Calcitonin

~HAPTER z

Calcltonln T. J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON St. Vincent's Institute of Medical Research and The University of Melboume Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia

I. II. III. IV. V.

Nature of Calcitonin Chemistry Biosynthesis Secretion and Metabolism Actions of Calcitonin

VI. Calcitonin Receptor VII. Calcitonin in Clinical Medicine VIII. Summary Acknowledgments References

When it became established that parathyroid hormone acted directly on bone to promote its resorption, the idea developed that parathyroid hormone was the major hormonal factor governing calcium homeostasis in the body. This was the basis of the McLean and Urist 1 hypothesis developed in the mid 1950s that the serum calcium was regulated by appropriate changes in the secretion rate of parathyroid hormone by a negative feedback control system. In 1961, Rasmussen 2 questioned this in suggesting that if the bone were the only means of regulating serum calcium level in conjunction with the parathyroids, the resulting feedback system would lead to wide fluctuations in serum calcium. It had been known for some time that parathyroid hormone lowered the urinary calcium, and Rasmussen made use of this in extending the McLean-Urist theory to involve the kidney. He considered that the kidney regulator was rapid to respond, sensitive to small fluctuations in hormone concentration, and of limited capacity; the bone regulator was slow to respond, relatively insensitive, but of nearly unlimited capacity. The renal action conserved serum calcium by rapidly inhibiting urinary secretion of calcium, the skeletal action by causing a less rapid dissolution of calcium from the bone matrix to the extracellular fluid. Although this seemed a more satisfactory METABOLIC BONE DISEASE

explanation of calcium control, Copp and his associates looked persistently for a better o n e . 3'4 In experiments in which they perfused the thyroparathyroid apparatus of dogs and sheep, they obtained evidence for the secretion, in response to a high calcium stimulus, of a factor that rapidly lowered systemic plasma calcium. 3 They called this calcitonin, and suggested that it was produced by the parathyroid glands. 4 After this discovery, Hirsch et al. looked afresh at some observations made in their laboratory. It had been noted that rats parathyroidectomized by cautery showed a more rapid and profound fall in serum calcium than rats parathyroidectomized by surgical excision. 5 Hirsch et al. then found that acid extracts of rat thyroid glands caused hypocalcemia when injected into r a t s . 6 They suggested that cautery of the thyroid gland during parathyroidectomy caused the release of a factor from the thyroid gland that provoked a greater fall in serum calcium than that which occurred following simple removal of the parathyroid glands (Fig. 4 - 1 ) . They called this activity "thyrocalcitonin" and showed that it could be extracted from the thyroid glands of several species, but not from other tissues. At the same time, Maclntyre's group provided support for Copp's work by demonstrating in thyroparathyroid perfusion studies in the dog that 95

Copyright 9 1998 by Academic Press. All fights of reproduction in any form reserved.

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FIGURE 4--2 Effect on systemic plasma calcium in the goat of high calcium perfusion of parathyroid (e) and of thyroid and parathyroid glands (o). (From Foster GV, Baghdiantz A, Kumar MA, et al: Thyroid origin of calcitonin. Nature 202:1303-1305, 1964. Copyright 1964, Macmillan Journals Limited.)

a potent, rapidly acting calcium-lowering hormone existed. 7 Subsequent perfusion studies in the goat showed that this activity was released from the thyroid gland. 8 In this species it was possible to perfuse either the external parathyroid alone or the thyroid plus parathyroid glands together (Fig. 4 - 2 ) . Furthermore, autogenous thyroid extract injected into the goats rapidly lowered the systemic serum calcium. It soon became clear that calcitonin and thyrocalcitonin were identical, were products of the thyroid gland in mammals, and probably represented an important new hormone involved in the regulation of calcium metabolism in the body.

logical assay contributed to the fact that calcitonins of several species were isolated and sequenced within a few years of its discovery. The recognition of calcitonin as a hypocalcemic hormone was hailed for several years as an answer to the tight control of plasma calcium. As information emerged about its action, however, it became apparent that this was not so, at least in the mature animal. The ability of calcitonin to inhibit osteoclastic bone resorption is beyond doubt, but the physiological significance of this seems likely to vary, for instance, during growth and development. Finally, the recognition of calcitonin's origin in the " C " or parafollicular cells of the mammalian thyroid drew attention to a second endocrine system in the thyroid gland, deriving its origin from the cells of the ultimobranchial bodies, which in birds and fish remain as discrete organs.

Comparison of the effects of thyroparathyroidectomy with parathyroidectomy by cautery and surgery in the rat. (From Hirsch PF, Gauthier GF, Munson PL: Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting response to parathyroidectomy in rats. Endocrinology 73:244-251, 1963.)

I. NATURE OF CALCITONIN Experiments in several species rapidly confirmed that calcitonin was a peptide released from the thyroid gland in response to a hypercalcemic stimulus and capable of rapidly lowering the plasma calcium and phosphorus levels. It was shown to produce this effect independent of the kidney or intestine, and it acted as an inhibitor of bone resorption. Evidence for this is summarized later in this chapter (see Section V.A). The calcium-lowering effect of calcitonin was the basis for its biological assay, and indeed the convenience and sensitivity of the bio-

A. Embryological Origin of Calcitonin-Producing Cells Much of the early work on the localization of calcitonin-producing cells comes from Pearse and his colleagues, who suggested that the "mitochondrion-rich" cells of the thyroid were responsible for calcitonin secretion. 9 When they observed electron microscopic

CHAPTER4 Calcitonin changes in the parafollicular cells of the dog thyroid following high calcium perfusion of the gland, they ascribed to these cells the function of either synthesizing or storing calcitonin, and gave them the name C cells. These cells are identical with the argyrophil parafollicular cells described in the dog by Nonidez, 1~ and in other species (e.g., the pig) they occupy epifollicular and follicular positions. 11 Pearse produced further evidence that the C cells may produce a polypeptide hormone by demonstrating their property of uptake of 5-hydroxytryptophan, 11 a faculty common to other polypeptide hormone-producing cells (e.g., the pancreatic islet cells) and pituitary corticotrophs and melanotrophs (APUD cells). At the same time he pointed out that there were two possibilities for the embryological origin of the C c e l l s n t h e ultimobranchial bodies and the neural crest h a n d he favored the former site of origin. Subsequent cytochemical studies confirmed the ultimobranchial origin of the parafollicular C cells in the rodent thyroid, 12 and it is considered that the ultimobranchial and C cells derived originally from the neural crest. The ultimobranchial body arises in the embryos of all vertebrates, with the exception of the cyclostomes, caudal to the last branchial arch, one on either side. Its fate in the adult varies across species. In lower vertebrates it remains as a distinct structure that may persist on both sides or unilaterally. In mammals it becomes fused with the thyroid during embryonic life and here gives rise to calcitoninsecreting C c e l l s . 13 No function had previously been ascribed to the ultimobranchial body, but seasonal changes in its structure had been observed in lizards, bats, and frogs. The presence of secretory material in the follicles of the ultimobranchial bodies of reptiles, amphibians, and fish suggested an endocrine function, but there was no indication as to its nature. 14-~6 It was thought that the ultimobranchial body might form reserves of parathyroid or thymic tissue in conditions of stress, or possibly that it had undergone regression during evolution and lost its original function. Observations of the teleost A s t y a n a x mexicanus revealed that the ultimobranchial body hypertrophied after the fish had been kept in the dark for periods of between 4 months and 2 years. TM These changes were associated with skeletal deformities, but the authors attributed them to a parathyroid function of the ultimobranchial body. Hypertrophy of the ultimobranchial body had also been observed in frogs 15 under conditions of calcium stress and during metamorphic climax when calcium is being transferred from stores in the paravertebral lime sacs for incorporation into bone. Thus there was some evidence for involvement of the ultimobranchial body in changes in calcium metabolism, but no specific mechanisms were known. Throughout their work, Pearse's group emphasized the variable ultimate fate of the ultimobranchial bodies

97 in different species. After originating from the neural crest, these bodies migrate forward during embryonic development. Although the ultimobranchial bodies remain as separate endocrine glands in birds, fish, and reptiles, in some submammalian species the cells can be found in other tissues of the neck and in the lung. In the lizard, for example, the main source of calcitonin is the lung, 16 although calcitonin-producing cells are present in other parts of the neck. Although in the rat the C cells are virtually all within the thyroid, this is not the case with several other mammals. In humans, for example, there is evidence for calcitonin-producing cells in the thymus and the lung, and it is therefore difficult to determine the result of calcitonin deficiency in mammals by experimentation or in humans by clinical observation. The association of calcitonin with granules in the parafollicular cells of the rat was suggested by experiments showing that administration of calcium to rats resuited in discharge from these cells of granules visible on electron microscopy. 17 In addition to the demonstration of the peptide hormone-secreting properties of C cells by histochemistry, calcitonin was identified in the C cells of the dog and the pig by immunofluorescence. This was later confirmed by in situ hybridization localization of calcitonin messenger ribonucleic acid (mRNA) to the C cells of the thyroid. TM

B. C a l c i t o n i n in Vertebrates a n d N o n v e r t e b r a t e s There has been considerable interest in the discovery that immunoreactive calcitonin-like material can be detected in the nervous systems of prevertebrate species including the sea squirt Ciona intestinalis, 19 the pond snail L y m n a e a stagnalis, 2~ a number of protochordates, and a cyclostome, Myxine. 21 These species lack bony skeletons, and the occurrence of human calcitonin-like molecules in their nervous systems suggests that the bone-regulating function of the calcitonins may be a later evolutionary development in vertebrates. Human calcitonin-like immunoreactivity (hCT-I) also exists in human cerebrospinal fluid, 22 although no direct evidence for the origin of this material is available. Low levels of hCT-I were found in extracts of postmortem human brain. 23"24 However, in addition to hCTI, low levels of material immunologically and chromatographically similar to salmon calcitonin sCT (sCT-I) also occurred. 24 The highest concentration of immunoreactivity was found in the hypothalamus, which is consistent with the hypothalamus containing the highest level of calcitonin receptors (see Section V.F). The concentrations of CT-I in the brain were ---10 times greater than those in serum and cerebrospinal fluid. 24 Similarly, extracts of rat brain diencephalon revealed the existence

98 of a biologically active sCT-like peptide 25 which was distinct from the low levels of hCT-I reported in earlier studies. 26 Although hCT-I has been detected in rat brain, there is only limited evidence for the presence of rat calcitonin (rCT) mRNA. In a Northern blot analysis of hypothalamus, a low level of rCT mRNA was observedZ7; however, S 1 nuclease assay of various brain regions failed to detect calcitonin mRNA expression, indicating that levels of calcitonin mRNA must be less than 0.2% of that of calcitonin gene-related peptide 28 (see Section II.B). The existence of multiple types of calcitonin within one species is sustained by the immunological detection of at least two forms of calcitonin in fish, reptiles, amphibia, mammals, and birds, 29-31 while the existence of a gene expressing a sCT-like peptide in humans is supported by hybridization of chicken calcitonin (cCT) cDNA to Northern blots of human medullary thyroid carcinoma tissue. 32 A role for a sCT-like peptide as a neurotransmitter or neuromodulator is supported by the presence of sCT-I within the central nervous system of lower vertebrates. In pigeon, the distribution and levels of peptide detected by a sCT-specific radioimmunoassay 33 are consistent with findings in rat b r a i n y with the highest amount detected in the hypothalamus followed by midbrain and low levels in brainstem. Immunohistochemical studies in lizard brain further demonstrated sCT-I, localized to varicosities and terminals in the diencephalon, although not in other brain regions. 34 Furthermore, the levels of calcitonin-like peptide detected in the rat diencephalon 25 were in accord with the reported levels of other neuropeptides, including CGRP and neuropeptide y,35.36 consistent with a potential role of a sCT-like peptide acting as a neurotransmitter or neuromodulator in mammalian brain. Intriguingly, the rat C lb isoform of the calcitonin receptor (CTR) (see Section VI-B) has very little interaction with the thyroidally derived form of calcitonin. 37'38 This receptor isoform is enriched in brain and may form a physiological target for endogenous sCT-like peptides. Furthermore, the C3-amylin receptor has high affinity for the teleost but not mammalian calcitonins in the rat, 39'4~ and as such this receptor phenotype is also a potential substrate for sCT-like peptides. Immunoreactivity equivalent to the thyroidally derived or ultimobranchial-derived form of calcitonin has also been found in the anterior pituitary of both mammals and lower vertebrates. 23'26'41-45 Although calcitoninlike immunoreactivity is also reported to occur in the intermediate pituitary, 41'43'44 this finding is not routinely reproducible. 42'44 The identity of the pituitary calcitonin remains to be determined. While reacting to specific hCT-antisera, the rat pituitary material does not react with all hCT-antisera, nor does preabsorption clearly

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

abolish pituitary staining. 43 This suggests that the pituitary calcitonin-like material is not equivalent to the thyroidal form of calcitonin. Consistent with this, Northern blot analysis of anterior pituitary, using cDNA to thyroidal calcitonin mRNA, failed to detect expression of calcitonin mRNA. 46'47 More recent data indicate that a sCT-like peptide may also be present in the anterior pituitary, with release of sCT-like peptide from cultured rat anterior pituitary cells. 48 Salmon CT-I also occurs in the mouse pituitary carcinoma cell line oL-TSH.49 The physiological significance of calcitonin-like immunoreactivity in the pituitary remains to be determined; however, calcitonin receptors are present in the intermediate pituitary 5~ and therefore the calcitonin-like material may act as a paracrine regulator of these receptors.

II. CHEMISTRY Several groups almost simultaneously published the sequence of pig calcitonin (pCT) 52'53 and within months pCT had been chemically synthesized. 54 It is a 32-aminoacid peptide with a carboxyl-terminal proline amide and a disulfide bridge between cysteine residues at positions 1 and 7. The sequence and synthesis of salmon ultimobranchial calcitonin were to follow shortly. 55 Copp had noted in making extracts of salmon ultimobranchial glands that the hormone was extremely potent. Indeed the pure or synthetic salmon hormone, at 3000 to 5000 MRC units/ mg, was 20 to 40 times more potent than calcitonins of mammalian origin. At the same time it was pointed out that medullary carcinoma of the thyroid was most likely a tumor of cells of ultimobranchial origin. This led to the extraction and purification of human calcitonin from medullary thyroid carcinoma tissue, 56 and subsequent synthesis of the human peptide. 57 Based on their amino acid sequence homologies, the different species (Fig. 4 - 3 ) have been classified into three groups as follows: 1. Artiodactyl, which includes porcine, bovine, and ovine, which differ by four amino acids. 2. Primate/rodent, which includes human and rat calcitonins, differing by two amino acids. 3. Teleost/avian, which includes salmon, eel, goldfish, and chicken, differing by four amino acids.

A. S t r u c t u r e / A c t i v i t y R e l a t i o n s h i p s The sequences of several mammalian and fish calcitonins are shown in Figure 4 - 3 . They are all 32-aminoacid peptides with a common 1 - 7 disulfide bridge and a proline amide at position 32, which is essential for

CHAPTER 4 Calcitonin 1 C G C C C C G

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activity. There are considerable differences in the sequences of the carboxy-terminal two thirds of these molecules. In several different biological assays, the order of potency of the calcitonins is teleost > artiodactyl > human, although it is important to note that the absolute efficacies vary greatly between CTRs of different species and of receptor isoforms within species. For example, hCT is 3- to 50-fold less potent than sCT at the human CTR (hCTR) 58-6~ and more than 1000-fold less potent at sheep brain membrane receptors. 61 Studies of substituted, deleted, and otherwise modified calcitonins have provided considerable information regarding structure/activity relationships of the calcitonin molecule. For example, it was deduced that the ring structure serves to protect and stabilize the molecule so that replacement of the disulfide bridge with an N - N bond in aminosuberic 1 - 7 eel calcitonin was found to provide increased biological stability and potency. 62 Despite this, linear analogs of sCT may retain full hypocalcemic activity and adenylate cyclase activation. 63 Circular dichroism studies indicate that sCT exhibits considerable secondary structure in the presence of lipid, consistent with the generation of an amphipathic oL-helix between residues 8 and 2 2 . 64,65 However, the less potent hCT has reduced propensity to form this secondary structure. Modifications of residues in the 8 - 2 2 sequence that alter the ability of sCT to form secondary structure have yielded conflicting results, in terms of the hypocalcemic potency of the analogs. 64-69 In some cases analogs with less secondary structure had correspondingly lower hypocalcemic activity, 65'67 whereas other analogs are fully active, 64'66'68 suggesting that conformational flexibility may also contribute to activity 67'69 and that factors other than secondary structure are also critical for ligand binding and receptor activation. Likely reasons for the conflicting results are first, the relative stability in vivo, compared with in vitro, of the various peptides; second, the use of different receptor types to assess the efficacy of the peptides; and third, a divergence between the relative potency of the peptides for

binding and activation of adenylate cyclase. These latter two points are illustrated in the following examples from our own work. The use of sCT analogs with varying capacities to form oL-helices revealed divergence in the responses of different receptors. 7~ This was most apparent for the stimulation of cyclic adenosine monophosphate (cAMP) production by the rat receptor isoforms C la and C l b (see Section VI.B). In cells expressing the C la receptor, helical analogs were equipotent with both sCT and analogs that have reduced or absent helical structure, consistent with their potencies in the rat hypocalcemic assay, and suggest that this latter function is mediated via interaction with C la receptors. In contrast, the nonhelical analogs were 100- to 1000-fold less potent than, for example, sCT at the C lb receptor. In addition, the general finding across receptors of several species was that reduction in the ability of sCT analogs to form helix structures had a greater impact on the potency of the analogs in competition for 125I-sCT binding than in cAMP accumulation. This disparity between the relative potencies of the peptides in studies of binding competition and cAMP accumulation was also seen in experiments to examine the basis of the high potency of sCT, relative to hCT, using chimeric sCT/hCT molecules. 7~These studies also highlighted the importance of the type of receptor used to study relative calcitonin peptide potencies. It was found that residues present in the carboxy-terminal half of sCT are more important for binding competition with the rat C la, rat C lb, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CTR.

III. BIOSYNTHESIS A. C a l c i t o n i n a n d Its P r e c u r s o r s The medullary carcinoma of the thyroid provided the source for studies of the amino acid sequence of hCT

100

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON The complete sequences of the cDNA for human, v4 rat, 75'76 chicken, 77 and sheep 78 calcitonins and the DNA sequence of the full human calcitonin gene, 79 have been determined. These show that the hormone is flanked in the precursor by N- and C-terminal peptides (Fig. 4 - 4 ) , but the biological significance of these peptides is unknown. The human calcitonin gene has been located in the p 14-qter region of chromosome 11.8~

and has allowed detailed analysis of the calcitonin gene in rat and humans. Thus calcitonin is synthesized as a large molecular-weight precursor (136 amino acids) with a leader sequence at the amino terminus, which is cleaved during transport of the molecule into the endoplasmic reticulum. The location of dibasic amino acid residues at either end of the (1-32) calcitonin sequence in the precursor molecule ensures cleavage of the prohormone to yield mature calcitonin (Fig. 4 - 4 ) . In addition to cleavage, however, it is essential, as discussed earlier, that the proline residue at position 32 is amidated. The residue immediately following the proline in the precursor is glycine, which has been suggested 71 to provide the amide group for the preceding amino acids. A potentially important posttranslational modification of calcitonin is that of glycosylation. 72 It had been noted that the tripeptide sequence, Asn-Leu-Ser, found within the amino-terminal ring structure of calcitonin (Fig. 4 3) is invariate among the calcitonins of different species. This sequence is an acceptor site for N-linked glycosylation. This, together with evidence for glycosylation of tumor calcitonin, led to detailed studies showing that the calcitonin precursor is indeed a glycoprotein 73 and that the only N-linked glycosylation site in the entire precursor was within the calcitonin portion itself. The biological significance of calcitonin glycosylation has yet to be determined.

B. C a l c i t o n i n G e n e - R e l a t e d P e p t i d e a n d A l t e r n a t i v e S p l i c i n g o f the P r i m a r y C a l c i t o n i n Gene mRNA Transcript It has been found that the calcitonin gene transcript actually encodes two distinct peptides, which arise by tissue-specific alternative splicing of the mRNA. The original observation was that serially transplanted rat medullary thyroid cancers changed from states of high to low or absent calcitonin production. 81 The low producers were found to produce different mRNA transcripts, which encoded a peptide termed "calcitonin gene-related peptide" (CGRP). The mature CGRP and calcitonin mRNAs predict proteins which share sequence identity in the amino terminal regions (Fig. 4 - 4 ) , but in the carboxy-terminal regions the nucleotide sequences

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CHAPTER 4 C

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are entirely different. The mature, secreted 32- and 37amino-acid CT and CGRP peptides, respectively, result from cleavage of both amino- and carboxy-terminal flanking sequences, at cleavage sites depicted in Figure 4 - 4 . 8z The pattem of expression of calcitonin and CGRP mRNA transcripts corresponds to the immunochemical analysis of the peptides. 83 Calcitonin mRNA is found almost exclusively in the thyroid and CGRP mRNA is found primarily in the nervous system. However, aberrant expression of CGRP may be seen in medullary thyroid carcinoma, as suggested above. Two different calcitonin/CGRP genes, oL and [3, have been identified in man and rat. 84 The calcitonin/CGRP gene was one of the first examples of a cellular gene exhibiting alternative, tissuespecific processing of its primary mRNA transcript and has served as an important paradigm to study the molecular mechanisms of RNA splicing. Processing of the pre-mRNA to the calcitonin mRNA transcript involves usage of exon 4 as a 3'-terminal exon with concomitant polyadenylation at the end of exon 4 (Fig. 4 - 4 ) . Processing to produce the CGRP mRNA involves the exclusion of exon 4 and direct ligation of exon 3 to exon 5, with polyadenylation at the end of exon 6. Much work has been done to illuminate the mechanisms by which this differential splicing is achieved, 85 although it remains to be fully elucidated. Briefly, however, the hCT/ CGRP exon 4 can be characterized as having weak processing signals, like many differential exons. Weak differential exons are frequently associated with special enhancer sequences that facilitate exon recognition in the presence of accessory factors that bind to the enhancer. 86 Indeed, such an enhancer, located in the intron downstream of exon 4, has been described for the calcitonin/ CGRP gene. In addition, sequences within exon 4 are necessary for the inclusion of exon 4. 85 Another hormone of the calcitonin family is amylin (Fig. 4 - 3 ) , a 37-amino-acid peptide produced by the pancreatic [3-cells which modulates glycogen synthesis and glucose uptake in skeletal muscle and can induce insulin resistance in this tissue in v i t r o . 87"88 Amylin has about 40% sequence homology with CGRP (Fig. 4 - 3 ) , and its gene has been localized to chromosome 12. Although amylin and CGRP have biological effects distinct from those of calcitonin, at high concentrations they may mediate calcitonin-like effects, such as inhibition of bone resorption. 89 A receptor distinct from calcitonin or CGRP receptors with high affinity for amylin has recently been identified. 39 However, it is important to note that sCT can also compete for binding with high affinity at amylin binding sites. 39 Since sCT has been the ligand primarily used for calcitonin binding and functional analysis of calcitonin action, it is possible that this has introduced a degree of ambiguity into interpretation of these studies.

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IV. SECRETION AND METABOLISM Calcitonin secretion from the C cells is clearly dependent on the prevailing serum calcium level. Any tendency toward lowering of the calcium level results in storage of calcitonin within the granules of the C cells; these stores are readily discharged as the serum calcium is elevated. Although there is little doubt that calcium is an important secretagogue for calcitonin in normal or tumor C cells, the exact mechanisms by which calcium provokes exocytosis of calcitonin have not been fully elucidated. It has recently been shown, however, that the same extracellular calcium-sensing receptor that is found in parathyroid cells, 9~ where its activation leads to decreased parathyroid hormone secretion, is also found in C cells and that its presence correlates with the extracellular calcium sensing function. 91 This calcium receptor is likely to represent the primary molecular entity through which C cells detect changes in extracellular calcium and control calcitonin release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types. Reduction in numbers of granules and vesicles occurred during induced calcitonin secretion in v i v o 17 and from thyroid s l i c e s , 92 but alteration of calcium levels did not influence the release of calcitonin from isolated pig thyroid granules, 93 consistent with the view that calcitonin release may be mediated at the cell membrane, and intracellular stores repleted from the storage granules as the intracellular concentration falls. Agents that elevate C cell cAMP may stimulate calcitonin secretion, since cAMP analogs have been shown to have this effect in v i v o 94 and in vitro. 95 Probably the most important calcitonin secretagogues apart from calcium, however, are the gastrointestinal hormones. In the pig, gastrin appears to be an effective physiological secretagogue, 96 providing part of the evidence that has led to a view of calcitonin's physiological role as a hormone important postprandially, capable of counteracting the effect of a calcium meal and of parathyroid hormone action by preventing the efflux of calcium from bone into blood. 97 Although there is some evidence in favor of this role in the pig and r a t , 97 it is difficult to envisage it as being important in adult humans. However, this awaits further study. Other gastrointestinal hormones, including glucagon, cholecystokinin, and secretin, are also capable of promoting calcitonin secretion. 97 The gastrin analog pentagastrin has been used clinically as a provocative test for calcitonin secretion in patients with medullary carcinoma of the thyroid. Other hormones that influence calcium homeostasis may also directly or indirectly influence calcitonin secretion. 1,25-dihydroxyvitamin D3 [1,25(OH)zD3] adminis-

102

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

tration has been reported to increase plasma calcitonin levels; this was suggested to occur via specific thyroid C cell receptors for 1,25(OH)2D3, which modify secretion of calcitonin. 98 Both calcitonin and 1,25(OH)2D3 levels are raised in pregnancy and lactation, 99 and it has been suggested that calcitonin may act to protect the skeleton in the face of increased calcium demand by the fetus. Most of this information on the regulation of calcitonin secretion comes from observations made during acute experiments with assays of calcitonin released into plasma or culture medium. The development of cDNA and oligonucleotide probes has allowed studies of the factors influencing calcitonin mRNA production. The serum and thyroid concentrations of calcitonin increase markedly with age in the rat, and this is associated with substantial increases in thyroid content of calcitonin mRNA. TM The mechanisms of this are undetermined. In the same study, TM increasing calcium concentrations did not alter hybridizable calcitonin mRNA in response to calcium. T M Thus, in normal rats subjected to acute calcium stimulation in vivo, thyroid calcitonin mRNA was increased as measured in hybridization experiments and by translatable preprocalcitonin. TM Furthermore, in a medullary thyroid carcinoma cell line, phorbol esters selectively increased calcitonin transcription while inhibiting cellular proliferation. 1~176 On current evidence it seems that calcium can stimulate both synthesis and secretion of calcitonin by thyroid C cells. Delineation of the molecular mechanisms by which translatable calcitonin is increased by calcium will be of considerable interest. Calcitonin is degraded by liver and kidney to inactive fragments, the half-life of the peptide in blood being only a few minutes. Teleost calcitonins are considerably

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more resistant to breakdown by tissue and serum enzymes than are the mammalian calcitonins. Injected salmon calcitonin, for example, has a much longer half life than either pig or human calcitonin. 1~ Although this might contribute to the greater biological potency in vivo of sCT, the more important factor is the greater affinity of sCT for receptors. Using the most specific and sensitive radioimmunoassays, the level of calcitonin in human blood appears to be less than 10 pg/ml in normal subjects.

V. ACTIONS OF CALCITONIN A. Bone Resorption Addition of calcitonin to resorbing bone in vitro inhibited bone resorption, 1~176 an effect that appeared to be explained by a direct action on osteoclasts, inhibiting their production and activity. Calcitonin treatment of resorbing bone in vitro resulted in rapid loss of osteoclast ruffled borders and decreased release of lysosomal enzymes. In vivo evidence was also consistent with an inhibitory action upon bone resorption. Thus, calcitonin infused into rats resulted in an immediate reduction in the rate of excretion of hydroxyproline, consistent with the action of the hormone inhibiting the breakdown of bone collagen. 1~ Furthermore, kinetic studies in rats led to similar conclusions, with no evidence to suggest any increase in the active uptake of calcium by bone. 1~176 For example, when calcitonin was infused into rats that had been injected 12 hours previously with 45Ca, hormone treatment lowered plasma calcium without affecting plasma 45Ca levels (Fig. 4 - 5 ) . Under these experi-

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mental conditions, disappearance of radioactivity from the plasma reflected uptake of 45Ca by the skeleton. The failure of calcitonin to influence this reflects an action of the hormone to prevent calcium efflux from bone and is not consistent with active stimulation of calcium uptake by bone. Studies of the actions of hormones on isolated bone cell populations have established that calcitonin acts directly on osteoclasts. Autoradiographic experiments using biologically active iodinated sCT as receptor ligand have pointed to osteoclasts as the only discernible bone cell targets. ~~ Consistent with this are the observations of its actions in organ culture, especially the demonstration that calcitonin-treated osteoclasts in cultured mouse calvaria rapidly lose their ruffled borders. TM A similar in vivo observation of loss of ruffled border in osteoclasts has been made in patients with Paget's disease, in w h o m bone biopsies were taken before and 30 minutes after an injection of calcitonin. 1'2 In the same clinical study, calcitonin was noted to decrease the number of osteoclasts in addition to altering their ultrastructure. Studies using isolated osteoclast preparations 1~3'114 point to a direct effect of calcitonin upon the osteoclast, in which the hormone rapidly inhibits the activity of osteoclasts. In further experiments 115 it was also noted that, while isolated osteoclasts remained quiescent in calcitonin as long as the hormone was present, they regained activity when osteoblasts were added to the culture. This escape of osteoclasts from inhibition by calcitonin took place at a rate proportional to the number of osteoblasts with which they were in contact. In other studies, Chambers showed that calcitonin reduced the cytoplasmic spreading of isolated osteoclasts in a dose-dependent manner. 115 Parathyroid hormone had no effect unless osteoblasts were co-cultivated with the osteoclasts, in which case addition of parathyroid hormone resulted in a marked increase in cytoplasmic spreading of osteoclasts. It cannot be assumed that these phenomena reflect the responses of cells in bone, but this work provided for the first time some useful direct observations of actions of hormones on isolated bone cell preparations containing osteoclasts. These observations are consistent with the view that the osteoblasts (or " l i n i n g " cells) might mediate the actions of bone-resorbing hormones by producing factors that stimulate the osteoclast 1~6 and also with the view that calcitonin acts directly upon the osteoclast. The molecular mechanisms by which calcitonin decreases osteoclast function have yet to be fully defined. The rapid effects of the hormone may be brought about through actions on a cytoskeletal function of osteoclasts, after initial events involving generation of several intracellular second messengers. Early events in calcitonin signal transduction have been studied in a variety of cell

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types and are described later (see Section VI.C). With the development of improved methods of studying isolated osteoclasts, it has been possible to establish clearly that mammalian osteoclasts possess abundant, specific, high-affinity receptors for calcitonin and that calcitonin stimulates cAMP formation in a sensitive and dose-dependent manner 117 as well as increases in intracellular Ca 2+ levels. 115 The other means by which calcitonin could inhibit resorption is through inhibition of osteoclast formation. In vivo data and results from calcitonin inhibition of resorption in organ culture are consistent with this. The development of methods of studying osteoclast formation in vitro from hemopoietic precursor cells 118 has allowed this question to be addressed directly. There were several reports of calcitonin inhibiting osteoclast-like cell formation in bone marrow cultures of human, 119 baboon, 12~ and mouse 118 origin. However, these experiments were all conducted at relatively high calcitonin concentrations. In recent studies using lower concentrations of calcitonin, which nevertheless reduced calcitonin receptor m R N A expression in developing mouse osteoclasts 12~ (Fig. 4 - 6 ) , there was no reduction in osteoclast formation. The multinucleated osteoclasts formed under calcitonin treatment, however, had fewer nuclei, and, notably in this and in another study, 122 evi-

FIGURE 4--6 Effectof continuous treatment of mouse bone marrow cultures with CT. Cultures were maintained for 8 days in the presence of 1,25(OH)2D3 with (+) or without (-) 10-1~ sCT. Reverse transcription/PCR of mRNA extracted at the times indicated, was amplified using primers specific for the mouse CTR sequence or murine GAPDH. In the presence of CT, osteoclast-like cells developed, which were deficient in CTR and CTR mRNA. (Data from the authors' group.)

104 dence was obtained for the generation of osteoclasts which are deficient in calcitonin receptor mRNA and protein, but nevertheless capable of resorbing bone. This observation may be relevant to the mechanism of "escape" from calcitonin action, which is considered in Section VI.D of this chapter. It should be stressed that the failure of calcitonin to inhibit osteoclast formation in such osteoclast-generating cell culture systems, except perhaps at very high calcitonin concentrations, does not exclude the possibility that inhibition of osteoclastogenesis contributes to calcitonin action in vivo. The emergence in vitro of osteoclasts deficient in calcitonin receptors complicates such experiments. Although it is interesting to consider that such a phenomenon might take place also in vivo, and even contribute to calcitonin resistance, it seems unlikely to be a consistent and major feature of in vivo responses. Although there has been no demonstration of calcitonin receptors in osteoblast-like cells, or of a direct biochemical effect of the hormone upon such cells, the administration of calcitonin has been observed to produce rapid changes in osteocytes, in which osteocyte shrinkage took place and was followed by the formation of hydroxyapatite crystals in the perilacunae and pericanaliculae. 123 The rapid changes in the bone lining cells produced by calcitonin were enhanced by phosphate, TM and could be related to the possible involvement of phosphate ions in the action of calcitonin, which has been argued by Talmage. 97 Most important, however, the proposal that calcitonin acts directly upon lining cells to reduce calcium efflux from bone (in direct opposition to the action of parathyroid hormone) implies that these cells should possess receptors for calcitonin, but there is no direct evidence for this. Parathyroid hormone and the other major bone-resorbing hormones have been shown to increase plasminogen activator production by osteoblast-like cells, both normal and malignant, 125 raising the possibility of a neutral protease derived from osteoblasts contributing to the process of matrix degradation, either directly or indirectly. This increase is not influenced by calcitonin. At present there is no convincing evidence for the existence of calcitonin receptors in cells of the osteoblast lineage, but the possibility cannot be excluded that a calcitonin-responsive subpopulation exists. It is of interest to note that calcitonin receptors and cAMP response have been found to occur in late passage cultures of a parathyroid hormone-responsive osteogenic sarcoma cell line that is phenotypically osteoblast. Subclones have been developed that respond to both parathyroid hormone and calcitonin. 126 Although it is possible that such cells reflect the existence within the osteoblast series of cells capable of a calcitonin response, it is also likely that this is a property of these malignant cells, with no relevance to normal osteoblasts.

T.J. MARTIN,D. M. FINDLAY,J. M. MosEI.F~u AND P. M. SEXTON B. B o n e F o r m a t i o n Although there is no good evidence for a stimulatory effect of calcitonin upon bone formation, some early evidence was obtained for a stimulatory effect on osteoblasts. In rats treated chronically with calcitonin, an increase in the number of osteoblasts was observed in the bones. 127 Furthermore, calcitonin treatment in vitro of cultures of mouse radius rudiments led to an increased net amount of bone tissue that was associated with an increased number of osteoblasts, 128 leading to the suggestion that calcitonin might have a stimulatory effect on bone formation in addition to its inhibition of resorption. It is difficult to explain such observations in the light of current views of the coupling of bone resorption to formation. It is considered that any change in bone resorption is rapidly followed by a change in formation rate in the same direction. Thus, inhibition of bone resorption by calcitonin would be expected to be accompanied by inhibition of bone formation. Indeed, this is the experience, for example, in the use of calcitonin in the treatment of Paget's disease. In in vivo experiments, no effect of calcitonin was detected on the incorporation of labeled proline into bone hydroxyproline in rats chronically treated with calcitonin. 129 To the present time, therefore, it cannot be concluded that calcitonin has an anabolic effect on bone, and indeed it seems more likely that it would be "antianabolic." In normal humans, calcitonin was shown to inhibit the excretion of hydroxyproline-containing peptides, consistent with its effect of inhibiting bone resorption, but treatment also inhibited the excretion of nondialyzable hydroxyproline, which reflects bone collagen synthesis. 13~ This can be interpreted as an inhibitory effect of calcitonin upon bone collagen synthesis, accompanying the decrease in breakdown in bone. It is interesting to note that in those acute experiments in humans, ~3~calcitonin decreased urinary hydroxyproline excretion acutely after injection, but several hours later the rates of excretion returned to pretreatment levels. With continued treatment, however, there was a gradual fall in hydroxyproline excretion, such as is seen in Paget's disease patients treated chronically with calcitonin. This is interpreted as a dual action of calcitonin, on the one hand acutely inhibiting the function of osteoclasts, and on the other a chronic effect of inhibiting the generation of new osteoclasts. Calcitonin treatment of rats led to increased matrixinduced bone formation TM which appeared to be associated with a stimulated proliferation of cartilage and bone precursor cells. The effect was seen provided the calcitonin treatment was begun before cartilage formation. After that time no effect was observed. It is not clear that this can be regarded as evidence for an ana-

CHAPTER 4

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bolic effect of calcitonin upon bone formation. The conclusion at present must be that there is no direct effect of calcitonin upon bone cells resulting in increased anabolic function. Rather, it seems likely that the reverse is the case, as an indirect consequence of inhibition of bone resorption by calcitonin.

C. Calcitonin, Bone, and Calcium Homeostasis It is worth considering how the discovery of calcitonin and its mechanism of action have influenced modern views of the regulation of the extracellular fluid calcium, and the contribution to this of bone. Older concepts of calcium homeostasis that considered only parathyroid hormone and bone were questioned with the suggestion that if bone were the only means of regulating the serum calcium level in conjunction with parathyroid hormone, control would be inadequate. Hence the suggestion that the parathyroid hormone action on the kidney might contribute. The arrival of a new calcium-lowering hormone seemed to solve the problem. However, events proved otherwise. Concepts of the role of bone in maintaining extracellular fluid calcium had relied upon observations made in the young, growing rat. Hence it was calculated that for the tibia in a young rat there was an accretion rate of 6.2% of the bone calcium per day, a resorption rate of 4.7% of the bone calcium per day, and an exchangeable fraction equivalent to 3.0% of the total calcium in the bone. 132 Thus it was clear that if accretion continued at the same rate and resorption was inhibited, the result would be a lowering of plasma calcium. The younger the animal, the more rapid the bone resorption rate. It would therefore be expected that the calciumlowering effect of calcitonin should be greater in younger than in older animals. This was indeed the case in the r a t 133 (Fig. 4 - 7 ) , in which it was noted that in the biological assay of calcitonin, which depends on the calcium-lowering effect of the hormone, the response became less marked with increasing age of the animals. It should be noted, however, that the ability of calcitonin to counteract the effect of a calcium load was not impaired in older animals, at least in the r a t , TM a n observation that has not been explained and that has not been extended to other species. Dependence of the hypocalcemic action of calcitonin upon the prevailing rate of bone resorption was also noted in other species, and it soon became clear that in normal adult humans even quite large doses of calcitonin had little effect on plasma calcium levels. In those subjects in whom bone turnover was increased (e.g., in thyrotoxicosis, Paget's disease), calcitonin treatment acutely inhibited bone resorption and resulted in a lowering of the plasma calcium. ~35

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It has been proposed that the hypocalcemic action of calcitonin is related to the availability of circulating inorganic phosphate. 9v In r a t s , 136 and in patients with chronic renal failure, 137 the degree of calcium lowering in response to calcitonin was found to be proportional to the initial blood phosphorus concentration. It has been claimed that no hypocalcemia followed calcitonin injection in rats maintained for several weeks on a phosphorus-deficient diet. 97 In contrast, Robinson et al. showed that in parathyroidectomized rats fed a low-phosphorus, high-calcium diet for 17 days, calcitonin lowered plasma calcium by a mechanism consistent with inhibition of bone resorption. 1~ It seems that the acute effect of calcitonin on serum calcium is related to the prevailing rate of bone resorption. If that is accepted, lack of a calcium-lowering effect of the hormone in the mature animal or human is not surprising, since the process of bone resorption is a slow one in maturity. It may be that the role of calcitonin in its effect on bone throughout life is that of a regulator of the bone resorptive process, whatever the overall rate of the latter. In the young, or in pathological states of increased bone resorption in maturity (e.g., Paget's disease, thyrotoxicosis), calcitonin inhibition of bone resorption can lower the serum calcium level, and there

106

T.J. MARTIN, D. M.

may even be a calcium homeostatic role for endogenous calcitonin in those circumstances. In a normal adult animal, however, when bone turnover is slow, no effect on serum calcium is obtained with calcitonin. The physiological function of calcitonin in maturity may nevertheless be to regulate the bone resorptive process, in either a continuous or intermittent manner. It follows that calcitonin should not necessarily be regarded as a "calcium-regulating hormone" in maturity, but may yet be shown to be such in stages of rapid growth (e.g., in the young or in states of increased bone turnover). It is nevertheless important that bone resorption be regulated, and calcitonin is the only hormone known to be capable of carrying out this function by a direct action on bone. Such a role might become more important in circumstances in which skeletal loss particularly needs to be prevented (e.g., in pregnancy and lactation). 138 Evidence in support of such an important physiological role for endogenous calcitonin in protecting against bone loss is provided by the experiments of Yamomoto e t al., 139 who showed that cancellous bone loss in thyroparathyroidectomized rats treated with parathyroid hormone was greater than that in similarly treated shamoperated controls.

D. Renal Effects of Calcitonin Although calcitonin lowered plasma calcium in the absence of the kidneys, it was noted that in parathyroidectomized rats with very low levels of plasma calcium, calcitonin had no effect on calcium but lowered phosphorus. 137 When this phosphate-lowering effect was found to be prevented by nephrectomy 1~ it was considered that in some circumstances the kidneys might be involved in the phosphate-lowering effect of calcitonin. Indeed, a phosphaturic effect of thyroid extract had been shown in intact r a t s , 14~ and infusion of calcitonin in parathyroidectomized rats led to a dose-dependent phosphat u r i a . 141 The effect on phosphate excretion was only a minor one in comparison with the phosphaturic effect of parathyroid hormone, and although it was demonstrated in human subjects also, 135 in several species calcitonin failed to have any effect on phosphate excretion. Thus, it has seemed unlikely that the phosphaturic effect is of any major physiological significance. The hormone was also noted to promote excretion of inorganic sulfate in r a t s 142 and humans, ~45 probably reflecting a shared renal tubular transport system between sulfate and phosphate. A number of other renal effects of calcitonin have been noted, including a transient increase in calcium excretion,135,143-145 due probably to inhibition of renal tubular calcium reabsorption. Although this has not usually been regarded as an important effect of calcitonin,

FINDLAY,

J. M.

MOSELEY, AND

P. M. SEXTON

recent observations link it to the calcium-lowering effect of calcitonin in patients with metastatic bone disease. The use of calcitonin in the treatment of hypercalcemia due to cancer has been based exclusively on the inhibition of osteolysis by calcitonin. Some evidence has been produced that failure of the kidneys to excrete the calcium load derived from bone breakdown is a major contributor to the hypercalcemia. 146 This prompted careful studies of the relative contributions to the hypocalcemic effect of calcitonin of its renal and skeletal components. 147 It was concluded that inhibition of renal tubular reabsorption by calcitonin can induce a rapid fall in serum calcium, and that the magnitude of this effect depends upon the correction of volume depletion, which inevitably accompanies hypercalcemia. Thus, the calciuretic action of calcitonin may assume greater importance than hitherto suspected. Calcitonin was found to produce a natriuretic effect in human subjects, 135''48 and study of the renal effects of calcitonin in rats pointed to striking increases in sodium excretion. 149 The effects were more marked with sCT than with calcitonins of mammalian origin, 149 raising the possibility that the natriuretic property of calcitonin from lower vertebrates might be of functional significance in those species, or that these effects were mediated by C3 receptors, which display high affinity for both sCT and amylin (see Section VI.B). Calcitonin receptors have been demonstrated clearly in rat kidney, 15~ and a further action on the kidney is to enhance 1-hydroxylation of 25-hydroxyvitamin D in the proximal straight tubule of the kidney. 15! Since autoradiographic studies ~52 and polymerase chain reaction (PCR) 152 analysis of calcitonin receptor mRNA expression have failed to localize calcitonin receptors in the proximal tubule, it seems unlikely that these actions are mediated by direct actions on calcitonin receptors. The action of calcitonin upon adenylate cyclase activity has been localized in the human nephron predominantly to the medullary and cortical portions of the thick ascending limb and to the early portion of the distal convoluted tubule. 154 The co-localization of the calcitonin receptor mRNA expression (Fig. 4 - 8 ) and cell surface receptors 152 with G-protein-sensitive adenylate cyclase is consistent with cAMP being an important mediator of calcitonin action in this organ (Fig. 4 - 8 ) . A further renal effect of calcitonin was noted as a result of studies of calcitonin effects upon a pig kidney cell line. 155 The hormone was found to stimulate greatly the production of the neutral protease, plasminogen activator. 156 This effect appeared to be related to calcitonin's actions on cAMP formation in the cells, and hormone treatment also was associated with marked inhibition of cell replication. In the same cells, calcitonin treatment enhanced the transcription of plasminogen ac-

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activation of C3 receptors, which have high affinity for amylin and sCT, but not hCT or rCT. These include inhibition of gastric acid secretion, inhibition of pancreatic enzyme secretion, and impairment of glucose tolerance.

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FIGURE 4--8 Distributionof Cla-receptor mRNAs along rat nephron. Each point represents mean value obtained from one animal (a total of 5 to 13 samples were analyzed for each structure). Dashed line indicates detection threshold of assay (50 molecules sample). (Redrawn from Firsov D, Bellanger A-C, Marsy S, et al: Quantitative RT-PCR analysis of calcitonin receptor mRNAs in the rat nephron. Am J Physiol 38:F702-F709, 1995.)

tivator mRNA, the data indicating that calcitonin was able to activate the plasminogen activator gene in those cells. 157 Subsequent studies in humans showed that calcitonin treatment resulted in increased urinary plasminogen activator activity. 158 The plasminogen activator/ plasmin system is an important local regulator of many functions, the nature of which depends on the local tissue environment. Its involvement in the local actions of calcitonin is an intriguing possibility that merits further study. The tissue effects of plasminogen activator depend on its location--thus, they are likely to be very different between kidney and bone. It is worth noting that the bone-resorbing hormones increased plasminogen activator production in osteoblast-like cells, 125 and that this effect was not influenced by calcitonin.

E. Calcitonin and the Gastrointestinal Tract It has been suggested that the function of calcitonin is to prevent rises in plasma calcium taking place after ingestion of calcium-containing m e a l s . 97'159 This is based on the observation made in pigs that calcium meals do not alter the plasma calcium, but that during the feeding period there is a rise in plasma calcitonin levels. This is a possible role for the hormone in humans, particularly in stages of growth, in which intermittent secretion in response to oral calcium loads could inhibit bone resorption and decrease the movement of calcium from bone to blood at a time when calcium was being absorbed from the intestine. However, much more study is needed in humans to define the relationship of calcitonin secretion to feeding and to gastrointestinal hormones. Other gastrointestinal effects of calcitonin are probably not physiological and, as discussed above, may relate to

F. Calcitonin in the Central Nervous System The origin of calcitonin-producing cells in the neural crest raised the possibility of calcitonin involvement in neural function. Both immunoreactive calcitonin and calcitonin receptors have been demonstrated in the brain and nervous system of rats, humans, and other species. 21'23'33 Immunoreactive calcitonin related antigenically to human calcitonin has been demonstrated in the nervous systems of protochordates, lizards, and pigeons. 21 Recent data, again obtained with radioimmunoassay, point to the presence of low levels of sCT-like peptide material in the human thyroid and in the paraventricular mesencephalic region of the brain. 16~ It has been suggested that this might be a vestige of a highly conserved gene for calcitonin. 16~ In addition to the well-characterized calcitonin receptors of bone and kidney, calcitonin receptors are also abundant in the central nervous system (CNS), 23'161-163 where central injection of calcitonin induces potent effects that include analgesia and inhibition of appetite and gastric acid secretion (reviewed in Sexton15~ Autoradiographic mapping of rat CT-binding sites revealed high densities associated with parts of the ventral striatum and amygdala, the hypothalamic and preoptic areas, as well as most of the circumventricular organs. High-density binding also occurs in parts of the periaqueductal gray, the reticular formation, most of the midline raphe nuclei, parabrachial nuclei, locus coeruleus, and nucleus of the solitary tract. 164-169 The central actions of calcitonin correlate well with the location of calcitonin-binding sites. The periaqueductal gray is important in central regulation of pain, and calcitonin binding within this region is likely to be involved in calcitonin-induced analgesia, 165 ' 170 ' 171 while the hypothalamic binding parallels the multiple hypothalamic actions of calcitonin, which include modulation of hormone r e l e a s e , 172'173 as well as decreased a p p e t i t e , 174-176 gastric acid secretion, 177'178 and intestinal motility. 179 Recently, and as also discussed in Section VI.B, cloning studies demonstrated that calcitonin receptors in rat brain are heterogeneous and exist as two distinct isof O r l T I S . 37'180 Calcitonin-specific binding sites in brain have been termed C1 sites 15~ and the two receptor isoforms are termed C la and C lb receptors. The two receptors are identical, except that the C lb sequence encodes a 37-amino-acid insert in the second extracellular domain, which confers altered ligand recognition. Both receptors

108

demonstrate high apparent affinity for sCT, but differ greatly in their affinity for hCT. C la receptors bind hCT with an apparent affinity two to three orders of magnitude less potent than sCT, whereas C lb receptors have negligible affinity for hCT. 37'38'18~PCR-based studies on the distribution of calcitonin receptor mRNA confirmed that message for both receptor isoforms is present in rat brain. 37,18~ Studies with helical and nonhelical analogs of sCT had previously suggested the existence of two potential subtypes of calcitonin-binding sites in rat brain membranes. 66 A calcitonin-linear (CT-L) type interacted with nonhelical sCT analogs with high affinity, and a calcitonin-helical (CT-H) type interacted with these analogs with a low affinity. While both types of receptors bind the helical sCT with high affinity, only the CT-L receptor binds with high affinity to hCT, which also has a reduced helical structure. The partial competition of 125I-sCT binding by hCT in the rat forebrain lsz supported the existence of multiple binding sites. These receptors are analogous to the C l a and C lb calcitonin receptor isoforms, with the specificity of interaction of the cloned receptors with helical and nonhelical sCT analogs equivalent to the CT-L and CT-H receptors, respectively. 7~ Receptor localization studies have predominantly used the fish-type calcitonins to identify receptors. 164-168'183Salmon CT, however, does not differentiate between rat C 1a and C lb receptors. Moreover, sCT also binds with high affinity to C3-amylin receptors, 39'4~ complicating interpretation of the binding distribution and its potential physiological significance. By utilizing the differences in ligand-specificity of the Cla, Clb, and C3-amylin receptors, Hilton and colleagues 51 demonstrated that distribution of C la and C lb receptors in brain was predominantly parallel. Brain regions containing C la but little or no C lb binding sites were restricted to the nucleus of the solitary tract, area postrema, and the intermediate lobe of the pituitary. In contrast, no nuclei containing exclusively C lb receptors were found, although parts of the mesencephalic and pontine reticular formation and the thalamic paraventricular nucleus were enriched in C lb receptors, relative to the density of C 1a receptors in other brain regions. The C3-amylin receptors are almost ubiquitously expressed in regions also expressing the C la receptor isoform but occur in only a subset of the nuclei labeled also with the C la-specific ligand 125IhCT. 4~ However, in restricted parts of the accumbens nucleus and fundus striati, the binding sites appear to be predominantly of the C3-receptor phenotype. 51 Only very limited structure/activity studies have been carried out in regard to the central actions of calcitonin. Indeed, in many experiments, sCT has been used to characterize either receptor localization or pharmacological action of calcitonin in the CNS. Consequently, in many

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

cases it is unclear whether the reported actions of calcitonin are mediated through the classic C 1-type calcitonin receptors or via C3-type amylin receptors of equivalent distribution. For example, although the analgesic action of calcitonin can be delineated as acting independently of C3-amylin receptors based both on receptor distribution studies and lack of an amylin-induced analgesic action, n~ the anorexic action of calcitonin may be primarily due to interaction with the C3 receptors. In support of the later assertion, the actions of amylin and calcitonin are paralleled (both are nonaversive agents) 184'185 and the mammalian calcitonins are relatively weaker at inhibiting appetite than lowering plasma calcium (a Cla-mediated action), 186'187 consistent with the specificity of C3-amylin receptors. 39'4~ Clearly, further studies need to be done to resolve the roles that the different receptors play in the central actions of calcitonin and related peptides. CGRP has been identified by immunocytochemistry in the central and peripheral nervous systems 188'189 and its receptors have been mapped in the brain, w~ It is uncertain whether CGRP has any significant effect on calcium metabolism, but its wide distribution and potent biological effects could indicate an important local function in several organ systems.

VI. CALCITONIN

RECEPTOR

Because preparation of osteoclasts suitable for biochemical study has only recently been possible, information on calcitonin receptor interactions was initially obtained from studies in other cell types. In mammals, calcitonin receptors were identified by direct binding studies in many cell types (e.g., in rat kidney, 192 human placenta, 193 human 23 and r a t 26'162'163 brain, human lymphoid cells, 194 cultured pig kidney cells, 156 human cancer cell lines derived from lung 195 and breast, 196 and a subclone from rat osteogenic sarcoma cells126). Calcitonin receptors have also been demonstrated in trout gill. w7

A. R e c e p t o r C l o n i n g Understanding of calcitonin receptor biology has undergone a dramatic change with the recent cDNA cloning of the calcitonin receptor of pig, 198 human, 58 rat, 37'18~ mouse, 199 and rabbit 2~176 origin. Based on amino acid homology, these receptors belong to a sub-family of the very large 7-transmembrane domain, G-protein-linked receptor class (Fig. 4 - 9 ) . This subfamily includes receptors for parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP), 2~ secretin, 2~ growth hormone releasing hormoneY 3 vasoactive intestinal

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polypeptide, TM glucagon-like peptide-I, 2~ pituitary adenylate cyclase activating peptide, 2~ gastric inhibitory polypeptide, 2~ and corticotropin releasing factor. 2~176 Nucleotide sequence analysis of the cloned calcitonin receptors indicated open reading frames that translate to polypeptides of around 500 amino acids, depending on the pattern of mRNA splicing. 37'58'198 Hydropathy plots indicate the presence of 7-transmembrane domains and a signal peptide at the far N-terminus. 37 There are four potential N-linked glycosylation sites in the hCTR and rCTR sequences, three of which are conserved in the porcine (p)CTR receptor. The presence of glycosylation was suggested by previous biochemical studies which indicated that the hCTR is associated with glycosyl moieties, the major contributors of which are N-acetyl-Dglucosamine residues. 21~ Posttranslational modification of the calcitonin receptor was confirmed by comparison of the predicted receptor molecular masses of--~50 kDa with those estimated from cross-linking 21~ or western blot 212 analysis, which suggest molecular masses of ---80 kDa.

B. R e c e p t o r I s o f o r m s Receptor cloning led to the discovery of calcitonin receptor isoforms (Fig. 4 - 9 ) , which arise from alternative splicing of the primary mRNA transcript and result in receptor heterogeneity. In the case of the rCTR, two isoforms were identified by cDNA cloning from a hypothalamic library. 37 These two forms, termed C la and C lb, differ structurally in that C lb contains a 37-aminoacid sequence in the second extracellular loop, which is not present in the C la form. The nomenclature here relates to that of Sexton et al., 181 who reported binding sites in rat brain, designated C1 (which bind calcitonin with high affinity), C2 (corresponding to high-affinity CGRP sites), and C3 (which interact with both peptides and which were later characterized as being high-affinity

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amylin receptors). 39 Functional studies of these isoforms have revealed interesting differences between C la and C lb. These isoforms displayed different ligand recognition, with the C lb form essentially not recognizing hCT o r r f T . 37'38'70 The kinetics of ligand binding were also very different; sCT binds to C la receptors essentially irreversibly, while binding to C lb receptors is rapidly and completely reversible. 38 Salmon CT activates intracellular signal transduction similarly via either receptor type (see below). Although the cloning data provided the first direct evidence for heterogeneity of the rCTR, there were some earlier data indicating that this might be the case. In studying calcitonin-binding sites in the brain, Nakamuta et a l . 66 found these to be heterogeneous with respect to recognition of analogs of calcitonin with different propensity for helix formation in solution. As discussed above (see Sections II.A and V.F), cells stably transfected with C la receptors have been used to show that helical calcitonin analogs are equipotent with analogs that have reduced or absent helical structure. In contrast, at C lb receptors, the nonhelical analogs were several orders of magnitude less potent than sCT.70 Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of rat tissues and cells, the distribution of mRNA encoding C l a and C l b receptor isoforms was determined. 37'18~ Both C l a and C l b mRNA are abundant in the hypothalamus, nucleus accumbens, cerebral cortex, and brainstem. The predominant mRNA species outside the central nervous system (e.g., in the kidney) is Cla. UMR106-06 osteogenic sarcoma cells express essentially C 1a receptor mRNA only. Osteoclasts express predominantly C l a receptor, 2~3 whereas no calcitonin receptor could be detected by RTPCR in rat calvarial osteoblasts. In general, these studies showed an unexpectedly wide tissue distribution of calcitonin receptor mRNA, and this was the case also with studies in human tissues. 214'215 The functional signifi-

FIGURE 4--9 Schematiclinear diagram illustrating known splice variations within the CTR. At least four different insertions or deletions into the coding region of the receptors have been described. (1) a 71-bp insertion into the 5' end of the receptor, which provides an upstream, in-frame, potential translation start site,2~8(2) a deletion of 125-bp located in the N-terminus of the receptor coding region, which is predicted to generate an N-terminal deletion of 47 amino acids in the mature protein,TM (3) a 48-bp insertion into the predicted first intracellular domain of the r e c e p t o r , 58'214'216 and (4) a lll-bp insertion into the predicted second extracellular domain of the r e c e p t o r . 37 e, extracellular domain; i, intracellular domain; TM, transmembrane domain.

110 cance of the calcitonin receptor in most tissues remains to be explored. It is likely that the structural variations of the rCTR species are the result of alternative splicing of the primary RNA transcript, although no classic splice consensus sequences have been found in the nucleotide sequences surrounding the insert sequence in C lb. However, Southern blot analysis results were consistent with a single rCTR gene. 37 Although the rCTR gene has not been isolated, the insert sequence of C lb does occur at an equivalent position to the junction of exons 8 and 9 in the pCTR gene. 216 The hCTR gene is located on chromosome band 7q21.2-q21.3 217 or band q22. 2~8 The mouse (m)CTR gene has been localized to the proximal region of chromosome 6,199 which is homologous to the 7q region of the human chromosome 7. The structural heterogeneity within the rCTR is also present in the mCTR, which possesses virtually the same insert in the second extracellular loop. 199 T h e pCTR gene was mapped to chromosome band 9ql 1-q12. 216 In the case of the hCTR, there is no compelling evidence for a receptor isoform corresponding to the rat and mouse C lb form, although its presence in some tissues was suggested by a minor PCR fragment in one study. 214 However, several forms of the hCTR have been identified (Fig. 4 - 9 ) . The human isoforms expressed most abundantly are, first, the form originally cloned an ovarian cancer cell line, possessing a 16-amino-acid insert in the intracellular domain58; and second, an isoform cloned from the T47D breast cancer cell line, in which the insert sequence is absent. 214 The insert-negative receptor appears to be much more abundant in most tissues than the insert-positive receptor; however, transcripts encoding the latter are well represented in ovary and placenta, 214 suggesting tissue-specific regulation of this receptor isoform. Our own studies, 219 and those by other groups, 217'218'22~have now investigated the functional significance of the receptor isoforms. We found that the binding affinity and kinetics were identical for the two forms. In contrast, the presence of the insert greatly reduced the ability of the receptor to couple to adenylate cyclase, with EDs0 values 100-fold higher than those of the insert-negative form. Calcitonin-induced stimulation of a transient intracellular Ca 2+ response, via activation of phospholipase C, was abolished for the insert-positive receptor isoform. 219 The rate of ligand-induced internalization of the insert-positive form of the receptor was significantly impaired relative to the insert-negative form, suggesting that this process may be dependent on intracellular signaling. 219 Unlike the pig calcitonin receptor gene, where the 16-amino-acid insert sequence was contiguous with exon 8, 216 nucleotide sequence encoding the 16-amino-acid insert in the hCTR has been located in a separate exon of the hCTR gene. 219'22~ An additional

T.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

hCTR isoform has been described in which the first 47 amino acids of the amino-terminal extracellular domain are truncated. 222 This transcript represents a minority mRNA species in many of the tissues expressing the fulllength calcitonin receptor mRNA. Comparison of the truncated and full-length molecules by transfection revealed similar binding constants as well as calcitoninmediated cAMP production. 222 Sequencing of cDNA clones from a giant cell tumor revealed the presence or absence of a 71-bp insert in the 5'-untranslated region of the mRNA. 218 The functional significance of this sequence remains unclear. In addition to intraspecies variants of the calcitonin receptor, there are primary structural differences between species, which are likely to influence tertiary structure and hence ligand recognition or signal transduction. Indeed, and as discussed above, studies to date have revealed several dramatic differences in the ligand recognition of the rCTR hCTR and pCTRs. 7~ For example, sCT and hCT were almost equipotent in activating the hCTR, while hCT was essentially inactive at the pCTR. Analysis of the predicted amino acid sequences of the pig, human, and rat receptors revealed 78% identity between the human receptor cloned from BIN 67 ovarian cancer cells 58 and the rat C l a receptor, 37 and 67% identity between the pig receptor cloned from LLC-PK1 cells 198 and the rat C la receptor. The availability of a large number of calcitonins and analogs continues to facilitate detailed investigation of functional differences among calcitonin receptors, now that it is possible to investigate properties of expressed recombinant receptors. Already there are a number of studies from which it is possible to derive a working model of calcitonin/calcitonin receptor interactions. Studies using receptor chimeras of hCTR and glucagon receptor provided evidence for a two-site hormonereceptor interaction model, the data suggesting that both the N-terminal extracellular extension and the extracellular loops cooperate to bind calcitonin, while activation of signaling is primarily via interaction with the extracellular loops. 212 Supportive of this model are the results of experiments using CTR/PTHR receptor chimeras together with CT/PTH peptide chimeras. This work suggested that the N-terminus of the calcitonin molecule interacts with the extracellular loops and/or the transmembrane domains to activate the receptor, while binding is stabilized by interactions between C-terminal portions of the calcitonin molecule and the extracellular extension of the receptor. 223 To date there are few studies to elucidate the structure/function of intracellular domains of the calcitonin receptor. As discussed above, the naturally occurring insert in the first intracellular loop of the hCTR blocks calcitonin-induced activation of phospholipase C and subsequent increases in intracellular Ca 2§ levels. 219 Deletion of the C-terminal intracellular

CHAPTER4 Calcitonin tail of the pCTR interfered with calcitonin-induced internalization of this receptor in transfected cells. TM

C. Signal Transduction Early experiments showed that osteoclast-rich cultures derived from mouse calvariae 2z5 show a rise in cAMP formation in response to calcitonin. Confirmation that the osteoclast is a direct target for calcitonin, and that calcitonin acts in these cells to increase cAMP levels, came from experiments using highly enriched rat osteoclasts 226 or human osteoclastoma cells. 227 Moreover, both dibutyryl cAMP 228 and forskolin, which directly activate adenylate cyclase, 229 inhibit bone resorption. Calcitonin was shown to increase adenylate cyclase activity in the medullary and cortical portions of the thick ascending limb of Henle's loop, in the early portion of the distal convoluted tubule, and to some extent in the collecting tubule. 154 Interestingly, recent quantitative RTPCR confirmed the selective expression of calcitonin receptor mRNA in the same regions of the nephron previously shown to be calcitonin responsive, ~53 which was consistent with our autoradiographic localization of renal calcitonin receptor. 152 Calcitonin induction of cAMP has now been documented in a large number of calcitonin receptor-beating cell lines in culture including LLC-PK~ pig kidney cells 23~ and cancers of lung, ~95 breast, 196 and bone, 126 the evidence suggesting that coupling of the calcitonin receptor to adenylate cyclase activation occurs via receptor interaction with the G proteins of the G~s family. It is now clear that many of the G-protein-coupled receptors interact with multiple G proteins. In the case of the calcitonin receptor, there is ample evidence that calcitonin may also induce increases in cytoplasmic calcium concentrations ([Ca2+]i). For example, it is apparent that in the osteoclast, signaling through both cAMP and changes in [Ca2+]i are important in calcitonin action. TM Although controversial and potentially species specific, retraction of osteoclasts by calcitonin, and calcitonin-induced cytoskeletal changes, at least in the mouse, appear to be mainly mediated by the protein kinase A pathway. 232 On the other hand, inhibition of osteoclastic bone resorption by calcitonin can be mimicked by both dibutyryl cAMP and phorbol esters or blocked by protein kinase C inhibitors. 233 These results suggest that alternative G protein coupling can mediate calcitonin activation of either adenylate cyclase or phospholipase C, leading in the latter case to raised intracellular inositol triphosphate levels and thence increased [Ca2+], which together with the co-liberated diacylglycerol, activate protein kinase C. Calcitonin apparently causes either no change, or an inhibition of adenylate cyclase activity, in

111 brain tissue. 234'e35 Despite this, calcitonin receptors cloned from brain are capable of coupling to G~s protein in other cell types. 37'18~ In hepatocytes, calcitonin-induced activation of adenylate cyclase has not been shown, but calcitonin, even at very low concentrations, is capable of increasing [Ca2+]i .236 There is a recent report that calcitonin prevented CCl4-induced oxyradical formation and cellular damage in hepatocytes by a C lb receptor-mediated activation of protein kinase C . 237 In LLC-PK~ pig kidney cells, calcitonin can, in a cell cycle-dependent manner, induce changes mediated by either cAMP or [cae+]i .e38 Finally, expression of cloned receptors in a variety of cell types has conclusively shown that calcitonin receptors of human, 2~4 rat, 28 and porcine 239'e4~origin are capable of signaling through both cAMP- and CaZ+-activated second-messenger systems. It is important to note that comparison of the calcium response in cell lines expressing different calcitonin receptor levels has suggested that the magnitude of the response is proportional to the receptor density. 241 This relationship has been clearly shown for the TSH 242 and PTH/PTHrP receptors 243 and it is possible that relative receptor density in target tissues may influence the signaling pathway(s) activated. Calcitonin-induced [cae+]i fluxes are rapid and sustained in the presence of extracellular calcium. TM In the absence of extracellular calcium, the sustained phase is not seen. These results suggest that the initial response is mediated by inositol 1,4,5-triphosphate (IP3), which stimulates the release of calcium from intracellular stores. Calcitonin stimulation of IP3, probably generated by Gq-mediated activation of phospholipase C, has been shown in the case of cloned pCTR 239'24~ and hCTRs. 214 The sustained phase of the Ca 2+ response is dependent on extracellular calcium, and current evidence, as discussed below, suggests that this is mediated by calcium inflow through plasma membrane calcium channels. An interesting "calcium-sensing" function of the hCTR was recently reported, whereby calcitonin receptor-bearing cells were rendered sensitive to extracellular calcium in terms of increased [Ca2+]i .241 Although initially reported to be independent of calcitonin, subsequent work in cells transfected with the hCTR, 245 rCTR, and pig CTRs TM showed that sensitivity to extracellular calcium is in fact dependent upon preexposure of cells to calcitonin. Thus calcitonin treatment of calcitonin receptor-bearing cells, in the presence of extracellular calcium, causes a sustained rise in [Ca2+]i, the extent of which is dependent on the concentration of the extracellular calcium. Since osteoclasts, which express high levels of calcitonin receptor, ~7 are reportedly exposed to calcium concentrations as high as 26 mM during bone resorption, 246 this phenomenon may have par-

1 12 ticular relevance for this cell type. In isolated osteoclasts, calcitonin and extracellular C a 2+ both produce intracellular C a 2+ transients. 247 Interestingly, calcitonin and [CaZ+]e greatly potentiate the signal produced by either agent alone. 248 The mechanisms underlying this phenomenon are not yet known but two possibilities are: (1) that this is analogous to the receptor-activated calcium entry seen for a number of other receptor systems where calcium inflow apparently results from emptying of intracellular calcium stores 249 and (2) that this involves a mechanism independent of these events, since there is now evidence that calcium "sensing" results from lower concentrations of calcitonin than those required to produce intracellular calcium transients. 245 It is becoming recognized that signals generated by cell surface receptors of various classes are subject to modulation by other receptors. An intriguing example of this "cross-talk" is a finding in osteoclasts that calcitonin can down-regulate the cell signals induced by a fragment of the bone extracellular matrix molecule, bone sialoprotein (BSP). 25~ BSP and osteopontin, a related molecule also found in bone extracellular matrix, can both bind to osteoclasts, via the OLv[33integrin receptor, and induce intracellular C a 2+ mobilization. TM Calcitonin was also shown to inhibit osteopontin mRNA in isolated rabbit osteoclasts. 252 Given that BSP and/or osteopontin can act as attachment molecules for osteoclasts, 253 reduction of osteopontin formation by calcitonin could be one means by which calcitonin inhibits osteoclast activity and thus bone resorption. Calcitonin can potently modulate the growth of some calcitonin receptor-beating cells. Calcitonin was shown to stimulate the growth of human prostate cancer cells, in which calcitonin increases intracellular C a 2+ and cAMP levels. TM On the other hand, calcitonin treatment inhibited the growth of T47D human breast cancer cells, an action believed to be mediated by the specific activation of the type II isoenzyme of the cAMP-dependent protein kinase. 255 Recently, several points of intersection of cAMP-mediated pathways and those of growth factor-stimulated proliferation pathways have been identified, 256'257 although none of these intracellular mechanisms have yet been explored with respect to calcitonin. Recent reports also support a role for Ca2+-ac tivated pathways in growth modulation, with depletion of intracellular C a 2+ stores by thapsigargin being implicated in inhibition of cell growth 258 while mitogenesis by thrombin and bradykinin appeared to involve Gq-mediated increases in [Ca2+]i .259 Again, the role of these pathways in calcitonin-mediated growth modulation remains to be determined, but the above considerations are of potential importance, given the demonstrated inhibition of growth, by calcitonin, of human breast cancer cells. 255

Y.J. MARTIN, D. M. FINDLAY, J. M. MOSELEY, AND P. M. SEXTON

D. Receptor Regulation: The "Escape" Phenomenon The molecular and cellular biology of the calcitonin receptor in osteoclasts has been amenable to study following the development of a means to derive osteoclasts in vitro, 118 in addition to the cloning of the calcitonin receptor. In osteoclasts generated in mouse bone marrow cultures treated with 1,25(OH)2D3, as well as in freshly isolated osteoclasts from newborn rat, the calcitonin receptor and calcitonin receptor mRNA are abundantly expressed. 213 In this cell type the C la receptor was found to be the isoform most abundantly expressed, with C lb detectable in lesser amounts. 213 In mouse marrow cultures, calcitonin receptor mRNA could be detected by RT-PCR very early, before receptor detection by autoradiography. Furthermore, at a stage when only very few calcitonin receptor-positive cells were seen against a background of mainly stromal cells in these cultures, it was even possible to detect calcitonin receptor m R N A by the less sensitive method of Northern blot hybridization. This observation implies that the developing osteoclasts in the marrow cultures are very rich in calcitonin receptor mRNA. The calcitonin receptor has been shown to be subject to both homologous and heterologous regulation, the latter by a number of factors including glucoc o r t i c o i d s , 26~ activators of protein kinase C, 262 and transforming growth factor ~.263 Calcitonin-induced down-regulation of the calcitonin receptor was first demonstrated in various transformed cell l i n e s , 264'266 and later in primary cultures of kidney cells. 267 Receptor loss from the cell surface was shown to be due to an energydependent cellular internalization of the ligand-receptor complex. 268 The molecular mechanisms involved in these events remain to be fully elucidated, but appear to be cell type dependent. As noted for the PTH/PTHrP receptor, 269 ligand-induced phosphorylation of the hCTR has recently been demonstrated 27~ and may be important in receptor regulation. Preincubation of calcitonin receptor-beating cell lines with calcitonin resulted also in persistent activation of adenylate cyclase, which was shown to be due to persistent occupancy of cell-surface receptors by poorly dissociable sCY. 271'272 Calcitonin treatment also resulted in desensitization of the cells to a subsequent challenge with calcitonin, which was thought to be due to receptor loss from the cell surface and possibly to an uncoupling of the remaining receptors from signal t r a n s d u c t i o n . 271'272 Although calcitonin inhibits bone resorption, it has been found in vitro and in vivo that calcitonin inhibition is followed by " e s c a p e . ''273'274 " E s c a p e " is defined as an increase in resorption in bones stimulated to resorb

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by a resorptive agent, despite the continued presence of concentrations of calcitonin that were maximally inhibitory. Furthermore, rats treated chronically with calcitonin become refractory to the hypocalcemic action of the peptide) 75 Data from the in vitro experiments suggested that "escape" was due to a change in responsiveness to calcitonin rather than a loss of activity of the hormone. An interesting feature of the phenomenon is that the development of escape in vitro can be prevented by concomitant treatment of the bones w i t h glucocorticoid, 276 which more recent experiments indicate might be due to antagonism by glucocorticoid of calcitonin-induced calcitonin receptor down-regulation in osteoclasts. 261 The phenomenon of "escape" is such an integral part of calcitonin action that it has to be borne in mind whenever the hormone is being used therapeutically. It helps to explain why calcitonin is less effective than might be expected in the treatment of such states of excessive bone resorption as hyperparathyroidism and malignant hypercalcemia. The biochemical and cellular mechanisms by which calcitonin induces refractoriness to its own action have been the subject of intense study in recent years. Studies of osteoclasts in c u l t u r e 276-278 have shown that calcitonin treatment results in down-regulation of the calcitonin receptor, which is nevertheless different from that seen in other cell types in that down-regulation is considerably prolonged. 276 These results were consistent with the hypothesis that the resistance to calcitonin that typifies the clinical phenomenon of "escape" might be due to reduced calcitonin sensitivity of osteoclasts. Evidence for this had previously been obtained by elegant experiments in bone organ culture. TM We have shown that either s h o r t 276'279 o r continuous 121 treatment of osteoclasts with calcitonin results in a rapid and prolonged downregulation of calcitonin mRNA, whereas in the nonosteoclastic human breast cancer T47D cells, there was no decrease in calcitonin receptor mRNA levels, z76 Significantly, calcitonin-treated osteoclasts regained the ability to resorb bone, 122 again suggesting that functional, calcitonin-resistant osteoclasts result from exposure to pharmacological doses of calcitonin. It was also found that in the continuous presence of calcitonin, resorptive osteoclast-like cells, that had very low levels of calcitonin receptor mRNA, formed in mouse bone marrow cultures TM (Fig. 4-6). These results, therefore, indicate that calcitonin-induced decreased responsiveness of osteoclasts to calcitonin is at least partly due to suppression of calcitonin receptor and its mRNA in existing osteoclasts and to the development of newly formed osteoclasts that have very low levels of calcitonin receptor. It is interesting to note that in bone organ culture, "escape" was prevented by irradiation, which would inhibit proliferation of osteoclast precursors, 28~ and that "es-

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cape" was accompanied by the formation of small, newly formed active osteoclasts. TM Down-regulation of calcitonin receptor and of calcitonin receptor mRNA in mature mouse osteoclasts, but not in nonosteoclastic cells, appears to be mediated by activation of cAMPdependent protein kinase. 282

VII. CALCITONIN IN CLINICAL MEDICINE The discussions in this chapter of the mechanism of action of calcitonin provide background to the use of calcitonin in therapy. Its specific uses are considered in detail elsewhere in this volume, but one of the most important unanswered questions concerning the role of calcitonin in physiology, pathology, and therapeutics is whether its role as an inhibitor of bone resorption is such that calcitonin deficiency can lead to the development of osteoporosis. The corollary would be that calcitonin could be used in the treatment of osteoporosis. Initial reports that basal calcitonin levels are lower in women than in men, 283'284 fall with age in both, 285 and are reduced in osteoporotic women 286 gave rise to considerable interest in the possible use of calcitonin in the treatment of postmenopausal osteoporosis. However, enthusiasm has been tempered by subsequent data showing no difference in basal calcitonin levels between postmenopausal osteoporotic and age-matched normal w o m e n . 287'288 Calcitonin has an established place in the treatment of Paget's disease of bone, and is increasingly used in osteoporosis, where its effectiveness in nasal spray form has been established. 289

VIII. SUMMARY Calcitonin is a polypeptide hormone whose major recognized effect in mammals, including humans, is to inhibit bone resorption. This it does by acutely inhibiting osteoclast activity, and perhaps also by inhibiting the generation of osteoclasts, although the in vitro evidence for the latter is conflicting. Because bone resorption is rapid enough to contribute to the maintenance of extracellular fluid calcium only in stages of growth or in certain disease states in maturity, in which bone resorption is increased, it follows that only in those circumstances does calcitonin injection result in a significant fall in plasma calcium. Thus, although calcitonin was discovered as a calcium-lowering hormone in experiments that were carried out in young animals, it may be that its function throughout life is that of a regulator of the bone resorption process. This might not necessarily contribute

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to acute maintenance of plasma calcium in maturity. Consideration of the "physiological" role of calcitonin often fails to take into account the need to regulate the bone resorptive process, however slowly that may be proceeding, and whether or not it contributes to maintenance of the plasma calcium level. Thus, the general role of calcitonin in skeletal metabolism may indeed be that of the most important inhibitor of the bone resorptive process, acting to prevent excessive skeletal loss throughout life, and especially at times at which skeletal loss becomes a risk, for example, in rapid growth, pregnancy and lactation, immobilization, and certain pathological states (Paget's disease of bone, thyrotoxicosis, hyperparathyroidism). This working model of calcitonin action and function can be applied to any discussion of the hormone's role in therapy or in pathogenesis of bone disease. The last few years have opened fascinating new aspects of calcitonin physiology. This includes particularly the cloning of the calcitonin receptor and the finding of a number of functionally different calcitonin receptor isoforms, the discovery of calcitonin and of calcitonin receptor sites in the brain, leading to the possible role of calcitonin as a neuropeptide. The most interesting areas we have to address now are the physiological significance of the receptor isoforms, the details of calcitonininduced intracellular signaling, and the potential role of calcitonin in cell growth and differentiation.

Acknowledgments These authors acknowledge the support of The National Health and Medical Research Council of Australia and The Anti-Cancer Council of Victoria in funding their work. Their thanks are also extended to Mrs. A. Carruthers for her help in the preparation of the manuscript.

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