Ca2+-activated K+ channels modulate membrane potential in the human parathyroid cell: Possible role in exocytosis.

The relationships between parathyroid hormone (PTH) secretion and parathyroid cell membrane potential, including the identities and roles of K+ channels that regulate and/or modulate membrane potential are not well defined. Here we have used Western blot/immunohistochemistry as well as patch-clamp and perifusion techniques to identify and localize specific K+ channels in parathyroid cells and to investigate their roles in the control of membrane potential and PTH secretion. We also re-investigated the relationship between membrane potential and exocytosis. We showed that in single human parathyroid cells K+ current is dependent on at least two types of Ca2+-activated K+ channels: a small-conductance Ca2+-activated K+ channel (KSK) and a large-conductance voltage and Ca2+-activated K+ channel (KBK). These channels were sensitive to specific peptide blocking toxins including apamin, charybdotoxin, and iberiotoxin. These channels confer sensitivity of the membrane potential in single cells to high extracellular K+, TEA, and peptide toxins. Blocking of KBK potently inhibited K+ channel current, and KBK was shown to be expressed in the plasma membrane of parathyroid cells. In addition, when using the capacitance technique as an indicator of exocytosis, clamping the parathyroid cell at -60 mV prevented exocytosis, whereas holding the membrane potential at 0 mV facilitated it. Taken together, the results show that human parathyroid cells have functional KBK and KSK channels but the data presented herein suggest that KBK/KSK channels likely contribute to the maintenance of the membrane potential, and that membrane potential, per se, modulates exocytosis independently of [Ca2+]i.

Comparison of communication skills between medical students admitted after interviews or on academic merits.

BACKGROUND: Selection of the best medical students among applicants is debated and many different methods are used. Academic merits predict good academic performance, but students admitted by other pathways need not be less successful. The aim of this study, was to compare communication skills between students admitted to medical school through interviews or on academic merits, respectively. METHODS: A retrospective cohort study. Communication skills at a surgical OSCE in 2008 were assessed independently by two observers using an evaluative rating scale. Correlations, t-tests and multivariate analyses by logistic regressions were employed. Academic merits were defined as upper secondary school grade point average (GPA) or scores from the Swedish Scholastic Assessment Test (SweSAT). RESULTS: The risk of showing unsatisfactory communicative performance was significantly lower among the students selected by interviews (OR 0.32, CI95 0.12-0.83), compared to those selected on the basis of academic merits. However, there was no significant difference in communication skills scores between the different admission groups; neither did the proportion of high performers differ. No difference in the result of the written examination was seen between groups. CONCLUSIONS: Our results confirm previous experience from many medical schools that students selected in different ways achieve comparable results during the clinical semesters. However, selection through interview seems to reduce the number of students who demonstrate inferior communication skills at 4th year of medical school.

Heterogeneous expression of SNARE proteins SNAP-23, SNAP-25, Syntaxin1 and VAMP in human parathyroid tissue.

In regulated exocytosis synaptosomal-associated protein of 25kDa (SNAP-25) is one of the key-players in the formation of SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) complex and membrane fusion. SNARE proteins are essentially expressed in neurons, neuroendocrine and endocrine cells. Whether parathyroid cells express these proteins is not known. In this study, we have examined the expression of the SNARE protein SNAP-25 and its cellular homologue SNAP-23, as well as syntaxin1 and VAMP (vesicle-associated membrane protein) in samples of normal parathyroid tissue, chief cell adenoma, and parathyroid carcinoma, using immunohistochemistry and Western blot analysis. SNAP-23 and VAMP were evenly expressed in all studied parathyroid tissues using immunohistochemistry and/or Western blot analysis. SNAP-25 (and Syntaxin1) was not expressed in normal parathyroid tissue, but in approximately 20% of chief cell adenomas, and in approximately 45% of parathyroid carcinoma samples. It is likely that the SNARE proteins SNAP-23 and VAMP play a role in the stimulus-secretion coupling and exocytosis of parathyroid hormone as these proteins were expressed in all of the parathyroid samples we studied. In particular, preferential expression of SNAP-23 rather than SNAP-25 provides an explanation of the high level of PTH secretion that occurs under conditions of low cytoplasmic free Ca(2+) concentration (around 0.1micromol/l). SNAP-25 (and Syntaxin1) appears to be a tumour-specific protein(s) in parathyroid tissues since its expression was restricted to pathological tissues.

Somatic and germ-line mutations of the HRPT2 gene in sporadic parathyroid carcinoma.

BACKGROUND: We looked for mutations of the HRPT2 gene, which encodes the parafibromin protein, in sporadic parathyroid carcinoma because germ-line inactivating HRPT2 mutations have been found in a type of familial hyperparathyroidism--hyperparathyroidism-jaw tumor (HPT-JT) syndrome--that carries an increased risk of parathyroid cancer. METHODS: We directly sequenced the full coding and flanking splice-junctional regions of the HRPT2 gene in 21 parathyroid carcinomas from 15 patients who had no known family history of primary hyperparathyroidism or the HPT-JT syndrome at presentation. We also sought to confirm the somatic nature of the identified mutations and tested the carcinomas for tumor-specific loss of heterozygosity at HRPT2. RESULTS: Parathyroid carcinomas from 10 of the 15 patients had HRPT2 mutations, all of which were predicted to inactivate the encoded parafibromin protein. Two distinct HRPT2 mutations were found in tumors from five patients, and biallelic inactivation as a result of a mutation and loss of heterozygosity was found in one tumor. At least one HRPT2 mutation was demonstrably somatic in carcinomas from six patients. Unexpectedly, HRPT2 mutations in the parathyroid carcinomas of three patients were identified as germ-line mutations. CONCLUSIONS: Sporadic parathyroid carcinomas frequently have HRPT2 mutations that are likely to be of pathogenetic importance. Certain patients with apparently sporadic parathyroid carcinoma carry germ-line mutations in HRPT2 and may have the HPT-JT syndrome or a phenotypic variant.

Distinction in gene expression profiles demonstrated in parathyroid adenomas by high-density oligoarray technology.

OBJECTIVES: Somatic deletion of chromosome 11q13 is the most frequent genetic aberration in parathyroid adenoma. To gain further insight into the genetic etiology of parathyroid tumor development, we examined a comprehensive gene expression profile of parathyroid adenomas and normal parathyroid tissues. The results were then evaluated with respect to differences between adenomas and normal parathyroid tissue, and to the presence of loss of heterozygosity (LOH) in chromosomal region 11q13. DESIGN AND METHODS: Sporadic parathyroid adenomas and normal parathyroids were hybridized against HG-U95Av2 oligonucleotide arrays (Affymetrix) containing a total of 12,625 probe sets. Quantitative real-time PCR (QRT-PCR) was performed in a larger series of parathyroid adenomas, in order to con-firm the microarray results. RESULTS: Cyclin D1 and c-Jun showed increased expression in adenomas vs normal parathyroids by microarray analysis and QRT-PCR, suggesting an oncogenic role of these genes in parathyroid tumor development. At unsupervised hierarchical clustering, the adenomas fell into two groups: Group I adenomas were characterized by 11q13 LOH, while Group II adenomas lacked this abnormality. In addition, a t-test analysis identified largely non-overlapping genes with differential expression in the tumors subgroups; e.g. in Group I tumors the putative oncogene ENC 1 was found highly over-expressed vs Group II adenomas. CONCLUSIONS: The microarray analyses revealed partly distinctive and partly common expression profiles in parathyroid adenomas with and without 11q13 LOH. In addition, approximately half of the under-expressed genes were mapped to chromosome 11, in agreement with a dose effect following loss of this chromosome.

Postoperative differentiation between unilateral adrenal adenoma and bilateral adrenal hyperplasia in primary aldosteronism by mRNA expression of the gene CYP11B2.

OBJECTIVE: Primary aldosteronism (PA) is characterized by hypertension, hypokalemia and suppressed renin-angiotensin system caused by autonomous aldosterone production. The aim of this study was to localize mRNA expression of the genes coding for steroidogenic enzymes in adrenals from a group of patients with PA and relate this to clinical work-up, histopathology and outcome of adrenalectomy. DESIGN: This was a retrospective study of 27 patients subjected to adrenalectomy for PA. METHODS: Clinical data were collected and follow-up of all patients was performed. Paraffin-embedded specimens were analyzed by the in situ hybridization technique, with oligonucleotide probes coding for the steroidogenic enzyme genes. RESULTS: The resected adrenals had the histopathologic diagnosis of adenoma (11), adenoma and/or hyperplasia (15) or hyperplasia (1). CYP11B2 expression (indicating aldosterone production) was found in a dominant adrenal nodule from 22 patients. Fourteen of these had additional CYP11B2 expression in the zona glomerulosa. All 22 patients were cured of PA by adrenalectomy. One of these patients, who had additional high expression of CYP11B2 in the zona glomerulosa, was initially cured, but the condition had recurred at follow-up. Two patients had a mass shown on computed tomography without CYP11B2 but with CYP11B1 and CYP17 expression (indicating cortisol production). Instead their adrenals contained small nodules with CYP11B2 expression. These patients were not cured. CONCLUSIONS: Clinical data, endocrinologic evaluation and histopathology in combination with mRNA in situ hybridization of steroidogenic enzyme genes provide improved opportunities for correct subclassification postoperatively of patients with primary aldosteronism. At present, the in situ hybridization method is of special value for analysis of cases not cured by adrenalectomy.

Inhibition of parathyroid hormone secretion by caffeine in human parathyroid cells.

CONTEXT AND OBJECTIVE: Caffeine is a highly consumed psychoactive substance present in our daily drinks. Independent studies have reported associations between caffeine consumption, low bone mineral density, and urinary calcium loss, as well as impaired bone development in vitro and in vivo. Calcium (Ca(2+)), vitamin D, and PTH are critical regulators of bone remodeling. A possible association between caffeine and parathyroid gland function has been suggested in the literature. DESIGN, SETTING, AND PATIENTS: Effects of caffeine on PTH secretion and Ca(2+) levels were determined by batch incubation and Fura-2, respectively, in pathological parathyroid cells. Protein expressions were studied by Western blot and immunohistochemistry in normal and parathyroid adenoma tissues. Alterations in gene expressions of adenosine receptor A1 (ADORA1) and A2 (ADORA2A) and PTH were quantified by PCR; intracellular cAMP levels and protein kinase A activity were analyzed by an antibody-based assay. RESULTS: We studied physiological concentrations of caffeine ranging from 1 to 50 µm and found that 50 µm caffeine caused a significant decrease of PTH secretion and PTH gene expression. This decrease occurred in parallel with a decrease of the intracellular cAMP level, protein kinase A activity, and ADORA1 gene expression, indicating a possible causal relationship. The intracellular level of Ca(2+) was unaffected even by high concentrations of caffeine. Protein expressions demonstrated two main targets for caffeine-ADORA1 and ADORA2A. CONCLUSION: A physiological high dose of caffeine inhibits PTH secretion in human parathyroid cells, possibly due to a decrease of the intracellular level of cAMP. The observation demonstrates a functional link between caffeine and parathyroid cell function.

Calmodulin and calmodulin-dependent protein kinase II inhibit hormone secretion in human parathyroid adenoma.

Intracellular calcium ([Ca(2+)](i)) is the most relevant modulator of parathyroid hormone (PTH) secretion. Uniquely, an increase in [Ca(2+)](i) results in an inhibition of PTH secretion, and it probably exerts its function via calcium-binding protein pathways. The ubiquitous calcium-binding proteins, calmodulin and calmodulin-dependent protein kinase II (CaMKII), have well-established roles in regulated exocytosis in neurons and neuroendocrine cells. However, their roles in parathyroid cells and PTH secretion are still unclear. Using reverse transcription-PCR and western blot analysis, we have demonstrated the expression of calmodulin and CaMKII in human normal parathyroid and parathyroid chief cell adenomas. Blocking of calmodulin and CaMKII activity by the specific antagonists calmidazolium and KN-62 respectively caused a rise in PTH secretion from parathyroid adenoma cells in spite of increased [Ca(2+)](i). The inhibitory effect of Ca(2+) calmodulin on PTH secretion may be due to the absence of synaptotagmin 1 protein in parathyroid adenomas, as demonstrated by western blot analysis. An increased extracellular calcium level acutely lowered the amount of active phosphorylated CaMKII (pCaMKII) in adenoma cells in vitro, indicating the physiological importance of this pathway. Moreover, a negative correlation between the levels of pCaMKII in parathyroid adenomas and serum calcium was found in 20 patients with primary hyperparathyroidism. Taken together, these results show that calmodulin negatively contributes to the regulation of PTH secretion in parathyroid adenoma, at least partially via a CaMKII pathway.

Expression and association of TRPC subtypes with Orai1 and STIM1 in human parathyroid.

The mechanism behind Ca(2)(+) entry into the parathyroid cells has been widely debated, and the molecular identities of the responsible ion channels have not been established yet. In this study, we show that the parathyroid cells lack voltage-operated Ca(2)(+) channels. Passive store depletion by thapsigargin, on the other hand, induces a large non-voltage-activated non-selective cation current. The increase in intracellular Ca(2)(+) caused by thapsigargin is attenuated by 2-aminoethoxydiphenyl borate, a blocker of store-operated Ca(2)(+) entry (SOCE). Candidate molecules for non-voltage-operated Ca(2)(+) signaling were investigated. These included members of the transient receptor potential canonical (TRPC) ion channel family, as well as Ca(2)(+) release-activated Ca(2)(+) modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1) that are key proteins in the SOCE pathway. Using RT-PCR screening, quantitative real-time PCR, and western blot, we showed expression of TRPC1, TRPC4, and TRPC6; Orai1; and STIM1 genes and proteins in normal and adenomatous human parathyroid tissues. Furthermore, co-immunoprecipitation experiments demonstrated a ternary complex of TRPC1-Orai1-STIM1, supporting a physical interaction between these molecules in human parathyroid.

High extracellular Ca2+ hyperpolarizes human parathyroid cells via Ca(2+)-activated K+ channels.

Membrane potential has a major influence on stimulus-secretion coupling in various excitable cells. The role of membrane potential in the regulation of parathyroid hormone secretion is not known. High K+-induced depolarization increases secretion from parathyroid cells. The paradox is that increased extracellular Ca2+, which inhibits secretion, has also been postulated to have a depolarizing effect. In this study, human parathyroid cells from parathyroid adenomas were used in patch clamp studies of K+ channels and membrane potential. Detailed characterization revealed two K+ channels that were strictly dependent of intracellular Ca2+ concentration. At high extracellular Ca2+, a large K+ current was seen, and the cells were hyperpolarized (-50.4 +/- 13.4 mV), whereas lowering of extracellular Ca2+ resulted in a dramatic decrease in K+ current and depolarization of the cells (-0.1 +/- 8.8 mV, p < 0.001). Changes in extracellular Ca2+ did not alter K+ currents when intracellular Ca2+ was clamped, indicating that K+ channels are activated by intracellular Ca2+. The results were concordant in cell-attached, perforated patch, whole-cell and excised membrane patch configurations. These results suggest that [Ca2+]o regulates membrane potential of human parathyroid cells via Ca2+-activated K+ channels and that the membrane potential may be of greater importance for the stimulus-secretion coupling than recognized previously.

Genetic and clinical characterization of sporadic cystic parathyroid tumours.

OBJECTIVE: The hyperparathyroidism--jaw tumour (HPT--JT) syndrome is one of the familial disorders characterized by primary hyperparathyroidism and has been linked to the chromosomal region of 1q32--q21. The parathyroid tumours related to this syndrome have shown loss of wild-type alleles at this locus suggesting that inactivation of a tumour suppressor gene might be responsible for the disease. In the majority of these tumours cysts are a prominent feature. By loss of heterozygosity (LOH) studies, we investigated the region of interest in an attempt to clarify its possible role in a series of cystic sporadic parathyroid adenomas. DESIGN AND SUBJECTS: A total of 30 patients diagnosed with sporadic hyperparathyroidism were included in the study, genotyped with 17 polymorphic microsatellite markers at chromosome 1q, and additional markers from 1p and 11q13 which are commonly involved in sporadic parathyroid tumours. The cystic parathyroid tumours were characterized clinically, and immunohistochemistry against PTH was carried out to confirm the parathyroid origin of the cysts. RESULTS: LOH was found in six of 30 tumours (20%) on 1q, six of 30 tumours (20%) on 1p and five of 30 tumours (17%) on 11q13. We found a significant correlation between allelic alterations and the clinical parameters, tumour weight and PTH. Furthermore, we found a significant difference between tumour weight and PTH in cases of cystic parathyroid tumours compared with unselected sporadic cases. CONCLUSIONS: These results suggest that cystic parathyroid tumours might represent a new subgroup among parathyroid tumours based on the genetic and clinical findings. Loss of heterozygosity at 1q further supports the presence of a tumour suppressor gene at this locus.