Vasoactive Effects of Acute Ergot Exposure in Sheep

Ergotism is a common and increasing problem in Saskatchewan’s livestock. Chronic exposure to low concentrations of ergot alkaloids is known to cause severe arterial vasoconstriction and gangrene through the activation of adrenergic and serotonergic receptors on vascular smooth muscles. The acute vascular effects of a single oral dose with high-level exposure to ergot alkaloids remain unknown and are examined in this study. This study had two main objectives; the first was to evaluate the role of α₁-adrenergic receptors in mediating the acute vasocontractile response after single-dose exposure in sheep. The second was to examine whether terazosin (TE) could abolish the vascular contractile effects of ergot alkaloids. Twelve adult female sheep were randomly placed into control and exposure groups (n = 6/group). Ergot sclerotia were collected and finely ground. The concentrations of six ergot alkaloids (ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) were determined using HPLC/MS at Prairie Diagnostic Services Inc., (Saskatoon, SK, Canada). Each ewe within the treatment group received a single oral treatment of ground ergot sclerotia at a dose of 600 µg/kg BW (total ergot) while each ewe in the control group received water. Animals were euthanized 12 h after the treatment, and the pedal artery (dorsal metatarsal III artery) from the left hind limb from each animal was carefully dissected and mounted in an isolated tissue bath. The vascular contractile response to phenylephrine (PE) (α₁-adrenergic agonist) was compared between the two groups before and after TE (α₁-adrenergic antagonist) treatment. Acute exposure to ergot alkaloids resulted in a 38% increase in vascular sensitivity to PE compared to control (Ctl EC₅₀ = 1.74 × 10⁻⁶ M; Exp EC₅₀ = 1.079 × 10⁻⁶ M, p = 0.046). TE treatment resulted in a significant dose-dependent increase in EC₅₀ in both exposure and control groups (p < 0.05 for all treatments). Surprisingly, TE effect was significantly more pronounced in the ergot exposed group compared to the control group at two of the three concentrations of TE (TE 30 nM, p = 0.36; TE 100 nM, p < 0.001; TE 300 nM, p < 0.001). Similar to chronic exposure, acute exposure to ergot alkaloids results in increased vascular sensitivity to PE. TE is a more potent dose-dependent antagonist for the PE contractile response in sheep exposed to ergot compared to the control group. This study may indicate that the dry gangrene seen in sheep, and likely other species, might be related to the activation of α₁-adrenergic receptor. This effect may be reversed using TE, especially at early stages of the disease before cell death occurs. This study may also indicate that acute-single dose exposure scenario may be useful in the study of vascular effects of ergot alkaloids.     

Characterization of 5-HT receptors mediating constriction of porcine carotid arteriovenous anastomoses; involvement of 5-HT1B/1D and novel receptors

1. It was previously shown that porcine cranial arteriovenous anastomoses (AVAs) constrict to 5-hydroxytryptamine (5-HT), ergotamine, dihydroergotamine, as well as sumatriptan and that sumatriptan acts exclusively via 5-HT_1B/1D receptors. The present study was devoted to establish the contribution of 5-HT_1B/1D receptors in the constriction of AVAs elicited by 5-HT (in presence of 0.5 mg kg⁻¹ ketanserin), ergotamine and dihydroergotamine in anaesthetized pigs.
2. Intracarotid infusion of 5-HT (2 μg kg⁻¹ min⁻¹) and intravenous doses of ergotamine (2.5–20 μg kg⁻¹) and dihydroergotamine (3–100 μg kg⁻¹) reduced AVA and increased nutrient blood flows and vascular conductances. The vasodilator response to 5-HT, observed mainly in the skin and ear, was much more prominent than that of the ergot alkaloids.
3. Treatment with the 5-HT_1B/1D receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (0.5 mg kg⁻¹, i.v.) significantly attenuated both ergot-induced AVA constriction and arteriolar dilatation, whereas {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 only slightly affected the carotid vascular effects of 5-HT.
4. The results suggest that 5-HT constricts carotid AVAs primarily via receptors, which seem to differ from those (5-HT_1B/1D) stimulated by sumatriptan. The ergot alkaloids produce AVA constriction for a substantial part via 5-HT_1B/1D receptors, but also stimulate unidentified receptors. Both these non-5-HT_1B/1D receptors may be targets for the development of novel antimigraine drugs.
5. The moderate vasodilator response to the ergot derivatives seems to be mediated, at least in part, by 5-HT_1B/1D receptors, whereas the arteriolar dilatation caused by 5-HT may be mediated by other, possibly 5-HT₇ receptors.

     

Evidence for various tryptamines and related compounds acting as substrates of the platelet 5-hydroxytryptamine transporter

Summary The aim of the present study was to answer the question whether amines other than 5-hydroxytryptamine (5-HT) and tryptamine act as substrates of the platelet 5-HT transporter. To this end, a large number of tryptamines, 5-HT receptor agonists and phenethylamines (which had IC₅₀ values for ³H-5-HT uptake inhibition of 145–24500 nmol l^–1) was examined in rabbit platelets in order to determine their ability to induce an outward transport of ³H-5-HT Platelets (the MAO of which was blocked) from reserpine-pretreated animals were loaded with ³H-5-HT and then exposed for 5 min to various concentrations (ranging from 0.25 to 40 times the IC₅₀) of each compound. The concentration-effect curves for the drug-induced increase in ³H-5-HT efflux served to determine values of E_max (maximum increase in efflux expressed in % of the ³H-5-HT content of cells) and EC₅₀ (drug concentration producing E_max/2).For the 24 compounds studied here (which included the 5-HT uptake inhibitors imipramine, citalopram, fluoxetine and cocaine) a linear correlation between EC₅₀ and IC₅₀ (r = 0.975) and a mean ratio of EC₅₀/IC₅₀ of 2.4 was found. Most of the compounds [e.g., (±)8-hy-ydroxy-2-(N,N-dipropylamino)tetralin, S(+)-methyl-5-HT, 5-carboxamidotryptamine and 5-methoxytryptamine] gave rise to E_max values (15.8–32.5%) that exceeded that brought about by imipramine (6.6%), indicating that they act as substrates of the 5-HT transporter; the ³H-5-HT outward transport observed in response to these substances was abolished in the presence of imipramine. Others (e.g., 2-methyl-5-HT and 5-methylurapidil) produced Emax values (3.4–14.3%) not significantly different from that of imipramine and, therefore, can be classified either as poor substrates or as inhibitors of the 5-HT transporter.Hence, many tryptamines and 5-HT receptor agonists are substrates of the platelet 5-HT transporter. The property of being substrates gives them the latent capacity to bring about release of endogenous 5-HT and, as a result, to cause indirect 5-HT receptor-mediated effects.Abbreviations MAO monoamine oxidase - 5-HT 5-hydroxytryptamine - 2-M-5-HT 2-methyl-5-HT - N-M-5-HT N-methyl-5-HT - N,N-DM-5-HT N,N-dimethyl-5-HT - S(+)-M-5-HT S(+)-methyl-5-HT - 5-CT 5-carboxamidotryptamine - 5-M-tryptamine 5-methyltryptamine - 5-MO-tryptamine 5-methoxytryptamine - 7-M-tryptamine 7-methyltryptamine - N-M-tryptamine N-methyltryptamine - N,N-DM-tryptamine N,N-dimethyltryptamine - N,N-DM-5-MO-tryptamine N,N-dimethyl-5-methoxytryptamine - (±)8-OH-DPAT (±)8-hydroxy-2-2-(N,N-dipropylamino)tetralin - 5-M-urapidil 5-methyl-urapidil Send offprint requests to R. Wölfel at the above address     

An inhibitory prejunctional 5-HT1-like receptor in the isolated perfused rat kidney

Summary The present study has identified a receptor for 5-hydroxytryptamine (5-HT) which functions to inhibit the stimulus-induced release of [³H] noradrenaline following sympathetic periarterial nerve stimulation to the isolated perfused rat kidney. In addition to 5-HT (IC₃₀=4.5×10^–8 mol/l), both 5-carboxamidotryptamine (IC₃₀=8×10^–9 mol/l) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl) indole (RU-24969, IC₃₀=2.5×10^–7 mol/l) acted as agonists whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was inactive. The inhibitory effect of 5-HT on the electrically-evoked release of tritium was antagonized in a concentration-dependent manner by methiothepin (IC₅₀=4×10^–9 mol/l), metergoline (IC₅₀=4×10^–8 mol/l) and methysergide (IC₅₀=1.3×10^–7 mol/l) but not by cyproheptadine, ketanserin, mesulergine, (–)-propranolol, (±)-pindolol, (±)-cyanopindolol, metoclopramide or phentolamine. It is concluded that the receptor to 5-HT conforms to general criteria defining 5-HT₁-like receptors but at the present time the receptor site cannot be fitted to the designated 5-HT_1A, 5-HT_1B or 5-HT_1C subtypes.Preliminary accounts of this work were presented to the British Pharmacological Society in London (December, 1984) and Southampton (July, 1985)     

Localization of leukaemia inhibitory factor to airway epithelium and its amplification of contractile responses to tachykinins

1. In neural tissue, leukaemia inhibitory factor (LIF) is an important trophic cytokine. In this investigation, we determined if LIF was present in human and guinea-pig airways and examined the role of this cytokine in modulating airway responses to endogenous and exogenous tachykinins as well as muscarinic receptor and β-adrenoceptor stimulation.
2. The presence of LIF in both human and guinea-pig airways was determined by immunohistochemistry. Guinea-pig tracheal explants were incubated in CRML-1066 media containing LIF (0.5, 5 or 50 ng ml⁻¹) for periods of 3, 6, 24 and 48 h. Tracheal rings were then transferred to organ baths for measurement of isometric force in response to carbachol, capsaicin, the neurokinin₁ (NK₁) receptor agonist [Sar⁹,Met(O₂)¹¹]-substance P (SP), the NK₂ receptor agonist neurokinin A (NKA) and isoprenaline.
3. LIF immunoreactivity was observed primarily in basally situated cells in the airway epithelium of both large and small airways. Less intense immunoreactivity was observed in vascular endothelium and glandular epithelium.
4. Treatment with LIF (0.5 ng ml⁻¹) for 3 and 6 h significantly increased contractile responses to capsaicin by 42% and 43%, respectively, compared to time controls, whereas higher concentrations of LIF (5 and 50 ng ml⁻¹) enhanced capsaicin-induced contractions only after 6 h. After 24 h, responses to capsaicin were not significantly different from 0 h control. Contractile responses to capsaicin following exposure to LIF at any concentration for 24 h were not significantly different from relative time control values.
5. Responses to [Sar⁹,Met(O₂)¹¹]-SP, carbachol and isoprenaline were not influenced by time in culture or by exposure to LIF for up to 48 h. Contractile responses induced by NKA were not influenced by 3 or 6 h exposure to LIF, but at 24 and 48 h the mean maximum contractile responses to NKA were significantly increased by 33% and 35%, respectively, compared to control.
6. These results demonstrate that LIF is present in guinea-pig and human airway epithelium, and modulates airway responses to tachykinins. In the acute setting LIF augments the capsaicin-induced release of endogenous tachykinins, whilst in the longer term (>24 h), LIF increases airway smooth muscle responses to tachykinins via an NK₂ receptor selective mechanism. We conclude that LIF may be an important effector molecule in the response of airways to injury or inflammation.

     

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).     

F 15845 inhibits persistent sodium current in the heart and prevents angina in animal models

Background and purpose:

Activation of the persistent sodium current in ischaemic myocardium results in calcium overload which is toxic for the cardiomyocyte. Thus, the activity of 3-(R)-[3-(2-methoxyphenylthio-2-(S)-methylpropyl]amino-3,4-dihydro-2H-1,5 benzoxathiepine bromhydrate (F 15845), a new selective persistent sodium current blocker, in protecting against the effects of cardiac ischaemia was examined, in both in vitro and in vivo models.

Experimental approach:

Electrophysiological studies using patch-clamp and conventional microlelectrode techniques, isolated perfused hearts and models of angina in anaesthetized animals were used to assess the protection afforded by F 15845 against ischaemia-induced changes.

Key results:

F 15845 reduced the persistent sodium current activated by veratridine (IC₅₀ 1.58 × 10⁻⁶ mol·L⁻¹). F 15845 blocked voltage-gated human cardiac sodium channels in a novel, voltage-dependent manner, selectively affecting steady-state inactivation. F 15845 did not affect action potential shape and basal function of guinea pig isolated perfused hearts but did reduce ischaemia-induced diastolic contracture in this model (IC₅₀ 0.64 × 10⁻⁶ mol·L⁻¹). In rabbits, F 15845 given i.v. (ED₅₀ 0.05 mg·kg⁻¹) or orally (ED₅₀ 0.13 mg·kg⁻¹) dose-dependently and powerfully inhibited regional myocardial ischaemia-induced ST segment elevation in the absence of haemodynamic effects, implying direct cardiac activity. In dogs, F 15845 dose-dependently inhibited epicardial ST segment changes (70 ± 8% at 0.63 mg·kg⁻¹) in an experimental angina model of demand ischaemia, again without haemodynamic effects, confirming a direct anti-anginal activity.

Conclusions and implications:

F 15845 is a selective, potent blocker of the persistent sodium current, generated by the human Na_v1.5 channel isoforms, and prevents cardiac angina in animal models.     

Methnaridine is an orally bioavailable,fast‐killing and long‐acting antimalarial agent that cures Plasmodium infections in mice

Background and PurposeMalaria is one of the deadliest diseases in the world. Novel chemotherapeutic agents are urgently required to combat the widespread Plasmodium resistance to frontline drugs. Here, we report the discovery of a novel benzonaphthyridine antimalarial, methnaridine, which was identified using a structural optimization strategy.Experimental ApproachAn integrated pharmacological approach was used to evaluate the antimalarial profile of methnaridine. The pharmacokinetic properties of methnaridine were investigated along with the associated safety profile. Host immune response patterns were also analysed.Key ResultsMethnaridine exhibited potent antimalarial activity against P. falciparum (3D7: IC₅₀ = 0.0066 μM; Dd2: IC₅₀ = 0.0056 μM). In P. berghei‐infected mice, oral administration effectively suppressed parasitemia (ED₅₀ = 0.52 mg·kg⁻¹·day⁻¹) and cured the established infection (CD₅₀ = 10.13 mg·kg⁻¹·day⁻¹). These results are equivalent to or better than those of other antimalarial agents in clinical use. Notably, a four‐dose oral regimen at a dosage of 25 mg·kg⁻¹ achieved a complete cure of P. berghei infection in mice. Methnaridine exhibited a rapid parasiticidal profile (PCT₉₉ = 36.0 h) and showed no cross‐resistance to chloroquine. Pharmacokinetic studies revealed that methnaridine is readily absorbed, long‐lasting and slowly cleared. The safety profile of methnaridine is also satisfactory (maximum tolerated dose = 1,125 mg·kg⁻¹). In addition, following methnaridine treatment, infection‐induced Th1 immune response was almost fully alleviated in mice.Conclusion and ImplicationsMethnaridine is an orally bioavailable, fast‐acting and long‐lasting agent with excellent antimalarial properties. Our study highlights the potential of methnaridine for clinical development as a promising antimalarial candidate.     

Effects of narcotic analgesics on the uptake and release of 5-hydroxytryptamine in rat synaptosomal preparations

1 The effects of various narcotic analgesics on the uptake and release of labelled 5-hydroxytryptamine (5-HT) in brain and spinal cord synaptosomes were investigated.

2 Methadone was most active in inhibiting 5-HT uptake (IC₅₀ 2.5 × 10^-7 M). Levorphanol also inhibited 5-HT uptake to a large extent (IC₅₀ 8.8 × 10^-7 M) while dextrophan, pethidine and pentazocine showed much less activity. Etorphine and morphine had virtually no such activity, with IC₅₀S higher than 10^-4 and 10^-3 M respectively.

3 The same order of potency as `5-HT releasers' was found when radioactivity was measured in [³H]-5-HT preloaded synaptosomal pellets incubated for 20 min with the various narcotics. Methadone, like chlorimipramine, showed a significant effect at a concentration of 10^-7 M while morphine, at a concentration of 10^-4 M, had no effect.

4 When 5-HT release was studied by a perfusion technique, which largely prevents reuptake of the released amine, only fenfluramine, an anorectic agent proposed as a 5-HT releaser, significantly increased spontaneous 5-HT release. These data suggest that the apparent 5-HT release induced by various narcotics in traditional incubation techniques may largely depend on their ability to interfere with neurotransmitter reuptake mechanisms.

5 The effects of the various narcotics on 5-HT uptake have no relationship to their relative potency as analgesics in the rat. In the light of their poor effectiveness as 5-HT releasers, it can be concluded that mechanisms other than 5-HT uptake inhibition and release are probably involved in the analgesic effects of these compounds in intact animals.

     

Blockade of 5-Hydroxytryptamine and noradrenaline uptake by venlafaxine: a comparative study with paroxetine and desipramine

1. Venlafaxine is an antidepressant agent which blocks in vitro the reuptake of both 5-HT and NA. The present in vivo electrophysiological studies were undertaken, in the rat, to compare the effects of venlafaxine on 5-HT and NA reuptake to those of the selective 5-HT reuptake inhibitor paroxetine and the selective NA reuptake inhibitor desipramine.
2. Administered acutely, venlafaxine dose-dependently prolonged the time required for a 50% recovery (RT₅₀) of the firing activity of dorsal hippocampus CA₃ pyramidal neurons from the suppression induced by microiontophoretic applications of 5-HT and NA. Venlafaxine and paroxetine increased with a similar potency the RT₅₀ values for 5-HT, while desipramine was more potent than venlafaxine at increasing the RT₅₀ values for NA. Moreover, venlafaxine demonstrated a greater potency at increasing the RT₅₀ values for 5-HT compared to that of NA.
3. A two-day treatment with venlafaxine (delivered s.c. by osmotic minipumps) increased the RT₅₀ values for both 5-HT and NA applications. The RT₅₀ values for 5-HT were significantly increased at a dose of 10 mg kg⁻¹ day⁻¹, whereas those for NA were increased at a dose of 20 mg kg⁻¹ day⁻¹, consistent with the data obtained following the acute administration of venlafaxine.
4. Taken together, these results indicate that, in vivo, venlafaxine blocks both reuptake processes, with a potency greater for the 5-HT than for the NA reuptake process. This dual action, combined with the differential potency of venlafaxine, might constitute the biological substratum responsible for its apparent unique clinical efficacy in major depression.

     

Risperidone inhibits 5-hydroxytryptaminergic neuronal activity in the dorsal raphe nucleus by local release of 5-hydroxytryptamine

1. The effects of risperidone on brain 5-hydroxytryptamine (5-HT) neuronal functions were investigated and compared with other antipsychotic drugs and selective receptor antagonists by use of single cell recording and microdialysis in the dorsal raphe nucleus (DRN).
2. Administration of risperidone (25–400 μg kg⁻¹, i.v.) dose-dependently decreased 5-HT cell firing in the DRN, similar to the antipsychotic drug clozapine (0.25–4.0 mg kg⁻¹, i.v.), the putative antipsychotic drug amperozide (0.5–8.0 mg kg⁻¹, i.v.) and the selective α₁-adrenoceptor antagonist prazosin (50–400 μg kg⁻¹, i.v.).
3. The selective α₂-adrenoceptor antagonist idazoxan (10–80 μg kg⁻¹, i.v.), in contrast, increased the firing rate of 5-HT neurones in the DRN, whereas the D₂ and 5-HT_2A receptor antagonists raclopride (25–200 μg kg⁻¹, i.v.) and MDL 100,907 (50–400 μg kg⁻¹, i.v.), respectively, were without effect. Thus, the α₁-adrenoceptor antagonistic action of the antipsychotic drugs might, at least partly, cause the decrease in DRN 5-HT cell firing.
4. Pretreatment with the selective 5-HT_1A receptor antagonist WAY 100,635 (5.0 μg kg⁻¹, i.v.), a drug previously shown to antagonize effectively the inhibition of 5-HT cells induced by risperidone, failed to prevent the prazosin-induced decrease in 5-HT cell firing. This finding argues against the notion that α₁-adrenoceptor antagonism is the sole mechanism underlying the inhibitory effect of risperidone on the DRN cells.
5. The inhibitory effect of risperidone on 5-HT cell firing in the DRN was significantly attenuated in rats pretreated with the 5-HT depletor PCPA (p-chlorophenylalanine; 300 mg kg⁻¹, i.p., day⁻¹ for 3 consecutive days) in comparison with drug naive animals.
6. Administration of risperidone (2.0 mg kg⁻¹, s.c.) significantly enhanced 5-HT output in the DRN.
7. Consequently, the reduction in 5-HT cell firing by risperidone appears to be related to increased availability of 5-HT in the somatodendritic region of the neurones leading to an enhanced 5-HT_1A autoreceptor activation and, in turn, to inhibition of firing, and is probably only to a minor extent caused by its α₁-adrenoceptor antagonistic action.

     

MDMA ‘ecstasy’ increases cerebral cortical perfusion determined by bolus-tracking arterial spin labelling (btASL) MRI

Background and Purpose

The purpose of this study was to assess cerebral perfusion changes following systemic administration of the recreational drug 3,4-methylendioxymethamphetamine (MDMA ‘ecstasy’) to rats.

Experimental Approach

Cerebral perfusion was quantified using bolus-tracking arterial spin labelling (btASL) MRI. Rats received MDMA (20 mg·kg⁻¹; i.p.) and were assessed 1, 3 or 24 h later. Rats received MDMA (5 or 20 mg·kg⁻¹; i.p.) and were assessed 3 h later. In addition, rats received MDMA (5 or 10 mg·kg⁻¹; i.p.) or saline four times daily over 2 consecutive days and were assessed 8 weeks later. Perfusion-weighted images were generated in a 7 tesla (7T) MRI scanner and experimental data was fitted to a quantitative model of cerebral perfusion to generate mean transit time (MTT), capillary transit time (CTT) and signal amplitude.

Key Results

MDMA reduces MTT and CTT and increases amplitude in somatosensory and motor cortex 1 and 3 h following administration, indicative of an increase in perfusion. Prior exposure to MDMA provoked a long-term reduction in cortical 5-HT concentration, but did not produce a sustained effect on cerebral cortical perfusion. The response to acute MDMA challenge (20 mg·kg⁻¹; i.p.) was attenuated in these animals indicating adaptation in response to prior MDMA exposure.

Conclusions and Implications

MDMA provokes changes in cortical perfusion, which are quantifiable by btASL MRI, a neuroimaging tool with translational potential. Future studies are directed towards elucidation of the mechanisms involved and correlating changes in cerebrovascular function with potential behavioural deficits associated with drug use.     

Agonistic properties of alniditan,sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT_1B/5-HT_1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT_1B receptor expressed at 200 fmol mg⁻¹ protein (B_max) in non-induced L929sA cells, at 740 fmol mg⁻¹ protein in HEK 293 and at 2300 fmol mg⁻¹ protein in mIFNβ-induced L929sA cells, and on the human cloned 5-HT_1D receptor expressed in C6 glioma cells (B_max 780 fmol mg⁻¹ protein).
2. Sodium butyrate treatment increased the expression level of human (h)5-HT_1B receptors in HEK 293 cells and h5-HT_1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [³H]-5-HT and [³H]-alniditan were unaffected.
3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5-HT_1B receptor (IC₅₀ values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5-HT_1D receptors (IC₅₀ values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT_1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).
4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT_1B receptor, and twice as potent at the h5-HT_1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT_1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5-HT_1D receptor.

     

Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT₁-like'' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996.
2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg⁻¹) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine.
3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg⁻¹) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg⁻¹) and mesulergine (300 and 1000 μg kg⁻¹) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg⁻¹), the 5-HT_1B/1D receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg kg⁻¹) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses.
5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT₇ receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht₇ gene product, the 5-HT₇ receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT₇ receptors.

     

Role of nitric oxide in the contractile response to 5-hydroxytryptamine of the basilar artery from Wistar Kyoto and stroke-prone rats

1. Isolated basilar arteries from spontaneously hypertensive stroke-prone rats (SHRSP) are more sensitive to the contractile effect of 5-hydroxytryptamine (5-HT) than those from normotensive Wistar Kyoto rats (WKY). This has been attributed to a different proportion of 5-HT receptor subtypes mediating these responses. In the present study we have examined if differences in nitric oxide release could also contribute to this difference in sensitivity to 5-HT.
2. At rest, the normalized internal diameter was significantly smaller in SHRSP (297.4±3.5 μm, n=88) than in WKY (375.1±4.0 μm, n=62, P<0.01) arteries. The contractile response to 100 mM KCl was higher in WKY (3.57±0.15 mN mm⁻¹, n=22) than in SHRSP arteries (2.32±0.20 mN mm⁻¹, n=28, P<0.01).
3. When added on the plateau of contraction to 5-HT (1 μM), acetylcholine (ACh, 3 μM) evoked significant relaxation in all preparations from WKY (n=20), but only in 15 out of 26 preparations from SHRSP. The mean relaxations were 55.4±5.2% in WKY and 20.6±4.6% in SHRSP (as % of the contractile tone evoked by 5-HT; P<0.01).
4. The NO synthase inhibitor N^ω-nitro-L-arginine (L-NOARG, 0.1 mM) produced a similar increase in tone in both WKY and SHRSP. This tone was equal (in % of the contractile response to 100 mM KCl) to 70.8±4.4% in WKY (n=20) and 67.6±5.9% in SHRSP (n=26) and was reversed by L-arginine (1 mM) and by 1,4-dihydropyridine calcium channel blockers (10 nM nisoldipine, 10 nM lacidipine, 100 nM nifedipine). The L-NOARG-induced tone was absent when the arteries were bathed in phosphate-free Krebs (pH 7.4).
5. EC₅₀ values of 5-HT were about four fold smaller in SHRSP than in WKY arteries (P<0.01). The maximal response to 5-HT (E_max) was higher than 100 mM KCl-contraction in SHRSP but not in WKY arteries. Removal of endothelium produced a shift to the left of the 5-HT curve in WKY, but not in SHRSP arteries.
6. When evoked in phosphate-free Krebs, the contractile responses to 5-HT showed tachyphylaxis, but the responses were reproducible by adding the agonist at 30 min intervals. In such conditions, EC₅₀ values of 5-HT were about two fold smaller in SHRSP than in WKY arteries (P<0.01). In phosphate-free Krebs, the blockade of NO synthase did not change the contractile response to 100 mM KCl; it reduced EC₅₀ and increased E_max of 5-HT in WKY, but not in SHRSP.
7. These results confirm that the sensitivity to 5-HT is higher in basilar artery isolated from SHRSP than in those from WKY. They show that endothelium-dependent vasorelaxation to ACh is impaired in SHRSP. The finding that removal of endothelium or blockade of NO synthase augmented the contractile response to 5-HT in WKY, but not in SHRSP basilar arteries indicates that the difference in responsiveness to 5-HT observed between WKY and SHRSP basilar arteries might be, at least in part, related to dissimilarities in NO release. Furthermore, the L-NOARG-induced contraction sensitive to calcium channel blockers indicates that, in basilar arteries, NO production might lower L-type calcium channel opening and thereby control the tone of the vessels.

     

Diadenosine pentaphosphate is a potent activator of cardiac ryanodine receptors revealing a novel high-affinity binding site for adenine nucleotides

Background and purpose:

Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2).

Experimental approach:

RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions.

Key results:

With10 µmol·L⁻¹ cytosolic Ca²⁺, a significant ‘hump’ or plateau at the base of the dose–response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol·L⁻¹–10 µmol·L⁻¹. High Po values were observed at >10 µmol·L⁻¹ Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose–response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC₅₀ of 16 µmol·L⁻¹ (Ap5A EC₅₀ = 140 µmol·L⁻¹). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2.

Conclusions and implications:

The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.     

Investigation of the mechanisms underlying the hypophagic effects of the 5-HT and noradrenaline reuptake inhibitor,sibutramine, in the rat

1. Sibutramine is a novel 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin- noradrenaline reuptake inhibitor, SNRI) which is currently being developed as a treatment for obesity. Sibutramine has been shown to decrease food intake in the rat. In this study we have used a variety of monoamine receptor antagonists to examine the pharmacological mechanisms underlying sibutramine-induced hypophagia.
2. Individually-housed male Sprague-Dawley rats were maintained on reversed phase lighting with free access to food and water. Drugs were administered at 09 h 00 min and food intake was monitored over the following 8 h dark period.
3. Sibutramine (10 mg kg⁻¹, p.o.) produced a significant decrease in food intake during the 8 h following drug administration. This hypophagic response was fully antagonized by the α₁-adrenoceptor antagonist, prazosin (0.3 and 1 mg kg⁻¹, i.p.), and partially antagonized by the β₁-adrenoceptor antagonist, metoprolol (3 and 10 mg kg⁻¹, i.p.) and the 5-HT receptor antagonists, metergoline (non-selective; 0.3 mg kg⁻¹, i.p.); ritanserin (5-HT_2A/2C; 0.1 and 0.5 mg kg⁻¹, i.p.) and SB200646 (5-HT_2B/2C; 20 and 40 mg kg⁻¹, p.o.).
4. By contrast, the α₂-adrenoceptor antagonist, RX821002 (0.3 and 1 mg kg⁻¹, i.p.) and the β₂-adrenoceptor antagonist, ICI 118,551 (3 and 10 mg kg⁻¹, i.p.) did not reduce the decrease in food intake induced by sibutramine.
5. These results demonstrate that β₁-adrenoceptors, 5-HT_2A/2C-receptors and particularly α₁-adrenoceptors, are involved in the effects of sibutramine on food intake and are consistent with the hypothesis that sibutramine-induced hypophagia is related to its ability to inhibit the reuptake of both noradrenaline and 5-HT, with the subsequent activation of a variety of noradrenaline and 5-HT receptor systems.

     

Functional interactions between 5-HT2A and presynaptic 5-HT1A receptor-based responses in mice genetically deficient in the serotonin 5-HT transporter (SERT)

Background and purpose:

Despite decreased presynaptic 5-HT_1A and altered 5-HT_2A receptor function in genetically-deficient serotonin (5-HT) transporter (SERT) mice, the 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY 100635) still induced head twitches in these mice, a well-established 5-HT_2A receptor-mediated response.

Experimental approach:

Interactions between 5-HT_1A and 5-HT_2A receptors were assessed using the head-twitch response following 5-HT_1A and 5-HT_2A receptor agonists and antagonists in SERT wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. The role of brain 5-HT availability in WAY 100635 induced head twitches was also examined.

Key results:

WAY 100635 induced head twitches in a SERT gene-dose dependent manner, inducing 5-fold more head twitches in SERT −/− versus SERT +/+ mice. In SERT −/− mice, inhibition of 5-HT synthesis with p-chlorophenylalanine (PCPA) markedly depleted tissue 5-HT in all five brain areas examined and abolished WAY 100635 induced head twitches. Further, the selective 5-HT reuptake inhibitor fluvoxamine increased WAY 100635 induced head twitches in SERT +/+ and +/− mice. Head twitches following the 5-HT_2A receptor agonist (+/−)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI) were robust in SERT +/+ and +/− mice but much reduced in SERT −/− mice. DOI-induced head twitches were decreased by the 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in SERT +/+ and +/− mice. All drug-induced head twitches were blocked by the 5-HT_2A receptor antagonist a-Phenyl-1-(2-phenylethyl)-4-piperidinemethanol (MDL 11,939).

Conclusions and implications:

These data show that indirect activation of 5-HT_2A receptors via blockade of presynaptic 5-HT_1A receptors potentiated head-twitch responses, suggesting functional interactions between these receptors, interactions affected by altered 5-HT availability. Our findings strongly support the correlation of WAY 100635 induced head twitches with increased 5-HT availability, induced genetically or pharmacologically.     

Stimulation of angiotensin AT2 receptors by the non-peptide agonist,Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:

Angiotensin type 2 receptor (AT₂ receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT₁ receptors). Recently, a novel non-peptide AT₂ receptor agonist, Compound 21, was described, which exhibited high AT₂ receptor selectivity.

Experimental approach:

Functional cardiovascular effects of the drug candidate Compound 21 were assessed, using mouse isolated aorta and rat mesenteric arteries in vitro and in conscious spontaneously hypertensive rats (SHR).

Key results:

Compound 21 evoked dose-dependent vasorelaxations in aortic and mesenteric vessels, abolished by the AT₂ receptor antagonist, PD123319. In vivo, Compound 21 administered alone, at doses ranging from 50 to 1000 ng·kg⁻¹·min⁻¹ over 4 h did not decrease blood pressure in conscious normotensive Wistar-Kyoto rats or SHR. However, when given in combination with the AT₁ receptor antagonist, candesartan, Compound 21 (300 ng·kg⁻¹·min⁻¹) lowered blood pressure in SHR only. Further analysis in separate groups of conscious SHR revealed that, at a sixfold lower dose, Compound 21 (50 ng·kg⁻¹·min⁻¹) still evoked a significant depressor response in adult SHR (∼30 mmHg) when combined with different doses of candesartan (0.01 or 0.1 mg·kg⁻¹). Moreover, the Compound 21-evoked depressor effect was abolished when co-infused (50 µg·kg⁻¹·min⁻¹ for 2 h) with the AT₂ receptor antagonist PD123319.

Conclusion and implications:

Collectively, our results indicate that acute administration of Compound 21 evoked blood pressure reductions via AT₂ receptor stimulation. Thus Compound 21 can be considered an excellent drug candidate for further study of AT₂ receptor function in cardiovascular disease.