Interactions of chromogranin A-derived vasostatins and monolayers of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine

Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.     

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.     

The tissue distribution of rat chromogranin A-derived peptides: evidence for differential tissue processing from sequence specific antisera

Summary The distribution of chromogranin A and related peptides in rat tissues was investigated using sequence specific antisera. N- and C-terminal antisera and a presumptive C-terminal rat pancreastatin antiserum immunostained an extensive neuroendocrine cell population throughout the gastro-entero-pancreatic tract, anterior pituitary, thyroid and all adrenomedullary cells. However, mid- to C-terminal antisera immunostained a subpopulation of chromogranin A positive cells. Most notable of these was with the KELATE antiserum (R635.1) which immunostained discrete clusters of adrenomedullary cells and antiserum A87A which immunostained a subpopulation of cells in the anterior pituitary and throughout the gastrointestinal tract. The present study has demonstrated the widespread occurrence of chromogranin A and related peptides in rat neuroendocrine tissues and provides evidence of tissue and cell specific processing.     

The tissue distribution of rat chromogranin A-derived peptides: evidence for differential tissue processing from sequence specific antisera.

The distribution of chromogranin A and related peptides in rat tissues was investigated using sequence specific antisera. N- and C-terminal antisera and a presumptive C-terminal rat pancreastatin antiserum immunostained an extensive neuroendocrine cell population throughout the gastro-entero-pancreatic tract, anterior pituitary, thyroid and all adrenomedullary cells. However, mid- to C-terminal antisera immunostained a subpopulation of chromogranin A positive cells. Most notable of these was with the KELTAE antiserum (R635.1) which immunostained discrete clusters of adrenomedullary cells and antiserum A87A which immunostained a subpopulation of cells in the anterior pituitary and throughout the gastrointestinal tract. The present study has demonstrated the widespread occurrence of chromogranin A and related peptides in rat neuroendocrine tissues and provides evidence of tissue and cell specific processing.     

Localization and characterization of evolutionarily conserved chromogranin A-derived peptides in the rat and human pituitary and adrenal glands

Chromogranin A (CgA) is a neuroendocrine protein that undergoes proteolytic cleavage in secretory granules. The aim of the present study was to characterize the peptides WE14 and EL35 that are derived from evolutionarily conserved regions of CgA in rat and human endocrine tissues. In the rat pituitary, HPLC analysis revealed that WE14 is present as a single immunoreactive peak, whereas EL35 elutes in two molecular forms. Authentic WE14 is also produced in both rat and human adrenal glands, while EL35 displays a variable elution profile depending on the tissue extract, indicating the existence of different forms of EL35 in these tissues. Immunohistochemical labeling of the rat pituitary showed that WE14 and EL35 occur in gonadotropes and melanotropes, but not in corticotropes. A strong immunoreaction for both peptides was also observed in rat adrenochromaffin cells. In the human adrenal gland, the WE14 and EL35 antisera revealed intense labeling of adrenomedullary cells in adult and nests of chromaffin progenitor cells in fetal adrenal. Finally, WE14 and EL35 immunoreactivity was detected in pheochromocytoma tissue where WE14 occurred as a single immunoreactive form, while EL35 displayed different forms. The observations that WE14 and EL35: (1). have been preserved during vertebrate evolution, (2). are processed in a cell-specific manner, and (3). occur during ontogenesis of the adrenal gland strongly suggest that these peptides play a role in endocrine tissues. In addition, the existence of differentially processed CgA-derived peptides in normal and tumorous tissues may provide new tools for the diagnosis and prognosis of neuroendocrine tumors.     

Expression of the chromogranin A-derived peptides pancreastatin and WE14 in rat stomach ECL cells

The ECL cells constitute the predominant endocrine cell population in the mucosa of the acid-secreting part of the stomach (fundus). They are rich in chromogranin A (CGA), histamine and histidine decarboxylase (HDC). They secrete CGA-derived peptides and histamine in response to gastrin. The objective of this investigation was to examine the expression of pancreastatin (rat CGA_266-314) and WE₁₄ (rat CGA_343-356) in rat stomach ECL cells. The distribution and cellular localisation of pancreastatin- and WE₁₄-like immunoreactivities (LI) were analysed by radioimmunoassay and immunohistochemistry with antibodies against pancreastatin, WE₁₄ and HDC. The effect of food deprivation on circulating pancreastatin-LI was examined in intact rats and after gastrectomy or fundectomy. Rats received gastrin-17 (5 nmol/kg/h) by continuous intravenous infusion or omeprazole (400 μmol/kg) once daily by the oral route, to induce hypergastrinemia. CGA-derived peptides in the ECL cells were characterised by gel permeation chromatography. The expression of CGA mRNA was examined by Northern blot analysis. Among all of the endocrine cells in the body, the ECL cell population was the richest in pancreastatin-LI, containing 20–25% of the total body content. Food deprivation and/or surgical removal of the ECL cells lowered the level of pancreastatin-LI in serum by about 80%. Activation of the ECL cells by gastrin infusion or omeprazole treatment raised the serum level of pancreastatin-LI, lowered the concentrations of pancreastatin- and WE₁₄-LI in the ECL cells and increased the CGA mRNA concentration. Chromatographic analysis of the various CGA immunoreactive components in the ECL cells of normal and hypergastrinemic rats suggested that these cells respond to gastrin with a preferential release of the low-molecular-mass forms.     

Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts

Vasostatins (VSs), i.e. the main biologically active peptides generated by the proteolytic processing of chromogranin A (CGA) N-terminus, exert negative inotropism in vertebrate hearts. Here, using isolated working eel (Anguilla anguilla) and frog (Rana esculenta) heart preparations, we have studied the role of the cytoskeleton in the VSs-mediated inotropic response. In both eel and frog hearts, VSs-mediated-negative inotropy was abolished by treatment with inhibitors of cytoskeleton reorganization, such as cytochalasin-D (eel: 10 nM; frog: 1 nM), an inhibitor of actin polymerisation, wortmannin (0.01 nM), an inhibitor of PI3-kinase (PI3-K)/protein kinase B (Akt) signal-transduction cascade, butanedione 2-monoxime (BDM) (eel: 100 nM; frog: 10 nM), an antagonist of myosin ATPase, and N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide (W7) (eel: 100 nM; frog: 1 nM), a calcium-calmodulin antagonist. These results demonstrate that changes in cytoskeletal dynamics play a crucial role in the negative inotropic influence of VSs on eel and frog hearts.     

Reprint of: Metabolic effects and mechanism of action of the chromogranin A-derived peptide pancreastatin

Pancreastatin is one of the regulatory peptides derived from intracellular and/or extracellular processing of chromogranin A, the soluble acidic protein present in the secretory granules of the neuroendocrine system. While the intracellular functions of chromogranin A include formation and maturation of the secretory granule, the major extracellular functions are generation of biologically active peptides with demonstrated autocrine, paracrine or endocrine activities. In this review, we will focus on the metabolic function of one of these peptides, pancreastatin, and the mechanisms underlying its effects. Many different reported effects have implicated PST in the modulation of energy metabolism, with a general counterregulatory effect to that of insulin. Pancreastatin induces glycogenolysis in liver and lipolysis in adipocytes. Metabolic effects have been confirmed in humans. Moreover, naturally occurring human variants have been found, one of which (Gly297Ser) occurs in the functionally important carboxy-terminus of the peptide, and substantially increases the peptide's potency to inhibit cellular glucose uptake. Thus, qualitative hereditary alterations in pancreastatin's primary structure may give rise to interindividual differences in glucose and lipid metabolism. Pancreastatin activates a receptor signaling system that belongs to the seven-spanning transmembrane receptor coupled to a Gq-PLCβ-calcium-PKC signaling pathway. Increased pancreastatin plasma levels, correlating with catecholamines levels, have been found in insulin resistance states, such as gestational diabetes or essential hypertension. Pancreastatin plays important physiological role in potentiating the metabolic effects of catecholamines, and may also play a pathophysiological role in insulin resistance states with increased sympathetic activity.     

Differential accumulation of catecholamines, proenkephalin- and chromogranin A-derived peptides in the medium after chronic nicotine stimulation of cultured bovine adrenal chromaffin cells

We have used an antiserum to a synthetic peptide fragment of bovine chromogranin A (ChrgA)[Tyr0] bovine ChrgA (306-313): YLSKEWEDA, together with antibodies to proenkephalin-derived peptides, to measure the release of immunoreactive peptides from nicotine-stimulated cultured bovine adrenal chromaffin cells. Over a period of 6 hr the accumulation of YLSKEWEDA immunoreactivity and Met-enkephalin Arg6Gly7Leu8 (MERGL) immunoreactivity in the medium of 10 microM nicotine-stimulated cells was shown to be biphasic; the initial phase occurred in the first 15-30 min and the second phase reached a peak after 4 hr. In contrast, catecholamine release occurred monophasically over the initial 15-30 min. Investigation of the second phase of peptide accumulation revealed that it was due in part to nicotine-evoked exocytosis and in part to extracellular processing of high molecular weight precursor proteins.     

Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery

Chromogranin A (CgA), an acidic granule protein of the regulated secretory pathway in the diffuse neuroendocrine system, is postulated to serve as a prohormone for regulatory peptides. Betagranin (rCgA(1-128)), the first N-terminal cleavage product of rat CgA, is 87% homologous to the bovine vasostatin I (bCgA(1-76)), previously shown to be vasoinhibitory in bovine resistance arteries. In this study the vasoactivity of homologous rat and bovine peptides was investigated in the rat posterior cerebral artery. Firstly, we examined the interaction of rhodamine (Rh)-labelled bCgA(7-40) and bCgA(47-70) with elements of the arterial wall by fluorescence microscopy. Secondly, rCgA(7-57), bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin) were studied for effects on arterial tone and intracellular calcium as function of pressure in an arteriograph. Although without dilator or constrictor responses at 60-150 mm Hg, the rat peptide (rCgA(7-57)) evoked a significant delay in the onset of forced dilatation at 170 mm Hg, in contrast to the bovine peptides bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin). Neither Rh-bCgA(7-40) nor Rh-bCgA(47-70) stained the endothelial layer, while Rh-bCgA(47-70) but not Rh-bCgA(7-40) stained the smooth muscle compartment. Analogously, bCgA(47-66) but not bCgA(7-40) reduced intracellular calcium, however without modifying the myogenic response. Thus, the betagranin peptide rCgA(7-57) and the two bovine chromofungin-containing peptides, highly homologous to the corresponding sequence (rCgA(47-66)), affected the rat cerebral artery without vasodilator effects, indicating significant species differences in vasoactivity of the N-terminal domain of CgA.     

Buforins: Histone H2A-derived antimicrobial peptides from toad stomach

Antimicrobial peptides (AMPs) constitute an important component of the innate immune system in a variety of organisms. Buforin I is a 39-amino acid AMP that was first isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. Buforin II is a 21-amino acid peptide that is derived from buforin I and displays an even more potent antimicrobial activity than its parent AMP. Both peptides share complete sequence identity with the N-terminal region of histone H2A that interacts directly with nucleic acids. Buforin I is generated from histone H2A by pepsin-directed proteolysis in the cytoplasm of gastric gland cells. After secretion into the gastric lumen, buforin I remains adhered to the mucous biofilm that lines the stomach, thus providing a protective antimicrobial coat. Buforins, which house a helix-hinge-helix domain, kill a microorganism by entering the cell without membrane permeabilization and thus binding to nucleic acids. The proline hinge is crucial for the cell penetrating activity of buforins. Buforins also are known to possess anti-endotoxin and anticancer activities, thus making these peptides attractive reagents for pharmaceutical applications. This review describes the role of buforins in innate host defense; future research paradigms; and use of these agents as human therapeutics.     

Effect of reserpine on the generation of the chromogranin A-derived neuropeptide WE-14 in rat oxyntic mucosa

WE-14, a post-translational product of the neuroendocrine protein chromogranin A (CgA), is generated in distinct subpopulations of endocrine cells. The objective of this study was to investigate the generation of WE-14 in the endocrine cell types of the oxyntic mucosa of the stomach, after treatment with reserpine, an irreversible inhibitor of vesicular monoamine uptake 2 (VMAT2). Reserpine (10 mg/kg) was administered subcutaneously and tissue analysed 1, 3, 5 and 18 h following treatment. The oxyntic mucosa was analysed immunohistochemically employing a site-specific WE-14 antiserum, a region-specific CgA antiserum and an antiserum against histidine decarboxylase (HDC), a marker of the histamine-producing ECL cells in the oxyntic mucosa. The number of oxyntic endocrine cells exhibiting WE-14 immunostaining increased more than 100-fold 18 h after reserpine administration relative to vehicle treated controls. Double immunostaining with HDC revealed that most, but not all, of the WE-14 positive cells were ECL cells. These results suggest that reserpine has the ability to influence the post-translational processing of CgA to generate WE-14 in rat stomach ECL cells, presumably as a consequence of reduced VMAT2-driven accumulation of histamine.     

VI. Measurement of proenkephalin A-derived peptides in biological tissues by high pressure liquid chromatography coupled with amperometric electrochemical detection

We describe an analytic method for the separation and quantitation of a number of proenkephalin A-derived peptides using high pressure liquid chromatography coupled with amperometric electrochemical detection (HPLC-AECD). Initially, we coupled our HPLC separation system with AECD in series with a UV detector for additional confirmation of peak specificity. AECD provided a 10⁶ - fold increase in sensitivity over UV detection for these peptides. In addition to Met-enkephalin (ME), ME-Arg, ME-Arg-Phe, ME-Arg-Gly-Leu, Leu-enkephalin (LE) and LE-Arg (Dyn 1–6), we separated and detected the sulfoxides of ME and its extended peptides. Subsequently, we used minor modifications of the isocratic mobile phase to separate and detect enkephalin-related peptides with greater sensitivity and shorter chromatographic run times; each of these mobile phases was used to separate and detect two to three peptides. We have applied this HPLC-AECD methodology to quantitate ME, ME-Arg-Phe, ME-Arg-Gly-Leu and LE in pheochromocytoma tumors.     

Peptides from the N-terminal domain of chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart

The negative inotropic effects of synthetic peptides derived from the N-terminus of chromogranin A (CgA) were studied in an avascular model of the vertebrate myocardium, the isolated working frog heart (Rana esculenta). The peptides were frog and bovine CgA(4-16) and CgA(47-66), and bovine CgA(1-40) with (CgA(1-40SS)) and without an intact disulfide bridge (CgA(1-40SH)). Under basal cardiac conditions, four of the peptides caused a concentration-dependent negative inotropism that was comparable to the negative inotropy reported for human recombinant vasostatin I (CgA(1-78)) and bovine CgA(7-57). By comparison of the structural characteristics of the bovine and frog sequences with their minimally effective concentrations ranging from 68 to 125 nM of peptide, the results were consistent with the natural structure (CgA(17-38SS)) being essential for the negative inotropism. In addition, the partial sequences of the frog and bovine vasostatin I were effective in counteracting the characteristic positive inotropism exerted by isoproterenol (1 nM) at minimally effective concentrations ranging from 45 to 272 nM. Taken together, these results extend the first evidence for a cardiosuppressive role of the N-terminal domain of chromogranin A known for its co-storage with catecholamines in the sympathoadrenal system of vertebrates.     

Processing of chromogranin A in the parathyroid: generation of parastatin-related peptides

Chromogranin A (CgA) is a glycoprotein present in secretory granules of endocrine cells. In the parathyroid, it is costored and cosecreted with parathormone (PTH) in response to hypocalcemia. CgA is the precursor of several bioactive peptides including pancreastatin and betagranin. Parastatin (PARA, pCgA(347-419)) is a novel peptide that we generated in vitro by enzymatic digestion of pCgA. In vitro, it inhibits low Ca(2+)-stimulated parathyroid secretion. Full activity resides in its first 19 residues. In order to determine if PARA or PARA-derived peptides are natural products of the parathyroid, we generated an antiserum directed against pCgA(347-359) corresponding to the bioactive N-terminal sequence of pPARA (pPARA(1-13) antiserum), and developed a specific radioimmunoassay that we used in conjunction with various chromatographic separations. We identified small peptides carrying the pPARA(1-13) immunoactivity in extracts and secretion medium of porcine parathyroid glands. Continuous and pulse-chase radiolabeling studies, along with immunoprecipitation using PARA(1-13) antiserum demonstrate that a newly-synthesized PARA-related peptide fraction with a Mr of 11 kDa is secreted by the parathyroid cells and accumulates in the secretion medium. Edman degradation of the 11 kDa PARA-related peptide band by Edman degradation yielded three major N-terminal sequences: S-K-M-D-R-L-A-K-E-L-(residues 313-322), D-R-L-A-K-E-L-T-A-E-(residues 316-325), and A-K-E-L-T-A-E-K-R-L-(residues 319-329), in a molar ratio of approximately 1:2:1. The peptide bonds required to be cleaved to yield these peptides, Trp-Ser, Met-Asp and Leu-Ala, suggest that a chymotrypsin-like endopeptidase participated in their formation. The molecular size and the results of amino acid compositional analysis, indicate that the C-termini of these peptides extended variably to residues 384-401 of pCgA. These results demonstrate that processing of CgA by the parathyroid gland generates bioactive PARA-related peptides that could affect the gland's secretory activity.     

Phytoecdysteroids: biological aspects

Phytoecdysteroids are a family of about 200 plant steroids related in structure to the invertebrate steroid hormone 20-hydroxyecdysone. Typically, they are C27, C28 or C29 compounds possessing a 14alpha-hydroxy-7-en-6-one chromophore and A/B-cis ring fusion (5beta-H). In the present review, the distribution, biosynthesis, biological significance and potential applications of phytoecdysteroids are summarised.     

Cellular localization of a chromogranin B-like derived peptides in leeches

Using immunocytochemistry techniques, we demonstrated specific immunostaining with antibodies rose against fragment (547-560) and (614-626) of bovine chromogranin B (CGB) at the level of brain and the tegument of the leech Theromyzon tessulatum. The used of these antibodies on leech sections revealed immunopositive labeling in neurons and glial cells of the central nervous system (CNS), and epidermal glandular cells of the tegument. Co-localization between two antibodies rose against different fragment of bovine CGB have been demonstrated in neurons and glial cells of leech CNS like in vertebrates. Finally, the whole of the data showed for the first time the presence in leeches of CGB like derived peptides.     

New aspects in the biological role of zinc: a stabilizer of macromolecules and biological membranes

It has long been known that zinc is essential to life as an integral part of a number of enzymes (1,2). Increasing evidence also suggests that zinc is important in the stability of macromolecules, particularly the components of various biological membranes. This brief review presents a personal and admittedly incomplete view of the latter aspect of the role of zinc. In this review, I shall combine experimental data with speculations in the hopes of stimulating research along new lines.