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The tachykinin family has recently been extended by the discovery of a third tachykinin gene encoding previously unknown mammalian tachykinins (hemokinin 1, endokinin A and endokinin B) that have a widespread peripheral distribution and a tachykinin NK(1) receptor selectivity. This and the identification of other tachykinin-like peptides such as C14TKL-1 and virokinin raise many questions about the roles played by tachykinins in peripheral tissues and render terms such as 'neurokinins' and 'SP receptor' inappropriate.

Kinins, generated in the extracellular matrix via the proteolytic cleavage of larger precursors (kininogens), are principal mediators of inflammation. Bradykinin (BK) B2 receptors, which are constitutively expressed by various cells, mediate many effects of newly formed kinins. By contrast, BK B1 receptors are generally absent in healthy tissues but are readily expressed following inflammation or injury. Activation of B2 receptors by kinins also plays a role in the physiological maintenance of homeostasis of the cardiovascular system, whereas B1 receptors have been considered, until recently, to be involved only in pathological events. Pharmacological studies using B1 receptor antagonists {[desArg9,Leu8]-BK and [desArg9,Leu8]-Lys0BK} have focused on models of inflammatory hyperalgesia, but results have been contradictory; this is probably due to the significant partial agonist activity that these ligands possess in rats and mice. However, the recent construction of transgenic mice that lack B1 receptors (B1R−/−) has provided new insights into the role of these receptors in models of inflammation and nociception1xHypoalgesia and altered inflammatory responses in mice lacking B1 receptors. Pesquero, J.B. et al. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8140–8145Crossref | PubMed | Scopus (282)See all References1.The first phase of the hypotension induced by endotoxin was reduced in B1R−/− mice, but this effect was attributed to a downregulation of endothelial nitric oxide synthase in these animals. Plasma extravasation, infiltration of mononuclear and polymorphonuclear leukocytes induced by the intrapleural injection of a B1 receptor agonist {[desArg9]-BK} was abolished in B1R−/− mice, whereas when similar inflammatory responses were induced by carrageenan, only the accumulation of polymorphonuclear leukocytes was inhibited compared with B1R+/+ mice. Unexpectedly, B1R−/− mice were hypoalgesic in tests of acute nociception, such as the hot plate test (for temperatures,58.58C) and intraplantar injection of capsaicin or formalin (both early and late phases of the response). Concomitantly, a selective reduction of the C fiber component of ventral root potentials elicited by the electrical stimulation of dorsal roots was observed in B1R−/− mice. However, neither the percentage of isolated primary afferent neurons responding to heat, nor the amplitude of the evoked current were different in B1R−/− mice from those recorded in B1R+/+ animals. Therefore, the sensory impairment observed in B1R−/− mice was attributed to a deficit in neurotransmission at the level of the spinal cord.These findings, in addition to confirming a role of B1 receptors in the accumulation of polymorphonuclear leukocytes in inflamed tissues, would suggest that a kinin B1 receptor agonist is generated at the spinal cord level during the synaptic activity that follows peripheral noxious stimulation. Furthermore, these results also imply that such an agonist would facilitate the painful response to heat and chemical irritants. Indeed, immunocytochemical studies have recently shown that small primary afferent neurons express plasma kininogen, and immunostained fibers (and neurons) have also been found in areas of the spinal cord involved in nociception. Immunostaining for B1 receptors was found in the spinal cord, localized to the substantia gelatinosa (lamina II) and intermediate grey matter. The presence of kininogen, kallikrein-like enzymes and membrane-bound carboxypeptidase provides a framework to speculate about a putative kinin-mediated neurotransmission at the level of the spinal cord. Testing of selective B1 receptor antagonists that are devoid of intrinsic activity is now required to check to what extent the acute administration of such a compound will reproduce the sensory deficit observed in B1R−/− mice and to exclude definitively that these deficits might involve genic adaptation during development.

Integration and crosstalk between the signals carried by different primary afferent neurones occurs mainly in the CNS. In its simplest forms, this integration occurs as a result of the convergence of different axon terminals on the same second-order afferent neurones, or through axo–axonic synapses mediating presynaptic inhibition or excitation. A new concept has emerged recently, following the discovery that the electrical stimulation of peripheral branches of dorsal root ganglion (DRG) cells induced excitation of the soma not only of the stimulated neurones but also of neighbouring DRG cells within the same ganglion, despite lack of evidence for synaptic contacts between the somata of DRG cells. This phenomenon has been defined as cross-depolarization (CD) or cross-excitation. A recent study [1xChemical communication between vagal afferent somata in nodose ganglia of the rat and the guinea-pig in vitro. Oh, E.J. and Weinreich, D. J. Neurophysiol. 2002; 37: 2801–2807See all References][1] now shows that ganglion cells associated with cranial nerves (e.g. the nodose ganglion associated with the vagal afferents) also display CD, demonstrating that this phenomenon is widespread among primary afferent neurones.Using current- or voltage-clamp methods, the activity of isolated nodose ganglion neurones of rats or guinea-pigs, containing the peripheral branches of the vagus nerve with or without laryngeal nerve (which inserts into the vagus) was recorded intracellularly. Three different preparations were studied: (1) the vagus nerve was stimulated proximal to the insertion of the laryngeal nerve, so both vagal and laryngeal axons could be stimulated; (2) the vagus nerve was stimulated distal to the insertion of the laryngeal nerve and therefore only vagal axons and not laryngeal axons were stimulated; and (3) the vagus nerve was stimulated distal to the insertion of the laryngeal nerve and, in a separate chamber, another stimulating electrode was placed to identify single airway afferents (this preparation allowed the researchers to stimulate the vagus and to record CD in an identified laryngeal neurone).Stimulation of the first preparation at just subthreshold current intensities for recording a somatic orthodromic action potential resulted in a long-lasting depolarization in the somata of nodose ganglion neurones. Following the stimulation of the second preparation at a fixed stimulus intensity, ‘spontaneous’ action potentials occurred in some of the nodose neurones. In other neurones the probability of evoked action potentials increased during, but not before or after, the CD. Similar results were obtained in the third preparation. Among nodose neurones, identified according to the conduction velocity of their axons, 64% of the total displayed CD, of which 7% were myelinated and 57% were unmyelinated. The investigation of the mechanisms underlying CD indicated that it was abolished by the removal of Ca2+ ions from the medium in 60% of neurones, suggesting that CD is mediated by the release of neurotransmitters from the somata of neurones directly excited by electrical stimuli. In the remaining 40% of neurones, CD was resistant to Ca2+ removal. In these neurones basal membrane conductance was so high that their membrane potential could follow the K+ potential, so that they could be depolarized by the increased concentration of extracellular K+ as a result of the excitation of neighbouring neurones. This interpretation was indirectly supported by the finding that ouabain, a blocker of the Na+–K+ exchanger, increased the amplitude and duration of the CD in neurones displaying Ca2+-independent CD.This study highlights a novel mechanism of neuronal integration that could affect both the efferent and the sensory function of primary afferent neurones. Because nodose ganglion neurones project to a variety of visceral organs, this mechanism could be involved in asthma associated with gastro-esophageal reflux. Therefore, the stimulation of esophageal afferents could spread out to airway afferents, which through the peripheral release of neuropeptides would induce airways inflammation and hyperreactivity. A further possibility concerns the involvement of CD in the genesis of referred pain. As an example, the epigastric pain occurring during myocardial infarction could involve CD spreading from afferents innervating the heart from those that project to the stomach. CD in vagal afferent ganglia should be considered as a concurrent mechanism of the axon reflexes that it has been claimed are involved in the above-mentioned pathologies.

Nociceptin, a 17-amino-acid neuropeptide, was originally named because of its pro-nociceptive or hyperalgesic effects in rodent models; however, subsequent studies determined that nociceptin also exerts antinociceptive effects in various paradigms, including capsaicin-induced allodynia. Although nociceptin is known to induce its effects by activating N/OFQ peptide (NOP) receptors (previously known as OP4 or ORL1 receptors), experimental evidence suggests the existence of nociceptin-induced, non-NOP-receptor-mediated nociception-related effects. In addition, it is unknown whether the inhibitory modulation of capsaicin-sensitive neurons by nociceptin can occur in humans. Two recent studies have addressed these issues, and have demonstrated that a therapeutic effect can be achieved by the peripheral modulation of capsaicin-sensitive neurons.Ko et al. [1xOrphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors. Ko, M.C.H. et al. Br. J. Pharmacol. 2002; 135: 943–950Crossref | PubMedSee all References[1] injected capsaicin subcutaneously, with or without nociceptin, in the presence or absence of selective opioid receptor antagonists (quadazocine for mu opioid peptide receptors, nor-binaltorphimine for kappa opioid peptide receptors, naltrindole for delta opioid peptide receptors, and J113397 for NOP receptors) into the tail of rhesus monkeys, and measured tail withdrawal latencies to thermal stimulation (water at 46°C). In an earlier study, Lazzeri et al. [2xUrodynamic and clinical evidence of acute inhibitory effects of intravesical nociceptin/orphanin FQ on detrusor overactivity in humans: a pilot study. Lazzeri, M. et al. J. Urol. 2001; 166: 2237–2240Abstract | Full Text | Full Text PDF | PubMedSee all References[2] infused nociceptin intravesically in either a group of nine patients with detrusor hyperreflexia owing to spinal cord diseases, or five normal subjects, and assessed urodynamic parameters 30 min before, during, or 15 days after nociceptin infusion.In monkeys, nociceptin did not evoke thermal analgesia per se, although it reversed capsaicin-induced thermal hyperalgesia in a dose-dependent manner. With the exception of J113397, none of the opioid receptor antagonists inhibited the effect of nociceptin when co-injected into the tail with nociceptin and capsaicin, indicating the involvement of NOP receptors in the anti-hyperalgesic action of the neuropeptide. Intriguingly, J113397 was inactive when injected subcutaneously into the back of rhesus monkeys, which indicates that local (tail) stimulation of NOP receptors is involved in the effect of nociceptin. In humans, intravesical infusion of nociceptin did not alter urodynamic parameters in normal subjects but increased bladder capacity in patients with detrusor hyperreflexia; this effect was not present 15 days after the intravesical infusion.The results obtained in monkeys indicate that the peripheral stimulation of NOP receptors does not alter thermal sensitivity per se but antagonizes thermal hyperalgesia induced by the stimulation of capsaicin-sensitive afferent neurons. The results obtained in normal subjects support the hypothesis that capsaicin-sensitive bladder afferent fibers are not involved in physiological micturition but peripheral inhibitory modulation of these fibers achieves a therapeutic effect on detrusor hyperreflexia. This therapeutic effect has already been demonstrated repeatedly by the use of intravesical infusion of high concentrations of vanilloid VR1 agonists, such as capsaicin and resiniferatoxin, but this was probably due to a neurotoxic effect on peripheral terminals of capsaicin-sensitive sensory neurons. These results indicate that capsaicin-sensitive (VR1-expressing) neurons are selectively recruited during pathological conditions, and reversible modulation of VR1-expressing sensory fibers can be obtained by stimulating NOP receptors, thus achieving therapeutic effects.

Unlike classical amine or amino acid transmitters, which are synthesized close to their sites of release, neuropeptides such as the 37-amino-acid calcitonin gene-related peptide (CGRP) are synthesized in the neuronal soma and transported through the axon to sites of release in the nerve terminals. The transport process is time- and energy-consuming, and an intense stimulation could deplete releasable pools of neuropeptides rendering the nerve terminals unresponsive (in terms of release) until this pool has been replenished by newly synthesized and transported peptide. Uptake mechanisms that remove neurotransmitters from the synaptic cleft and spare the synthesis of new molecules of neurotransmitters have been described for small amine or amino acid neurotransmitters. Now, Sams-Nielsen et al. 1xPharmacological evidence for CGRP uptake into perivascular capsaicin sensitive nerve terminals. Sams-Nielsen, A. et al. Br. J. Pharmacol. 2001; 132: 1145–1153Crossref | PubMedSee all References1 provide indirect evidence for a reuptake mechanism for CGRP into capsaicin-sensitive afferent neurones and suggest that this mechanism might have functional relevance for the refilling of a releasable neuropeptide pool in nerve terminals following CGRP release.Isolated segments of the guinea-pig basillary artery, precontracted with prostaglandin F2α (PGF2α), were challenged with capsaicin (10 μm), which caused a CGRP-dependent (blocked by the antagonist CGRP8–37), capsazepine-sensitive relaxation. When the capsaicin challenge was repeated twice again, the relaxation underwent a significant fading (from ∼60% to 20% of PGF2α contraction). At this point, preparations were incubated with CGRP (100 nm) for 20 min and the capsaicin challenges repeated another three times. Following this protocol, the fourth capsaicin challenge (the first after the CGRP incubation) caused a significant relaxation (capsazepine-resistant, but still CGRP-dependent), which showed a significant fading during the fifth and sixth capsaicin challenge. Control experiments showed that repeated challenges with exogenous CGRP induced reproducible relaxations, thus excluding the possibility that desensitization occurs at the smooth-muscle level. In another series of experiments, arterial segments were incubated with [125I]CGRP and [125I] outflow was monitored following relaxation induced by capsaicin in PGF2?-precontracted preparations. Capsaicin induced a significant radioactivity release, which suggests that [125I]CGRP had been taken up by capsaicin-sensitive nerve terminals, even in the absence of a previous capsaicin challenge. Intriguingly, the restoration of capsaicin-induced relaxation was inhibited when CGRP was co-incubated with its antagonist CGRP8–37, suggesting that prejunctional CGRP receptors are involved in the refilling of the neuropeptide in nerve terminals.These results provide indirect evidence for a CGRP reuptake mechanism in capsaicin-sensitive perivascular nerve terminals, possibly mediated through the activation of prejunctional CGRP receptors. Thus, following the activation of prejunctional CGRP receptors and the consequent internalization of the complex peptide ligand–receptor, the peptide ligand might be spared from metabolism and recycled locally for the preservation of physiological functions. In addition, these results highlight a novel role for peptide prejunctional receptors in neurones, as well as in the modulation of neurotransmitter release. Other examples for the transport of relatively large peptides across biological barriers include those mediated by multi-drug resistance protein, other peptide transporters, or specific peptide receptors located at the blood–brain barrier or the choriod plexus epithelium; however, this uptake mechanism for CGRP is the first example of a neuronal uptake of peptides. It will be interesting to assess whether this process represents an exception or similar mechanisms spare other neuropeptides.

Neuropetide Y (NPY) is a 36-amino-acid neuropeptide that is widely distributed in the CNS and PNS, where it is stored along with noradrenaline in sympathetic postganglionic fibres, and is also expressed in other autonomic (possibly parasympathetic) and sensory neurones. The physiological effects of NPY are mediated by at least six receptors (Y1–6), showing variable expression among mammalian species. An inhibitory modulation by NPY on the release of substance P (SP) from capsaicin-sensitive primary afferent neurones, from both central and peripheral nerve endings, was confirmed in many experimental conditions. Therefore, until recently, there was a general consensus that NPY had a putative anti-inflammatory effect at the peripheral level and an antinociceptive effect in the CNS. Y1, Y2 and possibly other NPY receptors are expressed by dorsal root ganglion neurones. The recent availability of Y1 receptor knockout (Y1−/−) mice was expected to elucidate whether this receptor is involved in the inhibitory effect of NPY on neurogenic inflammation and nociception.Naveilhan and colleagues 1xReduced antinociception and plasma protein extravasation in mice lacking a neuropeptide Y receptor. Naveilhan, P. et al. Nature. 2001; 409: 513–517Crossref | PubMed | Scopus (129)See all References1 showed that Y1−/− mice displayed a hyperalgesic phenotype in tests of thermal (hot plate and tail flick), chemical (first phase of formalin test, acetic-acid- and magnesium-sulfate-induced writhings), mechanical (Von Frey hairs) and neuropathic (partial nerve injury) nociception, and the antinociceptive effect observed following the intrathecal administration of NPY in wild-type animals was not evident in Y1−/− mice. Concomitantly, however, the capsaicin-induced, SP-mediated pro-inflammatory effects were blunted in Y1−/− mice. In fact, the administration of capsaicin in a paw did not induce plasma extravasation and oedema in Y1−/− mice, and the effects of mustard oil were also attenuated. By contrast, neither the inflammatory responses induced by carrageenan nor those induced by SP were modified in Y1−/− animals. Furthermore, the pro-inflammatory effects of capsaicin in wild-type mice were mimicked by a Y1 receptor agonist and were prevented by the selective Y1 receptor antagonist BIBP3226. In Y1−/− mice, tissue concentrations of SP were comparable with those found in normal mice, but capsaicin failed to induce SP release and subsequent neuropeptide depletion.These results led the authors to conclude that Y1 receptors exert a tonic physiological role in reducing nociceptive inputs at the spinal cord level, whereas at the peripheral level, the stimulation of these receptors is both necessary and sufficient for triggering neurogenic inflammation. An alternative hypothesis could reconcile previous and present findings on the role of NPY and NPY receptors in neurogenic inflammation: an opposing role of Y1 and Y2 receptors (or other Y receptors) in the excitability of capsaicin-sensitive nerve terminals could exist if both receptors are tonically stimulated by endogenous NPY. In wild-type animals, the exogenous administration of NPY has an inhibitory effect on capsaicin-induced inflammatory responses, possibly because of a preferential stimulation of Y2 receptors. In Y1−/− animals, endogenous NPY could be sufficient for the inhibition of the excitability of capsaicin-sensitive nerve terminals through the stimulation of Y2 receptors. This hypothesis could be tested either through the construction of Y2−/− mice, or with the use of already available selective Y2 receptor antagonists.