Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS.

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size-exclusion, immunoaffinity, and reversed-phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586-R602 (SR-17) and is phosphorylated at one or two serine residues. Another novel peptide H603-Q636 (HQ-34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin-like peptide fragment (KR-11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C-terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.

Postganglionic, direct axo-axonal contacts on the splenic nerve.

The splenic nerve is made up almost exclusively of adrenergic fibers. Consequently it was used as a model system in the study of autonomic sympathetic neurotransmission. The splenic nerve regulates the vasoconstriction and volume reduction of the spleen. Brain-immune interactions via modulation of the splenic nerve activity may regulate peripheral cellular immunity. An inhibition of noradrenaline release by alpha(2)-adrenoceptor activation has been reported. As we were interested in a structurally detailed distribution of synaptophysin, immunocytochemical methods were applied to splenic nerve axons. In 1 microm plastic sections a network of synaptophysin-positive varicosities could be observed all along the splenic nerve. They were also positive for dopamine-beta-hydroxylase and cytochrome b561. At the ultrastructural level the varicosities were seen to establish direct contact with the splenic axons. In normal morphology the varicosities revealed small synaptic vesicles and several dense granules. It is demonstrated that a network of direct symmetric contacts of adrenergic nature is present all along the nerve. These terminals may have an inhibitory effect on the splenic nerve activity via axonal receptors. This finding opens new perspectives for the study of the splenic nerve in general and more particularly for its role in the regulation of peripheral cellular immunity.

Changes in the expression of alpha(2)-adrenergic receptor subtypes during maturation of neuronal cells from fetal pig superior cervical ganglia.

The expression of presynaptic alpha(2)-adrenergic receptor (alpha(2)-AR) subtypes was investigated in cultured neurons from fetal pig superior cervical ganglion (SCG). Cells were incubated with chicken antibodies against alpha(2)A-, alpha(2)B- or alpha(2)C-AR subtypes either alone or together with antibodies against dopamine-beta-hydroxylase (DbetaH, a marker for adrenergic neurons) or against choline acetyl transferase (ChAT, a marker for cholinergic neurons). We found immunoreactivity for all three alpha(2)-AR subtypes in SCG-cells when cultured for 8-11 days. The relative expression of the alpha(2)A-subtype was approximately 1/3 of that of alpha(2)B- and alpha(2)C-AR. Co-localisation of all three alpha(2)-AR subtypes was observed in cells expressing DbetaH or ChAT. Increasing the potassium concentration in the culture medium increased the expression of DbetaH and decreased the expression of the alpha(2)A- and alpha(2)C-subtype without altering the expression of the alpha(2)B-subtype. Co-culture of neurons with pig splenocytes enhanced the expression of ChAT and decreased the expression of the alpha(2)B-subtype without altering the expression of alpha(2)A- and alpha(2)C-subtypes. Our results indicate that the three alpha(2)-receptor subtypes are expressed on both noradrenergic and cholinergic nerves. Induction of the noradrenergic phenotype favours the expression of the alpha(2)B-subtype over that of the alpha(2)A- and alpha(2)C-subtype. Conversely, enhancement of the cholinergic phenotype favours the expression of the alpha(2)A- and alpha(2)C-subtypes over that of the alpha(2)B-subtype. Our results suggest that the alpha(2)B-receptor is preferentially associated with noradrenergic nerve endings.

Secretory vesicle-specific antibodies in the confocal study of exo-endocytosis dynamics.

The study of secretory vesicle dynamics is a continuing challenge. Classically it was studied using biochemical techniques, such as subcellular fractionation and immunoprecipitation, combined with time-consuming electron microscopy studies. The recent development of confocal microscopy, giving in-focus optical section images throughout the thickness of a fluorescently labeled sample, allows scientists to study the key events in the secretory cycle at the level of light microscopy. This study demonstrates the use of specific antibodies against marker proteins of two different secretory vesicles (synaptic vesicles and large dense-cored vesicles) to follow their exo-endocytosis dynamics in peripheral adrenergic neurons. Only in recent years has insight grown regarding the presence of both exocytosis pathways in the same neuron. Confocal microscopy is a suitable technique to study aspects of exocytosis, endocytosis, and intracellular sorting and as such improves our knowledge on the interaction between both secretory pathways.

The processing of secretogranin II in the peripheral nervous system: release of secretoneurin from porcine sympathetic nerve terminals.

The distribution of secretoneurin (SN), a peptide derived from secretogranin II (SgII), in the coeliac ganglion, the splenic nerve and the spleen was examined by immunohistochemistry. In the ganglion, SN immunoreactivity (IR) was unevenly distributed. Positive nerve terminals densely surrounded some postganglionic perikarya in which also intense SN-IR was present. In the crushed splenic nerves, intense immunoreactivities appeared proximal (but to a less extent also distal) to the crush of the nerve. Analysis by cytofluorimetric scanning (CFS) demonstrated that SN-IR and neuropeptide Y immunoreactivity (NPY-IR) were predominant in the axons proximal to the crush representing anterogradely transported components. Using radioimmunoassay (RIA) we demonstrated that upon electrical stimulation (10 Hz, 1 min) of the splenic nerve, significant amounts of SN-IR (64.2+/-2.3 fmol) were released together with NA (4. 1x106+/-0.2 fmol) and NPY (330.0+/-7.2 fmol) from the isolated perfused porcine spleen. To evaluate the processing of SgII in sympathetic neurons, boiled tissue extracts (coeliac ganglia and splenic nerve) and boiled spleen perfusate (used as a suitable source for vesicle derived peptides) were analysed by gel filtration chromatography followed by SN-RIA. In all cases immunoreactivity was present solely as SN, indicating that SgII was fully processed to the free peptide. The evidence that SN is transported to the nerve terminals and is released from the porcine spleen upon nerve stimulation, suggests that it may modulate adrenergic neurotransmission and may also play a role in the neuroimmune communication.

Pig splenic nerve: peptides derived from chromogranins by proteolytic processing during axonal transport.

We have investigated the proteolytic processing of chromogranin A, chromogranin B and NESP55 (a novel chromogranin-like protein) during axonal transport using pig splenic nerve as a model. We have also studied the presence of chromogranin-derived peptides in the perfusate during electrical stimulation of this nerve. High-performance gel filtration chromatography followed by radioimmunoassay (RIA) revealed that chromogranins are proteolytically processed to varying degrees during axonal transport. For chromogranin A and NESP55, the precursor is still present in the proximal part of the nerve, whereas in the distal part and nerve terminals, intermediate-sized peptides and the free peptides GE-25 and GAIPIRRH dominate, respectively. For chromogranin B, the precursor has already been processed to an intermediate-sized peptide in the proximal part of the nerve, which is also present in the distal parts together with the free peptide PE-11. For chromogranin B and NESP55, only the free peptides PE-11 and GAIPIRRH, or in the case of chromogranin A, the free peptide GE-25 plus an intermediate-sized one, are released from the terminals into the splenic perfusate. These results demonstrate that chromogranins are processed to smaller peptides during axonal transport.

The storage of noradrenaline, neuropeptide Y and chromogranins in and stoichiometric release from large dense cored vesicles of the undifferentiated human neuroblastoma cell line SH-SY5Y.

Sucrose gradient centrifugation combined with electron microscopy revealed that undifferentiated SH-SY5Y cells contain predominantly one population of noradrenaline containing vesicles, i.e. large dense cored vesicles. These vesicles have been purified approximately twenty times using sucrose/D2O gradients. Electron microscopy of sucrose/D2O fractions confirms that large dense cored vesicles are enriched in the fractions containing predominantly dopamine- -hydroxylase, chromogranin A, noradrenaline and neuropeptide Y. The membranes of these vesicles contain the typical large dense cored vesicle markers dopamine- -hydroxylase, synaptotagmin, cytochrome b561 and rab 3. Stimulation of SH-SY5Y cells with carbachol and KCl shows that noradrenaline and neuropeptide Y are released in the same proportion as stored in the large dense cored vesicles. The immuno-blot pattern and intensity of chromogranin A and chromogranin B present in large dense cored vesicles and in the released material were definitely the same. This suggests that noradrenaline and the proteins/peptides are released in the same molar stoichiometry as they are stored in large dense cored vesicles. These data provide for the first time experimental evidence that the neuroblastoma cell line SH-SY5Y contains functionally active large dense cored vesicles similar to those of sympathetic neurons and indicate that this cell line is a suitable experimental cell model to study the exocytotic pathway of large dense cored vesicles.

Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve.

Synaptophysin and synapsin, closely correlated on synaptic vesicles in terminals, may show a differential distribution at synapse formation and maturation. In order to disclose the fine structural details of these differences, synapsin and synaptophysin distribution was studied by immunocytochemistry on ligated bovine splenic axons in vitro and compared with terminals in the vas deferens. In the synaptic differentiations taking place proximally synapsin could only be detected on the accumulating elements of the axonal reticulum. Large dense granules and clusters of small synaptic vesicles were negative. Synaptophysin was restricted to these clusters. In the vas deferens, co-localization of synapsin and synaptophysin could be seen on small vesicles. From their formation small synaptic vesicles carry synaptophysin. Synapsin may be involved in the dynamic membrane changes taking place at the ligation. At a functional terminal, synapsin shifts to small synaptic vesicles.

Retrieved constituents of large dense-cored vesicles and synaptic vesicles intermix in stimulation-induced early endosomes of noradrenergic neurons.

Two storage compartments in cultured noradrenergic neurons derived from the superior cervical ganglion from fetal pig have been defined using sucrose density gradient centrifugation and electron microscopy: (1) large dense-cored vesicles (LDV) contain noradrenaline and dopamine-beta-hydroxylase (DbetaH); (2) small electron-lucent vesicles contain acetylcholine and p38 and represent the noradrenergic small synaptic vesicles (SSV); no small dense-cored vesicles (SDV) could be detected. Our results demonstrate that internalized LDV membrane constituents are retrieved into early endosomes, as shown by the colocalization of retrieved DbetaH with the endosomal markers Rab5 and HRP in sucrose density gradients and on confocal microscopical images. Recycling of the SSV membranes via an endosomal intermediate is also confirmed in noradrenergic neurons. Finally, colocalization of retrieved DbetaH and retrieved p38 in stimulated neurons indicates that the two sets of constituents intermix. These data provide the first experimental evidence for a common early endosome in which SSV and LDV membrane constituents are internalized after exocytosis and imply that endosomal sorting is an important process for the generation of different secretory vesicles in the noradrenergic nerve terminal.

Rab3 dissociation and clathrin-mediated endocytosis, two key steps in the exo-endocytotic pathway of large dense-cored vesicles in primary cultures of superior cervical ganglia.

In this study we have used primary cultures of porcine superior cervical ganglia as a model system to study exo-endocytosis in sympathetic neurons. Pure neuronal cultures with a defined noradrenergic phenotype can be obtained when antimitotics are included in the culture medium, and the high yield from prenatal piglets allows a biochemical approach in addition to morphological studies. Release of large dense-cored vesicles (LDCVs) was visualized by exposing stimulated neurons to anti-dopamine-beta-hydroxylase (D beta H) prior to fixation. Double immunofluorescent staining revealed that synaptotagmin was colocalized with anti-D beta H labeled exocytotic spots, but not the small GTP-binding protein rab3. Density gradient centrifugation of a postmitochondrial supernatant of cultured cells confirmed the dissociation of rab3 from a population of mature LDCVs. As expected, rab3 did not reassociate with the lighter D beta H-positive membrane fraction in the gradient representing internalized LDCV membranes. The fluid phase marker horseradish peroxidase colocalized with retrieved LDCV membranes. Indirect immunofluorescence demonstrated the colocalization of clathrin with D beta H-positive exocytotic spots on the plasma membrane. At the ultrastructural level, depolarization of neurons resulted in the abrupt loss of LDCVs and concomitant appearance of clathrin-coated vesicles and coated omega profiles. The size distribution of coated structures overlapped strongly with that of LDCVs. Taken together, these data clearly suggest that two key regulatory events in the process of exo-endocytosis, i.e. rab3 dissociation and clathrin-mediated internalization, are not only reminiscent of small synaptic vesicles, but also are a feature of the LDCV pathway at the presynaptic plasma membrane of sympathetic neurons.

Noradrenaline storing vesicles in sympathetic neurons and their role in neurotransmitter release: an historical overview of controversial issues.

More than 25 years have passed since the original demonstration that proteins such as chromogranin A and dopamine-beta-hydroxylase, which are co-stored together with noradrenaline in large dense cored vesicles in adrenergic nerves, are released by exocytosis. Despite much evidence in favour, it was for a long time thought that large dense cored vesicles were not eminently involved in the release of noradrenaline. The present review attempts to demonstrate, making use of evidence from different approaches, that the release of noradrenaline from sympathetic neurons occurs ultimately from large dense cored vesicles. A model of the secretory cycle is proposed.

Isolation and identification of intact chromogranin A and two N-terminal processing products, vasostatin I and II, from bovine adrenal medulla chromaffin granules by chromatographic and mass spectrometric methods.

Chromogranin A (CGA) is the most abundant protein of the bovine adrenal medulla and plays an important role as precursor protein of several peptides that act as modulators for endocrine cell secretory activity. Furthermore, it is presumed to play a role in the targeting of peptide hormones and neurotransmitters to granules of the regulated pathway. However, its complete primary structure and proteolytic processing have not yet been identified. This study describes a rapid and efficient procedure for the high yield isolation of bovine CGA and its N-terminal processing products, vasostatin I and II. Using the lysate from bovine adrenal medulla chromaffin granules, the soluble proteins were purified by three consecutive HPLC steps, thereby avoiding the use of buffer solutions. The protein fractions were isolated and characterized by SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the latter analysis, the efficiency of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was demonstrated, enabling the unequivocal and sensitive characterization of proteins from crude mixtures. Sufficient amounts of pure protein were obtained by the present procedure to form the basis for detailed structural studies by spectroscopic methods and X-ray crystallography.

Noradrenergic neurons release both noradrenaline and neuropeptide Y from a single pool: the large dense cored vesicles.

In peripheral adrenergic nerve endings, noradrenaline is stored in two different types of vesicles, the large and the small dense cored vesicles. A systematic study was undertaken to examine the release of noradrenaline and neuropeptide Y from dog spleen and rat vas deferens under various conditions of stimulation, particularly those which previously have demonstrated a differential regulation of exocytosis of the different types of storage vesicles. Here we present evidence that noradrenaline is released by exocytosis exclusively from the large dense cored vesicles, in which it is stored together with neuropeptide Y. Upon a single stimulation (at frequencies varying from 2-20 Hz), the release of noradrenaline and neuropeptide Y from the dog splenic nerve increased with the frequency of stimulation, but the ratio of noradrenaline to neuropeptide Y remained constant. After repeated stimulation of the splenic nerve, both substances' overflow decreased gradually and in parallel to values of 12.5% and 11.1% of the first stimulation for noradrenaline and neuropeptide Y, respectively. Similarly, repeated stimulation of the rat vas deferens (of which only 2-10% is large dense cored vesicles, whereas in the dog splenic nerve the large dense cored vesicles make up 30-40% of the total vesicle population) with 120 mM K+, in the presence of phentolamine, caused a gradual and parallel decline in the release of noradrenaline and neuropeptide Y (31.6% and 34.0%, respectively). Moreover, omega-conotoxin (10(-8) M to 10(-5) M) had a similar inhibitory effect on the release of both substances, alpha-latrotoxin (10(-9) M) evoked a parallel release of both noradrenaline and neuropeptide Y. The results indicate that noradrenaline in peripheral noradrenergic nerves is released exclusively from large dense cored vesicles by an exocytotic mechanism.

Stimulation of T-cell proliferation by pancreastatin and its C-terminal fragment (33-49).

We have studied the effect of pancreastatin and its C-terminal fragment (33-49) on mitogen-stimulated T lymphocyte proliferation. In a concentration range from 10(-12) to 10(-8) M they exhibit a dose-dependent stimulatory effect on concanavalin A-induced response with the maximal effect at 10(-8) M concentration. They were inactive in response to a B-cell mitogen, lipopolysaccharide, which points to an involvement of T but not B lymphocytes in their response. Pancreastatin can still produce a stimulatory effect when added 18 h after incubation of cultures with concanavalin A and apparently uses a diacylglycerol independent mechanism. When cells were preincubated for 4, 16 or 24 h with pancreastatin or its fragment and then stimulated with concanavalin A, a ten times lower concentration of peptides was needed (10(-9) M) to obtain the maximal response. This suggests that resting cells are more sensitive to pancreastatin and its fragment. Both peptides exhibit a very similar pharmacological profile, indicating that the C-terminal part of the molecule is responsible for the effect on T-cell proliferation.

Mass spectrometric identification of phosphorylated vasostatin II, a chromogranin A-derived protein fragment (1-113).

Vasostatin II, an N-terminal chromogranin A-derived protein (CGA1-113), was purified from bovine chromaffin granule lysate and characterized by electrospray mass spectrometry (ES/MS) as being partially phosphorylated. The phosphorylation site was determined to be at the Ser81 position by mass spectrometric peptide mapping and tandem mass spectrometric analysis. This phosphorylation site is close to the processing site (...QKK78HSS(p)81...) yielding vasostatin I, an N-terminal CGA-derived peptide comprising residues 1-76, suggesting that phosphorylation at Ser81 is involved in the formation of vasostatin I in chromaffin cells.

Ultrastructural evidence for a separate, small synaptic vesicle (SSV) pathway in ligated bovine splenic nerves, incubated in vitro.

In sympathetic nerves the tubules of the axonal reticulum make up the immature elements of the neurosecretory apparatus. The formation of the mature large dense granules occurs via a less dense tubular intermediate, representing the maturing part. At a terminal small synaptophysin-positive vesicles are found intermingled with the dense granules. The biogenesis of these clear, small synaptic vesicles and their relationship with dense granules remains to be determined. In search for the small synaptic vesicles we undertook a careful ultrastructural examination of the axons proximal to a ligation in bovine splenic nerve incubated in vitro for 3 h. The distended axons were crowded with tubules, granulo-tubular elements and dense granules. Occasionally homogeneous clusters of small, uniform vesicles were detected. They were shown to be positive for synaptophysin and were negative for dopamine-beta-hydroxylase, a marker for the granular pathway. The clusters of small vesicles could be found in close spatial relationship with the maturing and mature elements of granular secretion. Our findings argue for the presence of two separate neurosecretory pathways in sympathetic nerves and favour the idea that both small synaptic vesicles and dense granules are a differentiation product of the axonal reticulum. This configuration can explain the biogenesis of small synaptic vesicles and dense granules both in the cell body and at the nerve terminal.

Ontogeny of epinephrine, norepinephrine, dopamine-beta-hydroxylase, and chromogranin A in the adrenal gland of pigs.

OBJECTIVE: To obtain data on the ontogeny of catecholamines and other chromaffin vesicle components, which could serve as a basis for the study of their role during fetal life in normal and pathologic conditions. DESIGN: Epinephrine, norepinephrine, dopamine-beta-hydroxylase, and chromogranin A contents were measured in the porcine adrenal gland during various stages of gestation. ANIMALS: 934 porcine fetuses representing 22 gestational ages between 43 and 108 days. PROCEDURE: Total homogenates of adrenal glands were extracted and contents of different neurochemical markers were measured, using high-performance liquid chromatography, immunoassays, and western blotting. Immunohistochemical studies also were performed. RESULTS: Epinephrine and norepinephrine contents as a function of gestational age can be represented by a sigmoidal curve. Norepinephrine content rises early in gestation, whereas epinephrine content increases later. Maximal increase was significantly higher for epinephrine content. A progressive appearance of separate epinephrine- and norepinephrine-storing cells was documented. Dopamine-beta-hydroxylase content as a function of gestational age can be adequately represented by a parabolic curve. No quantitative changes in chromogranin A concentration were observed, but western blotting revealed qualitative changes with progressing gestational age. CONCLUSIONS: Important changes occur in catecholamine formation around day 60 of gestation. The sharp increase in epinephrine/norepinephrine contents and the appearance of separate epinephrine- and norepinephrine-storing cells may be related to the progressive splanchnic innervation of the adrenal gland. The presence of chromogranin A early in gestation may indicate its necessity for catecholamine storage.

Two polypeptide toxins with opposite effects on calcium uptake in bovine chromaffin cells: isolation from the venom of the marine snail Conus distans.

Two polypeptide toxins which modulate the uptake of 45Ca2+ in bovine chromaffin cells were isolated from the venom of the marine snail Conus distans. The molecular weights were estimated by gel electrophoresis and gel filtration to be 25.5 and 24 kDa, respectively. The purified proteins were electrophoretically homogeneous. The 25.5 kDa-component caused a concentration-dependent increase of the initial rate of 45Ca2+ uptake, but it had no effect on the stimulation evoked uptake. The 24 kDa-component produced the opposite effects; it caused a concentration-dependent inhibition of the stimulation evoked 45Ca2+ uptake, but it did not affect the initial rate.

Umbilical cord dopamine beta-hydroxylase, chromogranin A and met-enkephalin after conditions associated with chronic intrauterine stress.

OBJECTIVE: To evaluate whether the markers of autonomic nervous system activity, dopamine beta-hydroxylase (DBH), chromogranin A (CGA) and met-enkephalin (E), are different in cord blood from neonates born after conditions associated with chronic intrauterine stress (CIUS) as compared to neonates born after a normal pregnancy. STUDY DESIGN: 61 newborns (median BW 2,840 g, range 617-4270 g) born after a pregnancy complicated by maternal hypertension, maternal smoking, maternal diabetes mellitus or intrauterine growth retardation (STR group) were compared with 88 neonates (median BW 2,910 g, range 4,00-4,370 g) who had not suffered from such intrauterine conditions. DBH, CGA and E were measured in the cord blood of both groups. RESULTS: When both groups were taken together, high DBH values were best related to maternal smoking (p = 0.004) and low E levels to maternal diabetes (p = 0.02). Within the STR group, high DBH values were best related with all conditions linked with CIUS (p = 0.008), E levels were best linked with the combination of intrauterine growth retardation (positive correlation) and maternal diabetes (negative correlation) (p = 0.03). For CGA there was only a weak positive relation with maternal smoking (p = 0.3). CONCLUSION: Certain intrauterine conditions associated with CIUS, especially maternal smoking, may lead to alterations of the autonomic nervous system as revealed by some of its markers in cord blood of neonates. This may be important in the pathogenesis of certain conditions after birth, such as the sudden infant death syndrome.