Combined effects of fungal alkaloids on intestinal motility in an in vitro rat model 1,2

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1 Published November 24, 2014 Combined effects of fungal alkaloids on intestinal motility in an in vitro rat model 1,2 J. E. Dalziel,* 3 K. E. Dunstan,* and S. C. Finch *Food Nutrition and Health Team, Food & Bio-based Products Group, AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand; and Plant-fungal Interactions Team, Forage Improvement Group, AgResearch Ruakura Research Centre, Private Bag 3123, Hamilton 3240, New Zealand ABSTRACT: Diarrhea is caused by factors that alter absorption and secretion of water and ions across the intestinal epithelium and disrupt motility. Parasitic infection, stress, poor nutrition, and exposure to plant or fungal toxins predispose livestock to noninfectious diarrhea. This is more prevalent in sheep that graze pastures infected with wild-type endophytic fungus, suggesting the involvement of fungal alkaloids. These increase smooth muscle contraction: ergovaline/ ergotamine (ergot alkaloid) activates serotonin (5-HT) receptors, and lolitrem B (indole diterpene) inhibits large-conductance Ca 2+ activated K + (BK) channels. Because of their separate mechanisms of action the objective of this study was to investigate whether they act synergistically to increase smooth muscle contraction. Effects of ergotamine (1 µm) and lolitrem B (0.1 µm) on the tension and frequency of spontaneous contractions were investigated in a longitudinal preparation of isolated distal colon. The compounds were dissolved in 0.1% dimethyl sulfoxide (DMSO) and applied separately or together for 1 h. Ergotamine increased contractile tension compared to the pretreatment control (P < 0.01) and produced a short-lived increase in frequency (P < 0.001). Lolitrem B increased contractile tension (P < 0.05) but had no effect on frequency. When applied together, the contractile tension was greater than the sum of the compounds applied separately (P < 0.05). The frequency of contractions was increased (P < 0.05) but was not significantly different from that for ergotamine alone. The increased contractile tension when both compounds were applied together indicates that ergotamine and lolitrem B acted synergistically to increase smooth muscle contraction, suggesting that they would alter motility in vivo. Key words: ergot alkaloid, fungal toxin, intestinal motility, ion channel, lolitrem B, smooth muscle 2013 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas Introduction Endophytic fungi confer increased insect pest resistance to perennial ryegrass in pastures through production of beneficial metabolites. However, the naturally occurring wild-type endophyte (Neotyphodium lolii) also produces compounds that are detrimental to animal health. Lolitrem B, an indole diterpene, is the main causative agent of a tremor syndrome of grazing animals known as 1 This work was supported by a grant from the Curiosity Fund, AgResearch, New Zealand. 2 We thank Ric Broadhurst and Bobby Smith for animal breeding and Hailey Gillespie for animal care. We thank Jonathan Simpson for assistance with data analysis and Catherine Lloyd-West for statistical analysis. 3 Corresponding author: julie.dalziel@agresearch.co.nz Received March 6, Accepted August 5, ryegrass staggers (Gallagher et al., 1981). Ergovaline, an ergot alkaloid, is associated with detrimental thermal stress responses in livestock (Strickland et al., 1993). In addition to the well-described deleterious physiological impacts of wild-type endophyte on animal production, it has also been observed that gastrointestinal tract associated problems are more common than for animals on nil-endophyte pastures. A field report noted that lambs grazing wild-type endophyte pastures had a 2- to 4-fold greater incidence of dags (dried feces adhered to hind quarter wool) compared with animals on nil-endophyte pastures (Fletcher et al., 1999). The association between endophyte toxins and dags is not understood. Both lolitrem B and ergovaline reach the intestine because they appear in the feces after ingestion in sheep (De Lorme et al., 2007; Cripps and Edwards, 2013). Previous studies have shown that ergot alkaloids

2 5178 (McLeay and Smith, 2006; Poole et al., 2009) and indole diterpenes (Smith et al., 1997; McLeay et al., 1999; McLeay and Smith, 2006) affect smooth muscle contraction in sheep. Indole diterpenes, including lolitrem B, increase intestinal motility (DeFarias et al., 1996; Wang et al., 2003) and inhibit K + channels (BK; Dalziel et al., 2005). The BK channel is highly expressed in smooth muscle (Knaus et al., 1995), including the colon (Sausbier et al., 2006), and are important for motility in the rat distal colon (France et al., 2012). The ability of ergot alkaloids to increase smooth muscle contraction via serotonin receptors is well described in the digestive tract (Franhuijzen, 1975; Radulovic et al., 1984) and in blood vessels (Muller Schweinitzer, 1983; MacLennan and Martin, 1990; Dyer, 1993; Klotz et al., 2009). The fact that lolitrem B and ergovaline are found together in wild-type endophyte and both increase smooth muscle contraction in vitro through separate mechanisms led us to investigate whether they might act synergistically to increase smooth muscle contraction. We used a rat model of colon function because it produces similar responses to those in sheep tissue for these compounds (Franhuijzen, 1975; Wang et al., 2003; Poole et al., 2009) and because these are naive animals. Materials and Methods This research was approved by the AgResearch Grasslands Animal Ethics Committee, established under the Animal Protection (Code of Ethical Conduct) Regulations Act, 1987 (New Zealand). Animals were killed by CO 2 inhalation. Three to four animals were used per experiment with duplicate preparations per animal. Methods were adapted from those described elsewhere for rat distal colon (Brighton et al., 2008; Gursoy et al., 2008; Jeong et al., 2009). Isolation of Rat Colon Male Sprague Dawley rats (7 12 wk of age) obtained from AgResearch Ruakura (Hamilton, NZ) were housed under a 12-h light/dark cycle and were fed Sharpes Diet 86 (Sharpes Stockfeeds Ltd., Carterton, New Zealand). After euthanasia, a 2-cm piece of distal colon was removed 1 cm distal to the striations of the mid colon. The tissue was placed in Kreb s buffer (118 mm NaCl, 4.7 mm KCl, 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 2.6 mm CaCl 2, 25 mm NaHCO 3, 11 mm glucose, ph 7.4) oxygenated with 95% O 2 /5% CO 2. The preparation was divided in half, and two distal colon pieces were mounted longitudinally on holders in an isolated tissue bath system (SI-MB4, SI-Heidelberg, World Precision Instruments, Sarasota, FL) at 37 C. Dalziel et al. Muscle Contractile Measurements Tissues were suspended under 1 g of tension and equilibrated for 1 h in Krebs buffer during which time the bath solution was exchanged every 15 min. Control spontaneous muscle contractions were measured during the last 15 min of a 1-h exposure to 0.1% dimethyl sulfoxide (DMSO) and compared with the response to the test compounds applied separately or in combination over 1 h. Ergotamine tartrate (1 µm) and lolitrem B (0.1 µm) were dissolved in DMSO and applied directly to the baths in a 1/10 dilution and reapplied every 15 min with the new bath solution (final DMSO concentration in bath of 0.1%). Muscle contraction data were measured using BAM21-E amplifiers integrated using Lab-Trax 4/24T hardware and acquired and analyzed using Labscribe 2 software (iworx Inc., Dover, NH). Data from the last 10 min of a 15-min recording period were analyzed, and the mean for each parameter was determined from the contractile responses recorded. Contractile tension or amplitude (g) was measured from the baseline to the maximum peak of the contractile response. Contractile frequency was measured as the number of contractions counted per min. The maximum tension response was compared to that for 1 µm acetylcholine (Sigma, St. Louis, MO), and preparations that gave little or no response were excluded. The response to tetrodotoxin, a neuronal voltage-gated sodium channel inhibitor (Alomone Labs, Jerusalem, Israel), was also assessed in spontaneously contracting preparations. Toxins Lolitrem B was extracted from ryegrass seed infected with Neotyphodium lolii and purified by chemical methods as previously described (Miles et al., 1994). A concentration of 0.1 µm lolitrem B was used because this concentration had the greatest effect in a concentrationresponse experiment (Suppl. Fig. 1; see online version of the article at and maximally inhibits BK ion channels in vitro (Dalziel et al., 2005; Imlach et al., 2008). Because of difficulty in obtaining sufficient quantities of ergovaline, the structurally related compound ergotamine was used instead. Ergotamine tartrate (Sigma Chemical Company, St Louis, MO) was used at a concentration of 1 µm ergotamine because this concentration had the greatest effect in a concentration-response experiment (Suppl. Fig. 1) and has shown effects in vitro in previous studies (Franhuijzen, 1975; Poole et al., 2009). Ergotamine has time-dependent and pre-exposure effects that are well described (Klotz et al., 2009). The effects of both compounds are not reversible in the time frame of an

3 Fungal alkaloids alter intestinal motility 5179 in vitro experiment (Dalziel et al., 2005; Poole et al., 2009); therefore, each tissue preparation was used for only one treatment. Statistical Analysis. Results are expressed as the mean ± SEM from 5 preparations (from 3 animals). Statistical comparisons were made using GenStat version 12.2 (VSN International Ltd., Hemel Hempstead, UK). Analysis of variance was used on repeated measurements to compare treatment effects over time. To compare between treatments, time was treated as a covariate. Results Spontaneous contractions were measured in the rat distal colon in vitro, and effects of lolitrem B and ergotamine compared, alone or in combination, on contractile tension and frequency. To determine the origin of the spontaneous contractions recorded from the 1 cm segments of isolated rat distal colon, 1 µm tetrodotoxin was applied to inhibit the neuronal voltagegated sodium channels. Tetrodotoxin did not inhibit the spontaneous phasic contractions (n = 3), indicating that they were not initiated by enteric neurons. Spontaneous contractile activity recorded from the isolated distal colon before and after addition of each compound is shown in Figs. 1a, 1b, and 1c. The effect of each treatment on spontaneous muscle contractions was measured relative to that in the 60 min control recording in Krebs with 0.1% DMSO (Jeong et al., 2009). Data comparing the effect of each treatment on contractile tension and frequency are shown in Figs. 2a and 2b. To account for variation in contractile activity among preparations, pretreatment controls were standardized to 1.0, and the effect of a treatment was determined as a difference from the control. Lolitrem B increased the contractile tension after 60 min of exposure (Fig. 2a) but had no effect on frequency (Fig. 2b). Ergotamine increased peak contractile tension (Fig. 2a) at all time points over the 60 min exposure period. The frequency of contractions was also increased over this time, but the effect was greatest in the first 15 min (Fig. 2b). Coapplication of lolitrem B and ergotamine greatly increased the contractile tension over 60 min of exposure (Fig. 2a). The magnitude of the increase in peak tension for the compounds in combination (1.35 g) was greater than the sum of the effect for each compound alone (0.90 g). The frequency of contractions was also significantly increased 15 min after application of both compounds but decreased on continued exposure and was not significantly different from the control at 60 min (Fig. 2b). All three treatment conditions were also able to induce contractions in quiescent preparations. Discussion The data indicate that the main effect of lolitrem B on the rat distal colon was to increase muscle tension, which occurred slowly over 1 h. This is the first report of lolitrem B affecting muscle contraction in the rat distal colon. This finding is consistent with that observed in the isolated sheep reticulorumen, where the same concentration of lolitrem B increased electrically stimulated contractions with 12 min of latency and the effect recorded over min thereafter (Wang et al., 2003). This supports the rat distal colon as a suitable small animal model for sheep. In contrast to lolitrem B, ergotamine induced a rapid increase in contractile tension and frequency of contraction. This is the first time an effect of ergotamine on frequency of contraction has been reported. Previous studies have used electrical stimulation protocols to induce the muscle to contract at a given frequency rather than measuring changes in spontaneous contractions. Ergotamine (and ergovaline) does, however, initiate spontaneous contractions in previously quiescent isolated sheep reticulum preparations (Poole et al., 2009), as we also observed for the rat colon. An effect on frequency as well as tension is relevant in the context of the endophyte alkaloids because it shows differences between the two classes of compound. This result is consistent with effects of serotonin agonist drugs that increase gastrointestinal motility via specific serotonin (5HT) receptor subtypes (5HT 2B, 5HT 2C, 5HT 3, and 5HT 4 ) in humans. When both toxins are applied together, the large increase in contractile tension compared with the sum of either applied alone was the most marked effect. This indicates that when the distal colon is exposed to both lolitrem B and ergotamine, the two compounds acted synergistically to increase contractile tension. It can be speculated that the mechanisms underlying this effect might involve calcium because ergotamine activation of serotonin receptors would be expected to increase intracellular calcium levels (via GPCR and IP 3 pathways or calcium-permeable 5-HT 3 ion channels) and BK channels are known to be activated by calcium released from IP 3 receptor operated stores in murine colonic muscle (Bayguinov et al., 2000). Ergovaline/ ergotamine (ergot alkaloid) activates serotonin (5- HT) receptors, leading to an increase in intracellular calcium, and lolitrem B (indole diterpene) inhibits large-conductance Ca 2+ activated K + channels that are activated by intracellular calcium and depolarizing membrane voltages. Although the mechanism of action of this effect is different for the ergot alkaloids and lolitrem B, both mechanisms involve calcium.

4 5180 Dalziel et al. Figure 1. Effect of endophyte compounds on spontaneous muscle contraction in the isolated rat distal colon. Examples of raw data recordings show changes in muscle tension over time for the control (0.1% dimethyl sulfoxide [DMSO]) and after addition of (a) 0.1 µm lolitrem B (LB), (b) 1 µm ergotamine (Erg), or (c) 0.1 µm lolitrem B and 1 µm ergotamine (LB + Erg). The BK ion channels are considered to have an important role in smooth muscle function in that they provide a safety mechanism to dampen excitation during times of high muscle or nerve activity. When activated by internal calcium and positive membrane potentials, BK channels allow K + ions to flow out of the cell, keeping the membrane potential closer to the resting potential. The BK channels appear to regulate the membrane potential in the mouse distal colon (France et al., 2012). When both ergotamine and lolitrem B are applied to colonic smooth muscle together, the anticipated increase in calcium mobilization due to serotonin receptor activation caused by ergotamine would be expected to increase muscle activity. Inhibition by lolitrem B resulting in removal of the safety mechanism in regulating the membrane potential that BK channels normally provide would make it more likely that action potential firing will occur, leading to a further increase in muscle contraction. The increased intestinal motility observed in response to a combination of endophyte alkaloids in this rat model suggests that in vivo gastrointestinal motility would be disrupted after dietary exposure to lolitrem B and ergovaline at concentrations in the range that may be ingested from wild-type endophyte-infected perennial ryegrass (De Lorme et al., 2007; Reed et al., 2011; Cripps and Edwards, 2013). This effect is consistent with the

5 Fungal alkaloids alter intestinal motility 5181 It is interesting to note that physiological effects of ergot alkaloids (serotonin agonists) on animals described by tall fescue toxicosis syndrome share symptoms in common with the clinical condition of serotonin syndrome in humans, including hyperthermia, hypertension, and hyperactive bowel (diarrhea; Boyer and Shannon, 2005). Serotonin syndrome occurs when serotonin receptors are overstimulated by agonists or excess serotonin release, as a consequence of taking more than one serotonergic medication (Birmes et al., 2003; Boyer and Shannon, 2005). It is possible that ingestion of other compounds that affect serotonin receptors through endophyte, other fungi, or plants might exacerbate the effects of ergotamine and lolitrem B to further increase intestinal motility. The findings of this study indicate that ergot alkaloids and indole diterpenes can act in a synergistic manner to increase smooth muscle contractility in mammalian intestinal tissue. This suggests that increased intestinal motility may occur in animals that ingest wild-type endophyte-infected grass containing a mixture of these compounds and other structurally related alkaloids and could contribute to the diarrhea observed in animals grazing these pastures. Figure 2. Summary of endophyte compound effects on contractile parameters of spontaneous muscle contraction in the isolated rat distal colon. Changes in (a) tension (g) and (b) frequency (number of contractions per min) were averaged over the preceding 10 min for each time point. The effect of treatments is shown as a difference relative to the control (0.1% dimethyl sulfoxide [DMSO]). Treatments were applied for 1 h each for either 0.1 µm lolitrem B (circles; n = 5), 1 µm ergotamine (squares; n = 7), or both (triangles; n = 6). Significance was tested using repeated measures analysis of variance to compare treatment effects over time. To compare between treatments, time was treated as a covariate. Asterisks indicate the significance of each treatment relative to controls or between treatments as indicated (*P < 0.05; **P < 0.01, ***P < 0.001). Data show mean ± SEM. intestinal dysfunction that occurs in diarrhea/scouring and subsequent dags observed in sheep grazing wildtype endophyte-infected pastures (Fletcher et al., 1999). Additional structurally related ergopeptide and indole diterpene compounds produced by endophytes, which activate 5-HT receptors and inhibit BK channels (Imlach et al., 2009), respectively, are likely to contribute to increased motility. It remains to be determined whether lolitrem B and ergotamine affect ion secretion in the colon to produce the increased fecal moisture that occurs in dags and subsequent fly-strike (fly larvae in wool of hindquarters). Furthermore, the urinary incontinence that occurs in mice lacking BK ion channels (Meredith et al., 2004) suggests that inhibition of BK channels by lolitrem B might produce a similar effect in sheep, exacerbating the probability of fly-strike. Literature Cited Bayguinov, O., B. Hagen, A. D. Bonev, M. T. Nelson, and K. M. Sanders Intracellular calcium events activated by ATP in murine colonic myocytes. Am. J. Physiol. Cell Physiol. 279:C126 C135. Birmes, P., D. Coppin, L. Schmitt, and D. Lauque Serotonin syndrome: A brief review. Can. Med. Assoc. J. 168: Boyer, E. W., and M. Shannon Current concepts: The serotonin syndrome. N. Engl. J. Med. 352: Brighton, P. J., A. Wise, N. B. Dass, and G. B. Willars Paradoxical behavior of neuromedin U in isolated smooth muscle cells and intact tissue. J. Pharmacol. Exp. Ther. 325: Cripps, M. G., and G. R. Edwards Fungal endophytes of a forage grass reduce faecal degradation rates. Basic Appl. Ecol. 14: Dalziel, J. E., S. C. Finch, and J. Dunlop The fungal neurotoxin lolitrem B inhibits the function of human large conductance calcium-activated potassium channels. Toxicol. Lett. 155: DeFarias, F. P., M. F. Carvalho, S. H. Lee, G. J. Kaczorowski, and G. Suarez-Kurtz Effects of the K + channel blockers paspalitrem-c and paxilline on mammalian smooth muscle. Eur. J. Pharmacol. 314: De Lorme, M. J. M., S. L. Lodge-Ivey, and A. M. Craig Physiological and digestive effects of Neotyphodium coenophialuminfected tall fescue fed to lambs. J. Anim. Sci. 85: Dyer, D. C Evidence that ergovaline acts on serotonin receptors. Life Sci. 53:PL223 PL228. Fletcher, L. R., B. L. Sutherland, and C. G. Fletcher The impact of endophyte on the health and productivity of sheep grazing ryegrass-based pastures. Grasslands Res. Pract. Ser. 7: France, M., Y. Bhattarai, J. J. Galligan, and H. Xu Impaired propulsive motility in the distal but not proximal colon of BK channel β1-subunit knockout mice. Neurogastroenterol. Motil. 24:e450 e459.

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