Stefan Just

The CGRP receptor antagonist BIBN4096BS peripherally alleviates inflammatory pain in rats.

Summary Calcitonin gene–related peptide (CGRP) antagonism reduces inflammatory pain. A peripheral site of action is assumed, as blockage of peripheral CGRP receptors by BIBN4096BS leads to pronounced reduction of spinal neuronal activity. Abstract Calcitonin gene–related peptide (CGRP) is known to play a major role in the pathogenesis of pain syndromes, in particular migraine pain. Here we focus on its implication in a rat pain model of inflammation, induced by injection of complete Freund adjuvant (CFA). The nonpeptide CGRP receptor antagonist BIBN4096BS reduces migraine pain and trigeminal neuronal activity. Here we demonstrate that the compound reduces inflammatory pain and spinal neuronal activity. Behavioural experiments reveal a reversal of the CFA‐induced mechanical hypersensitivity and monoiodoacetate (MIA)‐induced weight‐bearing deficit in rats after systemic drug administration. To further investigate the mechanism of action of the CGRP antagonist in inflammatory pain, in vivo electrophysiological studies were performed in CFA‐injected rats. Recordings from wide dynamic range neurons in deep dorsal horn layers of the lumbar spinal cord confirmed a reduction of neuronal activity after systemic drug application. The same amount of reduction occurred after topical administration onto the paw, with resulting systemic plasma concentrations in the low nanomolar range. However, spinal administration of BIBN4096BS did not modify the neuronal activity in the CFA model. Peripheral blockade of CGRP receptors by BIBN4096BS significantly alleviates inflammatory pain.

Responses of fine primary afferent nerve fibres innervating the rat knee joint to defined torque.

In total 23 rat knee joint afferent units with conduction velocities of 1.2-17 m/s were recorded extracellularly during inward and outward joint rotations of defined torque using a newly developed torque-meter. There was not a linear relation between the torque and the obtained angle of the joint. The mechanical thresholds of the units ranged from 10 to 60 mNm. During 10 s of stimulation with 60 mNm they responded with 12-300 action potentials. Two mechanosensitive nerve fibres could not be activated via knee joint rotation at any torque at all. The units could be classified according to their response pattern during stimulation: (1) phasic-tonic response behaviour; (2) constant discharge rate; (3) delayed onset of activity. In conclusion, the novel torque-meter allows a precise characterisation of the mechanical threshold and the corresponding response of fine afferent joint units during knee joint rotation.

Galanin influences the mechanosensitivity of sensory endings in the rat knee joint

The aim of the present study was to examine the effect of galanin on group III and IV afferent nerve fibres (n = 53) innervating normal and acutely inflamed knee joints in rats. They responded to local mechanical stimulation, movements of the joint and i.a. injections of KCl close to the joint. Single i.a. bolus injections of galanin (0.1 mm, 0.2 mL) caused no direct responses of the units. In normal and acutely inflamed joints, about half of the units did not change the responses to knee joint rotation. A significant reduction of the responses to noxious movements was found in ∼ 40% of the units reaching a mean value of 57% in normal joints and 70% in inflamed joints compared with control movements. In ∼ 10% the responses increased to 143% in normal joints and 120% in inflamed joints. Injection of a galanin receptor antagonist (M35) doubled the responses to noxious movements in 36% of the units in normal joints and reduced it in 18% to 86% of the control movements, indicating a tonic release and influence on the mechanosensitivity of a proportion of primary afferents by galanin. In conclusion, these data further support the hypothesis that the mechanosensitivity of fine afferent nerve fibres is regulated by a mixture of different substances being released into the innervated tissue. Besides the action of several pro‐inflammatory peptides there seems to exist a tonic inhibitory system.

The role of CGRP and nicotinic receptors in centrally evoked facial blood flow changes

The release of CGRP in humans is associated with the occurrence of migraine headaches. The vasoactive neuropeptide is released by afferent neurones originating in the peripherally located trigeminal ganglion supplying the dura mater. The role of CGRP in migraine is further supported by recently released data showing that the CGRP-antagonist BIBN4096BS is clinically effective for the treatment of migraine headaches. Yet, the trigger for CGRP release during migraine attacks is not identified. It is suggested that the peripheral CGRP release during a migraine attack might be either triggered by direct activation of afferent dural neurones, or, by indirect activation via the central nervous system. Recently, we were able to show that the CGRP-antagonist BIBN4096BS is able to inhibit vasodilation induced by trigeminal ganglion stimulation. Now, we extend our studies to the investigation of facial blood flow changes induced by electrical stimulation of the brainstem trigeminal nucleus caudalis (TNC). Here, we show that stimulation of the TNC leads to a pronounced increase of facial blood flow. The nicotinic antagonist Hexamethonium reduced the evoked flow by approximately 50% (30 mg/kg), while the muscarinic antagonist Atropin did not influence the stimulation evoked blood flow. Application of BIBN4096BS (0.3 mg/kg, i.v.) diminished the evoked flow almost completely. Therefore, we conclude that CGRP represents the key player in TNC-induced facial vasodilation, while activation of nicotinic receptors modulates centrally induced peripheral neurogenic vasodilation.

MECHANOPROTECTIVE ACTIONS OF ELASTOVISCOUS HYLANS ON ARTICULAR PAIN RECEPTORS

ABSTRACT The sensitivity of articular nociceptors can be influenced directly by drugs either applied systemically or locally, and this is a very promising way to exert analgesic actions. For instance, Pozo et al. reported in 19971 that an elastoviscous solution of hylan, a hyaluronan derivative, significantly reduced both the ongoing activity and the movement evoked responses of primary afferent units from inflamed knee joints of the cat. These results support the assumption that hylans injected into the knee joint cavity may act as an elastoviscous filter, reducing the transmission of stretch to the nociceptive terminals where the mechano-electrical transduction takes place. More recently we have extended these observation to the knee joint of the rat. So far, with no appreciable exception, the results from nociceptors with fine afferent fibers (A δ- and C-fibers) of the medial articular nerve of the rat correspond to those obtained in the cat. The mechanoprotective effects found in the knee joint of rat are in the same order of magnitude as those obtained by systemic application of peripherally acting analgesic drugs. This duplication allows a further generalization of the results obtained in the cat. In addition it has opened the possibility to test in a much more controlled and systematic manner the effects of hylans and other substances with different elastic and viscous properties (alone or in connection with various analgesic drugs) on the discharge characteristics of nociceptors innervating normal and acutely inflamed knee joints

Histological demonstration of voltage dependent calcium channels on calcitonin gene-related peptide-immunoreactive nerve fibres in the mouse knee joint.

The afferent (excitability) and efferent functions (release of neuropeptides) of primary afferent nerve fibres are based on Ca(2+)-influx. The aim of the present study was to examine the presence of L- and N-type Ca(2+)-channels at sensory nerve fibres in the mouse knee joint capsule. Specific fluorescent labelled channel blockers and antisera against these channel subtypes were combined with an immunohistochemical staining for calcitonin gene-related peptide (CGRP), a neuropeptide that is widely distributed in primary afferents. There was a nearly complete colocalisation of CGRP immunoreactivity and the binding of omega-conotoxin GVIA (toxin VIA of Conus geographus or BODIPY-verapamil (BODIPY(R) FL verapamil, hydrochloride) demonstrating the presence of N-type and L-type Ca(2+)-channels, respectively. These data were further confirmed by identical results obtained after an immunohistochemical demonstration of the two channel subtypes at the peptidergic nerve fibres.

Hyoscine butylbromide potently blocks human nicotinic acetylcholine receptors in SH-SY5Y cells

Hyoscine butylbromide (HBB; tradenames: Buscopan/Buscapina is an antispasmodic drug for the treatment of abdominal pain associated with gastrointestinal cramping. As a hyoscine derivative, this compound competitively inhibits muscarinic acetylcholine (ACh) receptors on smooth muscle cells in the gastrointestinal tract. Preliminary investigations suggested that it might also inhibit nicotinic ACh receptors. This study investigated the effect of HBB on nicotinic ACh receptor-mediated membrane currents in SH-SY5Y cells. ACh and nicotine application-induced comparable membrane currents with EC(50) values of 25.9+/-0.6 and 40.1+/-0.4microM, respectively. When coapplied with 100microM ACh, HBB concentration-dependently suppressed currents with an IC(50) value of 0.19+/-0.04microM, and was approximately seven-times more potent than the ganglionic blocker, hexamethonium (IC(50)=1.3+/-0.3microM). Increasing the agonist concentration to 5mM did not affect the amount of block by HBB, which suggests a non-competitive mode of action. These functional in vitro data demonstrate for the first time that HBB blocks neuronal nicotinic ACh receptors in the same concentration range as it inhibits muscarinic ACh receptors. If one hypothesizes that HBB might also affect nicotinic receptors in autonomic neurons in vivo (e. g. in the enteric nervous system), this effect could contribute to its spasmolytic activity.

Neuropeotide Y changes the excitability of fine afferent units in the rat knee joint

The aim of the present study was to examine the effects of the sympathetic co‐transmitter Neuropeotide Y on primary afferent nerve fibres of the rat knee joint. The responses to passive joint rotations at defined torque were recorded from 41 slowly conducting afferent nerve fibres (0.9 – 18.8 m s−1) innervating the knee joint capsule. About 70% of the joint afferents were significantly affected in their mechanosensitivity by topical application of Neuropeptide Y. Significant effects occurred at a concentration of 10 nM. Decreased mechanosensitivity was observed in about 40% of nerve fibres, whereas 30% of the units increased the mechanosensitivity. In addition, in about 35% of the fibres resting activity was induced or increased. Neither the conduction velocity nor the mechanical threshold of the units correlated with the described effects of Neuropeptide Y. NPY(13 – 36), a specific Y2‐receptor agonist, only modulated the mechanosensitivity, with no effect on the resting activity. The effects on the mechanosensitivity were similar to Neuropeptide Y, i.e. increase and decrease of the response. Studies with the Y1‐agonist (Leu31, Pro34)‐NPY showed that activation of the Y1‐receptor predominantly resulted in an enhanced mechanosensitivity and an induction or increase of a resting activity. The opposite effect was observed by application of BIBP 3226 BS, a Y1‐receptor antagonist. In conclusion, these data indicate that Neuropeptide Y affects the excitability of sensory nerve fibre endings.

The bradykinin B1 receptor antagonist BI113823 reverses inflammatory hyperalgesia by desensitization of peripheral and spinal neurons.

Bradykinin is a neuropeptide released after tissue damage which plays an important role in inflammatory pain. The up‐regulation of the bradykinin B1 receptor in response to inflammation makes it an attractive target for drug development. Aim was to investigate if the selective B1 receptor antagonist BI113823 reduces inflammation‐induced mechanical hyperalgesia and if the effect is mediated via peripheral and/or spinal B1 receptor antagonism.

Somatostatin 4 receptor activation modulates TPRV1 currents in dorsal root ganglion neurons

Somatostatin (sst) is a cyclic neuropeptide known to have inhibitory roles in the central nervous system. It exerts its biological effects via the activation of the 5 sst receptor subtypes, which belong to the family of G-protein coupled receptors (GPCR). This peptide has analgesic properties, specifically via the activation of the sst4 receptor subtype. Although this is established, the precise molecular mechanisms causing this have not yet been fully elucidated. This research aimed to identify a possible anti-nociceptive mechanism, showing functional links to the transient receptor potential vanilloid type 1 (TRPV1) within the pain processing pathway. Calcium imaging and whole cell voltage clamp experiments were conducted on DRG neurons prepared from adult rats, utilizing capsaicin stimulations and the sst4 receptor specific agonist J-2156. The complete Freunds adjuvant (CFA) inflammatory pain model was used to examine if effects are augmented in pain conditions. The sst4 receptor agonist J-2156 was able significantly to inhibit capsaicin induced calcium and sodium influx, where the effect was more potent after CFA treatment. This inhibition identifies a contributory molecular mechanism to the analgesic properties of sst4 receptor activation.