In situ hybridization: mRNA levels of secretogranin II,VGF and peptidylglycine alpha-amidating monooxygenase in brain of salt-loaded rats

The mRNA levels of secretogranin II (SgII), VGF and peptidylglycine alpha-amidating monooxygenase (PAM) were studied in brains of salt loaded rats by in situ hybridization. In these rats the levels of the message for secretogranin II and VGF were increased in the paraventricular, supraoptic and retrochiasmatic nuclei and in the subfornical organ. The increases ranged from 416 to 721% for SgII and from 778 to 890% for VGF. The PAM message was also elevated in these brain regions; however, the maximal increase was only 221%. We conclude that the message for all secretory peptides investigated so far, i.e. vasopressin, galanin, secretogranin II and VGF are upregulated to a similar degree in the hypothalamus of salt-located rats. The relative increase in mRNA for the enzyme peptidylglycine alpha-amidating monooxygenase occurred to a much lower extent, and was comparable to the limited changes previously seen for carboxypeptidase H.     

Large Dense-Core Vesicles in Rat Adrenal After Reserpine: Levels of mRNAs of Soluble and Membrane-Bound: Constituents in Chromaffin and Ganglion Cells Indicate a Biosynthesis of Vesicles with Higher Secretory Quanta

Abstract: Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense-core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense-core vesicles (chromogranins A and B., secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 215–857% after 1–3 days of drug treatment. For partly membrane-bound components (dopamine β-hydroxylase, prohormone convertase 2, carboxypeptidase H., and peptidylglycine α-amidating monooxygenase) the changes ranged from 182 to 315%, whereas for glycoprotein III and for intrinsic membrane proteins (cytochrome b ₆₆₁ and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y., secretogranin II, carboxypeptidase H., and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Long-lasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides. Key Words : ChromograninSecretogranin II—Monoamine transporter—Prohormone convertase 2—Carboxypeptidase H—Cytochrome b ₆₆₁-Clusterin.     

Effects of AF64A on Gene Expression of Choline Acetyltransferase (ChAT) in the Septo-Hippocampal Pathway and Striatum In Vivo

AF64A (ethylcholine mustard aziridinium ion) was stereotaxically administered bilaterally (1 nmol/side) into rat lateral cerebral ventricles. Choline acetyltransferase (ChAT) activity and ChAT mRNA levels were measured at predetermined time points in the septo-hippocampal pathway and striatum, both well identified as rich in cholinergic neurons. AF64A caused a rapid but transient increase in ChAT mRNA (167%, P < 0.05) and ChAT activity (164%, P < 0.01) in the septum. By day 7 post treatment, there was a significant decrease in ChAT mRNA (42.5% of control, P < 0.05) in the septum although the ChAT activity still stayed high. This decreased ChAT mRNA level in the septum lasted for at least four weeks, and was paralleled by a long-lasting decrease in ChAT activity in the hippocampus. In the striatum, on the other hand, there were no observed changes in either ChAT activity or ChAT mRNA. These data suggest that the long term effect of AF64A on the septo-hippocampal cholinergic pathway may, at least in part, be due to an action of AF64A on gene expression in the cholinergic neuron. The difference in the response to AF64A between the septo-hippocampal and striatal cholinergic systems might be due to their difference in neuron types.     

Chromogranins A and B and secretogranin II in hormonally identified endocrine cells of the gut and the pancreas

Chromogranins A and B and secretogranin II have been localized in a wide spectrum of gastroenteropancreatic endocrine/paracrine cells. Chromogranin A immunoreactivity showed the widest distribution and was displayed by glucagon-, PP-, gastrin-, gastrin-CCK-, secretin-immunoreactive cells, the most intense stainings being peculiar of enterochromaffin cells. Chromogranin B immunoreactivity was detected in gastrin- and glucagon cells and in some enterochromaffin cells containing also chromogranin A. Secretogranin II was paired to chromogranin A in glucagon cells of pancreatic islets or occurred alone in glycentin/PP cells of colonic mucosa. Neither of the chromogranins nor secretogranin II have been so far detected in somatostatin-, GIP-, or motilin-immunoreactive cells. Chromogranin A but not chromogranin B or secretogranin II has been detected in the gastric argyrophilic ECL cells.     

Sequence analysis, tissue distribution and regulation by cell depolarization, and second messengers of bovine secretogranin II (chromogranin C) mRNA

Secretogranin II is a very acidic, tyrosine-sulfated protein found in secretory granules of cells belonging to the diffuse neuroendocrine system. It gained more general importance recently as a universal immunohistochemical marker for endocrine neoplasms. Sequence information was obtained from secretogranin II isolated from bovine anterior pituitaries, allowing the isolation of cDNA clones and deduction of its primary structure. Bovine secretogranin II is a 586-amino acid protein of 67,455 Da which is preceded by a signal peptide of 27 residues and contains 9 pairs of basic amino acids in its sequence which are used as potential cleavage sites for generation of physiologically active peptides. Moderately abundant mRNA levels were found in adrenal medulla, pituitary, hippocampus, and caudate. Secretogranin II message was absent from parathyroid gland, adrenal cortex, kidney, liver, and spleen. Depolarization of isolated chromaffin cells by various secretagogues significantly up-regulated secretogranin II mRNA levels by mechanisms distinct from those established for chromogranins and neuropeptides, components maintained along with secretogranin II in neuroendocrine storage vesicles.     

Cholinergic Deficit Induced by Ethylcholine Aziridinium (AF64A) Transiently Affects Somatostatin and Neuropeptide Y Levels in Rat Brain

The question whether during the process of cholinergic degeneration somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and cortex react to the withdrawal of cholinergic function was addressed. After bilateral intracerebroventricular injection of the cholinotoxin ethylcholine aziridinium (AF64A; 1 or 2 nmol/ventricle) in rats, the activity of choline acetyltransferase (ChAT) started to decline in the hippocampus within 24 h. The reduction of ChAT activity reached its maximum within 4 days (34 and 55% after 1 and 2 nmol of AF64A/ventricle, respectively) and persisted during the observation period of 14 days. In the parietal cortex, ChAT activity decreased by 23% 4 days after 2 nmol of AF64A/ventricle. The loss in ChAT activity was accompanied by a transient decline in the levels of somatostatin and a transient increase in the levels of neuropeptide Y in both brain areas. In the hippocampus, the reduction in somatostatin content was most pronounced after 2 days (by 22 and 33% after 1 and 2 nmol of AF64A/ventricle, respectively). Within 14 days, somatostatin levels returned to control values. Neuropeptide Y levels increased slightly by approximately 25% of control values in the hippocampus. The changes described were present in both the dorsal and ventral subfields of the hippocampus. Similar but less pronounced changes in levels of both neuropeptides were observed in the parietal cortex. The present data provide further evidence for a close neuronal interrelationship between cholinergic and somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and parietal cortex.     

Photoperiod Regulates vgf-Derived Peptide Processing in Siberian Hamsters

VGF mRNA is induced in specific hypothalamic areas of the Siberian hamster upon exposure to short photoperiods, which is associated with a seasonal decrease in appetite and weight loss. Processing of VGF generates multiple bioactive peptides, so the objective of this study was to determine the profile of the VGF-derived peptides in the brain, pituitary and plasma from Siberian hamsters, and to establish whether differential processing might occur in the short day lean state versus long day fat. Antisera against short sequences at the C- or N- termini of proVGF, as well as against NERP-1, TPGH and TLQP peptides, were used for analyses of tissues, and both immunohistochemistry and enzyme linked immunosorbent assay (ELISA) coupled with high-performance liquid (HPLC) or gel chromatography were carried out. VGF peptide immunoreactivity was found within cortex cholinergic perikarya, in multiple hypothalamic nuclei, including those containing vasopressin, and in pituitary gonadotrophs. ELISA revealed that exposure to short day photoperiod led to a down-regulation of VGF immunoreactivity in the cortex, and a less pronounced decrease in the hypothalamus and pituitary, while the plasma VGF levels were not affected by the photoperiod. HPLC and gel chromatography both confirmed the presence of multiple VGF-derived peptides in these tissues, while gel chromatography showed the presence of the VGF precursor in all tissues tested except for the cortex. These observations are consistent with the view that VGF-derived peptides have pleiotropic actions related to changing photoperiod, possibly by regulating cholinergic systems in the cortex, vasopressin hypothalamic pathways, and the reproductive axis.     

Stimulation of rat adrenal medulla can induce differential changes in the peptide and mRNA levels of chromogranins, neuropeptides and other constituents of chromaffin granules

The levels of various components of chromaffin granules were determined in rat adrenals after treatment with several stimulants. After reserpine the levels of calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and chromogranin B but not those of chromogranin A and secretogranin II were elevated. On the other hand, the mRNA of chromogranins A, B and secretogranin II were significantly increased. Treatment with oxotremorine or nicotine (multiple injections for 2 or 3 days) induced analogous changes for peptide and mRNA levels, however, the increases were smaller and for the mRNA less consistent. A single injection of oxotremorine or nicotine raised only the levels of CGRP and NPY and of the NPY mRNA whereas those of the chromogranins and their respective mRNAs remained unaltered. Amongst the membrane proteins only the levels of dopamine beta-hydroxylase are increased after prolonged stimulation, whereas those of cytochrome b-561, carboxypeptidase H and synaptin/synaptophysin (SYN) remain unaltered. Thus, the biosynthesis of chromaffin granules can be regulated in quite sophisticated patterns.     

Effect of Nerve Growth Factor in Ethylcholine Mustard Aziridinium (AF64A)-Treated Rats: Sensitization of Cholinergic Enzyme Activity in the Septohippocampal Pathway

Abstract: In this study, we examined the effects of nerve growth factor (NGF) administration on cholinergic enzyme activity in both normal and ethylcholine mustard aziridinium (AF64A)-treated rats. Choline acetyltransferase (ChAT) and acetylcholinesterase activity were measured in the hippocampus and septum of rats chronically administered NGF (0.36–2.85 µg/day) into the lateral ventricle for 14 days. In both normal and AF64A-treated rats, NGF increased cholinergic enzyme activity in a dose-dependent manner. Furthermore, although NGF increased ChAT activity in normal rats by 147%, it had a greater effect in AF64A-treated rats, increasing ChAT activity as much as 273%. NGF increased acetylcholinesterase activity in normal rats by only 125% but produced a 221% increase in this activity in AF64A-treated rats. These data indicate that AF64A produces an increased sensitivity to NGF in cholinergic neurons.     

Selective Presynaptic Cholinergic Neurotoxicity Following Intrahippocampal AF64A Injection in Rats

Compound AF64A, ethylcholine mustard aziridinium ion (0.4-8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10-fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+-dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r = 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histochemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long-term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.     

The role of pancreatic chromogranins in islet physiology

Chromogranins are acidic secretory glycoproteins with a widespread but specific distribution in neuroendocrine tissues. The chromogranin family is heterogenous, consisting of propeptides such as chromogranin-A, chromogranin-B and secretogranin II, which can either elicit an effect themselves, or serve as precursors to a large number of peptides, which are biologically more active. Chromogranin processing varies in different neuroendocrine tissues. Furthermore, it is more marked in pancreatic islets than in many other tissues. Chromogranin-A and chromogranin-B are expressed in all types of pancreatic islet cells, whereas secretogranin II has not been found in pancreatic tissue. The aim of the present mini review is to focus on chromogranin-A, chromogranin-B and their derived peptides, in the function of pancreatic islets.     

Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas.

To investigate the constituents of the matrix of endocrine secretory granules, we analyzed endocrinoilogically silent ("non-functioning") human pituitary adenomas for the occurrence of the chromogranins/secretogranins (granins), a protein family normally stored together with many different hormones. When five non-functioning pituitary adenomas were analyzed by immunoblotting using polyclonal and monoclonal antibodies specific for individual members of the granin family, chromogranin A was detected in four cases and chromogranin B and secretogranin II were detected in all cases. The cellular distribution of the granins and of various hormones known to be expressed in the anterior pituitary was studied by immunocytochemistry in fixed, frozen tissue sections from five additional adenomas. Of the eight hormones investigated, only thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone were detected, occurring in only two of the five adenomas. In contrast, granins were found in all five tumors. Chromogranin B and secretogranin II were detected in each of the adenomas in virtually every cell studied, whereas chromogranin A exhibited such a widespread cell distribution in only three adenomas, being focally present in one and absent from the other tumor. The subcellular localization of the granins and the three glycoprotein hormones was investigated by double immunoelectron microscopy. Chromogranin A and chromogranin B were mainly co-localized in secretory granules, whereas secretogranin II was either co-localized with the other two granins or segregated to different secretory granules. When present, glycoprotein hormones were immunodetected in both the secretory granules containing all three granins and those containing mainly secretogranin II. Our data indicate that in non-functioning pituitary adenomas chromogranin A is differentially expressed from chromogranin B and secretogranin II. Moreover, the granins appear to be the most widespread constituents of endocrine secretory granules known, forming the dense-core matrix irrespective of the presence or absence of hormones.     

Cholinotoxicity induced by ethylcholine aziridinium ion after intracarotid and intracerebroventricular administration

The effect of ethylcholine aziridinium ion (AF64A) after an intracerebroventricular (icv) injection was compared to that obtained after an intravascular administration. Reductions in choline acetyltransferase (ChAT) and acetylcholinesterase activities in the hippocampus but not in the cerebral cortex or the corpus striatum were observed 10 days after bilateral injection of AF64A into the rat cerebroventricles (3 nmol/side). However, when AF64A was injected into the carotid artery (1 mumol/kg) following a unilateral opening of the blood-brain barrier by a hypertonic treatment, a significant decrease in ChAT activity was observed in the ipsilateral side of the cerebral cortex but not in hippocampus, corpus striatum, or cerebellum. High-affinity choline transport was reduced significantly 11 days after an icv injection of AF64A in all the above mentioned brain regions, and recovered 60 days post injection in the cerebral cortex and in the corpus striatum but not in the hippocampus. Our results suggest that in various brain regions, AF64A causes various degrees of damage to cholinergic neurons, depending on the quantity of the toxin that reaches the target tissue.     

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

Effects of AF64A on cholinergic neurotransmission in the sixth abdominal ganglion of the cockroach

1. The effects of ethylcholine mustard aziridinium ion (AF64A) on the cholinergic neurotransmission in the sixth abdominal ganglion of the cockroach were studied electrophysiologically and morphologically. 2. The pre- and post-synaptic compound action potentials (CAPs) elicited via electrical stimulation of the presynaptic fibers were recorded extracellularly. 3. The amplitude of both CAPs was depressed by AF64A (50-400 microM) in a concentration- and time-dependent manner. 4. At a high concentration, they were abolished but 100 microM of carbachol still evoked the postsynaptic event. 5. Electron microscopic observation of AF64A-treated ganglia showed that nerve terminals containing small lucent vesicles could not be observed but those containing dense core or large granular vesicles changed only slightly in shape. 6. These results suggest that AF64A is selectively neurotoxic for the presynaptic cholinergic neurons in the sixth abdominal ganglion of the cockroach.     

Selective impairment of acetylcholine release and content in the central nervous system following intracerebroventricular administration of ethylcholine mustard aziridinium ion (AF64A) in the rat

Ethylcholine mustard aziridinium ion (AF64A) was administered via intracerebroventricular injection to rats. Unilateral injection of 40 nmol AF64A resulted in pronounced toxicity with an 80% mortality rate. Administration of 10 nmol unilaterally resulted in a significant reduction in both acetylcholine content and ouabain stimulated acetylcholine release in the hippocampus 2, 4 and 7 days after treatment. Non-specific changes in hippocampal levels of dopamine, noradrenaline and 5-hydroxytryptamine were also observed.Bilateral injection of 5 nmol AF64A was more effective than a unilateral 10 nmol injection in reducing acetylcholine release from hippocampus 4 and 7 days after treatment. Hippocampal acetylcholine content was also reduced (to 35% of control). In contrast, there was less effect on acetylcholine content in striatum and frontal cortices, and acetylcholine release from these areas was not decreased. Although there was a transient reduction in hippocampal 5-hydroxytryptamine content 4 days after treatment, this had recovered to control levels within 7 days. 5-Hydroxytryptamine levels in striatum or cortex were not affected, nor were there any changes in noradrenaline or dopamine contents in the areas studied.This study indicates that, in the correct dose range, AF64A can exert selective effects on cholinergic systems, particularly in the hippocampus. The selective cholinotoxicity of this compound makes it a useful tool in developing animal models of cholinergic dysfunction.