Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate.

PURPOSE: The retinal pigment epithelium (RPE) and underlying Bruch's membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell-substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch's membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this "aged" substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. METHODS: Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. RESULTS: Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. CONCLUSIONS: This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch"s membrane could play a significant role in age-related dysfunction of the RPE.

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.

Characterisation of WE-14 in porcine ocular tissue.

WE-14 is derived from the cell-specific posttranslational processing of chromogranin A (CgA) in subpopulations of neuroendocrine cells and neurons. Region- and site-specific chromogranin A, pancreastatin and WE-14 antisera were employed to study the generation of WE-14 in porcine ocular tissues. No chromogranin A or pancreastatin immunostaining was detected in ocular tissue. Immunohistochemistry detected WE-14 immunostaining in a network of nerve fibre bundles and nerve fibres throughout the limbus, cornea, iris and ciliary body with sparse nerve fibres detected throughout the choroid and sclera. Retinal analysis detected intense WE-14 immunostaining in large ovoid cells in the ganglion cell layer with weak immunostaining in a population of small cells in the inner nuclear layer; weak immunostaining was detected within the fibre layers in the inner plexiform layer. Quantitatively, the highest WE-14 tissue concentration was recorded in aqueous retinal and corneal extracts with lower concentrations in the sclera, choroid and anterior uveal tissues. Chromatographic profiling resolved a minor chromogranin A-like immunoreactant and a predominant immunoreactant co-eluting with synthetic human WE-14. This is the first study to demonstrate that WE-14 is generated in neuronal fibres primarily innervating the anterior chamber and in select cell populations in the retina.

WE-14, a chromogranin a-derived neuropeptide.

The neuropeptide WE-14 is derived from the posttranslational processing of chromogranin A (CgA). While CgA is expressed in a preponderance of neuroendocrine cells, WE-14 is generated in a distinct subpopulation of CgA-immunopositive cells, most notably in the adrenal, pituitary, and parathyroid glands. Physiological and pharmacological studies have demonstrated that CgA is cleaved to generate WE-14 in the adrenal chromaffin cell population and in the enterochromaffin-like (ECL) cells of the oxyntic mucosa. Pathological analyses of neuroendocrine tumors have revealed a heterogeneous pattern of WE-14 immunostaining, with variable concentrations quantified and chromatographically resolved in tissue extracts. Phylogenetic surveys have demonstrated that WE-14 exhibits an ancient lineage, while ontogenetic examination has shown that it is generated at an early stage during fetal development. Putative WE-14 receptor binding sites have been identified in several tissues; however, the physiological role of WE-14 remains enigmatic.

Immunohistochemical localization of WE-14 in the developing porcine sympathoadrenal cell lineage.

Immunohistochemical investigation of the post-translational processing of chromogranin A (CgA) to generate WE-14 in the sympathoadrenal cell lineage of the developing porcine fetus (F) detected intense CgA and weak WE-14 immunoreactivity in migrating neuroblast cells of the diffuse sympathetic ganglia adjacent to the dorsal aorta and projecting toward the cortical mass at F24-27. F37-42; WE-14 immunoreactivity was detected in chromaffinoblasts at the periphery of the developing cortex and at F54-56 days gestation WE-14 immunoreactivity was detected in a large population of central medullary cells. From F74 to F76 days and thereafter the number of cells exhibiting intense WE-14 immunostaining decreased, and the majority of chromaffin cells exhibited uniform weak WE-14 immunostaining. At postnatal day 1 (P1) intense WE-14 immunoreactivity was primarily confined to clusters of chromaffin cells with weak immunostaining in the general population. The transitory neuroblasts, chromaffinoblasts, and maturing chromaffin cell population exhibited uniform intense CgA immunostaining through gestation and after birth. Additional observations detected intense CgA and WE-14 immunostaining in extrachromaffin tissue at P1 and in neuronal-like cells in vessels of the aortic arch at F37. This study has demonstrated that CgA is post-translationally processed to generate WE-14 during early fetal development in the migrating progenitor cells of the porcine sympathoadrenal lineage.

Analysis of the post-translational processing of chromogranin A in rat neuroendocrine tissue employing an N-terminal site-specific antiserum.

Chromogranin A (CgA) is a complex prohormone expressed as a constituent of the regulated secretory pathway of numerous neuroendocrine cells. Recent investigations have demonstrated that CgA is selectively cleaved to generate distinct peptides in different neuroendocrine tissues. This investigation employed a site-specific antiserum that detects residues 98-106 rat CgA to examine the amino-terminal processing of CgA to generate beta-granin and related peptides in rat neuroendocrine tissues. Immunohistochemistry revealed moderate to intense beta-granin-like immunostaining in cells scattered throughout the anterior pituitary, thyroid, in the islets of Langerhans and in the mucosa of the gastrointestinal tract. Variable intensities of immunostaining were observed in distinct clusters of chromaffin cells. Quantitatively, the highest concentration of beta-granin-like immunoreactivity was detected in pituitary extracts. Significantly lower concentrations were detected in adrenal and thyroid glands, brain, ventral and dorsal pancreatic lobes and gastrointestinal tissue extracts. Chromatography resolved three distinct beta-granin-like immunoreactants; a large CgA-like form, an intermediate molecular form presumably corresponding to beta-granin (rat CgA1-128) and small immunoreactants that coeluted with the synthetic peptide. Two beta-granin-like immunoreactants, 21 and 22 kDa, were detected following immunoblot analysis of pituitary extracts. This study has demonstrated that chromogranin A is subject to distinct amino-terminal patterns of tissue-and cell-specific processing to generate a beta-granin-like immunoreactant which is additionally cleaved in pancreatic, fundic and colonic tissue to generate previously unidentified peptides.

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach.

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine (b) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylase (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Immunohistochemical evaluation of the post-translational processing of chromogranin A in human pituitary adenomas.

Chromogranin A (CgA), pancreastatin (PST), intervening-peptide (IP) and WE-14 antisera were employed to investigate the proteolysis of CgA in 50 pituitary adenomas. All non-functioning (NF) pituitary tumours (n = 28) exhibited CgA immunoreactivity. PST, IP and WE-14 immunostaining was observed in 85%, 89% and 67%, respectively. CgA, PST and IP immunostaining were comparable in the majority of NF tumours, while less intense WE-14 immunoreactivity was detected in a subpopulation of NF tumour cells. Approximately half of the functioning pituitary tumours expressed CgA immunoreactivity. Six of nine ACTH-secreting tumours displayed CgA and IP immunostaining; four of these tumours displayed PST immunoreactivity. WE-14 immunoreactivity was detected in one corticotroph tumour. Three of six growth hormone (GH) secreting tumours displayed CgA immunostaining, two exhibited PST and IP, and one exhibited WE-14 immunoreactivity. Clusters of WE-14 immunopositive cells were detected in one GH tumour. One of seven prolactinomas exhibited weak CgA immunostaining, while weak IP and WE-14 immunostaining was detected in an additional tumour. No PST immunostaining was detected in prolactinomas. Therefore CgA is a valuable marker of NF pituitary tumours, however it is a more sporadic marker of functioning adenomas. In general, the cellular pattern and intensities of CgA, PST and IP immunoreactivity were comparable in the majority of pituitary adenomas. In contrast, WE-14 immunostaining was observed in a subpopulation of tumour cells. The pathophysiological significance of the proteolysis of CgA to generate bioactive peptides in both NF and functioning pituitary adenomas remains to be established.

Immunostaining for vasostatin I distinguishes between ileal and lung carcinoids.

Although chromogranin A (CgA) is a recognized marker of neuroendocrine tumours, little is known about the distribution of the CgA-derived peptides, vasostatin (VST) I or II, in these tumours. Rabbit polyclonal antiserum was raised to a fragment of VST I and used to immunostain sections (5 microns) of wax-embedded tumour tissue. Immunoreactivity (IR) was detected using swine anti-rabbit fluorescein secondary antibody and sections were viewed by fluorescence microscopy. Of 24 tumours from patients with lung carcinoids, one was weakly positive, while 23 of 26 ileal carcinoid tumours were immunoreactive. Metastatic deposits from patients with ileal carcinoids also tended to be immunoreactive (9/10). The difference in IR between lung and ileal carcinoid primary tumours did not appear to be related to the metastatic potential, since appendiceal tumours, which seldom metastasize, also tended to be immunoreactive (4/6) for VST I. The strongest IR was recorded in two patients with flushing as a result of ileal carcinoids; five other 'flushers' with ileal carcinoids were also immunopositive for VST I-like IR. By contrast, patients with flushing as a result of lung carcinoids were immunonegative for VST. In conclusion, VST I-like IR may assist in the identification of a secondary deposit from an unknown primary site.

Newer antiepileptic drugs: gabapentin, lamotrigine, felbamate, topiramate and fosphenytoin.

Twenty-five to 40 percent of patients with epilepsy continue to have seizures despite optimal treatment with traditional antiepileptic drugs. Treatment with standard anticonvulsants such as phenytoin, carbamazepine, valproic acid and phenobarbital is often complicated by side effects and by failure to adequately control seizures. Up to 61 percent of patients with seizures report having side effects with antiepileptic drugs. After a 15-year hiatus since the last new antiepileptic drug was marketed, five new drugs have been approved by the U.S. Food and Drug Administration for the control of seizures. Three of these, gabapentin, lamotrigine and topiramate, are approved for use in adults with partial seizures with or without generalization. Felbamate is approved for the above indication and also for use in children with Lennox-Gastaut syndrome, a rare childhood seizure disorder. Felbamate and lamotrigine have the potential of significant side effects and should be prescribed by physicians experienced in managing patients with complicated epilepsy. Fosphenytoin is a parenteral prodrug of phenytoin that is more tolerable than parenteral phenytoin.

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 CGA266-314) and WE14 (rat CGA343-356) 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 micromol/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.

Ontogenetic expression of chromogranin A and its derived peptides, WE-14 and pancreastatin, in the rat neuroendocrine system.

The ontogenetic expression of chromogranin A (CgA) and its derived peptides, WE-14 and pancreastatin (PST), was studied in the rat neuroendocrine system employing immunohistochemical analysis of fetal and neonatal specimens from 12.5-day embryos (E12.5), to 42-day postnatal (P42) rats. CgA immunostaining was first detected in endocrine cells of the pancreas, stomach, intestine, adrenal gland and thyroid at E13.5, E14.5, E15.5, E15.5 and E18.5, respectively. PST-like immunoreactivity was detected in endocrine cells of the pancreas at E13.5, stomach, intestine at E15.5, adrenal gland at E17.5 and thyroid at E18.5. WE-14 immunoreactivity was first observed in the immature pancreas at E15.5, mucosal cells of the stomach at E15.5, scattered chromaffin cells in the immature adrenal gland and mucosal cells of the intestine at E17.5 and thyroid parafollicular cells at E18.5. These data confirm that the translation of the CgA gene is regulated differentially in various neuroendocrine tissues and, moreover, suggests that the post-translational processing of the molecule is developmentally controlled.

Regulatory peptides and other neuroendocrine markers in medullary carcinoma of the thyroid.

Medullary thyroid carcinoma (MTC) is an APUDoma (APUD refers to amine precursor uptake and decarboxylation) arising from the parafollicular cells. Diarrhoea has been reported in some 30% of patients, variously attributed to excess production of calcitonin (CT), serotonin (5-HT), vasoactive intestinal peptide (VIP) or other factors. The regulatory factors in MTC were examined employing immunocytochemistry and RIA to tumours and their extracts. The patients were followed up for more than 15 years. CT and calcitonin gene-related peptide were universally expressed in all the tumours. The neuroendocrine markers chromogranin A (and its fragments pancreastatin and WE-14), neurone-specific enolase, protein gene product 9.5 and carcino-embryonic antigen were found in the majority of MTCs and might be useful as immunocytochemical markers. 5-HT, substance P, neurokinin A, glucagon and VIP could not be detected, excluding them as candidates in the diarrhoea of MTC.

Occurrence of WE-14 and chromogranin A-derived peptides in tissues of the human and bovine gastro-entero-pancreatic system and in human neuroendocrine neoplasia.

Antisera were generated to the synthetic peptides SREWEDS and KELTAE which correspond to residues 315-321 and 332-337 of human chromogranin A (CgA) respectively. KELTAE represents the C-terminal hexapeptide of WE-14, and SREWEDS (residue 316 human CgA Lys/Arg substitution) represents the C-terminal heptapeptide of the Intervening Peptide, located between pancreastatin and WE-14. The antisera were employed to study the occurrence of WE-14 and CgA-derived peptides in human and bovine gastro-entero-pancreatic (GEP) tissues and in a range of human GEP neuroendocrine tumours. Immunocytochemical analyses of normal human and bovine tissues demonstrated that each antiserum immunostained endocrine cells throughout the GEP tract, Variable intensities of immunostaining were detected in neoplastic tissues. Quantitatively, the highest levels of SREWEDS and KELTAE immunoreactivity were detected in pancreatic extracts, with lower levels in gastrointestinal tissues. Elevated levels of each immunoreactant were detected in neoplastic tissues. Chromatographic analysis resolved several SREWEDS-related peptides and a major KELTAE-related peptide that co-eluted with synthetic human WE-14. The present study has demonstrated that CgA is processed to generate distinct peptide products in normal and neoplastic tissues of the GEP system. A single molecular species co-eluting with synthetic human WE-14 was predominant and consistently detected in all the tissues studied.

PC12 cells show immunoreactivity to a number of proteins and peptides, including vasostatin.

N-terminal chromogranin A (CGA) contains peptides with vasoinhibitory properties, called vasostatin I (VST) and II [CGA (1-76) and (1-113) in human and bovine; (1-128) in rat]. Three fragments of VST were synthesized and antisera raised: human CGA (68-76) (VST I) rat CGA (121-128) (VST II fragment 2), and bovine/human CGA (83-91) (VST II, fragment 3). Strong immunoreactivity was observed in PC12 cells with antisera to VST II, fragment 3, VST I, and neuron-specific enolase. Little or no immunoreactivity was observed using antisera to synaptophysin, whole molecule CGA, pancreastatin, protein gene product 9.5, somatostatin, pancreatic polypeptide, or with antibodies 875 and 876 to VST II, fragment 2. Most of the VST antisera cross-reacted, with a species of molecular weight, 61 kDa but one, 874, cross-reacted with two species of molecular weights, 7.2 and 12 kDa. Our results show the presence of N-terminally processed CGA in PC12 cells.