Studies of the effect of natural and synthetic polypeptide type ergot compounds on a peripheral vascular bed

1. Six ergot alkaloids were tested for their effect on vascular resistance and for α-adrenergic blocking activity on the innervated perfused hind limb of the dog. The results were compared with those obtained earlier for three compounds of the ergotamine group.

2. Ergostine, dihydroergostine, 1-methylergostine and dihydroergocristine resembled ergotamine, dihydroergotamine and 1-methylergotamine in eliciting vasoconstriction at low vascular resistance and vasodilatation at high vascular resistance. The changeover occurred at the following “inversion points”: ergostine and dihydroergostine as with ergotamine and dihydroergotamine at about 4 R.U.; 1-methylergostine as with 1-methylergotamine at about 2·3 R.U.; dihydroergocristine at about 1·9 R.U. [1 R.U. = 1 resistance unit = 1 mm Hg/ml. per min.]

3. 1-methyldihydroergocristine consistently elicited vasodilatation (for initial vascular resistances down to 1·3 R.U.) and 5′-methylergoalanine always caused vasoconstriction (for initial values up to 5·8 R.U.).

4. Ergostine and 5′-methylergoalanine had the most powerful vasoconstrictor effect, which was of the same order of magnitude as that of ergotamine. Dihydroergostine, like dihydroergotamine, was considerably less active. Both 1-methylergostine and 1-methylergotamine elicited only weak vasoconstriction. Moreover, when the initial vascular resistance exceeded the critical inversion value, they elicited only weak vasodilatation. Dihydroergocristine and 1-methyldihydroergocristine had the least effect on vascular resistance.

5. The increase in vascular resistance by noradrenaline was inhibited in a dose-dependent manner by all the ergot alkaloids investigated. Ergostine, 5′-methylergoalanine and ergotamine had the greatest α-adrenergic blocking activity and 1-methylergostine, 1-methyldihydroergocristine and 1-methylergotamine the weakest. The activity of dihydroergostine, dihydroergocristine and dihydroergotamine fell between these two extremes.

6. No correlation was found between the qualitative effect of these ergot alkaloids on vascular resistance (inversion point) and (a) their quantitative effect on this parameter or (b) their α-adrenergic blocking activity. Determination of the inversion point thus provides additional pharmacological information on the vasoactive properties of the ergot alkaloids.

     

Effects of Heating,Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90–100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.     

A Targeted UHPLC-MS/MS Method Validated for the Quantification of Ergot Alkaloids in Cereal-Based Baby Food from the Belgian Market

Following pending new legislation in the European Union setting a maximum of 20 ng g⁻¹ for the total sum of ergot alkaloids in dry cereal-based baby food, a new UHPLC-MS/MS method was developed. It is suitable for the quantification of six ergot alkaloids: Ergocornine, ergocristine, ergometrine, ergosine, ergotamine, α-ergocryptine, and their corresponding epimers. The method is able to reliably detect individual ergot alkaloids at a level as low as 0.5 ng g⁻¹. The method uses a modified QuEChERS extraction approach before UHPLC-MS/MS analysis. The method showed good sensitivity, accuracy, and precision. It has been applied to 49 samples from the Belgian market. In 26 samples, not a single ergot alkaloid was detected while in 23 out of 49 samples at least one ergot alkaloid was detected with 2 samples containing 12 ergot alkaloids. Ergometrine was the alkaloid most frequently detected i.e., 16 out of 49 samples. Only one sample, testing positive for all 12 ergot alkaloids, would be non-conforming to the newly proposed Maximum Residue Level (MRL).     

Influence of Prolonged Serotonin and Ergovaline Pre-Exposure on Vasoconstriction Ex Vivo

Ergot alkaloid mycotoxins interfere in many functions associated with serotonergic neurotransmitters. Therefore, the objective was to evaluate whether the association of serotonin (5-hydroxytryptamine, 5-HT) and ergot alkaloids during a 24 h pre-incubation could affect the vascular contractile response to ergot alkaloids. To evaluate the effects of 24 h exposure to 5-HT and ergot alkaloids (ergovaline, ERV), two assays were conducted. The first assay determined the half-maximal inhibitory concentration (IC₅₀) following the 24 h pre-exposure period, while the second assay evaluated the effect of IC₅₀ concentrations of 5-HT and ERV either individually or in combination. There was an interaction between previous exposure to 5-HT and ERV. Previous exposure to 5-HT at the IC₅₀ concentration of 7.57 × 10⁻⁷ M reduced the contractile response by more than 50% of control, while the exposure to ERV at IC₅₀ dose of 1.57 × 10⁻¹⁰ M tended to decrease (p = 0.081) vessel contractility with a response higher than 50% of control. The 24 h previous exposure to both 5-HT and ERV did not potentiate the inhibitory response of blood vessels in comparison with incubation with each compound alone. These results suggest receptor competition between 5-HT and ERV. More studies are necessary to determine the potential of 5-HT to treat toxicosis caused by ergot alkaloids.     

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria

The natural occurrence of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers, were investigated in 60 cereal samples (barley and wheat) from Algeria. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and a QuEChERS extraction method were used for sample analysis. The results revealed that 12 out of 60 samples (20%) were contaminated with ergot alkaloids. Wheat was the most contaminated matrix, with an incidence of 26.7% (8 out of 30 samples). The concentration of total ergot alkaloids ranged from 17.8 to 53.9 µg/kg for barley and from 3.66 to 76.0 μg/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.     

Mycotoxins,Phytoestrogens and Other Secondary Metabolites in Austrian Pastures: Occurrences,Contamination Levels and Implications of Geo-Climatic Factors

Pastures are key feed sources for dairy production and can be contaminated with several secondary metabolites from fungi and plants with toxic or endocrine-disrupting activities, which possess a risk for the health, reproduction and performance of cattle. This exploratory study aimed to determine the co-occurrences and concentrations of a wide range of mycotoxins, phytoestrogens and other secondary metabolites in grazing pastures. Representative samples of pastures were collected from 18 Austrian dairy farms (one sample per farm) between April to October 2019. After sample preparation (drying and milling) the pastures were subjected to multi-metabolite analysis using LC-MS/MS. In total, 68 metabolites were detected, including regulated zearalenone and deoxynivalenol (range: 2.16–138 and 107–505 μg/kg on a dry matter (DM) basis, respectively), modified (3-deoxynivalenol-glucoside, HT-2-glucoside) and emerging Fusarium mycotoxins (e.g., enniatins), ergot alkaloids and Alternaria metabolites along with phytoestrogens and other metabolites. Aflatoxins, fumonisins, T-2 toxin, HT-2 toxin and ochratoxins were not detected. Of the geo-climatic factors and botanical diversity investigated, the environment temperature (average of 2 pre-sampling months and the sampling month) was the most influential factor. The number of fungal metabolites linearly increased with increasing temperatures and temperatures exceeding 15 °C triggered an exponential increment in the concentrations of Fusarium and Alternaria metabolites and ergot alkaloids. In conclusion, even though the levels of regulated mycotoxins detected were below the EU guidance levels, the long-term exposure along with co-occurrence with modified and emerging mycotoxins might be an underestimated risk for grazing and forage-fed livestock. The one-year preliminary data points out a dominant effect of environmental temperature in the diversity and contamination level of fungal metabolites in pastures.     

Cytotoxicity and Antiviral Properties of Alkaloids Isolated from Pancratium maritimum

Ten Amaryllidaceae alkaloids (AAs) were isolated for the first time from Pancratium maritimum collected in Calabria region, Italy. They belong to different subgroups of this family and were identified as lycorine, which is the main alkaloid, 9-O-demethyllycorine, haemanthidine, haemanthamine, 11-hydroxyvittatine, homolycorine, pancracine, obliquine, tazettine and vittatine. Haemanthidine was isolated as a scalar mixture of two 6-epimers, as already known also for other 6-hydroxycrinine alkaloids, but for the first time they were separated as 6,11-O,O′-di-p-bromobenzoyl esters. The evaluation of the cytotoxic and antiviral potentials of all isolated compounds was undertaken. Lycorine and haemanthidine showed cytotoxic activity on Hacat cells and A431 and AGS cancer cells while, pancracine exhibited selective cytotoxicity against A431 cells. We uncovered that in addition to lycorine and haemanthidine, haemanthamine and pancracine also possess antiretroviral abilities, inhibiting pseudotyped human immunodeficiency virus (HIV)−1 with EC50 of 25.3 µM and 18.5 µM respectively. Strikingly, all the AAs isolated from P. maritimum were able to impede dengue virus (DENV) replication (EC₅₀ ranged from 0.34–73.59 µM) at low to non-cytotoxic concentrations (CC₅₀ ranged from 6.25 µM to >100 µM). Haemanthamine (EC50 = 337 nM), pancracine (EC₅₀ = 357 nM) and haemanthidine (EC₅₀ = 476 nM) were the most potent anti-DENV inhibitors. Thus, this study uncovered new antiviral properties of P. maritimum isolated alkaloids, a significant finding that could lead to the development of new therapeutic strategies to fight viral infectious diseases.     

Effects of Sustained Administration of Quetiapine Alone and in Combination with a Serotonin Reuptake Inhibitor on Norepinephrine and Serotonin Transmission

Quetiapine is now used in the treatment of unipolar and bipolar disorders, both alone and in combination with other medications. In the current study, the sustained administration of quetiapine and N-Desalkyl quetiapine (NQuet) in rats in a 3 : 1 mixture (hQuetiapine (hQuet)) was used to mimic quetiapine exposure in patients because rats do not produce the latter important metabolite of quetiapine. Sustained administration of hQuet for 2 and 14 days, respectively, significantly enhanced the firing rate of norepinephrine (NE) neurons by blocking the cell body α₂-adrenergic autoreceptors on NE neurons, whether it was given alone or with a serotonin (5-HT) reuptake inhibitor. The 14-day regimen of hQuet enhanced the tonic activation of postsynaptic α₂- but not α₁-adrenergic receptors in the hippocampus. This increase in NE transmission was attributable to increased firing of NE neurons, the inhibition of NE reuptake by NQuet, and the attenuated function of terminal α₂-adrenergic receptors on NE terminals. Sustained administration of hQuet for 2 and 14 days, respectively, significantly inhibited the firing rate of 5-HT, whether it was given alone or with a 5-HT reuptake inhibitor, because of the blockade of excitatory α₁-adrenergic receptors on 5-HT neurons. Nevertheless, the 14-day regimen of hQuet enhanced the tonic activation of postsynaptic 5-HT_1A receptors in the hippocampus. This increase in 5-HT transmission was attributable to the attenuated inhibitory function of the α₂-adrenergic receptors on 5-HT terminals and possibly to direct 5-HT_1A receptor agonism by NQuet. The enhancement of NE and 5-HT transmission by hQuet may contribute to its antidepressant action in mood disorders.     

Cleaving Ergot Alkaloids by Hydrazinolysis—A Promising Approach for a Sum Parameter Screening Method

Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods—acidic esterification and hydrazinolysis—are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40‒60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.     

Ergot and Ergot Alkaloids in Cereal Grains Intended for Animal Feeding Collected in Slovenia: Occurrence,Pattern and Correlations

This four-year study reports the occurrence of ergot alkaloids (EAs) in cereals intended for animal feeding collected in Slovenia. A total of 517 samples of cereals were analysed using liquid chromatography-tandem mass spectrometry for the presence of EAs. The sample set included wheat, rye, triticale, oat, spelt and barley. The study revealed that 17% of the analysed cereal samples were contaminated with at least one ergot alkaloid. EAs have two epimeric forms: -ine and -inine. The incidence rates of the -ine and -inine forms in the analysed samples were 16% and 15%, respectively. The highest contamination rates were observed in rye (54%), oat (50%) and spelt (30%), where the highest mean concentrations of total EAs were also determined (502 µg/kg, 594 µg/kg and 715 µg/kg, respectively). However, the highest concentrations of total EAs were found in wheat and rye (4217 µg/kg and 4114 µg/kg, respectively). The predominant EAs were ergometrine, ergosine and ergocristinine. The occurrence of six or more ergot alkaloids was observed in 49% of the positive samples. A weak correlation (p = 0.284) in the positive samples was found between the mass of sclerotia and the total concentrations of EAs using the Spearman correlation coefficient.     

α1D-Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes

Aim:

It is unclear why α_1D-adrenergic receptors (α_1D-ARs) play a critical role in the mediation of peripheral vascular resistance and blood pressure in situ but function inefficiently when studied in vitro. The present study examined the causes for these inconsistencies in native α₁-adrenergic functional performance between the vascular smooth muscle and myocytes.

Methods:

The α₁-adrenergic mediated contraction, Ca²⁺ signaling and the subcellular receptor distribution were evaluated using the Fluo-4, BODIPY-FL prazosin and subtype-specific antibodies.

Results:

Rat aortic rings and freshly dissociated myocytes displayed contractile and increased intracellular Ca²⁺ responses to stimulation with phenylephrine (PE, 10 μmol), respectively. However, the PE-induced responses disappeared completely in cultured aortic myocytes, whereas PE-enhanced Ca²⁺ transients were seen in cultured rat cardiac myocytes. Further studies indicated that α_1D-ARs, the major receptor subtype responsible for the α₁-adrenergic regulation of aortic contraction, were distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, similar to the α_1A-AR subcellular localization in the cultured cardiomyocytes. In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for α_1D-ARs were found in the cytoplasm. Importantly, treating the culture medium with charcoal/dextran caused the reappearance of α_1D-ARs at the cell surface and a partial restoration of the Ca²⁺ signal response to PE in approximately 30% of the cultured cells.

Conclusion:

Reduction in α_1D-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to α₁-adrenergic receptor activation.     

The Usage of Ergot (Claviceps purpurea (fr.) Tul.) in Obstetrics and Gynecology: A Historical Perspective

In the past centuries consumption of bread made of ergot-infected flour resulted in mass poisonings and miscarriages. The reason was the sclerotia of Claviceps purpurea (Fr.) Tul.—a source of noxious ergot alkaloids (ergotamine and ergovaline). The authors have searched the 19th century medical literature in order to find information on the following topics: dosage forms of drugs based on ergot and their application in official gynecology and obstetrics. The authors also briefly address the relevant data from the previous periods as well as the 20th century research on ergot. The research resulted in a conclusion that applications of ergot in gynecology and obstetrics in the 19th century were limited to controlling excessive uterine bleeding and irregular spasms, treatment of fibrous tumors of the uterus, and prevention of miscarriage, abortion, and amenorrhoea. The most common dosage forms mentioned in the works included in our review were the following: tinctures, water extracts (Wernich’s and Squibb’s watery extract of ergot), pills, and powders. The information documented in this paper will be helpful for further research and helpful in broadening the understanding of the historical application of the described controversial crude drugs. Ergot alkaloids were widely used in obstetrics, but in modern times they are not used in developed countries anymore. They may, however, play a significant role in developing countries where, in some cases, they can be used as an anti-hemorrhage agent during labor.     

Characterization of α2-adrenoceptors mediating contraction of dog saphenous vein: identity with the human α2A subtype

1. In the dog saphenous vein α₁- and α₂-adrenoceptors mediate noradrenaline-induced contractions in vitro. In order to study the α₂-adrenoceptor in isolation, α₁-adrenoceptors were inactivated by treatment of tissues with the alkylating agent phenoxybenzamine (3.0 μM for 30 min) in the presence of rauwolscine (1 μM) to protect α₂-adrenoceptors.
2. Noradrenaline-induced contractions of tissues treated with phenoxybenzamine were antagonized competitively by the selective α₂-adrenoceptor antagonist rauwolscine, pK_B=8.63±0.07 (means±s.e.mean; n=3), consistent with an interaction at α₂-adrenoceptors.
3. Noradrenaline was a full agonist at α₂-adrenoceptors in dog saphenous vein. By use of the method of partial receptor alkylation and analysis of concentration-effect curve data by direct, operational model fitting methods, the affinity (pK_A) and efficacy (τ) were 5.74±0.07 and 7.50±1.05, respectively (n=6). Nine other agonists which were examined each had affinities higher than noradrenaline, but with the exception of the imidazoline, A-54741 (5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphthyl-imidazoline) had relatively lower efficacies.
4. To compare the α₂-adrenoceptor in dog saphenous vein to the human recombinant subtypes, the affinities of twenty-one compounds were estimated in functional studies in the dog saphenous vein and in radioligand binding studies for the human α_2A, α_2B and α_2C receptor subtypes expressed in Chinese hamster lung (CHL) cells.
5. Of twenty-one compounds examined in ligand binding studies, only nine had greater than ten fold selectivity for one human receptor subtype over either of the other two. These compounds were A-54741, oxymetazoline, guanfacine, guanabenz, prazosin, spiroxatrine, tolazoline, WB 4101 and idazoxan. In dog saphenous vein, their affinities (pK_A and pK_B for agonists and antagonists respectively) were: A-54741 (pK_A=8.03±0.05), oxymetazoline (pK_A=7.67±0.09), guanfacine (pK_A=6.79±0.03); guanabenz (pK_A=7.02±0.13); prazosin (pK_B=5.19±0.08), spiroxatrine (pK_B=6.59±0.04), tolazoline (pK_B=6.21±0.07), WB 4101 (pK_B=7.42±0.09) and idazoxan (pK_B=7.11±0.08).
6. Comparisons of affinity estimates for these nine compounds at the receptor in dog saphenous vein and at the human recombinant subtypes suggest that the vascular receptor is most similar to the h α_2A subtype; correlation coefficients (r) were 0.82 (h α_2A), 0.24 (h α_2B) and 0.04 (h α_2C).

     

Ergot Alkaloids Produced by Endophytic Fungi of the Genus Epichlo?

The development of fungal endophytes of the genus Epichloë in grasses results in the production of different groups of alkaloids, whose mechanism and biological spectrum of toxicity can differ considerably. Ergot alkaloids, when present in endophyte-infected tall fescue, are responsible for “fescue toxicosis” in livestock, whereas indole-diterpene alkaloids, when present in endophyte-infected ryegrass, are responsible for “ryegrass staggers”. In contrast, peramine and loline alkaloids are deterrent and/or toxic to insects. Other toxic effects in livestock associated with the consumption of endophyte-infected grass that contain ergot alkaloids include the “sleepy grass” and “drunken horse grass” diseases. Although ergovaline is the main ergopeptine alkaloid produced in endophyte-infected tall fescue and is recognized as responsible for fescue toxicosis, a number of questions still exist concerning the profile of alkaloid production in tall fescue and the worldwide distribution of tall fescue toxicosis. The purpose of this review is to present ergot alkaloids produced in endophyte-infected grass, the factors of variation of their level in plants, and the diseases observed in the mammalian species as relate to the profiles of alkaloid production. In the final section, interactions between ergot alkaloids and drug-metabolizing enzymes are presented as mechanisms that could contribute to toxicity.     

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor

1. The γ-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA_A receptors are favoured by β₂- or β₃- versus β₁-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (β_{2 N290}, β_{3 N289}, β_{1 S290}). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster.
2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at −60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC₅₀ for GABA of 20±0.4 μM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (Rdl_M314N) or a serine (Rdl_M314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC₅₀ of 4.8±0.2 μM.
3. Utilizing the appropriate GABA EC₁₀, etomidate (300 μM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at Rdl_M314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC₅₀ of 64±3 μM and an E_max of 68±2% (of the maximum response to GABA).
4. The actions of etomidate at Rdl_M314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 μM R-(+)-etomidate and S-(−)-etomidate enhancing the current induced by GABA (EC₁₀) to 52±6% and 12±1% of the GABA maximum respectively.
5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49±4% of the GABA maximum) and Rdl_M314N receptors (53±2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one (Rdl=25±4% of the GABA maximum) was not altered by this mutation (Rdl_M314N=18±3% of the GABA maximum).
6. Etomidate acting at β₁ (S290)-containing mammalian GABA_A receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at Rdl_M314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to Rdl_M314N receptors.
7. Etomidate acting at human α₆β₃γ_2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA_A receptor complex. Mutation of the human β₃ subunit asparagine to methionine (β_{3 N289M} found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10–300 μM) acting at α₆β_{3 N289M}γ_2L receptors.
8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

     

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant γ-aminobutyric acid_A (GABA_A) receptor subunits.
2. Currents mediated by recombinant receptors with the ternary subunit composition α_xβ_yγ_2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC₁₀, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms.
3. For the β₂-subunit containing receptors tested, the EC₅₀ for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 μM) was little affected by the nature of the α subunit present within the hetero-oligomeric complex. However, replacement of the β₂ by the β₁ subunit produced a 9–12 fold increase in the etomidate EC₅₀ (6 to 11 μM) for all α-isoforms tested.
4. For α₁, α₂ and α₆, but not α₃-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for β₂- than for β₁-subunit containing receptors. This was most clearly exemplified by receptors composed of α₆β₁γ_2L compared to α₆β₂γ_2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC₁₀ to 28±2% and 169±4% of the maximal GABA response, respectively.
5. For α₁ subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α-pregnan-3α-ol-20-one was marginally higher for β₁ rather than the β₂ subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms.
6. The GABA-mimetic action of etomidate was supported by β₂- but not β₁-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β₂-subunit containing receptors, both the agonist EC₅₀ and the maximal current produced by etomidate were additionally influenced by the α isoform.
7. It is concluded that the subtype of β-subunit influences the potency with which etomidate potentiates GABA-evoked currents and that the β isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the α-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

     

Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology.
2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists.
3. The EC₅₀ of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC₅₀ for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs.
4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit.

     

Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

1. The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT_3A(b)) heterologously expressed in Xenopus laevis oocytes.
2. At negative holding potentials, bath applied 5-HT (300 nM–10 μM) evoked a transient, concentration-dependent (EC₅₀=1.1±0.1 μM), inward current. The response reversed in sign at a holding potential of −2.1±1.6 mV.
3. The response to 5-HT was mimicked by the 5-HT₃ receptor selective agonists 2-methyl-5-HT (EC₅₀=4.1±0.2 μM), 1-phenylbiguanide (EC₅₀=3.0±0.1 μM), 3-chlorophenylbiguanide (EC₅₀=140± 10 nM), 3,5-dichlorophenylbiguanide (EC₅₀=14.5±0.4 nM) and 2,5-dichlorophenylbiguanide (EC₅₀= 10.2±0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 μM) of 5-HT.
4. Responses evoked by 5-HT at EC₅₀ were blocked by the 5-HT₃ receptor selective antagonist ondansetron (IC₅₀=231±22 pM) and by the less selective agents (+)-tubocurarine (IC₅₀=31.9± 0.01 nM) and cocaine (IC₅₀=2.1±0.2 μM).
5. The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT_3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT₃ receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT_3A subunit orthologues.

     

Biosynthetic Pathways of Ergot Alkaloids

Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes.     

Ergot Alkaloids in Feed for Pekin Ducks: Toxic Effects,Metabolism and Carry Over into Edible Tissues

Hardened sclerotia (ergots) of Claviceps purpurea contaminate cereal grains and contain toxic ergot alkaloids (EA). Information on EA toxicity in ducks is scarce. Therefore, the aim of the growth experiment (Day 0–49, n = 54/group) was to titrate the lowest observed adverse effect level (LOAEL) for total ergot alkaloids (TEA). A control diet was prepared without ergots, and the diets designated Ergot 1 to 4 contained 1, 10, 15 and 20 g ergot per kg diet, respectively, corresponding to TEA contents of 0.0, 0.6, 7.0, 11.4 and 16.4 mg/kg. Sensitivity of ducks to EA was most pronounced at the beginning of the experiment when feed intake decreased significantly by 9%, 28%, 41% and 47% in groups Ergot 1 to 4, respectively, compared to the control group. The experiment was terminated after two weeks for ducks exposed to Ergot 3 and 4 due to significant growth retardation. Ergot alkaloid residues in edible tissues were lower than 5 ng/g. Bile was tested positive for ergonovine (=ergometrine = ergobasine) with a mean concentration of 40 ng/g. Overall, the LOAEL amounted to 0.6 mg TA/kg diet suggesting that ducks are not protected by current European Union legislation (1 g ergot/kg unground cereal grains).