Current Medicinal Chemistry–Immunology, Endocrine & Metabolic Agents Volume 4, No. 3, 2004
Contents
Chromogranin
A and Its Derived Peptides as Potential Regulators of Immune, Endocrine and
Metabolic Systems – In Search of Its Physiological Role
Guest
Editors: K.B. Helle and G. Serck-Hanssen
Cellular Localisation of Chromogranins and
Processed Products in the Diffuse Neuroendocrine System and Related Tumours Pp.149-160
Chromogranin A in Tumors: More Than a Marker
for Diagnosis and Prognosis Pp.161-167
Angelo
Corti and Elisabetta Ferrero
The Innate Immunity: Roles for New Antifungal
and Antibacterial Peptides Secreted by Chromaffin Granules from the Adrenal Medulla
Pp.169-177
Marie-Helene
Metz-Boutigue, Karen B. Helle and Dominique Aunis
Aggregation, Sorting and Transport of Chromogranins
in the Regulated Secretory Pathway Pp.179-185
H.
-H. Gerdes, R. Rudolf and T. Kogel
New Insights About the Functional Role of
Chromogranins in the Latest Steps of Exocytosis Pp.187-193
R.
Borges, G. Delgado and J. Diaz
Vasostatins and Negative Inotropy in
Vertebrate Hearts Pp.195-201
B.
Tota, S. Imbrogno, C. Mannarino and R. Mazza
Pancreastatin, A Regulatory Peptide that
Modulates Energy Metabolism Pp.203-212
Víctor
Sanchez-Margalet, Carmen Gonzalez-Yanes and Souad Najib
Comparative Studies of the Chromogranin
A-Derived Neuropeptide WE-14 Pp.213-219
W.J.
Curry, J.G. Quinn, S. Brockbank, V. Miller, C. McVicar, K. Pogue, S. Barkatullah
and P. Norlen
Catestatin - The Catecholamine Release
Inhibitory Peptide: A Structural and Functional Overview Pp.221-234
Sushil
K. Mahata
The Role of Chromogranins and Other Statins
in Homeostasis: An Explanation of the Precise Regulation of Glucose and Ionised
Calcium in the Blood; An Overview Pp.235-249
Johan
H. Koeslag and Peter T. Saunders
Abstracts
[Back to top] Cellular Localisation of Chromogranins and
Processed Products in the Diffuse Neuroendocrine System and Related Tumours
Mats
Stridsberg, Eva T. Janson and Guida M. Portela-Gomes
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.
[Back to top] Chromogranin A in Tumors: More Than a Marker
for Diagnosis and Prognosis
Angelo
Corti and Elisabetta Ferrero
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.
[Back to top] The Innate Immunity: Roles for New Antifungal
and Antibacterial Peptides Secreted by Chromaffin Granules from the Adrenal
Medulla
Marie-Helene
Metz-Boutigue, Karen B. Helle and Dominique Aunis
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.
[Back to top] Aggregation, Sorting and Transport of
Chromogranins in the Regulated Secretory Pathway
H.
-H. Gerdes, R. Rudolf and T. Kogel
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.
[Back to top] New Insights About the Functional Role of
Chromogranins in the Latest Steps of Exocytosis
R.
Borges, G. Delgado and J. Diaz
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.
[Back to top] Vasostatins and Negative Inotropy in
Vertebrate Hearts
B. Tota, S. Imbrogno, C. Mannarino and R. Mazza
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 b-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.
[Back to top] Pancreastatin, A Regulatory Peptide that
Modulates Energy Metabolism
Víctor Sanchez-Margalet, Carmen Gonzalez-Yanes and Souad Najib
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-PLCb-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.
[Back to top] Comparative Studies of the Chromogranin
A-Derived Neuropeptide WE-14
W.J. Curry, J.G. Quinn, S. Brockbank, V. Miller, C. McVicar, K. Pogue, S. Barkatullah and P. Norlen
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.
[Back to top] Catestatin - The Catecholamine Release
Inhibitory Peptide: A Structural and Functional Overview
Sushil
K. Mahata
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 b-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, Pro370Leu, and Arg374Gln
that displayed the following rank order of potency for inhibition of
catecholamine secretion: Pro370Leu > 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.
[Back to top] The Role of Chromogranins and Other Statins
in Homeostasis: An Explanation of the Precise Regulation of Glucose and Ionised
Calcium in the Blood; An Overview
Johan
H. Koeslag and Peter T. Saunders
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 a-, and b-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.