Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides from the Diffuse Neuroendocrine System Karen B. Helle Abstract Chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) belong to a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These “granins” are characterized by numerous pairs of basic amino acids as potential sites for intra- and extragranular processing. In response to adequate stimuli, the granins are coreleased with neurotransmitters and hormones and appear in the circulation as potential modulators of homeostatic processes. This review is directed towards functional aspects of the secreted CgA, CgB, and SgII and their biologically active sequences. Widely different effects and targets have been reported for granin-derived peptides. So far, the CgA peptides vasostatin-I, pancreastatin, and catestatin, the CgB peptides CgB1–41 and secretolytin, and the SgII peptide secretoneurin are the most likely candidates for granin-derived regulatory peptides. Most of their effects fit into patterns of direct or indirect modulations of major functions, in particular associated with inflammatory conditions. 1 Introduction Chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) are well established as members of a family of uniquely acidic proteins that are ubiquitous in secretory cells of the nervous, endocrine, and immune system (Huttner et al. 1991; Winkler and Fischer-Colbrie 1992). Five, more selectively distributed, acidsoluble and heat-stable proteins of neuroendocrine origin are also included in this family. As reviewed elsewhere (Helle 2004), these are SgIII, SgIV (HISL-19 antigen), SgV (neuroendocrine secretory protein 7B2), SgVI (NESP55), and SgVII (the nerve growth factor inducible protein VGF). All granins, being products of distinct genes, K.B. Helle Department of Biomedicine, Division of Physiology, University of Bergen, Jonas Lies vei 91, 5009, Bergen, Norway e-mail: karen.helle@biomed.uib.no Results Probl Cell Differ, DOI 10.1007/400_2009_26 © Springer-Verlag Berlin Heidelberg 2010 21 22 K.B. Helle are characterized by numerous pairs of basic amino acids for cleavage by the costored prohormone convertases PC1/3 and PC2 and extracellular proteases such as plasmin (Parmer et al. 2000). The exocytotic corelease of CgA, CgB, and SgII with their costored amines and peptide hormones is by now a well-established concept (Feldman and Eiden 2003; Montesinos et al. 2008). This review is directed towards the extracellular effects of the granin cargo in relation to their postulated role as modulators of major functions, aiming at a coherent picture of CgA, CgB, and SgII and their derived peptides (Fig. 1a-c) in normal and pathophysiological conditions. 2 Granins and Granin-Derived Peptides Immunoreactive CgA-, CgB-, and SgII-like proteins are widespread among mammals and occur in lower vertebrates (Montero-Hadjadje et al. 2008). Within the CgA protein the vasostatin-I (VS-I) sequence is highly conserved across vertebrates from fish to man, while the sequences for pancreastatin (PST) and catestatin (CAT) are either lacking or poorly conserved in submammalian vertebrates (MonteroHadjadje et al. 2008). Although CgA and CgB are products of different genes, analyzes of their primary structure and gene organization have revealed a closer relationship between these two than between either protein with SgII and other members of the granin family. On the other hand, CgB, containing the highest number of potential cleavage sites, seems more extensively degraded in the bovine chromaffin granule extracts than CgA (Fischer-Colbrie et al.1985; Metz-Boutigue et al. 1993) while only three processed products of SgII have so far been reported (Montero-Hadjadje et al. 2008). 2.1 The Prohormone Concept The first reported peptide originating from a granin was the CgA-derived pancreastatin (PST), acting as an inhibitor of glucose-stimulated insulin secretion in the porcine pancreas (Tatemoto et al. 1986). This discovery formed the basis for the prohormone concept (Eiden 1987), implying that granins may serve as precursors of smaller peptides that, once released into the extracellular space, might serve some autocrine, paracrine, and/or endocrine function. The experimental support for this concept is steadily growing. There are numerous reports now on biological effects of graninderived peptides, notably from CgA and SgII. These peptides have been postulated to participate in a wide range of processes such as innate immunity, inflammatory reactions, cardiovascular modulations, and several homeostatic regulations (Metz-Boutigue et al. 1998; Koeslag et al. 1999; Helle and Aunis 2000; Helle 2004; Fischer-Colbrie et al. 2005; Helle et al. 2007). Notably CgA and SgII appear to be involved in mechanisms of disease, such as hypertension, heart failure, and inflammatory syndromes (Taupenot et al. 2003; Ceconi et al. 2002; Ferrero et al. 2004; Di Comite et Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 23 al. 2009; Zhang et al. 2008; 2009a) Although a coherent picture of the physiological impact of these granin-derived peptides is yet to be drawn, the available information lends substantial support for significant contributions of peptides derived from CgA, CgB, and SgII as modulators of normal and pathophysiological functions. 2.2 CgA CgA was the first granin to be isolated and characterized as a uniquely acidic protein costored and coreleased with the catecholamine hormones from the bovine adrenal medulla (Helle 2004). Contrary to earlier assumptions, CgA is not only a product of neuronal and glandular elements of the neuroendocrine system but appear also as a product of cardiocytes and polymorphonuclear neutrophils (PMNs). Accordingly, vertebrate hearts have proved fruitful as models for functional effects of not only VS-I (Corti et al. 2002, 2004; Tota et al. 2003; Imbrogno et al. 2004; Cerra et al. 2006, 2008; Cappello et al. 2007; Gallo et al. 2007) but also of CAT (Mazza et al. 2008; Angelone et al. 2008). The N-terminal peptides CgA1–76 and CgA1–113 obtained from the retrogradely stimulated bovine adrenal medulla (Helle et al. 1993) were named vasostatins, VS-I and VS-II, respectively (Fig. 1a), as a reflection of their suppressive effects in precontracted isolated human conduit vessels (Aardal and Helle 1992; Aardal et al. 1993). VS-I is a natural cleavage product of CgA in man and larger mammals (Stridsberg et al. 2000) but not in the rat (Glattard et al. 2006) due to the absence of a pair of dibasic amino acids in the position 77–78, giving rise to a glutaminerich, longer peptide, betagranin (rat CgA1–128, Hutton et al. 1988). As illustrated in Fig. 1a, prochromacin is the largest VS-I free CgA peptide in bovine chromaffin granules (Metz-Boutigue et al. 1993; 1998) and occurs also as the main CgA productin the urine of carcinoid patients (Gadroy et al. 1998). Prochromacin encompasses five other well-conserved domains of CgA, i.e. PST, WE-14, parastatin, catestatin (CAT), and GE-25 (Fig. 1a). In human plasma, PST occurs as a slightly elongated form and a substantially larger intermediate (Curry et al. 1990). In the pancreatic islet cells, PST is colocalized with insulin, glucagon and somatostatin (Schmidt and Creutzfeldt 1991), and histamine in the enterochromaffin cells of antrum of the stomach (Håkanson et al. 1995). Parastatin was first isolated as a 74 residues long fragment from the porcine parathyroid CgA and the name reflects its inhibitory effect on secretion of both parathormone (PTH) and CgA from the porcine parathyroid cells (Fasciotto et al. 1993). As illustrated in Fig. 1a, parastatin comprises not only the highly conserved CAT domain but also GE-25 (Kirchmair et al. 1994). Processing of CgA to CAT occurs by intraand extracellular processing (Parmer et al. 2000; Biswas et al. 2008). Biological activity has been assigned to CAT in a number of tissues such as bovine chromaffin cells (Mahata et al. 1997), the human baroreceptor centre of the nucleus tractus solitarius (Mahapatra 2008), porcine parathyroid cells (Fasciotto et al. 2000), rat mast cells (Krüger et al. 2003) in frog (Mazza et al. 2008) and rat heart (Angelone et al. 2008), and in Gram-positive and negative bacteria (Briolat et al. 2005; Radek et al. 2008). 24 K.B. Helle a 1 76 113 Vasostatin-I 248 Chromacin 344 431 Pancreastatin Catestatin Parastatin Vasostatin-II Prochromacin CHR WE14 GE25 b 1 420 550 CgB1−41 BAM GAWK 600 653 S.lytin CCB c 1 586 Manserin SN EM66 Fig. 1 Schematic illustration of peptides derived from (a) chromogranin A: vasostatin-I (bCgA1–76), chromofungin (CHR, CgA47–66), prochromacin (bCgA79–431), chromacin (bCgA173–194), pancreastatin (bgA248–296), parastatin (bCgA348–420), catestatin (bCgA344–366), WE14 (CgA316–330), GE25 (367–391); (b) chromogranin B; bCgB1–41, GAWK (bCgB420–493), BAM (bCgB547–560), CCB (bCgB597–653), secretolytin (S. lytin, bCgB614–626); and (c) secretogranin II: secretoneurin (SN, rat SgII154–186), EM66 (rat SgII189–256), manserin (rat SgII529–568) 2.3 CgB CgB is the largest and the least acidic of the granins, yet sharing with CgA not only the similar sized and structured disulfide-bridged loop at the N-terminus, but also the calcium binding capacity and aggregating properties (Huttner et al. 1991). Analogous to CgA, CgB is widespread in neuroendocrine cells of mammals, being expressed in species- and tissue-specific ratios relative to CgA and costored hormones (Rosa et al. 1985; Fischer-Colbrie et al. 1985). However, CgB, postulated to be released in constant ratio to insulin, has recently been shown to be largely segregated from CgA in the secretory granules, revealing that only 27% contained both CgA and CgB (Giordano et al. 2008). Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 25 As illustrated in Fig. 1b, cleavage at position 42–43 (Strub et al. 1995) results in the biologically active peptide CgB1–41 (Russell et al. 1994), while cleavage at the C-terminus results in the antimicrobial peptide, secretolytin (Strub et al. 1995). So far, no biological activity has been assigned to other CgB peptides such as GAWK and CCB, being abundant in human pituitary gland extracts (Benjannet et al. 1987) and BAM-1745 and particularly in the arcuate nucleus of the hypothalamus of the human brain (Marksteiner et al. 1999). 2.4 SgII SgII was initially identified as a sulfated secretory protein from the bovine anterior pituitary and the rat PC12 cell line, and the primary structures of bovine, rat, and human SgII were deduced from the respective cDNA sequences (Huttner et al. 1991). Three peptides have so far been identified in SgII, i.e. secretoneurin (SN), Fischer-Colbrie et al. 1995), EM66 (Anouar et al. 1998), and the 40 amino acid residues long peptide, manserin (Yajima et al. 2004), as illustrated in Fig. 1c. SN is the most highly conserved region in SgII (Montero-Hadjadje et al. 2008) and immunoreactive SN is widely distributed (Kirchmair et al. 1993, 1994), overlapping partly but not completely established neurotransmitter and neuropeptide systems (Marksteiner et al. 1993). The order of free SN is (by concentration): intestine > brain > anterior pituitary > pancreas, and adrenal (Wiedermann 2000). Moreover, the N- and C-terminal domains of SN have been immunodetected in all insulinpositive cells, most of the glucagon cells, and some of the pancreatic poloypeptide cells while no SgII peptide could be detected in the somatostatin cells (Stridsberg et al. 2008). A wide range of biological activities has been assigned to SN (Vaudry and Conlon 1991; Kirchmair et al. 1993; Kähler and Fischer-Colbrie 2000) and there are indications of a functional relevance for EM66 in the control of food intake and/or the stress associated with fasting (Boutahricht et al. 2005). 3 Functional Aspects 3.1 Compensatory Upregulation of CgB and SgII in CgA Null Mice Knockout technology has provided novel insight into granin functions. For instance, a compensatory increase in CgB has been demonstrated in the secretory granules of the adrenal medulla in CgA null mice, excreting elevated levels of catecholamines (Mahapatra et al. 2005; Hendy et al. 2006) despite reduced capacity for storage and exocytosis of catecholamines (Montesinos et al. 2008) and differences in developmental abnormalities in adrenomedullary morphology (Hendy et al. 2006). Moreover, a two- to threefold upregulated expression of CgB and other 26 K.B. Helle members of the granin family (SgII–SgVI) appeared to compensate for the CgA deficiency in the CgA null mice (Hendy et al. 2006). Also, pancreatic CgB and SgII epitopes were expressed in the CgA null mice, although in lower levels than in the wild type (Portela-Gomes et al. 2008; Stridsberg et al. 2008) and plasma insulin was decreased although plasma glucose and glucagon levels were normal, consistent with increased glucagon cell function in the absence of CgA (Portela-Gomes et al. 2008). Although essential hypertension is associated with high plasma CgA, an elevated blood pressure is also evident in the ChgA null mice characterized by a higher than normal catecholamine secretion (Mahapatra et al. 2005). Intriguingly, an alleviation of hypertension could be obtained by genetic humanization of the Chga null mice or by venous infusion of exogenous CAT (Mahapatra et al. 2005), suggesting a hypotensive effect of CgA via CAT. However, it remains to be clarified whether the hypotensive effect of CAT is secondary to a CAT-evoked histamine release from mast cells in mice, as is the case in the rat (Kennedy et al. 1998), or to a modulation of the baroreceptor centre of the nucleus tractus solitarius, as suggested for the human CAT variant (Gly364-Ser) (Mahapatra 2008). 3.2 Circulating Granins There is a relatively constant background of granins in the peripheral circulation and in the cerebrospinal fluid (CSF). Normal human serum contains low nanomolar concentrations not only of CgA (O’Connor et al. 1993) but also of CgB (Stridsberg et al. 1995; Aardal et al. 1996) and SgII (Kirchmair et al. 1994; Ischia et al. 2000). Taking into account the fact that all three granins occur in the brain and are released from the respective regions upon adequate stimuli, it is noteworthy that CgA, CgB, and SgII are represented in CSF by acidic domains largely devoid of biological activities (Stark et al. 2001; Helle 2004). The possibility that the basic and/or less acidic, biologically active peptides that may remain bound to their target tissues, might account for the unexpected and selective CSF patterns. A vast number of reports on pathologically high plasma CgA have accumulated since the first documentation of increased levels in patients with neuroendocrine tumors (O’Connor and Bernstein 1984). Plasma CgA is by now a commonly used diagnostic and prognostic marker for tumors of neuroendocrine origin, using antibodies raised to a range of epitopes along the CgA molecule (Stridsberg et al. 2004; Greenwood et al. 2006; Børglum et al. 2007). Plasma CgA is also elevated in patients with a range of systemic diseases including renal and hepatic failure, cardiac arrest, and essential hypertension (Taupenot et al. 2003) as well as in inflammatory conditions such as heart failure (Corti et al. 2000; Ceconi et al. 2002), acute coronary syndromes (Jansson et al. 2009), rheumatoid arthritis (Di Comite et al. 2006, 2009), systemic lupus erythematosis (Di Comite et al. 2006) and acute systemic inflammatory response syndrome (Zhang et al. 2009a). It seems well established that increased plasma CgA is predictive of shorter survival, not only in patients with metastatic neuroendocrine tumors (Arnold et al. 2008; Nikou et al. 2008), but also Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 27 in chronic heart failure (Ceconi et al. 2002) and in the critically ill, nonsurgical patients (Zhang et al. 2008; 2009a). Intriguingly, lower than normal serum CgA has been reported for patients suffering from self-reported food hypersensitivity in association with symptomatic carbohydrate malabsorption (Valeur et al. 2008). This points to circulating CgA being implicated in functional gastrointestinal disorders yet to be elucidated. Whether plasma CgA, CgB, and SgII serve solely as passive markers of the secretory state of the various elements of the diffuse endocrine system or, in addition, as active and functional contributors to homeostatic regulations of normal and clinical conditions, would depend on the ability of the prohormone and/or its derived peptides to activate or modulate relevant cellular functions. 3.3 Sources and Effects of Granin Peptides 3.3.1 Neurons and Chromaffin Cells CgA coreleased with the catecholamine cargo from the sympathoadrenal components appears to be the major source for the autocrine, negative feedback control of the adrenomedullary release exerted by the CAT domain (Mahata et al. 1997). The mechanism for this specific, noncompetitive inhibition involves the neuronal nicotinic acetylcholine receptors (nAChRs), suggesting the open state of the channel as the target (Herrero et al. 2002). In addition, CAT, like substance P (SP) also inhibited the nicotine-induced desensitization of the receptor (Mahata et al. 1999). SN occurs in high concentrations in several regions of the brain, the endocrine cells of the gastrointestinal tract, and in peripheral sympathetic and sensory nerves (FischerColbrie et al. 1995). In the terminals of sensory nerves, SN is colocalized with SP and calcitonin gene-related peptide (CGRP) (Klimaschewski et al. 1995). In response to mechanical or immunological injury, the release of these sensory peptides results in neurogenic inflammation characterized by chemotaxis of leucocytes and their transendothelial passage to the sites of injury (Kähler and Fischer-Colbrie 2000). 3.3.2 Extraneuronal Sources The heart, the gastrointestinal tract, and immune cells such as the polymorphonuclear neutrophils (PMNs), have recently attracted attention as extraneuronal sources of CgA in the rat and frog atrial myocytes (Steiner et al. 1989; Glattard et al. 2006) and the hypertrophied human ventricular myocardium (Pieroni et al. 2007). CgA is normally costored with ANP in classical secretory granules in the atrial myocardium while, in the hypertrophied human ventricular myocardium. CgA is expressed, colocalized and constitutively released together with BNP upon increased wall stress (Pieroni et al. 2007). Enterochromafin-like and enterochromaffin cells of the gastrointestinal tract also contain an abundance of CgA, costored and cosecreted with histamine (Håkanson 28 K.B. Helle et al. 1995) or serotonin (Cubeddu et al. 1995) upon adequate stimuli. A range of CgA-derived fragments immunoreactive containing VS-I and CAT are produced and secreted by human PMNs when stimulated by the leukocidin Panton-Valentin (Lugardon et al. 2000; Briolat et al. 2005). Wherever PMNs accumulate in response to invading microorganisms, tissue inflammation, and sites of mechanical injury, this source of CgA peptides may affect a wide range of cells involved in inflammatory responses, e.g. endothelial, endocardial and epithelial cells, other leucocytes, fibroblasts, cardiomyocytes, and vascular and intestinal smooth muscle. 4 Granin Peptides and Targets The first reported targets for granin peptides were the pancreatic b cells for the CgA-derived peptide PST (Tatemoto et al. 1986), the bovine parathyroid cells for CgB1–41 (Russell et al. 1994) and the rat striatum for SN (Saria et al. 1993). During the last decade, the spectrum of targets has increased exponentially, notably for the CgA-derived peptides and for SN. There is also accumulating support for the vascular endothelium as a pivotal target not only for the CgA peptide VS-I but also for SN. In the following, the target systems will be discussed in relation to functions modulated by one or more of the granin peptides. 4.1 Antimicrobial Potencies and Innate Immunity Among the different mechanisms integrated in the innate immunity, i.e. the inborn system of first defense against microorganisms, a range of natural cationic peptides have been isolated from insect lymph, skin of frogs, mammalian neutrophil granules, and plants as reviewed elsewhere (Helle et al. 2007). These peptides boost the innate immune responses by selectively modulating pathogen-induced inflammatory responses. During the last decade a range of natural antimicrobial peptides has been derived from the processing of granins, i.e. the CgA-derived VS-I, prochromacin, chromacin and CAT, and the CgB-derived secrelytin (Strub et al. 1995; Fig. 1a, b), implicating the adrenal medulla as a potential contributor to the innate immunity (Metz-Boutigue et al. 1998). Chromofungin (CHR, CgA47–66) is the most active of the antifungal VS-I-derived peptides (Lugardon et al. 2001; Zhang et al. 2009b), revealing an amphipathic helical conformation related to a destabilization of the plasma membrane, allowing the peptide to penetrate by pore formation into fungi and yeast cells. Subsequently, the internalized CHR has been assumed to interfere with intracellular targets such as calcium-dependent calmodulin (CaM) dependent systems including the phosphatase activity of calcineurin (Lugardon et al. 2001). Antimicrobial activity has also been assigned to the CgA-derived CAT and to the CgB-derived secretolytin. Consistent with an abundance of cationic charge, the active core of CAT, i.e. cateslytin (CgA344–358), inhibits growth of Grampositive and Gram-negative bacteria, a variety of filamentous fungi, and several Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 29 forms of yeasts (Briolat et al. 2005). Consistent with a role for CgA and CAT in immunoprotection, CAT penetration through human epidermis and inhibitory potencies against skin pathogens has more recently been demonstrated (Radek et al. 2008). Importantly, CgA was detected in keratinocytes and processed into CAT in human skin, while the expression of CAT in murine skin was increased in response to skin injury and infection. Secretolytin (Fig. 1b) displays potent antibacterial activity against Gram-positive species and reveals sequence homology with the lytic domain of the insect cecropins and the pigmyeloid antibacterial peptide (Strub et al. 1995, 1996). No antimicrobial activity has so far been assigned to the negatively charged SN or to other domains of SgII. Hence, evidence in favor of antimicrobial peptides derived from CgA and CgB is accumulating, seemingly providing protection against a wide variety of infections. These host defense peptides, notably VS-I, CAT, and secretolytin, have emerged as potential effectors for the innate immune system, suggesting roles in management of infections as antimicrobial peptides in their own right. 4.2 Inflammatory Conditions Neurogenic apoptosis, inflammatory pain, and neuronal inflammation appear as potential conditions involving granin peptides. In particular, the endothelial barrier between the circulation and the underlying tissues has emerged as a conspicuous target for the granin peptides VS-I and SN revealing however, striking, counteracting effects on endothelial (EC) permeability. Moreover, granular immunocytes such as mast cells and PMNs have to be included as targets for CAT. 4.2.1 VS-I and Nitrergic Neurons in Gatrointestinal Pain Inflammatory, somatovisceral pain may be induced experimentally by peritoneal application of acetic acid in vivo, abolishing the spontaneous contractile activity and decreasing the excitatory component of the tonic response to transmural nerve and reducing motility in human and rat colonic segments (Ghia et al. 2004a, b, 2005). Although without intrinsic activity, the very N-terminal domain of VS-I (CgA1–16) exerted a nociceptive effect similar to CGRP, and capsaicin but not SP. Moreover, CgA4–16 counteracted the acetic acid sensitive L-type of Ca2+ channels on both the colonic smooth muscle and the afferent nerve terminals. When intraluminal pressure was applied as the stimulus to rat proximal colon in vitro, low nanomolar concentrations of VS-1 and CgA7–57 produced a concentration-dependent, progressive decrease in the mean amplitude of the spontaneous contractions in the circular layer of smooth muscle without affecting the resting tone (Amato et al. 2005). Taken together these studies support the concept of suppressive effects of the entire VS-1 molecule on elements of the gastrointestinal tract, presumably via activation of primary inhibitory nitrergic afferents, in addition to a direct inhibition of smooth muscle contractility. 30 K.B. Helle 4.2.2 VS-I, Microglia, and Neurogenic Apoptosis As resident macrophages in the nervous system, microglial cells support neuronal survival and differentiation. By their release of neurotrophins, secretion, and responses to cytokines and by their stimulation of astrocytes, the microglial cells play a major role in the immune response (Ciesielski-Treska and Aunis 2000). CgA and VS-1 have been shown to activate cultured rat microglia in a manner analogous to but not identical to microbial toxins, triggering secretion of heat-stable, diffusible neurotoxins and accumulation of NO and TNFa (Taupenot et al. 1996; Ciesielski-Treska et al. 1998). The CgA induced reactive phenotype resulted in microglial apoptosis and death (Ciesielski-Treska et al. 2001). Moreover, a series of characteristic features, which forego neuronal apoptosis, were apparent in the CgA and VS-I stimulated cocultures of microglia and cortical neurons (Ciesielski-Treska et al. 2001). While the acute microglial activation by CgA and VS-I may be beneficial to the host, prolonged microbial activation cascades have been implicated in the inflammatory processes associated with degenerative disorders like Alzheimer’s, Pick’s, and Parkinson’s diseases (Kingham and Pocock 2000; Hooper and Pocock 2007). 4.2.3 CAT, VS-I-Derived CHR, and Activation of PMNs CAT has recently been reported to stimulate chemotaxis in human PMNs in a concentration-dependent manner with maximal potency at 1 nM, similar to that of the formylated chemoattradctant Met-Leu-Phe (fMLP; Egger et al. 2008). Intriguingly, the naturally occurring human variants of CAT varied in potencies, being highest for Pro370Leu and lowest for Gly364Ser. Moreover, CAT stimulated Akt- and extracellular signal related kinase (ERK) phosphorylation, and the effect was blocked by antagonists to a wide range of signaling pathways, indicating involvement of tyrosine kinase receptor-, G-protein-, and sphingosine-1-phosphate signaling. The authors conclude on a role for CAT as an inflammatory cytokine, of possible implications for the extensive microglial activation and neuronal damage in relation to the CgA-containing Alzheimer plaques. Moreover, CAT and the cationic and amphipathic CHR domain of VS-I (Fig. 1a) have most recently been shown to activate unstimulated human PMNs by provoking a transient influx of Ca2+ and leading to exocytosis of a series of relevant immunoregulating processes (Zhang et al. 2009b). The mechanism for this activation by CAT and CHR involves CaM binding and subsequent activation of Ca2+-independent phospholipase A2. Thus, CgA released from bacteriotoxin-stimulated PMNs might provide paracrine stimuli for unstimulated PMNs, thereby propagating their immunoregulatory contributions. 4.2.4 CAT and Histamine Release from Mast Cells As granular immunocytes, mast cells reside in the barrier tissues where they orchestrate inflammatory responses. In rat mast cells the N-terminal, biologically active domain Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 31 of CAT, i.e. cateslytin (CgA344–358), is a potent activator of histamine release (Krüger et al. 2003), accounting for the reduced pressor response to an intravenous injection of CAT in rats sensitive only to the H1 type of histamine blockers (Kennedy et al. 1998). Moreover, the potency and efficacy of cateslytin were higher than of the cationic wasp venom mastoparan and the sensory neuropeptide SP (Krüger et al. 2003). A pertussis-toxin (PTX)-sensitive, peptidergic, and receptor-independent pathway has already been established for mastoparan, SP, and other amphiphilic cationic neuropeptides on histamine release from rat mast cells (Jones and Howl 2006). Hence, it seems likely that the cateslytin domain of CAT may stimulate mast cell release by a similar PTX-sensitive pathway (Helle 2009), in marked contrast to the inhibitory, autocrine effect of CAT on adrenomedullary catecholamine release (Mahata et al. 1997). By comparison, neither the VS-I derived peptides nor WE-14 were able to elicit histamine release from the rat mast cells (unpublished observations). 4.2.5 VS-I, SN, and EC Integrity The vascular (ECs) and endocardial endothelia (EECs) form barriers against transvascular exchange of fluids, proteins, and blood cells. ECs and EECs may themselves be targets for granin-derived peptides, whether released locally or delivered via the circulation, affecting secretion, contractile properties, and transport of other cells and substances through gaps in the otherwise confluent monolayer. Notably, VS-I (Ferrero et al. 2004) and SN (Kähler et al. 2002b) may modulate transendothelial transport of leukocytes as part of the inflammatory response, however in opposite directions. In vivo and in vitro experiments strongly suggest that CgA via VS-I at pathophysio­ logical concentrations at and above 7 nM may prevent the TNFa-induced extravasation of macromolecules by targeting to EC in mouse liver in vivo and in cultured monolayers (HUVEC) in vitro (Ferrero et al. 2004). Analogously, VS-I also inhibited TNFainduced formation of gaps in cultured arterial EC (Blois et al. 2006). In addition, VS-1 also partially inhibited thrombin- and vascular endothelial growth factor (VEGF)induced permeability through confluent monolayers of HUVECs (Ferrero et al. 2004). Taken together, these findings suggest a role for VS-I in the protection of the EC barrier against the gap-forming, permeabilizing activity of TNFa by a mechanism involving cytoskeletal reorganization and downregulation of the transmembrane protein inter-cellular VE-cadherin responsible for for cell–cell adhesion (Ferrero et al. 2004). A pivotal role for VS-I as an inhibitor of the PTX- and TNFa activated p38MAP kinase phosphorylation was demonstrated in pulmonary arterial EC (Blois et al. 2006), implicating VS-1 in the protection of a Gai-coupled tonic inhibition of the p38MAPK activity in the PTX-sensitive pulmonary EC (Garcia et al. 2002; Helle 2009). In HUVECs, the protective effect of VS-1 on EC integrity is not limited to inhibition of gap formation induced by proinflammatory agents, but also appears to inhibit motility and basal ERK phosphorylation, leading to a more quiescent stage without apoptotic or necrotic effects (Belloni et al. 2007). Importantly, VS-1 also inhibited the VEGF-induced ERK phosphorylation, cell migration, proliferation, morphogenesis, and invasion of collagen gels in various in vitro assays (Belloni et al. 2007). 32 K.B. Helle Contrary to VS-I, SN has been reported to impair the integrity of the EC barrier by reducing the expression of Zona occuludens-1 and occludin and activating JNK and ERK1/2, but not p38MAPK in human coronary arterial EC (Yan et al. 2006). Of note, SN was almost as effective as TNFa in stimulating transmigration of PMNs via an EC pathway involving PTX, CTX, and staurosporine-sensitive signaling (Kähler et al. 2002a). In addition, SN may recruit immunocompetent monocytes and PMNs to the sites of injury. A selective SN-induced chemotaxis of human monocytes in vitro and in vivo (Reinich et al. 1993) and their adhesion to arterial and venous EC (Kähler et al. 2002a) appear to precede their transendothelial migration (Kähler et al. 1999; Kähler and Fischer-Colbrie 2000). Hence, with respect to of EC permeability, VS-I and SN appear to have striking, opposite effects. 4.3 Other Cardiovascular Functions A range of inhibitory effects by VS-I has been reported for blood vessels and elements of the heart. The first experimental models were isolated segments of human intrathoracic arteries and saphenous veins (Aardal and Helle 1992; Aardal et al. 1993; Angeletti et al. 1994), revealing suppressive effects on precontracted vessel segments. Most recently, several models of the vertebrate heart have been introduced (Corti et al. 2002; Imbrogno et al. 2004; Cerra et al. 2006). Common to the vertebrate hearts is a negative myocardial inotropy elicited not only by the highly conserved VS-I domain in CgA but also by CAT. 4.3.1 VS-I and Vasodilatation In human vessel segments, the natural bovine VS-I + VS-II and the synthetic CgA1–40 suppressed the ET-1 contractions independent of EC and extracellular calcium, affecting the maximal sustained tension response but not the potency for ET-1 (Aardal and Helle 1992; Aardal et al. 1993). Inhibitory effects of VS-I and CgA1–40 were also evident in isolated and pressurized bovine coronary resistance arteries (Brekke et al. 2002). Here, CgA1–40 evoked dilatation independent of other constrictors over a functional range of transmural pressures. Moreover, the intrinsic and concentration-dependent dilator effects persisted at moderately elevated extracellular K+ (Brekke et al. 2002), but was prevented by PTX and by antagonists to several subtypes of K+ channels, suggesting vasodilatation by a CgA1–40 and VS-I induced hyperpolarization via opening of K+ channels in the smooth muscle. 4.3.2 VS-I and CAT on Myocardial Inotropy The vertebrate heart, consisting of the epicardium, the myocardium, EEC, and the coronary blood supply, is a complex system to analyze for tissue-specific effects Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 33 of any given substance. As a simplistic first model, the avascular myocardium of the frog (Rana esculenta) was chosen for the assessment of myocardial effects of CgA peptides (Corti et al. 2002). A calcium-dependent negative inotropism was observed in response to the recombinant human STACgA1–78 (VS-1) This effect in the frog was independent of EEC, adrenergic, and muscarinic receptors and was completely antagonized by Ba2+, suggesting involvement of K+ channels and hyperpolarization in the cardiomyocytes (Corti et al. 2004). Moreover, VS-1 also counteracted the characteristic inotropism exerted by the b-adrenoceptor agonist isoprenaline (ISO). The natural loop structure of frog and bovine VS-I was essential for both the negative inotropism and the counteraction of the b adrenoceptor activation (Tota et al. 2003). In contrast, in the eel (Anguilla anguilla), VS-I derived peptides induced a negative basal and ISO stimulated myocardial inotropy that was dependent on EEC functions, notably the NO-cGMP-PKG pathway (Imbrogno et al. 2004). Analogously, in the Langendorff preparation of the nonworking rat heart, a perfusion with VS-1 caused a negative inotropic effect including inhibition of the inotropic response to ISO via EC-dependent NO production (Cerra et al. 2006, 2008), suggesting that, whatever the subcellular signaling route, VS-1 may exert negative inotropic effects on vertebrate hearts. Intriguingly, VS-I was ineffective on the basal contractility on rat papillary muscle while partially reducing the effect of ISO stimulation via EC-derived NO production (Gallo et al. 2007). Moreover, removal of EC and inhibition of NO synthesis and PI3K activity abolished the antiadrenergic effect of VS-1, indicating that the antiadrenergic effect in the rat heart is also due to a PI3Kdependent NO release from EC rather than to a direct action on the cardiomyocytes. Moreover, two different pathways appear to mediate the protective activity of VS-1 against ischemic insults in the rat heart, one via A1 receptors and the other by NO release, both converging on PKC (Cappellio et al. 2007). Enhancing NO production, either through a direct control of eNOS or through modulation of Gai/o proteins, is one alternative, another being PKG controlling intracellular calcium homeostasis and utilization. PKG may also exert a feedback regulation of Gai/o proteins, thereby generating a circuit of interactions converging to depress contractility. Taken together, the findings support the concept of a cardiosuppressive function of VS-I in vertebrates, which apart from the frog, appears to be mediated by EC-dependent NO production. Of note, not only VS-1 but also CAT has been shown to exert negative myocardial inotropy and to noncompetitively inhibit the b-adrenoceptor on the cardiomyocyte, presumably mediated by the relaxing effect of the EC-derived NO release mediated by Akt/PKB signaling to eNOS (Angelone et al. 2008). In addition, a noncompetitive inhibition of the ET-1 receptor in the rat cardiomyocyte has also been assigned to CAT (Angelone et al. 2008). However, in contrast to VS-I, CAT also increased heart rate and coronary pressure, suggesting significant peptide specific differences in coupling to some tissue responses. Moreover, the EC-dependent, PTX-sensitive negative inotropic responses to both VS-1 and CAT in the rat heart raise the question whether these two distinctly different CgA sequences may act competitively or synergistically, targeting to identical or different PTX-sensitive Gai/o subunits in the EC membrane. 34 K.B. Helle 4.3.3 VS-I and SN on Cell Motility Cell-adhesive effects of the intact human CgA and VS-1 have been observed in human and mouse fibroblasts and in human coronary artery smooth muscle cells, but not in neuroblastoma cells (Gasparri et al. 1997; Ratti et al. 2000). Importantly, the antiadhesive effect of the intact prohormone could be changed into a proadhesive effect upon limited tryptic treatment (Corti et al. 2004b). An indirect mechanism, probably dependent on stimulated synthesis of other cell surface proteins, was suggested from 3 to 4 h lag time for these antiadhesive effects. In contrast, SN actively stimulated cell motility in human skin fibroblasts but failed to induce proliferation (Kähler et al. 1997a). SN also induced a directed, selective migration of cultured rat aortic smooth muscle cells and stimulated cell proliferation and DNA synthesis (Kähler et al. 1997a, b; Kähler and Fischer-Colbrie 2000). 4.3.4 VS-I and SN in Angiogenesis and Vasculogenesis Angiogenesis is defined as the generation of new vessels by sprouting from the already existing vasculature, stimulated by VEGF and the basic fibroblast growth factor in vivo. Vasculogenesis implies, on the other hand, de novo formation of vessels from circulating endothelial progenitor cells in the embryo, from bonemarrow derived endothelial progenitor cells or from circulating precursor cells in postnatal neovasculogenization (Kirchmair et al. 2004a, b). There is to date only one report implicating a role for VS-1 in angiogenesis (Belloni et al. 2007). Here, an inhibitory effect of VS-1 on the formation of capillary-like structures could be demonstrated in a matrigel assay in a rat model. In addition, VS-1 inhibited the VEGF-induced migration, proliferation, morphogenesis, and invasion of collagen gels in HUVECs in vitro. Analogous to VS-1, SN inhibited the proliferation of HUVEC when stimulated by fibroblast growth factor (Kähler et al. 1997a). However, more recent reports have demonstrated that SN may act as an angiogenic cytokine comparable in potency to VEGF when assayed in a mouse cornea neovascularization model in vivo, stimulating a dose-dependent and specific capillary tube formation in a matrigel assay in vitro (Kirchmair et al. 2004a). Here, SN also stimulated proliferation and exerted antiapoptotic effects on EC. In a separate study with the same model, systemic injections of SN led to an increase in circulating stem cells and endothelial progenitor cells to sites of vasculogenesis in vivo, confirming stimulatory effects on proliferation and antiapoptotic effects (Kirchmair et al. 2004b). VEGF is an angiogenic cytokine that is enhanced by hypoxia like a range of other angiogenic factors. It has also been shown that SN is upregulated by hypoxia, however in a tissue-specific manner, being present in muscle cells but not in EC, vascular smooth muscle cells, or pituitary tumor cells (Egger et al. 2007). Hence, SN may play a role in hypoxia-driven induction of neovascularization in ischemic diseases like peripheral or coronary artery disease, diabetes, retinopathy, central ischemia, or in solid tumors (Fischer-Colbrie et al. 2005; Egger et al. 2007). Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 35 4.4 CgA Peptides as Modulators of Calcium and Carbohydrate Homeostasis The parathyroid hormone (PTH), being costored and coreleased with CgA (Cohn et al. 1982), is a primary homeostatic regulator of plasma Ca2+. While the release of PTH is stimulated at low plasma Ca2+, the hormone release is inhibited not only by elevated plasma Ca2+ via hyperpolarization (Välimäki et al. 2003) but also by the CgA peptide VS-I at low plasma Ca2+as effectively as by the physiologically high concentrations of Ca2+ (Angeletti et al. 2000). PST inhibition of the first phase of the glucose-stimulated secretion of insulin (Tatemoto et al. 1986) was sensational, implicating a novel role for CgA in regulation of carbohydrate metabolism. By now, it is well established that the islet cells of the endocrine pancreas together with the liver and adipose tissue represent essential targets for PST in the homeostatic regulation of plasma glucose. 4.4.1 VS-I, PST and Parastatin/CAT on PTH Secretion Both the natural and synthetic VS-I and the N-terminal CgA1–40 actively inhibit PTH release in the bovine parathyroid cells (Russell et al. 1994; Angeletti et al. 1996). Also CgB1–41, has been shown to be an active inhibitor of PTH release (Russell et al. 1994), suggesting that the N-terminal loop domains of CgA and CgB may perform the same function in this tissue, whether serving as autocrine (CgA) or endocrine (CgB) inhibitors of PTH secretion (Angeletti et al. 2000). A partial inhibition of PTH secretion by PST was regarded as physiologically irrelevant due to the low degree of CgA processing into PST in this tissue (Drees and Hamilton 1992). The CAT-containing parastatin (Fig. 1a) was also found to inhibit the cosecretion of PTH and CgA in the porcine parathyroid (Fasciotto et al. 1993, 2002), but with markedly lower potency than with VS-I in the bovine parathyroid cells. Hence, these findings suggest that three domains of CgA may contribute to modulation of PTH secretion and that VS-I via its N-terminal domain CgA1–40 appears as the most likely autocrine inhibitor of PTH release at low plasma Ca2+ in the bovine parathyroid cells. 4.4.2 PST on Carbohydrate Homeostasis CgA processing in the human gastrointestinal tract reveals cell and region-specific patterns (Portela-Gomes and Stridsberg 2001, 2002a, b; Portela-Gomes et al. 2008). Although the physiological relevance of PST in humans has been questioned due to the low degree of processing (Schmidt and Creutzfeldt 1991), a later study indicated that human PST (hP-16) corresponding to the amidated C-terminus of hCgA286–301, might be involved in reduction of elevated blood glucose and insulin levels after oral glucose load in nondiabetic humans (Siegel et al. 1998). The endocrine role of PST in humans has also been approached by a different experimental design 36 K.B. Helle (O’Connor et al. 2005). PST infusion into the brachial artery at a supranormal concentration was without intrinsic effects, yet it reduced the A-V glucose difference, and inhibited uptake of glucose and free fatty acids without affecting blood flow. The regulatory effects of PST on liver and adipose tissues are to date best documented in vitro, as extensively reviewed elsewhere (Sanchez-Margalet et al. 2000). In the rat, hepatocytes and adipocyte PST inhibit insulin-mediated glucose transport, glucose utilization, and lipid synthesis. A lipolytic effect has also been demonstrated in addition to a PTX stimulated basal and insulin-stimulated protein synthesis (Gonzalez-Yanes and Sanchez-Margalet 2002). These in vivo and in vitro results support the concept of hepatocytes and adipocytes as well pancreatic b cells as likely targets for PST in the rat. However, the postulated inhibitory role of PST on the first phase of glucose-stimulated insulin release from the human pancreas (Tatemoto et al. 1986) still awaits experimental support. 5 Towards a Unifying Concept for Extracellular Functions of CgA, CgB, and SgII The release of CgA, CgB, and SgII with costored amines and peptide hormones from elements of the diffuse neuroendocrine system upon adequate stimuli from the external environment and internal milieu is well-established. Although a coherent picture of the functional implications of CgA, CgB, and SgII and their derived peptides is still not complete, it is evident from the accumulated evidence that a wide range of processes associated with homeostasis appear to be modulated by one or several of the peptides derived from these granins. As illustrated in Table 1, CgA emerges notably as a multifunctional prohormone, giving rise to at least three peptides, modulating not only calcium and carbohydrate Table 1 Reported actions of granin-derived peptides with functions involved in homeostatic regulations CgA CgA CgA CgA CgB CgB SgII Calcium metabolism Carbohydrate metabolism EC integrity Heart, blood vessels Innate immunity Microglia, mast cell GI pain Cell motility etc. Angiogenesis Vasculogenesis Inhib Inhibition, Act demonstrated VS-I PST CAT Parastatin CgB1–41 Secretolytin SN Inhib – Inhib Inhib – – Inhib – Inhib – – Disrupt Prot – – – – Inhib – Inhib – – Anti – Anti – – Act – Act – – Inhib – – – – Inhib – Act – – Inhib activation, Prot protection, Disrupt disruption, – – – – – – Anti – – – – Act – Act Act Anti antimicrobial, not Chromogranins A and B and Secretogranin II as Prohormones for Regulatory Peptides 37 metabolism but also EC integrity, myocardial inotropy, microbial control, innate immunity gastrointestinal pain, cell adhesion, migration, and proliferation. Intriguingly, the N-terminal VS-I stands out as the most versatile among the CgA peptides, affecting all sectors but carbohydrate metabolism. While three CgA domains and the N-terminal CgB peptide may inhibit PTH release from the parathyroid, PST appears as the only granin peptide with modulating potentials on carbohydrate metabolism. Another aspect of considerable interest is the apparent convergence of the two structurally different CgA peptides, VS-I and CAT, on the heart, both inhibiting myocardial contractility via activation of PTX-sensitive EC production of NO in the rat heart. Nevertheless, the most striking aspect of the granin peptides is their association with inflammatory conditions. It seems likely that concerted effects of VS-I, CAT, and secretolytin may be relevant for the first-line host-defense against invading microorganisms. Moreover, several immunocompetent cells also respond to CgA peptides. For example, the rat microglia becomes activated by VS-I to cause neuronal apoptosis, while not only the rat mast cells but also human PMNs may respond to CAT, to evoke markedly different responses, i.e. histamine release and cellular migration and secretion, respectively. Furthermore, activation by VS-I of primary inhibitory nitrergic afferents in elements of the gastrointestinal tract points to a contribution to pain reduction during inflammatory conditions. Although being devoid of antibacterial potencies, one may regard SN as an indirect contributor to innate immunity in view of its activation of chemotaxis, transendothelial extravasation, and migration of leukocytes. In this context the oppositely directed effects of VS-I and SN on EC permeability are particularly important. Where SN appears to induce EC permeability for transendothelial transport of immunocompetent leucocytes, VS-I seemingly protects the integrity of the EC barrier against the disruptive effects of proinflammatory agents. It is by no means clear to what extent these opposite effects occur within the same frames of time and space. Rather than a direct competition between VS-I and SN on regulation of EC permeability, it is tempting to speculate that there may be a timelag between the release of SgII derived SN from sensory nerves in response to a mechanical or inflammatory injury and the release of CgA and VS-I from activated PMNs at site of inflammation. If SN initially triggers transendothelial passage of leukocytes including PMNs, a subsequent release of CgA-derived VS-I and CAT from activated PMNs might serve to combat the microbial invasion. In addition, the SN-induced EC leakage might be counteracted by VS-I to protect EC against further barrier disruption and transendothelial leakage of cells and solutes. In the case of CgA releasing tumors, VS-I may protect the host against transendothelial transport of tumor-derived products. Intriguingly, VS-I and SN also appear to exert opposite effects on new formation of blood vessels. While VS-I appears to inhibit VEGF-induced cell migration, proliferation, morphogenesis, and invasion of collagen gels inherent in angiogenesis, SN has, in contrast, been shown to activate EC chemotaxis, proliferation, angiogenesis, and vascularization while inhibiting EC apoptosis, suggesting a significant role for SN also in tissue repair. 38 K.B. Helle Two aspects remain presently unanswered, namely the question of receptors and concentrations needed to obtain the reported effects. For VS-I or SN, there is to date no reported extracellular receptor, while for CAT the nicotinic acethylcholin receptor in the sympatoadrenal system mediates only the autocrine inhibitory effect on the adrenal medulla. For VS-I and PST, there are reports on peptide-binding membrane proteins of the order of 70–80 kDa coupled to G-proteins. It has been postulated that hydrophobic and amphipathic properties of VS-I and CAT might allow for their receptor-independent penetration into and activation of cells (Helle 2009). However, a similar mechanism seems rather unlikely for the highly acidic SN. With respect to effective concentrations, many of the reported responses to the CgA peptides may come into play under pathophysiological conditions, e.g. during hypertension, cardiac heart failure, and inflammatory conditions in various organs. 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