; 10 mM CaCl₂n = 689 recordings from 12 intestine preparations from 6 individual females. D) Representative trace of a Ca²⁺ signal to 30 μM acetylcholine. Grey box indicates acetylcholine application. E) Representative trace of a Ca²⁺ signal to 30 μM levamisole. Grey box indicates levamisole application. F) Quantification of the time for the Ca²⁺ signal to reach peak after stimulus application for 10 mM CaCl₂ (Black bar), 30 μM acetylcholine (White bar) and 30 μM levamisole (Grey bar). **** Significantly different to CaCl₂ (CaCl₂ vs acetylcholine P < 0.0001, t = 33.62, df = 986; CaCl₂ vs levamisole P < 0.0001, t = 16.15, df = 916, unpaired t-tests). 10 mM CaCl₂n = 689 recordings from 12 intestinal preparations from 6 individual females; acetylcholine n = 280 recordings 7 intestine from 4 individual worms; levamisole n = 241 recordings 5 intestinal preparations from 4 females. G) Amplitudes of Ca²⁺ signals in intestines in response to 30 μM acetylcholine (White bar) and 30 μM levamisole (Grey bar). **** Significantly different to acetylcholine (P < 0.0001, t = 7.182, df = 519 unpaired t-test). H) Average percentage of recording regions of the intestine with positive increases in Fluo-3 fluorescence to 10 mM CaCl₂ (Black bar), 30 μM acetylcholine (White bar) and 30 μM levamisole (Grey bar). N.S. = not significant (acetylcholine vs levamisole P = 0.6037, t = 0.5359, df = 10, unpaired t-test). All values are represented as means ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Intestine Asu-gar-1 message and mecamylamine inhibition of levamisole mediated Ca²⁺ signals. A) RT-PCR analysis shows the presence of Asu-gar-1 in the intestine (1i, 2i, 3i, 4i, 5i) of five separate adult female A. suum. M = FastRuler Middle Range DNA Ladder (Thermo Fisher Scientific) and n.c = negative control. B) Representative trace to 10 μM mecamylamine. Grey box indicates application of the stimulus. C) Representative trace for simultaneous application of 30 μM levamisole (grey box) and 10 μM mecamylamine (black bar). D) Quantification of maximum changes in Ca²⁺ signal amplitude for untreated (Black bar), 10 μM mecamylamine (White bar) and 30 μM levamisole + 10 μM mecamylamine (Grey bar). N.S. = not significant to untreated (untreated vs mecamylamine P = 0.2278, t = 1.207, df = 578; untreated vs levamisole + mecamylamine P = 0.2365, t = 1.185, df = 506 unpaired t-test). Untreated n = 280 recordings from 10 intestinal preparations from 10 individual females; mecamylamine n = 300 recordings from 5 intestine taken from 3 individual worms; levamisole + mecamylamine n = 230 recordings from 4 intestinal preparations from 4 females. E) Representative trace of the response to 30 μM levamisole before co-application of 10 μM mecamylamine. Grey box indicates levamisole application; black represents mecamylamine. Note that mecamylamine inhibits the response to levamisole and produces a reduction in the Ca²⁺ fluorescence below the resting level due to a delayed uptake process that follows the rise in intracellular Ca²⁺. All values are represented as means ± SEM.