Chromogranin A Secretion Research Articles

Relationship between salivary Chromogranin-A and stress induced by simulated monotonous driving

The purpose of the present study was to evaluate the use of salivary Chromogranin-A (CgA), which is already used in general as a mental stress marker, for studying the stressful situation created by simulated monotonous driving. After informed consent, 25 healthy male and female subjects were studied under constant environment-controlled conditions. We measured the following physiological variables: blood pressure (BP), cardiac output, total peripheral resistance (TPR), normalized pulse volume (NPV) as an index of alpha-adrenergic sympathetic activity to the finger arteriolar vessels, levels of cortisol and CgA during monotonous driving. The induced stress led to the expected decreases in NPV and increases in TPR and BP caused by peripherally related sympathetic acceleration. However, CgA levels were found to fall gradually in accordance with the gradual increase of subjective rating of stress (SRS) and significantly (p < 0.01) decreased over the period of the simulated monotonous driving. Our hypothesis for the gradual decrease of CgA levels during the simulated monotonous driving is as follows. CgA, catestatin and catecholamines are co-released into the extra-cellular environment. Peripheral sympathetic activity was accelerated by stress resulting from the simulated monotonous driving. Upon peripheral vessel constriction, an increase in TPR then increased BP which, in turn, activated catestatin. Consequently, secretion of CgA was blocked by the co-secreted catestatin from chromaffin granules. The results obtained strongly indicate that, although CgA has been reported as a possible marker of stress, CgA levels are not increased in the stressful situation of simulated monotonous driving.

Change in salivary levels of chromogranin A in children by hearing dental air turbine noise

Dental fear in children can be a psychological barrier for maintenance of oral health, thus it is important to objectively evaluate the responses to stress stimuli produced during dental treatments. In this study, we analyzed the stress responses of 15 preschool (3- to 6-year) and 22 school-aged (6- to 12-year) children to dental air turbine noise by measuring salivary levels of chromogranin A (CgA), a marker of psychological stress. Salivary CgA was significantly increased during exposure to the noise in the school-aged subjects, especially in those who had previously experienced tooth excavation, while there was no significant difference in CgA levels before and during noise exposure in those without such experience. In the preschool children, the noise did not induce increased secretion of CgA even in those with previous excavation experience. In addition, the levels of dental fear were assessed using a dental subscale of children's fear survey schedule (CFSS-DS), while reactions during dental care were scored according to the Frankl Behavior Rating scale. In the school-aged children, change in salivary CgA level was not correlated with CFSS-DS ( r = 0), while there was a weak negative correlation with Frankl score ( r = −10.202). Our results indicate that air turbine noise causes psychological stress in school-aged children, especially in those who have previously received dental treatments.

Knockdown of Pdcd4 results in induction of proprotein convertase 1/3 and potent secretion of chromogranin A and secretogranin II in a neuroendocrine cell line

Pdcd4 (programmed cell death 4) is up-regulated during apoptosis and seems to play an important role as a tumour suppressor. To gain further insights into its biological functions, we suppressed Pdcd4 expression in the neuroendocrine cell line Bon-1 via siRNA (small interfering RNA) technology. Using this cell line, we found that suppression of Pdcd4 resulted in an increased release of CgA (chromogranin A) and Sg II (secretogranin II), and was accompanied by an up-regulation of intracellular PC1 (proprotein convertase 1/3). The enhanced secretion of CgA and Sg II seemed to be mediated by an activation of protein kinase Akt via PI3K (phosphoinositide 3-kinase). In accordance with this, inhibition of PI3K activity and, thereby, reduced phosphorylation of Akt was shown to enhance Pdcd4 expression. Neither the PKC (protein kinase C) signal transduction cascade nor the MAPK (mitogen-activated protein kinase) pathway seemed to play a role in the regulation of CgA and Sg II secretion by Pdcd4. CgA is considered to be a marker for neuroendocrine tumours, and up-regulation of PC1 has been reported in various types of cancers. The repression of PC1 by Pdcd4 may represent a novel mechanism for the function of Pdcd4 as a tumour suppressor. Our results are of particular interest, as we observed that pioglitazone, an oral medication used in the treatment of Type 2 diabetes, decreased Pdcd4 levels, activated Akt, increased CgA and Sg II secretion and augmented PC1 protein in Bon-1 cells. Enhanced PC1 levels, leading to improved processing of proinsulin and proglucagon, may contribute to the benefits of pioglitazone therapy. The in vivo relevance of our findings was highlighted by data indicating elevated CgA amounts in the sera of patients treated with pioglitazone. This is the first study connecting Pdcd4 levels, secretion behaviour of neuroendocrine cells and regulation of PI3K activity.

Changes in salivary physiological stress markers induced by muscle stretching in patients with irritable bowel syndrome

BackgroundPsychophysiological processing has been reported to play a crucial role in irritable bowel syndrome (IBS) but there has been no report on modulation of the stress marker chromogranin A (CgA) resulting from muscle stretching. We hypothesized that abdominal muscle stretching as a passive operation would have a beneficial effect on a biochemical index of the activity of the sympathetic/adrenomedullary system (salivary CgA) and anxiety.MethodsFifteen control and eighteen untreated IBS subjects underwent experimental abdominal muscle stretching for 4 min. Subjects relaxed in a supine position with their knees fully flexed while their pelvic and trunk rotation was passively and slowly moved from 0 degrees of abdominal rotation to about 90 degrees or the point where the subject reported feeling discomfort.Changes in the Gastrointestinal Symptoms Rating Scale (GSRS), State Trait Anxiety Inventory (STAI), Self-rating Depression Scale (SDS), ordinate scale and salivary CgA levels were compared between controls and IBS subjects before and after stretching. A three-factor analysis of variance (ANOVA) with period (before vs. after) as the within-subject factor and group (IBS vs. Control), and sex (men vs. female) as the between-subject factors was carried out on salivary CgA.ResultsCgA showed significant interactions between period and groups (F[1, 31] = 4.89, p = 0.03), and between groups and sex (F[1, 31] = 4.73, p = 0.03). Interactions between period and sex of CgA secretion were not shown (F[1, 3] = 2.60, p = 0.12). At the baseline, salivary CgA in IBS subjects (36.7 ± 5.9 pmol/mg) was significantly higher than in controls (19.9 ± 5.5 pmol/mg, p < 0.05). After the stretching, salivary CgA significantly decreased in the IBS group (25.5 ± 4.5 pmol/mg), and this value did not differ from that in controls (18.6 ± 3.9 pmol/mg).ConclusionOur results suggest the possibility of improving IBS pathophysiology by passive abdominal muscle stretching as indicated by CgA, a biochemical index of the activity of the sympathetic/adrenomedullary system.

Dimenhydrinate effect on cerebral oxygen status and salivary chromogranin-A during cognitive tasks

To investigate the effects of dimenhydrinate on cerebral oxygen status (COS; cerebral oxygenated hemoglobin concentration changes) and salivary chromogranin-A (CgA) during a cognitive test battery, a double-blind, placebo-controlled, randomized, crossover protocol was used to examine the effect of 50 mg of dimenhydrinate or placebo in 12 subjects. This test battery includes tests of both reaction time and fundamental cognitive ability and was used in the assessment of pilots. Poor cognitive performance was observed in the subjects taking dimenhydrinate. We used two-channel near-infrared spectroscopy to investigate the effects of dimenhydrinate on the COS. With the one exception of shifting attention task in the left forehead, no significant difference was found between dimenhydrinate and placebo during the tasks of the test battery. Under placebo treatment, on the other hand, CgA levels were significantly elevated during cognitive testing when compared with baseline. However, CgA levels were not significantly elevated above baseline following dimenhydrinate. The present study is one of the first to demonstrate that the first-generation antihistamine drugs altered the responses of salivary CgA during cognitive tasks. The changes in salivary CgA secretion, as a result of dimenhydrinate administration, may serve as a sensitive biomarker of a psychological status such as a drug-induced sedation during the performance of a cognitive test battery. Further studies, however, are required to examine the usefulness of this sensitive biomarker in investigation of psychological agents during cognitive tasks.

Salivary chromogranin-A as a marker of psychological stress during a cognitive test battery in humans

The purpose of the present study was to evaluate the salivary Chromogranin-A (CgA) response to the psychological stress induced by a cognitive test battery. The subjects were 14 healthy volunteers administered the cognitive test battery CogScreen Aeromedical Edition (CogScreen-AE). CogScreen-AE is a test of reaction time and fundamental cognitive ability in the assessment of aeroplane pilots. The subjects were given five batteries of the test (1st ∼ 5th) on separate days with 2 week intervals. Saliva samples were collected at 20 min before the test (BASE), immediately before the test (PRE), in the middle of the test (MID), and 5 min after the test (POS) for each subject. The concentration of CgA was determined by enzyme-linked immunosorbent assay. ANOVA revealed a significant by time interaction (BASE, PRE, MID and POS) without a significant effect of battery interaction (1st, 2nd, 3rd, 4th and 5th). The CgA concentration increased at PRE (2.46 ± 0.24 pmol·mg protein− 1) from BASE (1.19 ± 0.10 pmol·mg protein− 1). CgA level remained increased in the MID (2.90 ± 0.26 pmol·mg protein− 1) and remained high in the POS samples (2.81 ± 0.23 pmol·mg protein− 1). Salivary CgA remained at basal levels during a control study over the same time course without exposure to CogScreen-AE. The changes in salivary CgA secretion as a result of exposure to a cognitive task may indicate psychological stress in humans.

Role of H+-ATPase-mediated Acidification in Sorting and Release of the Regulated Secretory Protein Chromogranin A: EVIDENCE FOR A VESICULOGENIC FUNCTION

The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells. Selective aggregation of secretory granule constituents in an acidic, bivalent cation-rich environment is considered to be a prerequisite for sorting to the regulated secretory pathway. The effect of selective vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 on the pH gradient along the secretory pathway was used here to study the role of acidification on the trafficking of the regulated secretory protein chromogranin A (CgA) in PC12 cells. Sorting of CgA was assessed by three-dimensional deconvolution microscopy, subcellular fractionation, and secretagogue-stimulated release, examining a series of full-length or truncated domains of human CgA (CgA-(1-115), CgA-(233-439)) fused to either green fluorescent protein or to a novel form of secreted embryonic alkaline phosphatase (EAP). We show that a full-length CgA/EAP chimera is sorted to chromaffin granules for exocytosis. Inhibition of V-ATPase by bafilomycin A1 markedly reduced the secretagogue-stimulated release of CgA-EAP by perturbing sorting of the chimera (at the trans-Golgi network or immature secretory granule) rather than the late steps of exocytosis. The effect of bafilomycin A1 on CgA secretion depends on a sorting determinant located within the amino terminus (CgA-(1-115)) but not the C-terminal region of the granin. Moreover, examination of chromaffin granule abundance in PC12 cells exposed to bafilomycin A1 reveals a substantial decrease in the number of dense-core vesicles. We propose that a V-ATPase-mediated pH gradient in the secretory pathway is an important factor for the formation of dense-core granules by regulating the ability of CgA to form aggregates, a crucial step that may underlie the granulogenic function of the protein.

Effect of short-term treatment with low dosages of the proton-pump inhibitor omeprazole on serum chromogranin A levels in man.

Measurement of chromogranin A (CgA) levels in blood can be used to monitor neuroendocrine tumors (NETs). CgA levels may also be elevated in several other endocrine and non-endocrine diseases. It is well known that drugs affecting acid gastric secretion can increase gastrin. Proton-pump inhibitors are extensively used but only a few data have been reported on their effects on CgA secretion. The aim of the study was to evaluate the short-term effect of low dosages of omeprazole (OM) on CgA levels and to sensitize endocrinologists to possible false positive values of CgA in order to prevent expensive diagnostic work-up in searching for NETs. Thirty-five female and nine male in-patients (18-81 years) were studied. Mild or severe hypertension in 20 patients needed therapy. Endocrine and metabolic diseases were diagnosed in the majority of patients. CgA levels were evaluated before and during OM therapy (10 mg/day, orally). Without OM therapy, CgA levels were 64+/-6 microg/l. Elevated baseline CgA levels were found in nine subjects. CgA levels were significantly related to age (P<0.001), creatinine levels (P=0.03) and the severity of hypertension (P=0.002). On short-term OM therapy (n=42; 18.8+/-2.4 days; range 5-90 days) a significant (P<0.001) increase in CgA (145+/-22 microg/l) from baseline (63+/-7 microg/l) levels was found. The average net CgA increase on short-term OM therapy was 93+/-20 microg/l. There was a significant correlation between baseline CgA levels and CgA increase on short-term OM therapy (P=0.004) but not between the increase in CgA and the duration of the therapy. An increase in CgA levels quickly follows the start of low dosages of OM. This release is more pronounced when the baseline CgA levels are already increased by slight renal insufficiency or severe hypertension. In this common clinical situation an intensive work-up for NETs is not justified before reassessment of CgA after the withdrawal of OM.

R-type Ca(2+)-channel activity is associated with chromogranin A secretion in human neuroendocrine tumor BON cells.

This electrophysiological study was undertaken to investigate the role of voltage-operated Ca(2+) channels (VOCCs) in cultivated human neuroendocrine tumor (NET) cells. Patch-clamp techniques, measurements of intracellular Ca(2+) ([Ca(2+)](i)), and secretion analysis were performed using cultured human NET BON cells. Ba(2+) inward currents through R-type channels (Ca(V)2.3) were measured and identified by SNX-482 (10 n M), a novel voltage-sensitive R-type Ca(2+) channel antagonist. In the presence of nifedipine (5 micro M), omega-Conotoxin GVIA (100 n M) and omega-Agatoxin IVA (20 n M), R-type channel currents were also detectable. Release of Ca(2+) from intracellular Ca(2+) stores by intracellular application of inositol-1,4,5-trisphosphate (InsP(3); 10 micro M) via the patch pipette during whole-cell configuration as well as induction of capacitative Ca(2+) entry (CCE), a passive maneuver to release Ca(2+) from intracellular Ca(2+) stores, led to an increase in [Ca(2+)](i). This effect could be reduced by SNX-482 (20 n M). In addition, SNX-482 (25 n M) also decreased chromogranin A (CgA) secretion, whereas omega-Conotoxin GVIA (500 n M) and nifedipine (5 micro M) failed to reduce CgA secretion. We conclude that these data reveal neuronal R-type channel activity (Ca(V)2.3), for the first time associated with CgA secretion in BON cells. Influx of Ca(2+) by activation of R-type channels may lead to an increase of intracellular Ca(2+), which stimulates CgA secretion. Thus, R-type channels could play an important role in certain clinical characteristics of NETs, such as the hypersecretion syndrome.

Regulation of endothelial cell shape and barrier function by chromogranin A.

We have found that chromogranin A (CgA), a protein released in circulation by neuroendocrine cells and neurons, prevents the vascular leakage induced by tumor necrosis factor (TNF) in a mouse model. Studies of the mechanism of action showed that CgA and its NH(2)-terminal fragments inhibit TNF-induced vascular permeability by preventing endothelial cytoskeleton rearrangements. We propose that neuronal/endocrine secretion of CgA could contribute to the regulation of endothelial barrier function and the protection of vessels against plasma leakage in inflammatory diseases.

Chromogranin 'A' in normal subjects, essential hypertensives and adrenalectomized patients.

Chromogranin A (CgA) is an acidic glycoprotein co-stored in vesicles and co-released with catecholamines. Although currently used as a humoral marker of endocrine tumours, several aspects of CgA secretion still need to be clarified in humans. Fifty-four controls, 83 essential hypertensive and six adrenalectomized patients were studied. In the controls and hypertensive patients, CgA and catecholamines were measured before (supine position) and after changes in posture (2' upright position), insulin-induced hypoglycaemia (0.15 IU/kg i.v.) and glucagon injection (1 mg i.v.). In addition, blood samples were taken in the morning (0800 h) and in the afternoon (1800 h), and every 5 h for 24 h. In the adrenalectomized patients, blood samples were obtained in the morning and in the afternoon. CgA was measured by an immunoradiometric assay, and noradrenaline and adrenaline by high-performance liquid chromatography. In controls, posture slightly increased plasma catecholamines without affecting CgA levels. Hypoglycaemia evoked a rise in noradrenaline (P < 0.04), adrenaline (P < 0.01) and CgA (79.6 +/- 11.8 vs. 46.1 +/- 10.1 microg/l, P < 0.03). Glucagon injection increased plasma adrenaline (P < 0.01) but not noradrenaline or CgA levels. At variance with blood pressure and catecholamines, CgA increased significantly in the afternoon (51.1 +/- 4.0 vs. 45.0 +/- 3.9 microg/l, P < 0.05); it also had a circadian rhythm, with peak values during the night (at 2300 h, 65.4 +/- 9.0 microg/l) and a nadir in the morning (at 0800 h, 43.1 +/- 6.6 microg/l). In hypertensives, basal and stimulated CgA levels as well as diurnal/circadian variations of this peptide were similar to those in normal subjects. In adrenalectomized patients plasma CgA in the morning (34.3 +/- 6.5 microg/l) was lower (P < 0.03) than in all controls and hypertensives studied, but also showed an afternoon increment (46.4 +/- 6.6 microg/l, P < 0.003). No correlation was found between CgA and catecholamines or blood pressure in all subjects or in the subgroups. In normal humans, chromogranin A and catecholamines are not always co-secreted, and co-secretion occurs only for marked exocytotic adrenergic stimuli, such as hypoglycaemic stress. In addition, chromogranin A has a circadian rhythm unrelated to plasma catecholamines. Basal plasma concentrations and the secretory pattern of chromogranin A in hypertensives do not differ from the findings in controls. Finally, the adrenal glands contribute partially to circulating chromogranin A and are not involved in the circadian rhythm of this peptide in humans.

Polarized Secretion of the Regulated Secretory Protein Chromogranin A

Bovine chromogranin A (CgA), together with secreted alkaline phosphatase (SEAP) as an external control for apical secretion were expressed in MDCK cells to test if CgA contains sorting signals for polarized secretion. CgA, SEAP, and the endogenous apical marker GP80 were secreted 75–80% apically. Basolateral secretion of SEAP was inhibited 40% by ammonium chloride. Sulfate labeling and digestion with chondroitinase ABC revealed a 120 kDa proteoglycan-CgA and 75 kDa CgA. Inhibition of proteoglycan synthesis did not affect apical secretion of CgA. As CgA is not N-glycosylated, we used tunicamycin to test if cellular N-glycosylation is required for apical sorting. Tunicamycin reversed the polarity of secretion of CgA to the basolateral side. These results suggest that CgA contains dominant apical and recessive basolateral sorting information.

Phosphorylation of Chromogranin A and Catecholamine Secretion Stimulated by Elevation of Intracellular Ca2+ in Cultured Bovine Adrenal Medullary Cells

We have recently isolated a new endogenous substrate of 70 kDa for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from bovine adrenal medullary cells (Yanagihara, N., Toyohira, Y., Yamamoto, H., Ohta, Y., Tsutsui, M., Miyamoto, E., and Izumi, F. (1994) Mol. Pharmacol. 46, 423-430). Here we report the sequence analysis of the 70-kDa protein and examine its phosphorylation by various protein kinases in vitro and by depolarization of the cultured cells. Protein sequencing and immunoblotting revealed that the 70-kDa protein is chromogranin A (CgA) or a closely related protein. Partially purified CgA was phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C as well as CaM kinase II. Tryptic phosphopeptide mapping patterns of CgA differed among these protein kinases. In 32P-labeled bovine adrenal medullary cells, 56 mM K+ increased the phosphorylation of CgA and catecholamine secretion in similar time- and concentration-dependent manners, both of which were inhibited by 20 mM MgSO4, an inhibitor of voltage-dependent Ca2+ channels. These findings suggest that CgA serves as a substrate for several multifunctional protein kinases and that the elevation of the intracellular Ca2+ stimulates the phosphorylation of CgA associated with catecholamine secretion in cultured adrenal medullary cells.

Downregulation of Prohormone Convertase-1 by a Phorbol Ester

Acute TPA treatment (1h, 100nM) of a human pancreatic carcinoid cell line (BON) depletes cell contents of chromogranin A (CGA) and pancreastatin (PST), a peptide derived posttranslationally from CGA. Despite removal of TPA, BON cells continue to release CGA in an unregulated fashion whereas PST secretion is reduced substantially. TPA treatment also reduced prohormone convertase-1 (PC-1) protein and increased PC-1 mRNA levels. Together, these findings indicate that the TPA-induced switch from a regulated to unregulated pattern of CGA secretion is accompanied by a decrease in the processing of CGA to PST and a decrease in the active form of a processing enzyme potentially involved in processing CGA to a smaller peptide, PST.

Production and secretion of chromogranin A and pancreastatin by the human pancreatic carcinoma cell line QGP-1N on stimulation with carbachol

Chromogranin A (CGA) is thought to be a precursor of pancreastatin (PST). Carbachol (Cch) stimulated the secretion of CGA and PST from QGP-1N cells derived from a human pancreatic islet cell tumor. Atropine inhibited the secretion of both. Sodium fluoride, phorbol ester, and calcium ionophore also stimulated the secretion of both. Cch (10 −5 M) stimulated inositol 1,4,5-trisphosphate production in QGP-1N cells. Stimulation with Cch increased the total amount of PST in the cells and the medium 1.7-fold and decreased the amount of CGA in the cells and medium. QGP-1N cells were labelled with [ 35S]methionine, and then CGA and PST in the cells and medium were immunoprecipitated with specific antisera, and separated by electrophoresis in polyacrylamide gel. Stimulation with Cch resulted in an increase in the intensity of PST-immunoreactive bands and a decrease in those of CGA-immunoreactive bands. Cch did not increase the cellular level of CGA messenger RNA. These results suggested that (1) the secretion of CGA and PST from QGP-1N cells is regulated mainly through muscarinic receptors coupled with activation of polyphosphoinositide breakdown by a G protein, with intracellular calcium ion and protein kinase C playing a role in the stimulus-secretion coupling and that (2) Cch may induce the secretion of PST and CGA and processing from CGA to PST.

Pancreastatin, a chromogranin A-derived peptide, inhibits transcription of the parathyroid hormone and chromogranin A genes and decreases the stability of the respective messenger ribonucleic acids in parathyroid cells in culture.

Pancreastatin is a carboxyl-terminal amidated peptide derived from chromogranin A (CgA) corresponding to porcine CgA residues 240-288 (CgA240-288). CgA, in turn, is an acidic glycoprotein of about 450 amino acids that is costored and cosecreted with PTH in the parathyroid gland and with native hormones of many endocrine tissues. Among its effects on various endocrine glands, pancreastatin strongly inhibits the secretion of PTH and CgA by the parathyroid. The present report evaluates the action of pancreastatin on the regulation of cellular levels of PTH messenger RNA (mRNA) and CgA mRNA. After a 6-day preculture, dispersed porcine parathyroid cells were cultured for up to 48 h with or without pancreastatin in medium at stimulatory (0.5 mM) or inhibitory (3.0 mM) concentrations of Ca2+. Secretion of PTH and CgA in the absence of pancreastatin was 4- to 10-fold greater at 0.5 mM Ca2+ than at 3.0 mM Ca2+ as were the corresponding cellular levels of PTH and CgA mRNAs. Pancreastatin (10(-9)-10(-7) M) inhibited the secretion of PTH and CgA at 0.5 mM Ca2+ in a dose-dependent fashion by 93% and 75%, respectively, and decreased the cellular levels of PTH and CgA mRNAs 80% and 87%, respectively. Whereas pancreastatin inhibited the secretion of PTH and CgA by the fourth hour and beyond, changes in PTH and CgA mRNAs were not observed until 12 h. These decreases in mRNA were associated with a two-thirds reduction in the rates of transcription of the PTH and CgA genes. Additionally, the half-life of PTH mRNA decreased to 20 h from 38 h and that for CgA mRNA declined to 18 h from 33 h. It remains to be determined if the changes in transcription of the PTH and CgA genes and stability of their respective mRNAs are direct effects of pancreastatin or secondary actions due to the inhibition of secretion.

Parastatin (porcine chromogranin A347-419), a novel chromogranin A-derived peptide, inhibits parathyroid cell secretion.

Chromogranin A (CgA), previously referred to as secretory protein-I, is a 50-kilodalton protein present in secretory granules of many endocrine and neuroendocrine cells. In the parathyroid it is present and cosecreted with PTH in response to hypocalcemia. CgA appears to be a precursor of bioactive peptides including pancreastatin, beta-granin, vasostatin, and chromostatin. The presence of several highly conserved pairs of basic amino acids, putative cleavage sites, in the CgA molecule suggests that other yet unidentified bioactive peptides might exist within the molecule. We tested this speculation by subjecting porcine parathyroid CgA to digestion by endoproteinase Lys-C. Resulting CgA-derived peptides were isolated by reverse-phase C18 HPLC and tested for their ability to affect low-Ca2+ stimulated secretion by porcine parathyroid cells. We characterized one peptide, which we named parastatin, that inhibited secretion of both PTH and CgA in a dose-dependent fashion over the range of 0.2-0.6 microM. Parastatin migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mol wt of 11,000. Edman degradation yielded the sequence L-S-F-R-A-P-A-Y-G-F-R-G-P-G-L corresponding to residues 347-361 of porcine CgA. Amino acid analysis of endoproteinase Lys-C and endoproteinase Asp-N-generated fragments indicated that parastatin corresponds to residues 347-419 of CgA. A synthetic NH2-terminal fragment of rat parastatin corresponding to residues 1-19 was an inhibitory as intact porcine parastatin on parathyroid gland secretion. These results extend the concept that CgA is a precursor of biologically active peptides.

Dexamethasone and calcium interact in the regulation of parathormone and chromogranin-A secretion and messenger ribonucleic acid levels in parathyroid cells.

We investigated the interaction of dexamethasone and Ca2+ on the secretion of PTH and chromogranin-A (CgA) by porcine parathyroid cells in culture and on the cellular levels of PTH and CgA mRNA. Freshly dispersed parathyroid cells were cultured for 6 days in Ham's F-12 medium containing 10% fetal calf serum and 1 mM Ca2+. The experiments were initiated at 0.5 or 3.0 mM Ca2+ in 0.5% serum with or without dexamethasone for an additional 24 or 48 h. Secretion of PTH and CgA was 50-60% less, and the respective mRNAs levels were about 75% lower at 3.0 than at 0.5 mM Ca2+. Dexamethasone (10(-9)-10(-7) M) stimulated the secretion of PTH in a dose-dependent fashion about 2-fold at both 3.0 and 0.5 mM Ca2+ and increased the PTH mRNA level 15-fold at the high and 3-fold at the low Ca2+ concentration. In contrast, the direction of action of dexamethasone on the secretion of CgA and CgA mRNA was dependent upon the Ca2+ concentration. At 3.0 mM Ca2+, dexamethasone increased CgA secretion and mRNA levels about 2- and 8-fold, respectively. In contrast, at 0.5 mM Ca2+ dexamethasone decreased CgA secretion to one half and its mRNA to one tenth that cells without the glucocorticoid. The stimulatory action of dexamethasone on PTH and CgA mRNA could be noted in 12 h or less and continued through 24-48 h. These results demonstrate a decoupling of PTH and CgA secretion and gene regulation. Our data accord with a role for CgA as an autocrine regulator of parathyroid gland secretion.

Dexamethasone and calcium interact in the regulation of parathormone and chromogranin-A secretion and messenger ribonucleic acid levels in parathyroid cells

We investigated the interaction of dexamethasone and Ca2+ on the secretion of PTH and chromogranin-A (CgA) by porcine parathyroid cells in culture and on the cellular levels of PTH and CgA mRNA. Freshly dispersed parathyroid cells were cultured for 6 days in Ham's F-12 medium containing 10% fetal calf serum and 1 mM Ca2+. The experiments were initiated at 0.5 or 3.0 mM Ca2+ in 0.5% serum with or without dexamethasone for an additional 24 or 48 h. Secretion of PTH and CgA was 50-60% less, and the respective mRNAs levels were about 75% lower at 3.0 than at 0.5 mM Ca2+. Dexamethasone (10(-9)-10(-7) M) stimulated the secretion of PTH in a dose-dependent fashion about 2-fold at both 3.0 and 0.5 mM Ca2+ and increased the PTH mRNA level 15-fold at the high and 3-fold at the low Ca2+ concentration. In contrast, the direction of action of dexamethasone on the secretion of CgA and CgA mRNA was dependent upon the Ca2+ concentration. At 3.0 mM Ca2+, dexamethasone increased CgA secretion and mRNA levels about 2- and 8-fold, respectively. In contrast, at 0.5 mM Ca2+ dexamethasone decreased CgA secretion to one half and its mRNA to one tenth that cells without the glucocorticoid. The stimulatory action of dexamethasone on PTH and CgA mRNA could be noted in 12 h or less and continued through 24-48 h. These results demonstrate a decoupling of PTH and CgA secretion and gene regulation. Our data accord with a role for CgA as an autocrine regulator of parathyroid gland secretion.

Chromogranin A does not mediate glucocorticoid inhibition of adrenocorticotropin secretion.

It has recently been proposed that chromogranin A (CgA), a 50-kilodalton acidic glycoprotein that is costored and cosecreted with hormones and neurotransmitters in a variety of tissues, mediates glucocorticoid inhibition of ACTH secretion from AtT20/D16v mouse anterior pituitary corticotroph tumor cells by an undefined autocrine mechanism. We used AtT20/D16v cells, RIAs for murine CgA and ACTH, complementary DNA probes for CgA and POMC, the precursor of ACTH, antiserum that reacts with murine CgA, highly purified bovine CgA, and synthetic rat and porcine pancreastatin, a bioactive cleavage product of CgA in some systems, to study the kinetics of the effect of glucocorticoids on CgA and ACTH synthesis and secretion and of CgA's subsequent effects on ACTH secretion. Exposure to 100 nM dexamethasone (DEX) did not alter the size of CgA or POMC messenger RNA (mRNA) transcripts but increased cell CgA mRNA content 42% by 3 h and 192% by 48 h. DEX decreased cell POMC mRNA content 22% by 6 h and 57% by 48 h. These divergent effects of DEX on steady state mRNA levels were accompanied by similar divergent effects on the production of CgA and ACTH protein. Thirty-minute exposure to 10 nM ovine CRF increased CgA and ACTH release to 300% and 360% of basal levels, respectively. One-hour DEX pretreatment inhibited CRF-stimulated CgA and ACTH release 58% and 49% at 30 min and 67% and 66% at 60 min, respectively. There was a positive correlation between CgA and ACTH release under all conditions at both times (r = 0.976 and 0.964, respectively, P < 0.001), consistent with costorage and cosecretion of the two proteins. The ratio of secreted ACTH to CgA decreased progressively with DEX treatment. Purified bovine CgA (100 nM) had little or no effect on basal or CRF-stimulated ACTH secretion from AtT20/D16v cells, 100 nM synthetic pancreastatin had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells or dispersed normal male rat anterior pituitary cells, and anti-CgA sera had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells. These results indicate: 1) that DEX stimulates CgA synthesis, whereas it inhibits POMC synthesis; 2) that CgA and ACTH are cosecreted; 3) that DEX increases CgA secretion relative to ACTH secretion, but decreases the absolute secretion of both proteins; and 4) that neither CgA nor its proteolytic product, pancreastatin, inhibits ACTH secretion. Thus, CgA does not mediate the inhibitory effect of DEX on ACTH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)