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Keywords:

  • chromaffin vesicle;
  • molecular content;
  • stress response

Abstract

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Chromaffin vesicles (CV) are highly sophisticated secretory organelles synthesized in adrenal medullary chromaffin cells. They contain a complex mixture of structural proteins, catecholamine neurotransmitters, peptide hormones, and the relative processing enzymes, as well as protease inhibitors. In addition, CV store ATP, ascorbic acid, and calcium. During the last decades, extensive studies have contributed to increase our understanding of the molecular composition of CV. Yet, the recent development of biochemical and imaging procedures has greatly increased the list of CV-soluble constituents and opened new horizons as to the complexity of CV involvement in acute stress responses. Thus, a coherent picture of CV molecular composition is still to be drawn. This review article will provide a detailed account of the content of CV soluble molecules as it emerges from the most recent analytical studies. Moreover, this review article will attempt at focussing on the physiological and pathophysiological implications of the products released by CV. Anat Rec, 291:1587–1602, 2008. © 2008 Wiley-Liss, Inc.

Secretory granules in chromaffin cells are designated as chromaffin vesicles (CV). The terms “chromaffin cell” and “chromaffin vesicle” have been introduced because these structures exhibit histochemical affinity to chromate salts, the so-called chromaffin reaction (Henle,1865; Kohn,1902). During the first half of the last century, it was established that the chromaffin cells of the adrenal medulla are an integral part of the sympathetic nervous system and release catecholamines in response to acute stress (Carmichael,1983).

In 1953, two major articles were published which reported that, after differential centrifugation of homogenized chromaffin cells, catecholamine deposits were restricted to a distinct cell cytoplasmic fraction (Blaschko and Welch,1953; Hillarp et al.,1953). Shortly, thereafter, it was demonstrated by electron microscopy that this cytoplasmic fraction consisted of specialized organelles surrounded by an enclosing membrane (Hillarp et al.,1954). These structures were called chromaffin “vesicles” or “granules.”

CV are membrane-bound, 150 to 350 nm in diameter, moderately or strongly electron-dense organelles that originate from the Golgi network and belong to the class of dense-core vesicles, so called because of the opaque core evident in electron microscopy. They are specialized secretory organelles, which on an average account for 13.5% of the cytoplasmic volume of the chromaffin cell (Kryvi et al.,1979). Each chromaffin cell in the adrenal medulla contains about 10,000 CV.

The molecular structure of CV is surprisingly complex. Besides containing catecholamines, CV of the adrenal medulla contain a variety of other substances. They are made up of a matrix scaffolding mainly formed by special proteins belonging to the granin family that are partly responsible for the dense-core recognized in electron micrographs. Granins, in turn, also function as precursors to generate different biologically active peptides. CV, indeed, contain a large array of peptide hormones along with the relative processing enzymes and protease inhibitors likely involved in modulating the protease activity of prohormone-processing enzymes. In addition, these secretory organelles store some enzymes of the aminergic biosynthetic pathway, ascorbic acid, ATP, and calcium.

In the past, a number of excellent reviews dealing with the molecular organization of the soluble content of CV have been published (Winkler,1976; Winkler and Westhead,1980; Winkler and Carmichael,1982; Winkler et al.,1984). This review will principally focus on the novel aspects of CV structure as documented by the recent analytical studies on CV composition. The aim of this article is to provide a comprehensive picture of the complexity of CV involvement in acute stress responses and the potential role of these highly sophisticated secretory organelles in normal and physiopathological conditions.

BIOLOGY OF THE CHROMAFFIN VESICLE

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Morphology of Chromaffin Vesicles

In most species, two types of CV can be recognized by transmission electron microscopy: the adrenaline- and the noradrenaline-containing CV, which segregate to distinct chromaffin cell populations (Coupland,1965). CV can be distinguished on the basis of their different dimension, shape, and electron density. After glutaraldehyde fixation, followed by osmication, adrenaline-containing CV present rounded shape and fine granular, moderately electron-dense content (Fig. 1a), whilst noradrenaline-containing CV are irregular and exhibit solid, highly electron-dense, usually homogeneous content that is often eccentrically situated (Fig. 1b,c). The different ultrastructural features of CV mainly depend on glutaraldehye (Coupland,1965). During glutaraldehyde fixation, indeed, adrenaline is lost, whereas noradrenaline is precipitated in situ. Thus, adrenaline-containing CV appear less electron-dense. In nonmammalian species, CV often reveal more complex granular patterns with intragranular domains of different textures (Fig. 2a). Freeze-etching electron microscopy studies reveal that noradrenaline-containing CV display less asymmetry in the distribution of intramembrane particles than adrenaline-storing CV (Kryvi et al.,1979). Quick-freezing ultrastructural investigations show that 85% of isolated bovine CV and 60% of in situ CV present small vesicular structures bounded by unit membranes lying within their cores (Ornberg et al.,1986).

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Figure 1. Electron micrographs of chromaffin vesicles in the mouse adrenal medulla. (A) Adrenaline-containing chromaffin vesicles present rounded shape with a fine granular, moderately electron-dense content. Note that the limiting membrane is separated from the internal substance by a narrow and uniform peripheral halo. (B)(C) Noradrenaline-containing chromaffin vesicles exhibit variable shape and are provided with a solid, highly electron-dense, usually homogeneous content. The limiting membrane may be in direct contact with the inner material or may be separated by a prominent, lucent halo. Matrix constituents are often situated eccentrically with regard to their surrounding membranes. Bar = 0.2 μm (A)(B)(C).

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Figure 2. Electron micrographs of chromaffin vesicles in the chick embryo adrenal medulla (A) and Golgi complex in a murine chromaffin cell (B). (A) At embryonic day 12, chromaffin vesicles present a unique ultrastructure showing granular or thread-like peripheral arrangement and an inner condensing domain. (B) An image of the Golgi complex during biogenesis of chromaffin vesicles. Small vesicles of different density in the Golgi network are visible. Small electron-lucent secretory vesicles in continuity or separating from the Golgi saccules are observed. Note some dense-core vesicles near the Golgi stacks. Bar = 0.2 μm (A); 0.1 μm (B).

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Biogenesis of Chromaffin Vesicles

The mechanism underlying the initiation and regulation of CV biogenesis is poorly understood. CV formation initiates at the trans golgi network (TGN) (Fig. 2b). Protein aggregation and condensation may occur in the mildly acidic environment (pH 5.5–5.7) that exists in the TGN and within the immature CV and in the presence of millimolar concentrations of bivalent cations like Ca2+. Two basic models of sorting of proteins within the regulated secretory pathway have been proposed, viz. sorting for entry and sorting for retention (Dikeakos and Reudelhuber,2007). In the sorting-for-entry model, selective aggregation of the secretory protein may take place in the lumen of the TGN, followed by subsequent binding of a specific structural motif of the protein aggregate to the membrane of the nascent secretory granule or to a sorting receptor therein. This is actually what seems to occur in CV biogenesis, whereby structural proteins of the granin family, in particular, chromogranin A and chromogranin B, are believed to function as vesiculogenic determinants by providing distinct structural domains that drive CV formation in the TGN. In contrast, in the sorting-by-retention model, sorting takes place in the post TGN immature secretory vesicles, wherein selective aggregation and condensation occur, leading to preferential retention of regulated secretory proteins while unaggregated (nonretained) proteins are removed from maturing vesicles by clathrin-coated vesicles through constitutive-like secretory pathway. There is no definite proof in favor of such mechanism being operative in CV formation. All evidences indicate that maturation of CV continues once they have disengaged from the TGN. Catecholamine synthesis and storage in CV occur late in CV formation. Similarly, biosynthesis of transmitter and regulatory peptides take place in CV through complex proteolytic pathways starting from precursor structures. Once CV have concluded the process of formation and maturation, they may remain in the chromaffin cell until prompted for exocytotic fusion with the plasma membrane by a specific secretagogue.

Secretion From Chromaffin Vesicles

CV are prototype examples of regulated secretory vesicles. These organelles release their content by exocytosis in response to various stimuli, such as acetylcholine or splanchnic nerve stimulation. Indeed, like sympathetic ganglion cells, adrenal chromaffin cells are innervated by preganglionic sympathetic nerves. Substances stored in the CV are released into the space between the basal lamina and the plasma membrane, and thus, they have to pass through the basal lamina and capillary wall to appear in the venous effluents. This histological organization may account for the discrepancy between the time course of release of small transmitter molecules—like catecholamines—and large structural proteins—like dopamine β-hydroxylase (DβH)—and for the fast decomposition of ATP released from adrenal chromaffin cells. The different release kinetics of CV constituents may recognize various explanations. There is general consensus that CV secretion occurs by exocytosis, either in the form of “full-fusion” or “kiss-and-run” exocytosis (Aunis,1998). According to the first model, the limiting membrane of the chromaffin vesicle establishes a close contact with the cell plasma membrane and completely fuses with it, thus, entirely discharging the vesicle components into the extracellular space. “Kiss-and-run” exocytosis differs from “full fusion” exocytosis in that the limiting membrane of the CV does not completely merge with the cell plasma membrane during the exocytotic process but form a transient fusion pore that more or less rapidly recloses to pinch the granule back off from the cell membrane (Palfrey and Artalejo,2003). Unlike “full fusion” exocytosis, which is an “all-or-none” event, “kiss-and-run” exocytosis implies that only part of the vesicle-stored material can be released during transient pore formation. Docking to the plasma membrane and fusion of CV in chromaffin cells are mediated by a series of integral membrane proteins, membrane-associated proteins, and trafficking molecules, which are basically the same as those involved in the mobilization and exocytosis of large-dense core and clear synaptic vesicle in neurons (Takamori et al.,2006; Burré and Volknandt,2007). During exocytosis, CV extrude their catecholamine content completely within milliseconds. Larger molecules are released at different rates, depending upon their size and their propensity to aggregate with the granule matrix (Perrais et al.,2004; Felmy,2007). A similar mechanism is effective in synaptic vesicles. Neurotransmitters like acetylcholine and ATP are absorbed to an intravesicular proteoglycan matrix, which is mainly composed of the proteoglycan SV2 (Reigada et al.,2003). This intravesicular matrix controls and regulates the availability of free diffusible neurotransmitters in the early steps of vesicle fusion by an ion-exchange mechanism. Thus, secretion of substances contained in CV is subjected to different control by numerous factors, including the duration and size of the exocytotic pore and the package condition of transmitter molecules within CV. In addition, studies by Carmichael et al. (1990) suggest that the size of a molecule influences the route it takes following release from CV. Smaller molecules such as catecholamines may pass directly into the circulation, whereas larger molecules such as chromogranin A may be temporarily sequestered in the interstitial space before passing into the lymph and hence, into the circulation.

GRANINS

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

CV store and release chromogranins and secretogranins—also known as “granins”—a unique group of acidic, soluble secretory glycoproteins responsible in part for the characteristic electron-dense core of CV on electron microscopy. The three classic granins are chromogranin A (CGA), chromogranin B (CGB) (also called secretogranin I), and secretogranin II (SgII) (sometimes called chromogranin C). Granins are the major quantitative constituents of CV, accounting for more than 80% of soluble intravesicular proteins. They also function as prohormones, giving rise to bioactive peptides through proteolytic processing. In addition, they are key determinants of CV formation, and there is now substantial support for the concept that granins act as sorting proteins to the regulated secretory pathway. All granins consist of single-polypeptide chains, bearing an N-terminal signal peptide that directs the movement of the preproteins from ribosomes to the endoplasmic reticular lumen and, hence, the Golgi complex, where further post-translational modifications occur. Granins express the fundamental property of binding calcium with low affinity but high capacity (32–93 mol of calcium/mol) (Yoo et al.,2001). This property allows them to form in vitro aggregates at low pH in the presence of calcium. At the mildly acidic pH and high calcium concentration of the TGN environment, granins have a tendency to associate with each other and with other granule components, such as catecholamines, ATP, and other proteins, resulting in the formation of the dense cores characteristic of CV. All these features make granins essential protagonists in granule biogenesis.

Chromogranin A

CGA is the most abundant protein contained in bovine CV, accounting for 40% of all the soluble intravesicular proteins. First identified almost 4 decades ago, CGA is a highly acidic protein with 431–445 aminoacid residues, a molecular weight of 49–52 kD, and a pI of 4.5–5 (Taupenot et al.,2003). Bovine CGA is a glycoprotein containing 5.4% carbohydrate. In addition, it is a phosphoprotein containing five phosphoserine residues per molecule and also incorporates [35S] sulfate during protein biosynthesis, most linked to carbohydrates. CGA is a structural matrix protein that is involved in vesiculogenesis and the regulation of vesicle protein stability (Kim et al.,2001). CGA enters the low pH and high calcium concentration environment of the TGN where it tends to aggregate with other vesicle components. For its aggregation property, CGA has been proposed to function as the targeting of peptide hormones and neurotransmitters to CV. Active sequestration of newly synthesized CGA into CV is mediated by the vesicular monoamine transporter, which also keeps the concentration of CGA in CV constant by balancing out the passive leakage of CGA from these organelles (Eisenhofer et al.,2004).

Crucial experiments indicate that CGA is required in the process of vesiculogenesis. Indeed, depletion of CGA in the rat pheochromocytoma PC 12 cell line by antisense or small interfering RNAs leads to profound loss of dense-core secretory vesicles and reduction of other secretory vesicle proteins, such as CGB, carboxypeptidase E, and synaptotagmin (Kim et al.,2001; Huh et al.,2003). Evidence for a vesiculogenic function of CGA has also been documented in vivo. Indeed, genetic ablation of the mouse CGA gene (Mahapatra et al.,2005) or transgenic expression of antisense RNA against CGA in mice (Kim et al.,2005) impairs the formation of CV in adrenal chromaffin cells and reduces the content of other CV constituents such as catecholamines, CGB, and neuropeptide Y (Kim et al.,2005; Mahapatra et al.,2005). A contribution of CGA to the formation of secretory vesicles has also been documented across cell lineages. For instance, introduction of CGA into the non-neuroendocrine CV1 fibroblast cell line induces the formation of dense-core vesicles, allowing exocytosis of the secretory cargo in response to secretagogue stimulation (Kim et al.,2001; Huh et al.,2003; Beueret et al.,2004). Transfection of CGA into endocrine 6T3 cells, lacking CGA and dense-core secretory vesicles, not only recovers dense-core vesicle formation and regulated secretion but also prevents vesicle protein degradation by a mechanism that involves up-regulation of the serine protease inhibitor protease nexin-1 (PN-1), which stabilizes vesicle proteins against degradation (Kim and Loh,2006). Recently, CGA has been suggested to contain a vesiculogenic determinant located in the N-terminal [CGA (40–115)], but not C-terminal [CGA (233–439)], region (Courel et al.,2006). Inhibition of CGA expression by siRNA, disrupts regulated secretory protein trafficking by ∼65%, whereas targeted ablation of the CGA gene in the mouse reduces CV cotransmitter concentrations by ∼40%–80% (Mosley et al.,2007). In addition, CGA knock-out mice exhibit ∼30% reduction in the content and in the release of catecholamines compared with the wild type (Montesinos et al.,2008). These data suggest important roles for CGA in hormone and transmitter storage within CV, with implications for secretory cargo condensation (or dense core “packing” structure) within the regulated pathway.

CGA presents an extended “coiled-coil” tertiary structure. Electron microscopy with negative staining reveals an extended, filamentous CGA structure with a diameter of ∼9.4 ± 0.45 nm (Mosley et al.,2007). Extended, coiled-coil conformation is likely to permit protein “packing” in the CV at ∼50% higher density than a globular/spherical conformation. CV ultrastructure reveals a ∼10.8 ± 0.63 nm periodicity of electron density, suggesting nucleation of a binding complex by the CGA core.

CGA is a precursor protein for several peptides expressing autocrine, paracrine, and endocrine activities. Generation of bioactive peptides occurs as the result of post-translational proteolytic processing. CGA processing may take place intracellularly as well as extracellularly after secretion into the extracellular space. CGA-derived fragments identified in CV include the peptides vasostatins I and II, catestatin, cateslytin, and chromacin. Remarkably, the CGA content is increased in the CV of the spontaneously (genetic) hypertensive rat as well as in the CV of rats after induction of renovascular hypertension (Takiyyuddin et al.,1993).

Chromogranin B

First identified in 1984 (Winkler et al.,1984), CGB is also abundantly expressed in CV. In contrast to CGA, which is the prominent protein in bovine adrenomedullary CV, CGB is the most abundant component in rat and human chromaffin cells. It is an acidic protein (pI 5.1–5.2) with 626–657 aminoacid residues and a molecular weight of 48–52 kDa (Taupenot et al.,2003). Like CGA, this protein is subjected to post-translational modifications such as glycosylation, phosphorylation, and sulfation. CGA and CGB share some common biochemical properties by virtue of the sequence homologies in their molecular structure. There are indeed two conserved regions, one in the near N-terminal region and the other in the C-terminal region. Two cysteine residues that form a disulfide bond in each protein flank the conserved near N-terminal region of both chromogranins. These structural features distinguish CGA and CGB from the other granin proteins, such as secretogranins. CGB tends to associate with CGA and other matrix proteins in a pH- and calcium-dependent manner to form molecular aggregates, a property that is particularly important in secretory vesicle biogenesis and the packaging of vesicle constituents (Taupenot et al.,2003). Despite fundamental similarities with CGA, CGB presents also some differential features. It is at least two orders of magnitude more sensitive than CGA to calcium. This allows CGB to start aggregation at lower (micromolar) calcium concentration (Yoo,1995). In addition, CGB also localizes to the nucleus in bovine chromaffin cells, where it is believed to participate to transcription control (Yoo et al.,2002). As a result, it appears that the biosynthesis of CGA and CGB in CV is independently regulated. The extreme sensitivity of CGB to calcium and its capacity to interact with other soluble and membrane-bound proteins in the secretory vesicles make CGB another crucial element in the process of vesicle formation. In some experimental models, it appears that CGB may be even more efficacious than CGA to induce vesiculogenesis. For instance, expression of CGB in non-neuroendocrine cell lines NIH3T3 and COS-7 stimulates a higher number of vesicle formations than that induced by expression of CGA (Huh et al.,2003).

CGB is also a precursor protein for peptides. Secretolytin, a peptide fragment with potent activity against Gram-positive microorganisms, corresponds to the C-terminal sequence (614–626) of bovine CGB.

Secretogranin II

SgII, also called chromogranin C, is an acidic tyrosine-sulfated secretory protein found in secretory vesicles in a wide variety of endocrine cells and neurons. Although less abundant than CGA and CGB, SgII is found in adrenal medullary CV (Fischer-Colbrie et al.,1986a). It is an acidic protein (pI 5.0) with 586 aminoacid residues and a molecular weight of 86 kDa. The marked difference between apparent and nascent molecular weights reflects the high content of amino acid residues and post-translational modifications (phosphorylation, tyrosine sulphation, and sialylation). Similar to CGA and CGB, SgII binds calcium and may aggregate in the presence of this cation. SgII is processed by PC1/3 and PC2 enzymes to generate several polypeptides, among which are highly conserved 33-amino acid peptide called secretoneurin and the secretoneurin flanking-peptide EM66 (Vaudry and Conlon,1991).

Secretogranins III, V, and VI

Secretogranin III (SgIII), a 57 kDa protein of 468 aminoacid residues and a pI of 5.1, secretogranin V (SgV), a 23 kDa protein of 185 aminoacid residues and a pI of 5.0, and secretogranin VI (SgVI), a 241 aminoacid residues acidic protein with a molecular weight of 55 kDa and a pI of 5.0 have been found in bovine CV. Little information is currently available on the function of these secretogranins (Helle,2004).

GLYCOPROTEINS

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Five glycoproteins have been found in CV. Their molecular weights range in the unreduced form from 45 to 150 kDa. Glycoprotein I and IV are the enzyme DβH and the H+-ATPase subunit M45, respectively (Supek et al.,1994). DβH catalizes the conversion of dopamine into noradrenaline via a side chain hydroxylation using intragranular ascorbic acid as a cosubstrate. DβH is a tetrameric, 300 kDa, copper-containing glycoprotein, which exists in both soluble and membrane-bound forms. Accordingly, immunoelectron microscopic localization of DβH in CV showed a distribution both at the granule periphery and in the central matrix. The four subunits of about 75 kDa, which are arranged as pairs of disulphide-bonded dimers, are translated from a single gene (Lewis and Asnani,1992). In the membrane-attached enzyme, one of these dimers contains subunits of higher molecular mass (about 2 kDa) than the other. The activity of DβH has been found to be specifically and synergistically coregulated by ascorbic acid and Mg-ATP. The H+-ATPase subunit M45 is an integral protein (Supek et al.,1994).

Glycoprotein II is a heterogeneous membrane glycoprotein. It is not known whether it has a function in the CV membrane. Glycoprotein III is a heterogeneous band found in both the soluble and membrane fractions of bovine CV. Its physiological function is still unknown. No work specifically on glycoprotein V has been reported.

PROHORMONE-PROCESSING ENZYMES

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Biosynthesis of peptide hormones and neurotransmitters involves proteolysis of proprotein precursors by proteolytic enzymes. Prohormones are initially synthesized from their respective messenger RNAs at the rough endoplasmic reticulum, where the NH2-terminal signal peptide is removed by signal peptidases. The resultant prohormone is routed to the Golgi apparatus and packaged into newly formed secretory vesicles. Proteolytic processing of prohormones at dibasic residues as well as at monobasic residues (Lys and Arg) generates the smaller active peptide hormones and neuropeptides that are stored within secretory granules for subsequent secretion. Proteolytic processes of prohormones are accomplished by proteases localized to secretory granules. Seven of such proteases have been identified in CV (Hook et al.,2008). Their localization to CV and their ability to generate different biologically active peptides identify CV as the major subcellular site for peptide biosynthesis.

Cathepsin L

The cysteine protease cathepsin L has been identified as the enzyme responsible for the previously described proenkephalin thiol protease (PTP) activity involved in enkephalin and neuropeptide production (Hwang et al.,2007a). Cathepsin L is part of a high molecular mass complex of 180–200 kDa which has an intrinsic molecular weight of 26–28 kDa (Yasothornsrikul et al.,2003). This endopeptidase participates in the process of proenkephalin proteolysis to produce (Met)enkephalin. Cathepsin L cleaves proenkephalin and enkephalin-containing peptide substrates at the N-terminal site of dibasic sites or between the dibasic residues, generating peptide intermediates with basic residues at their N-termini (Yasothornsrikul et al.,2003). Cathepsin L has been identified in CV from bovine adrenal medulla by immuno-gold electron microscopy (Hwang et al.,2007a). Interestingly, cathepsin L is costored with aminopeptidase B, (Met)enkephalin and NPY in the same CV. It is remarkable that enzymes involved in enkephalin production, like cathepsin L and aminopeptidase B, localize to the same organelles.

Aminopeptidase B

The exopeptidase aminopeptidase B removes N-terminal Arg as well as Lys residues from enkephalin-related neuropeptide substrates, promoting the final conversion of proenkephalin to (Met)enkephalin. Remarkably, aminopeptidase B has been found to colocalize to CV with cathepsin L and the neuropeptides enkephalin and neuropeptide Y. The bovine form of this enzyme has a theoretical molecular weight of about 72 kDa and a predicted isoelectric point (pI) of 5.33 (Hwang et al.,2007b). Both the bovine and rat forms have optimum pH in the range of pH 5.5–6.5, with high activity in the range of pH 5.0–7, which indicates that aminopeptidase B is active within the CV environment (Hwang et al.,2007b).

Prohormone-Converting Enzymes PC1/3 and PC2

The prohormone processing proteases PC1/3 and PC2 belong to the family of mammalian subtilisin-related proprotein convertases (PC). PC1/3 and PC2 have been isolated from bovine CV, where they are implicated in the processing of the protein and neuropeptide precursors that are present in CV. Purified PC1/3 and PC2 each show a molecular mass of 66 kDa (Azaryan et al.,1995a). Both proteases cleave at the C-terminal side of dibasic sites, which then requires carboxypeptidase E for removal of C-terminal basic residues from peptide intermediates (Fugere and Day,2006). PC1/3 and PC2 are calcium-dependent proteases with pH optima of 6.5 and 7.0, respectively (Azaryan et al.,1995a). Both PC1/3 and PC2 are able to convert SgII to secretoneurin and EM66 (Laslop et al.,1998).

Carboxypeptidase E

The metallopeptidase carboxypeptidase E (CPE), also known as carboxypeptidase H, removes the C-terminal basic residues from peptide intermediates. This exopeptidase has been identified in lysates of CV from bovine chromaffin cells. CPE tends to aggregate with other granule proteins at the low pH and high calcium concentration of the TGN and immature granule lumen (Song and Fricker,1995). Mutant mice lacking CPE present multiple endocrine disorders, including hyperproinsulinemia, that have been interpreted as the consequence of missorting of peptide hormones to the constitutive rather than to the regulated secretory pathway accompanied by hormone release in an unregulated manner (Cool et al.,1997). Hence, CPE has been postulated to represent a “sorting receptor” having a direct role in chaperoning certain hormones to secretory granules (Shen and Loh,1997). The CPE knock-out mouse exhibits a wide range of neural and endocrine abnormalities, such as obesity, high plasma levels of glucose, insulin and leptin, diminished reactivity to stimuli, and reduced muscle strength (Cawley et al.,2004). This suggests that CPE may have additional physiological roles beyond those ascribed to peptide processing and sorting of prohormones in cells.

Aspartic Proteinase

This is an aspartic proteinase of 70 kDa isolated from bovine CV, which has been found to contribute to enkephalin precursor cleaving activity for ∼10% (Azaryan et al.,1995b). This proteinase shows optimal activity at pH 5.5 and cleaves preferentially the full-length preproenkephalin as well as proopiomelanocortin (POMC) substrates (Hook et al.,1996).

Tissue-Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) is a serine protease that is coreleased with catecholamines by chomaffin cells upon stimulations such as exercise, trauma, and stimulation of the nervous system (Parmer et al.,1997). Release of t-PA from CV has directly been visualized by real-time evanescent field fluorescence microscopy (Perrais et al.,2004; Felmy,2007). t-PA in chromaffin cells can participate in plasmin-dependent processing of CGA and other bioactive peptides, including peptides which modulate catecholamine release (Parmer et al.,2000; Jiang et al.,2001).

INHIBITORS OF ENDOGENOUS PROTEASES

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Different classes of endogenous proteases inhibitors regulate proteolytic processes that are required in the control of a variety of physiological functions. CV contain some protease inhibitors of the serpin class that are likely involved in modulating the protease activity of prohormone-processing enzymes. Thus, CV express both proteases and the molecular machinery for their fine-tuning.

Endopin 1

Endophin 1 is an endogenous protease inhibitor belonging to the class of serpins (serine protease inhibitors), which possesses homology to α1-antichymotrypsin. It has been demonstrated in bovine CV by Western blot analysis (Hwang et al.,1999). Immunofluorescence cytochemistry in cultured chromaffin cells shows a discrete, punctuate pattern of fluorescence staining that parallels the punctuate fluorescence of (Met)enkephalin, CGA (Hwang et al.,1999), and PTP. Experiments of cosecretion with PTP and (Met)enkephalin from cultured chromaffin cells demonstrate once more the functional localization of Endopin 1 to CV (Hwang et al.,1999). Endopin 1 (this name derives from its presence in neuroendocrine tissues and its serpin-like characteristic) is a 68–70 kD glycoprotein that possesses specificity for inhibiting basic residue-cleaving proteases. The functional activity in CV has been related to its strong inhibition of PTP.

Endopin 2

CV contain another serpin protease, endopin 2, related to α1-antichymotrypsin and possessing high homology to bovine elastase inhibitor (Hwang et al.,2000). Endopin 2 is a 72–73 kDa glycoprotein, which has been demonstrated to colocalize with enkephalin in in situ and isolated CV from bovine chromaffin cells (Hwang et al.,2002). Moreover, regulated secretion of endopin 2 from cultured chromaffin cells is induced by nicotine and KCl depolarization that once more demonstrates its intragranular localization.

TRANSMITTER PEPTIDES

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

As previously mentioned, chromaffin cells synthesize and store in their secretory granules different cytokines and peptide transmitters that are released by exocytosis upon cell stimulation. There is a marked species-specific variability in the cytokine and neuropeptide content of CV. In addition, discrete subpopulations of chromaffin cells within the adrenal medulla have the capacity to produce and release different combinations of these regulatory peptides. Here, we present a series of peptide transmitters, which have been identified in chromaffin cells and whose localization to CV has been conclusively demonstrated by microscopical and/or biochemical procedures.

Opioid Peptides

Enkephalins

CV contain enkephalins and release them upon stimulation. Enkephalin content is very elevated in human and cow CV (from 110 to 220 pmol/g) and low in rat CV (10–20 pmol/g). Enkephalins derive from a larger prohormone precursor, the preproenkephalin, by endopeptidase cleavage. Preproenkephalin consists of 267 amino acids and contains seven copies of the enkephalin peptide. The protein enters the endoplasmic reticulum as it is synthesized. There, it undergoes removal of an initial signal peptide and is transformed into proenkephalin-A. The proenkephalin-A then moves into the Golgi apparatus, where it is enclosed in CV. Here, proteolytic enzymes process it to yield free enkephalins. Proenkephalin-A degradation proceeds through an orderly series of steps. It appears to start initially with the removal of the C-terminal domain (Proenkephalin-A209–239) named peptide B. Several opioid peptides, including (Met)enkephalin and (Leu)enkephalin in the ratio 4:1, two C-terminally extended variants of enkephalins, the heptapeptide (Met)enkephalin-Arg-Phe, and the octapeptide (Met)enkephalin-Arg-Gly-Leu are liberated by cleavage of the precursor at pairs of basic residues (Goumon et al.,2000). Proenkephalin processing in vivo, however, is limited, because only 10% of (Met)enkephalin in adrenal medulla exists as completely processed pentapeptide and 90% of enkephalins in this tissue are present as high molecular mass intermediates of 10–25 kDa. A proenkephalin-A-derived peptide, corresponding to the biphosphorylated Proenkephalin-A209–237 and called enkelytin, is a potent inhibitor of Gram-positive bacteria (Goumon et al.,1998). Enkelytin is secreted by CV and released into the circulation in response to splanchnic nerve stimulation. As (Met)enkephalin has been shown to enhance immune reaction, the combined secretion of (Met)enkephalin and enkelytin by adrenal chromaffin cells appears as a highly beneficial survival strategy at the very beginning of a proinflammatory process (Goumon et al.,1998)

Endorphins

Whilst the majority of opioids in CV are enkephalin peptides, β-endorphin immunoreactivity represents a minor component, which is found principally in the form of β-endorphin1–31 (Nussdorfer,1996). This analgesic molecule has been demonstrated to be present in bovine and rat adrenal chromaffin cells as well as rat pheochromocytoma PC12 cells and eel chromaffin cells (Goumon and Stefano,2000). Recently, the highly analgesic morphine-derived molecule, morphine-6-glucuronide (M6G), has been detected in bovine CV and can be secreted by CV upon nicotinic stimulation (Goumon et al.,2006). Once secreted by CV into the circulation, M6G may mediate several systemic actions, for example, on the nervous system, immune cells and endothelial cells, based on its affinity for μ-opioid receptors. The analgesia observed upon acute stress, often related to enkephalin and corticoid release, might also be because of M6G secretion with PEBP acting as a transporter and molecular shield

Dynorphins

Analysis of dynorphin distribution among different subpopulations of bovine adrenal chromaffin cells demonstrates a prevailing localization in the noradrenaline enriched cell fraction (Dumont et al.,1983). It is costored with catecholamines and (Leu)enkephalin in CV whence it is released in response to acetylcholine, nicotine, and high potassium, but not muscarine (Dumont et al.,1983)

Neuropeptide Y

Neuropeptide Y (NPY) is a 36-amino acid peptide which, along with pancreatic polypeptide and peptide YY, belongs to the pancreatic polypeptide family. NPY derives from ProNPY, which undergoes cleavage at a single dibasic site Lys38-Arg39 resulting in the formation of 1–39 NPY that is further processed to NPY (Brakch et al.,1997). NPY has been detected in CV both by ultrastructural and biochemical procedures. Using immunogold electron microscopy, NPY has been found to colocalize with CGA, CGB, SgII, calcitonin gene-related peptide, and substance P in the CV of rat adrenal medulla (Laslop et al.,1989; Steiner et al.,1989; Murabayashi et al.,2007). Biochemical studies support immunohistochemical studies because NPY has been found to cosediment with catecholamines, (Met)enkephalin and bombesin in preparations of bovine CV. In this species, CV contain 1.9 μg NPY/mg protein, which gives 429 copies of NPY for a single granule (Fischer-Colbrie et al.,1986b). Recently, release of NPY by CV has directly been visualized by real-time evanescent field fluorescence microscopy (Perrais et al.,2004; Felmy,2007). Interestingly, NPY stimulates catecholamine secretion from chromaffin cells through the activation of NPY Y1 receptor (Cavadas et al.,2006).

Bombesin

Bombesin is a family of peptides firstly isolated in amphibians. Bombesin immunoreactivity has been detected in extracts from bovine adrenal medulla that also reacted with CGB and other CV components. Analysis of bombesin immunoreactivity in subpopulations of isolated bovine chromaffin cells reveals that the highest concentration of this peptide is to be found in a cell population enriched in noradrenaline (adrenaline/noradrenaline ratio of 0.6) (Lemaire et al.,1986). In bovine chromaffin cells, bombesin is mainly concentrated in CV (0.61 pmol/mg protein). Bombesin peptides have been implicated in the mediation and/or modulation of the stress response. Indeed, exogenous bombesin has been shown to exert trophic, antiulcerogenic, and anti-inflammatory actions that may minimize or reverse tissue damage against several injurious challenges (Yeğen,2003). For instance, bombesin improves IgA-mediated mucosal immunity with preservation of gut IL-4 in total parenteral nutrition-fed mice (Zarzaur et al.,2002). In addition, bombesin peptides are implicated in paracrine regulatory effects on the growth, structure and function of the adrenal cortex (Malendowicz and Markowska,1994).

Transforming Growth Factor-β

Transforming growth factor (TGF)-β is stored in CV and can be released by exocytosis. CV from bovine chromaffin cells contain ∼1 ng of TGF- β/10 ng of protein (Krieglstein and Unsicker,1995). TGF-β released by chromaffin cells is thought to operate via an autocrine/paracrine regulatory loop. Neutralization of endogenous TGF-β in quail embryos using a monoclonal antibody recognizing all three TGF-β isoforms (TGF-β1, -β2, and -β3) increases the number of TβH-immunoreactive adrenal chromaffin cells and TβH-positive cells incorporating 5′-bromo-2′deoxyuridine (Combs et al.,2000). Interestingly, Rahhal et al. (2004) observed a significant increase in the total number of TβH-positive cells in TGF-β2 (-/-) knockout mouse embryos at embryonic day (E) 18.5 compared with wild-type animals.

Neurotensin

Neurotensin (NT) is a 13-amino acid peptide, which exerts various central and peripheral effects, including hypotension, hypothermia, analgesia, and reduced locomotor activity. NT has been detected by immunocytochemistry in the chromaffin cells of rat adrenal medulla (Holgert et al.,1994). In the cat adrenal gland, a subpopulation of noradrenaline-storing cells has been shown to contain NT and its metabolites, the N-terminal octapeptide NT1–8, and NT1–12 (Rökaeus et al.,1984). In addition, subcellular fractionation of cat adrenal glands indicated that NT was stored in a large subcellular organelle, comigrating with CV, and was coreleased with noradrenaline after electrical stimulation of the splanchnic nerve (Rökaeus et al.,1984). NT directly inhibits aldosterone secretion of dispersed zona glomerulosa cells. When the integrity of the adrenal structure is preserved, NT increases mineralo- and glucocorticoid secretions, an effect that may be at least partly related to the increase in the gland blood flow (Nussdorfer,1996).

Secretoneurin

Secretoneurin is a highly conserved 33-amino acid peptide that derives from the processing of SgII. This peptide is stored in CV and, in bovine adrenochromaffin cells, its synthesis and release are regulated by PACAP liberated from splancnic nerve fibers innervating the adrenal medulla (Turquier et al.,2001). This peptide is implicated in angiogenesis and the modulation of inflammatory response (Fischer-Colbrie et al.,2005). It has chemoattractant effects on monocytes, eosinophils, fibroblasts, vascular smooth muscle cells, and endothelial cells (Wiedermann,2000).

EM66

EM66 is an evolutionary conserved secretoneurin flanking-peptide that also derives from cleavage of SgII. It has been found in the secretory granules of chromaffin cells in the rat, bovine, and human adrenal gland (Anouar et al.,1998; Montero-Hadjadje et al.,2003; Guillemot et al.,2006). Extensive studies in the rat adrenal medulla by electron microscopic immunocytochemistry reveal that EM66 occurs exclusively in adrenaline-synthesizing cells (Montero-Hadjadje et al.,2003). Both synthesis and release of EM66 are regulated by PACAP through activation of multiple signaling pathways (Guillemot et al.,2006). The occurrence of EM66 in islets of chromaffin cells within the adrenal cortex suggests that the peptide could function as a paracrine factor to regulate steroidogenic cells in the adrenal gland (Delarue et al.,2001).

Natriuretic Peptides

The natriuretic peptide family includes at least three peptides: ANF, the brain natriuretic peptide (BNP), and the C-type natriuretic peptide (CNP). Chromaffin cells produce, store in their secretory vesicles, and release all three members of the natriuretic peptide family (Nguyen et al.,1990; Babinski et al.,1995). Both the mature forms (ANF99–126, CNP82–103 and its elongated form CNP51–103, and BNP) and their precursor forms (the proANF (ANF1–126), the proCNP (CNP1–103), and PreBNP) have been identified in CV from bovine chromaffin cells and all molecules are cosecreted by cultured chromaffin cells in response to nicotinic activation or depolarizing agents (Nguyen et al.,1990). Natriuretic peptides exert an autocrine/paracrine inhibitory control on catecholamine secretion via the ANF-R2 receptor subtype expressed by chromaffin cells (Babinski et al.,1995). Natriuretic peptides inhibit aldosterone production by a direct action on the adrenal cortex both in vivo and in vitro. In addition, CNP exhibits more potent vasodilatatory effects in comparison to the other natriuretic peptides.

The Renin-Angiotensin System

Increasing evidence indicates that the adrenal medulla possesses an intrinsic renin-angiotensin system. Recently, subcellular fractionation approaches and ultrastructural immunocytochemistry have demonstrated that renin, angiotensin-converting enzyme, and angiotensin II are coexpressed in CV from cultured bovine chromaffin cells (Wang et al.,2002). Moreover, angiotensinogen mRNA has been detected within the same cultured cells. In addition, angiotensin Type 1 and angiotensin II receptors have been documented on bovine chromaffin cells (Wang et al.,2002). Angiotensin II has long been known as a stimulator of catecholamine release from adrenal chromaffin cells both in vivo and in vitro (Mazur-Ruder et al.,1979). Thus, adrenal medullary cells are targets for angiotensin II not only carried by the circulation but also generated locally by an intrinsic renin-angiotensin system.

Adrenomedullin

Adrenomedullin (AM) is a 52-amino acid multifunctional peptide with primarily hypotensive effect, which belongs to a peptide superfamily including calcitonin gene-related peptide and amylin. AM derives from a 185-amino acid prohormone called preproAM, which gives rise to another hypotensive peptide, proAM N-terminal 20 peptide (PAMP20). AM and PAMP20 are highly expressed in the mammalian adrenal medulla (Martinez and Cuttitta,1998). In addition to PAMP20, PAMP12, a 12-amino acid peptide with sequence identity to PAMP20 between amino acids 9–20, is also expressed in the adrenal medulla (Kobayashi et al.,2003). Both AM and PAMP peptides are released by regulated exocytosis along with catecholamines upon stimulation of adrenal chromaffin cells (Kobayashi et al.,2003). In addition, electron microscopy immunocytochemical procedures have clearly demonstrated that almost all the secretory granules of the adrenal medullary cells are specifically immunolabelled for AM (Yuchi et al.,2002). AM and PAMP peptides exert autocrine, paracrine, and endocrine effects. PAMP peptides modulate chromaffin cell function suppressing catecholamine release and synthesis by interfering with nicotinic cholinergic receptors. AM increases blood flow in the adrenal gland and causes a gradual release of catecholamines (Kobayashi et al.,2003). AM also expresses a mitogenic action on the zona glomerulosa of the adrenal cortex, probably through activation of CGRP-1 receptors. In addition, AM induces vasodilation through the activation of adenylate cyclase-coupled receptor of the CGRP-1 subtype (Samson,1999).

Calcitonon Gene-Related Peptide

Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide, which exerts a potent vasodilatory activity. In the rat and pig adrenal medulla, some chromaffin cells express CGRP immunoreactivity (Kuramoto et al.,1987; Kong et al.,1989). Double staining procedures suggest that CGRP-positive chromaffin cells contain adrenaline. In addition, all CGRP-storing cells also react for NPY. Ultrastructural immunocytochemistry localizes CGRP-immunoreactivity to CV. Thus, CGRP, NPY, and adrenaline are sequestered in the same CV. CGRP in adrenal gland may function as a local paracrine agent. Findings indicate that CGRP enhances both aldosterone and corticosterone release by adrenocortical cells (Mazzocchi et al.,1996).

Vasostatins

Vasostatins are two peptide fragments derived from post-translational proteolytic processing of CGA. The collective term of vasostatins refers to bovine N-terminal fragments CGA1–76 (vasostatin I) and CGA1–113 (vasostatin II). Both molecules have been purified from bovine CV (Lugardon et al.,2000). Vasostatins are predominantly generated in the matrix of CV and coreleased with catecholamines in the extracellular medium upon chromaffin cell stimulation. The name “vasostatins” was coined due to the vascular inhibitory activity of these molecules. Both vasostatin I and II, indeed, inhibit endothelin-induced contractions in human blood vessels. In addition, vasostatin I exerts autocrine inhibition on parathyroid cell secretion (Russell et al.,1994). Vasostatin I, which is liberated from stimulated chromaffin cells as well as immune cells upon stress, may also represent a new component of innate immunity. Indeed, it displays antibacterial activity against Gram-positive bacteria as well as the capacity to kill a large variety of filamentous fungi and yeast cells at micromolar concentrations (Lugardon et al.,2000). The C-terminal moiety of vasostatin I is essential for antifungine action. In addition, its disulfide bridge is crucial for vasostatin I antibacterial activity, whereas it is not required for antifungal activity. A recently discovered vasostatin I-derived fragment, “chromofungin” (bovine CGA47–66), expresses potent antifungal activity due to its ability to cross microorganism cell membranes and to inhibit the calmodulin-dependent activation of enzymes essential for the formation of cell wall and the fungal growth (Lugardon et al.,2001).

Catestatin and Cateslytin

Catestatin is a peptide fragment derived from proteolytic CGA cleavage. Processing of CGA to catestatin and catestatin storage takes place in CV (Taylor et al.,2000). A major form of catestatin is processed at dibasic sites (bovine CGA332–364 or human CGA340–372) while a smaller form with greater potency of catecholamine inhibition has also been identified (bovine CGA343–362) (Taylor et al.,2000). Catestatin is coreleased with catecholamines and other granule constituents upon chromaffin cell stimulation. This molecule blocks further catecholamine excretion because it acts as an antagonist of the nicotinic cholinergic receptor (Mahata et al.,1997). The name “catestatin” was just coined because of this profound catecholamine release-inhibitory property. Catestatin inhibits nicotinic release of not only endogenous catecholamines but also CGA, NPY, and ATP (Mahapatra et al.,2006). As catecholamine release increases blood pressure, this catestatin-induced mechanism of catecholamine inhibition is expected to exert profound systemic effects upon blood pressure with fundamental implications in the pathogenesis of essential hypertension (Kim and Loh,2005). Indeed, CVA gene knock-out mice develop hypertension, as the result of loss of catestatin inhibition upon blood pressure increase (Mahapatra et al.,2005). In addition, recovery of CGA gene knock-out mice through injection of catestatin or transgenic expression of human CGA clearly demonstrates a fundamental role for this peptide in the control of blood pressure (Mahapatra et al.,2005). Consistent with these data, the plasma concentration of catestatin in humans is diminished not only in established cases of essential hypertension but also in the still normotensive offsprings of patients with hypertension (O'Connor et al.,2002). Recently, the active domain of catestatin (bovine CGA344–358) has been shown to express a potent antimicrobial activity in the low micromolar range against bacteria, fungi, and yeasts (Briolat et al.,2005). This N-terminal sequence of catestatin has been named “cateslytin” and identified as a novel component of innate immunity (Briolat et al.,2005).

Chromacin

The term “chromacin” indicates a series of three antibacterial peptides, named chromacin-P, -G, and -PG, which derive from post-translational proteolytic processing of CGA (Strub et al.,1996). These peptides share the same 173–194 CGA sequence but differ in post-translational modifications. All express antibacterial activity against Gram-positive bacteria. These peptides originate from prochromacin, a large fragment corresponding to the bovine CGA79–431 segment. Prochromacin expresses antibacterial activity directed against both Gram-positive and Gram-negative bacteria (Strub et al.,1996).

Secretolytin

Secretolytin is a 13-residue peptide derived from proteolysis of CGB and corresponding to the precursor protein 614–626 C-terminal region (Strub et al.,1995). Secretolysin display potent antibacterial activity against Gram-positive microorganisms. Like other antimicrobial peptides, secretolytin is a positively charged molecule with net charge of +3 that allows binding to bacterial surfaces. This peptide is likely to lyse microorganisms by forming ion channels through the bacterial membrane (Strub et al.,1995).

Ubiquitin, Ubifungin, and Ubiquitin1–34

Ubiquitin is a peptide of 76 residues which can act as a regulated sorting signal at different steps of the endosomal and biosynthetic pathways (Hicke,2001). Recently, free ubiquinin has been identified in the granular matrix of CV from bovine adrenal medulla whence it is coreleased with catecholamines upon chromaffin cell stimulation (Kieffer et al.,2003). In general terms, it appears that ubiquitin may play a substantial role in responses to cell stress. Remarkably, ubiquitin displays in vitro antimicrobial properties. In particular, the C-terminal peptide fragment ubiquitin65–76, named “ubifungin,” displays potent antifungal activity at micromolar range (Metz-Boutigue et al.,2003). This cationic peptide interacts with the cell wall, destabilizes and crosses the fungal membrane, and eventually accumulates in the microorganism. In addition, another ubiquitin-derived fragment, the N-terminal peptide ubiquitin1–34, is blocked at the level of the cell wall and acts synergistically with ubiquitin65–76, killing fungi and yeasts (Kieffer et al.,2003).

MONOAMINES

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Catecholamines

Chromaffin cells store in and release from their CV three types of catecholamines: adrenaline, noradrenaline, and dopamine. According to the sequence postulated by Blaschko (1939), the biosynthesis of catecholamines involves hydroxylation of tyrosine into L-dihydroxyphenylalanine (L-DOPA), decarboxylation into dopamine, hydroxylation of dopamine into noradrenaline, and then methylation into adrenaline. The first two steps, that is, hydroxylation of tyrosine into L-DOPA and decarboxylation of L-DOPA to form dopamine, are reactions catalyzed by the cytosolic enzymes tyrosine hydroxylase (TH) and L-aromatic amino acid decarboxylase (AADC or DOPA decarboxylase), respectively. Dopamine is then transported into CV where the enzyme DβH converts it into noradrenaline via a side chain hydroxylation. Once synthesized, noradrenaline may remain stored within the CV in noradrenaline-containing chromaffin cells. Alternatively, noradrenaline is transported from the CV into the cytoplasm where it is methylated to form adrenaline by the enzyme phenylethanolamine-N-methyltransferase (PNMT). Once synthesized in the cytosol, adrenaline is then transported into the CV in the adrenaline-containing chromaffin cells.

The presence of catecholamines in chromaffin cells was first established in 1949 by Euler von and Hamberg (Euler von and Hamberg,1949). Quantitative estimation indicates that the catecholamine content within CV is 1.1–2.5 μmol/mg of protein, which corresponds to an intragranular concentration of 0.5–0.6 M, and a number of 3 × 106 molecules per granule (Winkler and Westhead,1980). Being the intragranular pH of 5.5–5.7, it means that 99% of the catecholamines are present in the protonated form (Winkler and Westhead,1980). The catecholamine content of CV is largely dependent upon the species examined. Purified CV from goat adrenal gland contain 51%–56% adrenaline, 42%–47% noradrenaline, and 1.3%–2% dopamine (Yamada et al.,1988) whilst in bovine CV adrenaline and noradrenaline account for 79.8% and 20.2%, respectively (Van Dyke et al.,1977). These values are almost the same as those found upon catecholamine release after stimulation by acetylcholine or nicotine or splanchnic nerve stimulation. In isolated bovine CV, catecholamine concentrations show marked seasonal variations, with lowest levels in the spring and highest levels in the winter (Bolstad et al.,1980).

Much has been investigated as to the way catecholamines are stored within CV. The low osmolality of CV in vivo suggests that catecholamines bind to the CV matrix through a weak binding. The whole observations support the view that the CV matrix functions as a weak cation-exchanger, with carboxyls as the cation-binding groups, and that catecholamines are stored in CV in a highly mobile aqueous environment.

Serotonin

Besides catecholamines, CV contain—at least in some species—another biogenic amine, serotonin. In the rat adrenal medulla, serotonin has been found in 75% of cells, that is, in adrenaline-storing cells (Holzwarth and Brownfield,1985). In this species, the adrenal serotonin content is 7.7 ± 0.1 μmol/Kg wet weight. Electron microscopy immunocytochemistry demonstrate that serotonin immunoreactivity is concentrated in the CV of adrenaline-containing cells (Brownfield et al.,1985). Indeed, serotonin colocalizes with the adrenaline synthesizing enzyme PNMT within CV. In an ontogenic perspective, serotonin immunostaining—calculated as number of positive cells and amount of serotonin immunostaining per cell—can be detected in the rat medullary cells at day 1 and progressively increases between 1 and 12 days of age. In the frog interrenal gland, serotonin immunostaining is localized in the CV of almost all adrenaline-producing cells, which represent about 90% of the total chromaffin cells (Delarue et al.,1988). Taken together, these results indicate that adrenaline-producing cells in the rat and frog adrenal glands contain significant amounts of serotonin. The observation of the storage of serotonin in CV of adrenaline-storing cells suggests that serotonin may be released with catecholamines under stress conditions.

OTHER CONSTITUENTS OF CHROMAFFIN GRANULES

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Some further constituents have been recognized in CV. Among these, peptides (such as arginin-vasopressin, substance P, vasoactive intestinal polypeptide, guanylin, and galanin), nucleotides, ascorbic acid, coenzyme A glutathione disulphide, Alzheimer amyloid precursors, mucopolysaccharides (chondroitin-4 and -6-sulfate), and ions, in particular calcium (Table 1).

Table 1. Minor components of chromaffin vesicles
ComponentFunction
Arginin-vasotocinStimulation of catecholamine secretion, stimulation of zona glomerulosa cell secretion
Substance PUnknown
Vasoactive intestinal polypeptideStimulation of catecholamine release, stimulation of adrenal steroid secretion
GuanylinInhibition of aldosterone secretion
GalaninStimulation of aldosterone secretion, trophism of zona fasciculata
Coenzyme A glutathione disulfideVasoconstriction, modulation of angiotensin II effects
Alzheimer amyloid precursorsUnknown
Nucleotides (ATP, GTP, UTP, ADP, AMP, GDP, and UDP)Formation of intravesicular complexes with catecholamines, intravesicular buffer function, reduction of intravesicular osmotic pressure, neuromodulation, modulation of inflammation, and regulation of blood flow
Ascorbic acidRegulation of dopamine-β-hydroxylase activity
Ions (Ca2+, Mg+2, K+, Na,+ and Cl)Mainly regulation of exocytosis
Mucopolysaccarides (chondroitin-4 and -6-sulfate)Structural function

CONCLUSION AND FURTHER PERSPECTIVES

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED

Chromaffin Vesicle Heterogeneity

CV exhibit an intrinsic variability of composition, not only among different species but even among distinct chromaffin cells in the same individual. We wish to report Kondo's words: “the coexistence of peptides and catecholamines is a constant feature of the chromaffin cells. The coexistence of multiple peptides is also seen in some chromaffin cells. This indicates that there are several subpopulations among the chromaffin cells in terms of bioactive substances contained” (Kondo,1985). For instance, differences in the quantitative composition of CV in adrenaline- and noradrenaline-containing cells have been reported. In bovine adrenal medulla, noradrenaline CV contain slightly more SgII but much less CGA than adrenaline CV (Weiss et al.,1996). Proteolytic processing of CGA and SgII is higher in noradrenaline cells, which is paralleled by a higher content of the prohormone convertase PC2. No differences have been found for DβH, prohormone convertase PC1, carboxypeptidase H and synaptophysin (Weiss et al.,1996). As previously mentioned, SgVI has been found to localize to CV in adrenaline-storing chromaffin cells of the bovine adrenal medulla but not noradrenaline-containing cells (Fischer-Colbrie et al.,2002). Enkephalins in the pig adrenal medulla are mostly localized to adrenaline-storing chromaffin cells, some of which also contain CGRP (Kong et al.,1989). By contrast, serotonin coincides with noradrenaline cells in this species. Thus, stimuli activating noradrenaline chromaffin cells would release serotonin while stimulation of adrenaline storage cells would release enkephalins and, to a lesser extent, CGRP. These results indicate that the secretory cocktail of peptides released from chromaffin cells may differ significantly between adrenaline- and noradrenaline-storing cells. The functional implications of such divergence in CV composition are to be carefully investigated in the future.

Distinct Control of Chromaffin Vesicle Release

Remarkably, individual components of CV are subjected to distinct control mechanisms. Nicotine and cAMP simultaneously increase enkephalins and vasoactive intestinal polypeptide (VIP) in cultured bovine chromaffin cells (Pruss et al.,1985). In the same experimental conditions, however, phorbol esters can specifically elevate VIP without changing the amount of enkephalins. In addition, interleukin (IL)-1α increases VIP content in chromaffin cells, whereas it leaves unaffected neurotensin and substance P levels and decreases (Met)enkephalin content (Eskay and Eiden,1992). Tumour necrosis factor-α also demonstrates a neuropeptide-specific pattern of modulation of chromaffin cell neuropeptide levels similar to those seen with IL-1α (Eskay and Eiden,1992). Although CGA is secreted in parallel with catecholamines and enkephalins in response to secretagogues, CGA levels are essentially unaffected by any of the agents which increase enkephalin and VIP levels (Waschek et al.,1987). This may indicate that the biosynthesis of proteins that are coupled with the production or assembly of the CV itself are expressed constitutively by the chromaffin cells in the presence or absence of positive regulators of other systems.

Thus, CV are heterogeneous structures. There are subtle variations in their content, which imply different regulation in their biosynthetic pathways as well as different response profile to distinct secretagogues. To make the picture even more complicated, CV have been found to express considerable seasonal variations in their releasable constituents, such as catecholamines, calcium, and enzymes (Bolstad et al.,1980). The concept of CV structural heterogeneity fits with physiological evidence of a heterogeneous population of CV. Recent experimental findings are emerging, which indicate a bimodal distribution of CV size and suggest a double granule population in mouse chromaffin cells (Grabner et al.,2005). If the large CV are less fusigenic, then weak stimulation would preferentially release small CV, whereas strong stimulation would release both large and small CV. In this way, chromaffin cells would have tight control of low levels of release, as each CV that fuses with the plasma membrane only releases a small amount of transmitter, but strong stimulation is able to release large amounts of catecholamines for rapid responses to stress or danger (Grabner et al.,2005). In addition, the use of fluorescent proteins specifically targeted to CV identifies distinct pools of CV, segregated according to age and distinguished by intracellular location, mobility, and priority in recruitment for exocytosis (Duncan et al.,2003). Newly assembled CV undergo rapid transport to the plasma membrane, where they are preferentially selected for exocytosis. CV that do not undergo exocytosis are eventually (in less than 16 hr) removed from the membrane, transported back into the cells and equilibrated with the large reserve CV pool, becoming indistinguishable from the pre-existing CV (Duncan et al.,2003).

Toward a Unifying Concept of Chromaffin Vesicle Function

The adrenal medulla has long been regarded as the effector arm of acute stress response. The picture we have here illustrated allows us to sketch some basic lineages concerning the biological significance of CV. The unique molecular composition of these secretory organelles implies different functional targets extending the complexity of the involvement of chromaffin cell in acute stress reactions (Fig. 3).

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Figure 3. Schematic drawing illustrating the multiplicity of functions provided by chromaffin vesicle constituents.

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Constituents of CV appear primarily implicated in the expression and/or modulation of the different aspects of acute stress response. CV contain molecules involved in the metabolic reaction to stress. Catecholamines are the major compounds but a hormonal or paracrine role for NPY and galanin in the response to hypoglycaemic shock has been proposed. In addition, CV content of ubiquitin supports the view that these secretory organelles may play a substantial role also in responses to cell stress.

As to the kind of response which accompanies infectious and inflammatory events, we have seen that CV contain a series of molecules actively involved in immunological reactions. Cell adhesion and chemotaxis, transendothelial extravasation, and migration of leukocytes are important endothelium-dependent functions of relevance for the fight of inflammation and tissue disruption. CGA-derived vasostatins and secretoneurin may interact in concerted patterns, which are still to be fully understood. Besides, the enhanced immune reactivity caused by the combined secretion of (Met)enkephalin and bombesin appears as a highly beneficial survival strategy during inflammation that may minimize or reverse tissue damage against several injurious challenges. Thus, modulation of inflammatory responses and activation of inflammation-related angiogenesis are integrated functions, which may be partly orchestrated by chromaffin cells during stress reactions caused by inflammatory processes. In addition, the large array of antimicrobial and antifungine peptides contained in CV suggests a profound involvement of the adrenal medulla in the responses to infective agents.

The analgesic activity of adrenal medulla is foundamental during stress-induced reactions. Highly analgesic molecules, like (Met)enkephalin, morphine, and NT has been demonstrated in CV. The recent discovery of the morphine-derived molecule M6G in bovine CV suggests that the analgesia observed upon acute stress, often related to enkephalin and corticoid release, might also be due to M6G secretion. Once secreted by CV into the circulation, M6G may mediate several systemic actions on the nervous system, immune cells, and endothelial cells based on its affinity for μ-opioid receptors.

Chromaffin cells intervene in the response to stress induced by physical exertion. The increase in circulating t-PA during physical exercise prompts some considerations. Catecholamines, primarily neradrenaline, stimulate t-PA release into the circulation, presumably by their action on vascular endothelium. During exercise, only ∼50% of the increase in plasma t-PA concentrations can be attributed to this mechanism. We have seen that CV contain t-PA. Therefore, release of CV contents during the sympathoadrenal activation that accompanies physical exercise may enhance the profibrinolytic capability of plasma by the direct release of t-PA and, secondarily, by catecholamine secretion (Parmer et al.,1997).

A number of molecules released by CV exerts important functions on blood pressure. Cathecolamines are implicated in different aspects of blood pressure regulation although hypertensive effects by and large prevail. As previously mentioned, augmented synthesis and storage of catecholamines have been found in CV from spontaneous hypertensive rats and normal rats after induction of renovascular hypertension (Takiyyuddin et al.,1993). The adrenal medulla possesses an intrinsic renin-angiotensin system, which also provides the molecular constituents capable of increasing blood pressure. Besides, coenzyme A glutathione disulfide has a direct vasopressor activity. It also potentiates the hypertensive effect of angiotensin II. By contrast, natriuretic peptides possess vasodilatatory effects. They also exert potent inhibitory action on the renin-angiotensin-aldosterone system promoting, at the same time, inhibition of catecholamine secretion. In addition, AM and CGRP induce vasodilation and hypotension through the activation of adenylate cyclase-coupled receptor of the CGRP-1 subtype. In humans, catestatin plasma levels are diminished at an early stage of hypertension (O'Connor et al.,2002). Moreover, low catestatin concentrations are inversely related to adrenergic pressor responses, suggesting that diminished catestatin levels might increase the risk of later development of hypertension. Likewise, NT and vasostatins have been implicated in hypotension. Both vasostatin I and II provide vascular inhibitory activity by inhibiting endothelin-induced contractions in human blood vessels.

The high concentration of regulatory peptides in CV suggests that chromaffin cells may exert paracrine, modulatory functions on both neighboring chromaffin cells in the adrenal medulla and steroidogenic cells in the adrenal cortex. NPY released by CV is capable to modulate catecholamine secretion from chromaffin cells. As we have seen, this peptide either decreases catecolamine release by blocking the nicotinic receptor ion channel, or stimulates catecholamine secretion through activation of NPY Y1 receptor. In addition, VIP appears to act as an autocrine neurotransmitter being a potent activator of catecholamine release. In contrast, the catestatin fragment of CGA has emerged as a novel regulatory peptide for autocrine, negative feed-back control of catecholamine release from chromaffin cells. Thus, CV include the molecular machinery for the fine-tuning of catecholamine secretion. The modulatory effect of different compounds stored in CV, such as bombesin, NT, guanylin and galanin implicates important activities of chromaffin cells on glucocorticoid and mineralcorticoid production.

In conclusion, CV appear as a versatile, multifunctional secretory organelle involved in the different aspects of stress response. Its assorted content of regulatory and transmitter peptides with important functions on the modulation of adrenal cortex activity and the regulation of blood pressure and salt balance makes CV an adaptable, multipurpose cell structure of expanding biological significance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. BIOLOGY OF THE CHROMAFFIN VESICLE
  4. GRANINS
  5. GLYCOPROTEINS
  6. PROHORMONE-PROCESSING ENZYMES
  7. INHIBITORS OF ENDOGENOUS PROTEASES
  8. TRANSMITTER PEPTIDES
  9. MONOAMINES
  10. OTHER CONSTITUENTS OF CHROMAFFIN GRANULES
  11. CONCLUSION AND FURTHER PERSPECTIVES
  12. LITERATURE CITED
  • Anouar Y,Desmoucelles C,Yon L,Leprince J,Breault I,Gallo-Payet N,Vaudry H. 1998. Identification of a novel secretogranin II-derived peptide (SgII (187–252)) in adult and fetal human adrenal glands using antibodies raised against the human recombinant peptide. J Clin Endocrinol Metab 83: 29442951.
  • Aunis D. 1998. Exocytosis in chromaffin cells of the adrenal medulla. Int Rev Cytol 181: 213320.
  • Azaryan AV,Krieger TJ,Hook VYH. 1995a. Purification and characteristics of the candidate prohormone processing proteases PC2 and PC1/3 from bovine adrenal medulla chromaffin granules. J Biol Chem 270: 82018208.
  • Azaryan AV,Schiller M,Mende-Müller M,Kook VYH. 1995b. Characteristics of the chromaffin granule apartic proteinase involved in proenkephalin processing. J Neurochem 65: 17711779.
  • Babinski K,Haddad P,Vallerand D,McNicoll N,De Lean A,Ong H. 1995. Natriuretic peptides inhibit nicotine-induced whole-cell currents and catecholamine secretion in bovine chromaffin cells: evidence for the involvement of the atrial natriuretic factor R2 recptors. J Neurochem 64: 10801087.
  • Beuret N,Stettler H,Renold H,Rutishauser J,Spiess M. 2004. Expression of regulated secretory proteins is sufficient to generate granule-like structures in constitutively secreting cells. J Biol Chem 279: 2024220249.
  • Blaschko H. 1939. The specific action of L-dopa decarboxylase. J Physiol Lond 96: 50P51P.
  • Blaschko H,Welch AD. 1953. Localization of adrenaline in cytoplasmic particles of the bovine adrenal medulla. Naunyn-Schmiedeberg's Arch Pharmacol 219: 1722.
  • Bolstad G,Helle KB,Serck-Hanssen G. 1980. Heterogeneity in the adrenomedullary storage of catecholamines, ATP, calcium and releasable dopamine beta-hydroxylase activity. J Auton Nerv Syst 2: 337354.
  • Brakch N,Rist B,Beck-Sickinger AG,Goenaga J,Wittek R,Bürger E,Brunner HR,Grouzmann E. 1997. Role of prohormone convertases in pro-neuropeptide Y processing: coexpression and in vitro kinetic investigations. Biochemistry 23: 1630916320.
  • Briolat J,Wu SD,Mahata SK,Gonthier B,Bagnard D,Chasserot-Golaz S,Helle KB,Aunis D,Metz-Boutigue MH. 2005. New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A. Cell Mol Life Sci 62: 377385.
  • Brownfield MS,Poff BC,Holzwarth MA. 1985. Ultrastructural immunocytochemical co-localization of serotonin and PNMT in adrenal medullary vesicles. Histochemistry 83: 4146.
  • Burré J,Volknandt W. 2007. The synaptic vesicle proteome. J Neurochem 101: 14481462.
  • Carmichael SW. 1983. The adrenal chromaffin vesicle: an historical perspective. J Auton Nerv Syst 7: 712.
  • Carmichael SW,Stoddard SL,O'Connor DT,Yaksh TL,Tyce GM. 1990. The secretion of catecholamines, chromogranin A and neuropeptide Y from adrenal medulla of the cat via the adrenolumbar vein and thoracic duct: different anatomic routes based on size. Neuroscience 34: 433440.
  • Cavadas C,Cefai D,Rosmaninho-Salgado J,Vieira-Coelho MA,Moura E,Busso N,Pedrazzini T,Grand D,Rotman S,Waeber B,Aubert JF,Grouzmann E. 2006. Deletion of the NPY Y1 receptor gene reveals a regulatory role of neuropeptide Y on catecholamine synthesis and secretion. Proc Natl Acad Sci USA 103: 1049710502.
  • Cawley NX,Zhou J,Hill JM,Abebe D,Romboz S,Yanik T,Rodriguiz RM,Wetsel WC,Loh YP. 2004. The carboxypeptidase E knockout mouse exhibits endocrinological and behavioral defects. Endocrinology 145: 58075819.
  • Combs SE,Krieglstein K,Unsicker K. 2000. Reduction of endogenous TGF-beta increases proliferation of developing adrenal chromaffin cells in vivo. J Neurosci Res 59: 379383.
  • Cool DR,Normant E,Shen FS,Chen HC,Pannell L,Zhang Y,Loh YP. 1997. Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpefat mice. Cell 88: 7383.
  • Coupland RE. 1965. Electron microscopic observations on the structure of the rat adrenal medulla. I. The ultrastructure and organization of chromaffin cells in the normal adrenal medulla. J Anat 99: 231254.
  • Courel M,Rodemer C,Nguyen ST,Pance A,Jackson AP,O'Connor DT,Taupenot L. 2006. Secretory granule biogenesis in sympathoadrenal cells. Identification of a granulogenic determinant in the secretory prohormone chromogranin A. J Biol Chem 281: 3803838051.
  • Delarue C,Contesse V,Lenglet S,Sicard F,Perraudin V,Lefebvre H,Kodjo M,Leboulenger F,Yon L,Gallo-Payet N,Vaudry H. 2001. Role of neurotransmitters and neuropeptides in the regulation of the adrenal cortex. Rev Endocr Metab Disord 2: 253267.
  • Delarue C,Leboulenger F,Morra M,Hery F,Verhofstad AJ,Berod A,Denoroy L,Pelletier G,Vaudry H. 1988. Immunohistochemical and biochemical evidence for the presence of serotonin in amphibian adrenal chromaffin cells. Brain Res 459: 1726.
  • Dikeakos JD,Reudelhuber TL. 2007. Sending proteins to dense-core secretory granules: still a lot to sort out. J Cell Biol 177: 191196.
  • Dumont M,Day R,Lemaire S. 1983. Distinct distribution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal chromaffin cells. Life Sci 32: 287294.
  • Duncan RR,Greaves J,Wiegand UK,Matskevich I,Bodammer G,Apps DK,Shipston MJ,Chow RH. 2003. Functional and spatial segregation of secretory vesicle pools according to vesicle age. Nature 422: 176180.
  • Eisenhofer G,Kopin IJ,Goldstein DS. 2004. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. Pharmacol Rev 56: 331349.
  • Eskay RI,Eiden LE. 1992. Interleukin-1 alpha and tumor necrosis factor-alpha differentially regulate enkephalin, vasoactive intestinal polypeptide, neurotensin, and substance P biosynthesis in chromaffin cells. Endocrinology 130: 22522258.
  • Euler von US,Hamberg U. 1949. 1-Noradrenaline in the suprarenal medulla. Nature (London) 163: 642643.
  • Felmy F. 2007. Modulation of cargo release from dense core granules by size and actin network. Traffic 8: 983997.
  • Fischer-Colbrie R,Diez-Guerra J,Emson PC,Winkler H. 1986b. Bovine chromaffin granules: immunological studies with antisera against neuropeptide Y, [Met]enkephalin and bombesin. Neuroscience 18: 167174.
  • Fischer-Colbrie R,Eder S,Lovisetti-Scamihorn P,Becker A,Laslop A. 2002. Neuroendocrine secretory protein 55: a novel marker for the constitutive secretory pathway. Ann NY Acad Sci 971: 317322.
  • Fischer-Colbrie R,Hagn C,Kilpatrick L,Winkler H. 1986a. Chromogranin C: a third component of the acidic proteins in chromaffin granules. J Neurochem 47: 318321.
  • Fischer-Colbrie R,Kirchmair R,Kahler CM,Wiedermann CJ,Saria A. 2005. Secretoneurin: a new player in angiogenesis and chemotaxis linking nerves, blood vessels and the immune system. Curr Protein Pept Sci 6: 373385.
  • Fugere M,Day R. 2006. Cutting back on pro-protein convertases: the last approaches to pharmacological inhibition. Trends Pharmacol Sci 26: 294301.
  • Goumon Y,Lugardon K,Gadroy P,Strub JM,Welters ID,Stefano GB,Aunis D,Metz-Boutigue MH. 2000. Processing of proenkephalin-A in bovine chromaffin cells. Identification of naturally derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Biol Chem 275: 3835538362.
  • Goumon Y,Lougardon K,Kieffer B,Lefevre JF,van Dorsselaer A,Aunis D,Metz-Boutigue MH. 1998. Characterization of antibacterial COOH-terminal proenkephalin-A-derived peptides (PEAP) in infectious fluids. Importance of enkelytin, the antibacterial PEAP209–237 secreted by stimulated chromaffin cells. J Biol Chem 273: 2984729846.
  • Goumon Y,Muller A,Glattard E,Marban C,Gasnier C,Strub GM,Chasserot-Golaz S,Rohr O,Stefano GB,Welters ID,Van Dorsselaer A,Schoentgen F,Aunis D,Metz-Boutigue MH. 2006. Identification of morphine-6-glucuronide in chromaffin cell secretory granules. J Biol Chem 281: 80828089.
  • Goumon Y,Stefano GB. 2000. Identification of morphine in the rat adrenal gland. Mol Brain Res 77: 267269.
  • Grabner CP,Price SD,Lysakowski A,Fox AP. 2005. Mouse chromaffin cells have two populations of dense core vesicles. J Neurophysiol 94: 20932104.
  • Guillemot J,Ait-Ali D,Turquier V,Montero-Hadjadje M,Fournier A,Vaudry H,Anouar Y,Yon L. 2006. Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells. Regul Pept 137: 7988.
  • Helle KB. 2004. The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects. Biol Rev 79: 769794.
  • Henle J. 1865. Über das Gewebeder Nebenniere und der Hypophyse. J Ration Med 24: 143152.
  • Hicke L. 2001. A new ticket for entry into budding vesicles-ubiquitin. Cell 106: 527530.
  • Hillarp NA,Hökfelt B,Nilson B. 1954. The cytology of the adrenal medullary cell with special reference to the storage and secretion of the sympathomimeticamines. Acta Anat 21: 155167.
  • Hillarp NA,Lagerstedt L,Nilson B. 1953. The isolation of a granular fraction from the suprarenal medulla, containing the sympathomimetic catecholamines. Acta Physiol Scand 29: 251263.
  • Holgert H,Dagerlind A,Hökfelt T,Lagercrantz H. 1994. Neuronal markers, peptides and enzymes in the nerves and chromaffin cells in the rat adrenal medulla during postnatal development. Brain Res Dev Brain Res 83: 3552.
  • Holzwarth MA,Brownfield MS. 1985. Serotonin coexists with epinephrine in rat adrenal medullary cells. Neuroendocrinology 41: 230236.
  • Hook V,Funkelstein L,Lu D,Bark S,Wegrzyn J,Hwang SR. 2008. Proteases for processing proneuropeptides into peptide neurotransmitters and hormones. Annu Rev Pharmacol Toxicol 48: 393423.
  • Hook VYH,Schiller MR,Azaryan AV. 1996. The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates. Arch Biochem Biophys 328: 107114.
  • Huh YH,Jeon SH,Yoo SH. 2003. Chromogranin B-induced secretory granule biogenesis. Comparison with the similar role of chromogranin A. J Biol Chem 278: 4058140589.
  • Hwang SR,Garza C,Mosier C,Toneff T,Wunderlich E,Goldsmith P,Hook V. 2007a. Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion. J Biol Chem 282: 95569563.
  • Hwang SR,O'Neill A,Bark S,Foulon T,Hook V. 2007b. Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules. J Neurochem 100: 13401350.
  • Hwang SR,Steineckert B,Hook VY. 2000. Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cysteine-374 for dithiothreitol-sensitive inhibition of elastase. Biochemistry 39: 89448952.
  • Hwang SR,Steineckert B,Toneff T,Bundey R,Logvinova AV,Goldsmith P,Hook VY. 2002. The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells. Biochemistry 41: 1039710405.
  • Hwang SR,Steineckert B,Yasothornsrikul S,Sei CA,Toneff T,Rattan J,Hook VYH. 1999. Molecular cloning of endopin 1, a novel serpin localized to neurosecretory vesicles in chromaffin cells. J Biol Chem 274: 3416434173.
  • Jiang Q,Taupenot L,Mahata SK,Mahata M,O'Connor DT,Miles LA,Parmer RJ. 2001. Proteolytic cleavage of chromogranin A (CGA) by plasmin. Selective liberation of a specific bioactive CGA fragment that regulates catecholamine release. J Biol Chem 276: 2502225029.
  • Kieffer AE,Goumon Y,Ruh O,Chasserot-Golaz S,Nullans G,Gasnier C,Aunis D,Metz-Boutigue MH. 2003. The N- and C-terminal fragments of ubiquitin are important for antimicrobial activities. FASEB J 17: 776778.
  • Kim T,Loh YP. 2005. Chromogranin A: a surprising link between granule biogenesis and hypertension. J Clin Invest 115: 17111713.
  • Kim T,Loh YP. 2006. Protease nexin-1 promotes secretory granule biogenesis by preventing granule protein degradation. Mol Biol Cell 17: 789798.
  • Kim T,Tao-Cheng JH,Eiden LE,Loh YP. 2001. Chromogranin A, an “on/off” switch controlling dense-core secretory granule biogenesis. Cell 106: 499509.
  • Kim T,Zhang CF,Sun Z,Wu H,Loh YP. 2005. Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis. J Neurosci 25: 69586961.
  • Kobayashi H,Yanagita T,Yokoo H,Wada A. 2003. Pathophysiological function of adrenomedullin and proadrenomedullin N-terminal peptides in adrenal chromaffin cells. Hypertens Res 26 Suppl: 7178.
  • Kohn A. 1902. Das chromaffine Gewebe. Ergebn Anat Entwick 12: 253348.
  • Kondo H. 1985. Immunohistochemical analysis of the localization of neuropeptides in the adrenal grand. Arch Histol Jpn 48: 453481.
  • Kong JY,Thureson-Klein A,Klein RL. 1989. Differential distribution of neuropeptides and serotonon in pig adrenal glands. Neuroscience 28: 765775.
  • Krieglstein K,Unsicker K. 1995. Bovine chromaffin cells release a transforming growth factor-beta-like molecule contained within chromaffin granules. J Neurochem 65: 14231426.
  • Kryvi H,Flatmark T,Terland O. 1979. Comparison of the ultrastructure of adrenaline and noradrenaline storage granules of bovine adrenal medulla. Eur J Cell Biol 20: 7682.
  • Kuramoto H,Kondo H,Fujita T. 1987. Calcitonin gene-related peptide (CGRP)-like immunoreactivity in scattered chromaffin cells and nerve fibers in the adrenal gland of rats. Cell Tissue Res 247: 309315.
  • Laslop A,Weiss C,Savaria D,Eiter C,Tooze SA,Seidah NG,Winkler H. 1998. Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases. J Neurochem 70: 374383.
  • Laslop A,Wohlfarter T,Fischer-Colbrie R,Steiner HJ,Humpel C,Saria A,Schmid KW,Sperk G,Winkler H. 1989. Insulin hypoglycaemia increases the levels of neuropeptide Y and calcitonin gene-related peptide, but not of chromogranin A and B, in rat chromaffin granules. Regul Pept 26: 191202.
  • Lemaire S,Chouinard L,Mercier P,Day R. 1986. Bombesin-like immunoreactivity in bovine adrenal medulla. Regul Pept 13: 133146.
  • Lewis EJ,Asnani LP. 1992. Soluble and membrane forms of dopamine β-hydroxylase are encoded by the same mRNA. J Biol Chem 267: 494500.
  • Lugardon K,Chasserot-Golaz S,Kieffer AE,Maget-Dana R,Nullans G,Kieffer B,Aunis D,Metz-Boutigue MH. 2001. Structural and biological characterization of chromofungin, the antifungal chromograninA-(47–66)-derived peptide. J Biol Chem 276: 3587535882.
  • Lugardon K,Raffner R,Goumon Y,Corti A,Delmas A,Bulet P,Aunis D,Metz-Boutigue MH. 2000. Antibacterial and antifungal activities of vasostatin-1, the N-terminal fragment of chromogranin A. J Biol Chem 275: 1074510753.
  • Mahapatra NR,O'Connor DT,Vaigankar SM,Hikim AP,Mahata M,Ray S,Staite E,Wu H,Gu Y,Dalton N,Kennedy BP,Ziegler MG,Ross J,Mahata SK. 2005. Hypertension from targeted abation of chromogranin A can be rescued by the human ortholog. J Clin Invest 115: 19421952.
  • Mahapatra NR,Mahata M,Mahata SK,O'Connor DY. 2006. The chromogranin A fragment catestatin: specificity, potency and mechanism to inhibit exocytotic secretion of multiple catecholamine storage vesicle co-transmitters. J Hypertens 24: 895904.
  • Mahata SK,O'Connor DT,Mahata M,Yoo SH,Taupenot L,Wu H,Gill BM,Parmer RJ. 1997. Novel autocrine feedback control of catecholamine release. A discrete chromogranin fragment is a noncompetitive nicotinic cholinergic antagonist. J Clin Invest 100: 16231633.
  • Malendowicz LK,Markowska A. 1994. Neuromedins and their involvement in the regulation of growth, structure and function of the adrenal cortex. Histol Histopathol 9: 591601.
  • Martinez A,Cuttitta F. 1998. Adrenomedullin. Amsterdam: IOS Press. p 1391.
  • Mazur-Ruder M,Feuerstein G,Roll D,Gutman Y. 1979. Selective reduction of adrenal medulla response to angiotensin induced by suppression of renin-angiotensin. Eur J Pharmacol 59: 261266.
  • Mazzocchi G,Musajo FG,Neri G,Gottardo G,Nussdorfer GG. 1996. Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: comparison with calcitonin gene-related peptide effects. Peptides 17: 853857.
  • Metz-Boutigue MH,Kieffer AE,Goumon Y,Aunis D. 2003. Innate immunity: involvement of new neuropeptides. Trends Microbiol 11: 585592.
  • Montero-Hadjadje M,Pelletier G,Yon L,Li S,Guillemont J,Magoul R,Tillet Y,Vaudry H,Anouar Y. 2003. Cytochemical localization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands. J Histochem Cytochem 51: 10831095.
  • Montesinos MS,Machado JD,Camacho M,Diaz J,Morales YG,Alvarez de la Rosa D,Carmona E,Castaneyra A,Viveros OH,O'Connor DT,Mahata SK,Borges R. 2008. The crucial role of chromogranins in storage and exocytosis revealed using chromaffin cells from chromogranin A null mouse. J Neurosci 28: 33503358.
  • Mosley CA,Taupenot L,Biswas N,Taulane JP,Olson NH,Vaingankar SM,Wen G,Schork NJ,Ziegler MG,Mahata SK,O'Connor DT. 2007. Biogenesis of the secretory granule: chromogranin A coiled-coil structure results in unusual physical properties and suggests a mechanism for granule core condensation. Biochemistry 46: 1099911012.
  • Murabayashi H,Kuramoto H,Kawano H,Sasaki M,Kitamura N,Miyakawa K,Tanaka K,Oomori Y. 2007. Immunohistochemical features of substance P-immunoreactive chromaffin cells and nerve fibers in the rat adrenal gland. Arch Histol Cytol 70: 183196.
  • Nguyen TT,Babinski K,Ong H,De Lean A. 1990. Differential regulation of natriuretic peptide biosynthesis in bovine adrenal chromaffin cells. Peptides 11: 973978.
  • Nussdorfer GG. 1996. Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev 48: 495530.
  • O'Connor DT,Kailasam MT,Kennedy BP,Ziegler MG,Yanaihara N,Parmer RJ. 2002. Early decline in the catecholamine release-inhibitory peptide catestatin in humans at genetic risk of hypertension. J Hypertens 20: 13351345.
  • Ornberg RL,Duong LT,Pollard HB. 1986. Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells. Cell Tissue Res 245: 547553.
  • Palfrey HC,Artalejo AR. 2003. Secretion: kiss and run caught on film. Curr Biol 13: R397R399.
  • Parmer RJ,Mahata M,Gong Y,Mahata SK,Jiang Q,O'Connor DT,Xi XP,Miles LA. 2000. Processing of chromogranin A by plasmin provides a novel mechanism for regulating catecholamine secretion. J Clin Invest 106: 907915.
  • Parmer RJ,Mahata M,Mahata S,Sebald MT,O'Connor DT,Miles LA. 1997. Tissue plasminogen activator (t-PA) is targeted to the regulated secretory pathway. Catecholamine storage vesicles as a reservoir for the rapid release of t-PA. J Biol Chem 272: 19761982.
  • Perrais D,Kleppe IC,Taraska JW,Almers W. 2004. Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells. J Physiol 560: 413428.
  • Pruss RM,Moskal JR,Eiden LE,Beinfeld MC. 1985. Specific regulation of vasoactive intestinal polypeptide biosynthesis by phorbol ester in bovine chromaffin cells. Endocrinology 117: 10201026.
  • Rahhal B,Dünker N,Combs S,Krieglstein K. 2004. Isoform-specific role of transforming growth factor-beta2 in the regulation of proliferation and differentiation of murine adrenal chromaffin cells in vivo. J Neurosci Res 78: 493498.
  • Reigada D,Diez-Perez I,Gorostiza P,Verdaguer A,Gomez de Aranda I,Pineda O,Vilarrasa J,Marsal J,Blasi J,Aleu J,Solsona C. 2003. Control of neurotransmitter release by an internal gel matrix in synaptic vesicles. Proc Natl Acad Sci USA 100: 34853490.
  • Rökaeus A,Fried G,Lundberg JM. 1984. Occurrence, storage and release of neurotensin-like immunoreactivity from the adrenal gland. Acta Physiol Scand 120: 373380.
  • Russell J,Gee P,Liu SM,Angeletti RH. 1994. Inhibition of parathyroid hormone secretion by amino-terminal chromogranin peptides. Endocrinology 135: 227342.
  • Samson WK. 1999. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 61: 363390.
  • Shen FS,Loh YP. 1997. Intracellular misrouting of abnormal secretion of adrenocorticotropin and growth hormone in cpefat mice associated with a carboxypeptidase E mutation. Proc Natl Acad Sci USA 94: 53145319.
  • Song L,Fricker LD. 1995. Calcium- and pH-dependent aggregation of carboxypeptidase E. J Biol Chem 270: 79637967.
  • Steiner HJ,Schmid KW,Fischer-Colbrie R,Sperk G,Winkler H. 1989. Co-localization of chromogranin A and B, secretogranin II and neuropeptide Y in chromaffin granules of rat adrenal medulla studied by electron microscopy immunocytochemistry. Histochemistry 91: 473477.
  • Strub JM,Garcia-Sablone P,Lonning K,Taupenot L,Hubert P,van Dorsselaer A,Aunis D,Metz-Boutigue MH. 1995. Processing of chromogranin B in bovine adrenal medulla. Identification of secretolytin, the endogenous C-terminal fragment of residues 614–626 with antibacterial activity. Eur J Biochem 229: 356368.
    Direct Link:
  • Strub JM,Goumon Y,Lugardon K,Capon C,Lopez M,Moniatte M,van Dorsselaer A,Aunis D,Metz-Boutigue MH. 1996. Antibacterial activity of glycosylated and phosphorylated chromogranin-A-derived peptide 173–184 from bovine adrenal medullary chromaffin granules. J Biol Chem 271: 2853328540.
  • Supek F,Supekova L,Mandiyan S,Pan YCE,Nelson H,Nelson N. 1994. A novel accessory subunit for vacuolar H+-ATPase from chromaffin granules. J Biol Chem 269: 2410224106.
  • Takamori S,Holt M,Stenius K,Lemke EA,Gronborg M,Riedel D,Urlaub H,Schenck S,Brügger B,Ringler P,Müller SA,Rammner B,Gräter F,Hub JS,De Groot BL,Mieskes G,Moriyama Y,Klingauf J,Grubmüller H,Heuser J,Wieland F,Jahn R. 2006. Molecular anatomy of a trafficking organelle. Cell 127: 831846.
  • Takiyyuddin MA,De Nicola L,Gabbai FB,Dinh TQ,Kennedy B,Ziegler MG,Sabban EL,Parmer RJ,O'Connor DT. 1993. Catecholamine secretory vesicles. Augmented chromogranins and amines in secondary hypertension. Hypertension 21: 674679.
  • Taupenot L,Harper KL,O'Connor DT. 2003. The chromogranin-secretogranin family. N Engl J Med 348: 11341149.
  • Taylor CV,Taupenot L,Mahata SK,Mahata M,Wu H,Yasothornsrikul S,Toneff T,Caporale C,Jiang Q,Parmer RJ,Hook VYH,O'Connor DT. 2000. Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules. J Biol Chem 275: 2290522915.
  • Turquier V,Yon L,Grumolato L,Alexandre D,Fournier A,Vaudry H,Anouar Y. 2001. Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-existing activator protein-1-like transcription factors. Mol Pharmacol 60: 4252.
  • Van Dyke K,Robinson R,Urquilla P,Smith D,Taylor M,Trush M,Wilson M. 1977. An analysis of nucleotides and catecholamines in bovine medullary granules by anion exchange high pressure liquid chromatography and fluorescence. Evidence that most of the catecholamines in chromaffin granules are stored without associated ATP. Pharmacology 15: 377391.
  • Vaudry H,Conlon JM. 1991. Identification of a peptide arising from the specific post-translation processing of secretogranin II. FEBS Lett 284: 3133.
  • Wang JM,Slembrouck D,Tan J,Arckens L,Leenen FHH,Courtoy PJ,De Potter WP. 2002. Presence of cellular renin-angiotensin system in chromaffin cells of bovine adrenal medulla. Am J Physiol Heart Circ Physiol 283: H1811H1818.
  • Waschek JA,Pruss RM,Siegel RE,Eiden LE,Bader MF,Aunis D. 1987. Regulation of enkephalin, VIP, and chromogranin biosynthesis in actively secreting chromaffin cells. Multiple strategies for multiple peptides. Ann NY Acad Sci 493: 308323.
  • Weiss C,Cahill AL,Laslop A,Fischer-Colbrie R,Perlman RL,Winkler H. 1996. Differences in the composition of chromaffin granules in adrenaline and noradrenaline containing cells of bovine adrenal medulla. Neurosci Lett 211: 2932.
  • Wiedermann CJ. 2000. Secretoneurin: a functional neuropeptide in health and disease. Peptides 21: 12891298.
  • Winkler H. 1976. The composition of adrenal chromaffin granules: an assessment of controversial resulta. Neuroscience 1: 6580.
  • Winkler H,Carmichael SW. 1982. The chromaffin granule. In: PoisnerAM,TrifaroJM, editors. The secretory granule. Amsterdam: Elsevier. p 379.
  • Winkler H,Falkensammer G,Patzak A,Fischer-Colbrie R,Schuler N,Weber A. 1984. Life cycle of the catecholaminergic vesicle: from biogenesis to secretion. In: VisiES,MagyarK, editors. Regulation of transmitter function: basic and clinical aspects. Budapest: Akademiai Kiado. p 6573.
  • Winkler H,Westhead E. 1980. The molecular organization of adrenal chromaffin granules. Neuroscience 5: 18031823.
  • Yamada Y,Nakazato Y,Ito S,Teraoka H,Ohga A. 1988. Catecholamines and dopamine-beta-hydroxylase secretion from perfused goat adrenal glands. Q J Exp Physiol 73: 113121.
  • Yasothornsrikul S,Greenbaum D,Medzihradszky KF,Toneff T,Bundey R,Miller R,Schilling B,Petermann I,Dehnert J,Logvinova A,Goldsmith P,Neveu JM,Lane WS,Gibson B,Reinheckel T,Peters C,Bogyo M,Hook V. 2003. Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter. Proc Natl Acad Sci USA 100: 95909595.
  • Yeğen BC. 2003. Bombesin-like peptides: candidates as diagnostic and therapeutical tools. Curr Pharm Des 9: 10131022.
  • Yoo SH. 1995. pH- and Ca(2+)-induced conformational change and aggragation of chromogranin B. Comparison with chromogranin A and implication in secretory vesicle biogenesis. J Biol Chem 270: 1257812583.
  • Yoo SH,Oh YS,Kang MK,Huh YH,So SH,Park HS,Park HY. 2001. Localization of three types of inositol 1,4,5-trisphosphate receptor/Ca(2+) channel in the secretory granules and coupling with the Ca(2+) storage proteins chromogranins A and B. J Biol Chem 276: 4580645812.
  • Yoo SH,You SH,Kang MK,Huh YH,Lee CS,Shim CS. 2002. Localization of the secretory granule marker protein chromogranin B in the nucleus. J Biol Chem 277: 1601116021.
  • Yuchi H,Suganuma T,Sawaguchi A,Ide S,Kawano JI,Aoki T,Kitamura K,Eto T. 2002. Cryofixation processing is excellent method to improve the retention of adrenomedullin antigenicity. Histochem Cell Biol 118: 259265.
  • Zarzaur BL,Wu Y,Fukatsu K,Johnson CD,Kudsk KA. 2002. The neuropeptide bombesin improves IgA-mediated mucosal immunity with preservation of gut interleukin-4 in total parenteral nutrition-fed mice. Surgery 131: 5965.