James W. Hamilton

Pancreastatin and bovine parathyroid cell secretion

Chromogranin A (CgA) is an acidic glycoprotein found in secretory granules of multiple peptidergic tissues and cosecreted with the resident peptide hormones. Pancreastatin is an amidated, biologically active peptide whose sequence is contained within CgA. We investigated the effect of the C-terminal fragment of bovine pancreastatin (bP32-47) on bovine parathyroid cell secretion. bP32-47 amide inhibited low-calcium-stimulated PTH secretion by 44% and chromogranin A (CgA) secretion by 33%. We were able to identify a pancreastatin-like peptide as a very minor component of the endogenous breakdown peptides from CgA. However, using several approaches, we were unable to detect pancreastatin in secretory granule extracts or in incubation media. We conclude that although exogenous bovine pancreastatin has inhibitory effects on secretion, detectable pancreastatin is not secreted under normal incubation conditions. Based on our current data, we would question the physiologic importance of pancreastatin in bovine parathyroid glands.

The isolation of identical thyroxine containing amino acid sequences from bovine, ovine and porcine thyroglobulins.

Identical, thyroxine containing tryptic peptides have been isolated from digests of bovine, ovine and procine thyroglobulins. This 19 residue hormone containing sequence, NH2-Asn-Ile-Phe-Glu-T4-Gln-Val-Asp-Ala-Gln-Pro-Leu-Arg-Pro-Cys-Glu-Leu-G in-Arg- COOH, is completely conserved across these three species, and it represents a principal site of thyroxine synthesis. HPLC maps of tryptic digests of the thyroglobulins have been monitored at several wavelengths and suggest that, in each case, only a small number of tryptic peptides are iodinated in vivo and that an even smaller number of tryptic peptides contain thyroid hormone. These data are consistent with a high degree of selectivity in iodination of tyrosines within thyroglobulin and the subsequent coupling of these selected tyrosines to form thyroid hormone.

Inhibition of parathormone-stimulated bone resorption by Type I interferon

The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation.

The structure of a naturally occuring 10K polypeptide derived from the amino terminus of bovine thyroglobulin

A combination of data derived from peptide sequencing and nucleic acid sequencing of cloned cDNA fragments has been used to define the complete amino acid sequence of a 10,000 M.W., thyroxine containing polypeptide derived from bovine thyroglobulin. This fragment, TG-F, which was obtained following reduction and alkylation, has been placed at the amino terminus of the parent protein with hormone located at residue 5 in the primary sequence of the thyroglobulin molecule. The carboxyl terminal sequence of this fragment -Cys-Gln-Leu-Gln is found on the N-terminal side of a lys residue, suggesting that the peptide bond cleavage which occurs to produce this 80 residue fragment from the parent (330K) thyroglobulin chain is a gln-lys. In addition, the amino acid sequence of this 10K fragment contains: No sequence which would be a substrate for glycosylation and no carbohydrate. Several repeated homologous amino acid sequences. A striking number of beta-bends predicted from Chou-Fasman analyses, particularly near its carboxyl terminus.

COOH-terminally extended secretins are potent stimulants of pancreatic secretion

Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and alpha-carboxyl amidated secretin. We used synthetic canine secretin analogs with COOH-terminal -amide, -Gly, or -Gly-Lys-Arg to examine the effects of COOH-terminal extensions of secretin on bioactivity and detection in RIA. Synthetic products were purified by reverse-phase and ion-exchange HPLC and characterized by reverse-phase isocratic HPLC and amino acid, sequence, and mass spectral analyses. Secretin and secretin-Gly were noted to coelute during reverse-phase HPLC. In RIA using eight different antisera raised against amidated secretin, COOH-terminally extended secretins had little or no cross-reactivity. Bioactivity was assessed by measuring pancreatic responses in anesthetized rats. Amidated canine and porcine secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had potencies of 81 +/- 9% (P > 0.05) and 176 +/- 13% (P < 0.01), respectively, compared with amidated secretin, and the response to secretin-Gly-Lys-Arg lasted significantly longer. These data demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not detect bioactive COOH-terminally extended forms of secretin in tissue extracts or blood, and 3) the secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and α-carboxyl amidated secretin. We used synthetic canine secretin analogs with COOH-terminal -amide, -Gly, or -Gly-Lys-Arg to examine the effects of COOH-terminal extensions of secretin on bioactivity and detection in RIA. Synthetic products were purified by reverse-phase and ion-exchange HPLC and characterized by reverse-phase isocratic HPLC and amino acid, sequence, and mass spectral analyses. Secretin and secretin-Gly were noted to coelute during reverse-phase HPLC. In RIA using eight different antisera raised against amidated secretin, COOH-terminally extended secretins had little or no cross-reactivity. Bioactivity was assessed by measuring pancreatic responses in anesthetized rats. Amidated canine and porcine secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had potencies of 81 ± 9% ( P > 0.05) and 176 ± 13% ( P < 0.01), respectively, compared with amidated secretin, and the response to secretin-Gly-Lys-Arg lasted significantly longer. These data demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not detect bioactive COOH-terminally extended forms of secretin in tissue extracts or blood, and 3) the secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.

Effects of isoproterenol and cycloheximide on parathyroid secretion.

Tissue slices or dispersed cells of bovine parathyroid gland were incubated with [3H]leucine to label the intracellular proteins and then tested for their secretory response to isoproterenol and cycloheximide at different calcium concentrations. Secretion of the newly synthesized as well as the older PTH and SP-I was stimulated by isoproterenol at all calcium levels tested, even when it was maximally enhanced by low calcium. Cycloheximide interfered with neither the secretory process nor the secretory response to different stimuli, but decreased the amount of PTH and SP-I secreted. We conclude that the inhibitor decreased the secretion by reducing the supply of PTH and SP-I. Calculations derived from the data reveal that, under most secretory conditions, newly synthesized PTH contributed a major portion of the total hormone secretion in bovine parathyroid cells.

Parathyroid hormone-like peptide and parathyroid hormone are secreted from bovine parathyroid via different pathways.

Parathyroid hormone-like peptide is a recently discovered protein which is thought to be responsible for the hypercalcemia of malignancy. Through the use of radioimmunoassay, Northern analysis and Western blot techniques this protein has been demonstrated to occur in a variety of tumor and normal cells. Its role in normal physiology is not established nor is there knowledge regarding its synthesis, secretion, and storage. We have investigated characteristics of the secretion of parathyroid hormone-like peptide in bovine parathyroid gland slices and cells to learn whether or not this protein is secreted in a manner similar to that of parathyroid hormone. We have used radioimmunoassays specific for PTH and PTH-rP to measure the secretion of each protein and have found that, unlike PTH, PTH-rP secretion was not influenced by the medium calcium concentration. Similarly, PTH-rP secretion was not influenced by other known PTH secretagogues such as c-AMP or isoproterenol. An examination of the subcellular distribution of PTH-rP revealed that 75-90% of it occurs in the soluble fraction of cell lysates. Analysis of isolated secretory granules demonstrated the presence of PTH while PTH-rP was undetectable in these organelles. We conclude that PTH-rP is not secreted from parathyroid cells via the regulated pathway utilizing PTH secretory granules.

Formation of parathormone 8–34 by cathepsin-D digestion of parathormone and its efficacy as a hormone antagonist

It previously has been shown that digestion of bovine parathormone (bPTH) with cathepsin-D results in rapid cleavage of the hormone between Phe34 and Val35 yielding PTH(1-34) and PTH(35-84). Since bPTH also contains a Phe at residue 7 we have conducted additional studies to determine whether cleavage at this position could occur. We have found that following longer incubation periods of hormone and enzyme, 2 additional peptides are generated; PTH(8-34) and PTH(1-7). Time course studies demonstrated that these 2 fragments are formed from the (1-34) peptide generated through the initial cleavage at Phe34-Val35 of PTH. The identification of the bPTH(8-34) was accomplished through amino acid analysis and N-terminal sequencing. bPTH(8-34) behaved as a PTH antagonist in an in vitro mouse calvarial bone resorption assay. Although bPTH(8-34) did not affect the PTH-stimulated cAMP response when added simultaneously with PTH, preincubation of bone cells with this peptide caused desensitization of the PTH-stimulated cAMP response.