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Background and purpose: Endothelin-1 (ET-1) is present in murine and human skin and causes itch (pruritus) when injected in humans. This behavioural study examined the scratch reflex evoked by ET-1 in mice. Experimental approach: An automated detector was used to determine whether ET-1 causes reflex scratching, the behavioural correlate of itching, in BALB/c mice. Selective agonists and antagonists were used to probe the ET receptor(s) involved. Key results: ET-1 evoked dose-related reflex scratching lasting up to 20 min following intradermal injection (0.1-100 ng; 0.04-40 pmol). The ED50 for ET-1 induced scratching was 2.1 ng and desensitization occurred with cumulative dosing. High doses of the ETB receptor agonist IRL1620 (10 μg; 5.5 nmol), also caused scratching (ED50 1.3 μg, 0.7 nmol). The ETA receptor antagonist BQ123 significantly reduced scratching evoked by ET-1 and IRL 1620, suggesting that both agonists caused scratching via an ETA receptor-dependent mechanism. The ETB receptor antagonist BQ788 significantly reduced scratching evoked by IRL1620 but had no effect on scratching evoked by ET-1. This indicated that activation of ETB receptors by high doses of ETB agonist, but not ET-1, can trigger scratching. Conclusion and implications: ET-1 is a potent endogenous activator of reflex scratching (itch). Mechanisms for ET-induced scratching are considered, including direct action of ET-1 on pruriceptive nerve endings and indirect actions via release of endogenous mediators such as histamine from mast cells. ET-1 and ETA receptors, possibly also ETB receptors, are potential targets for developing specific anti-pruritic drugs to treat pruritic skin disorders such as atopic dermatitis. British Journal of Pharmacology (2007) 151, 278–284. doi:10.1038/sj.bjp.0707216

We have designed an economical non-invasive movement detector for small animal studies and used it for monitoring and quantifying itch in mice. The system is based on a sensitive force transducer positioned below a recording platform holding a lightweight polystyrene recording box in which an animal is placed. A programmed micro-controller is used to discriminate between non-specific movement, grooming behaviour, and scratching movements made by the animal's hind limb. Following sub-dermal injection of histamine receptor agonists into the neck of a mouse, dose-related scratching occurred which was detected and quantified. There was 91% correlation between bouts of scratching as counted manually from playback of the video recording and recorded by the detector. The detector was also able rapidly to count the individual scratch movements of the hind limb that comprise a bout, with 95% accuracy in comparison with manual counting during slow motion playback of video tape, something that is impossible for an unaided observer to achieve because individual scratch movements are too fast to discriminate by eye. Separate detectors were used for the efficient non-invasive study of four animals simultaneously, and this number could easily be increased by adding more platforms. The system could also be modified to record the animal's position within the box, which would be of value in studies involving exploratory behaviour. In summary, the non-invasive multichannel repetitive movement detector will be very useful for accurate measurement of scratching during pruritus studies in small animals, with considerable savings in staff time and effort. It should therefore be a valuable tool for helping to investigate pruritus and in the evaluation of anti-pruritic drugs.

Neuropeptides synthesized in dorsal root ganglia (DRG) have been implicated in neurogenic inflammation and nociception in experimental and clinical inflammatory arthritis. We examined the very early changes in response to adjuvant injection in a rat model of unilateral tibio-tarsal joint inflammation and subsequent monoarthritis. Within 30 min of adjuvant injection ipsilateral swelling and hyperalgesia were apparent, and marked increases in β-preprotachykinin-A (β-PPT-A) and α-calcitonin gene-related peptide (CGRP)-encoding mRNAs were observed in small-diameter L5 DRG neurones innervating the affected joint. This response was augmented by recruitment of additional small-diameter DRG neurones expressing β-PPT-A and CGRP transcripts. The increased mRNA was paralleled by initial increases in L5 DRG content of the protein products, substance P and calcitonin gene-related peptide. Within 15 min of adjuvant injection there were increases in electrical activity in sensory nerves innervating a joint. Blockade of this activity prevented the rapid induction in β-PPT-A and CGRP mRNA expression in DRG neurones. Increased expression of heteronuclear (intron E) β-PPT-A RNA suggests that increases in β-PPT-A mRNA levels were, at least in part, due to transcription. Pre-treatment with the protein synthesis inhibitor cycloheximide had no effect upon the early rise in neuropeptide mRNAs. This and the rapid time course of these changes suggest that increased sensory neural discharge and activation of a latent modulator of transcription are involved.

If one drug, B, antagonizes another, A, by producing the opposite physiological effect, the antagonist concentration-effect curves should be affected by the recording system, which limits the range of agonist responses. With pieces of isolated guinea-pig ileum taken from adjacent parts of the same animal, one recorded isotonically, the other isometrically with the same load, the isotonic IC50 values for (−)isoprenaline opposing carbachol or histamine were lower than the isometric values (P

1. Experiments carried out on guinea-pig isolated ileum with carbachol as agonist and diphenyl-acetoxyethyl-dimethyl-ethyl-ammonium (DADMEA) bromide as antagonist gave results which fit the theoretical relation between fractional inhibition (Q) of the effects of an agonist ([A]) and the concentration of a competitive antagonist ([B]): this also involves the Hill coefficient (logistic slope factor, P) for the agonist concentration-response curve and the degree of agonist stimulation, [A]/[A]50, where [A]50 produces a half-maximum response. 2. Values of IC50 and an exponent, P', can be obtained by fitting Q to [B] using a logistic approximation to the relation. Both P' and IC50 should be greater with higher agonist stimulation but the increase in P' may be masked by errors in extreme values of Q. Estimates of IC50, however, invariably increased with higher agonist stimulation but with a steep concentration-response curve (P > 1) and low agonist stimulation ([A]/[A]50 < 1, IC50 can be less than KD. 3. KD was calculated from the results in three ways; (i) by a least-squares fit of Q to [B] using the values of P and [A]/[A]50 calculated from the control concentration-response curve; (ii) from the value of IC50 for each line and the values of P and [A]/[A]50 and (iii) by using the agonist concentration-response curve to calculate the dose-ratio and estimate of KD for each in the presence of the antagonist. The methods gave similar results (nM: 11 experiments), 12.4 +/- 1.1 (i), 11.7 +/- 0.9 (ii), 14.8 +/- 1.6 (iii) but there are advantages in using methods (i) or (ii) rather than (iii). 4. The method by which KD is calculated is less important than the experimental design: the plan used in this work, with alternative small and large responses from the tissue, is very suitable for estimating KD with low concentrations of antagonists and small dose-ratios. Although it is not a sensitive test for competitive behaviour because only a small range of concentrations of antagonist is tested, the estimate of affinity should be free from complications involved in the use of higher concentrations of antagonist (and agonist) and the nature of the antagonism can always be checked by doing further experiments in the presence of a known competitive antagonist.

1. 4-Diphenylacetoxy-N-cyclohexylmethyl-piperidine HCl (hexahydro-benz-4DAP) is more active as an antagonist of carbachol at receptors in guinea-pig isolated ileum, log K (pA2) = 6.64 +/- 0.14 (s.e. 7 results), than at receptors in guinea-pig isolated atria, log K = 5.43 +/- 0.14 (7). In the presence of neostigmine bromide (0.2 microM) the value for atria was 5.62 +/- 0.19 (4), so the lower activity on atria cannot be attributed to hydrolysis of the compound by cholinesterases present in this tissue. 2. The limit of solubility of the free base in Krebs solution (pH 7.6) is about 50 microM for both hexahydrobenz-4DAP and benzyl-fourdapine (benz-4DAP). 3. In experiments on guinea-pig isolated ileum with hexahydro-benz-4DAP given together with 4-DAMP methobromide, the combined dose-ratio was consistent with competition: similar results were obtained with benz-4DAP. 4. In rats anaesthetized with pentobarbitone, hexahydro-benz-4DAP antagonized the effects of bethanechol on blood-pressure in doses that had little effect on heart rate or airflow. There was a limit to the effect which could be obtained, however, and the slopes of the Schild plots were less than one. The effects on rat blood-pressure had a half-life of at least 30 min. 5. In similar experiments with zamifenacin the slopes of the Schild plots were close to 1 and the compound was 10 to 20 times as active on blood-pressure at it was on heart-rate. 6. The limited solubility of the base probably accounts for the flat Schild plots obtained with hexahydro-benz-4DAP, which had about 10 fold selectivity for effects on blood-pressure and was more active than expected from tests on isolated ileum.

Background: Reducing the excessive accumulation of amyloid β-protein (Aβ) in Alzheimer's disease (AD) is a key objective of most AD therapies. Several studies suggest that pharmacological inhibition of angiotensin-converting enzyme (ACE) or its by-product angiotensin II may delay onset or progression of dementia and it has been suggested that this occurs via regulation of Aβ. Intraneuronal oligomeric accumulation of Aβ is postulated to be one of the earliest pathological events. Thus this study investigated the effect of an ACE-inhibitor, captopril, and two angiotensin II receptor blockers (ARBs), eprosartan and valsartan, on intraneuronal Aβ pathology and oligomeric Aβ levels in a triple transgenic (3xTGAD) mouse model of AD. Methods: Male, adult (3-4 month old) 3xTgAD mice (n=39) were randomly assigned to 4 treatment groups: valsartan (0.17g/l), eprosartan (0.8g/l), captopril (5g/l) or normal drinking water and the drugs given ad libitum for 2 months. Mean arterial blood pressure (MABP) was measured at baseline, at 2 weeks and at 2 months when the mice were sacrificed and the brains hemisected for analysis. One hemisphere was processed for Aβ and amyloid precursor protein (APP) immunohistochemistry and the other for biochemical measurement of oligomeric Aβ and APP. ACE activity was measured in the brain and kidney. Results: MABP was significantly reduced at 2 weeks and 2 months in the ACE-I group (p=0.0006) but was unaltered in the ARB groups compared to vehicle. Neither ACE-I nor ARB treatment altered Aβ and APP immunolabelling or the level of Aβ or APP in brain tissue homogenates. Similarly neither ACE-I nor ARB treatment altered ACE activity in either brain or kidney compared to control tissue. Conclusions: ACE-I or ARB administration over 2 months did not affect APP levels or either intraneuronal Aβ or oligomeric Aβ levels in 3xTGAD mice. While ARBs did not alter MABP, captopril did mediate reductions in MABP in the 3xTGAD mice which appeared to be independent of ACE activity. Further studies are needed to examine the effects of these drugs over a longer term and in older mice (i.e. when AD-like changes are more pronounced).

1. 4-Diphenylacetoxy-1:1-dimethyl cyclohexane (carbo-4-DAMP) is the carbon analogue of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide. The compounds differ only in that the quaternary nitrogen atom in 4-DAMP methiodide is replaced by a quaternary carbon atom, which is uncharged. 2. Carbo-4-DAMP appears to act competitively at functional (M3) muscarinic receptors in guinea-pig ileum. Estimates of log affinity constant are 6.0 at 30 degrees C and 5.9 at 37 degrees C, i.e. the compound has 0.1% of the affinity of 4-DAMP methobromide. 3. The absence of charge makes little difference to the conformation as determined by X-ray crystallography. The bond lengths and angles are very similar, though the bonds in the cyclohexane ring of carbo-4-DAMP are consistently slightly longer than those in the piperidinium ring of 4-DAMP methiodide, and the presence of the charge slightly reduces the space between molecules. 4. The difference between the affinities of 4-DAMP methobromide and carbo-4-DAMP indicates that the contribution of coulombic forces to the binding between 4-DAMP methiodide and muscarinic (M3) receptors is at least 17 kJ mol-1 (4.1 kcal mol-1) at 37 degrees C. How much this is an underestimate depends upon how much hydrophobic binding is greater with the uncharged compound.

Chronic joint pain affects physical well being and can lead to severe psychological and social problems, therefore successful long-term management is highly sought-after. No current behavioural measures of pain used in pre-clinical models mimic the clinical dolorimeter, which provides an objective measure of joint hypersensitivity. In this study we aim to use a novel behavioural readout alongside an established measure to mimic the multifactorial measurements taken in the clinic. Using the pressure application measurement (PAM) device a gradually increasing squeeze was applied across the knee joint of rats until the animal gave an indication of pain or discomfort. PAM and the incapacitance tester were used to detect joint hypersensitivity in a well-established rodent model of adjuvant-induced arthritis. Subsequently, the analgesic effects of prednisolone (1, 3 or 10 mg kg(-1)), morphine (3 mg kg(-1)) and celecoxib (15 mg kg(-1)) were assessed. Both PAM and the incapacitance tester detected a reversal of hypersensitivity 1h post-drug administration. Furthermore, the two readouts were highly correlated, and power analysis indicated that PAM was highly reproducible. In conclusion, PAM provides a novel, accurate behavioural tool for detecting a primary mechanical hypersensitivity in a rat model of chronic inflammatory joint pain.

To investigate the role of the vagus nerve in acute inflammatory and cardiorespiratory responses to diesel particulate (DP) in the rat airway, we measured changes in respiration, blood pressure and neutrophils in lungs of urethane anesthetized Wistar rats 6-h post-instillation of DP (500 {mu}g) and studied the effect of mid-cervical vagotomy or atropine (1 mg kg{sup -1}) pre-treatment. In conscious rats, we investigated DP, with and without atropine pre-treatment. DP increased neutrophil level in BAL (bronchoalveolar lavage) fluid from intact anesthetized rats to 2.5 {+-} 0.7 x 10{sup 6} cells (n = 8), compared with saline instillation (0.3 {+-} 0.1 x 10{sup 6}, n = 7; P < 0.05). Vagotomy reduced DP neutrophilia to 0.8 {+-} 0.2 x 10{sup 6} cells (n = 8; P < 0.05 vs. intact); atropine reduced DP-induced neutrophilia to 0.3 {+-} 0.2 x 10{sup 6} (n = 4; P < 0.05). In conscious rats, DP neutrophilia of 8.5 {+-} 1.8 x 10{sup 6}, n = 4, was reduced by pre-treatment with atropine to 2.2 {+-} 1.2 x 10{sup 6} cells, n = 3. Hyperventilation occurred 6 h after DP in anesthetized rats with intact vagi, but not in bilaterally vagotomized or atropine pre-treated animals andmore » was abolished by vagotomy (P < 0.05, paired test). There were no significant differences in the other variables (mean blood pressure, heart rate and heart rate variability) measured before and 360 min after DP. In conclusion, DP activates a pro-inflammatory vago-vagal reflex which is reduced by atropine. Muscarinic ACh receptors in the rat lung are involved in DP-induced neutrophilia, and hence muscarinic antagonists may reduce airway and/or cardiovascular inflammation evoked by inhaled atmospheric DP in susceptible individuals.« less

The results of vasorespiratory studies in rats anaesthetised with pentobarbital show that (+/-) cannabidiol, a cannabinoid that lacks psychotropic actions and is inactive at cannabinoid (CB) receptors, does not affect respiration or blood pressure when injected (1-2000 microg; 3.2-6360 nmol i.a.). Cannabidiol in doses up to 2 mg (6360 nmol) i.a. or i.v. did not affect the fall in mean blood pressure or the increase in ventilation (respiratory minute volume) caused by capsaicin and high doses of anandamide, responses that are mediated by activation of vanilloid VR1 (TRPV1) receptors in this species. Similar results were obtained with (-) cannabidiol (30-100 microg i.a.; 95-318 nmol). It has previously been shown using human embryonic kidney (HEK) cells over-expressing vanilloid human VR1 (hVR1) receptors that cannabidiol is a full agonist at vanilloid VR1 receptors in vitro. However, in the intact rat cannabidiol lacked vanilloid VR1 receptor agonist effects. We conclude that there are substantial functional differences between human and rat vanilloid VR1 receptors with respect to the actions of cannabidiol as an agonist at vanilloid VR1 receptors. Studies in vivo show that cannabidiol lacks any significant effect on mean blood pressure or respiratory minute volume when injected i.a. or i.v., and that this cannabinoid does not modulate the vanilloid VR1 receptor-mediated cardiovascular and ventilatory changes reflexly evoked by capsaicin or anandamide in rats anaesthetised with pentobarbital.

1. We tested the hypothesis that activation of P2X receptors associated with vagal afferent nerves can evoke a Bezold-Jarisch (B-J) depressor reflex in anaesthetized rats. 2. Injection of alphabeta-methylene ATP (alphabeta-MeATP; 0.6-600 nmol i.v.) evoked a dose-dependent B-J reflex comprising bradycardia, hypotension and apnoea in rats anaesthetized with pentobarbitone. Apnoea was commonly preceded by hyperventilation. Bilateral vagotomy significantly reduced the bradycardia and most of the apnoeic response without affecting hyperventilation, and unmasked a vasopressor response. Hypotension and apnoea were subject to desensitization, and ATP was about 100 times less potent than alphabeta-MeATP in evoking the B-J reflex. 3. ED50 values for responses to alphabeta-MeATP were: bradycardia 14.6 +/- 3.8 nmol; apnoea 47.1 +/- 8.5 nmol; hyperventilation 23.3 +/- 6.0 nmol, n = 14. The ED50 for apnoea was significantly greater than that for bradycardia or hyperventilation (P0.05). Atropine (2.8 micromol (kg body wt)-1 i.v.) antagonized the reflex bradycardia and hypotension. 4. The P2 antagonists suramin (14 micromol (kg body wt)-1 i.v.) and PPADS (17 micromol (kg body wt)-1 i.v.) antagonized the bradycardic and apnoeic components of the reflex response to alphabeta-MeATP, without reducing the vasopressor or hyperventilatory responses to the agonist. 5. Recordings from vagal afferents showed that pulmonary inflation receptors were activated by alphabeta-MeATP in 62 % of units recorded (ED50 22 +/- 5 nmol) and this was blocked by PPADS (17 micromol (kg body wt)-1 i.v.); unidentified vagal afferents were also activated. 6. alphabeta-MeATP activated carotid chemoreceptor afferents (ED50 23 +/- 9 nmol), an action that was unaffected by PPADS or suramin. 7. The results support the hypothesis that P2X receptor subtypes for ATP are associated with specific sensory nerves that form part of the homeostatic mechanism for cardiovascular and respiratory regulation and these receptors therefore have physiological, pathological and therapeutic significance.

1 Baroreceptor and chemoreceptor reflex activity was studied in anaesthetized adult rats which had been treated neonatally with a single injection of capsaicin (50 mg/kg s.c.). 2 Pressor responses to bilateral carotid artery occlusion were significantly lower in capsaicin-treated rats compared with vehicle-treated controls. Pressor responses to intravenously injected noradrenaline were similar in the two groups of rats. 3 Resting respiratory minute volume and tidal volume were lower in anaesthetized capsaicin-treated animals than in vehicle-treated controls, but there was no significant difference in respiratory frequency. 4 The increases in respiration evoked by intravenous administration of the peripheral arterial chemoreceptor stimulant, sodium cyanide, or by breathing a hypoxic gas mixture, were significantly lower in capsaicin-treated rats compared with the controls. 5 It is concluded that baroreceptor and chemoreceptor reflex activity are significantly reduced in anaesthetized adult rats which had been treated neonatally with capsaicin, and that this is likely to result from the destruction of unmyelinated baro- and chemoreceptor afferent fibres.