Calcium regulates stanniocalcin mRNA levels in primary cultured rainbow trout corpuscles of Stannius

Molecular and Cellular Endocrinology, 99 (1994) 315-322

315

Elsevier Science Ireland Ltd. MCE 03117

Calcium regulates stanniocalcin mRNA levels in primary cultured rainbow trout corpuscles of Stannius Graham F. Wagner *‘l, Ewa Jaworski Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada N6A SC1

(Received 17 November 1993; accepted 22 November 1993)

Key words: Stanniocalcin; Calcium; mRNA; Gene expression; Cultured cell; Ainbow trout

Summary Stanniocalcin (STC) is an inhibitor of gill calcium transport produced by the corpuscles of Stannius (CS), endocrine glands in bony fishes. In previous studies we have described how STC secretion is regulated by calcium both in vitro and in vivo, using rainbow trout as a model system. In this report we have examined the effects of calcium on STC mRNA levels in primary cultured trout CS cells. The results show that message levels are positively regulated by extracellular calcium concentrations within the physiological range. The calcium response was also temporally-related as more prolonged exposures tended to have greater effects. Similar concentrations of magnesium had no effect on message levels. This represents another level at which calcium regulates the CS cell, in addition to its established effects on STC synthesis and secretion. The results are discussed in relation to the other known calciotropic hormones, calcitonin and parathyroid hormone.

Introduction The corpuscles of Stannius (CS) are endocrine glands on the kidneys of bony fishes (Stannius, 1839) that synthesize and secrete stanniocalcin (STC), a homodimeric, glycoprotein hormone with a unique primary structure (Wagner, 1993). In salmonids such as trout and salmon, STC secretion is positively regulated by extracellular levels of ionic calcium. This has been demonstrated both in vivo (Glowacki et al., 1990, Wagner et al., 1991) and in vitro (Wagner et al., 19891, but has been best characterized in primary cultured trout CS cells. The release of STC by cultured CS cells is exquisitely regulated by extracellular calcium within the physiological range, such that step-wise increases from 0.3 to 2.7 mM Ca2+ produce concomitant elevations in STC secretion (Wagner et al., 1989). The function of STC upon release is to restore normocalcemia, which is accomplished by reducing the rate of gill calcium transport (Fenwick and So, 1974; Lafeber et al., 1988a; So and Fenwick, 1977; 1979; Wagner et al., 1986, 1988a), increasing renal phosphate reabsorp-

* Corresponding author. Tel.: 519-661-3966; Fax: 519-661-3827.

* Medical Research Council of Canada Scholar. SSDI 0303-7207(93)E0287-5

tion (Lu et al., 1993) and in the case of marine fishes, by inhibiting intestinal calcium transport (Sundell et al., 1992). There are interesting parallels between STC in fish (Wagner, 1993) and calcitonin in mammals (Anast and Conway, 1972; Gage1 et al., 1980) with respect to stimulus-secretion coupling and function, in spite of the fact that they have different target organs. In view of the regulatory effects of calcium on STC secretion, it would be logical for calcium to have a role in hormone biosynthesis, a notion that is in fact supported by histophysiological studies. For instance, CS cells are more active in fish adapted to seawater which is rich in calcium (10 mM), than in freshwater fish. The CS cells in marine fishes have a more well developed network of endoplasmic reticulum and golgi, a higher content of secretory granules, and generally show increased nuclear and cytoplasmic volumes in comparison to CS cells from freshwater fishes (Krishnamurthy, 1976; Wendelaar Bonga and Pang, 1986). Stages in life history can also influence the activity of STC cells, especially if they involve changes in calcium metabolism. For instance, reproduction in fishes can be associated with increases in both serum calcium and the mean diameter of CS cell nuclei (Ahmad and Swarup, 19901, the latter of which may be indicative of increased STC gene expression. However, to our knowl-

edge there have been no studies on the regulation of STC gene expression. Consequently, the potential effects of calcium and other humoral factors are entirely unknown. As we have recently characterized cDNA clones encoding salmon STC (Wagner et al., 1992; Sterba et al., 1993) in this report we have examined the effects of calcium on STC mRNA levels in primary cultured trout CS cells. Materials

and methods

Reagents, biochemicals and supplies

Fine chemicals and electrophoretic reagents were enzymes from purchased from BDH, restriction Promega, chromatography supplies and labelling kits from Pharmacia, blotting membranes and radioisotopes from Amersham and tissue culture supplies were obtained from Gibco BRL. Preparation of cultured cells

Primary cultured trout CS cells were prepared as described previously (Gellersen et al., 1988; Wagner et al., 19891, except that cells were plated in Leibovitz media (L-15; Leibovitz, 1963). Adult, sexually maturing rainbow trout (0.25-0.4 kg) were obtained locally from a commercial dealer. The CS were dissected free of surrounding renal tissue and placed in ice-cold L-15 containing antibiotics (100 units each of penicillin/ streptomycin and 2.5 kg/ml of Fungizone). Glands from approximately 70 fish for used for each cell preparation. The CS were transported to the laboratory on ice where residual fat and renal tissue were removed with the aid of a dissecting microscope. The glands were then teased apart with fine forceps and digested overnight at 4°C with gentle agitation in L-15 containing antibiotics and 0.5% trypsin. The next day, DNase 1 (0.1%) was added and the digest was agitated for 15 min at room temperature. The remaining tissue fragments were then teased apart with fine forceps, residual connective tissue was removed and the dispersed cells were passed through a Nytex filter. Cell viability was estimated by Trypan blue exclusion. Cells were seeded in L-15 containing 10% fetal bovine serum and antibiotics (100 units each of penicillin/streptomycin per ml) at a density of 0.4 X 10h cells/ml, and maintained at 15°C in a normal atmosphere. All experiments commenced 5 days after plating. Histological characterization of cultured cells

Immediately prior to plating, aliquots of CS cells (0.5 x 10h cells) were attached to microscope slides by cytocentrifugation. The cells were fixed in methanol at -20°C for 30 min, rehydrated and subjected to tinctorial staining with periodic acid Schiff reagent or immunocytochemistry with a highly specific STC antiserum as previously described (Wagner et al., 1988b).

For immunocytochemistry, a two-step procedure was used whereby the cells were treated overnight at 4°C with a 1: 200 dilution of salmon STC antiserum, followed by a 1 : 100 dilution of peroxidase-coupled goat anti-rabbit gamma globulin at 22°C for 30 min. After each antiserum application the slides were washed 3 x 15 min in a diluent buffer consisting of 0.05 M Tris-HCl, pH 7.5, containing 0.15 M NaCl. The sites of antibody binding were visualized by treating the slides with 0.025% 3’3-diaminobenzidine HCl containing 0.01% H,O,. The percentage of cells positively stained by each of the staining procedures was estimated by manual cell counting. In addition, several whole CS glands were fixed in Bouin’s solution, dehydrated and embedded in paraffin, and tissue sections (4 PM) were subjected to immunocytochemistry as described above. Control procedures included preabsorption of the primary antiserum with salmon STC and the use of nonimmune rabbit serum (NRS) in lieu of primary antiserum. Experiments on cultured cells

Prior to experimentation, the cells were washed in serum-free L-15 containing 0.1% bovine serum albumin to remove traces of fetal bovine serum. They were then maintained in serum-free L-15 supplemented with antibiotics for all experiments. To start an experiment, aliquots of calcium chloride and magnesium chloride (10 Fl/ml of media) were added to cells from lOO-fold concentrates to achieve the desired final concentrations of each cation; 0.7-2.7 mM in the case of calcium and 2.3 mM in the case of magnesium. Cells were then maintained in a normal atmosphere (15’0 for 1, 3 or 6 days. Each experiment was conducted on triplicate wells of cells and was repeated on 3 successive cell preparations (September, October and November). RNA extraction and Northern blot analysis

Following aspiration of culture media, total RNA was isolated from each well of cells according to Chomczynski and Sacchi (1987) and resolved on 1% agarose/ formaldehyde gels. Resolved RNA was transferred to nylon membranes (Hybond N) by capillary action and cross-linked by UV irradiation. Following a 2 h pre-hybridization period, membranes were hybridized overnight to random primed, “2P-labelled CDNAs (pure insert; 1 X lo6 dpm/ml) under conditions of high stringency (50% formamide, 6 X standard saline citrate (SSC), 1.25 X Denhardt’s solution, 100 pg/ml salmon sperm DNA, 0.1% SDS at 42°C). Following hybridization, membranes were washed 4 X 15 min at room temperature in 2 X SSC/O.l% SDS, followed by 2 x 30 min in 0.1 X SSC/O.l% SDS at 65°C. Membranes were probed first with a 1490 bp fragment encoding exons 3 through 5 of the carp p-actin gene, kindly supplied by Dr. P. Hackett, University of Min-

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nesota (Liu et al., 1990) and which recognizes a single transcript of N 2 kilobases in total CS RNA. The membranes were then stripped and reprobed with a 412 bp fragment encoding residues Asn+29-Gln+165 of coho salmon STC which hybridizes to a 2 kilobase transcript in CS RNA from both salmon and trout (Wagner et al., 1992). Bands were visualized by autoradiography. Because of the size similarity of the two transcripts, the blots were always exposed to X-ray film after stripping and prior to reprobing with the second cDNA to ensure that the stripping procedure had been successful. Densitometric and statistical analysis

Individual STC and actin bands were quantified by scanning densitometry and expressed as STC/ Actin ratios. These ratios were converted to percentages for graphical display, using message levels in cells exposed to 1.1 mM Ca2+ as the 100% baseline value. For statistical testing, the data were arcsine transformed and subjected to analysis of variance (two-tailed) followed by Dunnet’s test, using cells in 1.1 mM Ca2+ as the control. Groups were considered to be significantly different than the controls if P < 0.05. Results Preparation and characterization of cultured cells

The yield of viable cells from rainbow trout CS glands ranged from 2-4 X lo6 cells/kg body weight and cell viability was 95% or greater for each of the three cell preparations reported on here. Equal numbers of cells - 57% - were stained by the PAS and immunocytochemical techniques in all three cell preparations. Irrespective of the staining technique used, positively stained cells appeared heavily granulated, whereas unstained cells contained no granules (Fig. 1). Few cells were intermediate in appearance in terms of granule content (i.e. containing some granules). In contrast, once the plated CS cells had attached to the culture dishes, a greater majority of cells appeared to be highly granulated when viewed by phase contrast microscopy. In trout CS glands that were fixed, embedded and processed for immunocytochemistry, almost all cells were positively stained with STC antiserum (Fig. 2). Cells that were not stained by the antiserum in tissue sections comprised only l-2% of the total and stood out in contrast to the darkly stained STC cells (arrows in Fig. 2). In view of these results, it appears that the cell preparation procedure causes an inevitable amount of stress and degranulation of some CS cells, much as surgical stress in trout causes a rise in STC secretion (Wagner et al., 1991). As far as the discrepancy in numbers of STC-positive cells in dispersed (57%) versus intact glands (> 90%) is concerned, it should be recognized that many of the un-

Fig. 1. Freshly dispersed rainbow trout corpuscle of Stannius cells stained with STC antiserum. Dispersed cells were attached to microscope slides by cytocentrifugation, fixed in methanol and subjected to peroxidase immunocytochemistry followed by counterstaining with hematoxylin. The darker, STC-immunoreactive cells (arrows) have a distinct granular appearance under the light microscope and it is these granules which are positively stained by both the antiserum and periodic Schiff reagent.

stained dispersed cells are bound to be endothelial in nature as the glands are highly vascularized. Hence, the real difference is probably less than it appears. The use of NRS and primary antiserum preabsorbed with STC resulted in no immunocytochemical staining reaction (results not shown).

Fig. 2. Rainbow trout corpuscles of Stannius (CS) and adjacent kidney tissue (K) stained with STC antiserum. Freshly dissected CS tissue was fixed and processed for light microscope immunocytochemistry as described in the Materials and methods. Note that the majority of CS cells are darkly stained and that only a few cells remain unstained (arrows).

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E -2 u u -a

cal range (0.7-2.7 mM Ca’+) to observe possible effects on STC mRNA levels. The experimental results revealed that STC mRNA levels were positively regulated by ionic Ca*+, such that both upward and downward changes in calcium concentrations resulted in same directional changes in message levels. However, the magnitude of the responses varied from month to month, especially in the case of longer exposures to calcium (3 and 6 days).

0.7 1.1 1.5 1.9 2.3 2.7 07 1:1 1.5 1.9 2.3 2.7

One day exposure of CS cells to calcium

0

50

loo

STUActin,

150

200

2;o

3&l

% Of Control

Fig. 3. Steady state STC mRNA levels in primary cultured rainbow trout CS cells following a 1 day exposure to graded calcium concentrations. October cells were the most responsive to calcium, whereas September cells were least responsive. Each data point represents the mean f SEM of 3 replicates (* P < 0.05 in comparison to controls in 1.1 mM Ca’+; two-tailed ANOVA and Dunnet’s test).

The effects of calcium on STC mRNA levels CS cells were exposed for different lengths of time

to calcium concentrations

encompassing the physiologi-

Cells prepared in October responded most readily to calcium (Fig. 3), with the peak response occurring between 2.3-2.7 mM, at which point message levels were more than double those in control cells (P < 0.05). The responses of September and November cells were not as great, but in both cases STC mRNA levels rose progressively in reaction to increasing calcium levels. In November cells, message levels peaked at 1.9 mM Ca2+ (P < 0.05) and then declined upon exposure to higher calcium concentrations. Three day exposure of CS cells to calcium

A three day exposure to calcium generally elicited greater effects and there was less variability in the

0.7 1.1 1.S

C

September

::‘3

**

October

*, l

-d u

*

0.7 1.1 1.S

November **

;3

**

217

STC/Actin,%

of Control

October

STC

Actin 1

0.7 mM i 1.1 mM 1 1.5 mM 1 1.9 mM \ 2.3 mM Calcium

1 2.7

mM i

Fig. 4. (A) Steady state STC mRNA levels in rainbow trout CS cells following a 3 day exposure to graded exposures result in steeper response curves between 0.7-1.9 mM calcium, less variability in the responses and levels between individual cell preparations. Each data point represents the mean f SEM of 3 replicates (* P to controls in 1.1 mM Ca’+; two-tailed ANOVA and Dunnet’s test). (B) Autoradiographs of cellular STC October experiment.

calcium concentrations. Three day more consistent effects on message < 0.05, * * P < 0.01 in comparison and actin mRNA levels from the

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September

STC November 300

0

600

900

1200

Actin

1500

STC/Actin,% of Control

Calcium

Fig. 5. A) Steady state STC mRNA levels in rainbow trout CS cells after a 6 day exposure to calcium. Six day exposures produced the steepest response curves and the greatest induction of message levels. September cells exhibited the greatest response to calcium ( > 13-fold), followed by October (ll-fold) and November cells (3-fold). Each data point represents the meanf SEM of 3 replicates (** P < 0.01 in comparison to controls in 1.1 mM Ca*+; ANOVA and Dunnet’s test). (B) Autoradiographs of cellular STC and actin mRNA levels from the September experiment.

responses of individual treatment groups (Fig. 4). Cells prepared in September were the most responsive and exhibited a maximal 3-fold rise in message levels compared to controls (P < 0.01). The concentration-dependent nature of the response was also more evident following a 3 day exposure to calcium. In October for instance, cells exposed to 0.7 mM Ca2+ had significantly lower message levels than the controls (P < 0.051, whereas message levels were significantly higher in 1.9-2.7 mM calcium. The cells responded optimally to 1.9-2.3 mM Ca2+ in all experiments, whereas at higher concentrations their responses were either the same or somewhat reduced in magnitude.

Six day exposure of CS cells to calcium

A six day exposure to calcium had the most marked effect on STC mRNA levels, especially in cells prepared in September and October (Fig. 5). September cells exhibited the greatest response, with the highest calcium concentration (2.3 mM Ca’+) invoking a 13.5 fold increase in STC mRNA levels in comparison to controls (P < 0.01). October cells responded maximally to 1.9 mM Ca2+ with an 11-fold increase over controls (P < O.Ol), whereas diminished responses were obtained with 2.3 mM (&fold; P < 0.01) and 2.7 mM Ca2+ (8.5-fold; P < 0.01). November cells were the least responsive to a 6 day exposure (3-fold maximum). The effects of magnesium on CS cells CS cells were exposed to 2.3 mM Mg2+ to see if this divalent cation could mimic the effects of calcium (Fig. 6). In these experiments, control cells were exposed to 1.2 mM caIcium. Whereas calcium significantly increased STC mRNA levels in both experiments (P < 0.011, Mg 2+ had only a minor effect on message levels that was not statistically significant.

1.2 mlU ca2+ 2.3 mM Mg2+ 2.3 mM Cr?’

1.2 mM ca2+ 2.3 mM Mg’+

Discussion

2.3 mM Ca’+ 0

50

loo

150

200

250

STUActin; % of Control Fig. 6. Magnesium does not stimulate STC mRNA levels in rainbow trout CS cells. Cultured cells were exposed to 1.2 mM Ca*+, 2.3 mM Ca’+, or 2.3 mM Mg’+ plus 1.2 mM Ca*+ for 3 and 6 days. High calcium prompted 1.8 and 2 fold inductions of STC mRNA at 3 and 6 days, respectively, whereas magnesium had no significant effects. Each data point represents ihe mean f SEM of 3 replicates (** P < 0.01 in comparison to controls in 1.2 mM Ca’+; two-tailed ANOVA and Dunnet’s test).

Stanniocalcin (STC), parathyroid hormone (PTH) and calcitonin are the principal polypeptide hormones controlling calcium homeostasis in vertebrates. Whereas PTH and calcitonin regulate plasma calcium levels in most higher vertebrates, there is a large body of evidence suggesting that calcium homeostasis in fishes is regulated by the corpuscles of Stannius (CS) and stanniocalcin. These endocrine glands were discovered by Stannius in 1839 and subsequently shown to

arise during embryogenesis from kidney tubule cells (Garret, 1942; Krishnamurthy, 1976; Kaneko et al., 1992). An association with calcium homeostasis was established when it was found that surgically removing the CS caused a form of hypercalcemia (Fontaine, 1964) later revealed to be due to accelerated gill calcium transport (Fenwick, 1974; So and Fenwick, 1977; 1979). The active principle was subsequently shown to be a homodimeric glycoprotein, now known as stanniocalcin, and proved to be a potent inhibitor of gill calcium transport (Lafeber et al., 1988b; Wagner et al., 1986, 1988a). Under normal circumstances, the rate of gill calcium transport is regulated by plasma STC levels which, in turn, are regulated by the levels of plasma calcium. The calcium-sensitivity of STC cells ensures that as plasma calcium levels rise above the set-point (- 1.2 mM ionic Ca2+) there is an increased rate of STC secretion and correspondingly greater inhibition of gill calcium transport (Wagner et al., 1989, 1991). Recent evidence suggests that STC also reduces intestinal calcium transport (Tagaki et al., 1985; Sundell et al., 1992) and increases renal phosphate reabsorption (Lu et al., 19931, both of which would contribute further to the maintenance of calcium homeostasis. The net result is the same precision in the regulation of plasma calcium as occurs in higher vertebrates, but employing entirely different mechanisms. To our knowledge, the present study is the first to examine the regulation of STC gene expression and to demonstrate the positive effects that calcium has on STC mRNA levels. As the results show, changes in media calcium concentration both upwards and downwards within the physiological range produced corresponding changes in the levels of STC mRNA. Irrespective of the month of study, STC mRNA levels rose in response to increasing Ca2+ concentrations until 1.9 mM/2.3 mM, beyond which message levels levelled off or in some cases declined. Precisely why mRNA levels decreased at the highest calcium concentrations cannot be explained under the present circumstances, however it is not unprecedented for bioactive compounds to have diminished effects at super-physiological levels. That magnesium did not regulate STC mRNA levels was entirely expected in view of the fact that it has no effects on hormone secretion (Wagner et al., 1989). It also strengthens the notion that of the major plasma electrolytes, calcium is the principal regulator of CS cell activity. Interestingly, the same calcium concentrations provoking the greatest level of message induction have also been shown to cause the highest level of secretion by trout CS cells (Wagner et al., 1989). That these processes are similarly regulated by calcium makes sense from a physiological standpoint. Above all, it ensures that the supply of template for hormone synthesis matches the prevailing rate of hormone output.

This would be especially important in marine environments which are high in calcium (10 mM) and where greater secretory demands are placed on CS cells (Glowacki et al., 1990; Mayer-Gostan et al., 1992). Previous studies in trout suggest that hypercalcemia also provokes de novo STC synthesis to a modest (1.7 fold), yet statistically significant extent (Flik et al., 1990). Consequently, it would seem that calcium regulates the STC biosynthetic pathway at two levels. This is also true in the case of parathyroid hormone (PTH) where low levels of plasma calcium stimulate PTH gene transcription (Naveh-Many et al., 1989, 1992; Naveh-Many and Silver, 1990), discourage newly synthesized hormone from entering a degradative pathway and at the same time stimulate PTH secretion (Brown et al., 1987). Hence, in the case of both hormones, secretion is tightly coordinated with renewed hormone synthesis, thereby ensuring that a constant supply of hormone is always available for release. There was also a temporal component to the response such that more prolonged exposures to calcium had correspondingly greater effects. One and three day exposures to calcium had 2-3-fold maximal effects on message levels. However, longer exposures revealed the extent to which the STC gene was capable of being activated, as message levels rose as much as 14-fold following a 6 day calcium treatment. As it is unlikely that changes of this magnitude are due solely to effects on message stability, calcium probably effects the rate of STC gene transcription over the long term. The modest effects obtained following 1 and 3 day calcium exposures suggests that in the short term, calcium may regulate STC synthesis by altering mRNA stability and rate of translation from extant message without altering transcriptional activity. Accordingly, the relative contributions of message stability and transcription to the observed effects of calcium are currently under study. CS cells are unique in their ability to modulate STC mRNA levels in a bi-directional manner in accordance with ambient calcium levels. Message levels rose in CS cells in response to increasing calcium concentrations, but fell when calcium levels were reduced to sub-physiological levels (i.e. 0.7 mM Ca’+). In contrast, the PTH gene is regulated in one direction by calcium in vivo and in vitro. In vivo, lowering plasma calcium causes a rise in the rate of PTH gene transcription and mRNA levels (Naveh-Many et al., 1989, 1992; Naveh-Many and Silver, 1990). Calcium also regulates the PTH gene in vitro, but again only in a unidirectional fashion and oddly enough, in the opposite manner to that observed in vivo. High calcium levels decrease PTH mRNA levels in cultured cells whereas low calcium levels have no effect (Brookman et al., 1987; Heinrich et al., 1983; Russel et al., 1983; Mouland and Hendy, 1991). This differential regulation of PTH gene expression by cal-

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cium under in vitro and in vivo conditions is a perfect example of why in vitro phenomena must be corroborated in vivo. Accordingly, it is important that the regulation of STC gene expression is characterized under in vivo conditions in future studies, to determine if message levels are similarly regulated by calcium in the whole animal. On the other hand, the calcitonin gene is not influenced by calcium under any circumstances (Naveh-Many et al., 1989, 1992). The calcium-responsiveness of CS cells varied considerably from month to month and this was most obvious following longer exposures to calcium. At present, it is difficult to conclude if this is of physiological significance and if so, in what respect. For each preparation of cells, we followed exactly the same procedures and obtained the same percentage of viable cells. However, two factors beyond our control were changing season and the sexual maturation of the fish, both of which are potential modifiers of STC gene activity. Fish reproduction is already known to be associated with changes in CS cell activity (Ahmad and Swarup, 1990). Likewise, there are temporal and seasonal influences governing calcium homeostasis in fish (Fenwick and Brasseur, 1992; Fleming et al., 19731, some of which are known to affect the corpuscles of Stannius and the biological activity of circulating STC (Milliken et al., 1990; Wagner et al., 1986, 1988, 1993). As a consequence, the variable responsiveness of CS cells to calcium could be due to any one of a number of influences. Future studies will hopefully clarify this issue. Acknowledgements Grant and Scholarship support from the Natural Sciences and Engineering Council of Canada and the Medical Research Council of Canada are gratefully appreciated. References Ahmad, N. and Swarup, K., 1990. Seasonal changes in structure and behavior of corpuscles of stannius in relation to the changes in serum calcium level and the reproductive cycle of a freshwater female catfish - Mystus vittatus (BLOCH). Eur. Arch. Biol. 101, 285-294. Anast, C.S. and Conway, H.H., 1972. Calcitonin. Clin. Orthop. 84, 207-262. Breimer, L.H., MacIntyre, I. and Zaidi, M., 1988. Peptides from the calcitonin genes: molecular genetics, structure and function. Biochem. J. 255, 377-390. Brookman, J.J., Farrow, S.M., Nicholson, L., O’Riordan, J.L.H. and Hendy, G.N., 1987. Regulation by calcium of parathyroid hormone mRNA in cultured parathyroid tissue. J. Bone Min. Res. 6, 5299537. Brown, E.M., LeBoff, MS., Oetting, M., Possilico, J.T. and Chen, C., 1987. Secretory control in normal and abnormal parathyroid tissue. Res. Prog. Horm. Res. 43, 337.

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