Presence and release of the chromogranin B-derived secretolytin-like peptide KR-11 from the porcine spleen

Chromogranin B (CgB) is a major matrix protein in secretory granules/large dense-cored vesicles and a precursor for smaller peptides. In an earlier study, we have identified a secretolytin-like peptide (KR-11, pCgB(637-647)) from porcine chromaffin granules. Further evidence is presented here to show the processing of chromogranin B to this peptide during axonal transport in the splenic nerve and its release in the spleen upon various conditions of stimulation. Immunohistochemical staining showed that in the porcine spleen chromogranin B and NPY completely colocalize in nerve fibres and varicosities in blood vessel walls and trabeculae, and along the loose network of smooth muscle cells in the red pulp, as identified by their alpha-smooth muscle cell actin content. No antibacterial activity was found for the porcine secretolytin-like peptide, KR-11. The change of one amino acid residue (Thr-->Asn) in the porcine homologous fragment of secretolytin appears to be responsible for its loss of antibacterial activity.     

Chromogranin A correlates with norepinephrine release rate.

Chromogranin A (CgA) is an acidic protein co-released with catecholamines during exocytosis from sympathetic nerve terminals and chromaffin cells. Previous work has demonstrated that large scale perturbations in sympathetic nervous system (SNS) functioning result in corresponding changes in CgA levels in plasma. Little is known about the physiologic significance of CgA. We hypothesized that, since CgA and catecholamines are co-released from the same storage vesicles, and since CgA is not subject to reuptake or enzymatic metabolism, plasma CgA should reflect norepinephrine release from sympathetic terminals. We therefore measured venous CgA, norepinephrine levels, and norepinephrine release rate in 30 unmedicated subjects. Although the correlation of CgA with plasma norepinephrine was only modest (r = 0.37, p less than 0.05), its correlation with norepinephrine release rate was highly significant (r = 0.58, p less than 0.001). Thus, CgA may offer a novel perspective on peripheral sympathetic activity.     

Isolation of peptides arising from the specific posttranslational processing of chromogranin A and chromogranin B from human pheochromocytoma tissue.

An extract of human adrenal medullary pheochromocytoma tissue was fractionated by gel permeation chromatography, and peptides of major abundance in the approximate molecular mass range 1000-4000 were purified to apparent homogeneity by reverse phase HPLC. Determination of the primary structures of four such peptides demonstrated that they were fragments of either chromogranin A or chromogranin B. The peptide WSKMDQLAKELTAE represents chromogranin A(324-337), the peptide LGELFNPYYDPLQWKSSHFE represents chromogranin B(498-517), the peptide NLARVPKLDL represents chromogranin B(568-577), and the peptide QYDRVAQLDQLLHY (isolated as the N-terminal pyroglutamyl derivative) represents chromogranin B(580-593). Analysis of the nucleotide sequences of cDNAs complementary to human chromogranin A and B messenger RNAs indicates that each of these peptide sequences is flanked by pairs or groups of basic residues, suggesting that these fragments are the products of specific posttranslational processing. In addition, a peptide identified as chromogranin B(496-517) was isolated from extract. This component represents the product of incomplete proteolytic cleavage at the Lys494-Arg495-Lys496-Arg497 processing site in chromogranin B. A minor component in the extract was identified as chromogranin B(508-517), but this component probably represents an artifact of the extraction procedure arising from the hydrolysis of the acid labile Asp507-Pro508 bond. The study has shown that chromogranin A and B in pheochromocytoma tissue function as the precursors of several small peptides that may have a regulatory role.     

Presynaptic inhibition of transmitter release from rat sympathetic neurons by bradykinin

Bradykinin is known to stimulate neurons in rat sympathetic ganglia and to enhance transmitter release from their axons by interfering with the autoinhibitory feedback, actions that involve protein kinase C. Here, bradykinin caused a transient increase in the release of previously incorporated [3H] noradrenaline from primary cultures of dissociated rat sympathetic neurons. When this effect was abolished by tetrodotoxin, bradykinin caused an inhibition of tritium overflow triggered by depolarizing K+ concentrations. This inhibition was additive to that caused by the alpha2-adrenergic agonist UK 14304, desensitized within 12 min, was insensitive to pertussis toxin, and was enhanced when protein kinase C was inactivated. The effect was half maximal at 4 nm and antagonized competitively by the B2 receptor antagonist Hoe 140. The cyclooxygenase inhibitor indomethacin and the angiotensin converting enzyme inhibitor captopril did not alter the inhibition by bradykinin. The M-type K+ channel opener retigabine attenuated the secretagogue action of bradykinin, but left its inhibitory action unaltered. In whole-cell patch-clamp recordings, bradykinin reduced voltage-activated Ca2+ currents in a pertussis toxin-insensitive manner, and this action was additive to the inhibition by UK 14304. These results demonstrate that bradykinin inhibits noradrenaline release from rat sympathetic neurons via presynaptic B2 receptors. This effect does not involve cyclooxygenase products, M-type K+ channels, or protein kinase C, but rather an inhibition of voltage-gated Ca2+ channels.     

Chromogranin B-induced secretory granule biogenesis: comparison with the similar role of chromogranin A

The two major proteins of secretory granules of secretory cells, chromogranins A (CGA) and B (CGB), have previously been proposed to play key roles in secretory granule biogenesis. Recently, CGA was reported to play an on/off switch role for secretory granule biogenesis. In the present study we found CGB being more effective than CGA in inducing secretory granule formation in non-neuroendocrine NIH3T3 and COS-7 cells. The mean number of dense core granules formed/cell of CGA-transfected NIH3T3 cells was 2.51, whereas that of CGB-transfected cells was 4.02, indicating the formation of 60% more granules in the CGB-transfected cells. Similarly, there were 55% more dense core granules formed in the CGB-transfected COS-7 cells than in the CGA-transfected cells. Moreover, transfection of CGA- and CGB-short interfering RNA (siRNA) into neuroendocrine PC12 cells not only decreased the amount of CGA and CGB expressed but also reduced the number of secretory granules by 41 and 78%, respectively, further suggesting the importance of CGB expression in secretory granule formation.     

Proteolytic processing of chromogranin A in cultured chromaffin cells

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.     

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach.

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine (b) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylase (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.     

Relaxation induced by N-terminal fragments of chromogranin A in mouse gastric preparations

A definitive role for chromogranin A (CGA)-derived fragments in the control of the gastrointestinal smooth muscle contractility has not been yet established. The purpose of the present study was to evaluate, in vitro, the effects of the recombinant vasostatin 1-78 (VS-1), CGA 7-57 and CGA 47-66 on the mouse gastric mechanical activity, recording the changes of intraluminal pressure. VS-1, CGA 7-57 and CGA 47-66 produced concentration-dependent relaxations. Mouse anti-vasostatin-1 monoclonal antibody 5A8, recognising the region 53-57, abolished the relaxation induced by VS-1, indicating the specificity of the effect. The relaxation was significantly reduced by tetrodotoxin (TTX), blocker of neuronal voltage-dependent Na(+) channels, l-NAME, inhibitor of nitric oxide (NO) synthase, or apamin, blocker of small conductance Ca(2+)-dependent K(+) channels. The joint application of TTX and l-NAME did not show any additive effects, whereas TTX plus apamin abolished the VS-1 response. The results suggest that the N-terminal CGA-derived peptides are able to relax mouse gastric muscle and, therefore, they point out an inhibitory role of vasostatin I in the gastrointestinal tract. The relaxation is mediated in part by neural mechanisms through NO production and in part by non-neural mechanisms involving the opening of small conductance Ca(2+)-dependent K(+) channels.     

Neuropeptide Y,enkephalin and noradrenaline coexist in sympathetic neurons innervating the bovine spleen

Summary The subcellular distribution of noradrenaline (NA), neuropeptide Y (NPY), Met and Leu-enkephalin (ENK), substance P (SP), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP) was investigated in homogenates of bovine splenic nerve. The distribution of noradrenergic peptide-containing nerves in the bovine celiac ganglion, splenic nerve and terminal areas in spleen was studied by indirect immunofluorescence histochemistry using antisera to tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), NPY, enkephalin peptides, SP, SOM, VIP and peptide HI (PHI).After density gradient centrifugation, high levels of NPY and ENK-like immunoreactivity (LI) were found in high-density gradient fractions, coinciding with the main NA peak. SP, SOM and VIP were found in fractions with a lower density, VIP being also enriched in a heavy fraction; the latter three peptides were present in low concentrations.Immunohistochemistry revealed that staining for NPYLI and ENK-LI partly overlapped that for TH and DBH in celiac ganglia, splenic nerve axons and terminal areas of spleen. Almost all principal ganglion cells were TH- and DBH-immunoreactive. Many were also NPY-immunoreactive, whereas a smaller number were ENK-positive. In the celiac ganglion patches of dense SP-positive networks and some VIP/PHI- and ENK-immunoreactive fibers were seen around cell bodies.The results indicate that NPY and ENK are stored with NA in large dense-cored vesicles in unmyelinated axons of bovine splenic nerve. SP, SOM and VIP appear in different organelles in axon populations separate from sympathetic noradrenergic nerves.     

Selective processing of chromogranin A in the different islet cells in human pancreas.

We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the four major neuroendocrine cell types of the human pancreas by using double immunofluorescence techniques. The antibodies raised to the N-terminal and midportions of CgA showed, on the whole, stronger immunoreactivity than did the C-terminal antibodies, with a few exceptions. Often the immunoreactivity was stronger in glucagon cells. Insulin cells expressed immunoreactivity to all region-specific antibodies, but glucagon cells were nonreactive to two antibodies. Somatostatin cells reacted only with the C-terminal antibodies (amino acid sequences CgA 411-424), while PP cells were stained with four CgA region-specific antibodies between amino acid sequences 63-195. The cause of these differences may be that the CgA molecule is cleaved, partly masked, or partly translated from CgA mRNA. Microwave treatment improved only the staining with the CgA 361-372 antibodies, which indicates that masking is not the sole or entire cause. Our findings may indicate that the CgA molecule is cleaved in different ways in the various pancreatic endocrine cell types, giving rise to a variety of biologically functional fragments.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal responses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuations of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Chromogranin A in neurons of the rat cerebellum and spinal cord: quantification and sites of expression.

Chromogranin A (CGA) is an abundant protein of dense-cored secretory vesicles in endocrine and neuronal cells. The present study, for the first time, compares CGA of neurons of the central nervous system with the CGA of adrenal origin. By S1 nucleus protection assay, we found that the 3' part of the CGA mRNA between exons 5-8 of the cerebellum and the spinal cord of the rat is homologous to that of the adrenal. In situ hybridization histochemistry revealed that CGA mRNA in the cerebellar cortex is present in cell bodies of Purkinje cells and in neurons of the deep cerebellar nuclei. The perikarya of these cells also exhibit CGA-like immunoreactivity. CGA mRNA and CGA-like immunoreactivity are also present in the motoneurons of the ventral, lateral, and dorsal horns of the rat spinal cord. The amounts of CGA, as determined by radioimmunoassay in cerebellum and spinal cord, were about one tenth of the amounts detected in the adrenal, adenohypophysis, or the olfactory bulb. The sites of CGA expression suggest that CGA may be involved in signal transduction in the motor system.     

Regulatory Peptides from Chromogranin A and Secretogranin II

This commentary is focusing on novel aspects on the secreted CgA- and SgII-derived peptides, vasostatin-I (bovine and human CgA_1–76, VS-I), WE-14 (CgA_316–329), catestatin (bovine CgA_344–366, human CgA_352–372, Cts) and the SgII-derived secretoneurin (SgII_180–204) as significant regulators of inflammatory reactions.     

Glutamine synthetase. IX. Purification and characterization of the enzyme from sheep spleen.

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) has been purified about 550-fold from sheep spleen. The subunit weight of the enzyme is estimated to be 48 000. Sedimentation coefficient determination by density gradient centrifugation gives a value of 15.0 S. The approximate molecular weight calculated from the S value is 378500. In addition, electron micrographs of the enzyme show an "H" shape. Hence, the protein appears to have eight subunits. In sheep spleen, the enzyme resides chiefly in the soluble fraction of the cell. The amino acid composition of the enzyme from spleen shows similarity to that from other sources. The enzyme activity is nearly five times as high in Mg2+ as in Mn2+. ATP inhibits the enzyme; the inhibition is competitive with respect to Mg2+ATP. A number of compounds, such as D-alanine, AMP, creatine phosphate, arsenite in combination with 2,3-dimercaptopropanol, and 2-amino-4-phosphonobutyrate, also inhibit the enzyme. The inhibition by the last compound is competitive with respect to glutamate. D-Glutamate and alpha-methyl-DL-glutamate can serve as substrates in the synthesis reaction, but N-methyl-DL-glutamate cannot. On the other hand, neither D-glutamine nor N-acetyl-L-glutamine can replace L-glutamine as a substrate in the gamma-glutamyl transfer reaction of the enzyme. Inhibition of Mn2+ and ATP and its reversal by Mg2+ have been discussed as a means of regulating the enzyme activity in mammalian tissues.     

Hydrocortisone and the release of prostaglandins from spleen

An infusion of noradrenaline (1 μg/ml/min) released a PGE-like substance (PGEs) from superfused splenic strips of rabbits and from perfused cat spleen. The release of PGEs from rabbit splenic strips was not inhibited by the treatment of strips with hydrocortisone (40 – 150 μg/ml), but it was completely abolished in strips obtained from animals pretreated with hydrocortisone (1 mg/kg). The release of PGEs from the perfused cat spleen was reduced by hydrocortisone and abolished by indomethacin. It is concluded that the route of administration of hydrocortisone is essential for an appearance of its inhibitory effect on the PG release.