heparitin-sulfate and Pheochromocytoma

To elucidate the mechanism of secretory granule formation, we here identify the first intermediate in this process, the immature secretory granule, in the neuroendocrine cell line PC12 and demonstrate the packaging of a regulated secretory protein, secretogranin II, to immature secretory granules in a cell-free system. The formation of immature secretory granules was as fast (t1/2 approximately 5 min) as that of constitutive secretory vesicles identified by the presence of a rapidly secreted heparan sulfate proteoglycan. Using the cell-free system, the formation of post-Golgi secretory vesicles was found to be dependent upon ATP. Two distinct populations of vesicles were formed: immature secretory granules containing secretogranin II and constitutive secretory vesicles containing the heparan sulfate proteoglycan. These results show that in a cell-free system, a constitutive and a regulated secretory protein are sorted upon exit from the trans-Golgi network.

Topics: Adenosine Triphosphate; Adrenal Gland Neoplasms; Animals; Cell Line; Cell-Free System; Chondroitin Sulfate Proteoglycans; Cytoplasmic Granules; Glycosaminoglycans; Golgi Apparatus; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kinetics; Microscopy, Electron; Pheochromocytoma; Protein Processing, Post-Translational; Proteoglycans

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.

Topics: Adrenal Gland Neoplasms; Animals; Chondroitin Sulfates; Electrophoresis, Cellulose Acetate; Glycosaminoglycans; Heparitin Sulfate; Pheochromocytoma; Rats; Sulfates; Sulfur Radioisotopes; Tumor Cells, Cultured

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.

Topics: Adrenal Gland Neoplasms; Animals; Cell Adhesion; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Glucosamine; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Molecular Weight; Nerve Growth Factors; Oligosaccharides; Pheochromocytoma; Proteoglycans; Rats; Sulfates; Tumor Cells, Cultured

We have examined the size, charge, and sulfation pattern of heparan sulfate in the cell-soluble fraction, membranes, and culture medium of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF) and compared the structural features of PC12 cell heparan sulfate to that of rat brain at several stages of early postnatal development. Nitrous acid degradation studies revealed significant differences in the distribution of N-sulfate and N-acetyl groups in heparan sulfate present in the PC12 cell-soluble fraction, membranes, and medium and demonstrated that NGF treatment led to an increased proportion of N-sulfated segments in the cell-associated heparan sulfate, although no such change was seen in that released into the culture medium. There was very little change in the N-sulfation of brain heparan sulfate during the first 30 days after birth. In brain, most of the heparan sulfate glucosamine residues are N-sulfated and yield predominantly di- and tetrasaccharide nitrous acid degradation products, whereas PC12 cell heparan sulfate contains large blocks of N-acetylglucosamine residues. There was very little difference in the overall charge or size (approximately 15,000 Da) of heparan sulfate chains between the different PC12 cell fractions or brain, although NGF treatment led to a decrease in the proportion of less-charged chains in the PC12 cell membranes and a small increase in molecular size. Our studies therefore demonstrate the presence in PC12 cells of several pools of heparan sulfate having different structural properties, and that significant alterations in the charge, size, and sulfation pattern of PC12 cell heparan sulfate accompany NGF-induced differentiation and neurite outgrowth.

Topics: Adrenal Gland Neoplasms; Age Factors; Animals; Brain; Cell Adhesion; Cell Differentiation; Cell Line; Glycosaminoglycans; Heparitin Sulfate; Molecular Weight; Nerve Growth Factors; Neuroblastoma; Nitrous Acid; Pheochromocytoma; Rats

We studied the distribution of the molecular forms of acetylcholinesterase (AChE) in a stable variant (F3) of the rat pheochromocytoma cell line, PC12, that lacks a heparan sulfate proteoglycan on the cell surface. After treatment with nerve growth factor F3 cells synthesize less 4S enzyme, and more 10S and 16S enzyme than normal PC12 cells. This distribution is similar to that seen in normal cells after incubation with beta-D-xylosides, molecules that interfere with proteoglycan assembly. Using collagenase treatment and membrane-permeable and -impermeable inhibitors of AChE, we determined the cellular location of the AChE forms. Although in normal cells greater than 90% of the 16S AChE is on the cell surface, approximately 60% is present in an internal pool in the variant. Following irreversible inhibition of all forms of AChE in the variant, the newly synthesized 16S AChE appears in the internal pool after a 1-h lag, but is not detected on the cell surface until after 2.5 h. Our results thus show that 16S AChE is assembled internally within neuronal cells and that alterations in the synthesis and distribution of proteoglycans affect the total amount and cellular localization of the 16S AChE form.

Topics: Acetylcholinesterase; Animals; Benzenaminium, 4,4'-(3-oxo-1,5-pentanediyl)bis(N,N-dimethyl-N-2-propenyl-), Dibromide; Cell Line; Cell Membrane; Cell Membrane Permeability; Centrifugation, Density Gradient; Cholinesterase Inhibitors; Chondroitin Sulfate Proteoglycans; Glycosaminoglycans; Glycosides; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Isoflurophate; Mutation; Nerve Growth Factors; Pheochromocytoma; Proteoglycans; Rats

We have identified a unique heparan sulfate (HeS) proteoglycan synthesized by the neuronal-like cell line PC12. The proteoglycan, purified with monoclonal antibodies from medium conditioned by PC12 cells, has an apparent molecular weight of 350,000, and it contains a Mr 80,000 core protein and HeS side chains of Mr 15,000 each. The purified molecule has the same apparent size and density as it has in conditioned medium. HeS proteoglycans that are indistinguishable antigenically but very difficult to solubilize are found on the external surface and in the interior of PC12 cells and neurons. Mild proteolysis converts the surface proteoglycan into a molecule closely resembling that found in the medium. The same surface antigens are also present on a subpopulation of T-cells and on a non-neuronal accessory cell found in dorsal root ganglion cultures. The PC12 cell line and the non-neuronal dorsal root ganglion cells secrete a factor into medium that, after adsorption to polylysine-coated surfaces, induces rapid neurite out-growth by primary sympathetic neurons. The monoclonal antibodies used to purify the neuronal HeS proteoglycan from PC12 cells are capable of depleting this conditioned medium of its neurite-promoting activity. These studies suggest that a HeS proteoglycan synthesized and secreted by neurons and certain accessory cells plays a role in regulating neurite outgrowth.

Topics: Animals; Antibodies, Monoclonal; Brain Chemistry; Cell Line; Chondroitin Sulfate Proteoglycans; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunologic Techniques; Neurons; Pheochromocytoma; Proteoglycans; Rats; Rats, Inbred Strains; Rodentia