Showing papers by "Anna Spada published in 2000"

Calcium-sensing receptor expression and signalling in human parathyroid adenomas and primary hyperplasia.

Abstract: OBJECTIVE Both in vivo and in vitro evidence indicates that primary hyperparathyroidism is characterized by a reduced sensitivity to extracellular calcium ([Ca2+]o) The existence of alterations in the expression and signalling of calcium sensing receptor (CaSR) in parathyroid neoplasia is still uncertain In order to clarify the role of CaSR in the reduced [Ca2+]o sensing of parathyroid neoplasia we investigated PTH secretion and intracellular effectors triggered by CaSR activation as well as the levels of expression of CaSR and CaSR coupled G proteins (Gq/G11) in parathyroid adenomas and primary hyperplasia MATERIALS AND METHODS The study included 27 parathyroid adenomas, 4 cases of primary hyperplasia and pools of normal parathyroid biopsies Tissues were either snap frozen in liquid nitrogen or placed in sterile medium for cell dispersion The effects of increasing [Ca2+]o on in vitro PTH release, intracellular cAMP levels and intracellular calcium ([Ca2+]i) in cells loaded with the Ca2 +indicator fura-2 were evaluated CaSR mRNA levels were assessed by semiquantitative RT-PCR analysis, using GAPDH as internal standard, while CaSR protein was detected by western blot analysis using a specific polyclonal antibody Purified antisera selective for G11α and Gqα were used to detect this class of proteins RESULTS In basal conditions (at 05 m m [Ca2+]o) in vitro PTH released ranged from 294 to 1186 pg/well/60 minutes Increasing [Ca2+]o from 05 to 1, 25 and 5 m m caused a variable effect One group (n = 7) showed a significant but partial reduction of PTH release (of 17 to 60% of basal levels) that occurred at physiological [Ca2+]o concentrations (1 m m) while the remainder showed either inhibition detectable only at 25 m m (n = 15) or total (n = 9) resistance to [Ca2+]o In the responsive cells, [Ca2+]o (1–5 m m) caused a pertussis toxin-insensitive [Ca2+]i rise (ranging from 10% to 260%), due to Ca2+ release from intracellular stores, and an inhibition of forskolin-stimulated cAMP levels By RT-PCR almost all tumours tested showed a substantial reduction in CaSR mRNA levels when compared to the normal tissue (CaSR/GAPDH ratio: 31 ± 05 vs 155 ± 31; P < 0001), which was confirmed by immunoblotting analysis demonstrating low levels of CaSR protein in tumour tissues Moreover, low amounts of G11α and Gqα, the G proteins involved in CaSR coupling, were observed in the majority of pathological tissues CONCLUSIONS The study shows that the activation of the calcium sensing receptors expressed in adenomatous parathyroid glands modulates intracellular effectors in a similar way to those operating in the normal parathyroid Although a reduction of calcium sensing receptor expression is probably involved in the poor inhibition of PTH release induced by [Ca2+]o, this is not the only factor altering [Ca2+]o sensing in parathyroid adenomas, since tumours characterized by different in vitro sensitivity to [Ca2+]o showed similar CaSR levels The low content of G proteins of the Gq subfamily might represent an additional alteration leading to a defective [Ca2+]o sensing

Induction of specific phosphodiesterase isoforms by constitutive activation of the cAMP pathway in autonomous thyroid adenomas.

Abstract: Thyrocytes largely depend on cAMP signaling for replication and differentiation. This pathway may be constitutively activated by mutations of the TSH receptor (TSHR) and Gsalpha in autonomous thyroid adenomas (ATAs). Because steady state cAMP results from production by adenylyl cyclase and degradation by phosphodiesterases (PDEs), we evaluated PDE activity and expression in ATAs with wild-type and mutant TSHR and Gsalpha. Activating mutations of TSHR and Gsalpha were identified in 7 and 1 of 18 ATAs, respectively. No difference was observed in the cAMP content in ATAs with or without activating mutants. In the surrounding normal thyroid tissue (NTs), PDE activity was 80% isobutylmethylxanthine sensitive, with the major contribution by PDE1 and a minor contribution by PDE4. No differences were observed in PDE activities between NTs and ATAs with wild-type TSHR and Gsalpha. In contrast, in the presence of mutant TSHRs or Gsalpha, total PDE activity was higher. This increase was primarily due to PDE4 induction (917 +/- 116% over NTs), associated with a minor PDE1 increase only in ATAs with mutant TSHR. By RT-PCR, increments of PDE4D and 4C messenger ribonucleic acids were found in the ATAs with mutant TSHR or Gsalpha, whereas messenger ribonucleic acids encoding other cAMP-specific PDEs were not significantly increased. This study provides a characterization of the PDEs expressed in human thyroid and demonstrates a dramatic PDE4 induction in the ATAs bearing mutant TSHR or Gsalpha genes. The increase in cAMP-degrading activity may represent a marker of constitutive adenylyl cyclase activation and constitutes an intracellular feedback mechanism with significant impact on the phenotypic expression of the activating mutations.

Mutational analysis of GNAS1 in patients with pseudohypoparathyroidism : Identification of two novel mutations

Abstract: Pseudohypoparathyroidism (PHP) refers to two major variants that generally coexist in the same family, PHP type Ia (PHP Ia), in which both PTH resistance and a constellation of physical features, termed Albright's hereditary osteodystrophy (AHO), are present, and pseudopseudohypoparathyroidism (PPHP), in which AHO occurs without PTH resistance. Most patients with PHP Ia show a partial deficiency (50%) of Gs activity, due to loss of function mutations in Gsalpha gene (GNAS1). The present study reports clinical, biochemical, and molecular data of 8 unrelated families with PHP Ia and PPHP. The 13 exons of GNAS1 were screened for mutations by PCR and direct sequencing of the amplified products. We detected heterozygous mutations in the affected members of the 4 families in which PHP Ia was present. In 2 families 2 previously reported deletions in exons 5 and 7 were found, whereas in the other 2 families, 2 novel frameshift deletions were identified in exons 1 and 11, causing a premature stop codon in the mutant allele. No mutation was detected in the families in which PPHP was the only clinical manifestation. In conclusion, we report the first mutational analysis of Italian patients with PHP Ia and PPHP, and we describe two novel deletions in GNAS1. Furthermore, we confirm that these mutations cannot be detected in families with isolated PPHP, suggesting that these forms of AHO are genetically distinct from PHP Ia.

Effect of aging on serum gonadotropin levels in healthy subjects and patients with nonfunctioning pituitary adenomas.

Abstract: Objective Nonfunctioning pituitary adenomas (NFPA), which represent about one-quarter of human pituitary tumors, occur in middle or old age. Determination of gonadotropin levels, which are not expected to be high during the early postmenopause in normal women and which are low in women with NFPA, is important to distinguishing hypogonadal status due to the normal decline of gonadal function from that due to hypothlalamic-pituitary dysfunction. The aim of the study was to verify whether this difference still persists in old subjects, despite the physiological decline of gonadotropins in the last decades of life. Design and methods The study included 154 healthy subjects (aged 50-104 years) and 47 patients with NFPA (aged 50-80 years). Blood samples were collected after an overnight fast and hormone levels were measured by two immunofluorimetric assays. Results In healthy women the highest serum levels of gonadotropins were present in the 50-60 year age group, with a slight but progressive age-associated decrease in serum FSH and LH being observed thereafter. In healthy men serum gonadotropin levels were stable up to 70 years, increased up to 75-85 years and thereafter gradually decreasing. Female patients with NFPA showed levels of gonadotropins which were far lower than controls. Only three patients had levels of both FSH and LH above the 2.5 centile for normal subjects. A high sensitivity and specificity of gonadotropin measurements (about 90%) for the diagnosis of NFPA was observed in female patients aged 50-80 years. In male subjects, a large overlap of gonadotropin values in NFPA and controls, namely over the 50-70 years age range, was observed. Conclusions Our study demonstrates that despite the gradual decline of gonadotropin levels in healthy postmenopsausal women, the reduction of both FSH and LH persists in old patients with NFPA, suggesting that measurement of gonadotropin levels could prove useful in the evaluation of pituitary lesions even in old women. More subtle differences seem to occur in male subjects.