David V. Cohn

Formation of bone by isolated, cultured osteoblasts in millipore diffusion chambers.

SummaryOsteoblast-like and osteoclast-like cells freed from neonatal calvaria by sequential enzymatic digestion after 6–7 days in culture were placed in diffusion chambers and implanted in the peritoneal cavities of CD-1 mice. About half of the chambers also contained a dead calvarium to test for the need of an “inducer.” After 20 days, 11 of 18 chambers containing the osteoblast-like cells formed large foci of mineralized bone that corresponded to alkaline phosphatase activity throughout the chambers. Moreover, only type I (i.e., bone) collagen was formed. Occasional deposits of bone were found in only 3 of 22 chambers containing the osteoclast-like cells. The presence of dead bone did not affect any of the results. These data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.

Biosynthesis of proparathyroid hormone and parathyroid hormone: Chemistry, physiology and role of calcium in regulation

The discovery of bovine proparathyroid hormone is reviewed, and recent findings regarding the structure of the prohormone are presented. Sequence analyses on two prohormone preparations have established that the NH2-terminal region-of the bovine prohormone is the hexapeptide, Lys-Ser-Val-Lys-Lys-Arg, attached to the NH*-terminal Ala of the hormone form. The sequence of the COOH-terminal region of the prohormone is not yet known. Our present data on the human prohormone suggest that it consists of a similar NH*-terminal hexapeptide. The newly synthesized peptides are separable from the bulk of the preexisting hormone by either deoxycholate extraction or sucrose gradient centrifugation, which permits studies of the subcellular distribution of the prohormone and hormone; from such studies a proposal is made concerning the intracellular locales of synthesis and conversion of prohormone to hormone. Glands from rats receiving a low calcium intake convert prohormone to hormone more efficiently than do glands from animals receiving a normal high calcium intake. A model depicting the various sites at which calcium may affect the production of hormone is proposed.

Biosynthesis of Proparathyroid Hormone and Parathyroid Hormone by Human Parathyroid Glands

Human parathyroid glands obtained at autopsy were incubated with [(3)H]leucine and [(3)H]lysine. After incubation, nonradioactive parathyroid tissue of either human or bovine origin was added. Radioactive parathyroid hormone and proparathyroid hormone were isolated from the gland and medium by organic solvent and salt fractionation, trichloroacetic acid precipitation, Sephadex G-100 gel filtration, and carboxymethyl cellulose column chromatography. The human hormonal peptides were identified in the ion-exchange column eluates by their relatively high levels of radioactivity, their elution positions, and their immunoreactivity to anti-PTH antiserum. The time-course of radioactive amino acid incorporation into these peptides and a brief incubation of the gland with radioactive amino acids, followed by various lengths of incubation with nonradioactive amino acids, indicated that a precursor-product relationship exists for the two peptides. An alternate method for isolation of the hormone and prohormone, which involves separation of peptides by urea-polyacrylamide gel electrophoresis, confirmed the identities of the human parathyroid hormone and proparathyroid hormone.

The migration behavior of proparathyroid hormone, parathyroid hormone, and their peptide fragments during gel filtration☆

Abstract The migration behavior of bovine proparathyroid and parathyroid hormones, as well as several hormonal peptide fragments, has been analyzed by gel filtration on thin-layer plates and by column chromatography. The two hormones migrated appreciably faster than was expected for their molecular weights. Their migration rates decreased with increasing pH and approached values more characteristic of their molecular weights at pH 11.0. Migration rates were the same over a concentration range of 2 × 10 −6 –0.9 × 10 −3 m . These results indicate that parathyroid hormone does not exist in solution as an aggregate at the concentrations used in these experiments. These studies suggest that the two hormones are asymmetrical, unfolded monomers in acid solution which become more folded or globular at alkaline pH values.

Intracellular Processing and Secretion of Parathyroid Gland Proteins

Publisher Summary This chapter focuses on parathyroid hormone (PTH), its synthesis, intracellular processing, and secretion, and provides current information on the chemistry and biology of secretory protein-I (SP-I) and its possible relationship to PTH. The parathyroid gland is important in fields related to calcium metabolism because of the role played by its hormone PTH plays in the regulation of body calcium. The chapter also focuses on the gland because of the realization that it also synthesizes and secretes a major glycoprotein termed SP-I that is similar to chromogranin A (CGA), a protein co-secreted with epinephrine by the adrenal. SP-I/CGA appears to be present in a large number of endocrine, but not exocrine, cells. PTH is unglycosylated and generally does not contain modified amino acids. PTH exhibits a complex conformation in solution that appears to reorder at different pHs. Images of PTH examined by dark-field electron microscopy suggest that the molecule consists of two domains connected by a short stalk. The fundamental mechanisms responsible for secretory control are at least as complex as those regulating intracellular formation and processing of the hormone.

Processing of chromogranin A in the parathyroid: generation of parastatin-related peptides

Chromogranin A (CgA) is a glycoprotein present in secretory granules of endocrine cells. In the parathyroid, it is costored and cosecreted with parathormone (PTH) in response to hypocalcemia. CgA is the precursor of several bioactive peptides including pancreastatin and betagranin. Parastatin (PARA, pCgA(347-419)) is a novel peptide that we generated in vitro by enzymatic digestion of pCgA. In vitro, it inhibits low Ca(2+)-stimulated parathyroid secretion. Full activity resides in its first 19 residues. In order to determine if PARA or PARA-derived peptides are natural products of the parathyroid, we generated an antiserum directed against pCgA(347-359) corresponding to the bioactive N-terminal sequence of pPARA (pPARA(1-13) antiserum), and developed a specific radioimmunoassay that we used in conjunction with various chromatographic separations. We identified small peptides carrying the pPARA(1-13) immunoactivity in extracts and secretion medium of porcine parathyroid glands. Continuous and pulse-chase radiolabeling studies, along with immunoprecipitation using PARA(1-13) antiserum demonstrate that a newly-synthesized PARA-related peptide fraction with a Mr of 11 kDa is secreted by the parathyroid cells and accumulates in the secretion medium. Edman degradation of the 11 kDa PARA-related peptide band by Edman degradation yielded three major N-terminal sequences: S-K-M-D-R-L-A-K-E-L-(residues 313-322), D-R-L-A-K-E-L-T-A-E-(residues 316-325), and A-K-E-L-T-A-E-K-R-L-(residues 319-329), in a molar ratio of approximately 1:2:1. The peptide bonds required to be cleaved to yield these peptides, Trp-Ser, Met-Asp and Leu-Ala, suggest that a chymotrypsin-like endopeptidase participated in their formation. The molecular size and the results of amino acid compositional analysis, indicate that the C-termini of these peptides extended variably to residues 384-401 of pCgA. These results demonstrate that processing of CgA by the parathyroid gland generates bioactive PARA-related peptides that could affect the glands secretory activity.

Autocrine inhibition of parathyroid cell secretion requires proteolytic processing of chromogranin A

Chromogranin A (CgA, Secretory Protein-I) is a protein of about 450 amino acids representing a major soluble component of the secretory granules of parathyroid and other endocrine and neuroendocrine cells. In the parathyroid, CgA is costored and cosecreted with parathormone (PTH). We earlier found that CgA and the derived peptide, pancreastatin, inhibited secretion of PTH and CgA by parathyroid cells in culture and that CgA antiserum stimulated secretion above the maximum achieved at low (0.5 mM) Ca2+. In the present study, porcine parathyroid cells were incubated at different cell concentrations at low Ca2+. The amount of secreted CgA increased over the 6-h incubation period at 1 x 10(6) to 4 x 10(6) cells/ml, but plateaued after 3 h at 6 x 10(6) cells/ml. Secretion did not plateau when antisera were added at 3 h. Conditioned medium contained a factor or factors that blocked secretion by fresh parathyroid cells at 0.5 mM Ca2+. Pulse-chase studies revealed that 40% of the secreted CgA was processed after 6 h of chase. alpha-2-macroglobulin, an inhibitor of proteolytic processing, increased the amount of CgA in the medium by 30% at 1 h of chase and decreased the amount processed to 20% by 6 h. Other protease inhibitors similarly enhanced the amount of CgA in the medium. These data indicate that proteolytic processing of intact CgA is requisite for its autocrine inhibitory activity.

Effects of pancreastatin and chromogranin A on insulin release stimulated by various insulinotropic agents.

The effects of porcine pancreastatin on insulin release stimulated by insulinotropic agents, glucagon, cholecystokinin-octapeptide (CCK-8), gastric inhibitory polypeptide (GIP) and L-arginine, were compared to those of bovine chromogranin A (CGA) using the isolated perfused rat pancreas. Pancreastatin significantly potentiated glucagon-stimulated insulin release (first phase: 12.5 +/- 0.9 ng/8 min; second phase: 34.5 +/- 1.6 ng/25 min in controls; 16.5 +/- 1.1 ng/8 min and 44.0 +/- 2.2 ng/25 min in pancreastatin group), whereas CGA was ineffective. The first phase of L-arginine-stimulated insulin release was also potentiated by pancreastatin (6.9 +/- 0.5 ng/5 min in controls, 8.4 +/- 0.6 ng/5 min in pancreastatin group), but not by CGA. Pancreastatin did not affect CCK-8 or GIP-stimulated insulin release. Similarly, CGA did not affect insulin release stimulated by CCK-8 or GIP. These findings suggest that pancreastatin stimulates insulin release in the presence of glucagon. Because pancreastatin can have multiple effects on insulin release, which are dependent upon the local concentration of insulin effectors, pancreastatin may participate in the fine tuning of insulin release from B cells.

Chromogranin-A secretion from individual parathyroid cells: effects of 1,25-(OH)2vitamin D3 and calcium

Chromogranin-A (CgA) is a 50-kDa protein located in and secreted by most endocrine and neuroendocrine cells along with native hormone. In the parathyroid gland, CgA is cosecreted with parathyroid hormone (PTH). Although these peptides are secreted together, recent evidence has suggested that they are processed differently in response to stimuli, such as 1,25-(OH)2 vitamin D3 and calcium. We have validated a reverse hemolytic plaque assay for studying CgA release from parathyroid cells. This assay allows for the detection of quantitative changes in hormone secretion from individual parathyroid cells. Bovine parathyroid cells were mixed with protein-A-linked ovine erythrocytes (oRBC) and plated in a monolayer in the presence of CgA antiserum. After incubation, complement was added to the cells to induce cell lysis. Lysis of oRBC around a parathyroid cell indicated the release of CgA from a cell. Results showed that plaque formation was dependent on assay reagents and that serial dilution of the antibody reduced plaque formation. CgA secretion was inhibited by increasing concentrations of calcium and stimulated by increasing concentrations of 1,25-(OH)2vitamin D3.

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells.