Calcitonin gene-related peptide: role in airway homeostasis Azzeddine Dakhama1, Gary L Larsen2 and Erwin W Gelfand3

The lung is an important source of sensory neuropeptides ily controlled by neural mechanisms, but underlying that modulate airway function in health and disease. Among inflammation and damage to airway structure can alter these neuropeptides, calcitonin gene-related peptide (CGRP) or interfere with this control [1]. plays a prominent role. CGRP is constitutively expressed in normal lungs where it localizes to a specialized subset of The lung is innervated by a rich supply of vagal nerve epithelial cells (neuroendocrine cells) and sensory C fibers afferent fibers, most of which are unmyelinated sensory C distributed to pulmonary airways, blood vessels and lymphoid fibers, with endings disseminated throughout the airways tissue. CGRP can mediate multiple effects, some of which have within the mucosa. Activation of afferent C fiber endings potential implications in airway homeostasis. These include by exposure to inhaled irritants or inflammatory stimuli vasoregulation, bronchoprotection, anti-inflammatory actions triggers the release of sensory neuropeptides and results and tissue repair. Targeting these effects of CGRP could be in the development of neurogenic inflammation in the a future avenue for modulation of certain aspects of airway airways [2]. This form of inflammation is characterized by diseases. increased vascular permeability, mucus hypersecretion and potentiation of airway smooth muscle contraction. Addresses These effects are normally terminated after proteolytic National Jewish Medical and Research Center, Department of degradation of effector sensory neuropeptides. Pediatrics, Denver, Colorado 80206, USA 1e-mail: [email protected] 2e-mail: [email protected] Among the variety of sensory neuropeptides that can be 3e-mail: [email protected] detected in the lung, calcitonin gene-related peptide (CGRP) is the most abundant. The fact that this neuro- peptide is constitutively expressed in normal airways Current Opinion in Pharmacology 2004, 4:215–220 implies a possible involvement in local homeostasis. This review comes from a themed issue on Recent studies suggest potential roles for CGRP in Respiratory pharmacology modulating airway function, vascular tone and inflam- Edited by Roy Goldie and Peter Henry matory immune responses. However, more studies are 1471-4892/$ – see front matter needed to further define the exact functions of CGRP in ß 2004 Elsevier Ltd. All rights reserved. health and disease and to prepare for evaluation of potential therapeutic targeting in human airway inflam- DOI 10.1016/j.coph.2004.01.006 matory diseases.

Abbreviations Synthesis and metabolism of CGRP AHR airway hyperresponsiveness CGRP belongs to a family of related neuropeptides that AM adrenomedullin includes calcitonin (CT), adrenomedullin (AM) and amy- CGRP calcitonin gene-related peptide CRLR calcitonin receptor-like receptor lin [3]. CGRP is a 37 amino acid neuropeptide produced CT calcitonin by tissue-specific alternative processing of the mRNA NEBs neuroepithelial bodies transcript encoded by CT/CGRP gene [4–6]. There are PNEC pulmonary neuroendocrine cell two known isoforms of CGRP (a-CGRP and b-CGRP), RAMP receptor activity-modifying protein which differ by one amino acid in rats, and three in mouse RCP receptor component protein and humans (Table 1). Analyses of mRNA expression levels showed that a-CGRP is the most abundant form Introduction (> 80%) in the nervous system. The two isoforms are Although seeming to be protected anatomically, the encoded by separate gene loci located on chromosome 11 mammalian lung contains the largest surface area of in humans; however, their biological activities are indis- the body directly exposed to the environment. Mainte- tinguishable [7]. CGRP is susceptible to degradation by nance of airway homeostasis is a constant challenge in the neutral endopeptidase, which is abundant in the airway face of multiple disturbances that can be induced by epithelium. However, CGRP degraded at about 1.2% of pathogens (e.g. bacteria and viruses) and a variety of the rate of degradation for in the presence of noxious environmental agents (e.g. allergens and pollu- recombinant neutral endopeptidase in vitro [8]. In the tants). These agents can reach the most distal parts of the presence of mast cell tryptase, CGRP degrades at faster lung and alter airway function, causing transient or per- rates comparable to those of other lung neuropeptides manent changes depending upon the susceptibility of the such as vasoactive intestinal peptide and peptide histi- exposed individual (Figure 1). Airway function is primar- dine-methionine [9]. www.sciencedirect.com Current Opinion in Pharmacology 2004, 4:215–220 216 Respiratory

Figure 1

Environmental factors (pollutant, allergen, pathogen) Host factors (susceptibility, atopy)

Damage Normal structure Altered structure Repair

mucus Neurogenic PNEC nerve endings Edema, Mucus NEB ASM NEP, SP, NKA Tryptase CGRP, VIP BV Immune inflammation Cells Cytokines Spinal Mediators cord Normal function Altered function Current Opinion in Pharmacology

Airway homeostasis is altered following exposure to noxious environmental factors in susceptible individuals. Damage inflicted to airway structure and activation of exposed sensory nerve endings by inhaled irritants and inflammatory mediators stimulate release of sensory neuropeptides, mediating neurogenic inflammation. Decreased neutral endopeptidase activity allows for unopposed actions by tackykinins (SP and NKA), causing increased mucus secretion, vascular leakage with plasma extravasation, and potentiation of airway smooth muscle contraction. Increased mast cell tryptase activity during an inflammatory response can cause degradation of CGRP and VIP, terminating their activities. Full recovery of homeostatic function requires complete and successful tissue repair. ASM, airway smooth muscle; BV, blood vessel; NKA, neurokinin-A; SP, substance P; VIP, vasoactive intestinal peptide.

CGRP receptors and their signalling transfected into HEK293 cells, and not in COS-7 cells, pathways suggesting that other proteins (or co-receptors) are Two CGRP receptors have been identified pharmacolo- required for receptor function. A breakthrough came with gically on the basis of their differential affinities for the the discovery of receptor activity-modifying proteins competitive peptide antagonist CGRP8–37 [7]. CGRP1 (RAMPs), a novel family of single transmembrane receptors are more sensitive than CGRP2 receptors to domain receptors [12]. Three RAMPs have been cloned the antagonist CGRP8–37. Alternatively, the linear a- and characterized. When co-expressed with RAMP1 in a CGRP analogs [Cys(ACM)2,7]- and [Cys(Et)2,7]-CGRP, variety of cells, including COS-7 cells, CRLR functions as which are agonists with greater potency for CGRP2 than the CGRP1 receptor. When co-expressed with RAMP2 or for CGRP1 receptors, have also been used to distinguish RAMP3, CRLR functions as a receptor for AM, another between the two receptors. From the sequence of the member of the calcitonin family that is related to CGRP. calcitonin receptor, a candidate receptor was initially In addition, an intracellular accessory protein termed cloned by PCR in rat lung and was named calcitonin CGRP-receptor component protein (RCP) has recently receptor-like receptor (CRLR) [10]. Human CRLR has been identified and shown to be required for signal subsequently been cloned and proposed as the CGRP1 transduction through CGRP and AM receptors [13].A receptor [11]. However, CRLR was functional only when recent study identified a 66 kDa protein as a new receptor

Table 1

Structure of CGRP isoforms in human, rat and mouse.

Human a CGRP A C D TATCVTHRLAGLLSRSGGV VKN NFVPTNVGSKAF b CGRP A C N TATCVTHRLAGLLSRSGGM VKS NFVPTNVGSKAF Rat a CGRP S C N T A T C V T H R L A G L L S R S G G V V K D N F V P T N V G S E AF b CGRP S C N T A T C V T H R L A G L L S R S G G V V K D N F V P T N V G S K AF Mouse a CGRP S C N T A T C V T H R L A G LLSRSGGVV KDNFVPTN VGSEAF b CGRP S C N T A T C V T H R L A D LLSRSGGVL KDNFVPTD VGSEAF Bold characters denote distinct amino acids for a-andb-CGRP.

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for CGRP, which is distinct from CRLR and RAMP1 of functions, including neurotransmission, vasodilation  [14 ], and proposed a new classification for CGRP1 and and immune regulation. CGRP is the most potent endo- CGRP2 receptors as CGRP-A and CGRP-B, respectively. genous vasodilator identified to date [3], and has been Binding of CGRP to its receptor is known to activate implicated in the regulation of vascular tone in the lungs, adenylyl cyclase and increase cAMP production, a path- where CGRP receptors are abundant in the bronchial way that involves Gas protein. Structure–function studies vessels [32]. CGRP attenuates the development of revealed that the first seven amino acids of the a-CGRP chronic hypoxic pulmonary hypertension in a rat model molecule interact with the transmembrane domain of [33]. In this animal model, chronic hypoxia has been CRLR and are required for receptor activation, whereas shown to increase cellular levels of CGRP in pulmonary residues 8–18 and 28–37 are necessary for high-affinity NEBs. In this context, pulmonary NEBs might function binding; residues 19–27 form a hinge region [15]. Struc- as chemoreceptors that sense altered oxygen levels dur- tural requirements for the interaction of CGRP with ing passage of air in the airways, with CGRP acting as a CGRP2 receptors are still unknown. regulatory mediator in the vascular response to hypoxia [34]. CGRP promotes epithelial cell proliferation in vitro Tissue distribution of CGRP and CGRP [27], and pulmonary NEBs can serve as an important receptors reservoir of progenitor cells during re-epithelialization of CGRP is distributed to central and peripheral nervous injured airways [35].AroleforCGRPintissuerepairis systems [16], and is present in sensory nerve fibers further supported by its capacity to induce migration and throughout the respiratory tract. In the lower respiratory proliferation of fibroblasts [28]. CGRP does not appear to tract, CGRP can be detected in pulmonary neuroendo- induce mucus secretion in the airways [36].Thisisnot crine cells (PNECs), singly disseminated within the air- surprising because no CGRP binding sites could be way epithelium, and in neuroepithelial bodies (NEBs), detected by autoradiography on mucous glands [24]. consisting of innervated clusters of PNECs localized at Unlike the tachykinin substance P, which plays an im- the branching points of the airways [17]. CGRP-contain- portant role in neurogenic airway inflammation, CGRP ing nerve fibers can be detected in close contact with does not induce vascular leakage or plasma protein blood vessels and smooth muscle. CGRP co-localizes extravasation [19]. with substance P in some but not all sensory C fibers located within the airways of several species, including Early studies found that CGRP was more potent than humans [18,19]. In some fibers, CGRP and substance P either substance P or carbachol in contracting isolated can co-exist within the same secretory granule [20]. Other human airways in vitro [37], and proposed an indirect important tissue locations include lymphoid organs such mechanism of action involving release of endogenous as spleen [21], thymus [22] and lymph nodes [23]. bronchoconstrictors. However, CGRP is known to acti- vate adenylyl cyclase and increase cAMP levels, a CGRP receptors are present in central and peripheral response usually associated with bronchodilation [38]. nervous systems. CGRP1 receptors are expressed on blood Even though CGRP is co-released with substance P from vessels in virtually all organs [7]. The distribution of guinea-pig airways after stimulation with , only CGRP2 receptors is less well known. However, owing substance P contracted the airway smooth muscle in one to the considerable lack of structural and biochemical data study [19]; CGRP did not contract or relax human or on these receptors, their exact anatomical distribution guinea-pig airways in vitro [19,39]. Furthermore, admin- remains largely undefined. CGRP receptors have initially istration of CGRP in vivo to normal guinea-pigs inhibited been mapped by radioligand binding to blood vessels in substance P-induced bronchoconstriction. When added in human airways, with sparse distribution in the airway vitro, CGRP inhibited substance P-evoked contraction in epithelium and smooth muscle; no binding was detected isolated guinea-pig airways. However, these inhibitory in submucosal glands [24]. A recent study detected CRLR effects of CGRP were lost in sensitized guinea-pigs [39]. immunoreactivity, suggestive of the presence of CGRP1 Similarly, CGRP inhibited carbachol-induced contraction receptor in the endothelium of human bronchial veinules of normal human airways — a protective effect that was but not in arterioles [25]. CGRP is known to produce direct lost in airways showing signs of inflammation. effects through its receptors present on vascular smooth muscle cells and endothelial cells [26], and on other Using quantitative morphometric analyses, we showed structural cells such as epithelial cells [27] and fibroblasts that CGRP was significantly depleted in pulmonary [28]. CGRP receptors are also expressed on a variety of NEBs and submucosal nerve fibers following airway inflammatory cells, including macrophages [29], B and T allergen challenge of sensitized mice [40]. These mice lymphocytes [30], and antigen-presenting cells [31]. developed a robust allergic airway inflammation and air- way hyperresponsiveness (AHR), measured as increases Effect of CGRP on airways in lung resistance to inhaled methacholine. Without As might be predicted from its wide anatomical distribu- sensitization, allergen challenge alone did not induce tion, CGRP has been implicated in a broad range AHR nor did it alter the levels of CGRP expression in www.sciencedirect.com Current Opinion in Pharmacology 2004, 4:215–220 218 Respiratory

the mouse airways. Similar observations of CGRP deple- tive mediator — the nature and source of which are tion have been reported in the guinea-pig following currently under investigation. sensitization and airway challenge with toluene-diisocya- nate [41]. Factors responsible for this depletion are not The presence of CGRP in lymphoid tissues suggests that known, but we believe that allergic inflammation CGRP may interact with key inflammatory cells expres- increases the sensitivity of sensory nerve fibers to inflam- sing its receptor and possibly influence the type of the matory stimuli and inhaled irritants, which then can cause response, impacting on airway function at multiple levels release of these neuropeptides. during the development, maintenance and resolution of an inflammatory response. CGRP inhibits T cell prolif- Mice lacking a-CGRP are either hypertensive [42,43] or eration and interleukin–2 production by direct interac- have normal cardiovascular function [44]; double knock- tions with T cells [48]. Recent studies demonstrated that out mice lacking a- and b-CGRP remain to be developed. CGRP also mediates T cell suppression by downregulat- A recent study evaluated the role of CGRP in the devel- ing antigen presentation [31]. Our recent work indicated opment of AHR in a-CGRP knockout mice [45], shown that, when administered during allergic sensitization previously to develop a hypertensive phenotype [43]. (at the time of T cell priming), CGRP prevents the The study described dramatic increases in CGRP expres- development of a Th2-dominated primary immune sion in the lungs of ovalbumin-sensitized and challenged response by inhibiting antigen-specific T cell activation wild-type mice, and reported an attenuated AHR in [49]. However, no inhibition could be observed when sensitized and challenged a-CGRP knockout mice. It these T cells were committed to a Th2 phenotype, was concluded that CGRP is a mediator of AHR in this which is consistent with our in vivo data showing phy- model [45]. However, the data should be carefully inter- siological effects on AHR but no effects on airway preted, as the pattern of CGRP immunoreactivity in the inflammation [40]. lungs of wild-type animals is unusual and further evalua- tions (i.e. using pharmacological approaches) are need- Conclusions ed to validate these findings and to exclude potential Most experimental observations agree that CGRP is not a compromising developmental issues resulting from the bronchoconstrictor of either human or animal airways, and phenotype of knockout mice. Studies that examined it is not a mediator of neurogenic inflammation. CGRP bronchial tissue from patients with did not detect has some bronchoprotective and anti-inflammatory prop- any increase in CGRP expression, although the size of erties and there is evidence that it might promote tissue tissue examined was limited [46,47]. repair following injury to the airways. Whether these functions prevail in vivo and under which circumstances In examining the involvement of CGRP in the develop- remain to be fully established. The wide distribution of ment of AHR, we used a pharmacological approach, CGRP and its receptors to multiple organs and cellular administering the CGRP1 receptor antagonist CGRP8–37 targets can at least suggest that its multiple actions are to sensitized mice either during or after completion of well coordinated in vivo. However, this might also indi- allergen airway challenge, followed by assessment of cate that its complete physiological functions are far from airway responsiveness to inhaled methacholine. The being established. Complete structural and biochemical antagonist did not inhibit development of AHR or airway characterization of CGRP receptor subtypes and the inflammation in these animals, clearly demonstrating that development of potent and highly selective pharmacolo- endogenous CGRP is not a mediator of AHR, nor does it gical agents are necessary for achieving these goals, and contribute to cellular inflammation in this model [40].To could help develop novel strategies aimed at modulating fully examine the role of CGRP in modulating AHR, aspects of airway diseases. sensitized mice were administered exogenous CGRP (systemically or by inhalation). In both cases, the devel- Update opment of AHR, but not airway inflammation, was com- Recent data indicate that CGRP can cause constriction, pletely inhibited by CGRP even when administered after although moderate, of isolated human airway segments completion of allergen challenge, and in spite of estab- precontracted with acetylcholine, but only in epithe- lished allergic airway inflammation. These findings sug- lium-denuded airways [50]. These observations led the gest an effect of CGRP downstream of the inflammatory authors to speculate that such constriction might occur response that could potentially involve direct interaction in pathological conditions such as asthma, where the with the smooth muscle. However, when directly tested epithelium is damaged in the airways. However, further on smooth muscle in vitro, CGRP did not inhibit carba- assessment using asthmatic airways are needed to vali- chol- or electrical field stimulation-induced tracheal date these findings. smooth muscle contraction, nor did it relax pre-contracted tracheal smooth muscle, indicating that the inhibitory Acknowledgements effects seen in vivo were probably mediated indirectly This work was supported by NIH grants HL-60015, HL-36577, HL-6718 and through mobilization of an endogenous bronchoprotec- Environmental Protection Agency grant R825702.

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