Monique Bassetti

A Regulated Secretory Pathway in Cultured Hippocampal Astrocytes

Glial cells have been reported to express molecules originally discovered in neuronal and neuroendocrine cells, such as neuropeptides, neuropeptide processing enzymes, and ionic channels. To verify whether astrocytes may have regulated secretory vesicles, the primary cultures prepared from hippocampi of embryonic and neonatal rats were used to investigate the subcellular localization and secretory pathway followed by secretogranin II, a well known marker for dense-core granules. By indirect immunofluorescence, SgII was detected in a large number of cultured hippocampal astrocytes. Immunoreactivity for the granin was detected in the Golgi complex and in a population of dense-core vesicles stored in the cells. Subcellular fractionation experiments revealed that SgII was stored in a vesicle population with a density identical to that of the dense-core secretory granules present in rat pheochromocytoma cells. In line with these data, biochemical results indicated that 40–50% of secretogranin II synthesized during 18-h labeling was retained intracellularly over a 4-h chase period and released after treatment with different secretagogues. The most effective stimulus appeared to be phorbol ester in combination with ionomycin in the presence of extracellular Ca2+, a treatment that was found to produce a large and sustained increase in intracellular calcium [Ca2+] i transients. Our findings indicate that a regulated secretory pathway characterized by (i) the expression and stimulated exocytosis of a typical marker for regulated secretory granules, (ii) the presence of dense-core vesicles, and (iii) the ability to undergo [Ca2+] i increase upon specific stimuli is present in cultured hippocampal astrocytes.

Microvasculature of human micro- and macroprolactinomas: a morphological study

A morphological study has been undertaken on the capillaries of 9 microprolactinomas and 9 macroprolactinomas, surgically removed from untreated patients. The study was carried out utilizing light and electron microscopic techniques and electron microscopic morphometry. The frequency of the capillaries and their structural appearance were taken into account. The frequency of capillaries was found to be very different in micro- and macroadenomas. In microadenomas 51.1 capillaries/0.1 mm2 of tissue section were observed; this value was not significantly different from that found in normal human pituitaries (62.0/0.1 mm2). In contrast, in macroprolactinomas a much lower degree of vascularization was found (9.3 capillaries/0.1 mm2 of tissue section). The capillary abnormalities previously reported for pituitary adenomas (endothelial thickening, swelling and blebbing, loss of fenestration, multilayered basal membrane, etc.) were observed in all prolactinomas studied, but no differences were found between the two types of tumors. In both types of tumors, the capillaries generally looked mature. Very rare sprouting capillaries were observed. Angiogenesis is likely to be slow, in agreement with the low frequency of capillaries in the more rapidly proliferating tumors such as macroprolactinomas. The different frequency of capillaries in micro- and macroprolactinomas could have some important consequences as to the regulation of the hormonal secretion. In fact, the different blood supply to the small and large tumors could result in a different availability of regulatory factors for the two types of tumors.

Bromocriptine treatment of macroprolactinomas: studies on the time course of tumor shrinkage and morphology

The effects of Bromocriptine (Brc) (7.5 2 - 15 mg/day for 45 days) on serum PRL levels and tumor size and morphology were investigated in 7 patients with macroprolactinomas (mean PRL ± SE = 4957 ± 920 ng/ml). Serial controls of serum PRL levels, CAT scan and visual field examination were carried out at the 5th, 10th, 20th and 45th day of Brc treatment. A rapid lowering in PRL concentrations (mean PRL ± SE = 23.1 ± 5.8 ng/ml) and a dramatic shrinkage of tumor mass were observed in 4 cases already at the 5th day of therapy. In 3 patients Brc was then withdrawn and a rapid rise of serum PRL concentration (mean PRL ± SE = 2618 ± 683 ng/ml) along with a reexpansion of the adenomas was documented within 15 days. These patients were then restarted on Brc and tumor regression was noticed again. In the remaining 3 cases no CAT scan variations occurred, though 2 of them had serum PRL in the normal range. Transsphenoidal adenomectomy was then performed in 4 patients upon Brc (1 with a marked reduction of the adenoma size and 3 with unmodified tumor mass) and tumor tissue examined. The histological picture of the 4 Brc-treated tumors was homogeneous and no relevant changes were seen in comparison with fragments of prolactinomas obtained from patients never treated with Brc. Though PRL cells appeared somewhat more closely associated, neither the tissue architecture, not the distribution of necrotic and hemorragic areas, nor the mitosis rate appeared modified by the treatment. At electron microscopy all the Brc-treated adenomas showed an increase of PRL secretory granules and a decrease of RER and Golgi apparatus. The morphometric analysis revealed in all cases a marked reduction of the cell size in comparison with the untreated tumors (73.56 μm2 ± 6.90 SE vs 148.41 μm2 ± 11.29 SE in untreated patients; p < 0.0001). In conclusion: i) the prolactinoma regression induced by Brc occurred only in patients whose serum PRL levels were markedly suppressed; however the PRL normalization was not constantly associated with tumor shrinkage; ii) all the Brc-treated adenomas showed a marked reduction in cell size and a decrease of the cytoplasmic structures responsible for PRL production; iii) cell size reduction was observed also in 3 patients who did not reduce their tumor size. This suggests that additional mechanisms should have been operating in the Brc-induced tumor shrinkage.

In vitro studies on TSH secretion and adenylate cyclase activity in a human TSH-secreting pituitary adenoma. Effects of somatostatin and dopamine

We have studied the in vitro TSH secretion and the adenylate cyclase (AC) activity of a human pituitary adenoma surgically removed from a hyperthyroid patient showing high serum TSH levels. The tumor appeared almost homogeneously constituted by cells positive for an anti-TSH-beta antiserum and showing the ultrastructural characteristics of the adenomatous thyrotrophs. Adenoma fragments released in vitro a large amount of TSH (148.4 μU/mg prot/30 min), alpha-subunit(35.5 ng/mg prot/30 min) and TSH-beta (10.1 ng/mg prot/30 min). The effects of somatostatin (GHRIH) and dopamine (DA) on the hormone release have been tested in vitro. Both agents markedly inhibited the release of intact TSH and TSH-beta whereas the release of alpha-subunit was less affected. The two agents were effective at concentrations higher than 10−8M. The ability of GHRIH and DA in modulating the AC activity was investigated in membrane fraction preparations. GHRIH inhibited AC at concentrations higher than 10 −7M. The maximal inhibition was 32% at 10−5 M. Conversely, DA slightly stimulated AC activity. This effects was not mimicked by the dopaminergic ergot CH 29–717, which was completely ineffective on the enzyme. These results suggest that: 1) in this TSH-secreting pituitary adenoma a normal secretory response to the inhibiting agents (GHRIH and DA) is present; 2) different mechanisms of transduction of the GHRIH and DA signals (cAMP dependent and cAMP independent) could be operating in this tumor.

Age-related changes in mitogen-induced β-endorphin release from human peripheral blood mononuclear cells

Abstract β-Endorphin is an opioid peptide synthesized in the pituitary, hypothalamus, and immunocytes, known to affect immune responses both when added in vitro and when its synthesis is increased in vivo (e.g., during stress). We show here that, similar to its concentrations in peripheral blood mononuclear cells, the release of the opioid peptide from these cells after stimulation with polyclonal mitogens such as PHA or Con-A is also age dependent. Moreover, the effect of both mitogens on Ca 2+ homeostasis changes with age. Finally, the ionophore ionomycin and the Ca 2+ ATPase blocker thapsigargin induce the same age related effect on β-endorphin release. For these reasons, we suggest that calcium homeostasis might be important for the differences observed in the release of the opioid from cells obtained from younger (≤30 years) or older (≥ 45 years) volunteers.

The silent corticotropinoma: is clinical diagnosis possible?

Up to now, the diagnosis of silent corticotropin cell pituitary adenomas has been made only on histopathological basis. In this paper we describe 6 women affected with pituitary adenomas, without evident clinical features of hypercortisolism, in whom retrospective data suggested the possibility of clinically diagnosing silent corticotropinomas in vivo. In all patients basal ACTH and Cortisol levels were normal, and the low-dose dexamethasone test constantly suppressed serum Cortisol and urinary 17-hydroxycorti-costeroid levels. The CRH and/or lysine-vasopressin tests, performed in five patients, always induced exaggerated ACTH/cortisol rises. In three cases the response to the opiate agonist loperamide was assessed and no inhibition of ACTH/cortisol levels was found. All patients underwent pituitary surgery. In five cases evidence of corticotropinoma was obtained by immunohistochemistry or immunofluorescence studies; moreover, in one adenoma ACTH was secreted into the culture medium, and in another one CRH and arginine-vasopressin induced a marked intracellular [Ca++] rise. Electron microscopy study of the adenoma, removed from three patients, showed the presence of adenomatous corticotropin cells. Finally, in another woman no hormonal abnormalities were initially observed and she was operated for a “nonfunctioning” pituitary adenoma, but four years later an overt Cushing’s disease appeared, suggesting that a silent corticotropinoma subsequently became functional, although the formation of a different adenoma cannot be excluded. In conclusion, the occurrence of ACTH/cortisol hyperresponsiveness to CRH and/or lysine-vasopressin and the lack of suppression of ACTH/cortisol secretion to opioid agonists in patients with apparently “nonfunctioning” pituitary tumors might allow the in vivo recognition of silent corticotropinomas.

In vitro detection of glycoprotein production and secretion by human nonfunctioning pituitary adenomas

This study, carried out on 9 nonfunctioning pituitary adenomas, was undertaken in order to evaluate the ability of these tumors to synthesize and release gonadotropins and/or free a-subunit (a-SU) of glycoproteins. The morphological study included electron microscopy and immunofluorescence analysis while hormone release was evaluated by the reverse hemolytic plaque assay (RHPA) and measurements in culture media. By electron microscopy in all tumors (6 null cell adenomas and 3 oncocytomas), it was possible to identify rough endoplasmic reticulum, Golgi apparatus and secretory granules. By immunofluorescence, 5 of 6 tumors were immunoreactive for one or more gonadotropin subunits; in particular, 5 adenomas were positive for α-SU and LH-β, and 3 for FSH-β. By the RHPA, about 1% of cells obtained from one single tumor formed plaques for LH-α and β-SU while the remaining tumors were negative. Similarly, the study of media concentrations of LH, FSH and α-SU in 2 h culture revealed very low amounts of released hormones. In these experimental conditions no modification was observed after the addition of stimulatory agents such as TRH, GnRH and VIP. The present study clearly indicates that although the large majority of nonfunctioning tumors are positive for gonadotropins their secretory capacity is very low in both basal and stimulated conditions.

Human pituitary adenomas. Recent advances in morphological studies

INTRODUCTION A large number of morphological studies have been carried out on human pituitary adenomas over the last two decades primarily in order to establish structure-function correlations so as to provide the clinical endocrinologist with information of diagnostic interest on the endocrine activity of tumors. Classifications were made, based on the morphological observations, supporting and integrating those based on clinical manifestations and blood hormone concentration measurements. Great advances in morphological diagnosis and classification have been made, mainly in the eighties, due to the considerable improvement in the morphological procedures. However the first approach, made by means of the histological staining methods, was far from adequate. Pituitary adenomas were divided into acidophilic, basophilic and chromophobic tumors according to the staining affinities of the cell cytoplasm. However, the tinctorial characteristics of the cells proved to give a poor indication of the type of their endocrine activity. This classification, therefore, has no diagnostic value (1, 2). Electron microscopic studies gave more insights into structure and activity of the tumor cells. The peculiarities of the cellular organelles were accurately considered. Thus, the development of the rough endoplasmic reticulum and the prominence of Golgi complex (the cytoplasmic organelles responsible for the synthesis and packaging of the hormones) typical of many adenoma cells were related to their high secretory activity. Number, size

Immunodetection of glycoprotein hormone subunits in nonfunctioning and glycoprotein hormone-secreting pituitary adenomas

While it is established that nonfunctioning pituitary adenomas (NFPA) produce a spectrum of glycoprotein hormones, the ability of glycoprotein hormone-secreting adenomas to synthesize hormones other than those in vivo hypersecreted has been poorly investigated so far. In this study the immunolocalization of the β-subunits of LH, FSH, CG and TSH and the common α-subunit was investigated in 10 NFPA, 3 gonadotropin-secreting adenomas (Gn-omas, 1 LH-oma, 1 FSH-oma and 1 LH/FSH-oma) and 3 TSH-se-creting adenomas (TSH-omas) using an immunohistochemical technique with specific antibodies to glycoprotein subunits. The percentage of positive cells was determined observing at least 5 photograph fields containing about 50 cells. Nine of the 10 NFPA were positive for FSHβ with a high percent of positive cells (≥30%), 6 for LHβ and 5 for TSHβ with a lower number of labelled cells (from 5 to 30%). Of the 8 NFPA tested, 5 showed a marked positivity for CGβ. In Gnomas, tumors were positive for the gonadotropin that was in vivo hypersecreted (10–60% positive cells) and negative for either TSHβ or CGβ. Similarly, in TSH-omas TSHβ was detected in a variable proportion of cells (10–30%) with no immunoreactivity for either LHβ or FSHβ or CGβ. This study indicates that the coex-pression of FSHβ, LHβ, CGβ and TSHβ molecules is characteristic of NFPA since Gn-omas were negative for CGβ and TSHβ and TSH-omas were negative for FSHβ, LHβ and CGβ. Moreover, the biological events that make NFPA ‘silent’ glycoprotein hormone-secreting adenomas remain unclear since the positivity for gonadotropins and TSH showed by NFPA was similar to that observed in Gn-omas and TSH-omas, respectively.

Mutations in the Alpha Subunit of the Stimulatory Regulatory Protein of Adenylyl Cyclase (Gs) in Human GH-Secreting Pituitary Adenomas: Biochemical, Clinical, and Morphological Aspects

Very recently a subset of human GH-secreting pituitary adenomas carrying a somatic mutation in the alpha subunit of the stimulatory regulatory protein of adenylyl cyclase (Gs) was identified. In all these tumors (Group 2; about 30% of all the GH secreting tumors studied) the alpha s cDNAs contained mutations; in 8 tumors mutations replaced Arginine 201 with either Cystein or Histidine while in the remaining tumors Glutamine 227 was replaced by either Arginine or Leucine. No mutations were observed in the remaining adenomas (Group 1). The two mutations caused a constitutive activation of adenylyl cyclase and a turn on of cAMP synthesis by inhibiting GTPase activity. The transformed phenotype was reflected in adenomatous cells with high rate of cAMP production and in vitro GH secretion. No difference in age, sex, clinical features, duration of the disease and cure rate were observed between the patients without (Group 1) or with alpha s mutation (Group 2), while higher serum GH levels and smaller tumor size were present in Group 2 patients. Moreover, hypersecretory activity in Group 2 tumors was also apparent at electron microscopy; cells of Group 2 tumors were densely granulated and showed prominent rough endoplasmic reticulum and Golgi complex. With respect to Group 1, Group 2 patients were less responsive to GH-releasing hormone (GHRH), while they were more sensitive to somastostatin. The former finding is in agreement with the hypothesis that the oncogenic proteins mimic the effects of extracellular growth factors, so removing the requirement for GHRH; the latter might explain the low rate of tumor growth as due to the counteracting role of endogenous inhibitory factors.