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Centre for Cardiovascular Biology and Medicine, King's College London, New Hunt's House, London, United Kingdom
ABSTRACT I. HISTORY AND DISCOVERY A. Calcitonin Gene-Related Peptide: A Novel Neuropeptide B. Adrenomedullin: A Tissue-Derived Peptide C. Amylin: A Pancreatic Peptide II. STRUCTURE III. DISTRIBUTION AND REGULATION OF GENES AND PEPTIDES A. CGRP: A Wide Distribution B. AM: Upregulation in Disease C. Amylin: Relevance to Diabetes IV. RECEPTORS A. Cloning of CGRP Receptors B. CL and RAMPs C. CGRP Receptor Antagonists V. VASODILATOR MECHANISMS A. CGRP: Multiple Mechanisms B. AM: Active on CGRP and AM Receptors C. Amylin: Active Via the CGRP1 Receptor VI. CARDIOVASCULAR REGULATION A. CGRP: Potent Vasodilator In Vivo B. AM: Role in Regulating Blood Pressure VII. MICROVASCULAR MECHANISMS A. CGRP: A Protective Factor? B. AM: Heterogeneity in Response VIII. INFLAMMATION AND VASCULAR BIOLOGY A. CGRP: Cellular Effects B. AM: Cellular Effects IX. INVOLVEMENT IN CARDIOVASCULAR DISEASE A. CGRP and Cerebral Conditions B. CGRP and AM: Heart Conditions C. AM and Hypertension D. CGRP and AM: Pulmonary Hypertension E. CGRP and AM: Sepsis F. CGRP and Raynaud's Disease G. CGRP and Blushing Syndromes X. CONCLUSION AND FUTURE PERSPECTIVES ACKNOWLEDGMENTS REFERENCES
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I. HISTORY AND DISCOVERY |
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Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that was identified in 1982 by molecular biological techniques. It was discovered when alternative processing of RNA transcripts from the calcitonin gene were shown to result in the production of distinct mRNAs encoding CGRP. The calcitonin mRNA predominates in the thyroid while the CGRP-specific mRNA appears to predominate in the nervous system (299). A human form of CGRP was isolated from thyroid tissue of patients with medullary thyroid carcinoma (259). CGRP is highly expressed in certain nerves (305) and is now known to belong to a family that includes the more recently discovered peptides adrenomedullin and amylin (see Fig. 1). This family belongs to a larger family of peptides that includes calcitonin. Calcitonin is a potent inhibitor of bone resorption, acting via receptor-mediated inhibition of osteoclast function (see Ref. 160). The overall effect of CGRP on bone resorption is unclear, although it can inhibit osteoclast activity (see Ref. 149), but it is best known for its potent cardiovascular effects.
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10-fold greater than the prostaglandins and 100–1,000 times greater than other classic vasodilators (e.g., acetylcholine, adenosine, 5-hydroxytryptamine, and substance P). This effect was first demonstrated in skin, where femtomole-picomole amounts of injected CGRP were able to induce reddening due to local microvascular dilation. In addition to its great potency, CGRP also differs from other vasodilator substances in that it has a particularly long duration of action. A dose of 15 pmol injected into human skin produces an erythema that lasts for 5–6 h (31). Further studies of CGRP have shown that its vasodilator activity extends to a wide variety of tissues and organs from other species, with particularly potent activity in the cerebral circulation, suggesting that it plays a role in the vasodilatation associated with the pathology of migraine (121). This in turn has highlighted a need to identify small nonpeptide receptor antagonists. B. Adrenomedullin: A Tissue-Derived Peptide
Adrenomedullin (AM) is a 52-amino acid peptide, which was discovered in 1993. It was isolated from human pheochromocytoma cells and was identified by its ability to stimulate cAMP production in platelets (197). It was soon realized that AM is produced by a wide range of cells including vascular endothelial and smooth muscle cells, especially upon stimulation with inflammatory cytokines (334–336). AM shares some of the cardiovascular activity of CGRP and thus may contribute to altered vascular function in disease. Only the terminal 40 amino acids of AM (AM13–52) are required for its biological activity. AM, or the active fragment AM13–52, has vasodilator and hypotensive effects but is 3–30 times less potent than CGRP (59, 132). Several reviews have discussed the clinical cardiovascular activities of AM in detail (94, 170), while the cellular and molecular biology of this peptide are reviewed by Lopez and Martinez (226). AM acts as a paracrine factor that can influence growth and development, renal effects, and endocrine (although it does not act as a hormone) activities. These properties have been extensively reviewed elsewhere (145).
A further peptide product encoded by the AM gene is pro-AM NH2-terminal 20 peptide (PAMP; Ref. 198). The precursor peptide pro-AM can be cleaved to produce AM and PAMP. PAMP is 30–100 times less potent than AM as a hypotensive agent in the rat (45, 325) but has been the subject of considerably less investigation. PAMP has been shown to be 60 times less potent than AM in the human forearm and is suggested to be of less importance in the regulation of blood pressure and flow (381).
C. Amylin: A Pancreatic Peptide
Amylin, or islet amyloid polypeptide (IAPP), is a 37-amino acid peptide that shares some structural homology with CGRP and AM (as shown in Fig. 1), as well as similarities in some of its biological activities and will be mentioned briefly in this review. Amylin was discovered as amyloid deposits in the pancreas of non-insulin-dependent diabetics (48, 65, 377). It has some vasodilator activity (32, 111), but its major physiological effect is regulating glucose metabolism. It is secreted with insulin from pancreatic 
-cells after meals. Amylin acts in the opposite manner to insulin with respect to glycogen synthesis and glucose uptake into muscle (64, 219; see Ref. 154 for review). Amylin has also been suggested to have roles in renal development and islet enlargement and may play a role in the development of the kidney (385). It is suggested that amylin decreases food intake in rats (47, 258) and that salmon calcitonin, which has a high affinity for amylin binding sites, can also mediate this effect (231). Furthermore, it has been proposed that amylin agonists could be beneficial as adjunct therapy in type 1 and certain cases of type 2 diabetes, where endogenous amylin is lacking (379).
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II. STRUCTURE |
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-type. The COOH and NH2 terminals of the peptide can interact independently with its receptors in that the CGRP8–37 fragment is an antagonist whilst CGRP1–7 is important for efficacy (see Ref. 20). There are two isoforms of CGRP for most species (
and ) that exhibit similar functional activities and differ by between one and three amino acids (7, 259, 305; see Fig. 1). AM13–52 has 25% structural similarity with CGRP, while amylin shares 46–50% structural homology (304). AM13–52 has 22% structural homology with amylin (see Fig. 1). |
III. DISTRIBUTION AND REGULATION OF GENES AND PEPTIDES |
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CGRP is widely distributed in the central and peripheral nervous systems (28, 83, 316, 383). It is primarily located in small, unmyelinated sensory C fibers and myelinated A
fibers in the periphery, where it is most usually found in nerves that are closely associated with blood vessels. CGRP is often colocalized with other peptides in C fibers, especially the tachykinins substance P and neurokinin A (230). It is considered that of the two forms, CGRP, encoded by the calcitonin gene, is the more abundant and found in discrete areas of the central and peripheral nervous system. In comparison, CGRP, which differs from CGRP by three amino acids in the human (see Fig. 1), is primarily located in the gut, in sites including those of enteric nerves (265) and the pituitary gland (288). CGRP is formed from a separate gene that does not produce calcitonin (5, 7). Both forms of CGRP possess similarly potent biological activity in terms of vasodilator activity (29), although there are proposed differences in their receptor-mediated effects (see sect. IVC). In general, it is the effects of CGRP that are discussed in this review.
CGRP has been identified at many sites complementary to its activity as a vasoactive mediator (see Ref. 150 for review). For example, CGRP-containing nerves innervate smaller arteries, where innervating nerve terminals can pass into the vascular smooth muscle layer. This allows CGRP to be released where it can have profound effects on arteriolar dilatation and on the microvasculature. CGRP-containing nerves also innervate venous tissues, but its activity on these tissues is less well documented. The distribution of CGRP-containing nerves has been studied in most tissues but has probably been most extensively reviewed with respect to pathophysiological function in the cerebral circulation (83). CGRP is released from sensory fibers originating in the trigeminal ganglia and acts to dilate cerebral vessels (119). The gut has also been intensively studied: here CGRP released from spinal afferents acts to dilate mucosal blood vessels and may protect against the acidic environment. It is possible that CGRP-containing vagal afferents, which originate from nodose ganglia, have a preferential nociceptive role (152).
CGRP has been suggested to be more abundant in common laboratory species than in humans (380). However, CGRP is localized in nerves in human coronary arteries and veins, especially at the adventitial-medial border, and has potent relaxant effects on arteries (127, 308). Low (picomolar) levels of immunoreactive CGRP have been detected in the plasma of healthy volunteers (117), with levels elevated during pregnancy (332). It is generally considered that the levels of CGRP detected in plasma are likely to be due to leakage after localized release rather than a specific systemic function. However, CGRP has the ability to decrease blood pressure and increase heart rate when given by intravenous administration to human volunteers (113), indicating that if sufficiently high plasma levels of CGRP are reached, systemic vasoactive effects can be triggered.
The regulation of CGRP production is poorly understood. Plasticity occurs at the level of the ganglia, for example, in models of peripheral axotomy, enteritis (190), and inflamed arthritic joints (77). In each case there is an associated increase in CGRP production in the ganglia. One factor of potential importance in influencing plasticity is nerve growth factor (NGF), which has an important role in the growth and maintenance of sensory nerve function (350). At a cellular level, NGF upregulates CGRP via a cAMP/ras responsive element (106), and via a constitutively active mitogen-activated protein kinase (MAPK) kinase (MEK; Ref. 82). In some experiments the upregulation of CGRP has been associated with nerve sprouting, an indication of NGF activity (56). Furthermore, upregulation of CGRP production in the dorsal root ganglia by NGF has been linked to restoration of the endogenous microvascular activity of CGRP in diabetic skin (72), and in promoting CGRP expression in the spontaneously hypertensive rat (341). It is possible that NGF acts as a rescue factor in times of vascular stress.
The release of CGRP from peripheral nerves was demonstrated at an early stage (73, 370). A classical mechanism leading to the release of sensory neuropeptides is that mediated by capsaicin, but the endogenous significance of this release mechanism remains unproven. CGRP immunoreactivity increases in the plasma after the administration of capsaicin, although the elevation is short lived (396). Capsaicin acts via vanilloid (TRPV1) receptors on sensory C and A fibers to increase permeability to cations (42). This leads to nerve depolarization, release of neuropeptides, and, in time, their depletion from sensory nerves. Indeed, long-term treatment with capsaicin to deplete the sensory neurogenic component has been exploited in a beneficial manner to treat a range of vascular conditions in humans (see Ref. 342). Low pH and heat are also associated with the activation of the capsaicin receptor, leading to a release of CGRP (e.g., Refs. 115, 193). This may be relevant to the release and contribution of CGRP in models of cardiovascular ischemic inflammation, where localized acidity and increased heat are observed. It has been recently suggested that a range of endogenous agents may also act to stimulate this receptor. These include anandamide (405) and leukotriene B4 (158). Other substances considered to be involved in mediating the release of CGRP include kinins and prostaglandins (9, 124, 168) and NO (22, 176). It has been suggested that under certain circumstances, such as septic shock, mediators can act in a synergistic manner to potentiate CGRP release (374). The direct influence of inflammatory cytokines remains unclear, although it is suggested that interleukin (IL)-1 can act in a time- and protein synthesis-dependent manner to increase CGRP release from dorsal root ganglion neurons via a protein kinase C-dependent mechanism (156). Proteinase-activated receptor-2 is a member of a novel subfamily of G protein receptors that are activated by proteolysis and present on sensory nerves where they stimulate CGRP release (331). The functional and pathological importance of this response is not yet known (367).
Presynaptic/prejunctional receptors on the sensory nerves themselves also play an important role in modulating CGRP release. Evidence indicates a range of such receptors that include those for opioids, 5-hydroxytryptamine (5-HT1 receptor), -aminobutyric acid (GABAB receptor), histamine (H3 receptor), neuropeptide Y, somatostatin, vasoactive intestinal polypeptide, purines, and galanin (see Refs. 15, 27, 234 for reviews). A recent study suggests that excitatory CGRP receptors are also present on sensory neurons within the dorsal root ganglia, coupled to an increase in intracellular calcium, and these may act as stimulatory autoreceptors (315). There is evidence in the rat mesentery that reciprocal interactions can occur between the noradrenergic constrictor system and the sensory system. It has been shown that stimulation of 2-adrenoceptors, located presynaptically on sensory neurons, acts to inhibit CGRP release (188), with CGRP also inhibiting the release of norepinephrine from sympathetic nerves (189). These results are indicative of an important role for CGRP in the regulation of peripheral blood flow.
In the circulation, CGRP has a half-life of 7–10 min in humans (205, 333). There is not an obvious mechanism for CGRP metabolism, and it is probably broken down via a number of routes. Mast cell tryptase has a potent effect in cleaving CGRP into inactive fragments, both in vivo and in vitro. This mechanism has been clearly demonstrated in extravascular sites, e.g., skin (30). In addition, CGRP can compete with substance P for breakdown by an enzyme in the central nervous system (279). In contrast to substance P, CGRP seems to be a poor substrate for neutral endopeptidase, so this pathway is probably less important as a route for CGRP degradation in peripheral tissues (185). A matrix metalloproteinase II has the ability to metabolize CGRP and remove its vasodilator activity (99). Finally, there has also been a suggestion that CGRP may be taken back up into sensory nerve terminals after repolarization, at least in vitro (313).
B. AM: Upregulation in Disease
The AM gene is located on chromosome 11 in humans (163). Unlike CGRP, AM is primarily produced by nonnervous tissue, especially endothelial (334) and vascular smooth muscle cells (336). AM mRNA is thus primarily found in most areas where abundant microvascular vessels exist (e.g., heart, lung, kidney, and cerebral vasculature; Refs. 159, 209). In particular, AM is produced in the atrium of the heart (170). The normal circulating levels of AM are in the low picomolar range, but levels are increased in disease and certain physiological conditions, including pregnancy (138). Studies have revealed increased circulating levels of AM in cardiovascular diseases such as hypertension and stroke, as well as septic shock, and it has been suggested that the increased levels are in proportion with disease severity (302, 375).
AM is constitutively released from endothelial cells, and its release is regulated entirely at the level of protein expression (334). The increase in AM plasma levels in disease is associated with an increase in AM gene expression in tissues, especially vascular and smooth muscle cells (161, 334–336), as discussed above. Increased AM production probably contributes to the vascular component of inflammatory disease, particularly as the major cytokines tumor necrosis factor (TNF)- and - and IL-1 and -1 are potent stimulators of upregulation of the AM gene in endothelial and smooth muscle cells (334–336). Interestingly, AM has been shown to potentiate IL-1-stimulated inducible nitric oxide synthase (NOS) and thus nitric oxide (NO) synthesis, although AM does not appear to have a direct effect on inducible NOS generation (139). Other factors shown to increase the mRNA and/or synthesis of AM in vascular smooth muscle cells include the adrenocortical steroids and thyroid hormones (252). In addition, the finding that shear stress promotes AM mRNA production in human endothelial cells is of relevance to vascular dysfunction (62). The regulation of the production and secretion of AM in the cardiovascular system has been recently reviewed (93). An adrenomedullin binding protein, originally known as complement factor H, has been discovered (89). This protein has been suggested to facilitate the presence of high concentrations of AM at receptor sites in tissues, and possibly also to modulate the degradation of AM (291).
The increase in tissue levels in disease has been most extensively studied in septic shock (147, 274). During infection, bacterial and viral products such as bacterial lipopolysaccharide (LPS) stimulate release of cytokines such as TNF- and IL-1. These cytokines in turn stimulate the host defense against microbial pathogens, and excessive production of these cytokines may be responsible in part for the fatalities observed during sepsis and systemic inflammatory response syndrome (SIRS; Ref. 17). Thus a link has been established between LPS, cytokines, and the increased levels of AM in sepsis and SIRS patients (see sect. IXE).
C. Amylin: Relevance to Diabetes
Amylin was primarily found as a major peptide constituent of amyloid deposits in the islet -cells in the pancreas of type 2 diabetics (65), and in the amyloid deposits associated with pancreatic tumors (378). Substantially lower amounts of amylin are found in nerves, but interestingly, it is constitutively expressed in CGRP-containing neurons. It is upregulated in a transient manner in a model of joint inflammation in the rat paw (264). This may be related to the early inflammatory component in this model. Amylin has also been localized to the pyloric antrum of the stomach, duodenum, jejunum, ileum, and colon in the rat (10).
Amylin is cosecreted with insulin in response to glucose, but the relative amount of each peptide may vary, depending on situation. Like insulin, it is lacking in type 1 diabetes, in keeping with the loss of the pancreatic cells. In obese subjects, amylin mirrors release of insulin as insulin resistance develops, but is deficient in type 2 diabetes (228), as insoluble amyloid fibrils are thought to develop. Evidence has recently been provided that a mutation in the enhancer region of the amylin promoter may be related to the development of type 2 diabetes (277).
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IV. RECEPTORS |
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The existence of two receptors, CGRP1 and CGRP2, was originally proposed in the late 1980s, with the CGRP1 receptor being the predominant mediator of cardiovascular effects. This receptor classification was developed as a consequence of pharmacological studies carried out with different agonists and antagonists in a range of tissue preparations, especially the positive inotropic effect in the guinea pig or rat atrium for determination of CGRP1 receptor activity, and the inhibition of electrically evoked twitch responses in the rat vas deferens for determination of CGRP2 receptor activity (69, 70, 81). The 30-amino acid fragment of CGRP, CGRP8–37, is an antagonist showing preference for the CGRP1 receptor (54). In contrast, linearized CGRP analogs such as diacetoamidomethyl cysteine CGRP {[Cys(ACM)2,7]hCGRP} are considered to show preferential agonist potency for the CGRP2 receptor. [Cys(ACM)2,7]hCGRP is formed by reduction of the disulfide bond of CGRP. In general, receptors that can be antagonized by CGRP8–37 with an approximate pKb value of 7.0 are designated as CGRP1 receptors, while those that CGRP8–37 block with a pKb of 6 or less are classified as CGRP2 receptors (293, 299). However, more recent studies have questioned the selectivity of [Cys(ACM)2,7]hCGRP for the CGRP2 receptor and suggested that it also exhibits potent activity at the CGRP1 receptor (79). This classification of receptors still holds true today in that experiments with peptidase inhibitors have not revealed reasons for the differences (173). However, the classification is not universally accepted, as recently debated by Poyner and Marshall (294). They describe how mean pA2 values that range between 8.1 and <5 in 11 different rat tissues do not allow themselves to be readily divided into two receptor groups (294).
Evidence for an AM receptor was initially obtained from functional studies where the responses were not inhibited by CGRP8–37 (e.g., in the rat; Ref. 270). Eguchi et al. (88) suggested that human AM22–52 is an antagonist of the AM receptor. There is some confusion in the literature, in that some studies (e.g., in the perfused hindlimb vascular bed of the cat) show that AM22–52 did not antagonize vasodilator responses to AM, but did inhibit CGRP responses (46). However, in later studies, AM22–52 was found to selectively antagonize rat cerebral vasodilatation (76) and has slowly become established as a weak antagonist of AM responses (see Ref. 112), thus strengthening the concept of a specific AM receptor.
Several members of the CGRP family of receptors have been cloned in recent years. In 1995 an orphan receptor commonly known as L1 (see Ref. 13) was suggested to be an AM receptor (183), although little supporting evidence has been provided to date (192). A second receptor, a putative CGRP receptor, RDC, that had originally been shown to increase cAMP in response to CGRP (184) has also not been supported in the literature (173, 248).
Rat calcitonin receptor-like receptor (CL) was cloned in 1993 (275). Human CL was cloned 2 years later and consists of 461 amino acids with 7 transmembrane domains; 91 and 56% of the amino acids were found to be identical to the rat orphan calcitonin receptor-like sequence and the human calcitonin receptor, respectively (103). However, the receptor did not bind CGRP in the cells studied and was considered an orphan receptor. A major breakthrough was made in 1996 when Aiyar et al. (4) cloned and characterized cDNA encoding the hCGRP1 receptor. Interestingly, the cloned receptor demonstrated significant peptide sequence homology with CL. Furthermore, the cDNA was expressed in a stable manner in human embryonic kidney 293 cells (HEK293) with associated specific, high-affinity binding sites for CGRP that displayed functional properties very similar to the human CGRP1 receptor: CGRP induced a 60-fold elevation in cAMP production that was inhibited in a competitive manner by CGRP8–37 (4). These results were this time confirmed by two other groups using rat (134) and porcine CL (90). However, in the same study, Han et al. (134) found that CL expressed in COS-7 cells failed to produce a functional receptor, so they concluded that HEK293 cells must also possess an extra intrinsic factor necessary for the production of a functional receptor. It was not until the work of McLatchie et al. (248) was published that it was realized that a receptor activity-modifying protein (RAMP; a 148-amino acid peptide with a single transmembrane domain) was required to associate with CL to confer receptor activity (248).
The CGRP/AM receptors that have been cloned and characterized to date consist of a seven-transmembrane G protein-coupled CL in association with one of three single membrane-spanning RAMPs (see Fig. 2). There is strong evidence from studies in cultured cells that CL, in combination with an appropriate RAMP, acts as a receptor for CGRP and adrenomedullin (e.g., Ref. 55). CL is a member of the B family of seven transmembrane G protein-coupled receptors. Members include, in addition to the calcitonin receptor, receptors for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide, and parathyroid hormone (318).
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CGRP is 15 times less potent than AM at activating the rat CL/RAMP3 receptor in COS-7 cells, CGRP is only 2.5 times less potent. It is not clear whether CGRP signaling via the AM2 receptor plays an important role in vivo. The nomenclature for these receptor types has been recently discussed and standardized (295). The less widely distributed calcitonin receptor acts alone as a calcitonin (hCTR2) receptor but can also interact with RAMPs to produce a high-affinity receptor for amylin/calcitonin (58, 262) and CGRP (220). It is now suggested that RAMPs can also interact more widely with G protein-linked receptors that include the vasoactive intestinal polypeptide VPAC-1 receptor (57). RAMP1, -2, and -3 have been identified in humans, rats, and mice. The same RAMPs in different species show >60% homology, but <30% homology exists between different RAMPs in the same species (330). The RAMPs interact with CL to provide an active receptor in the cell membrane and are essential in determining receptor specificity (23, 317). The extracellular NH2 terminus of the RAMP is important for ligand binding. The deletion of residues 93–99 from RAMP2 and 58–64 from RAMP3 have been each shown to substantially inhibit AM binding (211). In comparison, the short COOH terminus is not essential to activity, although loss of the W and Q amino acid residues adjacent to the membrane abolishes signaling activity (330). CL is widely distributed (130), so the expression pattern of the RAMPs is likely to determine the expression of functional CGRP and AM receptors. However, the regulation and functional relevance of the CL/RAMP interactions in vivo are still poorly understood. Studies from transfected endothelial cell lines indicate that the dynamic interactions between RAMPs can lead to competition between different RAMP types (263). It has been proposed that RAMP1 is dominant, leading to the preferential expression of a functional CGRP receptor when both RAMP1 and RAMP2 are present in a cell (36). In some cell types, such as cultured macrovascular (61) and dermal microvascular endothelial cells (181), RAMP2 is expressed to a much greater degree than RAMP1, producing functional AM receptors. Indeed, RAMP1 was not detected in human cerebral vascular tissue endothelial cells (281). Thus a substantial amount is known about the CGRP and AM receptors from elegant molecular and cell-based studies.
Studies of CGRP/AM receptor expression in various pathological conditions have revealed alterations in receptor components such as sepsis, indicating that receptor plasticity may play a role in pathophysiological states. Ono et al. (282) provide preliminary evidence in representative results from a model of sepsis, that the normally high expression of CL and RAMP2 in the mouse lung is substantially decreased at 12 h after induction of sepsis by LPS, but this has not been confirmed by Ornan et al. (284) in rat sepsis. In addition, RAMP3 levels have been shown to be elevated in the late stages of sepsis (282, 284) and in a rodent model of chronic heart failure (67). In the latter model RAMP1 upregulation was also observed.
It is now accepted that while the CL is important for ligand binding, the RAMP proteins have roles in determining receptor phenotype and species selectivity. The trafficking activity of RAMP1 has been studied, and it is now realized that RAMP1, when expressed alone, is located in the endoplasmic reticulum and the Golgi mainly as a disulfide-linked homodimer (144). However, when found at the cell surface it is present as a heterodimer with CL. Recent evidence suggests that the RAMP protein is stablized at the cell surface when complexed with CL (57; see Fig. 2). The association of RAMP with CL leads to a poorly characterized noncovalent interaction. The coexpression of RAMP1 with CL as heterodimers at the cell surface in HEK-293 cells and studies of deletion mutants have revealed that residues 91–103 are important for high-affinity CGRP binding, although no individual residue was critical, while the deletion of residues 78–80 or 88–90 reduced AM binding (210). These authors suggest that the RAMPs probably influence binding by influencing the formation of a "receptor pocket" or by allosteric modulation of the conformation of the receptor. In elegant studies with hybrid CGRP receptors, Kane and co-workers (240) have described how chimeric RAMP constructs reveal that RAMP1 determines the species selectivity for receptor antagonists, and that a specific amino acid residue (tryptophan at position 74) is responsible. The binding of CGRP ligand and activation of the receptor is then associated with phosphorylation of CL and receptor internalization. The internalization process is typical of that associated with seven transmembrane receptors involving -arrestin and clathrin-coated pit-mediated endocytosis (143). Interestingly, there is a lack of CL in some tissues, such as the cerebellum of certain species, where there is substantial CGRP binding and thus evidence for further receptors (52). In reality, the functional information has to be viewed alongside the molecular data, and this is not easy at the present stage.
The CGRP-receptor component (RCP; see Fig. 2) is a 17-kDa intracellular membrane protein that was cloned and shown to provide CGRP receptor activity to Xenopus oocytes (229). Antisense studies in NIH3T3 cells and immunocoprecipitation studies have implicated a role for RCP in driving the receptor-mediating response, by involvement in receptor coupling and stimulation of adenylate cyclase (96). However, this concept awaits further experimental confirmation, for example, through development of RCP knockout mice.
The CGRP antagonist CGRP8–37 has been used since 1989 as a pharmacological tool to block CGRP1 responses (54). The peptide nature of this antagonist has limited its use, and there has been a requirement among researchers for a more stable nonpeptide antagonist. This requirement has accompanied the need to develop nonpeptide antagonists for possible treatment of pathological conditions, particularly migraine. Progress has been slow in that until 2000 the novel antagonists that had been developed had been considered either too difficult to use or, due to solubility problems, of insufficient potency for use. However, Boehringer patented a group of compounds as CGRP antagonists in 1998 and published results on the activity of one of these compounds BIBN4096BS in February 2000. BIBN4096BS, 1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)-methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazoli-nyl)-,[R-(R*,S*)], is a competitive nonpeptide antagonist with potent antagonistic activity at the human CGRP1 receptor (79). It has an affinity (Ki) of 14.4 ± 6.3 pM for human CGRP1 receptors in SK-N-MC cells (a human neuroblastoma cell line) and 3.4 ± 0.5 nM for CGRP1 receptors in spleen. This compares to 1.3 nM on SK-N-MC cells for CGRP8–37 (87), so it is apparent that BIBN4096BS is much more potent. BIBN4096BS also displays potent selective antagonism of CGRP receptors in human and marmoset tissues with species selectivity (200-fold greater affinity compared with its binding in rodent tissues) when compared with common laboratory species. This was found to be due to a single amino acid difference between primate and rodent receptors, as recently described (240). Furthermore, there is evidence that this compound antagonizes human CGRP more readily than CGRP-induced positive inotropic effects, whereas CGRP8–37 is a similar antagonist of both (386). BIBN4096BS has been used to learn more about the classification of receptors into the CGRP1 and -2 subclasses. Interestingly, like CGRP8–37, BIBN4096BS shows an 10-fold higher affinity for CGRP1 (blockade of positive inotropy in the rat left atrium) than CGRP2 receptors (inhibition of CGRP-evoked twitch in the rat vas deferens; Ref. 386). Furthermore, the authors also suggested that a novel receptor may exist in the rat vas deferens that is not blocked by CGRP8–37 but at which AM and the linearized CGRP analog [Cys(Et)2,7]hCGRP have potent activity. BIBN4096BS was found to block this receptor (386). More recently, studies using all-rat and rat-human combination AM1 and AM2 receptors in several different cell lines found that BIBN4096BS was unable to antagonize AM responses at doses up to 10 µM (140).
Experiments with BIBN4096BS have also aided in identifying and clarifying additional biological roles for CGRP. BIBN4096BS has been suggested as a potential therapy for sufferers of migraine, a condition linked to elevated secretion of CGRP. It potently antagonized CGRP-induced dilation of human temporal arteries (368), bovine middle artery and human meningeal, cerebral, and pial arteries (255). Dilation of these vessels is believed to be at least partially responsible for the pain suffered during a migraine attack (255).
A second compound from Boehringer patent WO98/11128, (4-(2-oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1–3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide), Compound 1, has also been synthesized and studied. It is a weak antagonist of CGRP receptors. Binding data with SK-N-MC cells revealed pKi values of 7.8, compared with 8.9 for CGRP8–37 in displacing CGRP. It also weakly antagonized CGRP responses in human cerebral and guinea pig basilar arteries (87). However, this compound failed to inhibit the vascular relaxation induced by CGRP, AM, and amylin in porcine coronary arteries (137), but does so in human coronary artery (136), whereas CGRP8–37 is an effective antagonist in both tissues. Once again, these results underline the species and tissue selectivity that is now becoming associated with CGRP receptor antagonists.
An alternative nonpeptide CGRP receptor antagonist has been developed by GlaxoSmithKline (2). SB-273779, [N-methyl-N-(2-methylphenyl)-3-nitro-4-(2-thiazolylsulfinyl)nitrobenzanilide], is selective for the CGRP receptor but is less potent than BIBN4096BS, with a Ki value of 310 ± 40 nM on SK-N-MC cells. In addition this compound was weak in competition studies with CGRP in rat and porcine lungs. The authors describe it as the first "cross-species" (i.e., comparable affinities in human, porcine, and rat tissues) nonpeptide CGRP antagonist (2).
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There are several mechanisms by which CGRP produces vascular relaxation, as discussed in earlier reviews (20, 28, 242). It is accepted that vasodilatation is mediated via the CGRP1 receptor and blocked in a competitive manner by CGRP8–37. Current evidence points to the existence of an NO- and endothelium-independent pathway, where CGRP administration correlates closely with a rise in intracellular cAMP ([cAMP]i) (see Fig. 3). This mechanism is observed in the majority of tissues that have been studied to date (e.g., rat perfused mesentery, Ref. 133; cat cerebral artery, Ref. 86; porcine coronary artery, Ref. 393). The ability of CGRP to relax these tissues in the absence of an endothelium implies that it acts directly on the smooth muscle cells to stimulate adenylate cyclase, and this has been demonstrated in cultured smooth muscle cells (66, 148). The resulting rise in [cAMP]i activates protein kinase A (PKA), which probably phosphorylates and opens K+ channels, leading to relaxation. Nelson et al. (271) first suggested an involvement of ATP-sensitive potassium channels in the vasodilator mechanism of CGRP in 1990. They showed that glibenclamide (an ATP-sensitive K+ channel blocker) acted selectively to block the CGRP-induced response and that CGRP hyperpolarizes arterial smooth muscle. CGRP indirectly activates ATP-sensitive potassium currents via adenylate cyclase activation and cAMP generation in smooth muscle from porcine coronary artery (376) and guinea pig ureter (237). In other tissues, a role for K+ channel activation in the relaxation to CGRP is evident, although coupling through activation of adenylate cyclase and cAMP generation has not been confirmed. In rat pial arterioles, vasodilatation to CGRP was inhibited in the presence of glibenclamide or charybdotoxin (a large-conductance Ca2+-activated K+ channel blocker; Ref. 153). In the mouse aorta (292) and rat renal microvasculature (301), vasodilatation to CGRP was partially inhibited by glibenclamide. Also, in the renal microvasculature, the vasorelaxant activity of CGRP could be mimicked by the application of pinacidil (an opener of ATP-sensitive K+ channels) (301). Administration of bolus doses of CGRP to rats produced significant hypotension, which was potentiated by cromakalim (an opener of ATP-sensitive K+ channels) and attenuated by glibenclamide (310). These data all indicate a role for K+ channel activation in the relaxation to CGRP by vascular smooth muscle.
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Both the signaling pathways described above are mediated through stimulation of adenylate cyclase to produce cAMP. This implies that the CGRP receptor is coupling to GS proteins. The CGRP receptor may also be able to stimulate intracellular activity through a different G protein. Aiyar et al. (3) reported that CGRP was able to activate phospholipase C (PLC) in HEK293 cells, leading to an increase in intracellular calcium via inositol trisphosphate (IP3) activity. This increase in calcium occurred concurrently with the stimulation of adenylyl cyclase and accumulation of cAMP. Activation of PLC is considered to occur through Gq/11, rather than through GS, suggesting that the activated CGRP receptor is able to interact with both types of G protein. If this mechanism is present in endothelial cells, it provides an alternative explanation for CGRP activation of eNOS (which is traditionally considered to be dependent on Ca2+/calmodulin for activation) independently of cAMP accumulation. The possibility that CGRP receptors may be coupled to phosphatidylinositol turnover is supported by another study that found this secondary messenger pathway in skeletal muscle (216).
B. AM: Active on CGRP and AM Receptors
The mechanisms via which AM can elicit vascular relaxation are heterogeneous with respect to both species and vascular bed. They are incompletely understood, but known to involve both the CGRP and AM receptors as AM has been shown to induce vascular relaxation via either CGRP8–37-sensitive or AM22–52-sensitive mechanisms. Furthermore, in some tissues, there is evidence that AM can act via both endothelium-dependent (NO-dependent) and K+ channel-dependent mechanisms (214, 307, 349, 393). In addition, there is some controversy in that different results have been obtained using the same tissue in different laboratories. One problem may be that the AM antagonist AM22–52 (88), while being the best available, has been criticized for being weak and lacking in specificity (145). Thus the following acts as an overview, based on assessment of published information.
AM, like CGRP, has been shown to increase both cAMP (161, 186) and intracellular calcium levels in endothelial cells (324, 393). A similar effect of AM on cAMP levels has been reported in vascular smooth muscle cells, with a decrease in intracellular calcium levels, although results may depend on experimental conditions and tissue (14, 88, 393). There is evidence that AM acts to relax tissues via a CGRP receptor-dependent mechanism in the porcine coronary artery preconstricted with U46619 (393), in a range of arterial vessels from the dog, where the effect was largely unaffected by endothelial removal (268), in the rat coronary artery (324), rat cutaneous microvasculature (132), the rat isolated perfused kidney (142), and the rat mesenteric bed (278) to name a few. In addition, CGRP8–37-resistant responses have been found in other tissues, where the weak AM antagonist AM22–52 blocked responses (e.g., rat cerebral microvessels, Ref. 202; rat smooth muscle cells, Ref. 88; and in the human coronary artery, Ref. 349).
A further mechanism that may be involved in AM signaling is suggested by Nishimatsu et al. (273). They examined the NO-dependent relaxation to AM in the rat aorta and identified the involvement of a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent pathway in the stimulation of eNOS. The results showed that AM stimulated Akt activation in aortic endothelium via the Ca2+/calmodulin-dependent route, an established stimulant of eNOS (366). This protein kinase was also implicated in the increased production of NO stimulated by shear stress, demonstrated by Dimmeler et al. (75). They found that serine phosphorylation of eNOS increased its Ca2+/calmodulin sensitivity, and thus stimulated production of NO. Nishimatsu et al. (273) did not investigate the receptor involved in mediating NO-dependent relaxation in the rat aorta. However, in the human coronary artery, AM-induced, NO-dependent relaxation has been shown to be selectively blocked by AM22–52, but not CGRP8–37, indicating a role for a specific AM receptor. In this tissue it was suggested that AM stimulates vasodilatation via both NO and K+ channels, with only a minor contribution of cAMP (349). The relevance of adenylate cyclase to NO production in response to AM stimulation is unclear, in that there is evidence for either a requirement for cAMP accumulation during relaxation in canine coronary blood vessels (400) or no requirement in bovine aortic endothelial cells (324).
There are thus a variety of mechanisms via which AM may induce vascular relaxation, and further research is required to fully understand them. However, it is clear that there is an overall similarity in the mechanisms of CGRP- and AM-induced relaxation. This is presumably related to the fact that both AM and CGRP are known to act via the same G protein-linked receptor (i.e., CL). The majority of the differences in intracellular signaling pathways activated by CGRP and AM are probably related to the different RAMPs that form their receptors. For example, RAMP2 and RAMP3 may be involved in modulating the migration of endothelial and smooth muscle cells, via cAMP-independent mechanisms. AM has been recently shown to inhibit the migratory activity of vascular smooth muscle cells, in a model where CGRP was inactive (107).
C. Amylin: Active Via the CGRP1 Receptor
The ability of amylin to act as a vascular relaxant of both macro- and microvessels is well established, although it is in the region of 100-fold less active than CGRP in vitro (16, 32, 131, 167) and is not considered to be involved in the regulation of blood pressure. The vasodilator effects of amylin appear to be mediated by the CGRP1 receptor as they are blocked by CGRP8–37. The vascular relaxant effects of CGRP, AM, and amylin have been compared in porcine coronary arteries. The responses were all blocked by CGRP8–37, supporting the above findings. In this study a distinct amylin receptor (consisting of calcitonin interacting with a RAMP; see sect. IVB) was sought, without success (137).
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VI. CARDIOVASCULAR REGULATION |
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The vasodilator activity of CGRP has been studied extensively in the vasculature in vitro, and the section above highlights the importance of these studies for the elucidation of vasodilator mechanisms. Studies in vivo are also essential to determine the role of CGRP in cardiovascular regulation. The contribution of the various vasodilator mechanisms of CGRP to the vasodilatation in humans has been examined in a study in the human forearm where the ability of intra-arterial infusion of CGRP to stimulate a decrease in forearm vascular resistance due to vasodilatation is well established. The CGRP-induced vasodilatation induced by this intravascular source of CGRP was shown to be dependent, at least in part, on NO and not activation of K+ channels (68). The role of cAMP was inconclusive, primarily as adenylate cyclase inhibitors are too toxic to administer in vivo. The intravenous administration of CGRP is associated with hypotension and positive inotropic and chronotropic responses in the rat (8, 19, 111). In comparison, intracerebroventricular injection of CGRP, as well as other members of the calcitonin family of peptides causes an increase in blood pressure in rats, due to sympathetic nerve stimulation and release of the vasoconstrictor norepinephrine (102). However, the available evidence, as summarized below, suggests that CGRP does not have a role in the regulation of blood flow in the normal rat or mouse.
Studies with intravenously injected CGRP8–37 in rodents have shown a lack of effect on basal blood pressure (111). A recent investigation in the anesthetized rat and conscious dog supports these results and shows that CGRP8–37, at doses that antagonized the effects of CGRP, had no effect on either systemic blood pressure or regional vascular beds (323). This supports the hypothesis that endogenous CGRP acts in a local rather than a systemic manner to modulate blood flow. In addition to studies using CGRP receptor antagonists, elucidation of the role of CGRP in cardiovascular regulation has been aided by the creation of CGRP knockout mice. However, the results from these mice have already caused some controversy. The first strain of CGRP knockout to be created did not show any change in basal blood pressure (227). In contrast, CGRP knockout mice produced by a different group showed pronounced increases in both systolic and mean arterial pressure, as measured by the tail cuff technique, or after implantation of a carotid artery cannula in conscious mice (108). In the two cases cited above, the knockout mice exhibited no obvious phenotypic differences from their wild-type counterparts, but the nature of the mode of deletion of CGRP differed. The mice studied by Lu et al. (227) were created through the insertion of a stop codon into exon 5 of the CGRP/calcitonin gene, so calcitonin was still expressed normally (227). In contrast, the other strain of mice had exons 2–5 of the CGRP/calcitonin gene replaced with a PGK neoBPA cassette, which also knocked out the calcitonin gene. Although no evidence for calcitonin regulation of the cardiovascular system has been found to date, the loss of this protein may be relevant to the increased blood pressure observed in the knockout mice (108). A third strain of CGRP knockout mouse, produced more recently, has added to the confusion surrounding the role of CGRP in the maintenance of basal blood pressure (280). These mice expressed calcitonin normally, but still showed an increase in both mean arterial pressure and heart rate, compared with their wild-type counterparts. These cardiovascular changes were suggested to be a result of increased sympathetic nervous activity, which was measured as an increased level of urinary catecholamine metabolites (280). The reasons for the observed differences in these strains of mice remain to be elucidated.
The studies using the CGRP knockout mice have been important in the absence of a well-characterized, potent, and selective antagonist for the CGRP receptor(s). They have revealed some inconsistencies compared with each other and to the antagonist studies, and these require further investigation. In comparison, it is established that CGRP8–37 has little potency as a systemic vasoconstrictor, although effects observed on regional blood flow (110) suggest that CGRP may play a local modulatory/homeostatic role in the control of blood pressure. Furthermore, it has been suggested that the attenuated release of CGRP in spontaneously hypertensive rats may contribute to the observed hypertension (341).
B. AM: Role in Regulating Blood Pressure
AM has been shown to act as a vasodilator in a range of vascular beds and species (e.g., cat, rat, and sheep; Refs. 46, 51, 112) leading to decreased blood pressure and increased heart rate. In addition, AM has been shown to act via NO-dependent mechanisms to dilate the renal vasculature and to mediate diuretic and natriuretic responses in the kidney (239). The vascular relaxant effects of AM administration in the cerebral and systemic circulations are blocked by AM22–52 in the rat (76, 112). However, in a model of endotoxemia in the conscious rat, evidence was provided for a hypotensive role for CGRP, but not AM (112). In comparison, Mazzocchi et al. (245) have provided evidence for a role for AM, in a model involving intraperitoneal administration of endotoxin. The hypotensive responses were blocked by AM22–52, but not CGRP8–37. Others have suggested that AM may have a role in maintaining renal blood flow in septic shock (274). This is further discussed in section IXE.
Development of AM knockout mice has also been attempted, but surprisingly deletion of the AM gene in mice is lethal. The reason is thought to be due to insufficient development of blood vessels, umbilical arteries, and the formation of hydrops fetalis (40, 141). Studies of pregnant subjects have revealed that AM levels are raised in plasma, amniotic fluid, and cord blood from 8 weeks of gestation onwards (74). It is thought that AM may have an essential role in fetal development as well as the regulation of placental and fetal circulation (232, 254, 394).
Studies with transgenic mice overexpressing AM, and heterozygote AM knockout mice have provided evidence that AM may play a role in the regulation of basal blood pressure. The blood pressure in heterozygotes was elevated, with suppressed NO production (327), while the blood pressure in AM overexpressing transgenics was lower (326). In addition, these mice were more resistant to septic shock than wild-type mice (326), as discussed in section IXE. Furthermore, it has been clearly shown that in a model of cardiovascular injury, induced by salt loading, the heart weight-to-body weight ratio was significantly increased in heterozygotes. In addition, the perivascular coronary artery in heterozygotes contained inflammatory cells with a fibrotic appearance, and transforming growth factor- (TGF-) mRNA was upregulated. Thus it is suggested that AM possesses protective action against angiotensin II-induced coronary artery injury and cardiomegaly (95, 141).
The infusion of AM via the brachial artery in human volunteers is associated with dose-dependent vasodilatation and increased blood flow (63). Indeed, at low doses AM was considered to be more potent than CGRP. The response to administration in humans appears dose-dependent in that doses that produce a low plasma AM concentration (<12 pM) induce a long-lasting decrease in blood pressure without affecting plasma renin and catecholamine levels, or urine sodium content (212). However, infusions of higher amounts, leading to plasma levels of 50 pM or greater, are associated with tachycardia and increased prolactin levels.
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VII. MICROVASCULAR MECHANISMS |
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The intravenous infusion of subvasodepressor doses into the conscious rat led to specific relaxant effects in a range of tissues, for example, a reduction in hindquarters vascular resistance (109). The concept of CGRP as a highly targeted vasodilator is enhanced by the observation of increased microvascular blood flow induced in the ipsilateral, but not contralateral, skin of the hindleg of the anesthetized rat after stimulation of CGRP-containing nerves, demonstrating that its activity is primarily at the site of release (92). It is also in keeping with the observation of selective facial flushing observed after intravenous CGRP administration in humans (113). Furthermore, in studies with genetically transformed mice, Champion et al. (43) have developed elegant techniques to study the vascular responses in the lung and hindquarters of the mouse. The wild-type and CGRP knockout mice used exhibited similar blood pressure, but vascular resistance was increased in knockout mice, suggesting that CGRP normally acts as a "braking system" for vascular resistance in the microvasculature.
The potent microvascular vasodilator activity of CGRP and its wide distribution in the periphery ensures that it is in a prime position to protect tissues from injury, in addition to regulating tissue blood flow under physiological conditions. Indeed, an involvement of CGRP-containing sensory nerves in the maintenance of tissue homeostasis has been proposed for some years. Evidence from a variety of tissues is given below. Of particular interest is the observation of both harmful and protective roles, depending on the circumstance of CGRP release. The administration of capsaicin to the rat at a neonatal stage leads to a selective destruction of capsaicin-sensitive sensory nerves, and thus a loss of their contents (e.g., CGRP). This is associated with an increased likelihood of the appearance of cutaneous lesions (236, 352). The beneficial role of sensory nerves is considered to be due to the vasodilator activity of CGRP. Peripheral vascular conditions associated with a deficit of CGRP-containing nerves, vascular dysfunction, and slow wound healing include diabetes (21) and Raynaud's disease (37), where a lack of reflex vasodilatation is observed. In comparison, excess release of CGRP is associated with blushing syndromes (388).
A rat skin flap model has allowed the demonstration that transcutaneous electrical stimulation is associated with increased blood flow and tissue perfusion in normal rats, but not in rats depleted of sensory nerves with capsaicin (200). Perhaps more importantly, there was a clear decrease in survival of the sensory nerve-depleted skin flaps, while addition of exogenous CGRP was found to increase skin blood flow and flap survival (199). In support of these studies, Knight et al. (201) demonstrated that infusion of a CGRP analog appears to rescue ischemic skin flaps. They demonstrated that CGRP infusions were associated with a restoration of tissue ATP levels and reduced thromboxane (a constrictor eicosanoid) levels. In comparison, CGRP did not affect either lipid peroxidation or neutrophil accumulation (201). However, a beneficial role of CGRP was clear.
In many species, the coronary arteries and left anterior descending artery receive innervation from a high density of CGRP-containing nerve fibers (104). CGRP is released from the heart in laboratory species in response to ischemia and low pH (105), with evidence that endothelium-derived prostacyclin (PGI2) may also play a role in CGRP release (178). Infusion of CGRP was shown to attenuate and delay ischemia reperfusion arrythmias in the anesthetized rat (397), and these beneficial effects have been shown to be inhibited by CGRP8–37 in vitro (222). After occlusion of the left anterior descending coronary artery and subsequent reperfusion, CGRP was found to improve the contractile function of the heart in an ischemia model in the dog (12). Indeed, there is also evidence for a protective role of endogenous CGRP in a myocardial infarction model in the pig (179); however, exogenously administered CGRP caused hypotension but no cardioprotection, as observed by a lack of reduction in the size of infarct size (180). The term preconditioning is used to describe the ability of a tissue to withstand severe ischemic attacks after exposure to previous brief ischemic episodes. Li et al. (221) suggest that CGRP is involved in ischemic preconditioning, possibly via protection of microvascular endothelial cells, and that the protective role of nitroglycerin may be related to stimulation of CGRP release (403; see also sect. IXB).
Evidence suggests that ischemic insult leads to the release of CGRP in the rat intestine (250, 348), which contributes in a proinflammatory manner to the reperfusion injury. It has been demonstrated that the CGRP contributes both to the mesenteric injury, especially the systemic hypotension and plasma leakage, in combination with endogenous kinins (233). Interestingly, CGRP8–37 was able to attenuate leukocyte infiltration. In comparison, it has been shown in the rat stomach that either sensory nerve activation, or infusion of CGRP, acts with other mediators to mimic the protection against ischemia observed with preconditioning (287). Furthermore, it has been suggested that the delayed cardioprotection observed after intestinal ischemic preconditioning is mediated by endogenous CGRP in an NO-dependent manner as an NOS inhibitor abrogated the response (390).
B. AM: Heterogeneity in Response
The complexities of the mechanisms of AM-induced vascular dilation, as discussed in section V with respect to mechanisms and section VI with respect to cardiovascular regulation, are mirrored in the microvasculature. AM can act on either the CGRP or AM receptors and then mediate relaxation via one of several mechanisms, dependent on activation of adenylate cyclase or potassium channels, or through NO release.
The intravenous administration of AM is associated with skin flushing (249), highlighting the ability of AM to selectively mediate increased microvascular blood flow in the cutaneous microcirculation in a similar manner to CGRP. The microvascular activity of AM has been compared with that of CGRP in a variety of studies including in the rat mesentery (278), rat pulmonary circulation (71), and rat skin (132). In most cases, AM was found to be 10–30 times less potent than CGRP, and responses were found to be antagonized by CGRP8–37, suggesting that microvascular vasodilatation is mediated by the CGRP receptor (e.g., in rat cutaneous microcirculation and rat isolated heart, Refs.
