Medical Encyclopedia

The acute secretory response of parathyroid hormone (PTH) is strictly regulated by the extracellular calcium concentration (Ca2+ o), and the G protein-coupled, calcium-sensing receptor (CaR) located on the chief cells of the parathyroid glands mediates this process.

Abnormalities of the Ca2+ o-sensing system lead to diseases that show hypo-/hypersecretion of PTH in addition to relative hyper-/hypocalciuria.

Novel signaling pathways, e.g., mitogen-activated protein kinases (MAPK), have been shown to be involved in CaR signaling in addition to “classical” CaR-regulated pathways, e.g., phospholipase C (PLC) and adenylate cyclase.

We will discuss the following topics in this chapter: (1) the regulatory mechanisms of Ca2+ o-sensing and PTH secretion, (2) disorders due to mutations in the CaR gene, abnormal CaR expression, or the production of antibodies against the CaR, and (3) the promising utility of drugs acting on the CaR.

In mammals, the extracellular calcium (Ca2+ o) concentration is held nearly constant at ~1 mM by the Ca2+ o-regulating hormones, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and calcitonin (CT), which, in turn, modulate the functions of their target organs, bone, kidney and intestine.

Conversely, Ca2+ o, in turn, regulates the secretion of PTH and CT from the chief cells of the parathyroid gland and the thyroidal C cells, respectively, as well as the production of 1,25(OH)2D3 by the kidney.

While fish living in the ocean do not need to raise Ca2+ o in their bodily fluids because the surrounding seawater contains ~8 mM Ca2+ o, mammals are always exposed to the risk of calcium deficiency.

Thus, the Ca2+ o-elevating hormones, PTH and 1,25(OH)2D3, play pivotal roles in calcium homeostasis in mammals. When mammals are exposed to calcium deficiency, parathyroid cells recognize and respond to the slight decrease in Ca2+ o by secreting PTH.

This acute secretory response of preformed PTH occurs within seconds and can last for 60-90 minutes.

Secreted PTH increases renal tubular reabsorption of Ca2+ and augments the release of Ca2+ from bone. PTH also enhances the synthesis of 1,25(OH)2D3 in the renal proximal tubules, which increases intestinal Ca2+ o absorption and renal Ca2+ conservation. Raising Ca2+ o also stimulates the secretion of the Ca2+ o-lowering hormone, CT, although CT has little impact on Ca2+ o homeostasis in normal adult humans.

Therefore, the coordinated interactions between Ca2+ o and these Ca2+ o-regulating hormones maintain Ca2+ o homeostasis.

Our understanding of how these cells sense Ca2+ o has progressed greatly following the molecular cloning of a Ca2+ o-sensing receptor (CaR). The CaR is expressed mainly on the parathyroid glands, distal tubules of the kidney and the C-cells.

Although the receptor is also present in intestinal epithelial cells, bone-forming osteoblasts, several nephron segments other than the distal tubule, and many other tissues and cell lines, we will focus on the role of the CaR in the control of PTH secretion in this chapter.

Expression cloning in Xenopus laevis oocytes enabled isolation of a 5.3 kb cDNA from bovine parathyroid gland encoding a CaR with pharmacological properties similar to those of the Ca2+ o-sensing mechanism in parathyroid cells.

Following the isolation of this novel receptor, nucleic acid hybridization-based techniques enabled the cloning of additional full-length CaRs from human and chicken parathyroid; rat, human and rabbit kidney; rat C-cells; and striatum of rat brain.

All of these subsequently cloned CaRs were highly homologous to the bovine parathyroid receptor (more than 90% identical in their amino acid sequences), indicating that they all derive from a common ancestral gene.

The human CaR gene resides on chromosome 3 (3q13.3-21) as previously predicted by linkage analysis of a disorder of Ca2+ o– sensing, familial hypocalciuric hypercalcemia.

The CaR belongs to family C of the G protein-coupled receptors (GPCRs), which comprises at least three different subfamilies that share more than 20% amino acid identity over their seven membrane-spanning domains.

Recent work has shown that there is some homology with the gamma-aminobutyric acid B, taste and vomeronasal odorant receptors as well as metabotropic glutamate receptors.

The CaR exhibits three principal structural domains: a very large NH2-terminal extracellular domain, seven transmembrane domains, and an intracellular COOH-terminal tail.

The CaR expressed on the cell surface comprises principally a dimeric form of the receptor when transfected into human embryonic kidney (HEK293) cells.

The two monomers within the dimeric CaR interact with one another, as documented by the fact that co-expressing two individually inactive mutant CaRs reconstitutes substantial biological activity.

In addition to intermolecular covalent disulfide bonds mediated by Cys-129 and Cys-131, noncovalent, possibly hydrophobic, interactions also contribute to the formation of the dimeric CaR.

Since the metabotropic glutamate receptor belongs to the same family of GPCRs and the structure of its extracellular domain has been solved, the CaR probably has a similar overall structure.

Binding to its ligand presumably induces a conformational change in the dimerized CaR. This current model has been termed the “Venus-flytrap model”.

This structure consists of two lobes each with alpha-helices and beta-sheets connected by a hinge region of three strands.

The glycosylation of at least three out of eight sites seems to be essential for the efficient cell surface expression of the CaR.

Although treatment with concanavalin A interferes with Ca2+ o-induced CaR signaling, perhaps by cross-linking receptors and interfering with their mobility within the plasma membrane, glycosylation is not critical for ligand binding and signal transduction.

The CaR has several protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites within its intracellular domains (the PKA sites are present in all species studied to date except the bovine CaR).

Phosphorylation at amino acid 888 in the CaR C-terminal tail reduces its coupling to mobilization of intracellular calcium (Ca2+ i) by interfering with receptor-mediated activation of phospholipase C (PLC).

This indicates that the action of PKC on the CaR could contribute, at least in part, to the PKC-mediated inhibition of high Ca2+ o-induced suppression of PTH.

Familial hypocalciuric hypercalcemia (FHH) is a generally benign, autosomal dominantly inherited condition, which usually presents as asymptomatic hypercalcemia accompanied by an inappropriately low rate of urinary calcium excretion (a calcium to creatinine clearance ratio of <0.01).

At least two-thirds of all FHH families harbor heterozygous inactivating mutations within the coding region of the CaR gene on chromosome 3.

The chromosomal location of the CaR gene was suggested by earlier linkage analyses of families with FHH.

Two families, however, despite having clinical features similar to those of FHH, have disease genes located at two additional chromosomal locations one on chromosome 19p13.3 and the other on chromosome 19q13.

These additional chromosomal locations for an FHH-like condition indicate the existence of factors modulating the expression/function of the CaR or of additional calcium sensing mechanisms.

In most FHH cases, serum intact PTH levels are within the normal range or slightly elevated, and are accompanied by mild hypercalcemia.

In some FHH cases, however, there can be a greater degree of hypercalcemia (over 3 mM) or an absence of hypocalciuria, which has been accompanied by enlargement of the parathyroid glands in some cases.

FHH families exhibit a mild increase in set point in in vivo Ca2+ o-PTH dynamic studies, indicating that the CaR plays a central role in setting the level of Ca2+ o.

Most of the mutations in the CaR gene are missense mutations that result in variable degrees of inactivation of the receptor.

Some missense or truncation mutations, however, totally inactivate the receptor. When co-transfected with the wild type receptor, some mutant CaRs can interfere with the normal CaR’s function, exerting a so-called dominant negative action that is mediated by the formation of wild type-mutant heterodimers with reduced function.

Such dominant negative mutations can cause elevations in serum calcium concentration that are greater than those observed with mutant receptors that do not exert such an effect on the wild type CaR.

Neonatal severe hyperparathyroidism (NSHPT) generally presents within the first few weeks postnatally with symptoms of severe hypercalcemia.

The marked hypercalcemia is due to severe primary hyperparathyroidism accompanied by the enlargement of all four parathyroid glands.

NSHPT, in some cases, can be fatal unless parathyroidectomy is performed in the neonatal period.

Functional examination of parathyroid glands resected from infants with NSHPT have revealed markedly abnormal Ca2+ o-regulated PTH release, with substantial increases in set point and, in some cases, severely impaired inhibition of secretion even at levels of Ca2+ o (e.g., 4 mM) higher than those encountered in vivo.

Cases of true NSHPT (e.g., as opposed to milder cases of neonatal hyperparathyroidism) are caused by the presence of homozygous or compound heterozygous mutations in the CaR and, as a result, the lack of any normal CaRs.

NSHPT has also been seen in infants heterozygous for CaR mutations in some families with mutations that exert a dominant negative effect of the wild type CaR.

Ablation of the CaR produces clinical and biochemical findings similar to those of FHH and NSHPT in mice heterozygous or homozygous, respectively, for knockout of the CaR gene.

In the homozygous CaR knockout mice, severe hypercalcemia is caused by high levels of PTH in association with parathyroid hyperplasia.

These mice generally die within the first week of life, similar to infants with NSHPT resulting from homozygous or compound heterozygous inactivating mutations in the CaR.

The heterozygous CaR knockout mice also exhibit findings similar to those of patients with FHH.

In these heterozygous mice, the expression level of the normal CaR is decreased in parathyroid and kidney, indicating that a normal level of expression of a normal CaR is essential for normal calcium homeostasis.

In contrast to inactivating mutations of the CaR, gain-of-function mutations in the CaR gene produce hypocalcemia.

Autosomal dominant hypoparathyroidism (ADH) is characterized by autosomal dominant inheritance of hypocalcemia accompanied by relative hypercalciuria and inappropriately low-normal or low serum PTH levels.

Some sporadic hypoparathyroidism cases caused by de novo mutations of the CaR gene have been also reported.

When vitamin D is administered to these patients, they are prone to the development of marked hypercalciuria, nephrocalcinosis, and renal stone formation, even while still hypocalcemic.

Therefore, it is important to monitor them carefully during therapy and to correct their hypocalcemia only to the point when their hypocalcemic symptom(s) is relieved and not beyond.

So far, more than 30 activating mutations have been found in the CaR gene, most of which are located in the extracellular domain.

When appropriate dynamic studies of parathyroid function are carried out and the curve relating PTH to calcium is drawn, inactivating mutations cause a rightward shift in the set point (e.g., the level of calcium at which PTH is halfway between the maximum and minimum of the curve).

Recent reports suggest that activating mutations of the CaR with high activity can develop Bartter’s syndrome through the inhibition of a renal outer medullary potassium channel.

In primary hyperparathyroidism as well as secondary hyperparathyroidism due to chronic renal failure, an increase in PTH release and increased cell proliferative activity are usually seen.

Although the possibility that an abnormality in the CaR gene could produce this pathological progression seemed reasonable, no mutations in the CaR gene were found in human parathyroid tumors.

So far, however, CaR expression is known to decrease at the mRNA and protein levels in the parathyroid glands from patients with primary and secondary hyperparathyroidism.

This alteration in CaR expression could potentially be related to the excessive PTH release and parathyroid cell proliferation seen with these conditions.

Although the factors regulating CaR expression are not fully understood, vitamin D is thought to be a candidate as suggested by an in vivo study of a renal failure model.

In fact, the promoter of the human CaR gene possesses the vitamin D responsive elements through which vitamin D stimulates transcription of the gene. At least in the rat, a single intraperitoneal injection of vitamin D lead to a 2-2.5 fold increase in CaR mRNA in parathyroid, thyroid, and kidney.

In addition, since there is a positive correlation between CaR and vitamin D receptor expression in both primary and secondary hyperparathyroidism, decreased expression of the CaR can be explained by a reduction in serum 1,25(OH)2D3 level and/or vitamin D receptor expression.

However, overexpression of cyclin D1 in the mouse parathyroid gland leads to increases in serum calcium and PTH, as well as enhanced parathyroid cell proliferation, associated with a decrease in CaR expression.

In uremic rats after switching from low phosphate diet to high phosphate diet, parathyroid cell proliferation is observed before reduction of the CaR.

Taken together, these findings indicate that CaR expression may be related to the cell cycle status of the parathyroid cells.

Other types of disorders related to the CaR have been described. Li et al found autoantibodies to the extracellular domain of the CaR in patients with autoimmune hypoparathyroidism.

The antibodies were present in 14 out of 25 patients with acquired hypoparathyroidism, 17 of whom had type I autoimmune polyglandular syndrome and 8 of whom had autoimmune hypothyroidism.

These authors did not find any evidence that these antibodies altered the function of the CaR, however, suggesting that the hypoparathyroidism was not caused by an activating effect of the antibodies on the CaR, analogous to that seen with ADH.

Recently, autoimmune hypocalciuric hypercalcemia caused by anti-CaR antibodies has been documented and must be added to the differential diagnosis of hypercalcemia.

In this report, four individuals from two kindreds presented with PTH-dependent hypercalcemia.

These patients showed findings similar to those in FHH (e.g., hypercalcemia accompanied by relative or absolute hypocalciuria), However, an autoimmune etiology was suspected since one of the patients had sprue and the other three had autoimmune thyroid disease.

The patients’ sera reacted with the cell surface of bovine parathyroid cells and stimulated PTH release from human parathyroid in vitro.

Since autoimmune hypocalciuric hypercalcemia is presumably acquired, the hypercalcemia might cause symptoms, unlike the generally asymptomatic nature of FHH.

Activation of PLC transiently increases the cytosolic calcium concentration (Ca2+ i) as a result of IP3-mediated release of intracellular calcium stores, and then produces a sustained increase in Ca2+ i due to calcium influx from the extracellular fluid. High Ca2+ o induces a pertussis toxin-sensitive inhibition of cAMP accumulation in bovine parathyroid cells, suggesting that CaR-mediated inhibition of adenylate cyclase could involve one or more isoforms of the inhibitory G protein, Gi.

However, this inhibitory action can result from an indirect mechanism mediated by transient increases in Ca2+ i, which then inhibits a calcium-sensitive adenylate cyclase, in some kidney cells.

Recent work has demonstrated that activation of the CaR leads to phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2), members of one of the mitogen-activated protein kinase (MAPK) families.

This stimulation of ERK1/ 2 was partially blocked by inhibitors of PKC as well as by pertussis toxin, suggesting the involvement of Gq/11-mediated activation of PLC and a Gi-dependent pathway, respectively.

Activation of this MAPK pathway was also inhibited by tyrosine kinase inhibitors, demonstrating the involvement of tyrosine kinases as well.

In normal human parathyroid cells, pretreatment with a specific inhibitor of the ERK1/2 pathway did not modify PTH release at low Ca2+ o but totally abrogated the inhibition of PTH secretion at high Ca2+ o, indicating that the ERK1/2 pathway could play a major role in high Ca2+ o-induced PTH suppression.

These workers also showed that CaR-induced ERK activation is mediated by PKC and, to a lesser extent, phosphoinositide 3 kinase (PI3K).

CaR-induced MAPK activation is also seen in HEK cells stably transfected with the CaR and in H-500 rat leydig cancer cells, and MAPK activation stimulates the secretion of parathyroid hormone-related protein (PTHrP) production in these cells.

Furthermore, MAPK activation is involved in high Ca2+ o-induced cell proliferation in rat-1 fibroblasts and other cell types.

Thus, the CaR can modulate cellular proliferation as well as hormonal secretion through MAPK activation.

The pharmacology of the CaR is divided into calcimimetics and calcilytics. Calcimimetics mimic or potentiate the action of Ca2+ o.

They can be subdivided into type I and type II ligands; type I ligands can activate the CaR in the absence of Ca2+ o and behave as true agonists, while type II ligands act as allosteric activators. Calcilytics are CaR antagonists.

The CaR can be activated by a surprising variety of ligands. These include inorganic diand trivalent cations (La3+, Gd3+, Ca2+, Ba2+, Sr2+, and Mg2+) as well as organic polycations including spermine, neomycin, polylysine and protamine.

These all behave as type I calcimimetics. In contrast, the phenylalkylamine, NPS R-568, selectively activates the CaR only in the presence of Ca2+ o.

Type II calcimimetic compounds like NPS R-568 shift the concentration-response curve for Ca2+ o to the left without affecting the maximal or minimal response.

Furthermore, the R-enantiomer is 10- to 100-fold more potent than the corresponding S-enantiomer.

The R-enantiomer, NPS R-568, increases Ca2+ i and inhibits PTH secretion from bovine parathyroid cells in vitro at concentrations between 3 and 100 nM, whereas NPS S-568 has no effect on either response at concentrations less than 3 µM.

Additional differences between the type I and type II calcimimetic ligands are the regions of the CaR on which they act.

The type I calcimimetic ligands are thought to act predominantly in the extracellular domain of the CaR, whereas type II calcimimetics of the phenylalkylamine class bind within the transmembrane domain.

Recent studies have shown that certain amino acids, particularly aromatic amino acids, can also potentiate the actions of calcium on the CaR.

Since they are markedly less effective at 1.0 mM calcium and below, they can be considered to be type II calcimimetics.

In contrast to the phenylalkylamines, however, amino acids have been shown to have a binding site on the extracellular domain of the CaR.

Oral administration of NPS R-568 to normal rats causes a rapid fall in plasma PTH levels associated with hypocalcemia.

Since calcimimetic induced-hypocalcemia results mostly from the ability of the compounds to inhibit PTH secretion, these compounds were recognized to have applicability for the treatment of hypercalcemic disorders caused by PTH excess.

In a pilot study, oral administration of NPS R-568 was effective in reducing plasma levels of PTH and Ca2+ in patients with primary HPT.

The inhibitory effects of NPS R-568 were readily reversible, and plasma PTH returned to pre-dose levels within 2 to 8 hours in a dose dependent manner.

In rats with secondary HPT resulting from chronic renal failure, oral administration of NPS R-568 caused a dose dependent decrease in plasma PTH levels.

The magnitude and rate of change in plasma PTH levels were similar to what was observed in normal animals.

Moreover, activation of the CaR with NPS R-568 markedly lowers circulating PTH levels irrespective of the severity of the secondary HPT or the magnitude of the hyperphosphatemia. Results from studies of calcimimetics in patients with secondary HPT are similar to those in the partially nephrectomized rat model.

In seven patients receiving hemodialysis who had secondary HPT, serum PTH level decreased by 40 to 60% 1 to 2 hours after administration of a single dose of NPS R-568 in both the low-dose group (40-80 mg) and the high-dose group (120-200 mg).

Although the PTH level increased gradually thereafter, it remained considerably lower than the basal level after 24 hours. In the high-dose group, there was a significant decrease in PTH level even 48 hours after administration.

The effects of NPS R-568 were also confirmed by a randomized, double-blind, placebo-controlled study in 21 patients undergoing hemodialysis.

Interestingly, NPS R-568 inhibited not only PTH secretion but also parathyroid growth in rats with renal insufficiency.

In addition, NPS R-568 prevented osteitis fibrosa, which is caused by an excessive action of PTH on bone, in the rat model of renal failure.

Despite these encouraging results, NPS R-568 (or R-467) has a couple of problems in terms of their potential commercialization.

The major problems with these compounds are their low bioavailability and differences in their metabolism depending on the genotypes within the general population.

Since these agents are metabolized primarily by CYP2D6, one of the cytochrome P-450 enzymes, much higher serum concentrations were reached in individuals with a form of this enzyme that has reduced biological activity.

This compound is not metabolized by CYP2D6 and has increased bioavailability. In ongoing clinical trials, AMG073 has produced results similar as those observed previously with NPS R-568 in patients with primary and secondary HPT.

PTH is known to have an anabolic action on bone, and PTH derived-peptides have been shown to be effective for the treatment of osteoporosis.

PTH has shown this anabolic effect only when administered intermittently (e.g., once daily subcutaneously).

Since calcilytics produce a transient increase in PTH secretion by “tricking” the parathyroid into thinking that the serum calcium concentration is low, they could provide another mechanism for producing the transient increase in PTH needed for obtaining the associated anabolic effect.

Moreover, calcilytic agents have an advantage over PTH in that they can be taken orally, whereas PTH must be injected.

Therefore, an in vivo study was performed to examine the effects of NPS 2143, a calcilytic compound, on bone.

In aged ovariectomized (OVX) rats, plasma PTH levels increased 5 fold or more within 30 min after administering NPS 2143 orally, which was followed by a remarkable increase in bone turnover.

When NPS 2143 was administered with or without 17β-estradiol, however, OVX rats treated with both agents showed a significant increase in BMD at the distal femur 5 weeks after treatment, compared with the groups treated with either NPS 2143 or 17β-estradiol alone.

These findings suggest that calcilytic compounds could increase bone mass in the presence of an antiresorptive agent.

Its most important functions to date are in maintaining calcium homeostasis, as demonstrated by the human diseases that result from activating or inactivating mutations in the CaR gene or mice with ablation of the gene.

The recent development of mice in which the homozygous genotype has been “rescued” by deletion of the PTH gene or the parathyroid glands should provide a useful model for understanding the CaR’s role in the numerous tissues expressing it.

Further studies are also required to understand the biochemical characteristics of the CaR and its signaling pathways, including MAPK.

Finally, calcimimetics will undoubtedly provide a very effective way of controlling hyperparathyroidism in patients with primary or uremic hyperparathyroidism, and calcilytics provide an effective means of producing pulses of PTH that could be useful in the treatment of osteoporosis.