Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.     

Immunolocalization of secretogranin II, chromogranin A, and chromogranin B in differentiating human neuroblastoma cells.

In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.     

A denaturant-insoluble form of tyrosine hydroxylase in PC12 pheochromocytoma cells

A 6M urea-insoluble form of tyrosine hydroxylase (TH_i) was detected in PC12 pheochromocytoma cells by western blotting immunodetection methods, and the characteristics and mechanisms of formation of this insoluble species were investigated. TH_i accounts for about 4% of the immunodetectable tyrosine hydroxylase in exponentially dividing pheochromocytoma cells. It is unlikely that a subpopulation of dead or dying cells is the source of TH_i since essentially no changes in TH_i levels were detected when cell death was intentionally increased. To measure the kinetics of formation of cellular TH_i, exponentially dividing cells were metabolically labeled first with [³H]leucine and then with [¹⁴C]leucine, and though both³H and¹⁴C were incorporated into soluble tyrosine hydroxylase, the near absence of¹⁴C in TH_i demonstrated that a lag period of at least a day exists between biosynthesis of tyrosine hydroxylase and the accumulation of measurable TH_i. The cellular accumulation of TH_i can evidently be regulated by the cell, since upon nerve growth factor (NGF) treatment of cells the total content of tyrosine hydroxylase increased and the content of TH_i decreased to yield, overall, a fivefold lower proportion of TH_i after 4 days. A large increase in urea-insoluble enzyme was found upon sublethal exposure of cells to ferrous ion and hydrogen peroxide, indicating that oxidative damage via metal-ion-catalyzed formation of hydroxide free radical can yield an enzyme that is similar in its insolubility to TH_i.Abbreviations DOPA 3,4-dihydroxyphenylalanine - NGF nerve growth factor - TH_i denaturant-insoluble tyrosine hydroxylase - EDTA ethylene diamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)-aminomethane - LLPM low-leucine pulse medium - WS water-solubilized protein - US 6 M urea-solubilized protein - UI 6 M urea-insoluble protein     

Identification of a chromogranin A domain that mediates binding to secretogranin III and targeting to secretory granules in pituitary cells and pancreatic beta-cells

Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca(2+)-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48-111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214-373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells.     

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4C1 cells

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.     

Chromogranin B (secretogranin I), a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in PC12 cells.

Chromogranin B (CgB, also called secretogranin I) is a secretory protein sorted to secretory granules in a wide variety of endocrine cells and neurons. Unexpectedly, after stimulation of regulated secretion in the neuroendocrine cell line PC12, a fraction of the exocytosed CgB was not released into the medium but remained associated with the plasma membrane. The addition of exogenous CgB to unstimulated cells did not result in the appearance of cell surface CgB, suggesting that the presence of cell surface CgB could not be accounted for by adsorption of released CgB to the cell surface. Upon further incubation of stimulated PC12 cells, the surface CgB was internalized by the cells and largely degraded. The surface CgB was not released by exposure to pH 11, yet it partitioned in the aqueous phase upon Triton X-114 phase separation. Subcellular fractionation and differential extraction studies showed that the membrane-associated CgB constituted at least 10% of the total cellular CgB. These observations suggest that (a) the appearance of CgB at the cell surface is due to fusion of secretory granules with the plasma membrane and (b) a fraction of CgB is present in tight association with the secretory granule membrane. We propose a model in which membrane-associated CgB, by virtue of its ability to interact in a homophilic manner with soluble CgB, plays a key role in the sorting and targeting of CgB to the regulated pathway.     

Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi- intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.     

Pharmacological profile of adenosine A2 receptor in PC12 cells

The PC12 cell line, a clone isolated from a pheochromocytoma tumor of rat adrenal medulla, was shown to exclusively contain stimulatory adenosine (A2) receptors linked to adenylate cyclase (AC). AC was stimulated 6-7 fold by several agonists with a rank order of potency of 5'-N-Ethyl carboxamidoadenosine (NECA) greater than 2-Chloroadenosine (2-CADO) greater than (R)-N-Phenylisopropyladenosine (R-(-)-PIA) greater than N6-Cyclopentyladenosine (CPA) greater than N6-Cyclohexyladenosine (CHA) greater than S-(+)-PIA. AC activity was antagonized by a variety of adenosine receptor antagonists with a potency order of 1,3,-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) greater than 1,3,-Diethyl-8-phenylxanthine (DPX) greater than 8-Phenyltheophylline greater than 3-Isobutyl-1-methylxanthine (IBMX) greater than 8-(p-sulfophenyl)theophylline (PST) greater than 7-(beta-chloroethyl)theophylline greater than theophylline = enprofylline = caffeine. Under conditions known to favour receptor-mediated Ni-coupled inhibition of AC, R-(-)-PIA failed to inhibit both basal and forskolin stimulated AC activity in PC12 cells, confirming the absence of an A1 mediated response. On the other hand, adenosine agonists inhibited AC activity in rat cortical membranes with a rank order of potency of CPA greater than R-(-)-PIA greater than CHA greater than NECA greater than S-(+)-PIA greater than 2-CADO. These findings suggest that PC12 cells are functionally deficient in an A1 receptor linked AC response but are efficiently coupled to A2 stimulatory receptors. The cells should prove useful for further study of A2 adenosine receptors and to establish selectivity profiles of compounds acting at both A1 and A2 receptors.     

Glucocorticoid- and nerve growth factor-induced changes in chromogranin A expression define two different neuronal phenotypes in PC12 cells

The regulation of chromogranin A mRNA was examined in PC12 cells after treatment with nerve growth factor, dexamethasone, or a combination of the two agents. PC12 cells have low levels of chromogranin A mRNA, and this does not change upon treatment with nerve growth factor. Dexamethasone treatment of these cells results in a 4-fold increase in the amount of chromogranin A mRNA. The dexamethasone-stimulated increase in chromogranin A mRNA is not apparent until at least 16 h after the addition of the drug and is maintained only with continuous culture in the presence of the drug. Dexamethasone and nerve growth factor together increase chromogranin A mRNA to the level seen with dexamethasone alone. Immunohistochemistry shows a similar pattern of protein accumulation within individual cells. Chromogranin B mRNA levels are unaltered by any of the drug treatments described. Treatment with dexamethasone plus NGF seems to be required for full expression of the adrenergic, neuronal phenotype in PC12 cells. Measurement of chromogranin A mRNA provides more specific delineation of neural differentiation and how it is influenced by hormones and growth factors.     

Immunoreactivities for chromogranin A and B, and secretogranin II in the guinea-pig endocrine pancreas

The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

Immunoreactivities for chromogranin A and B,and secretogranin II in the guinea-pig endocrine pancreas

Summary The chromogranins are acidic proteins present in various endocrine cells and organs. They consist of chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). In the pancreas, these proteins or their breakdown products are possibly involved in the regulation of pancreatic hormone secretion. The guinea-pig endocrine pancreas was now investigated immunohistochemically for the presence of the chromogranins in five endocrine cell types. CgA is a regular constituent of insulin (B-), pancreatic polypeptide (PP-) and enterochromaffin (EC-) cells. In addition, a minority of somatostatin (D-) cells were immunoreactive for CgA. CgB immunoreactivities were very faint and exclusively observed in B-cells. SgII was found in B- and PP-cells; a faint immunostaining for SgII was also seen in a few glucagon (A-) cells. Typically, the densities of CgA or SgII immunoreactivities varied among the members of a given cell population, e.g. among individual B- or PP-cells. The present findings about the heterogeneities of immunoreactivities for the chromogranins are in line with findings obtained in pancreatic endocrine cells of other species. The true reasons for these heterogeneities are enigmatic. It seems probable, however, that the corresponding immunoreactivities depend on the intracellular processing of the chromogranins which in turn might be related to the metabolic state of endocrine cells. This has to be examined in future by experimental investigations.     

L-dopa and dopamine enhance the formation of aggregates under proteasome inhibition in PC12 cells

The formation of inclusion bodies in dopaminergic neurons is associated with the pathogenesis of Parkinson's disease. In order to clarify the role of dopamine/L-dopa in the formation of protein aggregates, we investigated dopamine/L-dopa-related aggregation using an experimental inclusion model. The inhibition of tyrosine hydroxylase (TH) by alpha-methyltyrosine dramatically decreased MG132-induced aggregate formation. In addition, the inhibition of TH caused the upregulation of proteasomes in cultured cells and the dopamine/L-dopa induced non-enzymatic polymerization of ubiquitin. This inhibition did not affect cell viability. These results suggest that dopamine/L-dopa might enhance aggregate formation, and that intracellular aggregates may not be toxic to cells.     

A cell-free system for regulated exocytosis in PC12 cells

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.     

Synaptotagmin VII is targeted to secretory organelles in PC12 cells, where it functions as a high-affinity calcium sensor

Synaptotagmin (syt) I is thought to act as a Ca2+ sensor that regulates neuronal exocytosis. Fifteen additional isoforms of syt have been identified, but their functions are less well understood. Here, we used PC12 cells to test the idea that different isoforms of syt impart cells with distinct metal (i.e., Ca2+, Ba2+, and Sr2+) requirements for secretion. These cells express syt's I and IX (syt IX sometimes referred to as syt V), which have low apparent metal affinities, at much higher levels than syt VII, which we show has a relatively high apparent affinity for metals. We found that syt I and VII partially colocalize on large dense core vesicles and that upregulation of syt VII produces a concomitant increase in the divalent cation sensitivity of catecholamine release from PC12 cells. Furthermore, RNA interference-mediated knockdown of endogenous syt VII reduced the metal sensitivity of release. These data support the hypothesis that the complement of syt's expressed by a cell, in conjunction with their metal affinity, determines the divalent cation sensitivity of exocytosis.     

Identification of IRAS/Nischarin as an I1-imidazoline receptor in PC12 rat pheochromocytoma cells

The I1-imidazoline receptor (I1R) is a proposed target for drug action relevant to blood pressure and glucose control. The imidazoline receptor antisera-selected (IRAS) gene, also known as Nischarin, has several characteristics of an I1R. To test the contribution of IRAS to I1R binding capacity and cell-signaling function, an antisense probe targeting the initiating codon of rat IRAS gene was evaluated in PC12 rat pheochromocytoma cells, a well-established model for I1R action. The density of I1R was significantly reduced by antisense compared with control transfection (Bmax = 400 +/- 16 vs. 691 +/- 29 fmol/mg protein), without significantly affecting binding affinity (Kd = 0.30 +/- 0.04 vs. 0.39 +/- 0.05 nmol/L). Thus, IRAS expression is necessary for high-affinity binding to I1R. Western blots with polyclonal anti-IRAS showed reduced IRAS expression in the major 85-kDa band relative to an actin reference, paralleling the reduction in binding site density. To determine whether reduced IRAS expression attenuated I1R cell signaling, PC12 cells transfected with antisense or sense oligo-DNA were treated with moxonidine, an I1R agonist, then cell lysates were analyzed by western blot. Dose-dependent activation of extracellular signal-regulated kinase was attenuated without affecting the potency of the agonist. In contrast, extracellular signal-regulated kinase activation by insulin was unchanged. The IRAS gene is likely to encode an I1R or a functional subunit.     

p75 neurotrophin receptor functions as a survival receptor in brain-metastatic melanoma cells

The p75 neurotrophin receptor (p75(NTR)), a common receptor for members of the neurotrophins (NT) family, was previously identified as a molecular determinant of brain metastasis. We have also reported that NT treatment of murine and human brain-metastatic melanoma cells affects their invasive capacities and increases the production of heparanase, an important and unique extracellular matrix (ECM) degradative enzyme. Neurotrophism can be a survival-support mechanism for brain-metastatic cells and a survival assay was devised to mimic the growth limiting conditions of rapidly expanding metastatic tumors prior to neoangiogenesis. We report that p75(NTR) promoted the survival of brain-metastatic melanoma cells but not melanocytes in stress cultures conditions. Secondly, melanoma cells fluorescently sorted for high p75(NTR) expression (p75(NTR-H) cells) had an up to a 15-fold greater survival than those sorted for low p75(NTR) expression (p75(NTR-L) cells). Thirdly, cells overexpressing p75(NTR) associated with the growth fraction and provided these cells with an inherent growth advantage. Finally, we observed an increased survival of sorted p75(NTR-L) cells, dependent upon treatment of NT members whose functional receptors are present on these cells. Together, these results delineate that p75(NTR)-mediated trophic support profoundly affects competitive melanoma-cell survival when the tumor cell microenvironment becomes growth limiting.     

The beta-PDGF receptor induces neuronal differentiation of PC12 cells.

Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.