Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells.
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Reelin regulates telencephalic and cerebellar lamination during mammalian development and is expressed in several structures of the adult brain; however, only traces of reelin were believed to be in peripheral tissues. Because reelin structurally resembles extracellular matrix proteins, and because many of these proteins are expressed in blood, we hypothesized that reelin also might be detectable in the circulation. Reelin (420 kDa) and two reelin-like immunoreactive bands (310 and 160 kDa) are expressed in serum and platelet-poor plasma of rats, mice, and humans, but these three bands were not detectable in serum of homozygous reeler (rl/rl) mice. Reelin plasma levels in heterozygous (rl/+) mice were half of those in wild-type littermates. Western blotting and immunocytochemistry using antireelin mAbs indicated that reelin-like immunoreactivity was expressed in a subset of chromaffin cells within the rat adrenal medulla and in a subset of cells coexpressing alpha-melanocyte-stimulating hormone within the pituitary pars intermedia. However, surgical removal of adrenal or pituitary failed to decrease the amount of reelin (420-kDa band) expressed in serum. Adult liver expressed one-third of the reelin mRNA concentration expressed in adult mouse cerebral cortex. Full-length reelin protein was detectable in liver extracts in situ; acutely isolated liver cells also secreted full-length reelin in vitro. Liver appears to be a prime candidate to produce and maintain the circulating reelin pool. It now becomes relevant to ask whether circulating reelin has a physiologic role on one or more peripheral target tissues. (+info)
Leukocyte common-antigen-related tyrosine phosphatase receptor: altered expression of mRNA and protein in the New England Deaconess Hospital rat line exhibiting spontaneous pheochromocytoma.
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Regulation of cell proliferation by protein tyrosine phosphatases (PTPs) suggests that PTPs are important tumor suppressor genes. The gene encoding the leukocyte common-antigen-related (LAR) PTP receptor maps to chromosome 1p32-33, a region in which loss of heterozygosity is associated with human pheochromocytoma and other neuroectodermal tumors. The rat pheochromocytoma PC12 cell line was originally derived from the transplantable P259 tumor originating from the New England Deaconess Hospital (NEDH) line of Wistar inbred rats. Compared with their Wistar counterparts, 1-2-year-old NEDH rats exhibit a high incidence of spontaneous pheochromocytomas. This study investigates whether levels of LAR transcripts and protein are altered in NEDH adrenal tissue prior to tumor onset. In addition, alternative splicing of an LAR extracellular domain [LAR alternatively spliced element-c (LASE-c)], regulating LAR interaction with extracellular matrix components, was examined. These changes in LAR expression and alternative splicing were hypothesized to be more pronounced in tumor tissue and PC12 cells. Northern blot analysis demonstrated the presence of the approximately 5 kb LAR transcript in all cell lines examined, except PC12. In adrenal medulla tissue harvested from 2-3-month-old rats, LAR approximately 8 and approximately 5 kb transcript expression was decreased in NEDH compared with Wistar samples. RT-PCR demonstrated increased splicing of the LASE-c 27 bp alternatively spliced insert in the LAR extracellular domain in NEDH adrenal medulla tissue. Even greater LASE-c splicing was detected in adrenal medulla tumor tissue derived from 12-month-old NEDH rats and in PC12 cells. Western blot analysis demonstrated decreased levels of LAR protein and increased levels of LASE-c containing LAR protein isoforms in NEDH adrenal medulla tissue. These studies demonstrate that patterns of altered LAR expression present in PC12 cells and in pheochromocytoma tumor tissue are also present in adrenal tissue predisposed to a high incidence of spontaneous pheochromocytoma. (+info)
Comparison of cysteine string protein (Csp) and mutant alpha-SNAP overexpression reveals a role for csp in late steps of membrane fusion in dense-core granule exocytosis in adrenal chromaffin cells.
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Assembly of the SNARE complex and its disassembly caused by the action of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) and NSF is crucial for the maintenance of vesicular traffic, including fusion of regulated exocytotic vesicles. Various other proteins may also have important roles in the processes leading to membrane fusion via interaction with the SNARE proteins, including the secretory vesicle cysteine string protein (Csp). Here we have examined the effect of overexpression of a dominant negative alpha-SNAP mutant or Csp on exocytosis of dense-core granules in single chromaffin cells monitored using amperometry to detect released catecholamine. Exocytosis of trans-Golgi network (TGN)-derived dense-core granules was substantially inhibited by expression of alpha-SNAP(L294A). The amplitude and characteristics of the individual release events were unaffected by expression of alpha-SNAP(L294A), consistent with an essential role for alpha-SNAP in early steps of priming but not in the fusion process. In contrast, Csp overexpression, which also inhibited the extent of exocytosis, also modified the kinetics of the individual release events seen as an increase in the rise time and a broadening of the residual amperometric spikes in Csp-transfected cells. These results suggest that unlike alpha-SNAP, Csp plays a key role in the protein interactions close to the fusion process or fusion pore opening during Ca(2+)-regulated exocytosis. (+info)
Subcellualr distribution of protein carboxymethylase and its endogenous substrates in the adrenal medulla: possible role in excitation-secretion coupling.
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Protein carboxymethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24) transfers a methyl group from S-adenoxyl-L-methionine to carboxyl side chains of proteins to form labile protein-methyl esters which, thus, neutralize negative charges. This enzyme was examined for its possible participation in excitation-secretion coupling in the adrenal medulla. Protein carboxymethylase has a specific activity several times higher in the adrenal medulla than in the adrenal cortex; also, the medulla has a higher concentration of methyl-acceptor proteins. In the adrenal medulla, 97% of the enzyme was localized in the cytosol. Of the various subcellular fractions of the medulla, the catecholamine-containing chromaffin vesicles had the highest concentrations of substrat(s) for protein carboxymethylase. Carboxymethylation of proteins in intact chromaffin vesicles results in stripping of methylated protein(s) from the membranes. Thus, protein carboxymethylase appears to be involved in the neutralization of charges on the surface of chromaffin vesicles and in the release of surface proteins; both phenomena are likely to be required for exocytosis. (+info)
A forward-facilitating influence of cortisol on catecholamines assessed during the work of garbage collectors.
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OBJECTIVES: In this study, the hypothesis was tested of whether part of the hypothalamic-pituitary-adrenal system can have a facilitating influence on mean adrenomedullary reactivity during a workday. METHODS: Data of a field study on 115 male garbage collectors were used for this purpose. Catecholamines and cortisol were assessed by urinary sampling during a workday and during a day off. The within-subjects baseline levels of the catecholamines, personal factors, physical work load, and work characteristics were controlled for in the analyses. RESULTS: The results showed that there is reason to assume a "forward-facilitating influence" between the aforementioned systems. The morning cortisol excretion rate explained a reasonable amount of the mean catecholamine excretion rates during a workday. The morning cortisol level explained more variance in the adrenaline than in the noradrenaline values, and for both catecholamines the influence of cortisol was more pronounced than the influence of psychosocial stress factors like autonomy and job demands. CONCLUSIONS: The morning level of cortisol proved to be a more powerful predictor of the reactivity of adrenaline than that of noradrenaline during work. The association between the pituitary adrenocortical system and the adrenomedullary system could therefore be a "forward-facilitating influence". It is recommended that future research should focus on cortisol as the predictor of neuroendocrine reactivity and on determining whether this predictive power is expandable to work-induced health complaints. (+info)
Desensitisation of calcitonin gene-related peptide responsiveness but not adrenomedullin responsiveness in vascular smooth muscle cells.
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Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) are distantly related peptides. Both act through G protein-coupled receptors on vascular smooth muscle cells to increase intracellular cAMP concentrations, causing vasorelaxation. Recent evidence suggests that both peptides bind to a common heptahelical receptor, with specificity for each peptide being determined by a receptor activity modifying protein (RAMP). This hypothesis predicts that each peptide should desensitise the cellular response to subsequent stimulation by the other. We have studied the patterns of desensitisation of ADM/CGRP receptors in rat aortic vascular smooth muscle cells. Cells were incubated for 20 min in either serum free medium (SFM), alone (control) or in SFM containing vasoactive agonist (e.g. ADM 10(-8) M, CGRP 10(-7) M, angiotensin II 10(-9) M or isoproterenol 10(-6) M). Cells were then washed and incubated for a further 20 min in SFM containing a second agonist and 1 mM isobutyryl methyl xanthine. Cells were harvested and assayed for cAMP. Pre-exposure of cells to CGRP, isoproterenol, angiotensin II or ADM, decreased cAMP generation in response to subsequent stimulation with CGRP by 84% (+/-5), 66% (+/-18), 45% (+/-5) and 60% (+/-10) respectively (mean+/-s.d.). Pre-incubation of cells with 100 nM H-89, a protein kinase A (PKA) inhibitor, abolished the desensitisation of CGRP by itself, implying that this desensitisation was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with ADM by pre-exposure to ADM and all other agonists tested. Identical results were seen with or without PKA inhibition by H-89. These results indicate that the ADM receptor does not desensitise over this time period in RAVSMCs, in contrast to the CGRP receptor, which is desensitised by pre-exposure to CGRP and other vaso-active agonists. These data also suggest that ADM and CGRP act through separate receptors in these cells. (+info)
Central injection of nitric oxide synthase inhibitors increases peripheral interleukin-6 and serum amyloid A: involvement of adrenaline from adrenal medulla.
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1. Accumulating evidence suggests that plasma levels of interleukin-6 (IL-6), a major cytokine stimulating the synthesis of acute phase proteins, are intimately regulated by the central nervous system (CNS). 2. In the present study, effects of intracerebroventricular (i.c. v) injection of N(G)-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole, nitric oxide synthase (NOS) inhibitors, on plasma IL-6 levels and peripheral IL-6 mRNA expression were examined in mice. 3. L-NAME (0.1 - 2 microg per mouse i.c.v.) and 7-nitroindazole (0.2 - 2 microg per mouse i.c.v.) induced a dose-dependent increase in plasma IL-6 levels and a subsequent increase in circulating serum amyloid A, a liver acute-phase protein. In contrast, an intraperitoneal (i.p.) injection of L-NAME up to the dose of 25 microg per mouse had no effect. 4. Pretreatment with yohimbine (alpha(2)-adrenergic antagonist; 1 mg kg(-1) i.p.), or ICI-118,551 (beta(2)-adrenergic antagonist; 2 mg kg(-1) i.p.), but not with prazosin (alpha(1)-adrenergic antagonist; 1 mg kg(-1) i.p.), nor betaxolol (beta(1)-adrenergic antagonist; 2 mg kg(-1) i.p.), significantly inhibited the central L-NAME-induced plasma IL-6 levels. 5. I.c.v. (50 microg per mouse) or i.p. (100 mg kg(-1)) pretreatment with 6-hydroxydopamine had no effect on central L-NAME-induced plasma IL-6 levels. However, intrathecal (i.t.) pretreatment with 6-hydroxydopamine (20 microg per mouse) markedly inhibited central L-NAME-induced plasma IL-6 levels. Both yohimbine (1.5 microg per mouse i.t.) and ICI-118,551 (1.5 microg per mouse i. t.) were effective in inhibition of central L-NAME-induced plasma IL-6 levels. 6. There was an elevation of base-line plasma IL-6 levels in adrenalectomized animals. The adrenalectomy-enhanced levels were not further increased by central L-NAME. 7. L-NAME (2 microg per mouse i.c.v.) induced an increase in IL-6 mRNA expression in liver, spleen, and lymph node. 8. These results suggest that NOS activity in the brain tonically down-regulates peripheral IL-6 by inhibiting adrenaline release from the adrenal medulla. (+info)
Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules.
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The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide. (+info)