Acta Biologica Hungarica 69(2), pp. 135–143 (2018) DOI: 10.1556/018.69.2018.2.3
EFFECTS OF THE VENOM OF THE BROWN BULLHEAD CATFISH (AMEIURUS NEBULOSUS) ON ISOLATED SMOOTH MUSCLES
LORÁND BARTHÓ,1 1 and 2
1Department of Pharmacology, Faculty of Medicine, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary 2Department of Pharmacognosy, Faculty of Pharmacy, University of Pécs, Rókus u. 2, H-7624 Pécs, Hungary
(Received: December 5, 2017; accepted: March 19, 2018)
+| [ .Ameiurus nebulosus Lesueur, 1819) caused contraction of the guinea-pig small intestine in vitro, a widely-used preparation in pharmacology. The action is dependent on extracellular Ca2+, and probably takes place on the smooth muscle cells. Mouse 9 [ causes a painful sensation in man. We tested the effect of an extract of spines in isolated organ experi- ments on innervated smooth muscle preparations. In the guinea-pig ileum, the response to the extract was abolished by the Ca2+-channel blocker nifedipine, but only slightly reduced by atropine (a muscarine receptor antagonist) or tetrodotoxin (TTX; a blocker axonal conduction) or antagonists for P2X purinocep- tors. Blocking of serotonin or histamine H1 receptors, tachykinin NK1 receptors, functional impairment 6 6 contractile effect of the extract. No inhibitory action of the extract was detected on the ileum subject to electrical motor nerve stimulation.
Keywords: [ M M
INTRODUCTION
* [ (Ameiurus nebulosus) was studied on innervated smooth muscle preparations. This [ > %W > " pannonicus has been formed and is widely distributed both in natural waters and ] [ " " [ * character of pain caused by the stings is indicative of the presence of venom, not only * [ .Ictalurus catus) has been analyzed [9]. The fraction toxic to mice has been found to be heat-resistant and not destroyed by trypsin, in spite of a high estimated molecular weight (around %!"!!!5 qWx Q [ " qJx [ .Schilbeoides
Q 6 ]
0236-5383/$ 20.00 © 2018 Akadémiai Kiadó, Budapest 136 LORÁND BARTHÓ et al. exilis and S. leptanthus, Ictalurus melas and I. punctatus5 [ - ied [5], but the tests performed were quite different from those of the current study. The aim of our experiments was to investigate the biological effects of an aqueous extract of spines of Ameiurus nebulosus (ssp. pannonicus) in isolated organ experi- ments on the isolated small intestine of the guinea-pig, a tissue containing smooth muscle and nerves and a most widely-used and thoroughly-characterized preparation in experimental pharmacology (see Table 1). Gastrointestinal preparations of the mouse were also used.
MATERIALS AND METHODS
The experiments have been approved by the Regional Committee for Animal Research, as well as by the respective committee of University of Pécs. These exper- iments were of the “ex vivo” type and are in accordance with the European Guidelines on Laboratory Animal Care. 3 6 [ * ] | nearly 1 year (from a size of a few mm) and now weighed 14 g on average. They were | [ . 6 [ plus dried Daphnia). Fish were killed by a strong blow to the head. All three spines were removed, soaked in Krebs solution (50 mg wet tissue/ml solution) for 10 min at room temperature, with intermittent vortexing. The tissue was then removed from the solu- tion (that will hereafter be called extract). Samples of the extract were pooled to make %! ] " %!! 3 ] M ! °C. An [ < 6 . 6 " 5 " #B!%B$! stunned by a blow to the occiput and bled out. Whole segments of the ileum (approx- imately 2 cm in length) were suspended in organ baths containing 7 ml of oxygen- ated (95% O2, 5% CO25 M> ?@ Q / were recorded auxotonically, using Experimetria (Budapest) force transducers and [ * [ [ to contract. The initial load was 6 mN. All experiments commenced after 40 min equilibration period. A maximum of 2 preparations from the same animal were used 9 [ [27]; cholinergic „twitch” responses elicited by single electrical pulses with 20 min intervals, delivered through a pair of ring electrodes. Parameters of the pulses were ?! z ! % * 6 pig ileal preparation is given in Table 1. Adult female C57 mice were killed by a strong blow to the head. Ileum (whole ileum segments in the longitudinal direction) and gastric fundus preparations were set up as described above, with a resting tone of 5 mN. Each stomach yielded one single fundus preparation, in which the isolated gastric fundus was opened along both the lesser and the greater curvatures. Contractions to 2 μl/ml of the extract were reproduc- . " ? JM$ 5"
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Table 1 Spectrum of detection of the guinea-pig ileum tests
Selected No. Tests Activities detected by the test references 1 Guinea-pig ileum at Ganglionic stimulants; serotonin (to a greater extent); 4, 7, 8, 11, basal tone: contrac- cholecystokinin-like drugs, tachykinin NK? receptor agonists, 16, 17, tors via nerve stimu- P2X . ] "!6 +* 5" " %WM %" " lation PACAP, CGRP, some eicosanoids, endocannabinoids B" ?
2 Guinea-pig ileum at Muscarinic receptor agonists, histamine (at H1 receptors), 1, 7, 8, 12, basal tone: contrac- serotonin (to a lesser extent), tachykinins (substance P, %$" ? tors via smooth mus- neurokinin A, through NK1 and NK2 receptors), some other cle stimulation polypeptides, mast cell activators; spasmogenic effect by [
? Guinea-pig ileum, Acetylcholine muscarine M? receptor antagonists; %!" %?" %B" contractions due to Na+ channel blocking (TTX or local anesthetic-like); 24, 26, 28, [ - N-type Ca2+-channel blocking effect; W" ?% lation of cholinergic negative modulators of intestinal cholinergic nerves (e.g., nerves 6 2 agonist-like, nociceptin-, cannabinoid-, adenosine-like) L-type Ca2+-channel blocker, K+-channel opener or any other smooth-muscle relaxant (e.g., enhancers of intracellular cAMP or cGMP levels)
#! .* ?5 - tions received TTX (0.5 μM) 20 min before the second exposure to the extract. Drugs used were apamin, atropine sulfate, capsaicin, histamine dihydrochloride, indomethacin, pyridoxalphosphate-6-azophenyl-2’,4’-disulphonic acid (PPADS), " .**¤ 9 5" +* " "!6 +* " (2S"?S)-N6. 6 5 6 6 6%6 j q x 6?6 .Q WJ?#B5" " " .%6 6#6 5 6$6 6 7-chloro-1,4-benzodioxane-5-carboxylate (SB 204 070), suramin, tropisetron, %" "?"J6 6%"?6 66q#6.%6 5 x6 "J6 6@>6 6 @6 " 6.%"?6 6 "J6 6%" "?"J6 6@>6 6@6 566 .#6 5 .>Q !?!!?%" * 5" (Egis Pharma, Hungary).
RESULTS
The extract showed contractile activity on the guinea-pig ileum (Fig. 1A). Q 6 ! B B 3 z " ?$ ? / W @# % / B ! . #5" " maximal spasm evoked by histamine (10 μM). One μl/ml of the extract was used for pharmacological analysis. Contractions were quick in onset, reached their maximum
Acta Biologica Hungarica 69, 2018 138 LORÁND BARTHÓ et al.
! B ? observation period. If an interval of 40 min was allowed to the preparations as wash- out time, the responses to 1 μl/ml extract were reproducible (Table 2). Pretreatments that partially diminished responses to exposure to the extract were the Na+ channel blocker TTX, the muscarinic acetylcholine receptor antagonist atropine, or a combi- 2+ nation of the two P2 purinoceptor antagonists PPADS and suramin. The Ca channel blocker nifedipine abolished the response (Table 2). The effect of the extract was not inhibited by the histamine H1 receptor antagonist chloropyramine, a combination of serotonin receptor antagonists, the tachykinin NK1 Q WJ?#B" cyclooxygenase inhibitor indomethacin, and capsaicin desensitization (Table 2). The 2+ TRPA1 z >Q !?!!?% " Q sensitive, small conductance K+ channel blocker apamin moderately enhanced the response to the extract (Table 2). Table 2 [ .% 3 z 5 6
Contraction Contact time Pretreatment (% of maximum) of pretreatment 1st administration: no pretreatment #W $ / @ %# 2nd administration: no pretreatment B? J / W 1st: control (solvent: saline) B? W / # 20 min %! 2nd: tetrodotoxin (0.5 μM) # J / J 1st: control (solvent: saline) #J W / B ! 20 min $ 2nd: atropine (0.5 μM) #% / B @ 1st: control (solvent: saline) B% ? / # W 20 min @ .! ? 3'5 B? ! / B # 1st: control (solvent: saline) B# @ / # $ ?! $ 2nd: PPADS (50 μM) + suramin (100 μM) #% $ / B !% 1st: control (solvents: DMSO) B % / @ $ 20 min @ 2nd: serotonin receptor antagonists# BB W / # 1st: control (solvent: alcohol) B! ? / B 20 min @ 2nd: nifedipine (1 μM) B % / % 1st: control (solvent: DMSO) B / # 45 min $ Q WJ?#B .% B 3'5 #! W / ? W 1st: control (solvent: saline) #J ? / ? ! 20 min J 2nd: apamin (0.1 μM) JB W / # @ 1st: control: (solvent: alcohol) ## % / B B 45 min @ 2ns: indomethacin (5 μM) ?$ $ / # B 1st: control (solvent: alcohol) #B % / ? % (see below) J 2nd: capsaicin desensitization## B# ! / J B 1st: control (solvent: DMSO) #@ W / # ?! $ >Q !?!!?% .?! 3'5 B# $ / % @
# ' .! ? 3'" B6>*2 " 5" .? 3'" 5-HT? " 5 9 !# !@! .? 3'" B6>*4 receptors, dissolved in DMSO). ##Capsaicin, 10 μM for 10 min, followed by 60 min washout period. ' / [ 6 (Wilcoxon test).
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Fig. 1. 3 [ . 5 on the guinea-pig ileum (A; 1 μl/ml extract), mouse ileum (B; 2 μl/ml extract) and mouse gastric fundus strip (C; 2 μl/ml extract). Calibrations, vertical: 50% of maximal contraction to histamine (10 μM, A) or .%! 3'" " Q5 j ?
Two concentrations of the extract (0.25 and 1 μl/ml) were added while the prepara- [ * cholinergic “twitch” response, with only a small and transient elevation of the base- line, while the larger one elevated the baseline without modifying the “twitch” %B . # 5 " * %" ? not activated by the extract. + [ . 3 z 5 .!MJ " % " B5 Positive contols from previous and the current experiments in the ileum for the inhibitors (in the concentrations used) were as follows. TTX and atropine practically
Acta Biologica Hungarica 69, 2018 140 LORÁND BARTHÓ et al. abolish half-maximal, cholinergic „twitch” responses to electrical stimulation; chlo- ropyramine blocks near-maximal contractions to histamine (0.1 μM), without inhibit- O P + 9 +* .?!! 3'5 "!6 +* .%! 3'5 .% 3'5 ] 6 - tions to exogenous acetylcholine or histamine (also the cholinergic „twitch”); the combination of serotonin receptor antagonists blocks near-maximal ileum contrac- .! ?M% 3'5 ] 1 Q WJ?#B .% B 3'5 ?!6 6 .! ?MJ '5 Q j ] .! ?M% 3'5 * ?M%! [ q %" ?x * " *~ +% z >Q !?!!?% 6 " . - lished observations). The extract (2 μl/ml) also contracted the mouse gastric fundus and mouse ileum preparations. The effect of TTX on these responses was similar to that seen in the 6 . %" Q * ?5 Table 3 [ . 3 z 5
Contraction Contact time Pretreatment (% of maximum) of pretreatment Mouse ileum 1st administration: no pretreatment J / B $ 2nd administration: no pretreatment B ! / # 1st: control (solvent: saline) # # / 20 min @ 2nd: tetrodotoxin (0.5 μM) %W ? / % Mouse gastric fundus 1st administration: no pretreatment #@ ! / J ? B 2nd administration: no pretreatment B! ? / @ ! 1st: control (solvent: saline) #B $ / B 20 min # 2nd: tetrodotoxin (0.5 μM) #W W / B B ' /
DISCUSSION
* . [ 5 [ caused smooth contraction on the guinea-pig small intestine; no inhibitory action has . " [ 6 5 from a pharmacological analysis as follows. The strongest (practically full) inhibition of the contractile response to the extract was found with the Ca2+ channel blocker nifedipine, which indicates an essential role of voltage-dependent, L-type Ca2+ channels in this response. For comparison, acetyl-
Acta Biologica Hungarica 69, 2018 ! 141 choline- or histamine-evoked responses of similar size are also strongly inhibited by nifedipine, which points to the involvement of voltage-dependent Ca2+ channels in the course of the Gq-protein-mediated response to these agonists. Whether or not a release of intracellular Ca2+ is also involved in the effect of the extract cannot be decided on the basis of the current study. Tetrodotoxin and atropine caused a rather slight reduction in the effect of the extract. Given that both pretreatments are able to fully inhibit the “twitch” responses (tetrodotoxin pre- and atropine postjunctionally), the modest effect of these drugs on the response to the extract might be attributed to an overall reduction of the excitabil- " [ This indicates that mechanisms, listed in Table 1, 1st row are unlikely to mediate the contraction caused by the extract. The moderate enhancement of the effect of the extract by the K+ channel inhibitor apamin [see 1] may also be a result of an overall increase of the excitability of the preparations. Q WJ?#B ] 1 receptor antagonist with an IC50 below 100 nM [14]. On the longitudinal muscle of the guinea-pig small intestine the main tachykinin receptor is the NK1 Q WJ?#B .% B 3'5 - [ the extract indicates that a substance P-like peptide is not involved in the contractile effect of the extract. Cyclo-oxygenase products seem not to be involved, as shown by ] + " +* 6 ] " ] - yramine (histamine H1 receptor antagonist), or serotonin receptor antagonists, and a modest inhibition by PPADS + suramin, respectively. The lack of inhibitory effect of the extract on the electrically-induced “twitch” contractions of the guinea-pig ileum indicates that no major involvement of smooth muscle-relaxant mediators (e.g., the cGMP enhancer nitric oxide, NO) takes place. Taken together, these data indicate that the extract acts on the ileum directly on the smooth muscle, in a Ca2+-dependent manner. Yet, a tetrodotoxin-insensitive release of . [ 5 as mechanism. Although the pharmacological sensitivity of the effect of the extract does not match the excitatory effect of the sensory stimulant drug capsaicin in this q ?x" - zation, a procedure that renders capsaicin-sensitive sensory nerve endings function- ally inactive, i.e. insensitive not only against capsaicin, but also any type of stimulus, q " ?" ?!x 6 producing effect of Ameiurus stings, no involvement of capsaicin-sensitive sensory 6 [ * " " does not exclude other ways of sensory stimulation by the venom. Further studies will [ Experiments on mouse preparations yielded rather similar results to those obtained 6 " . [ 5 the response in the presence of TTX.
Acta Biologica Hungarica 69, 2018 142 LORÁND BARTHÓ et al.
" [ Q 2+-dependent smooth muscle contraction. This effect, as well as the contractile action on mouse preparations seem not to involve Na+-dependent nerve action potentials. As far as the mechanism of this effect is concerned, the roles of a number of important mediators have been excluded. The stimulatory mechanism(s) encountered might contribute to the toxicity and pain produced by the venom.
ACKNOWLEDGEMENTS
The authors express their thanks to the Faculty of Medicine, University Medical School for providing a postdoctoral grant to Timea Bencsik and supporting this research in other ways as well.
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