Susan M. Bond

Adjuvant-induced joint inflammation causes very rapid transcription of β-preprotachykinin and α-CGRP genes in innervating sensory ganglia

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.

Endothelin-1 activates ETA receptors to cause reflex scratching in BALB/c mice

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.

INFLUENCE OF NEONATALLY ADMINISTERED CAPSAICIN ON BARORECEPTOR AND CHEMORECEPTOR REFLEXES IN THE ADULT RAT

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.

Pressure application measurement (PAM): A novel behavioural technique for measuring hypersensitivity in a rat model of joint pain

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.

Antagonist inhibition curves and the measurement of dissociation constants

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. 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. K D 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 response 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). 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.

A repetitive movement detector used for automatic monitoring and quantification of scratching in mice

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 animals 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 animals 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.

A comparison of effects measured with isotonic and isometric recording: I. Concentration-effect curves for agonists

Concentration‐effect curves were obtained with carbachol tested on isolated preparations of guinea‐pig ileum taken from adjacent sites in the same animal, one recorded isotonically, the other isometrically: similar experiments were made with histamine as agonist and with carbachol on rat uterus (in oestrus). The position and steepness of the curves was expressed as the values of [EC50] and the exponent, P: with carbachol or histamine on guinea‐pig ileum the curves were significantly steeper with isotonic recording (P<0.02, sign test) and displaced towards lower concentrations (P<0.005) but there were significant correlations (P<0.05) between values obtained with tissues from the same animal. The curves for carbachol on the rat uterus were very steep: with isotonic recording the exponent (often eight or more) was consistently higher than with isometric (P<0.001): there was no significant displacement but there was a significant correlation (P<0.05) between values of [EC50] obtained with tissues from the same animal. Although the results obtained by the two methods are different, they are correlated. These effects are to be expected because with isotonic recording there can be no change in length until the tension exceeds the load and the tissue bulk sets an upper limit to shortening: the range within which an effect can be measured (the ‘operational window’) is smaller. The observed effects on [EC50] and P have been reproduced with theoretical data.

A comparison of effects measured with isotonic and isometric recording: II. Concentration-effect curves for physiological antagonists.

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<0.01) but there was a significant correlation between them (P<0.01): the isotonic curves were steeper (P<0.01) and there were wider shifts in IC50 before increasing the agonist reduced the maximum relaxation. In similar experiments with pieces of rat uterus in oestrus from the same animal, the concentration‐effect curves for carbachol opposed by increasing concentrations of (−)isoprenaline or (−)adrenaline had slightly lower EC50 values with isometric recording but there was a significant correlation (P<0.01) with isotonic values. The antagonist effect (ratio of the EC50 relative to that for the control) was higher with isotonic recording (P<0.01 for (−)isoprenaline, P<0.025 for (−)adrenaline) and all (27) curves were steeper than the corresponding isometric curve (P<0.001). The influence of the method of recording on the results is expected from the narrower operational window and smaller upper limit to relaxation with isotonic recording. A way of obtaining measurements of IC50 against a standard agonist effect is suggested in an Appendix .

The contribution of charge to affinity at functional (M3) muscarinic receptors in guinea-pig ileum assessed from the effects of the carbon analogue of 4-DAMP methiodide.

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 quarternary 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°C and 5.9 at 37°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°C. How much this is an underestimate depends upon how much hydrophobic binding is greater with the uncharged compound.

Selective blockade of M2 and M3 muscarinic receptors by hexahydrobenzyl-fourdapine and a comparison with zamifenacin.

1 4‐Diphenylacetoxy‐N‐cyclohexylmethyl‐piperidine HC1 (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 μm) 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 μm for both hexahydro‐benz‐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.