Obstructive sleep apnea induced bilateral tonic- clonic seizure of unknown origin: A case series of a novel association.

Background: Obstructive sleep apnea is a highly prevalent disorder, characterized by recurrent events of upper airway obstruction during sleep and associated with recurrent cycles of desaturation and re-oxygenation, sympathetic hyperactivity, and intra-thoracic pressure fluctuations, resulting in fragmentation of sleep and subsequent daytime fatigue with excessive sleepiness. Obstructive sleep apnea-induced bilateral tonic-clonic seizures are unheard of. We aimed to report 3 patients with previously undiagnosed obstructive sleep apnea who presented to the emergency department with new onset bilateral tonic-clonic seizure without any evidential neurological or metabolic cause. Methods: Patient data were obtained from medical records from the Department of Internal Medicine, IPGMER and SSKM Hospital, Kolkata, and Belle Vue Clinic, Kolkata, India. Results: Three male patients (67, 58, and 44 years old) presented with bilateral tonic-clonic seizure disorder without any underlying cause of seizures after rigorous investigations except for moderate to severe obstructive sleep apnea on polysomnography. All 2 patients were seizure-free after being treated with levetiracetam, chronic continuous positive airway pressure therapy in 2, and only continuous positive airway pressure in the other. The patients remained seizure-free on continuous positive airway pressure, even when levetiracetam was withdrawn, suggesting obstructive sleep apnea's causality in their new-onset acute seizures. Conclusion: Although further investigation is required to clarify this association, underlying obstructive sleep apnea should be ruled out in patients with a first-ever bilateral tonic-clonic seizure. Whether or not continuous positive airway pressure alone could effectively treat hypoxia and deranged cortical excitability, which may lead to seizures in cases with longstanding obstructive sleep apnea, is yet to be explored.

Introducción: La apnea obstructiva del sueño es una enfermedad con una alta prevalencia que se caracteriza por episodios recurrentes de obstrucción de las vías respiratorias altas durante el sueño, lo que conlleva ciclos repetidos de hipoxia y reoxigenación, hiperactividad simpática y fluctuaciones en la presión intratorácica. Todos estos procesos dan lugar a una fragmentación del sueño, lo que provoca fatiga diurna y somnolencia excesiva. Las crisis tónico-clónicas bilaterales inducidas por apnea obstructiva del sueño son poco conocidas. Presentamos los casos de tres pacientes con apnea obstructiva del sueño sin diagnosticar previamente que acudieron a urgencias por crisis tónico-clónicas de nueva aparición sin causa neurológica o metabólica aparente. Métodos: Los datos de nuestros pacientes se recogieron de los historiales médicos del servicio de Medicina Interna del Institute of Post-Graduate Medical Education and Research and Seth Sukhlal Karnani Memorial Hospital y de la Belle Vue Clinic, ambos en Kolkata (India). Resultados: Tres pacientes varones de 67, 58 y 44 años de edad presentaron convulsiones tónico-clónicas bilaterales sin causa identificada tras examen riguroso, exceptuando una apnea obstructiva del sueño de gravedad moderada a grave observada en la polisomnografía. Los tres pacientes recibieron tratamiento con levetiracetam durante el ingreso; al alta, se pautó tratamiento crónico con presión positiva continua de las vías respiratorias más levetiracetam en dos pacientes, y en el tercero solo presión positiva continua de las vías respiratorias. Ninguno presentó nuevas crisis tras la retirada de levetiracetam, lo que sugiere que la causa de las convulsiones era la apnea obstructiva del sueño. Conclusión: Aunque es necesario realizar más estudios para aclarar esta asociación, debemos descartar la apnea obstructiva del sueño en pacientes con crisis tónico-clónicas bilaterales de nueva aparición. Queda aún por determinar si la presión positiva continua podría tratar de forma efectiva la hipoxia y las alteraciones en la excitabilidad cortical, que podrían provocar crisis en casos de apnea obstructiva del sueño de larga evolución.

Enhanced temperature sensing response of upconversion luminescence in ZnO-CaTiO3: Er(3+)/Yb(3+) nano-composite phosphor.

Upconversion emission and temperature sensing of the Er(3+)/Yb(3+) doped ZnO-CaTiO3 nano-composite phosphor is studied by varying the ZnO concentration. The XRD and EDX studies reveal the formation of composite phase when ZnO doping exceeds above 10 mw%. Five prominent upconversion emission bands at 410, 492, 524, 545 and 662 nm corresponding to (2)H9/2→(4)I15/2, (4)F3/2→(4)I15/2, (2)H11/2→(4)I15/2, (4)S3/2→(4)I15/2 and (4)F9/2→(4)I15/2 transitions, respectively are found under 980 nm excitation from a diode laser. On the basis of rise time analysis it was found that energy transfer process is responsible for the intense upconversion emission. Large reduction in decay time of (4)S3/2 level is observed on the ZnO incorporation in host matrix. Moreover, the absolute sensor sensitivity, relative sensor sensitivity and calculated color coordinates of the samples are also determined. These results indicate the potentiality of this composite phosphor for various applications.

Genetic regulation of catecholamine synthesis, storage and secretion in the spontaneously hypertensive rat.

Understanding catecholamine metabolism is crucial for elucidating the pathogenesis of hereditary hypertension. Here we integrated transcriptional and biochemical profiling with physiologic quantitative trait locus (eQTL and pQTL) mapping in adrenal glands of the HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and normotensive Brown Norway (BN.Lx). We found simultaneous down-regulation of five heritable transcripts in the catecholaminergic pathway in young (6 weeks) SHRs. We identified cis-acting eQTLs for Dbh, Pnmt (catecholamine biosynthesis) and Vamp1 (catecholamine secretion); enzymatic activities of Dbh and Pnmt paralleled transcripts, with pQTLs for activities mirroring eQTLs. We also detected trans-regulated expression of Vmat1 and Chga (both involved in catecholamine storage), with co-localization of these trans-eQTLs to the Pnmt locus. Pnmt re-sequencing revealed promoter polymorphisms that result in decreased response of the transfected SHR promoter to glucocorticoid, compared with BN.Lx. Of physiological pertinence, Dbh activity negatively correlated with systolic blood pressure in RI strains, whereas Pnmt activity was negatively correlated with heart rate. The finding of such cis- and trans-QTLs at an age before the onset of frank hypertension suggests that these heritable changes in biosynthetic enzyme expression represent primary genetic mechanisms for regulation of catecholamine action and blood pressure control in this widely studied model of hypertension.

Common genetic variants in the chromogranin A promoter alter autonomic activity and blood pressure.

Chromogranin A (CHGA) is stored and released from the same secretory vesicles that contain catecholamines in chromaffin cells and noradrenergic neurons. We had previously identified common genetic variants at the CHGA locus in several human populations. Here we focus on whether inter-individual variants in the promoter region are of physiological significance. A common haplotype, CGATA (Hap-B), blunted the blood pressure response to cold stress and the effect exhibited molecular heterosis with the greatest blood pressure change found in Hap-A/Hap-B heterozygotes. Homozygosity for three minor alleles with peak effects within the haplotype predicted lower stress-induced blood pressure changes. The G-462A variant predicted resting blood pressure in the population with higher pressures occurring in heterozygotes (heterosis). Using cells transfected with CHGA promoter-luciferase reporter constructs, the Hap-B haplotype had decreased luciferase expression compared to the TTGTC (Hap-A) haplotype under both basal conditions and after activation by pre-ganglionic stimuli. The G-462A variant altered a COUP-TF transcriptional control motif. The two alleles in transfected promoters differed in basal activity and in the responses to COUP-II-TF transactivation and to retinoic acid. In vitro findings of molecular heterosis were also noted with the transfected CHGA promoter wherein the diploid combination of the two G-462A alleles gave rise to higher luciferase expression than either allele in isolation. Our results suggest that common genetic variants in the CHGA promoter may regulate heritable changes in blood pressure.

Proteolytic cleavage of chromogranin A (CgA) by plasmin. Selective liberation of a specific bioactive CgA fragment that regulates catecholamine release.

Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism.

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.

Primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A(344-364)): identification of amino acid residues crucial for activity.

The novel chromogranin A fragment catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, approximately 0.2-0.3 microM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A(344-364). Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358. We conclude that a small, 15-amino acid core of catestatin (chromogranin A(344-364)) is sufficient to exert the peptide's typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.

Neuroendocrine cell type-specific and inducible expression of the chromogranin B gene: crucial role of the proximal promoter.

Chromogranin B, a soluble acidic secretory protein, is widely distributed in neuroendocrine and neuronal cells, although not in other cell types. To identify the elements governing such widespread, yet selective, expression of the gene, we characterized the isolated mouse chromogranin B promoter. 5'-Promoter deletions localized neuroendocrine cell type-specific expression to the proximal chromogranin B promoter (from -216 to -91 bp); this region contains an E box (at [-206 bp]CACCTG[-201 bp]), four G/C-rich regions (at [-196 bp]CCCCGC[-191 bp], [-134 bp]CCGCCCGC[-127 bp], [-125 bp]GGCGCCGCC[-117 bp], and [-115 bp]CGGGGC[-110 bp]), and a cAMP response element (CRE; at [-102 bp]TGACGTCA[-95 bp]). A 60-bp core promoter region, defined by an internal deletion from - 134 to -74 bp upstream of the cap site and spanning the CRE and three G/C-rich regions, directed tissue-specific expression of the gene. The CRE motif directed cell type-specific expression of the chromogranin B gene in neurons, whereas three of the G/C-rich regions played a crucial role in neuroendocrine cells. Both the endogenous chromogranin B gene and the transfected chromogranin B promoter were induced by preganglionic secretory stimuli (pituitary adenylyl cyclase-activating polypeptide, vasoactive intestinal peptide, or a nicotinic cholinergic agonist), establishing stimulus-transcription coupling for this promoter. The adenylyl cyclase activator forskolin, nerve growth factor, and retinoic acid also activated the chromogranin B gene. Secretagogue-inducible expression of chromogranin B also mapped onto the proximal promoter; inducible expression was entirely lost upon internal deletion of the 60-bp core (from 134 to -74 bp). We conclude that CRE and G/C-rich domains are crucial determinants of both cell type-specific and secretagogue-inducible expression of the chromogranin B gene.

Processing of chromogranin A by plasmin provides a novel mechanism for regulating catecholamine secretion.

Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is also distributed widely in secretory vesicles throughout the neuroendocrine system. CgA contains the sequences for peptides that modulate catecholamine release, but the proteases responsible for the release of these bioactive peptides from CgA have not been established. We show here that the major fibrinolytic enzyme, plasmin, can cleave CgA to form a series of large fragments as well as small trichloroacetic acid-soluble peptides. Peptides generated by plasmin-mediated cleavage of CgA significantly inhibited nicotinic cholinergic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells. We also show that the zymogen, plasminogen, as well as tissue plasminogen activator bind saturably and with high capacity to catecholaminergic (PC12) cells. Occupancy of cell surface binding sites promoted the cleavage of CgA by plasmin. Positive and negative modulation of the local cellular fibrinolytic system resulted in substantial alterations in catecholamine release. These results suggest that catecholaminergic cells express binding sites that localize fibrinolytic molecules on their surfaces to promote plasminogen activation and proteolytic processing of CgA in the environment into which CgA is secreted to generate peptides which may regulate neuroendocrine secretion. Interactions between CgA and plasmin(ogen) define a previously unrecognized autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion.

Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules.

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.

Time-dependent effects of the neuropeptide PACAP on catecholamine secretion : stimulation and desensitization.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. We previously reported that PACAP coupled to the PAC1 receptor to evoke dihydropyridine-sensitive early (15 to 20 minutes) catecholamine secretion and cAMP response element binding protein-mediated trans-activation of the secretory protein chromogranin A promoter in PC12 pheochromocytoma cells. In this report, we studied whether the secretory and transcriptional responses elicited by PACAP were subject to desensitization. We found that PACAP evoked distinct immediate (initial, 0 to 20 minutes) and long-lasting (20 to 180 minutes) effects on catecholamine secretion. Initial secretory and chromogranin A trans-activation responses induced by PACAP were desensitized in a dose-dependent fashion after preexposure of cells to PACAP, and the IC(50) doses of PACAP for desensitization were approximately 18- to approximately 32-fold lower than the EC(50) activating doses for secretion or transcription. Desensitization of the initial secretion response was associated with decreased Ca(2+) influx through L-type voltage-operated Ca(2+) channels. Acute exposure to PACAP also triggered long-lasting (up to 3 hours), extracellular Ca(2+)-dependent, pertussis toxin-insensitive catecholamine secretion; indeed, even after short-term (20 minutes) exposure to PACAP and removal of the secretagogue, PC12 cells continued to secrete norepinephrine up to 76.9+/-0.22% of cellular norepinephrine content after 3 hours. A phospholipase C-beta inhibitor (U-73122) blocked this extended secretory response, which was dependent on low-magnitude Ca(2+) influx resistant to several L-, N-, P/Q-, or T-type Ca(2+) channel antagonists, but sensitive to Zn(2+), Ni(2+), Cd(2+), or to the store-operated Ca(2+) channel blocker SKF96365. A less than additive effect of the sarco-endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin plus PACAP on this sustained secretion also supported a contribution of store-operated Ca(2+) entry to the sustained secretory response. We propose that PACAP-evoked secretion and transcription are subject to homologous desensitization in PC12 cells; however, PACAP also induces long-lasting secretion, even under dose and time circumstances in which acute, dihydropyridine-sensitive secretion has been desensitized. Although initial secretion is mediated by an L-type voltage-operated Ca(2+) channel, extended secretion may involve a store-operated Ca(2+) channel that is activated through a G(q/11)/phospholipase C-beta/phosphoinositide signaling pathway.

Neurotrophin activation of catecholamine storage vesicle protein gene expression: signaling to chromogranin a biosynthesis.

Nerve growth factor differentiates precursor cells into sympathetic neurons. Does acquisition of a "neuronal" phenotype after nerve growth factor involve biosynthesis of chromogranin A, the major soluble protein in chromaffin granule cores? Nerve growth factor activated chromogranin A gene expression 7.6-fold in PC12 pheochromocytoma cells, and similarly activated PC12-transfected mouse, rat or human chromogranin A promoter/reporter constructs. Chromogranin A promoter 5'-deletions narrowed the nerve growth factor response element to a region from - 77 to - 61 bp upstream of the cap site, a region containing the chromogranin A cyclic AMP response element (TGACGTAA). Three different site-directed mutations of the cyclic AMP response element each reduced the nerve growth factor effect by >90%. Transfer of the cyclic AMP response element to a heterologous (thymidine kinase) promoter activated that promoter approximately 5-fold after nerve growth factor, while transfer of a cyclic AMP response element point-gap mutant (TGA-GTAA) to a heterologous promoter abolished the nerve growth factor effect. These findings indicate that the cyclic AMP response element in cis is, at least in part, both necessary and sufficient to activate the chromogranin A gene. Chemical blockade of the nerve growth factor receptor TrkA or the mitogen-activated protein kinase pathway component MEK substantially diminished nerve growth factor-induced expression of chromogranin A. By contrast, the response of chromogranin A to nerve growth factor was not impaired after blockade of phospholipase C-gamma or phosphoinositide-3 kinase. Chemical blockade of TrkA, Ras, MEK or mitogen-activated protein kinase similarly inhibited nerve growth factor activation of chromogranin A. Expression of constitutively activated Ras, Raf or MEK mutants increased chromogranin A promoter activity. Expression of dominant negative (inhibitory) mutants of Sos, Ha-Ras, Rafl, mitogen-activated protein kinase, ribosomal protein S6 serine kinase II (CREB kinase) or CREB (KCREB) each inhibited the nerve growth factor-induced increase in chromogranin A promoter activity. Thus, each component of the mitogen-activated protein kinase pathway is crucially involved in relaying the nerve growth factor signal in trans to the chromogranin A gene, in the following proposed sequence: nerve growth factor --> TrkA --> Shc/Grb2/Sos --> Ras --> Raf --> MEK --> mitogen-activated protein kinase --> ribosomal protein S6 serine kinase II --> CREB cyclic AMP response element.

Neuroendocrine cell type-specific and inducible expression of the secretogranin II gene: crucial role of cyclic adenosine monophosphate and serum response elements.

Secretogranin II, an acidic protein in the chromogranin/secretogranin family, is widely distributed in neuroendocrine secretory granules. What factors govern such widespread, yet selective, expression? The 5' deletions localized neuroendocrine cell type-specific expression to the proximal mouse secretogranin II promoter: such expression was abolished after deletion past the cAMP response element (CRE; [-67 bp]TGACGTCA[-60 bp]), and transfer of the CRE to a neutral promoter conferred 3.4- to 5.3-fold neuroendocrine selectivity. Thus, the CRE is, at least partly, sufficient to confer tissue-specific expression. Substantial (48-59%) loss of cell type-specific expression also occurred upon deletion past the serum response element (SRE; [-302 bp]GATGTCC[-296 bp]), and transfer of the SRE to a neutral promoter also conferred neuroendocrine selectivity. Expression of both the endogenous gene and the transfected secretogranin II promoter was up-regulated after secretagogues, and the degree of trans-activation of the transfected promoter (2.2- to 5.4-fold) paralleled activation of the endogenous gene (1.8- to 3.2-fold). The 5' promoter deletions revealed complete loss of secretagogue responses after deletion past the CRE. Transfer of the CRE to a neutral promoter conferred secretagogue responses (by 2.2- to 18.6-fold). Substantial (59-74%) falls in secretagogue responses also occurred after deletion past the promoter's SRE. Transfer of the SRE to a neutral promoter conferred secretagogue responses (by 2.7- to 8.3-fold). We conclude that the CRE is a crucial determinant of cell type-specific constitutive and secretagogue-inducible expression of the secretogranin II gene and that the SRE also plays a substantial role in both processes.

Desensitization of catecholamine release. The novel catecholamine release-inhibitory peptide catestatin (chromogranin a344-364) acts at the receptor to prevent nicotinic cholinergic tolerance.

Nicotinic cholinergic receptors undergo desensitization upon repeated or prolonged exposure to agonist. We investigated the effects of a novel chromogranin A catecholamine release-inhibitory fragment, catestatin (chromogranin A344-364), on agonist-induced desensitization of catecholamine release from pheochromocytoma cells. In a dose-dependent fashion, the nicotinic antagonist catestatin blocked agonist desensitization of both catecholamine release (IC50 approximately 0.24 microM) and 22Na+ uptake (IC50 approximately 0.31 microM), the initial step in nicotinic cationic signal transduction; both secretion inhibition and blockade of desensitization were noncompetitive with agonist. Desensitizing effects of the nicotinic agonists nicotine and epibatidine were blocked. This antagonist action was specific to desensitization by nicotinic agonists, since catestatin did not block desensitization of catecholamine release induced by agents which bypass the nicotinic receptor. Hill plots with slopes near unity suggested noncooperativity for catestatin effects on both nicotinic responses (secretory antagonism and blockade of desensitization). Human, bovine, and rat catestatins (as well as substance P) had similar potencies. IC50 values for secretion inhibition and blockade of desensitization paralleled each other (r = 0.76, n = 10 antagonists, p = 0.01) for several noncompetitive nicotinic antagonists. Peptide nicotinic antagonists (catestatins, substance P) were far more potent inhibitors of both secretion (p = 0.019) and desensitization (p = 0.005) than nonpeptide antagonists (trimethaphan, hexamethonium, procaine, phencyclidine, cocaine, or clonidine), and the peptides displayed enhanced selectivity to block desensitization versus secretion (p = 0.003). We conclude that catestatin is a highly potent, dose-dependent, noncompetitive, noncooperative, specific inhibitor of nicotinic desensitization, an effect which may have implications for control of catecholamine release.

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.

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.