Susan D. Brain

Sensory neuropeptides: their role in inflammation and wound healing

Vasoactive neuropeptides including substance P and calcitonin gene-related peptide (CGRP) are localised in sensory nerves which innervate blood vessels. These are the major vasoactive neuropeptides released from sensory nerve endings and both have been suggested to have roles in inflammatory and cardiovascular disease. The neuropeptides have potent effects on microvascular tone and permeability, which are seen soon after release from perivascular nerves. There is also evidence that neuropeptides can affect various activities of inflammatory cells and that sensory nerves play a role in the recovery of the healthy microcirculation during wound healing phases. This review concentrates on evidence that the neuropeptides substance P, acting via tachykinin NK1 and NK2 receptors, and CGRP, acting via CGRP1 receptors, play a pro-inflammatory role in disease and a beneficial role in wound healing. In addition, results from clinical trials of recently developed neuropeptide antagonists are discussed.

Evidence that endogenous nitric oxide modulates oedema formation induced by substance P.

The possibility that nitric oxide (NO) could have a role in the modulation of inflammatory oedema formation was investigated in rat skin using selective inhibitors of NO synthesis. Intradermally injected substance P (0.03-1 nmol) induced oedema which was inhibited by concurrent administration of the inhibitor of NO synthesis L-NG-nitro arginine methyl ester (L-NAME), but not by the enantiomer D-NAME. L-Arginine reversed the inhibitory effect of L-NAME. A second inhibitor of NO formation, L-NG-monomethyl arginine (L-NMMA), had a similar inhibitory effect on substance P-induced oedema. The results suggest that endogenous NO has a modulatory role in oedema formation induced by mediators of increased microvascular permeability.

Calcitonin Gene-Related Peptide: Physiology and Pathophysiology

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide. Discovered 30 years ago, it is produced as a consequence of alternative RNA processing of the calcitonin gene. CGRP has two major forms (α and β). It belongs to a group of peptides that all act on an unusual receptor family. These receptors consist of calcitonin receptor-like receptor (CLR) linked to an essential receptor activity modifying protein (RAMP) that is necessary for full functionality. CGRP is a highly potent vasodilator and, partly as a consequence, possesses protective mechanisms that are important for physiological and pathological conditions involving the cardiovascular system and wound healing. CGRP is primarily released from sensory nerves and thus is implicated in pain pathways. The proven ability of CGRP antagonists to alleviate migraine has been of most interest in terms of drug development, and knowledge to date concerning this potential therapeutic area is discussed. Other areas covered, where there is less information known on CGRP, include arthritis, skin conditions, diabetes, and obesity. It is concluded that CGRP is an important peptide in mammalian biology, but it is too early at present to know if new medicines for disease treatment will emerge from our knowledge concerning this molecule.

Neuropeptides and their receptors: innovative science providing novel therapeutic targets

This review examines our current understanding of the roles of some of the best known neuropeptides that have played major roles in our combined research programmes. Evidence obtained from over 75 years of research shows involvement of these transmitters in a wide range of organs relevant to cardiovascular, respiratory, cutaneous, neuronal and intestinal systems. There is an increasing understanding of the mechanisms involved in the release of the peptides (substance P and calcitonin gene‐related peptide (CGRP)) from sensory nerves or, neuropeptide Y (NPY) from sympathetic, parasympathetic and nonadrenergic, noncholinergic (NANC) neurons. Responses in target tissues result from interactions of the neuropeptides, or related forms, with specific G‐protein coupled receptors (GPCRs or 7 transmembrane‐spanning, 7TM proteins) that belong to either rhodopsin‐like, class 1 (neurokinin (NK) and NPY Y receptors) or secretin‐like, class 2 GPCRs (CGRP receptors). The majority of receptors activated by our chosen neuropeptides are now cloned, with knowledge of preferred agonists and selective antagonists for many receptor subtypes within these families. The study of neuropeptides in animal models has additionally revealed physiological and pathophysiological roles that in turn have led to the ongoing development of new drugs, through utilization predominantly of antagonist activities.

Nerve growth factor induced hyperalgesia in the rat hind paw is dependent on circulating neutrophils

Abstract The mechanisms by which nerve growth factor (NGF) induces thermal hyperalgesia and neutrophil accumulation have been investigated in the rat. Thermal nociceptive thresholds in rat hind paw were measured as the time taken for paw withdrawal from a heat source and neutrophil accumulation was measured in hind paw and dorsal skin samples using a myeloperoxidase assay. NGF (23–80 pmol intra‐plantar (i.pl.) injection) induced a significant (P<0.05, n=6–16) thermal hyperalgesia at 5 h after injection and significant neutrophil accumulation (P<0.05, n=6) was observed with NGF (40 pmol). In dorsal skin, where multiple samples can be assessed, intradermal (i.d.) NGF was 10–30 times less potent than interleukin‐1&bgr; in inducing neutrophil accumulation. The 5‐lipoxygenase inhibitor ZM230487 (10 nmol co‐injected with NGF) significantly attenuated neutrophil accumulation and hyperalgesia induced by NGF; unlike the histamine and 5‐hydroxytryptamine antagonists (mepyramine and methysergide) which were without effect at the times measured. Furthermore, depletion of circulating neutrophils (using a rabbit anti‐rat neutrophil antibody) abolished NGF induced hyperalgesia. These results indicate that neutrophils, which accumulate in response to a 5‐lipoxygenase product, play a crucial role in NGF‐induced hyperalgesia.

Effect of a calcitonin gene-related peptide antagonist (CGRP8-37) on skin vasodilatation and oedema induced by stimulation of the rat saphenous nerve

1 The effect of the calcitonin gene‐related peptide antagonist (CGRP8–37, 400 nmol kg−1, i.v.) on the increased blood flow induced by calcitonin gene related peptide (CGRP), vasodilator prostaglandins, and topical capsaicin was measured with a laser Doppler blood flow meter in rat abdominal skin. 2 The saphenous nerve was electrically stimulated and the effect of CGRP8–37 (400 nmol kg−1, i.v.) on the increased blood flow (measured by laser Doppler flowmetry) and oedema formation (measured by the extravascular accumulation of [125I]‐albumin) was investigated in the rat hind paw. 3 CGRP8–37 (400 nmol kg−1, i.v.) had no effect on basal cutaneous blood flow at uninjected sites and sites injected with Tyrode buffer, but acted selectively to inhibit the increased blood flow induced by intradermal CGRP (10 pmol/site, P < 0.05), but not that induced by prostaglandin E2 (PGE2, 300 pmol/site) or carba‐prostacyclin (cPGI2, 100 pmol/site). 4 Capsaicin (0.1–33 mm), applied topically, acted in a dose‐related manner to increase blood flow. CGRP8–37 (400 nmol kg−1, i.v.) almost totally inhibited blood flow induced by capsaicin (10 mm; P < 0.05) but did not significantly inhibit blood flow induced by a higher dose of capsaicin (33 mm). 5 The increased blood flow induced by short stimulation of the saphenous nerve (10 V, 1 ms, 2 Hz for 30 s) was inhibited by 76%, 5 min after i.v. CGRP8–37 (400 nmol kg−1, i.V., P < 0.05). 6 A longer (5 min) electrical stimulation of the saphenous nerve caused oedema formation, in addition to increased blood flow. The oedema formation was significantly inhibited by CGRP8–37 (400 nmol kg−1, i.v., P < 0.05). 7 The results suggest that the potent microvascular vasodilator neuropeptide, CGRP, is responsible for the increased blood flow observed after short stimulation of the saphenous nerve and that endogenous CGRP contributes in a pro‐inflammatory manner to neurogenic oedema formation in the rat hind paw.

A calcitonin gene-related peptide (CGRP) antagonist (CGRP8-37) inhibits microvascular responses induced by CGRP and capsaicin in skin

1 The effect of the calcitonin gene‐related peptide (CGRP) antagonist CGRP8–37 on responses to CGRP and other mediators was investigated in rabbit dorsal skin. 2 Blood flow changes at intradermally‐injected sites were measured by a multiple site 133xenon clearance technique. CGRP8–37 had little effect on blood flow at doses up to 0.3 nmol/site, when injected alone, although a significant increase in blood flow was observed at the highest dose tested (1 nmol/site). 3 CGRP8–37 dose‐dependently inhibited the increased blood flow induced by human αCGRP and human βCGRP, but had no effect on equivalent vasodilator responses induced by vasoactive intestinal peptide (VIP) and prostaglandin E1 (PGE1). CGRP8–37 showed a preferential ability to inhibit αCGRP (IC50 0.04 nmol), when compared with βCGKP (IC50 ≥ 0.3 nmol). 4 Capsaicin, which selectively activates sensory nerves, caused a dose‐dependent increase in blood flow when injected intradermally into rabbit skin. The effects of capsaicin (0.01–0.1 μmol/site) were inhibited by CGRP8–37 (0.3 nmol/site), with a partial but significant attenuation of blood flow induced by the highest dose of capsaicin. 5 Oedema formation, induced by intradermal histamine injection (3 nmol/site), was measured in rabbit skin by the local accumulation of intravenously‐injected 125I‐labelled albumin. Vasodilator doses of CGRP, PGE1 and capsaicin potentiated, in a synergistic manner, oedema formation induced by histamine. CGRP8–37 totally inhibited the potentiating effect of CGRP, partially inhibited the synergistic effect of capsaicin, but did not affect PGE1‐induced responses. 6 The results suggest that capsaicin acts to release a rabbit form of CGRP in skin and that CGRP8–37 is a useful antagonist for investigating the potential of CGRP as a neurogenic mediator of inflammation.

Neurokinin-1 Receptor Agonists Are Involved in Mediating Neutrophil Accumulation in the Inflamed, But Not Normal, Cutaneous Microvasculature: An In Vivo Study Using Neurokinin-1 Receptor Knockout Mice

We have used tachykinin neurokinin-1 receptor (NK1 receptor) knockout mice to learn of the link between NK1 receptors and neutrophil accumulation in normal naive skin, as compared with inflamed skin. Intradermal substance P (300 pmol) induced edema formation in wild-type mice, but not in NK1 knockout mice, as expected. However, in contrast to IL-1β (0.3 pmol), substance P did not induce neutrophil accumulation in wild-type mice. IL-1β-induced neutrophil accumulation was similar in wild-type and knockout mice, but a significant (p < 0.05) contributory effect of added NK1 agonists, which by themselves have no effect on neutrophil accumulation in normal skin, was observed. The results support the concept that NK1 agonists such as substance P cannot act on their own to mediate neutrophil accumulation in naive skin and provide direct evidence that in inflamed skin, under certain circumstances, the NK1 receptor can play a pivotal role in modulating neutrophil accumulation during the ongoing inflammatory process. We investigated responses to two inflammatory stimuli (carrageenin and zymosan). Neutrophil accumulation was significantly attenuated (p < 0.001) in carrageenin- but not zymosan-induced inflammation in NK1 knockout mice. The carrageenin (500 μg)-induced response was inhibited (p < 0.05) by a NK1 receptor antagonist, SR140333 (480 nmol/kg i.v. at −5 min), in the wild-type group. The bradykinin B1 and B2 receptor antagonists (desArg9[Leu8]bradykinin and HOE 140) each reduced neutrophil accumulation to carrageenin in wild-type animals (p < 0.05), but did not cause further reduction of the suppressed response of knockout mice. The results provide evidence that kinin receptors participate in NK1 receptor-dependent neutrophil accumulation in inflamed mouse skin.

A distinct role for transient receptor potential ankyrin 1, in addition to transient receptor potential vanilloid 1, in tumor necrosis factor α–induced inflammatory hyperalgesia and Freund's complete adjuvant–induced monarthritis

OBJECTIVE To investigate the involvement of transient receptor potential ankyrin 1 (TRPA1) in inflammatory hyperalgesia mediated by tumor necrosis factor α(TNFα) and joint inflammation. METHODS Mechanical hyperalgesia was assessed in CD1 mice, mice lacking functional TRP vanilloid 1 (TRPV1-/-) or TRPA1 (TRPA1-/-), or respective wildtype (WT) mice. An automated von Frey system was used, following unilateral intraplantar injection of TNFα or intraarticular injection of Freunds complete adjuvant (CFA). Knee swelling and histologic changes were determined in mice treated with intraarticular injections of CFA. RESULTS TNFα induced cyclooxygenase-independent bilateral mechanical hyperalgesia in CD1 mice. The selective TRPV1 receptor antagonist SB-366791 had no effect on mechanical hyperalgesia when it was coinjected with TNFα, but intrathecally administered SB- 366791 attenuated bilateral hyperalgesia, indicating the central but not peripheral involvement of TRPV1 receptors. A decrease in pain sensitivity was also observed in TRPV1-/- mice. Intraplantar coadministration of the TRPA1 receptor antagonist AP-18 with TNFα inhibited bilateral hyperalgesia. Intrathecal treatment with AP-18 also reduced TNFα-induced hyperalgesia. CFA-induced mechanical hyperalgesia in CD1 mice was attenuated by AP-18 (administered by intraarticular injection 22 hours after the administration of CFA). Furthermore, intraarticular CFA–induced ipsilateral mechanical hyperalgesia was maintained for 3 weeks in TRPA1 WT mice. In contrast, TRPA1-/- mice exhibited mechanical hyperalgesia for only 24 hours after receiving CFA. CONCLUSION Evidence suggests that endogenous activation of peripheral TRPA1 receptors plays a critical role in the development of TNFα-induced mechanical hyperalgesia and in sustaining the mechanical hyperalgesia observed after intraaarticular injection of CFA. These results suggest that blockade of TRPA1 receptors may be beneficial in reducing the chronic pain associated with arthritis.

Calcitonin gene-related peptide: Vasoactive effects and potential therapeutic role

1. The cardiovascular biology of calcitonin gene-related peptide (CGRP) and the structurally related peptides amylin and adrenomedullin are briefly reviewed. 2. CGRP is a potent and long-lasting vasodilator; its possible role in disease, and the therapeutic potential of CGRP receptor agonists and antagonists is discussed.