Purinoceptor‐mediated,Capsaicin‐resistant Excitatory Effect of Allyl Isothiocyanate on Neurons of the Guinea‐Pig Small Intestine

Allyl isothiocyanate (AITC; 200 μM) caused atropine‐ and tetrodotoxin‐sensitive longitudinal muscle contraction on the guinea‐pig small intestine. The response was not influenced by hexamethonium, a functional blockade of capsaicin‐sensitive neurons or by antagonists acting at TRPV1 or TRPA1, but was abolished by the P₂ purinoceptor antagonist PPADS (50 μM). It is concluded that cholinergic motoneurons are activated by a purinergic mechanism in the course of the AITC response, independently of capsaicin‐sensitive processes or even TRPA1.     

Modulation of Capsaicin‐Sensitive Nerve Activation by Low pH Solutions in Guinea‐Pig Lung

Abstract: We have studied the stimulation of airways sensory nerves by low pH solutions and concomitantly induced bronchoconstriction. The effect of low pH buffer and lactic acid solutions at the same pH (5 and 6) were compared and the influence of low pH on the capsaicin effect was recorded. We have used the isolated guinea‐pig perfused lung model taking the insufflation pressure as an indicator of bronchial smooth muscle tone while the calcitonin gene‐related peptide‐like immunoreactivity measured in the lung perfusate represented sensory nerves activation. Low pH buffer and lactic acid solution (3 and 4.1 mM) at the same pH of 5 and 6 induced pH‐dependent bronchoconstriction and peptides release which were completely abolished after systemic pretreatment with capsaicin. Both responses were significantly inhibited after Ca²⁺‐free infusion. Capsazepine (10^?6 M), a selective capsaicin antagonist, significantly reduced the calcitonin gene‐related peptide‐like immunoreactivity overflow evoked by all the solutions studied. Diclofenac (10^?5 M), a cyclooxygenase blocker, inhibited pH 5, pH 6 and lactic acid 3 mM (pH 6)‐evoked peptide release, but not lactic acid 4.1 mM (pH 5). The functional response was not significantly modified after diclofenac while only the lactic acid 3 mM response was significantly reduced by capsazepine. There was a synergistic interaction between capsaicin and low pH buffer on calcitonin gene‐related peptide‐like immunoreactivity release and an additive effect on bronchoconstriction. It is concluded that in the isolated perfused guinea‐pig lung, lactic acid and low pH buffer induced calcitonin gene‐related peptide‐like immunoreactivity release and bronchoconstriction by stimulation of capsaicin‐sensitive C fibres via a pathway partly dependent of extracellular Ca²⁺. The mechanism of calcitonin gene‐related peptide‐like immunoreactivity release seems to be the same at pH 6, while differences are evident at pH 5 between low pH buffer and lactic acid. Our results also suggest that proton activity could exert a modulatory role on the capsaicin‐sensitive sensory nerves by a mechanism which remains to be clarified.     

Role of Pre‐Junctional CB1, But not CB2, TRPV1 or GPR55 Receptors in Anandamide‐Induced Inhibition of the Vasodepressor Sensory CGRPergic Outflow in Pithed Rats

Stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. Interestingly, endocannabinoids such as anandamide (which interacts with CB₁, CB₂, TRPV1 and GPR55 receptors) can regulate the activity of perivascular sensory nerves in dural blood vessels by modulating CGRP release. Yet, as no publication has reported whether this mechanism is operative in the healthy systemic vasculature, this study has specifically analysed the receptors mediating the potential inhibitory effects of the cannabinoid (CB) receptor agonists anandamide (non‐selective), JWH‐015 (CB₂) and lysophosphatidylinositol (GPR55) on the rat vasodepressor sensory CGRPergic outflow (an index of systemic vasodilatation). Healthy pithed rats were pre‐treated with consecutive i.v. continuous infusions of hexamethonium, methoxamine and the above agonists. Electrical spinal (T₉–T₁₂) stimulation of the vasodepressor sensory CGRPergic outflow or i.v. injections of α–CGRP produced frequency‐dependent or dose‐dependent vasodepressor responses. The infusions of anandamide in a dose‐dependent manner inhibited the vasodepressor responses by electrical stimulation (remaining unaffected by JWH‐015 or lysophosphatidylinositol), but not those by α–CGRP. After i.v. administration of antagonists, the inhibition by 3.1 μg/kg min anandamide was: (i) potently blocked by 31–100 μg/kg NIDA41020 (CB₁), (ii) unaffected by 180 μg/kg AM630 (CB₂), 31 μg/kg cannabidiol (GPR55) or 31–100 μg/kg capsazepine (TRPV1) and (iii) slightly blocked by 310 μg/kg AM630. The above doses of antagonists were enough to block their respective receptors. These results suggest that anandamide‐induced inhibition of the vasodepressor sensory CGRPergic outflow is mainly mediated by pre‐junctional activation of CB₁ receptors, with no pharmacological evidence for the role of CB₂, TRPV1 or GPR55 receptors.     

Donepezil‐ or rivastigmine‐induced acetylcholinesterase inactivation is not modulated by neramexane in rat brain

In Alzheimer's disease (AD), the involvement of cholinergic deficit and overstimulation of N‐methyl‐D‐aspartate (NMDA) receptors by excessively released glutamate has been proposed. Since no existing drug currently addresses both pathologies, a combination therapy may be used clinically to target both mechanisms. The objective of the current study was to determine whether the NMDA receptor antagonist, neramexane affects the acetylcholinesterase (AChE) inhibition produced by donepezil and rivastigmine, two drugs used in the treatment of AD, or a prototype organophosphate compound diisopropylphosphorofluoridate (DFP) in rat cortex, hippocampus, striatum, and brainstem. Neramexane, at therapeutically relevant or higher doses (3.1, 6.2, or 12.4 mg/kg, ip), did not produce any apparent behavioral toxicity. Donepezil (0.75 mg/kg, ip), rivastigmine (0.35 mg/kg, ip) or DFP (1.5 mg/kg, ip) were administered at doses that produced approximately 50% inhibition of AChE and were well tolerated. Also, neramexane in combination with either of the AChE inhibitors did not produce any behavioral toxicity. This magnitude of AChE inhibition has been shown to increase extracellular acetylcholine (ACh) concentrations to therapeutically relevant levels. Neramexane (12.4 mg/kg) did not inhibit AChE activity in any of the brain regions tested when evaluated over a 24‐h period. Rats receiving neramexane alone or in combination with an AChE inhibitor (donepezil, rivastigmine, or DFP) were sacrificed 60 min after neramexane or DFP, 15 min after donepezil, and 30 min after rivastigmine administration. The times at which drug (donepezil, rivastigmine, or DFP) effects were assessed were based on maximal AChE inhibition established from previously reported time course studies. The current findings indicate that neramexane alone did not affect AChE activity at any dose tested nor did it influence the AChE inhibition produced by donepezil or rivastigmine in any brain region examined. However, a low dose of neramexane (3.1 mg/kg) significantly attenuated the inhibition of AChE produced by DFP in the hippocampus, striatum and brainstem, while higher doses (6.2 or 12.4 mg/kg) attenuated DFP‐induced AChE inhibition in all four of the brain regions evaluated. These results suggest that AChE inhibition produced by donepezil or rivastigmine is not altered by the co‐administration of neramexane. Drug Dev Res 68:253–260, 2007. © 2007 Wiley‐Liss, Inc.     

Inhibition of platelet aggregation by vanilloid‐like agents is not mediated by transient receptor potential vanilloid‐1 channels or cannabinoid receptors

Vanilloid‐like agents, including capsaicin, N‐arachidonoyl‐dopamine and N‐oleoyldopamine inhibit platelet aggregation, however little is known about the precise mechanism(s) of action. The authors have previously shown that blocking of the capsaicin receptor, transient receptor potential vanilloid‐1 (TRPV1), does not interfere with capsaicin action during adenosine diphosphate (ADP)‐induced aggregation. This research is extended to investigate the effect of these vanilloid‐like‐agents on platelet count, and to test whether the effect of these agents is mediated through TRPV1 and/or cannabinoid (CB1 and CB2) receptors in the presence of other agonists, including collagen and arachidonic acid. Incubation of platelets with each of the individual vanilloids, or with receptor antagonists of TRPV1 (SB452533), CB1 (AM251) and CB2 (AM630), for up to 2 h did not significantly affect the platelet count. Similarly, the effect of individual vanilloids on the inhibition of platelet aggregation was not significantly different in the presence of receptor agonists compared to control, irrespective of the agonist used, suggesting that the inhibitory effect of vanilloids on platelet aggregation is independent of TRPV1, CB1 and CB2 receptors. Further research on the antiplatelet activity of vanilloids should focus on mechanisms other than those associated with vanilloid receptors.     

Conditioned hyperkinesia induced by cocaine in mice is dose-dependent but not correlated with the unconditioned response or the contextually-sensitized response

The aims of the study were to test whether drug dose is positively related to the magnitude of the conditioned response following sensitization to the behavioural effects of cocaine and to investigate the relationship between the conditioned response and cocaine-induced sensitization. Male mice (C57BL/6J) were first injected over seven successive days with either saline or cocaine at 2.5, 5, 10 or 20 mg/kg s.c., in the testing room. On the test day, 24 h after the last injection, mice from all conditions were challenged with saline in the testing room to test for conditioned cocaine effects. Mice were video-recorded and various behaviours were later scored using a time-sampling technique. Cocaine-elicited orofacial stereotypy was significantly sensitized at the two highest doses and dose-dependently conditioned at the three highest doses. Cocaine-increased locomotion was sensitized at the three highest doses and significantly conditioned at 10 and 20mg/kg. Cocaine-increased sniffing did not change over pretreatment at any dose, and was conditioned only at 10 mg/kg. Cocaine-decreased immobility also did not change over pretreatment at any dose, but was conditioned at 10 and 20mg/kg. Concomitantly, rearing was reduced by cocaine at 10 and 20mg/kg, without sensitization being induced, and it was reduced under saline challenge after 5 mg/kg cocaine, while cocaine-decreased grooming was sensitized at the three highest doses and conditioned at 10 and 20 mg/kg cocaine. There was a positive relation between the size of the conditioned response for orofacial stereotypy and the magnitude of the unconditioned stimulus (the doses), a result conforming to the Pavlovian account of the placebo effect. This could also be concluded from considering the behaviour patterns as components of a unique placebo effect (hyperkinetic syndrome), since orofacial stereotypy, very apparent at 20 mg/kg cocaine, interfered at that dose with the full-blown expression of locomotion and sniffing, both yielding (approximately) inverted U-shaped dose-effect curves. However, no correlation was found between the magnitude of the conditioned response and the amplitude of sensitization (the difference between the initial unconditioned non-sensitized response and the last unconditioned sensitized response), a finding which indicates that conditioned responding does not participate in the generation of the sensitized effects, contrary to the 'excitatory conditioning model of contextual sensitization'.     

The Prejunctional Inhibitory Effect of α-Methylnoradrenaline in the Rat Vas Deferens is Not Mediated via α2-Adrenoceptors

In field-stimulated rat vas deferens, clonidine (10^?9–10^?7 m ) and α-methylnoradrenaline (10^?7–3 times 10^?5 m ) concentration-dependently inhibited twitch response to electrical field stimulation. The inhibitory effects of clonidine and α-methylnoradrenaline were similarly reduced by phenoxybenzamine (10^?6 m ). However, idazoxan (3 times 10^?7 m ) antagonized clonidine but not α-methylnoradrenaline-induced responses. The inhibitory effect of α-methylnoradrenaline was also not antagonized by phentolamine (3 times 10^?7 m ), SK&F 104856 (2-vinyl-7-chloro-3,4,5,6-tetrahydro-4-methylthienol[4,3,2 ef][3]benzazepine) 3 times 10^?6 m ) or yohimbine 3 times 10^?7 m ). These antagonists however, attenuated the twitch-inhibiting effect of clonidine. The -logK_B values were 8·02 ± 0·05, 6·91 ± 0·10, and 7·58 ± 0·07 for phentolamine, SK&F 104856 and yohimbine respectively. In-vivo treatment of rats with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (6 mg kg^?1), to inactivate prejunctional α₂-adrenoceptors, attenuated clonidine, but not α-methylnoradrenaline-induced responses. It was concluded that adrenoceptors are not involved in the prejunctional inhibitory effects of α-methylnoradrenaline in the rat vas deferens.     

The genetic ablation or pharmacological inhibition of TRPV1 signalling is beneficial for the restoration of quiescent osteoclast activity in ovariectomized mice

Background and Purpose

Osteoporosis is a condition characterized by a decrease in bone density, which decreases its strength and results in fragile bones. The endocannabinoid/endovanilloid system has been shown to be involved in the regulation of skeletal remodelling. The aim of this study was to investigate the possible modulation of bone mass mediated by the transient receptor potential vanilloid type 1 channel (TRPV1) in vivo and in vitro.

Experimental Approach

A multidisciplinary approach, including biomolecular, biochemical and morphological analysis, was used to investigate the involvement of TRPV1 in changes in bone density in vivo and osteoclast activity in vitro, in wild-type and Trpv1^−/− mice, that had undergone ovariectomy or had a sham operation.

Key Results

Genetic deletion of Trpv1 as well as pharmacological inhibition/desensitization of TRPV1 signalling dramatically reduced the osteoclast activity in vitro and prevented the ovariectomy-induced bone loss in vivo, whereas the expression of cannabinoid type 2 (CB₂) receptors was increased.

Conclusions and Implications

These findings highlight the pivotal role TRPV1 channels play in bone resorption and suggest a possible cross-talk between TRPV1 and CB₂ receptors. Based on these results, hybrid compounds acting on both TRPV1 and CB₂ receptors in an opposite manner could provide a future pharmacological tool for the treatment of diseases associated with disturbances in the bone remodelling process.

Linked Articles

This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10     

Fentanyl Pharmacokinetics is not Dependent on Hepatic Uptake by Organic Anion‐Transporting Polypeptide 1B1 in Human Beings

A recent study investigating the pharmacokinetics of fentanyl in Sprague–Dawley rats suggested fentanyl to be a substrate of rat organic anion‐transporting polypeptide Oatp. In human beings, the most important OATP for the pharmacokinetics of many drugs is OATP1B1. Therefore, genetic variants of OATP1B1 (SLCO1B1) might modulate fentanyl pharmacokinetics and efficacy in human beings. Sixteen healthy male and female volunteers, homozygous for SLCO1B1*1a (genetic wild‐type) (n = 11) or *15 (deficient haplotype carrying the single‐nucleotide polymorphisms rs2306283 and rs4149056 and exhibiting altered transport activity; n = 5), were included in this randomized crossover study. The participants received fentanyl (5 μg/kg) intravenously alone or with the OATP inhibitor rifampicin (600 mg single oral dose). The pharmacokinetics of fentanyl and norfentanyl were determined by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). In addition, fentanyl uptake in vitro was evaluated in OATP1B1 overexpressing HEK293 cells and compared to a mock‐transfected cell line. In the clinical trial, fentanyl clearance was 18.8 ± 8.2 mL/min. kg in SLCO1B1*1a and 19.5 ± 1.8 mL/min/kg in SLCO1B1*15 carriers and not significantly different between the genotypes. During rifampicin, fentanyl clearance was 15.0 ± 4.4 mL/min/kg in SLCO1B1*1a and 16.7 ± 5.9 mL/min/kg in SLCO1B1*15 carriers (p > 0.5). In addition, in vitro data also indicate that fentanyl is not transported by OATP1B1. In conclusion, our data indicate that OATP1B1 has no impact on fentanyl pharmacokinetics in human beings.     

'Compulsive' lever-pressing in rats is attenuated by the serotonin re-uptake inhibitors paroxetine and fluvoxamine but not by the tricyclic antidepressant desipramine or the anxiolytic diazepam

Rats undergoing extinction of lever-pressing for food after the attenuation of an external feedback for this behavior, exhibit excessive lever-pressing unaccompanied by an attempt to collect a reward, which may be analogous to the excessive and unreasonable behavior seen in obsessive-compulsive disorder (OCD). Given that one of the most salient features of OCD is its selective response to treatment with serotonin re-uptake inhibitors (SRIs), the present study compared the effects of the SRIs paroxetine and fluvoxamine on compulsive lever-pressing, with those of the tricyclic antidepressant, desipramine, and the benzodiazepine, diazepam, which are not effective in the treatment of OCD. Paroxetine (1-15 mg/kg) and fluvoxamine (10-20 mg/kg) dose-dependently reduced the number of compulsive lever-presses and the number of lever-presses followed by an attempt to collect a reward; desipramine (5-15 mg/kg) dose-dependently reduced only the number of lever-presses followed by an attempt to collect a reward; diazepam (2-10 mg/kg) did not affect either type of lever-pressing, except for the highest dose (10 mg/kg), which almost completely abolished lever-press responding. When administered in an extinction session not preceded by signal attenuation, paroxetine, fluvoxamine and desipramine affected only the number of lever-presses followed by an attempt to collect a reward, whereas diazepam (4-8 mg/kg) decreased both types of lever-presses. The present findings strengthen the suggestion that compulsive lever-pressing may serve to model compulsive behavior in OCD, and lends the model predictive validity.     

Alpha1-receptor or adenosine A1-receptor dependent pathway alone is not sufficient but summation of these pathways is required to achieve an ischaemic preconditioning effect in rabbits

1. It is reported that alpha1-receptors and adenosine A1-receptors are involved in the ischaemic preconditioning (PC) effect on infarct size (IS). However, it is still unclear to what extent alpha1-receptors and adenosine A1-receptors contribute to the mechanism of PC. Therefore, we investigated the extent of the contribution of alpha1-receptors and adenosine A1 receptors to the PC effect on IS and examined the relationship between these receptors and protein kinase C. 2. Infarct size was measured in rabbits subjected to 30 min ischaemia and 48 h reperfusion. Tyramine (Tyr) was intravenously administered before 30 min ischaemia in the absence or presence of bunazosin (BN, alpha1-receptor blocker) and staurosporine (ST), a protein kinase C inhibitor, respectively. R(-)N6-(2-phenylisapropyl)-adenosine (PIA), a selective adenosine A1 agonist, was intravenously administered before 30 min ischaemia in the absence or presence of 8-p-sulphophenyltheophylline (8SPT), an adenosine blocker and ST, respectively. In the PC groups, BN, BN + PIA, 8SPT, 8SPT + Tyr or placebo saline was injected before or during PC. 3. Both Tyr and PIA reduced the IS, which was blocked by BN and 8SPT, respectively. The IS-reducing effect of Tyr or PIA was blocked by ST. The IS-reducing effect of PC was completely blocked by BN and 8SPT, respectively. The blocking effect of BN on the IS-reducing effect of PC was abolished by adding PIA during PC ischaemia. The blocking effect of 8SPT on the IS-reducing effect of PC was abolished by adding Tyr before PC ischaemia. 4. These data suggest that an alpha1-receptor dependent pathway exists and an adenosine A1-receptor dependent pathway, stimulation of both of which activates protein kinase C, then reduces the IS. However, exclusive stimulation of a single alpha1-receptor dependent pathway or a single adenosine A1-receptor dependent pathway alone is not sufficient but the summation of these pathways is required to achieve a PC effect on IS in rabbits.     

Interaction of morphine but not fentanyl with cerebral α2‐adrenoceptors in α2‐adrenoceptor knockout mice

Objectives α₂‐Adrenergic and μ‐opioid receptors belong to the rhodopsin family of G‐protein coupled receptors and mediate antinociceptive effects via similar signal transduction pathways. Previous studies have revealed direct functional interactions between both receptor systems including synergistic and additive effects. To evaluate underlying mechanisms, we have studied whether morphine and fentanyl interacted with α₂‐adrenoceptor‐subtypes in mice lacking one individual α₂‐adrenoceptor‐subtype (α₂‐adrenoceptor knockout). Methods Opioid interaction with α₂‐adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of α_2A‐, α_2B‐ or α_2C‐adrenoceptor deficient mice. Displacement of the radiolabelled α₂‐adrenoceptor agonist [¹²⁵I]paraiodoclonidine from α₂‐adrenoceptors in different brain regions by increasing concentrations of morphine, fentanyl and naloxone was analysed. The binding affinity of both opioids to α₂‐adrenoceptor subtypes in different brain regions was quantified. Key findings Morphine but not fentanyl or naloxone provoked dose‐dependent displacement of [¹²⁵I]paraiodoclonidine from all α₂‐adrenoceptor subtypes in the brain regions analysed. Binding affinity was highest in cortex, medulla oblongata and pons of α_2A‐adrenoceptor knockout mice. Conclusions Our results indicated that morphine interacted with α₂‐adrenoceptors showing higher affinity for the α_2B and α_2C than for the α_2A subtype. In contrast, fentanyl and naloxone did not show any relevant affinity to α₂‐adrenoceptors. This effect may have an impact on the pharmacological actions of morphine.