Current Medicinal Chemistry - Immunology, Endocrine & Metabolic Agents - Volume 4, Issue 3, 2004

Chromogranin A (CgA) and chromogranin B (CgB) are proteins present in secretory granules of the diffuse neuroendocrine system, with multiple pairs of basic amino acids, which are potential cleavage sites for production of biologically active peptides. We have developed antibodies against 12 defined epitopes of the CgA and 16 defined epitopes of CgB. With these, we have shown that the pancreatic neuroendocrine cells express different epitopes of the CgA and the CgB molecules. The insulin and glucagon producing cells express almost all examined epitopes of CgA and CgB, while the pancreatic polypeptide cells express some defined parts of the chromogranins and the somatostatin cells express only one CgA epitope and two CgB epitopes. Malignant endocrine pancreatic tumours show varied expression of chromogranin epitopes. The normal and neoplastic neuroendocrine cells of the gastrointestinal tract show various expressions of CgA epitopes. Thus, gastrin cells show a different expression of CgA epitopes than the enterochromaffin cells. One of the antibodies, CgA176-195, immunostained a larger cytoplasmic area than the other CgA antibodies, including the commercially available monoclonal antibody (LK2H10). In the adrenal glands, all chromaffin cells showed immunoreactivity to all region-specific chromogranin antibodies tested. By radioimmunoassay measurements we have shown that circulating plasma concentrations of different chromogranin epitopes differ between various types of tumours, indicating specific processing. We have also shown that the region-specific antibodies can be used to study biological processes. We conclude that the region-specific antibodies presented in this review are important tools to further study the biological role of chromogranins.

Chromogranin A (CgA), a protein stored in the secretory granules of many neuroendocrine cells and neurons, is believed to play intracellular and extracellular functions as a regulator of secretory granules biogenesis and as a precursor of various regulatory peptides of the endocrine and the metabolic systems. CgA is abnormally expressed by various neuroendocrine tumors and is released in high amounts in the blood stream. Detection of CgA in tumor tissues and in biological fluids has proven useful for tumor diagnosis and for monitoring tumor progression / regression after therapy. However, little is known on the effect of excessive production of CgA on tumor behavior. A growing body of evidence suggests that tumor-derived CgA is not only an important diagnostic and prognostic marker, but that it could also play a role in tumor / host interaction.

In our search of a function for the chromogranin-derived peptides present within secretory granules of chromaffin cells we observed that a large number of them displayed potent antibacterial and antifungal activities. These peptides are released by exocytosis into the circulation together with catecholamines and are also found in inflammatory fluids together with a variety of other antimicrobial peptides such as defensins. Vasostatin I, the natural N-terminal domain of chromogranin A (1-76), and the vasostatin-derived peptide chromofungin (chromogranin A47-66) are, in addition to fragments of proenkephalin (enkelytin) and ubiquitin (ubifungin), potent antimicrobial peptides of adrenomedullary origin. These peptides interact with the cell wall of fungi and yeast cells and may within minutes penetrate and accumulate within the cells. Once inside, the peptides may target to enzymes that are crucial for the growth of the microorganisms, inhibiting their activities and thus causing the rapid death of the affected species. These properties lead us to propose that these peptides participate in a primary and early line of host defence, acting as a shield against invading microorganisms during stress and infection, linking the neuroendocrine and nervous tissues to the immune system. On the basis of structure, absence of a specific receptor, ability to cross membranes and to inhibit the calmodulin-dependent enzyme calcineurin in vitro, we propose that the vasostatin-derived chromofungin should be recognised as a cell penetrating peptide.

Chromogranin A (CgA) and chromogranin B (CgB) belong to the granin family of acidic secretory proteins with widespread distribution in neuroendocrine tissue. They are co-packaged with other proneuropeptides and prohormones into exocytotic carriers, the secretory granules, and are processed to signalling molecules in this storage organelle. The analysis of the sorting of CgB revealed two key domains involved in sorting to the regulated secretory pathway. These domains are also conserved in CgA. One is an acidic domain inducing Ca2+ / pH-dependent aggregate formation in the trans-Golgi network. The other is a N-terminal loop structure, which is sufficient to direct secretory cargo to secretory granules. Furthermore, it appears that lipid micro-domains play an important role in cargo-membrane interaction. Recently, green fluorescent protein (GFP)-based imaging, employing fusion proteins of CgB and other regulated secretory proteins, has provided new insights into the dynamic complexity of the transport of secretory granules from the TGN to the F-actin-rich cortex. The resolution of this transport in time and space in conjunction with biochemical analyses led to the identification of motor proteins and cytoskeletal elements, which are part of the underlying molecular machinery. This knowledge greatly facilitated the interpretation of physiological studies on the regulated exocytosis of neuropeptides and hormones at the molecular level.

Chromaffin granules are organelles similar to the large dense cored vesicles (LDCV) found in many neurons. They accumulate catecholamines and other soluble components at concentrations that may result in a hypertonic intravesicular medium. However, they maintain isotonicity with cytosol probably as result of aggregation of intravesicular components. Chromogranins could play a role in that isotonic equilibrium as well as in the control of the free diffusion of catecholamines to the external media once granule fusion occurs. Although the release of soluble components upon exocytosis has been considered as a merely passive phenomenon, we have demonstrated that it is finely modulated by second messengers and, therefore, interfered with drugs. Our data suggest that the main regulatory target for these signals is not the fusion pore but most probably intravesicular factors like pH and chromogranins. We also present results suggesting that chromogranins are involved in the mechanisms that modulate the latter steps of exocytosis. Of importance, changes in the kinetics of exocytosis or/and in the quantal size can result in profound modifications to synaptic performance, at least in those processes mediated by LDCV.

We review our recent work on the cardiotropic actions of vasostatins (VS) with emphasis on their putative role in cardiac homeostasis. Studies on the isolated and perfused eel (Anguilla anguilla), frog (Rana esculenta) and rat (Langendorff preparation) hearts, as paradigms of fish, amphibian and mammalian hearts, highlight two important cardiotropic features of VS, i.e. their intrinsic negative myocardial inotropy (in terms of reduced haemodynamic parameters of mechanical performance) and their counteracting action against β-adrenergic (i.e. isoproterenol; ISO)-mediated positive inotropism. This uniform cardiotropic behaviour is consistent with an ubiquitous cardiac role of VS in vertebrates. Comparison of VS-mediated negative inotropy in the eel and frog hearts illustrates aspects of uniformity and speciesspecific differences in the mechanism of action of the peptides. In both eel and frog hearts, VS-mediated inotropy is abolished by pre-treatment with Ca++ and K+ channels antagonists. In contrast, while VS-mediated inotropy in the eel requires an endocardial endothelium (EE) and G protein-nitric oxide (NO)-cGMP signalling, it is independent from this mechanism in frog. By using the isolated working frog heart as a bioassay system, the structural characteristics of several sequences of synthetic VS I-derived peptides (e.g. bovine and frog CgA4-16 and CgA47-66, intact and reduced bovine CgA1-40) have been functionally compared and evaluated in terms of their intrinsic inotropy and “anti-adrenergic” action. This analysis supports the phylogenetic conservation of CgA1-76 and the importance of the intact disulfide bridge-loop CgA1-40SS. On the basis of these data, we suggest that VS I and peptides thereoff are novel cardio-inhibitory agents acting as cardiostatins, i.e. principles able to protect the heart by locally counteracting excessive sympathetic stimulation.

Pancreastatin (PST) is a chromogranin A (CgA)-derived peptide that was first isolated from the porcine pancreas. Although the physiological role of PST has yet to be fully established, a multitude of effects have implicated PST in the modulation of secretion and the control of energy metabolism, with a general counter-regulatory effect to that of insulin. Thus, PST has a glycogenolytic effect in the liver and a lipolytic effect in the adipocyte. Besides, PST inhibits leptin and increases UCP-2 expression in isolated adipocytes. In liver and adipose tissue, specific PST receptors and PSTinduced signal transduction have been characterized, involving a Gq-PLCβ-calcium-PKC pathway, providing a basis for the molecular mechanisms of the metabolic effects of PST, and the cross-talk to the insulin receptor signalling. Increased levels of PST, which correlate with those of catecholamines, have been found in insulin resistance states, such as gestational diabetes, and essential hypertension. Our current hypothesis is that PST plays a role in the physiology of stress, by regulating the supply of energy to the body. In this line, PST could play a role potentiating the metabolic effects of catecholamines, and therefore playing also a possible role in insulin resistant states with increased sympathetic activity.

The primary amino acid sequence of WE-14, corresponding to residues 324-337 of human CgA, was deduced following purification from a liver metastasis of an ileal carcinoid and a phaeochromocytoma, respectively. WE-14 is flanked by conserved Lys-Arg proteolytic sites and its cDNA deduced sequence displays a high degree of inter-species sequence conservation. WE-14 immunostaining was evident in distinct subpopulations of CgA immunopositive neuroendocrine cells. This observation was most obvious in the adrenal and pituitary glands. Immunohistochemical studies of the central and peripheral nervous system have shown that WE-14 immunostaining is localised to nerve fibres in all organs studied. Mapping studies in the eye have revealed that WE-14 is generated in nerve fibres innervating the anterior uvea and in nerve fibres and cell bodies in the retina. Ontogenetic studies have demonstrated that WE-14 was differentially generated in neuroendocrine cell populations at an early stage of rat and pig foetal development, whilst phylogenetic examination has revealed that WE-14-like molecules exhibit an ancient lineage. Qualitative studies of diverse neuroendocrine tumour types revealed heterogeneous patterns of WE-14 immunostaining; quantitative analyses consistently detected significantly elevated tissue concentrations while chromatographic profiling detected an immunoreactant that co-eluted with synthetic human WE-14. Analysis of chromaffin and oxyntic mucosal endocrine cells revealed that pharmacological manipulation had a significant impact on chromogranin A proteolysis to generate WE-14. Collectively, these studies have shown that the neuropeptide WE-14 exhibits a widespread distribution in multiple neuronal elements and neuroendocrine cells and in resultant neuroendocrine neoplasia, throughout ontogeny and that it also has an ancient lineage.

We have identified a novel peptide, catestatin (bovine chromogranin A [CgA]344-364: RSMRLSFRARGYGFRG PGLQL; human CgA352-372: SSMKLSFRARGYGFRGPGPQL), which is a potent inhibitor of nicotinic cholinergicstimulated catecholamine secretion, desensitization of catecholamine release and transcription of chromogranin A (Chga) gene. Although the homology modeling of bovine catestatin suggested a β-strand / loop / b-strand active conformation, 2-D 1H-NMR studies of linear human catestatin (hCgA352-372) exhibited a loosely coiled loop conformation in solution. This peptide is stored within and secreted from chromaffin granules. Catestatin displays characteristic inhibitory effects on nicotinic cationic (Na+, Ca 2+) signal transduction and the effects are specific to the neuronal nicotinic receptor. Results from artificial mutants of catestatin defined an active catestatin core (bovine CgA344-358) as well as the roles of crucial amino acid residues for inhibition of nicotinic cholinergic-stimulated catecholamine secretion and agonist desensitization. Utilizing systematic polymorphism discovery at the human CgA locus we discovered three human variants of catestatin: Gly364Ser, Pro 370Leu, and Arg374Gln that displayed the following rank order of potency for inhibition of catecholamine secretion: Pro 370Leu > wild-type > Gly364Ser > Arg374Gln. Treatment with nicotine augmented expression of the Chga / luciferase transgene and the increment was inhibited >90% by catestatin, establishing an entirely new role for the peptide on gene expression in vivo. Diminished catestatin is observed in hypertensive individuals as well as early-normotensive offspring of patients with hypertension, suggesting that an early deficiency of catestatin might play a pathogenic role in the subsequent development of the disease.

Pairs of counter-regulatory hormones achieve very precise control of several plasma constituents. This precision implies integral control. Locally acting chromogranins and other statins within individual endocrine glands cause endocrine tissues to respond to the time integral of the error in the controlled variable, and not merely to the magnitude of that error. This produces integral control. Pairs of integral controllers need, however, to be linked to avoid homeostatic conflict. Thus the pancreatic islets consist of α-, and β-cells linked by gap junctions to form heterologous functional syncytial units. These probably act as flip-flop mechanisms which secrete either insulin or glucagon. Insulin is therefore always secreted at the expense of glucagon, and vice versa, allowing the two controllers to operate as an Integral Rein Control unit. When counterregulatory hormones are secreted by anatomically remote tissues then the co-secretion of a common inhibitory “link hormone” replaces the gaps junctions of the pancreatic islets. Co-secreted chromogranin A provides the link between the calcitonin-PTH pair, while somatostatin from the pancreas and hypothalamus allows growth hormone to operate in conjunction with insulin and glucagon without conflict.