Clinical lessons from the calcium-sensing receptor

The extracellular calcium ion (Ca(2+)(e))-sensing receptor (CaR) enables key tissues that maintain Ca(2+)(e) homeostasis to sense changes in the Ca(2+)(e) concentration. These tissues respond to changes in Ca(2+)(e) with functional alterations that will help restore Ca(2+)(e) to normal. For instance, decreases in Ca(2+)(e) act via the CaR to stimulate secretion of parathyroid hormone-a Ca(2+)(e)-elevating hormone-and to increase renal tubular calcium reabsorption; each response helps promote normalization of Ca(2+)(e) levels. Further work is needed to determine whether the CaR regulates other parameters of renal function (e.g. 1,25-dihydroxyvitamin D(3) synthesis, intestinal absorption of mineral ions, and/or bone turnover). Identification of the CaR has also elucidated the pathogenesis and pathophysiology of inherited disorders of mineral and electrolyte metabolism; moreover, acquired abnormalities of Ca(2+)(e)-sensing can result from autoimmunity to the CaR, and reduced CaR expression in the parathyroid may contribute to the abnormal parathyroid secretory control that is observed in primary and secondary hyperparathyroidism. Finally, calcimimetics-allosteric activators of the CaR-treat secondary hyperparathyroidism effectively in end-stage renal failure.     

L-amino acid sensing by the extracellular Ca2+-sensing receptor

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) recognizes and responds to (i.e., "senses") Ca(2+)(o) as its principal physiological ligand. In the present studies, we document that the CaR is activated not only by extracellular calcium ions but also by amino acids, establishing its capacity to sense nutrients of two totally different classes. l-Amino acids, especially aromatic amino acids, including l-phenylalanine and l-tryptophan, stereoselectively mobilized Ca(2+) ions in the presence of the CaR agonists, Ca(2+)(o), gadolinium (Gd(3+)(o)), and spermine in fura-2-loaded human embryonic kidney (HEK-293) cells stably transfected with the human CaR. l-amino acid-dependent effects were observed above, but not below, a threshold level of Ca(2+)(o) of approximately 1.0 mM. l-Amino acids, particularly aromatic amino acids, also stereoselectively enhanced the sensitivity of the CaR to its agonists, Ca(2+)(o) and spermine. Branched-chain amino acids were almost inactive, and charged amino acids, including arginine and lysine, were much less effective than aromatic and other amino acids. l-amino acid mixtures emulating the amino acid composition of fasting human plasma reproduced the effects of high concentrations of individual l-amino acids on Ca(2+) mobilization and enhanced the sensitivity of the CaR to Ca(2+)(o). The data presented herein identify the CaR as a molecular target for aromatic and other l-amino acids. Thus, the CaR can integrate signals arising from distinct classes of nutrients: mineral ions and amino acids. The actions of l-amino acids on the CaR may provide explanations for several long recognized but poorly understood actions of dietary protein on calcium metabolism.     

Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones

We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca2+]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca2+]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3beta, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor alpha1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [Ca2+]o, as was mineralized nodule formation. Alkaline phosphatase activity was maximal for [Ca2+]o between 1.2 and 1.8 mM. Inhibition of CaR by NPS 89636 blocked responses to the CaR agonists. In conclusion, we show that small deviations of [Ca2+]o from physiological values have a profound impact on bone cell fate, by means of the CaR and independently of systemic calciotropic peptides.     

Decreased expression of caveolin-1 and altered regulation of mitogen-activated protein kinase in cultured bovine parathyroid cells and human parathyroid adenomas

Caveolins are key components of caveolae membranes. The calcium-sensing receptor (CaR) resides within caveolin-rich membrane domains in bovine parathyroid (PT) cells. Recent studies reported reduced CaR expression, and abnormal calcium-sensing in PT tumors. To examine this altered CaR signaling, we investigated ERK activation after CaR stimulation in human and bovine PT cells. In freshly prepared bovine PT cells, high extracellular calcium (Ca(2+)(0)) stimulates ERK1/2 phosphorylation, and activated ERK1/2 colocalizes with caveolin-1 at the plasma membrane but fails to translocate to the nucleus, and cell proliferation is low. In cultured bovine PT cells, CaR and caveolin-1 levels are reduced; activated ERK1/2 localizes in the cell periphery at 10 min and in the perinuclear and nuclear regions at 60 min after exposure to high Ca(2+)(0), and cell proliferation is increased. In PT cells from adenomas, there are high levels of caveolin-2, variably reduced caveolin-1, and hyperactivation of ERK1/2, which colocalizes with caveolin-1 in some cells, but localizes in the cytosol and nucleus in others. Finally, caveolin-1 negative human PT cells exhibit reduced suppressibility of PTH secretion by high Ca(2+)(0). Thus, CaR and caveolin-1 colocalize in PT cells, and reduced levels of caveolin-1 could participate in the abnormal cellular function and proliferation of cultured bovine PT cells and PT adenomas.     

A syndrome of hypocalciuric hypercalcemia caused by autoantibodies directed at the calcium-sensing receptor

Antibodies to cell surface receptors can cause endocrine dysfunction by mimicking or blocking the actions of their respective hormones. We sought patients with autoantibodies to the extracellular calcium (Ca(2+)(o))-sensing receptor (CaR), which sets the normal level of blood calcium, that mimic the genetic disorder, familial hypocalciuric hypercalcemia, caused by heterozygous inactivating mutations of the CaR. Four individuals from two kindreds were identified with PTH-dependent hypercalcemia, who had other autoimmune manifestations: one with sprue and antigliadin and antiendomyseal antibodies and three with antithyroid antibodies. Three of the patients also had relative or absolute hypocalciuria. The patients' sera contained antibodies that reacted with the cell surface of bovine parathyroid cells in a manner similar to an authentic polyclonal anti-CaR antibody, stained bands on Western analysis of sizes similar to those labeled by the anti-CaR antiserum, and reacted with several synthetic peptides derived from sequences within the CaR's extracellular amino terminus. The patients' sera also stimulated PTH release from dispersed human parathyroid cells compared with the effect of sera from normocalcemic control subjects. This stimulation could be blocked by preabsorbing serum with membranes from CaR-transfected, but not nontransfected, human embryonic kidney (HEK293) cells. Finally, in two of the patients, antibodies affinity-purified using a synthetic peptide from within the CaR's extracellular domain inhibited high Ca(2+)(o)-stimulated, CaR-mediated accumulation of inositol phosphates and activation of mitogen-activated protein kinase in CaR-transfected HEK293 cells. DNA sequencing revealed no mutations within the index patients' CaR genes in the two families. Therefore, a biochemical phenotype of PTH-dependent hypercalcemia resembling that caused by heterozygous inactivating mutations of the CaR in familial hypocalciuric hypercalcemia can be observed in patients with antibodies to the CaR's extracellular domain that stimulate PTH release, probably by inhibiting activation of the CaR by Ca(2+)(o). Autoimmune hypocalciuric hypercalcemic is an acquired disorder of Ca(2+)(o) sensing that should be differentiated from that caused by inactivating mutations of the CaR.     

Functional desensitization of the extracellular calcium-sensing receptor is regulated via distinct mechanisms: role of G protein-coupled receptor kinases, protein kinase C and beta-arrestins

The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca(2+)(e)) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to beta-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and beta-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca(2+)(e)-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G alpha(q) binding (D110A) was without major effect. Overexpression of GRK 4-6 did not reduce Ca(2+)(e)-dependent inositol phosphate accumulation. Overexpression of beta-arrestin 1 or 2 revealed a modest inhibitory effect on Ca(2+)(e)-dependent inositol phosphate production (20-30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10-15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to beta-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to G alpha(q).     

Calcium-sensing receptor induces proliferation through p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase but not extracellularly regulated kinase in a model of humoral hypercalcemia of malignancy

Using H-500 rat Leydig cancer cells as a model of humoral hypercalcemia of malignancy (HHM), we previously showed that high Ca(2+) induces PTH-related peptide (PTHrP) secretion via the calcium-sensing receptor (CaR) and mitogen- and stress-activated kinases, e.g. MAPK kinase 1 (MEK1), p38 MAPK, and stress-activated protein kinase 1/c-Jun N-terminal kinase. Because cellular proliferation is a hallmark of malignancy, we studied the role of the CaR in regulating the proliferation of H-500 cells. Elevated Ca(2+) has a mitogenic effect on these cells that is mediated by the CaR, because the calcimimetic NPS R-467 also induced proliferation. Inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 MAPK but not MEK1 abolished the mitogenic effect. Activation of PI3K by elevated Ca(2+) was documented by phosphorylation of its downstream kinase, protein kinase B. Because protein kinase B activation promotes cell survival, we speculated that elevated Ca(2+) might protect H-500 cells against apoptosis. Using terminal uridine deoxynucleotidyl nick end labeling staining, we demonstrated that high Ca(2+) (7.5 mM) and NPS R-467 indeed protect cells against apoptosis induced by serum withdrawal compared with low Ca(2+) (0.5 mM). Because the CaR induces PTHrP secretion, it is possible that the mitogenic and antiapoptotic effects of elevated Ca(2+) could be indirect and mediated via PTHrP. However, blocking the type 1 PTH receptor with PTH (7-34) peptide did not alter either high Ca(2+)-induced proliferation or protection against apoptosis. Taken together, our data show that activation of PI3K and p38 MAPK but not of MEK1/ERK by the CaR promotes proliferation of H-500 cells as well as affords protection against apoptosis. These effects are likely direct without the involvement of PTHrP in an autocrine mode.     

Physiology and pathophysiology of the extracellular calcium-sensing receptor

The system governing extracellular calcium (Ca2+o) homeostasis maintains near constancy of Ca2+o so as to ensure continual availability of calcium ions for their numerous intracellular and extracellular roles. In contrast to the intracellular ionized calcium concentration (Ca2+i), which varies substantially during intracellular signaling via this key second messenger, Ca2+o remains nearly invariant. Yet there must be a mechanism that senses small changes in Ca2+o so as to set into motion the homeostatic responses that return Ca2+o to its normal level. The recent identification and molecular cloning of the mechanism through which parathyroid cells and a number of other cell types sense Ca2+o, a G protein-coupled Ca2+o-sensing receptor (CaR), has proven unequivocally that extracellular calcium ions serve in an informational capacity. The CaR permits Ca2+o to function in a hormone-like role as an extracellular first messenger through which parathyroid, kidney, and other cells communicate with one another via the CaR. The identification of inherited human hypercalcemic and hypocalcemic disorders arising from inactivating and activating mutations of the CaR, respectively, has provided additional proof of the essential, nonredundant role of the CaR in mineral ion homeostasis. Moreover, CaR-active drugs are currently in clinical trials for the treatment of primary and uremic hyperparathyroidism, disorders in which there are acquired, tissue-specific reductions in CaR expression and, in turn, defective Ca2+o-sensing by pathological parathyroid cells. No doubt further studies of Ca2+o-sensing by the CaR will reveal additional functions of Ca2+o, not only as a systemic "hormone" but also in local, paracrine, and autocrine signaling through this novel Ca2+o-sensing receptor.     

Ca(2+) receptor from brain to gut: common stimulus, diverse actions.

An extracellular Ca(2+)-sensing receptor (CaR) plays central roles in Ca(2+) homeostasis by regulating parathyroid hormone (PTH)secretion and renal Ca(2+) handling. The CaR is also expressed in intestine and bone, where its functions in mineral metabolism are not yet well defined. The receptor is also present in various types of cells seemingly uninvolved in systemic mineral ion homeostasis (such as neuronal and glial cells in the brain and various epithelial cells), where its actions are poorly understood but might involve the regulation of local ionic homeostasis and/or diverse cellular processes, such as cellular differentiation and proliferation.     

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.     

Calcium sensing in cultured chondrogenic RCJ3.1C5.18 cells

The availability of Ca2+ in the extracellular fluid plays an important role in regulating cartilage and bone formation. We hypothesized that chondrocytes detect changes in the extracellular [Ca2+] ([Ca2+]o) and modify their function. The effects of changing [Ca2+]o on the expression of matrix proteins were quantified by staining of cartilage nodules with alcian green and assessing RNA levels of cartilage-specific genes in chondrogenic RCJ3.1C5.18 (C5.18) cells. Alcian green staining in these cells decreased with increasing [Ca2+]o in a dose-dependent and reversible manner (ID50, approximately 2 mM Ca2+). RNA levels for aggrecan and type II collagen decreased with increasing [Ca2+]o (ID50, approximately 2.0 and 4.1 mM Ca2+, respectively). RNA levels for type X collagen and alkaline phosphatase were also reduced by high [Ca2+]o with ID50 values of approximately 2.9 and 1.6 mM Ca2+, respectively. These responses were rapid, in that increasing [Ca2+]o from 1.0 to more than 6 mM suppressed aggrecan RNA levels by about 50%, and lowering [Ca2+]o from 2.9 to 1.0 mM increased aggrecan RNA levels by about 300% within 4 h. As Ca2+ receptors (CaRs) mediate extracellular Ca2+ sensing in parathyroid and kidney, we assessed the expression of CaRs in these cells. C5.18 cells stained positively for CaR protein with an anti-CaR antiserum and for CaR RNA by in situ hybridization. An approximately 150-kDa protein was detected by immunoblotting with anti-CaR antiserum. CaR antisense oligonucleotides suppressed the expression of CaR protein and enhanced RNA levels of aggrecan in C5.18 cells. These data support the idea that CaRs are expressed in this cell system and may be involved in regulating chondrogenic gene expression.     

Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells

The calcium-sensing receptor (CaR) activation has recently been shown to modulate the ERK1 and ERK2 cascade in different cell lines. The present study investigated this pathway in human normal and tumoral parathyroid cells. In cells from normal parathyroids and almost all hyperplasia increasing extracellular calcium concentrations (Ca(o)(2+)) induced a significant activation of ERK1 and -2, the percent stimulation over basal activity (at 0.5 mM Ca(o)(2+)) being 545 +/- 140 and 800 +/- 205 in normal cells and 290 +/- 71 and 350 +/- 73 in hyperplasia at 1 and 2 mM Ca(o)(2+), respectively. This effect was mediated by CaR because it was mimicked by the receptor agonist gadolinium and neomycin. Basal and Ca(o)(2+)-stimulated ERK1 and -2 activity was nearly abolished by the PKC inhibitor calphostin C, and PKA changes did not affect ERK1 and -2 activity. PI3K blockade by wortmannin, known to prevent G protein betagamma subunit effect on ERK1 and -2, induced a 30% reduction of the Ca(o)(2+)-stimulated ERK1 and -2 activity. Adenomatous cells showed high PKC-dependent ERK1 and -2 activity in resting conditions that was unresponsive to high Ca(o)(2+). A role of MAPK on PTH secretion was suggested by the finding that PD98059, a specific MEK inhibitor, abolished the inhibitory effect of 1.5 mM Ca(o)(2+) on PTH release from normal parathyroid cells. In conclusion, these data first demonstrate that CaR activation, through the PKC pathway and, to a lesser extent, PI3K, increases ERK1 and -2 activity in normal parathyroid cells and this cascade seems to be involved in the modulation of PTH secretion by Ca(o)(2+). Interestingly, this signaling pathway is disrupted in parathyroid tumors.     

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.     

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.     

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.     

Parathyroid gland calcium receptor mRNA levels are unaffected by chronic renal insufficiency or low dietary calcium in rats

Extracellular ionized calcium (Ca(2+)) is the primary physiological regulator of parathyroid hormone (PTH) secretion and the G protein-coupled receptor (CaR) that mediates this response has been cloned from bovine and human parathyroid glands. The Ca(2+) set-point for the regulation of PTH secretion is right-shifted in primary hyperparathyroidism (1°HPT), but whether there is a similar shift in 2°HPT is unclear. Additionally, the molecular defects associated with such changes in the set-point remain uncharacterized. These experiments were designed to determine (1) if changes in set-point occur in rats with 2°HPT induced by chronic renal insufficiency (CRI) or dietary Ca deficiency, and (2) whether any changes in set-point are mirrored by changes in steady-state mRNA levels for the parathyroid CaR. CaR mRNA levels were quantified in pairs of glands from individual rats using a solution hybridization assay. Blood urea nitrogen and PTH levels were ～ 4-fold higher in rats with CRI induced by 5/6 nephrectomy 7 weeks earlier. Rats with CRI were also significantly hypocalcemic and hyperphosphatemic. The setpoint was unchanged in CRI rats and CaR mRNA levels were also unaffected. Normal rats fed a 0.02% Ca diet for 6 weeks were markedly hypocalcemic, and had 10- and 15-fold increases in plasma PTH and 1,25-dihydroxyvitamin D(3) levels, respectively. Technical problems prevented assessment of the set-point in these animals, but parathyroid gland CaR mRNA levels were identical in both dietary groups. Thus, neither alterations in mRNA levels for the CaR nor changes in the set-point play demonstrable roles in the pathogenesis of 2°HPT in these models.     

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.