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The complex ergot alkaloids, ergovaline and ergotamine, cause dysregulation of physiological functions, characterised by vasoconstriction as well as thermoregulatory and cardiovascular effects in grazing livestock. To assess the effect of the mycotoxins, blood pressure and heart rate of male mice were measured, and metabolite profiling undertaken to determine relative abundances of both ergotamine and its metabolic products in body and brain tissue. Ergotamine showed similar cardiovascular effects to ergovaline, causing elevations in blood pressure and reduced heart rate. Bradycardia was preserved at low-levels of ergovaline despite no changes in blood pressure. Ergotamine was identified in kidney, liver and brainstem but not in other regions of the brain, which indicates region-specific effects of the toxin. The structural configuration of two biotransformation products of ergotamine were determined and identified in the liver and kidney, but not the brain. Thus, the dysregulation in respiratory, thermoregulatory, cardiac and vasomotor function, evoked by ergot alkaloids in animals observed in various studies, could be partially explained by dysfunction in the autonomic nervous system, located in the brainstem.

Drugs active at G protein–coupled receptors (GPCRs) can differentially modulate either canonical or noncanonical signaling pathways via a phenomenon known as functional selectivity or biased signaling. We report biochemical studies showing that the hallucinogen lysergic acid diethylamide, its precursor ergotamine (ERG), and related ergolines display strong functional selectivity for β-arrestin signaling at the 5-HT 2B 5-hydroxytryptamine (5-HT) receptor, whereas they are relatively unbiased at the 5-HT 1B receptor. To investigate the structural basis for biased signaling, we determined the crystal structure of the human 5-HT 2B receptor bound to ERG and compared it with the 5-HT 1B /ERG structure. Given the relatively poor understanding of GPCR structure and function to date, insight into different GPCR signaling pathways is important to better understand both adverse and favorable therapeutic activities.

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT 1B G protein–coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT 2B receptor, the 5-HT 1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

G-protein-coupled receptors (GPCRs)—the largest family of cell-surface membrane proteins—mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT 1B and AT 2 R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.

Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT1B and 5-HT2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and β-arrestin signaling at the 5-HT1B receptor but clearly favoring β-arrestin over G protein coupling at the 5-HT2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT1B receptor, helix 6 movement in the 5-HT2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity.

Ergot and pyrrolizidine alkaloids, either extracted from endophyte-infected tall fescue, synthesized, or purchased commercially, were evaluated in cultured cells to estimate their binding to the D2 dopamine receptor and subsequent effects on cyclic AMP production in GH4ZR7 cells, transfected with a rat D2 dopamine receptor. Ergopeptide alkaloid (alpha-ergocryptine, bromocryptine, ergotamine tartrate, and ergovaline) inhibition of the binding of the D2-specific radioligand, [3H]YM-09151-2, exhibited inhibition constants (K(I)) in the nanomolar range, whereas dopamine was less potent (micromolar). The lysergic acid amides (ergine and ergonovine) were 1/100th as potent as the ergopeptide alkaloids. Ergovaline and ergotamine tartrate were equally effective in inhibiting vasoactive intestinal peptide (VIP )-stimulated cyclic AMP production, with consistent nanomolar effective concentration (EC50) values. The remaining ergopeptide alkaloids (alpha-ergocryptine and bromocryptine), lysergic acid amides (ergonovine and ergine), and dopamine were 1/100th as potent. Two representative pyrrolizidines, N-formylloline and N-acetylloline, exhibited no binding activity at the D2 dopamine receptor or effects on the cyclic AMP system within the concentration ranges of nanomolar to millimolar. Our results indicate that the commercially available ergot alkaloids ergotamine tartrate and ergonovine may be used interchangeably in the D2 dopamine receptor system to simulate the effects of extracted ergovaline and ergine and to examine responses in receptor binding and the inhibition of cyclic AMP.

The objectives of this study were to evaluate degradation of ergovaline in a tall fescue [ (Schreb.) Darbysh.] seed extract by rumen microbiota ex vivo and to identify specific bacteria capable of ergovaline degradation in vitro. Rumen cell suspensions were prepared by harvesting rumen fluid from fistulated wether goats ( = 3), straining, and differential centrifugation. Suspensions were dispensed into anaerobic tubes with added Trypticase with or without extract (∼10 μg kg ergovaline). Suspensions were incubated for 48 h at 39°C. Samples were collected at 0, 24, and 48 h for ergovaline analysis and enumeration of hyper-ammonia producing (HAB) and tryptophan-utilizing bacteria. Ergovaline values were analyzed by repeated measures using the mixed procedure of SAS. Enumeration data were log transformed for statistical analysis. When suspensions were incubated with extract, 11 to 15% of ergovaline disappearance was observed over 48 h ( = 0.02). After 24 h, suspensions with added extract had 10-fold less HAB than controls ( = 0.04), but treatments were similar by 48 h ( = 1.00). However, after 24 h and 48 h, suspensions with extract had 10-fold more tryptophan-utilizing bacteria ( < 0.01) that were later isolated and identified by their 16S RNA gene sequence as . The isolates and other known rumen pure cultures ( JB1, B159, HD4, B, F, MD1, SR) were evaluated for the ability to degrade ergovaline in vitro. Pure culture cell suspensions were incubated as described above and samples were taken at 0 and 48 h for ergovaline analysis. Data were analyzed using the ANOVA procedure of SAS. All HAB, including the isolates, tested degraded ergovaline (54 to 75%; < 0.05). B14 was also able to degrade ergovaline but to a lesser capacity (12%; < 0.05), but all other bacteria tested did not degrade ergovaline. The results of this study indicate which rumen bacteria may play an important role in ergovaline degradation and that microbiological strategies for controlling their activity could have ramifications for fescue toxicosis and other forms of ergotism in ruminants.

Ergot alkaloids from endophyte-infected (Epichloë coenophiala) tall fescue (Lolium arundinaceum) induce vasoconstriction. Previous work has shown that serotonin receptor subtype, 5HT2A, is present in bovine ruminal (R) and mesenteric (M) vasculature, plays a role in vasoconstriction, and could be influenced by ergot alkaloids. To determine the influence of ergot alkaloids on 5HT2A, the vasoactivity of an agonist selective for 5HT2A, (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine HCl (TCB-2), was evaluated using bovine ruminal and mesenteric arteries and veins (RA, RV, MA, MV) that were exposed to ergovaline (ERV) prior to or during the TCB-2 additions. Ruminal and mesenteric blood vessel segments were collected, cleaned, and cut into 2- to 3-mm cross-sections. Vessel segments were incubated in Krebs-Henseleit buffer containing 0, 0.01 or 1 µM ERV for 2 h prior to TCB-2 dose response or exposed to ERV concentrations simultaneously during TCB-2 dose response. For the dose response portion of the study, vessels were suspended in a multimyograph containing 5 mL of continuously oxygenated Krebs-Henseleit buffer and equilibrated to 1 g tension for 90 min. Vessels were exposed to increasing concentrations of TCB-2 every 15 min and contractile response data were normalized as a percentage of the maximum contractile response induced by 120 mM KCl reference. Analysis of variance was evaluated separately for each vessel and each ERV exposure experiment using the mixed models procedure of SAS for effects of TCB-2 and ERV concentrations. All blood vessels with previous ERV exposure had significantly lower contractile responses to TCB-2 (P < 0.01). All blood vessels with simultaneous exposure to 1 µM ERV had higher (P < 0.01) contractile responses at lower concentrations of TCB-2. Simultaneous ERV addition at 1 × 10-4 M TCB-2 did not affect contractility of RV, MA, MV (P > 0.05), but decreased contractility of RA (P < 0.01). These results indicate that ergopeptine alkaloid exposure influences contractility of bovine ruminal and mesenteric blood vessels through serotonin receptor subtype 5HT2A by acting as both an agonist and antagonist. Additional work is needed to determine if ergot alkaloids like ERV simply occupy receptor binding sites competitively, or influence receptor internalization to cause the observed divergent responses.

Ergovaline (EV) produced by symbiotic association of Epichloë coenophiala with tall fescue (Lolium arundinaceum) causes toxicoses in livestock. In this study, 16 lactating ewes (BW 76.0 ± 0.6 kg) were used to determine the effects of feeding endophyte-infected (FE+) or endophyte free (FE−) tall fescue hay on animal health and performances and to investigate the putative mechanisms of action of EV. The mean EV concentrations in FE+ and FE− diets were 497 ± 52 and <5 µg/kg DM, respectively. Decreased hay consumption and BW were observed in the FE+ group. Prolactin (PRL) concentrations decreased (P < 0.02) in the FE+ group from d 3 to 28 of the study compared to the FE− group, but no consequences were observed on milk quantity or quality. Skin temperature and the thermocirculation index were lower (P < 0.05) in the FE+ than in the FE− group from d 3 to 7, but this effect disappeared from d 14 to 28. Hematocrit, mineral and biochemical, and enzymatic analyses of plasma revealed no differences between the 2 groups. Measurement of oxidative damage and antioxidant enzyme activities revealed a decrease in the activities of plasma catalase (P < 0.05), kidney glutathione reductase and peroxidase and in kidney total glutathione and malo-ndialdehyde contents (P < 0.02) in ewes fed FE+. Hepatic flavin monooxygenase enzyme activities decreased (P < 0.01) in ewes fed FE+, except for a marked increase in the demethylation of erythromycin. This activity is linked to cytochrome P4503A content and is known to be involved in ergot alkaloid metabolism. Glutathione S-transferase activity in the kidneys decreased (P < 0.02) in the FE+ group, whereas no difference was observed in uridine diphosphate-glucuronosyltransferase activity in the liver or kidneys. The reversibility of the effect of FE+ hay on skin temperature and the increase in erythromy-cin N-demethylase activity may contribute to the relative resistance of ewes to EV toxicity.

Ergot alkaloids have been associated with vasoconstriction in grazing livestock affected by the fescue toxicosis syndrome. Previous in vitro investigations studying how ergot alkaloids caused vasoconstriction have shown that ergovaline has a distinct receptor affinity and sustained contractile response. A similar contractile response has not been noted for lysergic acid. The objectives of this study were to determine if repetitive in vitro exposure of bovine lateral saphenous vein to lysergic acid or ergovaline would result in an increasing contractile response and if a measurable bioaccumulation of the alkaloids in the vascular tissue occurs over time. Segments of vein were surgically biopsied from healthy, Angus x Brangus cross-bred, fescue-naïve yearling heifers (n = 16) or collected from healthy mixed breed and sex cattle immediately after slaughter (n = 12) at a local abattoir. Veins were trimmed of excess fat and connective tissue, sliced into cross-sections, and suspended in a myograph chamber containing 5 mL of oxygenated Krebs-Henseleit buffer (95% O₂/5% CO₂; pH = 7.4; 37°C). Contractile responses to repetitive additions of ergovaline (1 x 10⁻⁹ and 1 x 10⁻⁷ M) and lysergic acid (1 x 10⁻⁵ and 1 x 10⁻⁴ M) were evaluated using the biopsied veins. For the bioaccumulation experiments, veins collected at the abattoir underwent repetitive additions of 1 x 10⁻⁷ M ergovaline and 1 x 10⁻⁵ M lysergic acid and the segments were removed after every 2 additions and media rinses for alkaloid quantification via HPLC/mass spectrometry. Contractile data were normalized as a percentage of contractile response induced by a reference dose of norepinephrine (1 x 10⁻⁴ M). Repetitive additions of 1 x 10⁻⁹ M ergovaline and 1 x 10⁻⁵ and 1 x 10⁻⁴ M lysergic acid resulted in contractile response with a negative slope (P < 0.02). In contrast, repetitive addition of 1 x 10⁻⁷ M ergovaline resulted in a contractile response that increased with each addition (P < 0.01). Lysergic acid and ergovaline were detected at all 4 exposure levels (2x to 8x), but only the 1 x 10⁻⁷ M ergovaline treatment resulted in increased tissue content as the number of exposures increased (P < 0.05). These data indicate that ergovaline, but not lysergic acid, bioaccumulates with repetitive exposure in vitro. These results suggest that ergovaline may have a greater potential for inducing toxicosis in grazing animals than lysergic acid because of its potential to bioaccumulate at the cellular site of action.