Parathyroid hormone (PTH) secretion, PTH mRNA and calcium-sensing receptor mRNA expression in equine parathyroid cells, and effects of interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha on equine parathyroid cell function

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid gland in response to changes in ionized calcium (Ca(2+)) concentrations. In this study, we measured PTH secretion, and PTH mRNA and calcium-sensing receptor (CaR) mRNA expression by equine parathyroid chief cells in vitro. We also evaluated the effects of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha on PTH secretion, and PTH and CaR mRNA expression. The relationship between PTH and Ca(2+) was inversely related. PTH secretion decreased from 100% (day 0) to 13% (day 30). PTH mRNA expression declined from 100% (day 0) to 25% (day 30). CaR mRNA decreased from 100% (day 0) to 16% (day 30). Chief cells exposed to high (2.0 mM) Ca(2+) concentrations had a lower PTH mRNA expression compared with low Ca(2+) concentrations. Ca(2+) concentrations had no effect on CaR mRNA expression. The inhibitory effect of high Ca(2+) concentrations on PTH secretion also declined over time. After day 10, there was no significant difference in PTH secretion between low and high Ca(2+ )concentrations. IL-1beta decreased both PTH secretion (75%) and PTH mRNA expression (73%), and resulted in a significant overexpression of CaR mRNA (up to 142%). The effects of IL-1beta were blocked by an IL-1 receptor antagonist. IL-1beta decreased the Ca(2+) set-point from 1.4 mM to 1.2 mM. IL-6 decreased PTH secretion (74%), but had no effect on PTH and CaR mRNA expression. TNF-alpha had no effect on PTH secretion, and PTH and CaR mRNA expression. In summary, the decreased responsiveness of parathyroid cells to Ca(2+) from 0 to 30 days can be explained, in part, by the reduced CaR expression. IL-1beta and IL-6 but not TNF-alpha affected parathyroid function in vitro and may be important in influencing PTH secretion in the septic horse.     

Parathyroid expression of calcium-sensing receptor protein and in vivo parathyroid hormone-Ca(2+) set-point in patients with primary hyperparathyroidism

A reduced expression of calcium-sensing receptor (CaR) messenger ribonucleic acid and protein accompanied by abnormalities in parathyroid cell proliferation and PTH secretion are present in primary hyperparathyroidism. We studied the expression of CaR protein by immunohistochemistry in 36 sporadic parathyroid adenomas and investigated the relationship between CaR expression and several preoperative clinical parameters, including the set-point of Ca(2+)-regulated PTH secretion (measured in vivo). The adenomas were classified in 4 categories according to the intensity of immunohistochemical staining: 5 (14%) showed a CaR staining intensity similar to that of normal parathyroid ( ), 10 (27%) showed moderate staining (++), 16 (45%) showed weak staining (+), and 5 (14%) were negative (-). The intensity of CaR staining was not related to preoperative serum Ca(2+), PTH levels or adenoma volume. Twenty-nine patients underwent preoperatively the calcium infusion test to evaluate the PTH-Ca(2+) set-point. Individual values of PTH-Ca(2+) set-point ranged from 1.38-1.93 mmol/L and were significantly correlated with basal Ca(2+) levels (r = 0.96; P: = 0. 0001) and adenoma volume (r = 0.5; P: = 0.01). The mean PTH-Ca(2+) set-point values were significantly different in the 4 groups of patients classified according to immunohistochemical staining intensity of their adenoma (P: = 0.025; F = 3.78); the mean PTH-Ca(2+) set-point was significantly higher in the groups classified as negative than in those classified as weak or moderate. No correlation was observed between the PTH-Ca(2+) set-point and basal PTH levels or between the percent maximal PTH inhibition and adenoma volume and basal PTH or Ca(2+) levels. In summary, our data suggest that there is a relationship between apparent CaR protein expression and PTH-Ca(2+) set-point abnormality, suggesting that a reduced receptor content might have an important role in the pathogenesis of primary hyperparathyroidism.     

Drugs inhibiting parathyroid hormone (PTH) secretion by control of the calcium receptor (calcimimetics)--effect on the set point of calcium-regulated PTH secretion

Calcimimetics are positive allosteric modulators that activate the parathyroid calcium receptor (CaR) and thereby immediately suppress parathyroid hormone (PTH) secretion. Preclinical studies have demonstrated that calcimimetics inhibit PTH secretion and parathyroid gland hyperplasia and ameliorates bone qualities in rats with chronic renal insufficiency. Clinical trials with cinacalcet hydrochloride, a calcimimetic compound, have shown that calcimimetics possess lowering effects not only on serum PTH levels but also on serum phosphorus levels in dialysis patients with secondary hyperparathyroidism (2HPT). Thus, calcimimetics have considerable potential as an innovative medical approach to manage 2HPT. In this review, the similarities are extrapolated between the pharmacological effect of calcimimetics on the set point of Ca-regulated PTH secretion and clinical observations in affected subjects with activating CaR mutations.     

Relationship between parathyroid calcium-sensing receptor expression and potency of the calcimimetic, cinacalcet, in suppressing parathyroid hormone secretion in an in vivo murine model of primary hyperparathyroidism

Cinacalcet HCl, an allosteric modulator of the calcium-sensing receptor (CaR), has recently been approved for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on dialysis, due to its suppressive effect on parathyroid hormone (PTH) secretion. Although cinacalcet's effects in patients with primary and secondary hyperparathyroidism have been reported, the crucial relationship between the effect of calcimimetics and CaR expression on the parathyroid glands requires better understanding. To investigate its suppressive effect on PTH secretion in primary hyperparathyroidism, in which hypercalcemia may already have stimulated considerable CaR activity, we investigated the effect of cinacalcet HCl on PTH-cyclin D1 transgenic mice (PC2 mice), a model of primary hyperparathyroidism with hypo-expression of CaR on their parathyroid glands. A single administration of 30 mg/kg body weight (BW) of cinacalcet HCl significantly suppressed serum calcium (Ca) levels 2 h after administration in 65- to 85-week-old PC2 mice with chronic biochemical hyperparathyroidism. The percentage reduction in serum PTH was significantly correlated with CaR hypo-expression in the parathyroid glands. In older PC2 mice (93-99 weeks old) with advanced hyperparathyroidism, serum Ca and PTH levels were not suppressed by 30 mg cinacalcet HCl/kg. However, serum Ca and PTH levels were significantly suppressed by 100 mg/kg of cinacalcet HCl, suggesting that higher doses of this compound could overcome severe hyperparathyroidism. To conclude, cinacalcet HCl demonstrated potency in a murine model of primary hyperparathyroidism in spite of any presumed endogenous CaR activation by hypercalcemia and hypo-expression of CaR in the parathyroid glands.     

Calcium Receptor and Regulation of Parathyroid Hormone Secretion

The cloning of the CaR has made it possible to show definitively that the CaR is a critical mediator of the inhibitory effect of high Cao2+ on PTH secretion and parathyroid cellular proliferation. The receptor may also mediate the suppressive action of high Cao2+ on PTH gene expression, while its involvement in several other known actions of Cao2+ on parathyroid function remain to be examined. Alterations in CaR expression and/or function are clearly involved in hyper- and hypocalcemic disorders caused by inactivating or activating CaR mutations, respectively, and could contribute to the deranged Cao(2+)-sensing in primary and uremic secondary HPT. Finally, CaR activators offer promise as the first truly effective mode of medical therapy for these latter two conditions.     

The search for calcium receptor antagonists (calcilytics)

The Ca(2+) receptor on the surface of parathyroid cells is the primary molecular entity regulating secretion of parathyroid hormone (PTH). Because of this, it is a particularly appealing target for new drugs intended to increase or decrease circulating levels of PTH. Calcilytic compounds are Ca(2+) receptor antagonists which increase the secretion of PTH. The first reported calcilytic compound was NPS 2143, an orally active molecule which elicits rapid, 3- to 4-fold increases in circulating levels of PTH. These rapid changes in plasma PTH levels are sufficient to increase bone turnover in ovariectomized, osteopenic rats. When administered together with an antiresorptive agent (estradiol), NPS 2143 causes an increase in trabecular bone volume and bone mineral density in osteopenic rats. The magnitude of these changes are far in excess of those caused by estradiol alone and are comparable with those achieved by daily administration of PTH or a peptide analog. These anabolic effects of NPS 2143 on bone are not associated with hyperplasia of the parathyroid glands. Calcilytic compounds can increase endogenous levels of circulating PTH to an extent that stimulates new bone formation. Such compounds could replace the use of exogenous PTH or its peptide fragments in treating osteoporosis.     

The calcimimetic NPS R-568 decreases plasma PTH in rats with mild and severe renal or dietary secondary hyperparathyroidism

NPS R-568 is a Ca²⁺ receptor agonist (“calcimimetic”) compound that reduces circulating parathyroid hormone (PTH) levels in rats and humans with mild secondary hyperparathyroidism (2°HPT) resulting from chronic renal insufficiency (CRI). These studies extend those observations to show that NPS R-568 is equally effective in decreasing plasma PTH and Ca²⁺ levels in rats with mild or severe 2°HPT, resulting either from CRI or from dietary calcium deficiency. Male rats were 5/6 nephrectomized and fed either normal chow or a high-phosphorus diet; other normal rats were fed a low-calcium diet. When 2°HPT had developed, NPS R-568 was administered and blood samples were collected for up to 6 h. PTH levels decreased to a minimum level within 30 min in both CRI and calcium deficiency models of 2°HPT. PTH and Ca²⁺ levels remained significantly depressed for >3 h after dosing. The percentage decrease in PTH levels was unaffected by the severity of 2°HPT or the basal plasma Ca²⁺ or phosphate levels. In rats with severe 2°HPT, the minimum plasma PTH level after NPS R-568 was greater than the basal level in mild 2°HPT. Thus, NPS R-568 is equally effective in suppressing plasma PTH and Ca²⁺ levels in rats with mild or severe renal or nutritional 2°HPT.     

Hyperparathyroidism is associated with reduced expression of a parathyroid calcium receptor mechanism defined by monoclonal antiparathyroid antibodies

Parathyroid tissue from patients with hyperparathyroidism (HPT) exhibited reduced immunohistochemical reactivity with monoclonal antiparathyroid antibodies, previously shown to stain intensely the surface of normal human parathyroid cells and to interfere with a receptor mechanism of these cells which is involved in the sensing and gating of Ca2+. Parathyroid hormone (PTH) release and cytoplasmic Ca2+ concentrations (Ca2+i) of dispersed cells from the pathological parathyroid glands had right-shifted dependencies on extracellular Ca2+, and exposure to the antibodies rendered both Ca2+i and PTH release almost completely insensitive to changes in ambient Ca2+. The results suggest that reduced expression of a parathyroid calcium receptor mechanism may be an important cause for the aberrant PTH release in HPT.     

Reduced parathyroid vitamin D receptor messenger ribonucleic acid levels in primary and secondary hyperparathyroidism

Vitamin D, via its receptor (VDR), inhibits the hormone secretion and proliferation of parathyroid cells. Vitamin D deficiency and reduced parathyroid VDR expression has been associated with development of hyperparathyroidism (HPT) secondary to uremia. VDR polymorphisms may influence VDR messenger RNA (mRNA) levels and have been coupled to an increased risk of parathyroid adenoma of primary HPT. VDR mRNA relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels were determined by RNase protection assay in 42 single parathyroid adenomas of patients with primary HPT, 23 hyperplastic glands of eight patients with uremic HPT, and 15 normal human parathyroid glands. The adenomas and hyperplasias demonstrated similar VDR mRNA levels, which were reduced (42 +/- 2.8% and 44 +/- 4.0%) compared with the normal glands (P < 0.0001). Comparison of parathyroid adenoma with a normal-sized parathyroid gland of the same individual (n = 3 pairs) showed a 20-58% reduction in the tumor. Nodularly enlarged glands represent a more advanced form of secondary HPT and showed greater reduction in the VDR mRNA levels than the diffusely enlarged glands (P < 0.005). The reduced VDR expression is likely to impair the 1,25(OH)2D3-mediated control of parathyroid functions, and to be of importance for the pathogenesis of not only uremic but also primary HPT. Circulating factors like calcium, PTH, and 1,25(OH)2D3 seem to be less likely candidates mediating the decreased VDR gene expression in HPT.     

The influence of the progression of secondary hyperparathyroidism on the set point of the parathyroid hormone-calcium curve

The influence of secondary hyperparathyroidism (2 HPT) on the set point of the parathyroid hormone (PTH)-Ca(2+) curve is controversial. In vitro experiments have shown an increase in the set point. However, clinical studies with hemodialysis patients have provided a variety of results (increases, decreases and no changes in the set point have been reported). The present study was designed to investigate the influence of the progression of 2 HPT on the set point of the PTH-Ca(2+) curve. The PTH-Ca(2+) curve and the expression of parathyroid calcium receptor (CaR mRNA) and vitamin D receptor (VDR mRNA) have been studied in normal rabbits (group I, n=9) and in nephrectomized rabbits (group II, n=18) at two stages after inducing 2 HPT: 2-3 weeks (group IIA) and 5-6 weeks (group IIB). In group I, the set point of the PTH-Ca(2+) curve was 1.63+/-0.03 mM. A progressive hypocalcemia was detected during the evolution of 2 HPT (groups IIA and IIB). Rabbits from group IIA had a significant (P<0.001) decrease in the set point to values of 1.45+/-0.02 mM. However, the set point increased significantly in group IIB (P<0.001) to 1.56+/-0.03 mM. CaR mRNA was similarly decreased in groups IIA (39+/-12%) and IIB (48+/-7%). No changes were detected in VDR mRNA. In conclusion, a reduction in the set point of the PTH-Ca(2+) curve in response to decreased extracellular Ca(2+) was detected in the early phases of 2 HPT. However, with the progression of 2 HPT the set point tended to increase even though extracellular Ca(2+) was markedly decreased. The increase in the set point in the course of 2 HPT seems to be a complex process that cannot be fully explained by changes in parathyroid CaR mRNA or VDR mRNA.     

Familial hypercalcemia and hypercalciuria caused by a novel mutation in the cytoplasmic tail of the calcium receptor

Familial hyperparathyroidism (HPT), characterized by hypercalcemia and hypercalciuria, and familial benign hypocalciuric hypercalcemia (FHH) are the most common causes of hereditary hypercalcemia. The calcium-sensing receptor (CaR) regulates PTH secretion and renal calcium excretion. Heterozygous inactivating mutations of the gene cause FHH, whereas CaR gene mutations have not been demonstrated in HPT. In a kindred with 20 affected individuals, the hypercalcemic disorder segregated with inappropriately higher serum PTH and magnesium levels and urinary calcium levels than in unaffected members. Subtotal parathyroidectomy revealed parathyroid gland hyperplasia/adenoma and corrected the biochemical signs of the disorder in seven of nine individuals. Linkage analysis mapped the condition to markers flanking the CaR gene on chromosome 3q. Sequence analysis revealed a mutation changing phenylalanine to leucine at codon 881 of the CaR gene, representing the first identified point mutation located within the cytoplasmic tail of the CaR. A construct of the mutant receptor (F881L) was expressed in human embryonic kidney cells (HEK 293), and demonstrated a right-shifted dose-response relationship between the extracellular and intracellular calcium concentrations. The hypercalcemic disorder of the present family is caused by an inactivating point mutation in the cytoplasmic tail of the CaR and displays clinical characteristics atypical of FHH and primary HPT.     

Effects of calcium and cyclic nucleotides on rat calcitonin and parathyroid hormone secretion.

Recently we developed a system for studying concurrent secretion of calcitonin (CT)and parathyroid hormone(PTH)in vitro from single rat thyroparathyroid gland complexes. In the present study, mechanisms involved in secretion of CT and PTH were explored by altering the medium [Ca ] and by using the Ca antagonist, verapamil. We also re-examined the idea that cyclic nucleotides may help regulate secretion of these hormones and attempted to determine if effects of cyclic nucleotides might be altered by changes in medium [Ca]. Thyroparathyroid glands from 8-day-old rats were incubated in serum-free medium for 8h, and CT and PTH levels in the medium were measured by radioimmunoassays. We show for the first time that: (1) although low [Ca] is well known to promote PTH release, some extracellular Ca is needed for PTH secretion to occur at a maximal rate; (2) inhibition of Ca entry into cells with verapamil mimics the effects of low medium Ca on both CT and PTH release; and (3) cyclic nucleotides may exert their effects on secretion of CT and PTH at least in part via effects on Ca entry into cells.     

Compounds modulating parathyroid hormone (PTH) secretion

The control of parathyroid hormone (PTH) secretion is strictly regulated by the parathyroid Ca receptor (CaR). Calcimimetics and calcilytics selectively act on the parathyroid CaR to inhibit and enhance PTH secretion, respectively. According to the recent pharmacological two-state model, calcimimetics act on the CaR as allosteric agonists to stabilize an active conformation of CaR. Conversely, calcilytics act on the CaR as allosteric inverse agonists to stabilize an inactive conformation of CaR. These compounds that can alter circulating levels of PTH and bone turnover might provide novel treatments for adynamic bone disease in patients with chronic renal failure.     

Familial hypocalciuric hypercalcemia and other disorders with resistance to extracellular calcium.

Cloning of the CaR has increased understanding of the normal control of mineral ion homeostasis and has clarified the pathophysiology of PTH-dependent hypercalcemia. Cloning of the CaR has enabled identification of FHH and NSHPT as inherited conditions with generalized resistance to Ca2+o, which is caused in many cases by inactivating mutations in the CaR gene. In most kindreds with FHH, there is resetting of Ca2+o to a mildly elevated level that does not require an increase in the circulating level of PTH above the normal range to maintain it. FHH is not accompanied by the usual symptoms, signs, and complications of hypercalcemia. The kidney participates in the genesis of the hypercalcemia in FHH by avidly reabsorbing Ca2+; consequently, there is no increased risk of forming urinary calculi in most cases. Generally, there is no compelling rationale for attempting to lower the level of Ca2+o in these patients to a nominal normal level. In contrast, in primary hyperparathyroidism, the Ca2+o resistance is limited to the pathologic parathyroid glands, and the rest of the body suffers the consequences of high circulating levels of calcium, PTH, or both. In this condition, removal of the offending parathyroid glands is often the treatment of choice. Parathyroidectomy may also be appropriate in disorders with generalized resistance to Ca2+o owing to inactivating CaR mutations in the following special circumstances: in selected families with FHH in which there is unusually severe hypercalcemia, frankly elevated PTH levels, or atypical features such as hypercalciuria; in cases of NSHPT with severe hypercalcemia and hyperparathyroidism; and in the occasional mild case of homozygous FHH owing to CaR mutations that confer mild-to-moderate resistance to Ca2+o that escapes clinical detection in the neonatal period. As discussed elsewhere in this issue, selective calcimimetic CaR activators are being tested in clinical trials, which potentiate the activation of the CaR by Ca2+o, thereby resetting the elevated set point for Ca2+o-regulated PTH release in primary and secondary hyperparathyroidism toward normal. It is hoped that these agents may become an effective medical therapy for the acquired Ca2+o resistance in primary and secondary hyperparathyroidism and perhaps for that present in the unusual cases of FHH and NSHPT, resetting the "calciostat" downward and thereby reducing Ca2+o and PTH toward normal.     

Hysteresis and calcium set-point for the calcium parathyroid hormone relationship in healthy horses

Abnormalities in calcium (Ca(2+)) homeostasis are reported in horses with several pathological conditions; however, there is little information on Ca(2+) regulation in horses. The objectives of the present study were to determine the Ca(2+) set-point in healthy horses, to determine whether the Ca(2+)/parathyroid hormone (PTH) response curves were characterized by hysteresis, and to determine if the order of experimentally induced hypocalcemia or hypercalcemia had an effect on PTH secretion. The Ca(2+) set-point and hysteresis were determined in 12 healthy horses by infusing Na(2)EDTA and calcium gluconate. The Ca(2+) set-point was 1.37 +/- 0.05 mmol/L, which is higher than values reported for humans and dogs (1.0-1.2 mmol/L). Hysteresis was present during hypocalcemia and hypercalcemia. Horses in which hypocalcemia was followed by hypercalcemia secreted more PTH (7440 +/- 740 pmol min/L) than horses in which hypercalcemia was followed by hypocalcemia (5990 +/- 570 pmol min/L). This study has demonstrated that the Ca(2+) set-point in the horse is higher than in other domestic animals and man. We have shown that the Ca(2+)/PTH relationship in horses is sigmoidal and displays hysteresis during both hypocalcemia and hypercalcemia, and that extracellular Ca(2+) concentrations may affect the response of the parathyroid gland to hypocalcemia.     

A novel calcium-sensing receptor antagonist transiently stimulates parathyroid hormone secretion in vivo

Circulating calcium (Ca(2+)) is a primary regulator of bone homeostasis through its action on PTH secretion. Extracellular Ca(2+) modulates PTH secretion through a cell surface G protein-coupled receptor, the calcium-sensing receptor (CaR). The expression of the CaR suggests a critical role in cellular regulation by calcium in various organs, including parathyroid gland, bone, and kidney. Despite an obvious pharmacological utility for CaR antagonists in the treatment of disease, only a limited number of such classes of compounds exist. We have identified a novel class of small molecules with specific activity at the CaR. This class of compounds is represented by compound 1. It possesses potent antagonist activity at the human CaR with IC(50) values of 64 nm and 230 nm in inhibiting intracellular Ca(2+) flux and inositol phosphate generation in vitro, respectively. When administered to male rats in vivo, compound 1 robustly increased serum PTH levels. The stimulation of PTH secretion was rapid and transient when administered either iv or orally. The pharmacokinetic profile of compound 1 after oral administration revealed that maximal plasma levels of compound were reached within 1 h and the half-life of the compound to be approximately 2 h in rats. These data describe a representative compound of a novel chemical class than previously described allosteric modulators that offer a new avenue for the development of improved treatments of osteoporosis.     

Expression of calcium-sensing receptor gene by avian parathyroid gland in vivo: relationship to plasma calcium

Calcium-sensing receptor (CaR) gene expression and parathyroid hormone (PTH) content were evaluated in situ in chicken parathyroid glands (PG) in relation to changes in plasma calcium. The CaR gene is expressed by the parathyroid chief cells, the same cells that store and secrete PTH. An increase in plasma calcium, achieved by repletion of vitamin D-deficient chicks with a normal diet, by PTH injection, or during eggshell formation, increased the expression of the CaR gene. Low plasma calcium concentration in vitamin D-deficient chicks or in layers, before or after eggshell formation, was associated with decrease in CaR gene expression in the PG. The level of CaR gene expression was inversely correlated with the PTH content of the PG. The results of this study demonstrate for the first time that, in contrast to mammals, the CaR gene expression in the PG of the chicken is inversely associated with changes in plasma calcium.     

Regulation of the calcium-sensing receptor. Influence of secondary hyperparathyroidism

The parathyroid glands have a great sensitivity to small changes in the extracellular ionic calcium. The calcium-sensing receptor (CaR) is a G protein-coupled receptor that responds to extracellular ionic calcium changes activating several intracellular signalling systems (phospholipases C, A2 and D) finally inhibiting the PTH secretion. In addition to calcium, there are some other agonists and modulators such as the Mg2+, spermine, amyloid beta-peptides, a variety of aminoacids, especially aromatic aminoacids and ionic strength. In the uraemia, the sensitivity of the parathyroid glands to calcium is altered and higher values of calcium are necessary to suppress the PTH. In the secondary hyperparathyroidism the CaR expression is reduced. It has been found a negative correlation between cellular proliferation and the expression of the CaR in hyperplasic glands. Despite it is a calcium receptor, the expression of the CaR does not seem to be regulated by calcium and there is some controversy about the role of calcitriol regulating its expression. On the other hand, the phosphorous induces hyperplasia of the parathyroid gland increasing the cellular proliferation and a decrease of the CaR expression.