European Society of Endocrinology: European Society of Endocrinology Clinical Guideline: treatment of chronic hypoparathyroidism in adults

Hypoparathyroidism (HypoPT) is a rare (orphan) endocrine disease with low calcium and inappropriately low (insufficient) circulating parathyroid hormone levels, most often in adults secondary to thyroid surgery. Standard treatment is activated vitamin D analogues and calcium supplementation and not replacement of the lacking hormone, as in other hormonal deficiency states. The purpose of this guideline is to provide clinicians with guidance on the treatment and monitoring of chronic HypoPT in adults who do not have end-stage renal disease. We intend to draft a practical guideline, focusing on operationalized recommendations deemed to be useful in the daily management of patients. This guideline was developed and solely sponsored by The European Society of Endocrinology, supported by CBO (Dutch Institute for Health Care Improvement) and based on the Grading of Recommendations Assessment, Development and Evaluation (GRADE) principles as a methodological base. The clinical question on which the systematic literature search was based and for which available evidence was synthesized was: what is the best treatment for adult patients with chronic HypoPT? This systematic search found 1100 articles, which was reduced to 312 based on title and abstract. The working group assessed these for eligibility in more detail, and 32 full-text articles were assessed. For the final recommendations, other literature was also taken into account. Little evidence is available on how best to treat HypoPT. Data on quality of life and the risk of complications have just started to emerge, and clinical trials on how to optimize therapy are essentially non-existent. Most studies are of limited sample size, hampering firm conclusions. No studies are available relating target calcium levels with clinically relevant endpoints. Hence it is not possible to formulate recommendations based on strict evidence. This guideline is therefore mainly based on how patients are managed in clinical practice, as reported in small case series and based on the experiences of the authors.(AU)

Changes in vertebral bone marrow fat and bone mass after gastric bypass surgery: A pilot study.

Bone marrow fat may serve a metabolic role distinct from other fat depots, and it may be altered by metabolic conditions including diabetes. Caloric restriction paradoxically increases marrow fat in mice, and women with anorexia nervosa have high marrow fat. The longitudinal effect of weight loss on marrow fat in humans is unknown. We hypothesized that marrow fat increases after Roux-en-Y gastric bypass (RYGB) surgery, as total body fat decreases. In a pilot study of 11 morbidly obese women (6 diabetic, 5 nondiabetic), we measured vertebral marrow fat content (percentage fat fraction) before and 6 months after RYGB using magnetic resonance spectroscopy. Total body fat mass declined in all participants (mean ± SD decline 19.1 ± 6.1 kg or 36.5% ± 10.9%, p<0.001). Areal bone mineral density (BMD) decreased by 5.2% ± 3.5% and 4.1% ± 2.6% at the femoral neck and total hip, respectively, and volumetric BMD decreased at the spine by 7.4% ± 2.8% (p<0.001 for all). Effects of RYGB on marrow fat differed by diabetes status (adjusted p=0.04). There was little mean change in marrow fat in nondiabetic women (mean +0.9%, 95% CI -10.0 to +11.7%, p=0.84). In contrast, marrow fat decreased in diabetic women (-7.5%, 95% CI -15.2 to +0.1%, p=0.05). Changes in total body fat mass and marrow fat were inversely correlated among nondiabetic (r=-0.96, p=0.01) but not diabetic (r=0.52, p=0.29) participants. In conclusion, among those without diabetes, marrow fat is maintained on average after RYGB, despite dramatic declines in overall fat mass. Among those with diabetes, RYGB may reduce marrow fat. Thus, future studies of marrow fat should take diabetes status into account. Marrow fat may have unique metabolic behavior compared with other fat depots.

Postmenopausal women treated with combination parathyroid hormone (1-84) and ibandronate demonstrate different microstructural changes at the radius vs. tibia: the PTH and Ibandronate Combination Study (PICS).

SUMMARY: In postmenopausal women receiving combination parathyroid hormone (PTH) (1-84) therapy and ibandronate, we evaluated bone microarchitecture and biomechanics using high-resolution peripheral quantitative computed tomography (HR-pQCT). Cortical and trabecular changes were different at the nonweight-bearing radius vs. the weight-bearing tibia, with more favorable overall changes at the tibia. INTRODUCTION: PTH therapy and bisphosphonates decrease fracture risk in postmenopausal osteoporosis, but their effects on bone microstructure and strength have not been fully characterized, particularly during combination therapy. PTH increases trabecular bone mineral density (BMD) substantially but may decrease cortical BMD, possibly by stimulating intracortical remodeling. We evaluated bone microarchitecture and biomechanics with HR-pQCT at the radius (a nonweight-bearing site) and tibia (weight bearing) in women receiving combination PTH(1-84) and ibandronate. METHODS: Postmenopausal women with low bone mass (n = 43) were treated with 6 months of PTH(1-84) (100 µg/day), either as one 6- or two 3-month courses, in combination with ibandronate (150 mg/month) over 2 years. HR-pQCT was performed before and after therapy. RESULTS: Because changes in HR-pQCT parameters did not differ between treatment arms, groups were pooled into one cohort for analysis. Trabecular BMD increased at both radius and tibia (p < 0.01 for each). Cortical thickness and BMD decreased at the radius (p < 0.01), consistent with changes in dual-energy X-ray absorptiometry, while these parameters did not change at the tibia (p ≤ 0.02 for difference between radius and tibia). In contrast, cortical porosity increased at the tibia (p < 0.01) but not radius. Stiffness and failure load decreased at the radius (p < 0.0001) but did not change at the tibia. CONCLUSIONS: Cortical and trabecular changes in response to the PTH/ibandronate treatment combinations utilized in this study were different at the nonweight-bearing radius vs. the weight-bearing tibia, with more favorable overall changes at the tibia. Our findings support the possibility that weight bearing may optimize the effects of osteoporosis therapy.

Cinacalcet hydrochloride reduces the serum calcium concentration in inoperable parathyroid carcinoma.

BACKGROUND: Management of inoperable parathyroid carcinoma presents a challenge because until recently, effective medical therapy was not available. Morbidity and mortality result primarily from severe hypercalcemia. We assessed the ability of the calcimimetic cinacalcet HCl to reduce serum calcium in patients with parathyroid carcinoma as well as its effect on PTH concentrations, bone turnover markers, safety, and health-related quality of life variables. METHODS: Twenty-nine patients with parathyroid carcinoma were enrolled in this open-label, single-arm study consisting of titration and maintenance phases. Cinacalcet doses were titrated (30 mg twice daily to 90 mg four times daily) for 16 wk or until serum calcium was no more than 10.0 mg/dl. The study endpoint was the proportion of patients with at least a 1 mg/dl reduction in serum calcium at the end of the titration phase (responders). RESULTS: Mean (+/- se) serum calcium (14.1 +/- 0.4 mg/dl) and PTH (697 +/- 94 pg/ml) were markedly elevated at baseline. At the end of the titration period, serum calcium was reduced by at least 1 mg/dl in 62% of patients (mean decline to 12.4 +/- 0.5 mg/dl). In the 18 responders, serum calcium fell from 15.0 +/- 0.5 to 11.2 +/- 0.3 mg/dl (P < 0.001). The greatest reductions in serum calcium were observed in patients with highest baseline calcium levels. PTH levels decreased, but not significantly, to 635 +/- 73 pg/ml (-4.6%). Adverse events included nausea, vomiting, headache, and fracture. CONCLUSIONS: Cinacalcet effectively reduces hypercalcemia in approximately two thirds of patients with inoperable parathyroid carcinoma and may represent an important new treatment option for these patients.

Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation.

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca(2+)] ([Ca(2+)](o)) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca(2+)](o) increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca(2+)](o) (up to 30 mm) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser(866) and Val(895) using tandem-Ala and single-site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca(2+)](o)-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha-helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.

Parathyroid cells express dihydropyridine-sensitive cation currents and L-type calcium channel subunits.

Parathyroid cells express Ca2+ -conducting currents that are activated by raising the extracellular Ca2+ concentration ([Ca2+]o). We investigated the sensitivity of these currents to dihydropyridines, the expression of voltage-dependent Ca(2+) channel (VDCC) subunits, and the effects of dihydropyridines on the intracellular free [Ca2+] ([Ca2+]i) and secretion in these cells. Dihydropyridine channel antagonists dose dependently suppressed Ca2+ -conducting currents, and agonists partially reversed the inhibitory effects of the antagonists in these cells. From a bovine parathyroid cDNA library, we isolated cDNA fragments encoding parts of an alpha(1S)- and a beta(3)-subunit of L-type Ca(2+) channels. The alpha(1S)-subunit cDNA from the parathyroid represents an alternatively spliced variant lacking exon 29 of the corresponding gene. Northern blot analysis and immunocytochemistry confirmed the presence of transcripts and proteins for alpha(1)- and beta(3)-subunits in the parathyroid gland. The addition of dihydropyridines had no significant effects on high [Ca2+]o-induced changes in [Ca2+]i and parathyroid hormone (PTH) release. Thus our studies indicate that parathyroid cells express alternatively spliced L-type Ca2+ channel subunits, which do not modulate acute intracellular Ca2+ responses or changes in PTH release.

Analysis of inositol phospholipid turnover during lymphocyte activation.

Receptor-mediated activation of phospholipase C (PLC) leads to the hydrolysis of membrane inositol phospholipids, generating diacylglycerol (DAG) and water-soluble inositol phosphates. This signaling mechanism is used by antigen receptors on T and B cells that have been implicated as mediators of receptor-induced influx of extracellular Ca(2+). This unit provides protocols that describe the resolution of InsP by Dowex anion-exchange chromatography. This technique provides a reliable means of separating inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, but does not resolve isomers of these. An Alternate Protocol describes the separation of inositol phosphates by anion-exchange HPLC. A protocol for resolution of inositol phospholipids by thin-layer chromatography (TLC) is also provided.

Paget's disease of bone: diagnosis and treatment update.

Paget's disease is a metabolic bone disease characterized by excessive bone resorption and formation due to activated osteoclasts. Although Paget's disease is a high bone turnover state, the excess bone that is formed lacks the structural stability of normal bone. Complications from Paget's disease include deformity, fracture, and pain. Although still unclear, both prevalence and severity of Paget's disease seem to be declining. Recent progress has focused on the environmental as well as genetic etiologies for this disease. Many studies indicate a role for viral infectious agents, whereas others point to a recently identified candidate gene on chromosome 18q. Therapy with bisphosphonate drugs is the treatment of choice. With newer and more powerful agents from this family now available, the majority of patients affected by Paget's disease can achieve sustained remission and avoid complications.

Amino acids in the second and third intracellular loops of the parathyroid Ca2+-sensing receptor mediate efficient coupling to phospholipase C.

To determine the role of amino acids in the second and third intracellular (IC) loops of the Ca(2+)-sensing receptor (CaR) in phospholipase C (PLC) activation, we mutated residues in these loops either singly or in tandem to Ala and assessed PLC activity by measuring high extracellular [Ca(2+)] ([Ca(2+)](o))-induced inositol phosphate accumulation and protein expression by immunoblotting and immunocytochemistry in human embryonic kidney 293 cells. Two CaR constructs in the second IC loop, F707A CaR and to a lesser extent L704A CaR, demonstrated reduced activation of PLC, despite levels of protein expression comparable with the wild-type (wt) CaR. Substitution of Tyr or His for Phe-707, but not Leu, Val, Glu, or Trp, partially restored the ability of high [Ca(2+)](o) to activate PLC. Eight residues in the third IC loop were involved in PLC signaling. The responses to high [Ca(2+)](o) in cells expressing CaRs with Ala substitutions at these sites were <35% of the wt CaR. The L798A, F802A, and E804A CaRs were dramatically impaired in their responses to [Ca(2+)](o) even up to 30 mm. Substitutions of Leu-798 with other hydrophobic residues (Ile, Val, or Phe), but not with acidic, basic, or polar residues, produced reduced responses compared with wt. Phe-802 could be replaced with either Tyr or Trp with partial retention of the ability to activate PLC. Glu-804 could only be substituted with Asp or Gln and maintain its signaling capacity. Cell surface expression of the CaRs mutated at Leu-798 and Phe-802 appeared normal compared with wt CaR. Cell surface CaR expression was, however, reduced substantially in cells expressing several mutants at position Glu-804 by confocal microscopy. These studies strongly implicate specific hydrophobic and acidic residues in the second and third IC loops of the parathyroid CaR (and potentially larger stretches of the third loop) in mediating efficient high [Ca(2+)](o)-induced PLC activation and or CaR expression.

Expression and signal transduction of calcium-sensing receptors in cartilage and bone.

We previously showed that Ca2+-sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca2+]([Ca2+]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (approximately 140-190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca2+]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca2+]o reduced PTH-induced cAMP formation. Raising [Ca2+]o also increased intracellular free [Ca2+] in SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca2+]o may couple to signaling pathways important in skeletal metabolism.

Clinical effects of raloxifene hydrochloride in women.

PURPOSE: To review clinical data on raloxifene hydrochloride, a selective estrogen receptor modulator that was recently approved for the prevention of osteoporosis in postmenopausal women. DATA SOURCES: English-language articles published from 1980 to May 1998 were identified through MEDLINE searches. Bibliographies, book chapters, and meeting abstracts were reviewed for additional relevant publications. STUDY SELECTION: Publications that contained information on the background of development, structure, mechanism of action, tissue-selective effects, and adverse effects of raloxifene hydrochloride were included. DATA EXTRACTION: Data in selected articles were reviewed, and relevant clinical information was extracted. DATA SYNTHESIS: Raloxifene hydrochloride was developed in an effort to find a treatment for breast cancer and osteoporosis. It binds to the estrogen receptor and shows tissue-selective effects; thus, it belongs to a class of drugs recently described as selective estrogen receptor modulators. Tissue selectivity of raloxifene may be achieved through several mechanisms: the ligand structure, interaction of the ligand with different estrogen receptor subtypes in various tissues, and intracellular events after ligand binding. Raloxifene has estrogen-agonistic effects on bone and lipids and estrogen-antagonistic effects on the breast and uterus. An increase in bone mineral density at the spine, total hip, and total body has been reported with raloxifene but seems to be less than that seen with estrogen or alendronate therapy. Raloxifene has been shown to produce a reduction in total and low-density lipoprotein cholesterol concentrations similar to that produced by estrogen therapy, but high-density lipoprotein cholesterol and triglyceride concentrations do not increase during raloxifene therapy. In the uterus, raloxifene does not stimulate the endometrium. Long-term data on the effects of raloxifene in reduction of risk for fracture; prevention of cardiovascular events; cognitive function; and the incidence of breast, ovarian, and uterine cancer are not available. The most common adverse effect of raloxifene is hot flashes. CONCLUSIONS: Raloxifene has been shown to have beneficial effects in selected organs in postmenopausal women. Although estrogen remains the drug of choice for hormonal therapy in most postmenopausal women, raloxifene may be an alternative in certain groups of women at risk for osteoporosis.

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.

Protein kinase C activation blocks calcium receptor signaling in Xenopus laevis oocytes.

We examined whether calcium receptor (CaR) signaling is affected by protein kinase C (PKC) activation by assessing the effects of phorbol-12-myristate-13-acetate (PMA) on 45Ca2+ efflux from Xenopus laevis oocytes expressing wild-type (WT) and mutant bovine parathyroid CaRs. Raising extracellular [Ca2+] ([Ca2+]0) from 0.5 to 5.5 mM increased 45Ca efflux (26 +/- 3-fold) in oocytes expressing full-length and C-terminally truncated receptor (amino acid 1-895). These increases in 45Ca efflux were blocked by 88 +/- 3% after PMA treatment for 20 min. Three consensus PKC phosphorylation sites (Thr-647, Ser-795, and Thr-889) were mutated in the context of the full-length and truncated CaR. PMA treatment inhibited high [Ca2+]0-induced responses in oocytes expressing the Ser795Ala CaR (1-895), Thr889Ala CaR (1-895), and Ser795Ala/Thr889Ala CaR (1-895) by 30-40% compared with untreated controls (P < 0.05). A triple mutant of the full-length CaR demonstrated similarly reduced susceptibility to inhibition of 45Ca efflux by PMA. Thus, these sites are important in mediating the effects of PKC activation on CaRs, but other residues and effector molecules are likely to participate in the effects of PKC on CaR-induced signal transduction in target cells.

Hypercalcemia in malignant paraganglioma due to parathyroid hormone-related protein.

A 15-year-old boy had hypercalcemia in association with malignant retroperitoneal paraganglioma. He had suppressed circulating levels of intact parathyroid hormone, whereas parathyroid hormone-related protein (PTHrP) immunoreactivity was elevated in plasma. Both the serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels were normal. Preoperatively the patient required control of hypercalcemia with intravenous pamidronate therapy. His circulating calcium and PTHrP concentrations became normal after a successful surgical resection of the primary retroperitoneal tumor. To our knowledge, this is the first reported case of elevated PtHrP levels in a patient with paraganglioma which resolved postoperatively.

Regulation of extracellular calcium-activated cation currents by cAMP in parathyroid cells.

Parathyroid cells express Ca2+-sensing receptors that couple changes in the extracellular Ca2+ concentration ([Ca2+]o) to increases in the intracellular free Ca2+ concentration ([Ca2+]i) and to the suppression of parathyroid hormone secretion. Using whole cell patch clamping, we previously identified voltage-independent Ca2+-conducting currents in bovine parathyroid cells that increased with rising [Ca2+]o and were blocked by Cd2+ and nifedipine. Because cAMP-dependent phosphorylation regulates dihydropyridine-sensitive Ca2+ channels in other systems, we tested whether cAMP modulates these currents. At 0.7 mM Ca2+, nonselective Ca2+-conducting currents were suppressed by 30-50% when the recording pipette was perfused with cAMP. High-[Ca2+]o-induced increases in membrane currents were also abrogated. The effects of cAMP were reversible and dose dependent (3 x 10(-9) to 3 x 10(-3) M) and required ATP in the pipette solution. Perfusion of the cell interior with the catalytic subunit of protein kinase A mimicked the effects of cAMP, as did perfusion of the bath with the adenylate cyclase activator forskolin. These findings support the idea that cAMP-dependent phosphorylation suppresses high-[Ca2+]o-induced cation currents and may play a role in regulating ion fluxes in parathyroid cells.

Clinical profile of primary hyperparathyroidism in adolescents and young adults.

OBJECTIVE: Primary hyperparathyroidism (PHPT) is an uncommonly diagnosed condition among adolescents and young adults. We review the clinical characteristics of these patients based on our institutional experience. SUBJECTS: Patients aged 12-28 years treated for PHPT at our institution from 1990 to 1996 were evaluated by a review of medical records and current follow-up data. This consisted of 22 patients (8M:14F), constituting approximately 3% of all patients operated for PHPT during this period. MEASUREMENTS: Serum and urinary calcium concentrations, renal function, and serum intact parathyroid hormone (IPTH) levels were measured in all patients. After biochemical confirmation of diagnosis, the patients completed a questionnaire to evaluate the presence of symptoms and/or conditions associated with PHPT. All the patients underwent parathyroidectomy and their tumour characteristics were evaluated. Surgical outcome was determined by measurements of serum calcium and IPTH levels postoperatively and during long-term follow-up. RESULTS: A third of the patients were diagnosed by routine serum chemistry whereas two-thirds presented with symptoms or conditions associated with hypercalcaemia. Non-specific complaints such as fatigue or exhaustion, and weakness or lethargy constitute the most common findings on questionnaire review. A family history of PHPT was present in only 2 patients. The preoperative peak serum calcium levels ranged from 2.67 to 4.19 mmol/l (norm: 2.10-2.54 mmol/l), with a median of 3.07 mmol/l. Surgical pathologies revealed 59% solitary adenoma, 27% hyperplasia, 9% multiple adenomas and 5% carcinoma. Comparison between the adolescents (aged 12-18 years) and young adults (aged 19-28 years) revealed no differences in the clinical, pathological or laboratory profiles, except for a male predominance in adolescent patients. Fifteen patients had resection of one or more adenomas while 7 underwent subtotal parathyroidectomy. Six patients (27%) were reoperated cases, all received primary treatment elsewhere. All patients with benign PHPT were cured surgically, with a median follow-up of 47 months (range 3-77 months). One reoperated patient developed permanent hypocalcaemia. One patient with carcinoma underwent several operations for recurrence; he is now eucalcaemic despite persistent disease at 80 months from diagnosis. CONCLUSIONS: We found a high incidence of multiglandular disease and relatively non-specific symptomatology in our adolescent and young adult patients with primary hyperparathyroidism. In view of the heterogeneous clinical expression noted in young patients, one should consider primary hyperparathyroidism in the differential diagnosis of unexplained non-specific complaints, and perform serum calcium estimations more readily in these subjects. Our experience suggests that primary hyperparathyroidism can be a serious disease with significant morbidity if left untreated, whereas parathyroidectomy provides successful results.

Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera.

Ca2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell-surface Ca2+-sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca2+ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in different expression systems. In Xenopus laevis oocytes injected with bovine parathyroid CaR cRNA, the addition of extracellular cations to 1.5 mM Ca2+, 5.5 mM Mg2+, or 10 microM Gd3+ significantly increased 45Ca efflux (p < 0.01). InsP accumulation also increased dramatically when adding these cations to human embryonic kidney (HEK) 293 cells stably transfected with wild-type bovine parathyroid CaR cDNA. Raising the extracellular [Ca2+] ([Ca2+]o) from 0.1 to > 1.4 mM in oocytes and to > 1.0 mM in HEK 293 cells stimulated significant increments in 45Ca efflux and InsP accumulation, respectively (p < 0.05). In contrast, Ca2+ and Mg2+ increased InsPs to a lesser extent in COS 7 cells transiently transfected with CaR cDNA. In HEK 293 cells stably expressing CaR cDNA, there were significant reductions in cAMP content when adding high Ca2+, Mg2+, Gd3+, or the CaR modulator NPS R-467. Three region-specific anti-CaR peptide antisera immunoblotted bands of approximately 140 and 155 kDa in membranes from CaR-transfected HEK 293 cells and bovine parathyroid tissue. Immunocytochemistry demonstrated strong cell-surface staining in CaR-transfected HEK 293 cells and parathyroid tissue, which was absent when antisera were preabsorbed with CaR peptides. These results indicate that the activation of the recombinant CaR by extracellular Ca2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca2+]o.

Parathyroid Ca(2+)-conducting currents are modulated by muscarinic receptor agonists and antagonists.

Parathyroid cells express Ca(2+)-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration approximately equal to 10(-8) M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution (+)-Muscarine enhanced the adenosine 3',5'-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed > 90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.