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S K Mahata

Publications and source records attributed to S K Mahata.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a beta-strand/loop/beta-strand structure secured by hydrophobic interactions and predictive of activity.

A novel fragment of chromogranin A, known as 'catestatin' (bovine chromogranin A344-364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide's structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin's primary structure bore significant (29-35.5% identity, general alignment score 44-57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, IPKM, and 2IG2. Each of these sequences exists in nature as a beta-strand/loop/beta-strand structure, stabilized by hydrophobic interactions between the beta-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic beta-strand. Circular dichroism in the far ultraviolet showed substantial (63%) beta-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was 'docked' with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the beta and delta subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist.

Animals↗
PubMed1998

Secretoneurin and chemoattractant receptor interactions.

Secretoneurin (SN) is a 33-amino acid peptide derived from secretogranin II (chromogranin C) which induces chemotaxis of monocytes but not neutrophils. In this study, we found that SN interacted with specific cell surface binding sites on human monocytes. The chemoattractants MCP-1, MCP-2 or fMLP could not compete for SN binding sites suggesting SN may bind to a novel chemotactic receptor. Additional studies showed that neither SN nor MCP-2 induced a rise in cytosolic Ca2+, and chemotaxis to SN was inhibited by cholera toxin (CT) and pertussis toxin (PT). Chemotactic desensitization studies demonstrated that fMLP, MCP-1, SN, and MCP-2 could all desensitize monocytes to subsequent SN stimulation. Our results indicate that SN binds to a cell surface receptor expressed on monocytes and activates signaling pathways which are sensitive to CT and PT.

Binding Sites↗
PubMed1998

Peptidergic activation of transcription and secretion in chromaffin cells. Cis and trans signaling determinants of pituitary adenylyl cyclase-activating polypeptide (PACAP).

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. Chromogranin A is the major soluble core component in secretory vesicles. Since chromogranin A is secreted along with catecholamines, we asked whether PACAP regulates expression of the chromogranin A gene in PC12 rat chromaffin cells, so as to resynthesize the just-secreted protein, and whether such biosynthetic regulation is coupled mechanistically to catecholamine secretion. PACAP activated the endogenous chromogranin A gene by four- to fivefold. Proportional results (seven- to eightfold activation) were obtained with a transfected 1,200-bp mouse chromogranin A promoter/luciferase reporter construct. A series of chromogranin A promoter 5' deletion mutant/luciferase reporter constructs narrowed down the PACAP response element to a proximal region containing the cAMP response element (CRE box), at (-71 bp)5'-TGACGTAA-3'(-64 bp). Site-directed point mutations of the CRE site suppressed PACAP-induced trans-activation of the promoter. Thus, the proximal CRE box is entirely necessary for the chromogranin A promoter response to PACAP. Transfer of the CRE box to a neutral, heterologous promoter also conferred activation by PACAP, suggesting that the CRE domain is also sufficient to mediate the transcriptional response to PACAP. Expression of a dominant-negative mutant (KCREB) of the CRE-binding factor CREB markedly diminished trans-activation of the chromogranin A promoter by PACAP. Cotransfection of expression plasmids encoding the protein kinase A inhibitor, or an inactive protein kinase A (PKA) catalytic beta subunit, inhibited both forskolin and PACAP activation of chromogranin A transcription, revealing that PACAP-induced trans-activation is highly dependent on PKA. By contrast, inhibition of protein kinase C (by chronic exposure to phorbol ester) had no effect on transcriptional activation by PACAP. The potent PACAP/vasoactive intestinal peptide (VIP) type I receptor antagonist PACAP6-38 impaired both chromogranin A transcription or catecholamine secretion triggered by PACAP38, while the PACAP/VIP type II receptor antagonist (p-Chloro-D-Phe6, Leu17)-VIP had little or no ability to antagonize the PACAP38 effect. The agonist VIP was approximately 100- to 1,000-fold less potent than PACAP in stimulating either secretion or transcription. Thus, PACAP-evoked chromogranin A transcription and catecholamine secretion are likely mediated by the PACAP/VIP type I receptor isoform. Although the calcium channel antagonists Zn2+ (100 microM), nifedipine (10 microM), or ruthenium red (10 microM), or the cytosolic calcium chelator BAPTA-AM (50 microM) each strongly impaired PACAP-induced secretion, transcriptional activation of chromogranin A remained unaltered. Therefore, we propose that PACAP signals to chromogranin A transcription through the CRE in cis, and through PKA and CREB in trans. By contrast, a pathway involving cytosolic calcium entry through L-type voltage-dependent channels is required for PACAP to evoke catecholamine secretion.

Animals↗
PubMed1998

Mechanism of cardiovascular actions of the chromogranin A fragment catestatin in vivo.

Catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL), reduces catecholamine secretion from chromaffin cells in vitro. We investigated the effects of this peptide on catecholamine release and blood pressure in vivo. Intravenous catestatin reduced pressor responses to activation of sympathetic outflow by electrical stimulation in rats, and the catestatin effect persisted even after adrenergic (alpha plus beta) blockade. Catestatin did not alter plasma norepinephrine levels, but increased plasma epinephrine 11-fold. Catestatin also blunted pressor responses to exogenous neuropeptide Y agonists. A control peptide (chromogranin A(141-160)) did not alter pressor or catecholamine responses to electrical stimulation. Pretreatment with a histamine H1 receptor antagonist blocked both the vasodepressor response to catestatin and the elevation in plasma epinephrine. Catestatin elevated endogenous circulating histamine 21-fold, and exogenous histamine mimicked both the epinephrine elevation and the vasodepressor actions of catestatin. We conclude that catestatin is a potent vasodilator in vivo whose actions appear to be mediated, at least in part, by histamine release and action at H1 receptors.

Adrenergic Antagonists↗
PubMed1998

A crucial role for the mitogen-activated protein kinase pathway in nicotinic cholinergic signaling to secretory protein transcription in pheochromocytoma cells.

The mitogen-activated protein kinase (MAPK) pathway plays a pivotal role in intracellular signaling, and this cascade may impinge on cAMP response elements (CREs) of target genes. Both the MAPK pathway and chromogranin A expression may be activated by cytosolic calcium influx, and calcium-dependent signals map onto the chromogranin A promoter proximal CRE. We therefore probed the role of the MAPK pathway in chromogranin A biosynthesis after secretory stimulation of PC12 pheochromocytoma cells by the nicotinic cholinergic pathway, the physiological secretory trigger. Chemical inhibition of either MAPK or MAPK kinase blocked the response of a transfected chromogranin A promoter to nicotine or protein kinase C activation [by phorbol-12-myristate-13-acetate (PMA)], although nicotine-evoked catecholamine secretion was unaffected. Activation of the MAP kinase cascade (Ras, Raf, MAPK, or CREB kinase) by cotransfection of pathway components stimulated the chromogranin A promoter. Cotransfection of MAPK pathway dominant negative mutants (for Raf, MAPK, or CREB kinase) blocked nicotinic or PMA activation of chromogranin A, although a dominant negative Ras mutant was without effect. MAPK pathway enzymatic activity was stimulated by both nicotine and PMA. Point mutations of the chromogranin A CRE suggested that this element was necessary in cis for stimulation by nicotine, PMA, or chemical activation of the MAPK pathway. Transfer of the CRE to a heterologous promoter conferred inducibility by not only nicotine or cAMP but also MAPK activation. Expression of the CREB antagonist KCREB blocked the response of the chromogranin A promoter to nicotine, cAMP, or MAPK pathway activation by either chemical stimulation or cotransfection of active cascade components. Chromogranin A mRNA responded to MAPK pathway manipulation in a fashion similar to the transfected chromogranin A promoter, in both direction and magnitude. We conclude that the MAPK pathway is a necessary intermediate in signaling from the nicotinic receptor to secretory protein transcription, although not to catecholamine secretion. In trans, this response seems to involve the following signal cascade: protein kinase C --> Raf --> MAPK kinase --> MAPK --> CREB kinase --> CREB. In cis, activation by the cascade maps onto the chromogranin A promoter proximal CRE, which is both necessary and sufficient to confer the response.

Adrenal Gland Neoplasms↗
PubMed1998

Proadrenomedullin N-terminal 20 peptide: minimal active region to regulate nicotinic receptors.

Proadrenomedullin N-terminal 20 peptide (PAMP-[1-20]; ARLDVASEFRKKWNKWALSR-amide) is a potent hypotensive and catecholamine release-inhibitory peptide released from chromaffin cells. We studied the mechanism of PAMP action and how its function is linked to structure. We tested human PAMP-[1-20] on catecholamine secretion in PC12 pheochromocytoma cells and found it to be a potent, dose-dependent (IC50 approximately 350 nmol/L) secretory inhibitor. Inhibition was specific for nicotinic cholinergic stimulation since PAMP-[1-20] failed to inhibit release by agents that bypass the nicotinic receptor. Nicotinic cationic (22Na+,45Ca2+) signal transduction was disrupted by this peptide, and potencies for inhibition of 22Na+ uptake and catecholamine secretion were comparable. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. N- and C-terminal PAMP truncation peptides indicated a role for the C-terminal amide and refined the minimal active region to the C-terminal 8 amino acids (WNKWALSR-amide), a region likely to be alpha-helical. PAMP also blocked (EC50 approximately 270 nmol/L) nicotinic cholinergic agonist desensitization of catecholamine release, as well as desensitization of nicotinic signal transduction (22Na+ uptake). Thus, PAMP may exert both inhibitory and facilitatory effects on nicotinic signaling, depending on the prior state of nicotinic stimulation. PAMP may therefore contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release.

Adrenomedullin↗
PubMed1997

Novel autocrine feedback control of catecholamine release. A discrete chromogranin a fragment is a noncompetitive nicotinic cholinergic antagonist.

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.

Amino Acid Sequence↗
PubMed1997

Stimulus coupling to transcription versus secretion in pheochromocytoma cells. Convergent and divergent signal transduction pathways and the crucial roles for route of cytosolic calcium entry and protein kinase C.

How do chromaffin cell secretory stimuli program resynthesis of secreted peptides and amines? We previously showed that the physiologic nicotinic cholinergic signal for secretion also activates the biosynthesis of chromogranin A, the major protein released with catecholamines. Here, we examine signal transduction pathways whereby secretory stimuli influence exocytotic secretion versus chromogranin A transcription. Both secretion and transcription depended on initial nicotinic-triggered sodium entry into the cytosol, followed by calcium entry through -type voltage-gated channels. When calcium entered through -type channels, activation of secretion paralleled activation of transcription (r = 0.897, P = 0.002). Calcium entry from intracellular stores or through calcium ionophore channels activated secretion, though not transcription. Nicotinic-stimulated transcription depended upon protein kinase C activation; nicotine caused translocation of protein kinase C to the cell membrane fraction, and inhibition of protein kinase C blocked activation of transcription, while activation of protein kinase C mimicked nicotine effects. Transcriptional responses to both nicotine and protein kinase C mapped principally onto the chromogranin A promoter's cAMP response element (TGACGTAA; CRE box). KCREB, a dominant negative mutant of the CRE-binding protein CREB, blunted activation of chromogranin A transcription by nicotine, phorbol ester, or membrane depolarization. We conclude that activation of chromogranin A transcription by secretory stimulation in chromaffin cells is highly dependent upon precise route of calcium entry into the cytosol; transcription occurred after entry of calcium through -type channels on the cell surface, and was mediated by protein kinase C activation. The trans-acting factor CREB ultimately relays the secretory signal to the chromogranin A promoter's CRE box in cis.

Animals↗
PubMed1996

Stimulus-transcription coupling in pheochromocytoma cells. Promoter region-specific activation of chromogranin a biosynthesis.

To explore stimulus-transcription coupling in pheochromocytoma cells, we studied the biosynthetic response of chromogranin A, the major soluble protein co-stored and co-released with catecholamines, to chromaffin cells' physiologic nicotinic cholinergic secretory stimulation. Chromogranin A mRNA showed a time-dependent 3.87-fold response to nicotinic stimulation, and a nuclear run-off experiment indicated that the response occurred at a transcriptional level. Transfected chromogranin A promoter/luciferase reporter constructs were activated by nicotinic stimulation, in time- and dose-dependent fashions, in both rat PC12 pheochromocytoma cells and bovine chromaffin cells. Cholinergic subtype agents indicated that nicotinic stimulation was required. Promoter deletions established both positive and negative nicotinic response domains. Transfer of candidate promoter domains to a heterologous (thymidine kinase) promoter conferred region-specific nicotinic responses onto that promoter. A proximal promoter domain (from -93 to -62 base pairs) was activated in copy number- and distance-dependent fashion, and thus displayed features of a promoter element. Its activation was sufficient to account for the overall positive response to nicotine. Within this proximal region, a cAMP response element (CRE) was implicated as a major nicotinic response element, since a CRE point-gap mutation decreased nicotinic induction, transfer of CRE to a thymidine kinase promoter augmented the promoter's response to nicotine, and nicotine activated the CRE-binding protein CREB through phosphorylation at serine 133. We conclude that secretory stimulation of pheochromocytoma cells also activates the biosynthesis of the major secreted protein (chromogranin A), that the activation is transcriptional, and that a small proximal domain, including the CRE box, is, at least in part, both necessary and sufficient to account for the positive response to nicotine.

Animals↗
PubMed1996

Dispersion of chromogranin/secretogranin secretory protein family loci in mammalian genomes.

Chromogranin A, chromogranin B, and secretogranin II, members of the chromogranin/secretogranin secretory protein family, are overexpressed in some human hereditary maladies and may have arisen, in part, from common ancestor genes. To understand better the mammalian chromosomal dispersion of this gene family and to facilitate studies of these genes in human illnesses and their animal models, we positioned the locus of each member in the rat, mouse, and human genomes. Our results indicate that each locus lies in a region of locally syntenic chromosomal homology across the three species.

Animals↗
PubMed1996

Vesicular monoamine transport inhibitors. Novel action at calcium channels to prevent catecholamine secretion.

Vesicular monoamine transport (VMAT) inhibitors, such as reserpine and tetrabenazine, impair vesicular catecholamine storage in chromaffin cells and sympathetic neurons, thereby lowering blood pressure. Here we describe a novel action of VMAT inhibitors-blockade of L-type voltage-gated calcium channels-that may also influence catecholamine release from both PC12 rat pheochromocytoma cells and bovine adrenal chromaffin cells. When given alone, VMAT inhibitors acutely release catecholamines from chromaffin cells in a dose-dependent fashion. However, VMAT inhibitors block catecholamine secretion stimulated by either nicotinic cholinergic agonists or cell membrane depolarization, each of which rely on the opening of L-type channels; the inhibition was more potent after long-term exposure to VMAT inhibitors (IC50 < 100 nmol/L). Reserpine blocked nicotinic-stimulated catecholamine release from neurite-bearing PC12 cells. Reserpine also antagonized catecholamine release triggered by combined membrane depolarization and the dihydropyridine L-type channel agonist Bay K8644, and reserpine blocked cellular uptake of extracellular 45Ca2+ in response to nicotine. Taken together, these results indicate that VMAT inhibitors are also antagonists at L-type voltage-gated calcium channels. Classic L-type channel antagonists (verapamil or nifedipine) also exhibited the reciprocal actions; acutely, they released norepinephrine from chromaffin cells, and chronically, they depleted cellular catecholamine stores, albeit with inferior molar potency to reserpine (IC50 < 1 nmol/L). We conclude that VMAT inhibitors and L-type calcium channel antagonists exert reciprocal inhibitory actions on each other's more classic pharmacological targets. Furthermore, these novel actions are seen at concentrations of these compounds frequently taken to be specific in vitro and likely to occur during antihypertensive treatment in vivo.

Adrenal Glands↗
PubMed1995

Effects of haloperidol, clozapine and citalopram on messenger RNA levels of chromogranins A and B and secretogranin II in various regions of rat brain.

We have measured the messenger RNA levels of chromogranins A and B and secretogranin II in various brain regions of rats subchronically treated with various antipsychotic drugs. Since, as shown previously, the messenger RNA levels of these peptides are increased when neurons are stimulated, we hoped to identify by this approach those nuclei which are subchronically influenced by these drugs. The drugs chosen were the neuroleptic halperidol, a blocker of dopamine receptors, the atypical antipsychotic clozapine, which in addition to blocking dopamine receptors also blocks those for serotonin, and citalopram, a specific serotonin reuptake inhibitor. In agreement with previous data on neuropeptide messenger RNAs, we found in the dorsolateral striatum an increase of the secretogranin II messenger RNA levels after haloperidol and a much smaller one after clozapine. In the nucleus accumbens and in the bed nucleus of the stria terminalis, both compounds had a comparable positive effect. These differential effects can be attributed to a different action of these drugs on dopamine receptor subtypes. In the zona incerta, clozapine decreased the secretogranin II and chromogranin A message, whereas in the dorsal raphe it led to an increase. On the other hand, citalopram induced exactly the opposite effects in these two brain regions. This phenomenon can be explained by the differential interaction of these drugs with serotonergic mechanisms. Additional, relatively small changes of the mRNAs were seen in several other brain regions. These results establish that changes in the mRNA levels of the chromogranins are good indicators for the effect of drugs on certain brain nuclei. The concomitant action of haloperidol and clozapine on the limbic regions, i.e. the nucleus accumbens and the bed nucleus of the stria terminalis, points to these brain regions for the antipsychotic action of these two neuroleptics.

Animals↗
PubMed1995

A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type specificity of the promoter. The functional activity of the CRE site was confirmed through studies of the endogenous chromogranin A gene. Northern mRNA analysis showed that expression of the endogenous chromogranin A gene was stimulated seven- to eightfold by cAMP in PC12 cells, whereas no induction occurred in the NIH3T3 cells. Similar cAMP induction was obtained with the transfected chromogranin A promoter in PC12 cells, and abolition of the CRE site (by deletion or point mutation) eliminated the induction. Thus, the CRE site in the chromogranin A proximal promoter is functional and plays a crucial, indeed indispensable, role in neuroendocrine-specific expression of the gene. These results also provide insight into transcriptional mechanisms governing acquisition of the neuroendocrine secretory phenotype.

3T3 Cells↗
PubMed1994

Hormone storage vesicle proteins. Transcriptional basis of the widespread neuroendocrine expression of chromogranin A, and evidence of its diverse biological actions, intracellular and extracellular.

Chromogranin A (CgA) is an acidic soluble protein found in the core of secretory vesicles throughout the neuroendocrine system, from which it is coreleased by exocytosis with a variety of amine and peptide hormones and neurotransmitters. Much has now been learned about the structure of CgA, and there is emerging evidence that it plays several biological roles, both within secretory granules and after release from neuroendocrine cells. Factors governing its gene's widespread yet restricted (neuroendocrine) pattern of expression are only now being explored. In an attempt to understand how cells throughout the neuroendocrine system (but not exocrine or other nonendocrine cells) turn on and control the expression of CgA, we have isolated and begun to characterize functional 5' promoter elements from the rodent CgA genes. Within the sympathoadrenal system, interest focuses on a recently proposed (though as yet incompletely investigated) function of CgA: its ability to suppress catecholamine release from adrenal chromaffin cells when such cells are stimulated by their usual physiologic secretagogue. We anticipate that such studies will contribute to an understanding of this abundant, yet previously mysterious protein's role in neuroendocrine function.

Adrenal Medulla↗
PubMed1994

Human and rat primary C-fibre afferents store and release secretoneurin, a novel neuropeptide.

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre-treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C-) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin-immunoreactivity (IR) in the spinal cord and, lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post-mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin-IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (< 25 microns) SgII-mRNA-containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin-IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin-treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin-IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin-sensitive, primary afferent (C-fibre) neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Distribution of secretoneurin immunoreactivity in the spinal cord and lower brainstem in comparison with that of substance P and calcitonin gene-related peptide.

Secretoneurin is a peptide of 33 amino acids generated in brain by proteolytic processing of secretogranin II. The distribution of this newly characterized peptide was investigated by means of immunocytochemistry and in situ hybridization in the spinal cord and lower brainstem of the rat. The staining pattern of secretoneurin immunoreactivity (IR) was compared to that of substance P (SP) and calcitonin gene-related peptide (CGRP) in adjacent sections. A high density of secretoneurin-IR fibers and terminals was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord at all levels, around the central canal, and in the sympathetic and parasympathetic areas of the lateral cell columns. The ventral horn displayed a low to moderate density of secretoneurin-IR. The highest number of secretogranin II mRNA-containing cells was found in lamina II of the dorsal horn and in neurons of the dorsal root ganglia. In the white matter, secretoneurin-IR was most prominent in the dorsolateral part of the lateral funiculus and in the tract of Lissauer. The distributions of secretoneurin-IR and SP-IR were strikingly similar. CGRP-IR and secretoneurin-IR overlapped in the outer laminae of the dorsal horn, in the lateral cell column, and probably in some motoneurons. This study establishes that, like SP and CGRP, secretoneurin is a peptide highly concentrated in the terminal field of primary afferents and in sympathetic and parasympathetic areas. Thus secretoneurin might be involved in the modulation of afferent transmission.

Animals↗
PubMed1994

Large dense-core vesicles in rat adrenal after reserpine: levels of mRNAs of soluble and membrane-bound constituents in chromaffin and ganglion cells indicate a biosynthesis of vesicles with higher secretory quanta.

Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense-core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense-core vesicles (chromogranins A and B, secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 215-857% after 1-3 days of drug treatment. For partly membrane-bound components (dopamine beta-hydroxylase, prohormone convertase 2, carboxypeptidase H, and peptidylglycine alpha-amidating monooxygenase) the changes ranged from 182 to 315%, whereas for glycoprotein III and for intrinsic membrane proteins (cytochrome b561 and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y, secretogranin II, carboxypeptidase H, and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Long-lasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides.

Adrenal Glands↗