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Biosynthesis of parathyroid hormone
2 Biosynthesis of Parathyroid Hormone
T. J. M A R T I N
For many years the importance of a low plasma calcium level in stimulating parathyroid gland function has been recognised. De Robertis (1941) showed that parathyroid hyperplasia could be produced by a low calcium diet, and Albright (1941) suggested that the parathyroid hyperfunction in vitamin D deficiency arose from the stimulus of a low plasma calcium, which in turn was secondary to impaired calcium absorption. In a series of experiments in which they infused intact and thyroparathyroidectomised dogs with low calcium-containing blood, Patt and Luckhardt (1942) obtained evidence that a low blood calcium was a direct stimulus for the parathyroid glands to produce more hormone. However, there was no unified concept of control of parathyroid hormone (PTH) secretion until 1955 when McLean and Urist formulated their negative feedback hypothesis. Direct evidence for the validity of this hypothesis was provided by Raisz (1963) who showed an inverse relationship between ambient calcium concentration and the release of parathyroid hormone in in vitro studies of PTH release from rat and chick parathyroid glands. Parathyroid hormone was measured by its bone-resorbing activity on cultured embryonic bone rudiments. Direct in vivo evidence for a negative feedback control came from Sherwood et al (1966) when they measured parathyroid hormone by radioimmunoassay directly in the plasma of goats and cattle during infusions of calcium and EDTA. The negative feedback control of parathyroid hormone secretion therefore seemed firmly established as a physiological concept. With the development of methods of assaying parathyroid hormone, and as knowledge of its chemistry began to accumulate, considerable interest centred on studies of its biosynthesis. Although initially a major aim may have been to determine whether calcium ions affect PTH synthesis as well as secretion, the overall results of biosynthetic studies have been far-reaching; indeed understanding of the biosynthetic pathways is clearly essential for the clarification of the role of parathyroid hormone in disease. Clinics in Endocrinology and Metabolisrn--Vol. 3, No. 2, July 1974.
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7. J. MARTIN CONTROL OF BIOSYNTHESIS
Hamilton and Cohn (1969) incubated parathyroid gland slices and showed that the cells in the slices synthesised parathyroid hormone. They found that the degree of incorporation of radioactive amino acids into PTH was inversely related to calcium ion concentration, and that this effect of calcium was directed more towards the biosynthesis of parathyroid hormone than of the general proteins of the tissue. Au et al (1970) reached similar conclusions regarding the role of calcium in regulating the synthesis of parathyroid hormone in cultured rat parathyroids, whilst Raisz (1967) had shown previously that lowering the calcium concentration of culture media stimulated afiaino acid uptake by parathyroid tissue, and that a high calcium concentration inhibited it. Changes in magnesium concentration have no effect on the synthesis of parathyroid hormone (Hamilton et al, 1971a), whereas magnesium has been shown to have a calcium-like action on PTH secretion both in vitro (Sherwood, Hermann and Bassett, 1970) and in vivo experiments (Care et al, 1966; Gitelman, Kubolj and Welt, 1968; Buckle et al, 1968). This suggested that calcium may have a dual role, affecting both the synthesis of parathyroid hormone and its secretion, whereas magnesium may affect secretion alone. Hamilton et al (1971a) compared the radioactively-labelled parathyroid hormone in the tissue with that in the medium after incubation of parathyroid gland slices with radioactive amino acids, and found that the two behaved identically on gel filtration and electrophoresis. They noted also that when 14Clabelled PTH was added to media containing parathyroid slices, the 14CPTH was degraded to fragments, but the degradation process was not affected by changes in calcium concentration. This phenomenon may explain their further observation that dilutions of incubation media containing the secreted parathyroid hormone did not parallel pure PTH standards in the PTH radioimmunoassay. The other possible explanation of this latter observation was that another form of parathyroid hormone was being secreted, in addition to the 'native', 84-amino-acid-containing molecule that is normally obtained when parathyroid glands are extracted and purified. Such a possibility was being considered by other workers~ at that time, and in the following section this work will be reviewed. N A T U R E OF H O R M O N E RELEASED F R O M P A R A T H Y R O I D
GLANDS Interest in the nature of parathyroid hormone released from the parathyroid cell arose from the observations of Berson and Yalow (1968) that circulating endogenous PTH is immunochemically heterogeneous. They suggested that plasma may contain metabolic fragments of the parathyroid hormone molecule which would be detected to a variable extent by different antisera to PTH. Subsequently Arnaud, Tsao and Oldham (1970) and Arnaud et al (1971) detected differences between parathyroid hormone present in human hyperparathyroid serum and that in extracts of parathyroid adenomata. Furthermore, the parathyroid hormone liberated into the culture medium from parathyroid glands incubated in vitro resembled that of serum rather
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BIOSYNTHESIS OF PARATHYROID HORMONE
than the hormone extracted from the gland. This led to the postulate of a 'glandular' and a 'secreted' form of PTH, the former being a precursor of the latter. These findings were similar to those of Sherwood, Rodman and Lundberg (1970) who studied PTH synthesis in cultures of bovine parathyroid glands.
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TUBE NUMBER Figure 1. Gel filtration elution patterns of medium (upper) and tissue extract (lower) of bovine parathyroid glands incubated with ]4C-leucine for 72 hours. Patterns observed with 1 '5 mM and 3.5 mM cation (calcium and magnesium) are indicated. Immunoreactive material from the medium was consistently eluted later than that from the tissue. Reproduced from Sherwood, L. Rodman, J. S. and Lundberg, W. B., Jr (1970). Proceedings of the National Academy of Sclences of the U.S.A., 67, 1631, by courtesy of the authors and the publisher.
These workers, however, made further observations which led them to conclude that the parathyroid hormone in the gland was indeed a prohormone, and that conversion to the hormonal, or circulating form, took place when the gland was stimulated. Figure 1 illustrates this finding.
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If the interpretation of these experiments was correct, some important problems would arise. First, it would clearly be inappropriate to use parathyroid hormone extracted from glands as an antigen for the development of PTH radioimmunoassays. Furthermore, the corollary was that the circulating form of PTH would need to be purified and characterised from serum in order to determine precisely the nature of the circulating hormone and to attempt to measure it--surely a formidable task. However, two lines of evidence became available to indicate that the above mechanism did not operate to bring about the secretion of PTH. In vivo evidence was provided by Habener et al (1971) who used gel filtration and radioimmunoassay to determine the molecular size and immunoreactivity of PTH in peripheral plasma, in parathyroid effluent blood obtained from catheter specimens, and in gland extracts. Both in cattle and in man the parathyroid hormone emerging directly from the gland was indistinguishable from the 84-amino-acid-peptide (mol. w. 9500) extracted from the parathyroid glands. However, PTH in the peripheral plasma in each species differed in its immunochemical properties (Figure 2), and on gel filtration its major immunoreactive component appeared to have a molecular weight of about 7000. The conclusion reached was that the 'glandular' 84-amino-acidcontaining-peptide was indeed the secreted form of the hormone, but that it underwent cleavage peripherally to a smaller component, which differed in immunological reactivity with some antisera. 0.5 0.~
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ng of parathyroid hormone Figure 2. Demonstration of immunochemical dissimilarity of parathyroid hormone in peripherally obtained plasma, as compared with plasma from parathyroid gland effluent and with hormone extracted from parathyroid adenomas. O, superior vena cava; A, inferior thyroid vein; O, human PTH standard. Reproduced from Habener et al (1971) Proceedings of the National Academy of Sciences of the U.S.A., 68, 2986, by courtesy of the authors and the publisher.
The second line of evidence which questioned the validity of proposing 'glandular' PTH as a prohormone was that obtained from a study of human parathyroid hormone synthesis and secretion by monolayer cell cultures. These cultures secrete parathyroid hormone into the media for periods of several months (Martin et al, 1971). Secreted PTH was studied in media that were free of serum or contained very low (less than 1 per cent) amounts of
203
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serum, to avoid proteolytic degradation of the hormone. Gel filtration of samples of medium obtained under these conditions revealed that the secreted parathyroid hormone was of the same molecular size as the marker 1251. PTH. Radioactive PTH was prepared by incorporation of laC-amino acids, and the 14C-PTH purified from culture media was found to be of the same size and charge as that of highly purified bovine PTH, and of human PTH extracted from glands (Figure 3). The conclusion from these two different approaches was therefore that the parathyroid hormone secreted from human parathyroid cells was indistinguishable from the hormone which could be extracted and purified from the glands. "--" HPTH °--o c.p.m. bPTH
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Figure 3. Disc gel electrophoresis of 14C-labelled PTH synthesised by human parathyroid cells. Its behaviour is identical with that of highly purified bovine PTH (upper part of diagram) and of human PTH (HPTH) extracted from parathyroid adenomata. Reproduced from Martin, T. J., Greenberg, P. B. and Melick, R. A. (1972) Journal of Clinical Endocrinology and Metabolism, 34, 436, by courtesy of the publisher.
DISCOVERY OF PROPARATHYROID HORMONE
The subsequent discovery of a biosynthetic precursor of parathyroid hormone must be attributed in no small measure to the careful studies of Cohn, Hamilton and their colleagues on the: biosynthesis of parathyroid hormone by bovine parathyroid slices. In the'course of this work (Hamilton et al, 1971b) they noted that although the bulk of the immunoreactivity of the parathyroid hormone liberated into the incubation media eluted from Sephadex GI00 columns corresponded in molecular size to an 84-aminoacid-containing-PTH, there was some immunoreactivity found preceding the major peak, and some following i~. The smaller molecular weight material could be explained by breakdown of PTH which they had shown to occur in the medium, but they pursued the larger molecular weight protein, which
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they realiged could represent a precursor molecule. They noted also that in purifying 14C-PTH from extracts of slices after biosynthesis they obtained, in addition to a labelled peptide behaving as 'authentic P T H ' , another peptide which eluted late from carboxymethylcellulose ion exchange columns. From its behaviour on Sephadex chromatography (Hamilton et al, 1971b), this peptide appeared to be slightly larger than PTH. Moreover, in the biosynthetic studies this peptide became labelled to a high specific activity (Hamilton and Cohn, 1969). Subsequently (Hamilton et al, 1971b) they achieved a substantial purification of the peptide which they called 'calcaemic fraction-A' (Figure 4). Under standard conditions of biological assay calcaemic fraction-A was found to be about one-third as active as parathyroid hormone in raising plasma calcium in the parathyroidectomised rat. Moreover, it was only approximately one half as active as P T H in an in vitro bone bioassay system. In their early experiments, using only one antiserum to parathyroid hormone, the immunological reactivity of calcaemic fraction-A mMho,
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was found to be approximately 5 per cent that of P T H (Hamilton et al, 1971b). Their observations and characterisation of calcaemic fraction-A were crucial in stimulating subsequent work demonstrating a precursorproduct relationship between ' p r o P T H ' and PTH. In addition to the features already discussed, a particular point of their experiments which led them to suggest that calcaemic fraction-A might be a biological precursor of P T H (Cohn et al, 1972a) was the fact that its synthesis, like that of PTH, was inversely related to calcium concentration in the incubation medium (Hamilton et al, 1971b).
BIOSYNTHESIS OF PARATHYROID HORMONE
205
That such a precursor of PTH did indeed exist was soon shown by Kemper et al (1972) and by Cohn et al (1972b) in pulse-chase experiments with bovine gland slices, showing early labelling of the precursor and formation of PTH without any evidence of an intermediate step. The precursor was called proPTH, and it seemed clear that it was identical with calcaemic fraction-A. Similar experiments have recently clearly demonstrated the existence of proPTH in chicken (MacGregor et al, 1973a), rat (Chu et al, 1973a), and human glands (Habener et al, 1972a). The proPTH of bovine origin has been the most extensively studied up to the present time; chemical and enzymatic cleavage of biosynthesised radioactive proPTH allowed Habener et al (1973) to conclude that the additional peptide sequence of the molecule occurs at the amino-terminal end of the hormone. Hamilton et al (1973) showed that the additional N-terminal sequence was lys - ser - val - lys - lys - arg, hence the increased basic charge of the prohormone. The reduced biological activity of the prohormone is not surprising as the N-terminal alanine of the PTH molecule is important for activity, at least in the rat kidney adenyl cyclase assay (Tregear et al, 1973), and extending the sequence by several residues beyond the amino terminus lowers activity appreciably. SYNTHESIS Formation of P T H from ProPTH
Subcellular fractionation of bovine parathyroid glands indicates that over 90 per cent of the hormone and prohormone is in particulate fractions (MacGregor et al, 1973b), consistent with the view that the membrane-bound granules seen on electron microscopy are secretory granules (Munger and Roth, 1963). Present evidence suggests that the synthesis and packaging of PTH in the parathyroid cell do not differ remarkably from those processes for other secreted proteins. MacGregor et al (1973b) suggest that newly synthesised proPTH is released from ribosomes to the cisternal space of the endoplasmic reticulum, where conversion to parathyroid hormone may begin. The newly formed hormone is then slowly transferred to the secretory granules. Control of the conversion process has been studied by the same group of workers, and is clearly of considerable importance, especially as proPTH differs from PTH in its biological properties. Chu et al (1973) studied some factors which might influence the glandular synthesis of proPTH and its conversion to parathyroid hormone. A low calcium diet in rats doubled the rate of PTH synthesis in the parathyroid glands without any change in rate of synthesis of proPTH or in the half-life of its cellular pool. The conclusion was that the parathyroid glands adapted to the low calcium diet by increasing the efficiency of conversion of proPTH to PTH, and that perhaps the control of PTH biosynthesis by calcium may occur at the level of intracellular turnover and degradation of the hormone. It may be relevant that a calciumdependent degradative process in the parathyroid cell has been proposed by Fischer, Oldham and Sizemore (1972). A role for cyclic 3'5' adenosine monophosphate in PTH release has been postulated by Dufresne and Gitelman
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T.J. MARTIN
(1972) and by Abe and Sherwood (1972). If this is the case, it will be interesting to determine at what point the nucleotide is acting in the synthesis-secretion pathway, and how it relates to calcium effects. The hypothesis of Chu et al (1973) is illustrated in Figure 5. It implies that calcium ions exert effects either directly or indirectly at several points along the biosynthetic and secretory pathways of parathyroid hormone. Raisz (1967) has shown that conditions of low calcium can stimulate amino acid uptake by the parathyroid cell (step A), but Hamilton and Cohn (1969) found that low calcium stimulated the synthesis of parathyroid hormone under conditions in which no effect on the uptake of amino acid was observed (steps B, C and D). The effect of calcium ions on net PTH secretion (step E) is well established. Perhaps the most interesting aspect of the hypothesis of Chu et al (1973) is their proposal of a role for calcium ions in regulating the intracellular turnover of proPTH and PTH to smaller fragments, and thereby influencing the amount of parathyroid hormone available for secretion. It would be of great intrinsic interest to cell biologists to analyse such a process, which may be applicable to other secretory cells, as for example the [3-cell of the pancreas. Also it would be of interest to determine whether the function of the secretory machinery of glandular cells is to protect hormone from excessive exposure to intracellular degradation.
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Human PTH The conversion of proPTH to PTH in the human parathyroid gland is of particular interest. Habener et al (1972a) studied the ability of several parathyroid adenomata to convert proPTH to PTH and found considerable variation in this property (Table 1). Chu et al (1973) pointed out that this variability could simply reflect differences in physiological state immediately before study of the gland in vitro; nevertheless, the results suggest the interesting possibility that some parathyroid tumours may accumulate and even release proPTH into the circulation. Indeed, this is implied by earlier in vivo experiments in which some patients with primary hyperparathyroidism released into the circulation immunoreactive PTH which was slightly larger than the normal 84-amino-acid-containing-molecule (Habener et al, 1971). Furthermore, although in most cell cultures established from human parathyroid glands the form of PTH released into the media was indistinguishable
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from that of normal 1-84 PTH, in cultures from two adenomata evidence has been obtained for the synthesis and release of a basic peptide resembling proPTH in its behaviour on gel filtration, disc electrophoresis and ion exchange chromatography (Marlin, Greenberg and Michelangeli, 1973); Figure 6 illustrates this. Table 1. Relative amounts of P T H and proPTH synthesised by various parathyroid tissues in short-term incubations
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The most likely conclusion from these various experiments is that normally and in the case of most parathyroid adenomata, the conversion of proPTH proceeds rapidly and that only parathyroid hormone containing 84amino acids is released from the gland. Occasj0nally, however, the capacity of the converting process is exceeded, and proPTH will be released. Clearly, the situation resembles closely that for insulinomata, which may secrete proinsulin (Melani et al, 1970). This property of some adenomata should be recalled as a potential contributor to the difficulty of measuring parathyroid hormone in plasma by radioimmunoassay, since proPTH would be recognised with difficulty by some N-terminal specific antisera. The other important implication for the syndrome of hyperparathyroidism in man is related to the biological activity of human proPTH. If, as seems likely, the human precursor molecule resembles its'.bovine counterpart by possessing extra amino acids at the amino-terminal end of the molecule, human proPTH would be expected to be less active than 1-84 PTH, and the clinical features of primary hyperparathyroidism may be modified in those patients secreting a significant amount of proPTH. It will be important to have this point clarified by prospective studies in patients with primary hyperparathyroidism, in whom proPTH and PTH may eventually be assayed specifically. Synthesis of P T H by non-parathyroid cancer in man
It seems likely that malignant hypercalcaemia unassociated with bony metastases is most frequently due to hnmoral factors other than parathyroid
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MARTIN
hormone. Nevertheless, the syndrome of 'ectopic' hyperparathyroidism is now well recognised. The capacity of non-parathyroid cancers to produce parathyroid hormone was established by the demonstration of a gradient of hormone concentration across the tumour-bearing organ in the case of a hypernephroma (Buckle, McMillan and Mallinson, 1970) and of a hepatoma (Knill-Jones et al, 1970). Some differences have been found between circulating immunoreactive parathyroid hormone in primary and in ectopic hyperparathyroidism, which have raised questions regarding the nature of the PTH synthesised and released by the tumours. Riggs et al (1971) and Roof et al (1971) noted immunological differences between the plasma PTH of primary hyperparathyroidism and of hyperparathyroidism due to cancer. + ~ -
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Figure 6. Disc gel electrophoresis of 14C-labelled peptides with PTH-like immunoreactivity, synthesised and secreted by a human parathyroid adenoma in culture. Peak I migrates identically with pure bovine PTH (shown as heavy band in upper part of diagram), and peak II is faster moving and therefore more basic. Reproduced from Martin, T. J., Greenberg, P. B. and Michelangeli, V. (1973) Clinical Science, 44, 1-8, by courtesy of the publisher.
The explanation most commonly offered for ectopic peptide hormone production is that derepression occurs in the cancer cell, leading to the transcription of normally repressed DNA. If this is correct, the cancer cell should produce a hormone identical to that synthesised by the appropriate gland cell. There is increasing evidence, already referred to, that this may not be the case with ectopic PTH production. Although secretion of proPTH by a cancer is a potential contributor to immunochemical heterogeneity, it is unlikely to be the sole explanation. In one instance ofectopic PTH production by a hypernephroma, Greenberg, Martin and Sutcliffe (1973) showed that cell cultures of the cancer synthesised a peptide with properties of proPTH. However, they suggested another possible explanation for the 'abnormal' PTH in the cancer syndrome in their case, because evidence was obtained for the formation of immunoreactive fragments of PTH within the cancer cells during culture (Figure 7). In extensive studies with human parathyroid
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BIOSYNTHESIS OF PARATHYROID HORMONE
cells these authors found no evidence for the formation of intracellular PTH fragments (Martin et al, 1971 ; Martin et al, 1973). Although they did not exclude the possibility that partial sequences of PTH were made by the tumour cells, they suggested that the immunoreactive PTH fragments found in the cells and released into the media may result from rapid and potent proteolytic processes within the cells, leading to a rapid turnover of newly synthesised hormone and the generation of fragments of the molecule; some of these would have biological activity, whereas their immunological reactivity would depend on the particular antisera being used. If such a mechanism is B.W.211/32 GPC3
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Figure 7. Gel filtration of aqueous extract of cultured cells from a clear cell carcinoma of the kidney associated with ectopic PTH production. The cells had been incubated for 24 hours with 14C-leucine. Pool III corresponds in molecular size to pure bovine PTH (shown as BPTH). The inset shows the results of examining pools I to V for the presence of 14C-peptide with PTH-like immunoreactivity. Double antibody precipitation was carried out with two different antisera to PTH. Results show that the ceils synthesised peptides with PTH-like immunoreactivity, corresponding in size to 1-84 PTH, but also some peptides of smaller size (pools IV and V). Reproduced from Greenberg, P. B., Martin, T. J. and Sutcliffe, H. S. (1973) Clinical Science and Molecular Medicine, 45, 183-191, by courtesy of the publisher.
found to be a common occurrence in cancers, it would be interesting to speculate whether the increased susceptibility of the peptide to proteolysis might be due to the absence of the normal packaging and secretory mechanisms from the cancer cell. Although there is evidence for the generation of peptide fragments in other cancer syndromes--for example ectopic ACTH production (Orth et al, 1973), it is unlikely that such a mechanism is operative for all ectopic humoral syndromes. Indeed, there is evidence for the production by a lung cancer of vasopressin, which was identical with the neurohypophyseal hormone in all systems studied (George, Capen and Phillips, 1972). Very careful s,tudy of the nature of parathyroid hormone produced by individual cancers will be needed to resolve the questions surrounding the ectopic PTH syndrome, since it is by no means impossible that several different abnormalities may exist.
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x.J. MARTIN 'SECRETED' AND 'CIRCULATING' PTH
Although the emphasis in this discussion has been on the biosynthesis of parathyroid h o r m o n e and the nature of the h o r m o n e released from the gland, it is appropriate to consider those properties of P T H in the peripheral circulation which distinguish it from h o r m o n e directly leaving the parathyroid gland cell. On the present evidence, one must conclude that parathyroid h o r m o n e normally enters the circulation as the 84-amino-acid-containing-molecule, but in some cases of parathyroid adenoma secretion of p r o P T H m a y occur, whereas in some cases of 'ectopic' hyperparathyroidism there may be a release of PTH, p r o P T H , fragments of the hormone, or perhaps other forms, as yet uncharacterised. Even under normal conditions of secretion of parathyroid hormone, however, there are recognisable immunological differences between "secreted' parathyroid h o r m o n e and P T H in the peripheral circulation. Recent studies with well characterised antisera (reviewed by Parsons and Potts, 1972) indicate that the suggestion of Berson and Yalow (1968) is probably correct, namely that the immunochemical heterogeneity of circulating P T H m a y be due to circulating immunoreactive fragments of the hormone. i
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Figure 8. Gel filtration of parathyroid hormone in the general circulation of a patient with primary hyperparathyroidism. The interrupted line indicates the elution volume of the marker, lzSI-labelled PTH. C and N refer to immunoassays carried out on column fractions, using two different antisera, one C-terminal and one N-terminal specific. The C-terminal immunoreactivity is of slightly lower molecular weight (approximately 7500) than 1-84 PTH, but the small amount of N-terminal immunoreactivity probably indicates that intact 1-84 PTH is remaining in the circulation. Reproduced from Habener et al (1972b) Nature New Biology, 238, t52-154, by courtesy of the authors and the publisher.
BIOSYNTHESIS OF PARATHYROID HORMONE
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The precise nature of the circulating fragments is not known, but some useful working proposals can be made on the basis of the experiments of Habener et al (1972b). Using N- and C-terminal specific radioimmunoassays for PTH, they assayed peripheral and thyroid vein plasma from patients with primary hyperparathyroidism and found that whereas the two assays gave equal results in the thyroid vein plasma, the peripheral plasma levels were very much lower with the N-assay than with the C-assay. Figure 8 illustrates results of gel filtration of a peripheral plasma sample and applying the two assays. The small amount of N-terminal activity coincided in elution volume with native PTH, whereas the C-terminal reactivity emerged slightly later from the column, consistent with a peptide of molecular weight of approximately 7500. Habener et al (1972b) suggest that cleavage of secreted PTH occurs in some organ, giving rise to a fairly large C-terminal fragment of the hormone. The persisting N-terminal immunoreactivity seen in Figure 8 would represent secreted PTH as yet undegraded. There are several interesting questions which arise from this observation. First: is the peripheral cleavage a constant, reproducible process occurring at some particular point along the polypeptide chain ? If so, does it yield an N-terminal sequence of sufficient length to possess biological activity in addition to the presumably inactive C-terminal fragment ? Finally, in what organ does peripheral metabolism take place predominantly? No definite answers are available to these questions. Canterbury, Levey and Reiss (1973) have extracted peripheral plasma of patients with hyperparathyroidism and found PTH-like biological activity in low molecular weight fractions, suggesting that a low molecular weight biologically active form of parathyroid hormone may be circulating in the blood. In view of the evidence for a role for the kidney in the metabolism of parathyroid hormone in the rat (Orimo and Fujita, 1966; Martin, Melick and de Luise, 1969), and in the dog and in man (Melick and Martin, 1969), the kidney seems a likely site for the peripheral cleavage of parathyroid hormone, although there is evidence in the rat for a significant role also for the liver in the metabolism of parathyroid hormone (Vajda, Martin and Melick, 1969; Fang and Tashjian, 1972). CONCLUSIONS Recent studies on the biosynthesis of parathyroid hormone have been reviewed, and the experiments described which have led ultimately to the discovery of proPTH, the biosynthetic precursor of PTH. Careful further study of the control of PTH biosynthesis should yield valuable information to the cell biologist, and may help in understanding the biological purpose of 'prohormones' and the functions of hormone storage mechanisms in glandular cells. There are a number of clinical implications which arise. It has been pointed out that proPTH may circulate in the plasma of some patients with primary hyperparathyroidism. In view of the relatively low biological activity of proPTH, it is now important to the understanding of the hyperparathyroid syndrome that such cases be studied in great detail. It should be clear from
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T. J. MARTIN
this review that there are m a n y other possible causes of the ' i m m u n o c h e m i c a l heterogeneity' of plasma PTH, and we can readily u n d e r s t a n d the difficulties which have been experienced in measuring circulating levels of P T H in man. The need for well characterised antisera is evident. ACKNOWLEDGEMENTS
Work referred to from the author's laboratory was supported by the Anti-Cancer Council of Victoria and the National Health and Medical Research Council. Drs D. V. Cohn and J. W. Hamilton kindly provided details of work prior to publication. REFERENCES Abe, M. & Sherwood, L. M. (1972) Regulation of parathyroid hormone secretion by adenyl cyclase. Biochemical and Biophysical Research Communications, 48, 396~401. Albright, F. (1941) The parathyroids--physiology & therapeutics. Journal of the American Medical Association, 117, 527-535. Arnaud, C. D., Tsao, H. S. & Oldham, S. B. (1970) Native human parathyroid hormone: an immunochemical investigation. Proceedings of the National Academy o f Sciences of the U.S.A., 67, 415--422. Arnaud, C. D., Sizemore, G. W., Oldham, S. B., Fischer, J. A., Tsao, H. S. & Littledike, T. (1971) Human parathyroid hormone: glandular and secreted molecular species. American Journal of Medicine, 50, 630-638. Au, W. Y. W., Poland, A. P., Stern, P. H. & Raisz, L. G. (1970) Hormone synthesis and secretion by rat parathyroid glands in tissue culture. Journal of Clinical Investigation, 49, 1639-1646. Berson, S. A. & Yalow, R. S. (1968) Immunochemical heterogeneity of parathyroid hormone. Journal of Clinical Endocrinology and Metabolism, 28, 1037-1047. Buckle, R. M., Care, A. D., Cooper, C .W. & Gitelman, H. J. (1968) The influence of plasma magnesium concentration on parathyroid hormone secretion. Journal of Endocrinology, 42, 529-534. Buckle, R. M., McMillan, M. & Mallinson, C. (1970) Ectopic secretion of PTH by a renal adenocarcinoma in a patient with hypercalcaemia. British Medical Journal, iv, 724-726. Canterbury, J. M., Levey, G. S. & Reiss, E. (1973) Activation of renal cortical adenylate cyclase by circulating immunoreactive parathyroid hormone fragments. Journal of Clinical Investigation, 52, 524-527. Care, A. D., Sherwood, L. M., Potts, J. T. Jr & Aurbach, G. D. (1966) Evaluation by radioimmunoassay of factors controlling the secretion of parathyroid hormone. Nature, 209, 55-57. Chu, L. L. H., MacGregor, R. R., Anast, C. S., Hamilton, J. W. & Cohn, D. V. (1973) Studies on the biosynthesis of rat parathyroid hormone and proparathyroid hormone : adaptation of the parathyroid gland to dietary restriction of calcium. Endocrinology, 93, 915-924. Cohn, D. V., MacGregor, R. R., Chu, L. L. H. & Hamilton, J. W. (1972a) Studies on the biosynthesis in vitro of parathyroid hormone and other calcemic polypeptides of the parathyroid gland. In Calcium, Parathyroid Hormone and the Calcitonins, Proceedings of the Fourth Parathyroid Conference, ed. R. V. Talmage & P. L. Munson, pp. 173-182. Amsterdam: Excerpta Medica. Cohn, D. V., MacGregor, R. R., Chu, L. L. H., Kimmel, J. R. & Hamilton, J. W. (1972b) Calcemic fraction-A: Biosynthetic peptide precursor of parathyroid hormone. Proceedings of the National Academy of Seiences of the U.S.A., 69, 1521-1525. de Robertis, E. (1941) The cytology of the parathyroid and thyroid glands of rats with experimental rickets. Anatomical Record, 79, 417-434. Dufresne, L. R. & Gitelman, H. J. (1972) A possible role of aderiyl cyclase in the regulation of parathyroid activity by calcium. In Calcium, Parathyroid Hormone and the Calcitonins, Proceedings of the Fourth Parathyroid Conference, ed. R. V. Talmage & P. L. Munson, pp. 202-206. Amsterdam: Excerpta Medica.
BIOSYNTHESIS OF PARATHYROID HORMONE
213
Fang, V. S. & A. Tashjian, A. H. Jr (1972) Studies on the role of the liver in the metabolism of parathyroid hormone. Endocrinology, 90, 1177-1184. Fischer, J. A., Oldham, S. B., Sizemore, G. W. & Arnaud, C. D. (1972) Calcium-regulated parathyroid hormone peptidase. Proceedings of the National Academy of Sciences of the U.S.A., 69, 2341-2345. George, J. M., Capen, C. C. & Phillips, A. S. (1972) Biosynthesis of vasopressin in vitro and ultrastructure of a bronchogenic carcinoma. Journal of Clinical Investigation, 51, 141-146. Gitelman, H. J., Kubolj, S. & Welt, L. G. (1968) Inhibition of parathyroid gland activity by hypermagnesaemia. American Journal of Physiology, 215, 483-485. Greenberg, P. B., Martin, T. J. & Sutcliffe, H. S. (1973) Synthesis and release of parathyroid hormone by a renal carcinoma in cell culture. Clinical Science and Molecular Medicine, 45, 183-191. Habener, J. F., Powell, D., Murray, T. M., Mayer, G. P. & Potts, J. T. Jr (1971) Parathyroid hormone: secretion and metabolism in vivo. Proceedings of the National Academy of Sciences of the U.S.A., 68, 2986-2991. Habener, J. F., Kemper, B., Potts, J. T. Jr & Rich A. (1972a) Proparathyroid hormone: biosynthesis by human parathyroid adenomas. Science (New York), 178, 630-633. Habener, J. F., Segre, G. V., Powell, D., Murray, T. M. & Potts, J. T. Jr (1972b) Immunoreactive parathyroid hormone in circulation in man. Nature New Biology, 238, 152-154. Habener, J. F., Kemper, B., Potts, J. T. Jr & Rich, A. (1973) Bovine parathyroid hormone: structural analysis of radioactive peptides formed by limited cleavage. Endocrinology, 92, 219-226. Hamilton, J. W. & Cohn, D. V. (1969) Studies on the biosynthesis in vitro of parathyroid hormone. Journal of Biological Chemistry, 244, 5421-5429. Hamilton, J. W., Spierto, F. W., MacGregor, R. R. & Cohn, D. V. (1971a) Studies on the biosynthesis in vitro of parathyroid hormone. Journal of Biological Chemistry, 246, 3224-3233. Hamilton, J. W., MacGregor, R. R., Chu, L. L. H. & Cohn, D. V. (1971b) The isolation and partial purification of a non-parathyroid hormone calcemic fraction from bovine parathyroid glands. Endocrinology, 89, 1440-1447. Hamilton, J. W., NialI, H. D., Keutmann, H. T., Potts, J. T. Jr & Cohn, D. V. (1973) Amino terminal sequence of bovine proparathyroid hormone (calcemic fraction-A). Federation Proceedings, 32, 269. Kemper, B., Habener, J. F., Potts, J. T. Jr & Rich, A. (1972) Proparathyroid hormone: identification of a biosynthetic precursor of parathyroid hormone. Proceedings of the National Academy of Sciences of the U.S.A., 69, 643-647. Knill-Jones, R. P., Buckle, R. M., Parsons, V., Calne, R. Y. & Williams, R. (1970) Hypercalcaemia and increased parathyroid hormone activity in a primary hepatoma. New England Journal of Medicine, 282, 704-708. MacGregor, R. R., Chu, L. L. H., Hamilton, J. W. & Cohn, D. V. (1973a) Partial purification of parathyroid hormone from chicken parathyroid glands. Endocrinology, 92, 13121317. MacGregor, R. R., Chu, L. L. H., Hamilton, J. W. & Cohn, D. V. (1973b) Studies on the subcellular localization of proparathyroid hormone and parathyroid hormone in the bovine parathyroid gland: separation of newly synthesized from mature forms. Endocrinology, 93, 1387-1397 McLean, F. C. & Urist, M. R. (1955) An Introduction to the Physiology of Skeletal Tissue. University of Chicago Press. Martin, T. J., Melick, R. A. & de Luise, M. (1969) The effect of nephrectomy on the metabolism of labelled parathyroid hormone. Clinical Science, 37, 137-142. Martin, T. J., Greenberg, P. B., Melick, R. A. & Pilczyk, R. (1971) Synthesis of human parathyroid hormone by cells in culture. In Endocrinology, ed. S. Taylor, pp. 363-373. London: Heinemann. Martin, T. J., Greenberg, P. B. & Melick, R. A. (1972) Nature of human parathyroid hormone secreted by monolayer cell cultures. Journal of Clinical Endocrinology and Metabolism, 34, 437-440.
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Martin, T. J., Greenberg, P. B. & Michelangeli, V. (1973) Synthesis of human parathyroid hormone by cultured cells: evidence for release of prohormone by some adenomata. Clinical Science, 44, 1-8. Martin, T. J., Greenberg, P. B., Beck, C., Burger, H. G. & Johnston, C. I. (1973) Synthesis and secretion of peptide hormones by endocrine and non-endocrine tumours. In Endocrinology 1973, ed. S. Taylor. London: Heinemann (in press). Melani, F., Ryan, W. G., Rubenstein, A. H. & Steiner, D. F. (1970) Proinsulin secretion by a pancreatic beta-cell adenoma. New England Journal of Medicine, 283, 713-719. Melick, R. A. & Martin, T. J. (1969) Parathyroid hormone metabolism in man. Effect of nephrectomy. Clinical Science, 37, 667-674. Munger, B. L. & Roth, S. J. (t963) The cytology of the normal parathyroid glands in man and Virginia deer; a light and electron microscopic study with morphologic evidence of secretory activity. Journal of Cell Biology, 16, 379-400. Orimo, H. & Fujita, T. (1966) Inactivation in vitro of parathyroid hormone activity by kidney slices. Endocrinology, 76, 255-258. Orth, D. N., Nicholson, W. E., Mitchell, W. M., Island, D. P. & Liddle, G. W. (1973) Biologic and immunologic characterization and physical separation of ACTH and ACTH fragments in the ectopic ACTH syndrome. Journal of Clinical hlvestigation, 52, 1756-1769. Parsons, J. A. & Potts, J. T. Jr (1972) Physiology and chemistry of parathyroid hormone. Clinics in Endocrinology and Metabolism, 1, 33-78. Part, H. M. & Luckhardt, A. B. (1942) Relationship of a low blood calcium to parathyroid secretion. Endocrinology, 31, 384-392. Raisz, L. G. (1963) Regulation by calcium of parathyroid growth and secretion in vitro. Nature, 197, 1115-1116. Raisz, L. G. (1967) Effect of calcium on uptake and incorporation of amino acids in the parathyroid glands. Biochimica Biophysica Acta, 148, 460-468. Riggs, B. L., Arnaud, C. D., Reynolds, J. C. & Smith, L. H. (1971) Immunological differentiation of primary hyperparathyroidism from hyperparathyroidism due to nonparathyroid cancer. Journal of Clinical Investigation, 50, 2079-2083. Roof, B. S., Carpenter, B., Fink, D. J. & Gordan, G. S. (1971) Some thoughts on the nature of ectopic parathyroid hormone. American Journal of Medicine, 50, 686-691. Sherwood, L. M., Potts, J. T. Jr, Care, A. D., Mayer, G. P. & Aurbach, G. D. (1966) Evaluation by radioimmunoassay of factors controlling the secretion of parathyroid hormone. Nature, 209, 52-55. Sherwood, L. M., Hermann, J. & Bassett, C. A. (1970) Parathyroid hormone secretion in vitro: regulation by calcium and magnesium ions. Nature, 225, 1056-1058. Sherwood, L. M., Rodman, J. S. & Lundberg, W. B. Jr (1970) Evidence for a precursor to circulating parathyroid hormone. Proceedings of the National Academy of Sciences of the U.S.A., 67, 1631-1638. Tregear, G. W., van Rietschoten, J., Greene, E., Keutmann, H. T., Niall, H. D., Reit, B., Parsons, J. A. & Potts, J. T. Jr (1973) Bovine parathyroid hormone: minimum chain length of synthetic peptide required for biological activity. Endocrinology, 93, 1349-1353. Vajda, F. J. E., Martin, T. J. & Melick, R. A. (1969) Destruction of bovine parathyroid hormone labelled with 131I by rat kidney tissue. Endocrinology, 84, 162-164.
T. J. M A R T I N
For many years the importance of a low plasma calcium level in stimulating parathyroid gland function has been recognised. De Robertis (1941) showed that parathyroid hyperplasia could be produced by a low calcium diet, and Albright (1941) suggested that the parathyroid hyperfunction in vitamin D deficiency arose from the stimulus of a low plasma calcium, which in turn was secondary to impaired calcium absorption. In a series of experiments in which they infused intact and thyroparathyroidectomised dogs with low calcium-containing blood, Patt and Luckhardt (1942) obtained evidence that a low blood calcium was a direct stimulus for the parathyroid glands to produce more hormone. However, there was no unified concept of control of parathyroid hormone (PTH) secretion until 1955 when McLean and Urist formulated their negative feedback hypothesis. Direct evidence for the validity of this hypothesis was provided by Raisz (1963) who showed an inverse relationship between ambient calcium concentration and the release of parathyroid hormone in in vitro studies of PTH release from rat and chick parathyroid glands. Parathyroid hormone was measured by its bone-resorbing activity on cultured embryonic bone rudiments. Direct in vivo evidence for a negative feedback control came from Sherwood et al (1966) when they measured parathyroid hormone by radioimmunoassay directly in the plasma of goats and cattle during infusions of calcium and EDTA. The negative feedback control of parathyroid hormone secretion therefore seemed firmly established as a physiological concept. With the development of methods of assaying parathyroid hormone, and as knowledge of its chemistry began to accumulate, considerable interest centred on studies of its biosynthesis. Although initially a major aim may have been to determine whether calcium ions affect PTH synthesis as well as secretion, the overall results of biosynthetic studies have been far-reaching; indeed understanding of the biosynthetic pathways is clearly essential for the clarification of the role of parathyroid hormone in disease. Clinics in Endocrinology and Metabolisrn--Vol. 3, No. 2, July 1974.
199
200
7. J. MARTIN CONTROL OF BIOSYNTHESIS
Hamilton and Cohn (1969) incubated parathyroid gland slices and showed that the cells in the slices synthesised parathyroid hormone. They found that the degree of incorporation of radioactive amino acids into PTH was inversely related to calcium ion concentration, and that this effect of calcium was directed more towards the biosynthesis of parathyroid hormone than of the general proteins of the tissue. Au et al (1970) reached similar conclusions regarding the role of calcium in regulating the synthesis of parathyroid hormone in cultured rat parathyroids, whilst Raisz (1967) had shown previously that lowering the calcium concentration of culture media stimulated afiaino acid uptake by parathyroid tissue, and that a high calcium concentration inhibited it. Changes in magnesium concentration have no effect on the synthesis of parathyroid hormone (Hamilton et al, 1971a), whereas magnesium has been shown to have a calcium-like action on PTH secretion both in vitro (Sherwood, Hermann and Bassett, 1970) and in vivo experiments (Care et al, 1966; Gitelman, Kubolj and Welt, 1968; Buckle et al, 1968). This suggested that calcium may have a dual role, affecting both the synthesis of parathyroid hormone and its secretion, whereas magnesium may affect secretion alone. Hamilton et al (1971a) compared the radioactively-labelled parathyroid hormone in the tissue with that in the medium after incubation of parathyroid gland slices with radioactive amino acids, and found that the two behaved identically on gel filtration and electrophoresis. They noted also that when 14Clabelled PTH was added to media containing parathyroid slices, the 14CPTH was degraded to fragments, but the degradation process was not affected by changes in calcium concentration. This phenomenon may explain their further observation that dilutions of incubation media containing the secreted parathyroid hormone did not parallel pure PTH standards in the PTH radioimmunoassay. The other possible explanation of this latter observation was that another form of parathyroid hormone was being secreted, in addition to the 'native', 84-amino-acid-containing molecule that is normally obtained when parathyroid glands are extracted and purified. Such a possibility was being considered by other workers~ at that time, and in the following section this work will be reviewed. N A T U R E OF H O R M O N E RELEASED F R O M P A R A T H Y R O I D
GLANDS Interest in the nature of parathyroid hormone released from the parathyroid cell arose from the observations of Berson and Yalow (1968) that circulating endogenous PTH is immunochemically heterogeneous. They suggested that plasma may contain metabolic fragments of the parathyroid hormone molecule which would be detected to a variable extent by different antisera to PTH. Subsequently Arnaud, Tsao and Oldham (1970) and Arnaud et al (1971) detected differences between parathyroid hormone present in human hyperparathyroid serum and that in extracts of parathyroid adenomata. Furthermore, the parathyroid hormone liberated into the culture medium from parathyroid glands incubated in vitro resembled that of serum rather
201
BIOSYNTHESIS OF PARATHYROID HORMONE
than the hormone extracted from the gland. This led to the postulate of a 'glandular' and a 'secreted' form of PTH, the former being a precursor of the latter. These findings were similar to those of Sherwood, Rodman and Lundberg (1970) who studied PTH synthesis in cultures of bovine parathyroid glands.
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TUBE NUMBER Figure 1. Gel filtration elution patterns of medium (upper) and tissue extract (lower) of bovine parathyroid glands incubated with ]4C-leucine for 72 hours. Patterns observed with 1 '5 mM and 3.5 mM cation (calcium and magnesium) are indicated. Immunoreactive material from the medium was consistently eluted later than that from the tissue. Reproduced from Sherwood, L. Rodman, J. S. and Lundberg, W. B., Jr (1970). Proceedings of the National Academy of Sclences of the U.S.A., 67, 1631, by courtesy of the authors and the publisher.
These workers, however, made further observations which led them to conclude that the parathyroid hormone in the gland was indeed a prohormone, and that conversion to the hormonal, or circulating form, took place when the gland was stimulated. Figure 1 illustrates this finding.
202
T . J . MARTIN
If the interpretation of these experiments was correct, some important problems would arise. First, it would clearly be inappropriate to use parathyroid hormone extracted from glands as an antigen for the development of PTH radioimmunoassays. Furthermore, the corollary was that the circulating form of PTH would need to be purified and characterised from serum in order to determine precisely the nature of the circulating hormone and to attempt to measure it--surely a formidable task. However, two lines of evidence became available to indicate that the above mechanism did not operate to bring about the secretion of PTH. In vivo evidence was provided by Habener et al (1971) who used gel filtration and radioimmunoassay to determine the molecular size and immunoreactivity of PTH in peripheral plasma, in parathyroid effluent blood obtained from catheter specimens, and in gland extracts. Both in cattle and in man the parathyroid hormone emerging directly from the gland was indistinguishable from the 84-amino-acid-peptide (mol. w. 9500) extracted from the parathyroid glands. However, PTH in the peripheral plasma in each species differed in its immunochemical properties (Figure 2), and on gel filtration its major immunoreactive component appeared to have a molecular weight of about 7000. The conclusion reached was that the 'glandular' 84-amino-acidcontaining-peptide was indeed the secreted form of the hormone, but that it underwent cleavage peripherally to a smaller component, which differed in immunological reactivity with some antisera. 0.5 0.~
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The second line of evidence which questioned the validity of proposing 'glandular' PTH as a prohormone was that obtained from a study of human parathyroid hormone synthesis and secretion by monolayer cell cultures. These cultures secrete parathyroid hormone into the media for periods of several months (Martin et al, 1971). Secreted PTH was studied in media that were free of serum or contained very low (less than 1 per cent) amounts of
203
BIOSYNTHESIS OF PARATHYROID HORMONE
serum, to avoid proteolytic degradation of the hormone. Gel filtration of samples of medium obtained under these conditions revealed that the secreted parathyroid hormone was of the same molecular size as the marker 1251. PTH. Radioactive PTH was prepared by incorporation of laC-amino acids, and the 14C-PTH purified from culture media was found to be of the same size and charge as that of highly purified bovine PTH, and of human PTH extracted from glands (Figure 3). The conclusion from these two different approaches was therefore that the parathyroid hormone secreted from human parathyroid cells was indistinguishable from the hormone which could be extracted and purified from the glands. "--" HPTH °--o c.p.m. bPTH
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DISCOVERY OF PROPARATHYROID HORMONE
The subsequent discovery of a biosynthetic precursor of parathyroid hormone must be attributed in no small measure to the careful studies of Cohn, Hamilton and their colleagues on the: biosynthesis of parathyroid hormone by bovine parathyroid slices. In the'course of this work (Hamilton et al, 1971b) they noted that although the bulk of the immunoreactivity of the parathyroid hormone liberated into the incubation media eluted from Sephadex GI00 columns corresponded in molecular size to an 84-aminoacid-containing-PTH, there was some immunoreactivity found preceding the major peak, and some following i~. The smaller molecular weight material could be explained by breakdown of PTH which they had shown to occur in the medium, but they pursued the larger molecular weight protein, which
204
T.J. MARTIN
they realiged could represent a precursor molecule. They noted also that in purifying 14C-PTH from extracts of slices after biosynthesis they obtained, in addition to a labelled peptide behaving as 'authentic P T H ' , another peptide which eluted late from carboxymethylcellulose ion exchange columns. From its behaviour on Sephadex chromatography (Hamilton et al, 1971b), this peptide appeared to be slightly larger than PTH. Moreover, in the biosynthetic studies this peptide became labelled to a high specific activity (Hamilton and Cohn, 1969). Subsequently (Hamilton et al, 1971b) they achieved a substantial purification of the peptide which they called 'calcaemic fraction-A' (Figure 4). Under standard conditions of biological assay calcaemic fraction-A was found to be about one-third as active as parathyroid hormone in raising plasma calcium in the parathyroidectomised rat. Moreover, it was only approximately one half as active as P T H in an in vitro bone bioassay system. In their early experiments, using only one antiserum to parathyroid hormone, the immunological reactivity of calcaemic fraction-A mMho,
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was found to be approximately 5 per cent that of P T H (Hamilton et al, 1971b). Their observations and characterisation of calcaemic fraction-A were crucial in stimulating subsequent work demonstrating a precursorproduct relationship between ' p r o P T H ' and PTH. In addition to the features already discussed, a particular point of their experiments which led them to suggest that calcaemic fraction-A might be a biological precursor of P T H (Cohn et al, 1972a) was the fact that its synthesis, like that of PTH, was inversely related to calcium concentration in the incubation medium (Hamilton et al, 1971b).
BIOSYNTHESIS OF PARATHYROID HORMONE
205
That such a precursor of PTH did indeed exist was soon shown by Kemper et al (1972) and by Cohn et al (1972b) in pulse-chase experiments with bovine gland slices, showing early labelling of the precursor and formation of PTH without any evidence of an intermediate step. The precursor was called proPTH, and it seemed clear that it was identical with calcaemic fraction-A. Similar experiments have recently clearly demonstrated the existence of proPTH in chicken (MacGregor et al, 1973a), rat (Chu et al, 1973a), and human glands (Habener et al, 1972a). The proPTH of bovine origin has been the most extensively studied up to the present time; chemical and enzymatic cleavage of biosynthesised radioactive proPTH allowed Habener et al (1973) to conclude that the additional peptide sequence of the molecule occurs at the amino-terminal end of the hormone. Hamilton et al (1973) showed that the additional N-terminal sequence was lys - ser - val - lys - lys - arg, hence the increased basic charge of the prohormone. The reduced biological activity of the prohormone is not surprising as the N-terminal alanine of the PTH molecule is important for activity, at least in the rat kidney adenyl cyclase assay (Tregear et al, 1973), and extending the sequence by several residues beyond the amino terminus lowers activity appreciably. SYNTHESIS Formation of P T H from ProPTH
Subcellular fractionation of bovine parathyroid glands indicates that over 90 per cent of the hormone and prohormone is in particulate fractions (MacGregor et al, 1973b), consistent with the view that the membrane-bound granules seen on electron microscopy are secretory granules (Munger and Roth, 1963). Present evidence suggests that the synthesis and packaging of PTH in the parathyroid cell do not differ remarkably from those processes for other secreted proteins. MacGregor et al (1973b) suggest that newly synthesised proPTH is released from ribosomes to the cisternal space of the endoplasmic reticulum, where conversion to parathyroid hormone may begin. The newly formed hormone is then slowly transferred to the secretory granules. Control of the conversion process has been studied by the same group of workers, and is clearly of considerable importance, especially as proPTH differs from PTH in its biological properties. Chu et al (1973) studied some factors which might influence the glandular synthesis of proPTH and its conversion to parathyroid hormone. A low calcium diet in rats doubled the rate of PTH synthesis in the parathyroid glands without any change in rate of synthesis of proPTH or in the half-life of its cellular pool. The conclusion was that the parathyroid glands adapted to the low calcium diet by increasing the efficiency of conversion of proPTH to PTH, and that perhaps the control of PTH biosynthesis by calcium may occur at the level of intracellular turnover and degradation of the hormone. It may be relevant that a calciumdependent degradative process in the parathyroid cell has been proposed by Fischer, Oldham and Sizemore (1972). A role for cyclic 3'5' adenosine monophosphate in PTH release has been postulated by Dufresne and Gitelman
206
T.J. MARTIN
(1972) and by Abe and Sherwood (1972). If this is the case, it will be interesting to determine at what point the nucleotide is acting in the synthesis-secretion pathway, and how it relates to calcium effects. The hypothesis of Chu et al (1973) is illustrated in Figure 5. It implies that calcium ions exert effects either directly or indirectly at several points along the biosynthetic and secretory pathways of parathyroid hormone. Raisz (1967) has shown that conditions of low calcium can stimulate amino acid uptake by the parathyroid cell (step A), but Hamilton and Cohn (1969) found that low calcium stimulated the synthesis of parathyroid hormone under conditions in which no effect on the uptake of amino acid was observed (steps B, C and D). The effect of calcium ions on net PTH secretion (step E) is well established. Perhaps the most interesting aspect of the hypothesis of Chu et al (1973) is their proposal of a role for calcium ions in regulating the intracellular turnover of proPTH and PTH to smaller fragments, and thereby influencing the amount of parathyroid hormone available for secretion. It would be of great intrinsic interest to cell biologists to analyse such a process, which may be applicable to other secretory cells, as for example the [3-cell of the pancreas. Also it would be of interest to determine whether the function of the secretory machinery of glandular cells is to protect hormone from excessive exposure to intracellular degradation.
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Figure 5. Possible sites of regulation by calcium of PTH biosynthesisand secretion. For explanation see text. Reproduced from Chu et al (1973) Endocrinology, 93, 915-924 by courtesy of the authors and the publisher.
Human PTH The conversion of proPTH to PTH in the human parathyroid gland is of particular interest. Habener et al (1972a) studied the ability of several parathyroid adenomata to convert proPTH to PTH and found considerable variation in this property (Table 1). Chu et al (1973) pointed out that this variability could simply reflect differences in physiological state immediately before study of the gland in vitro; nevertheless, the results suggest the interesting possibility that some parathyroid tumours may accumulate and even release proPTH into the circulation. Indeed, this is implied by earlier in vivo experiments in which some patients with primary hyperparathyroidism released into the circulation immunoreactive PTH which was slightly larger than the normal 84-amino-acid-containing-molecule (Habener et al, 1971). Furthermore, although in most cell cultures established from human parathyroid glands the form of PTH released into the media was indistinguishable
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BIOSYNTHESISOF PARATHYROIDHORMONE
from that of normal 1-84 PTH, in cultures from two adenomata evidence has been obtained for the synthesis and release of a basic peptide resembling proPTH in its behaviour on gel filtration, disc electrophoresis and ion exchange chromatography (Marlin, Greenberg and Michelangeli, 1973); Figure 6 illustrates this. Table 1. Relative amounts of P T H and proPTH synthesised by various parathyroid tissues in short-term incubations
Tissue sample (no.) Human adenoma
PTH (%)
proPTH (%)
I
13
2 3 4 5 6 Human clear cell hyperplasia
22 23 45 50 83
87 78 77 55 50 17
51
49
42 46 55 57
58 54 45 43
1
Bovine glands 1
2 3 4 Data from Habener et al (1972a).
The most likely conclusion from these various experiments is that normally and in the case of most parathyroid adenomata, the conversion of proPTH proceeds rapidly and that only parathyroid hormone containing 84amino acids is released from the gland. Occasj0nally, however, the capacity of the converting process is exceeded, and proPTH will be released. Clearly, the situation resembles closely that for insulinomata, which may secrete proinsulin (Melani et al, 1970). This property of some adenomata should be recalled as a potential contributor to the difficulty of measuring parathyroid hormone in plasma by radioimmunoassay, since proPTH would be recognised with difficulty by some N-terminal specific antisera. The other important implication for the syndrome of hyperparathyroidism in man is related to the biological activity of human proPTH. If, as seems likely, the human precursor molecule resembles its'.bovine counterpart by possessing extra amino acids at the amino-terminal end of the molecule, human proPTH would be expected to be less active than 1-84 PTH, and the clinical features of primary hyperparathyroidism may be modified in those patients secreting a significant amount of proPTH. It will be important to have this point clarified by prospective studies in patients with primary hyperparathyroidism, in whom proPTH and PTH may eventually be assayed specifically. Synthesis of P T H by non-parathyroid cancer in man
It seems likely that malignant hypercalcaemia unassociated with bony metastases is most frequently due to hnmoral factors other than parathyroid
208
T.J.
MARTIN
hormone. Nevertheless, the syndrome of 'ectopic' hyperparathyroidism is now well recognised. The capacity of non-parathyroid cancers to produce parathyroid hormone was established by the demonstration of a gradient of hormone concentration across the tumour-bearing organ in the case of a hypernephroma (Buckle, McMillan and Mallinson, 1970) and of a hepatoma (Knill-Jones et al, 1970). Some differences have been found between circulating immunoreactive parathyroid hormone in primary and in ectopic hyperparathyroidism, which have raised questions regarding the nature of the PTH synthesised and released by the tumours. Riggs et al (1971) and Roof et al (1971) noted immunological differences between the plasma PTH of primary hyperparathyroidism and of hyperparathyroidism due to cancer. + ~ -
1000I
II (o) I
500
0
20
~0
Distancemigrated(mm)
60
Figure 6. Disc gel electrophoresis of 14C-labelled peptides with PTH-like immunoreactivity, synthesised and secreted by a human parathyroid adenoma in culture. Peak I migrates identically with pure bovine PTH (shown as heavy band in upper part of diagram), and peak II is faster moving and therefore more basic. Reproduced from Martin, T. J., Greenberg, P. B. and Michelangeli, V. (1973) Clinical Science, 44, 1-8, by courtesy of the publisher.
The explanation most commonly offered for ectopic peptide hormone production is that derepression occurs in the cancer cell, leading to the transcription of normally repressed DNA. If this is correct, the cancer cell should produce a hormone identical to that synthesised by the appropriate gland cell. There is increasing evidence, already referred to, that this may not be the case with ectopic PTH production. Although secretion of proPTH by a cancer is a potential contributor to immunochemical heterogeneity, it is unlikely to be the sole explanation. In one instance ofectopic PTH production by a hypernephroma, Greenberg, Martin and Sutcliffe (1973) showed that cell cultures of the cancer synthesised a peptide with properties of proPTH. However, they suggested another possible explanation for the 'abnormal' PTH in the cancer syndrome in their case, because evidence was obtained for the formation of immunoreactive fragments of PTH within the cancer cells during culture (Figure 7). In extensive studies with human parathyroid
209
BIOSYNTHESIS OF PARATHYROID HORMONE
cells these authors found no evidence for the formation of intracellular PTH fragments (Martin et al, 1971 ; Martin et al, 1973). Although they did not exclude the possibility that partial sequences of PTH were made by the tumour cells, they suggested that the immunoreactive PTH fragments found in the cells and released into the media may result from rapid and potent proteolytic processes within the cells, leading to a rapid turnover of newly synthesised hormone and the generation of fragments of the molecule; some of these would have biological activity, whereas their immunological reactivity would depend on the particular antisera being used. If such a mechanism is B.W.211/32 GPC3
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Figure 7. Gel filtration of aqueous extract of cultured cells from a clear cell carcinoma of the kidney associated with ectopic PTH production. The cells had been incubated for 24 hours with 14C-leucine. Pool III corresponds in molecular size to pure bovine PTH (shown as BPTH). The inset shows the results of examining pools I to V for the presence of 14C-peptide with PTH-like immunoreactivity. Double antibody precipitation was carried out with two different antisera to PTH. Results show that the ceils synthesised peptides with PTH-like immunoreactivity, corresponding in size to 1-84 PTH, but also some peptides of smaller size (pools IV and V). Reproduced from Greenberg, P. B., Martin, T. J. and Sutcliffe, H. S. (1973) Clinical Science and Molecular Medicine, 45, 183-191, by courtesy of the publisher.
found to be a common occurrence in cancers, it would be interesting to speculate whether the increased susceptibility of the peptide to proteolysis might be due to the absence of the normal packaging and secretory mechanisms from the cancer cell. Although there is evidence for the generation of peptide fragments in other cancer syndromes--for example ectopic ACTH production (Orth et al, 1973), it is unlikely that such a mechanism is operative for all ectopic humoral syndromes. Indeed, there is evidence for the production by a lung cancer of vasopressin, which was identical with the neurohypophyseal hormone in all systems studied (George, Capen and Phillips, 1972). Very careful s,tudy of the nature of parathyroid hormone produced by individual cancers will be needed to resolve the questions surrounding the ectopic PTH syndrome, since it is by no means impossible that several different abnormalities may exist.
210
x.J. MARTIN 'SECRETED' AND 'CIRCULATING' PTH
Although the emphasis in this discussion has been on the biosynthesis of parathyroid h o r m o n e and the nature of the h o r m o n e released from the gland, it is appropriate to consider those properties of P T H in the peripheral circulation which distinguish it from h o r m o n e directly leaving the parathyroid gland cell. On the present evidence, one must conclude that parathyroid h o r m o n e normally enters the circulation as the 84-amino-acid-containing-molecule, but in some cases of parathyroid adenoma secretion of p r o P T H m a y occur, whereas in some cases of 'ectopic' hyperparathyroidism there may be a release of PTH, p r o P T H , fragments of the hormone, or perhaps other forms, as yet uncharacterised. Even under normal conditions of secretion of parathyroid hormone, however, there are recognisable immunological differences between "secreted' parathyroid h o r m o n e and P T H in the peripheral circulation. Recent studies with well characterised antisera (reviewed by Parsons and Potts, 1972) indicate that the suggestion of Berson and Yalow (1968) is probably correct, namely that the immunochemical heterogeneity of circulating P T H m a y be due to circulating immunoreactive fragments of the hormone. i
0.8
~'"~,¢/
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Figure 8. Gel filtration of parathyroid hormone in the general circulation of a patient with primary hyperparathyroidism. The interrupted line indicates the elution volume of the marker, lzSI-labelled PTH. C and N refer to immunoassays carried out on column fractions, using two different antisera, one C-terminal and one N-terminal specific. The C-terminal immunoreactivity is of slightly lower molecular weight (approximately 7500) than 1-84 PTH, but the small amount of N-terminal immunoreactivity probably indicates that intact 1-84 PTH is remaining in the circulation. Reproduced from Habener et al (1972b) Nature New Biology, 238, t52-154, by courtesy of the authors and the publisher.
BIOSYNTHESIS OF PARATHYROID HORMONE
21 l
The precise nature of the circulating fragments is not known, but some useful working proposals can be made on the basis of the experiments of Habener et al (1972b). Using N- and C-terminal specific radioimmunoassays for PTH, they assayed peripheral and thyroid vein plasma from patients with primary hyperparathyroidism and found that whereas the two assays gave equal results in the thyroid vein plasma, the peripheral plasma levels were very much lower with the N-assay than with the C-assay. Figure 8 illustrates results of gel filtration of a peripheral plasma sample and applying the two assays. The small amount of N-terminal activity coincided in elution volume with native PTH, whereas the C-terminal reactivity emerged slightly later from the column, consistent with a peptide of molecular weight of approximately 7500. Habener et al (1972b) suggest that cleavage of secreted PTH occurs in some organ, giving rise to a fairly large C-terminal fragment of the hormone. The persisting N-terminal immunoreactivity seen in Figure 8 would represent secreted PTH as yet undegraded. There are several interesting questions which arise from this observation. First: is the peripheral cleavage a constant, reproducible process occurring at some particular point along the polypeptide chain ? If so, does it yield an N-terminal sequence of sufficient length to possess biological activity in addition to the presumably inactive C-terminal fragment ? Finally, in what organ does peripheral metabolism take place predominantly? No definite answers are available to these questions. Canterbury, Levey and Reiss (1973) have extracted peripheral plasma of patients with hyperparathyroidism and found PTH-like biological activity in low molecular weight fractions, suggesting that a low molecular weight biologically active form of parathyroid hormone may be circulating in the blood. In view of the evidence for a role for the kidney in the metabolism of parathyroid hormone in the rat (Orimo and Fujita, 1966; Martin, Melick and de Luise, 1969), and in the dog and in man (Melick and Martin, 1969), the kidney seems a likely site for the peripheral cleavage of parathyroid hormone, although there is evidence in the rat for a significant role also for the liver in the metabolism of parathyroid hormone (Vajda, Martin and Melick, 1969; Fang and Tashjian, 1972). CONCLUSIONS Recent studies on the biosynthesis of parathyroid hormone have been reviewed, and the experiments described which have led ultimately to the discovery of proPTH, the biosynthetic precursor of PTH. Careful further study of the control of PTH biosynthesis should yield valuable information to the cell biologist, and may help in understanding the biological purpose of 'prohormones' and the functions of hormone storage mechanisms in glandular cells. There are a number of clinical implications which arise. It has been pointed out that proPTH may circulate in the plasma of some patients with primary hyperparathyroidism. In view of the relatively low biological activity of proPTH, it is now important to the understanding of the hyperparathyroid syndrome that such cases be studied in great detail. It should be clear from
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T. J. MARTIN
this review that there are m a n y other possible causes of the ' i m m u n o c h e m i c a l heterogeneity' of plasma PTH, and we can readily u n d e r s t a n d the difficulties which have been experienced in measuring circulating levels of P T H in man. The need for well characterised antisera is evident. ACKNOWLEDGEMENTS
Work referred to from the author's laboratory was supported by the Anti-Cancer Council of Victoria and the National Health and Medical Research Council. Drs D. V. Cohn and J. W. Hamilton kindly provided details of work prior to publication. REFERENCES Abe, M. & Sherwood, L. M. (1972) Regulation of parathyroid hormone secretion by adenyl cyclase. Biochemical and Biophysical Research Communications, 48, 396~401. Albright, F. (1941) The parathyroids--physiology & therapeutics. Journal of the American Medical Association, 117, 527-535. Arnaud, C. D., Tsao, H. S. & Oldham, S. B. (1970) Native human parathyroid hormone: an immunochemical investigation. Proceedings of the National Academy o f Sciences of the U.S.A., 67, 415--422. Arnaud, C. D., Sizemore, G. W., Oldham, S. B., Fischer, J. A., Tsao, H. S. & Littledike, T. (1971) Human parathyroid hormone: glandular and secreted molecular species. American Journal of Medicine, 50, 630-638. Au, W. Y. W., Poland, A. P., Stern, P. H. & Raisz, L. G. (1970) Hormone synthesis and secretion by rat parathyroid glands in tissue culture. Journal of Clinical Investigation, 49, 1639-1646. Berson, S. A. & Yalow, R. S. (1968) Immunochemical heterogeneity of parathyroid hormone. Journal of Clinical Endocrinology and Metabolism, 28, 1037-1047. Buckle, R. M., Care, A. D., Cooper, C .W. & Gitelman, H. J. (1968) The influence of plasma magnesium concentration on parathyroid hormone secretion. Journal of Endocrinology, 42, 529-534. Buckle, R. M., McMillan, M. & Mallinson, C. (1970) Ectopic secretion of PTH by a renal adenocarcinoma in a patient with hypercalcaemia. British Medical Journal, iv, 724-726. Canterbury, J. M., Levey, G. S. & Reiss, E. (1973) Activation of renal cortical adenylate cyclase by circulating immunoreactive parathyroid hormone fragments. Journal of Clinical Investigation, 52, 524-527. Care, A. D., Sherwood, L. M., Potts, J. T. Jr & Aurbach, G. D. (1966) Evaluation by radioimmunoassay of factors controlling the secretion of parathyroid hormone. Nature, 209, 55-57. Chu, L. L. H., MacGregor, R. R., Anast, C. S., Hamilton, J. W. & Cohn, D. V. (1973) Studies on the biosynthesis of rat parathyroid hormone and proparathyroid hormone : adaptation of the parathyroid gland to dietary restriction of calcium. Endocrinology, 93, 915-924. Cohn, D. V., MacGregor, R. R., Chu, L. L. H. & Hamilton, J. W. (1972a) Studies on the biosynthesis in vitro of parathyroid hormone and other calcemic polypeptides of the parathyroid gland. In Calcium, Parathyroid Hormone and the Calcitonins, Proceedings of the Fourth Parathyroid Conference, ed. R. V. Talmage & P. L. Munson, pp. 173-182. Amsterdam: Excerpta Medica. Cohn, D. V., MacGregor, R. R., Chu, L. L. H., Kimmel, J. R. & Hamilton, J. W. (1972b) Calcemic fraction-A: Biosynthetic peptide precursor of parathyroid hormone. Proceedings of the National Academy of Seiences of the U.S.A., 69, 1521-1525. de Robertis, E. (1941) The cytology of the parathyroid and thyroid glands of rats with experimental rickets. Anatomical Record, 79, 417-434. Dufresne, L. R. & Gitelman, H. J. (1972) A possible role of aderiyl cyclase in the regulation of parathyroid activity by calcium. In Calcium, Parathyroid Hormone and the Calcitonins, Proceedings of the Fourth Parathyroid Conference, ed. R. V. Talmage & P. L. Munson, pp. 202-206. Amsterdam: Excerpta Medica.
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Fang, V. S. & A. Tashjian, A. H. Jr (1972) Studies on the role of the liver in the metabolism of parathyroid hormone. Endocrinology, 90, 1177-1184. Fischer, J. A., Oldham, S. B., Sizemore, G. W. & Arnaud, C. D. (1972) Calcium-regulated parathyroid hormone peptidase. Proceedings of the National Academy of Sciences of the U.S.A., 69, 2341-2345. George, J. M., Capen, C. C. & Phillips, A. S. (1972) Biosynthesis of vasopressin in vitro and ultrastructure of a bronchogenic carcinoma. Journal of Clinical Investigation, 51, 141-146. Gitelman, H. J., Kubolj, S. & Welt, L. G. (1968) Inhibition of parathyroid gland activity by hypermagnesaemia. American Journal of Physiology, 215, 483-485. Greenberg, P. B., Martin, T. J. & Sutcliffe, H. S. (1973) Synthesis and release of parathyroid hormone by a renal carcinoma in cell culture. Clinical Science and Molecular Medicine, 45, 183-191. Habener, J. F., Powell, D., Murray, T. M., Mayer, G. P. & Potts, J. T. Jr (1971) Parathyroid hormone: secretion and metabolism in vivo. Proceedings of the National Academy of Sciences of the U.S.A., 68, 2986-2991. Habener, J. F., Kemper, B., Potts, J. T. Jr & Rich A. (1972a) Proparathyroid hormone: biosynthesis by human parathyroid adenomas. Science (New York), 178, 630-633. Habener, J. F., Segre, G. V., Powell, D., Murray, T. M. & Potts, J. T. Jr (1972b) Immunoreactive parathyroid hormone in circulation in man. Nature New Biology, 238, 152-154. Habener, J. F., Kemper, B., Potts, J. T. Jr & Rich, A. (1973) Bovine parathyroid hormone: structural analysis of radioactive peptides formed by limited cleavage. Endocrinology, 92, 219-226. Hamilton, J. W. & Cohn, D. V. (1969) Studies on the biosynthesis in vitro of parathyroid hormone. Journal of Biological Chemistry, 244, 5421-5429. Hamilton, J. W., Spierto, F. W., MacGregor, R. R. & Cohn, D. V. (1971a) Studies on the biosynthesis in vitro of parathyroid hormone. Journal of Biological Chemistry, 246, 3224-3233. Hamilton, J. W., MacGregor, R. R., Chu, L. L. H. & Cohn, D. V. (1971b) The isolation and partial purification of a non-parathyroid hormone calcemic fraction from bovine parathyroid glands. Endocrinology, 89, 1440-1447. Hamilton, J. W., NialI, H. D., Keutmann, H. T., Potts, J. T. Jr & Cohn, D. V. (1973) Amino terminal sequence of bovine proparathyroid hormone (calcemic fraction-A). Federation Proceedings, 32, 269. Kemper, B., Habener, J. F., Potts, J. T. Jr & Rich, A. (1972) Proparathyroid hormone: identification of a biosynthetic precursor of parathyroid hormone. Proceedings of the National Academy of Sciences of the U.S.A., 69, 643-647. Knill-Jones, R. P., Buckle, R. M., Parsons, V., Calne, R. Y. & Williams, R. (1970) Hypercalcaemia and increased parathyroid hormone activity in a primary hepatoma. New England Journal of Medicine, 282, 704-708. MacGregor, R. R., Chu, L. L. H., Hamilton, J. W. & Cohn, D. V. (1973a) Partial purification of parathyroid hormone from chicken parathyroid glands. Endocrinology, 92, 13121317. MacGregor, R. R., Chu, L. L. H., Hamilton, J. W. & Cohn, D. V. (1973b) Studies on the subcellular localization of proparathyroid hormone and parathyroid hormone in the bovine parathyroid gland: separation of newly synthesized from mature forms. Endocrinology, 93, 1387-1397 McLean, F. C. & Urist, M. R. (1955) An Introduction to the Physiology of Skeletal Tissue. University of Chicago Press. Martin, T. J., Melick, R. A. & de Luise, M. (1969) The effect of nephrectomy on the metabolism of labelled parathyroid hormone. Clinical Science, 37, 137-142. Martin, T. J., Greenberg, P. B., Melick, R. A. & Pilczyk, R. (1971) Synthesis of human parathyroid hormone by cells in culture. In Endocrinology, ed. S. Taylor, pp. 363-373. London: Heinemann. Martin, T. J., Greenberg, P. B. & Melick, R. A. (1972) Nature of human parathyroid hormone secreted by monolayer cell cultures. Journal of Clinical Endocrinology and Metabolism, 34, 437-440.
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Martin, T. J., Greenberg, P. B. & Michelangeli, V. (1973) Synthesis of human parathyroid hormone by cultured cells: evidence for release of prohormone by some adenomata. Clinical Science, 44, 1-8. Martin, T. J., Greenberg, P. B., Beck, C., Burger, H. G. & Johnston, C. I. (1973) Synthesis and secretion of peptide hormones by endocrine and non-endocrine tumours. In Endocrinology 1973, ed. S. Taylor. London: Heinemann (in press). Melani, F., Ryan, W. G., Rubenstein, A. H. & Steiner, D. F. (1970) Proinsulin secretion by a pancreatic beta-cell adenoma. New England Journal of Medicine, 283, 713-719. Melick, R. A. & Martin, T. J. (1969) Parathyroid hormone metabolism in man. Effect of nephrectomy. Clinical Science, 37, 667-674. Munger, B. L. & Roth, S. J. (t963) The cytology of the normal parathyroid glands in man and Virginia deer; a light and electron microscopic study with morphologic evidence of secretory activity. Journal of Cell Biology, 16, 379-400. Orimo, H. & Fujita, T. (1966) Inactivation in vitro of parathyroid hormone activity by kidney slices. Endocrinology, 76, 255-258. Orth, D. N., Nicholson, W. E., Mitchell, W. M., Island, D. P. & Liddle, G. W. (1973) Biologic and immunologic characterization and physical separation of ACTH and ACTH fragments in the ectopic ACTH syndrome. Journal of Clinical hlvestigation, 52, 1756-1769. Parsons, J. A. & Potts, J. T. Jr (1972) Physiology and chemistry of parathyroid hormone. Clinics in Endocrinology and Metabolism, 1, 33-78. Part, H. M. & Luckhardt, A. B. (1942) Relationship of a low blood calcium to parathyroid secretion. Endocrinology, 31, 384-392. Raisz, L. G. (1963) Regulation by calcium of parathyroid growth and secretion in vitro. Nature, 197, 1115-1116. Raisz, L. G. (1967) Effect of calcium on uptake and incorporation of amino acids in the parathyroid glands. Biochimica Biophysica Acta, 148, 460-468. Riggs, B. L., Arnaud, C. D., Reynolds, J. C. & Smith, L. H. (1971) Immunological differentiation of primary hyperparathyroidism from hyperparathyroidism due to nonparathyroid cancer. Journal of Clinical Investigation, 50, 2079-2083. Roof, B. S., Carpenter, B., Fink, D. J. & Gordan, G. S. (1971) Some thoughts on the nature of ectopic parathyroid hormone. American Journal of Medicine, 50, 686-691. Sherwood, L. M., Potts, J. T. Jr, Care, A. D., Mayer, G. P. & Aurbach, G. D. (1966) Evaluation by radioimmunoassay of factors controlling the secretion of parathyroid hormone. Nature, 209, 52-55. Sherwood, L. M., Hermann, J. & Bassett, C. A. (1970) Parathyroid hormone secretion in vitro: regulation by calcium and magnesium ions. Nature, 225, 1056-1058. Sherwood, L. M., Rodman, J. S. & Lundberg, W. B. Jr (1970) Evidence for a precursor to circulating parathyroid hormone. Proceedings of the National Academy of Sciences of the U.S.A., 67, 1631-1638. Tregear, G. W., van Rietschoten, J., Greene, E., Keutmann, H. T., Niall, H. D., Reit, B., Parsons, J. A. & Potts, J. T. Jr (1973) Bovine parathyroid hormone: minimum chain length of synthetic peptide required for biological activity. Endocrinology, 93, 1349-1353. Vajda, F. J. E., Martin, T. J. & Melick, R. A. (1969) Destruction of bovine parathyroid hormone labelled with 131I by rat kidney tissue. Endocrinology, 84, 162-164.