G. Giannattasio

Dopamine-inhibited adenylate cyclase in female rat adenohypophysis

Abstract A dopamine-inhibited adenylate cyclase has been demonstrated in anterior pituitary gland of adult female rats, lactating and not lactating. This inhibitory effect was completely GTP dependent. In contrast, in the adenohypophysis of male rats, dopamine had no detectable effect on adenylate cyclase activity. In female rats the inhibition of the enzyme appears mediated by specific dopaminergic receptors: the effect of dopamine was mimicked by the dopaminergic agonists apomorphine and the ergot derivative CH 29–717, while norepinephrine was much less potent. On the other hand, the dopaminergic antagonists trifluoperazine and sulpiride competitively antagonized the dopamine inhibition of the adenylate cyclase. The possibility that the dopamine-inhibited enzyme is located in mammotrophs appears supported 1) by its observation in the female rat pituitary, which contains this type of cells in much larger proportion than the male gland (33–38% vs.

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.

Presence of sulfated proteoglycans in prolactin secretory granules isolated from the rat pituitary gland

The composition of the segregated content of rat prolactin granules was investigated taking advantage of the fact that these organelles, isolated as a pure fraction, retain their structural organization after solubilization of their limiting membrane by mild detergent treatment. We found that these membraneless granules contain not only the hormone, but also a number of minor macromolecular components including sulfated glycosaminoglycans, which are labeled when pituitary slices are incubated in vitro with [35S] sulfate. In order to characterize the latter components, the isolated radioactive granules were solubilized (by treatment with either a high ionic strength solution orNaOH) and 35S-labeled acidic glycosaminoglycans precipitated by complexing with cetylpirydinium chloride. A high degree of heterogeneity was observed when the ensuing precipitates were analyzed by cellulose acetate electrophoresis: different components were found to co-migrate with authentic heparin and chondroitin sulfate A and C standards. Another component, which accounts for approx. 50% of the glycosaminoglycan-bound radioactivity, might be heparin sulfate. These acidic glycosaminoglycans are linked to peptide moieties to form proteoglycans.

Effects of vasoactive intestinal polypeptide (VIP) in human prolactin (PRL) secreting pituitary adenomas. Stimulation of PRL release and activation of adenylate cyclase.

Vasoactive intestinal polypeptide (VIP) might be involved in the regulation of pituitary function [l-4]. Some of the peptide action is probably effected at the level of pituitary cells, as suggested by its presence at high concentration in the hypophyseal portal blood [S] and by the existence of a VIP-sensitive adenylate cyclase in rat pituitary gland [6,7]. In particular, although the available experimental evidence is still incomplete and controversial, a physiological role of VIP in the direct control of prolactin (PRL) release has been envisaged [I ,3]. These results support this hypothesis and give some insight on the mechanisms of action of VIP on pituitary mammotroph cells. In fact, working on human PRL-secreting adenomas, i.e., on homogeneous populations of lnammotroph cells, we demonstrate that: (1) VIP stimulates the release of PRL from tissue fragments incubated in vitro; (2) A VIP-sensitive adenylate cyclase is present in these pituitary tumors.

VIP stimulates ACTH release and adenylate cyclase in human ACTH-secreting pituitary adenomas

The effect of vasoactive intestinal polypeptide (VIP) on in vitro ACTH release and adenylate cyclase activity was investigated in human ACTH-secreting pituitary adenomas from 4 patients with Cushings disease and 2 patients with Nelsons syndrome. In all the tumors tested, VIP elicited a dose-dependent stimulation of hormone release from adenoma fragments (90-247% at 10-7 M VIP) and of cAMP formation in membrane preparations (75-140% at 3 X 10-6 M VIP). Therefore a role of VIP in the control of ACTH secretion in human ACTH-secreting adenomas is suggested; a cAMP-dependent mechanism of action can also be hypothesized.

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.

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

Complex Carbohydrates of Secretory Organelles

Over the last few years, considerable attention has been devoted to the role played by complex carbohydrates in the structure and function of secretory granules and vesicles. These intracellular storage organelles, which are delimited by a single membrane, are endowed with the ability to discharge their content into the extracellular space by exocytosis. Since each secretory system is characterized by the specificity of its secretion products, the various granules and vesicles constitute an extremely heterogenous family of organelles. In some of them, complex carbohydrates represent secretion products of known physiological significance. This is the case, for instance, with the gonadotropin and thyrotropin granules of the anterior pituitary, the B granules of follicular thyroid cells, and the immunoglobulin vesicles of plasmocytes. Other secretory granules (e.g., those of the exocrine pancreas, parotid gland, and liver) contain a mixture of many physiologically important secretion products, some of which are complex carbohydrates. Finally, in a variety of other systems, the occurrence of complex carbohydrates (often in small amounts) in the segregated content of secretory organelles has also been reported, even if no clear information is yet available on whether these components have any function after their discharge. However, complex carbohydrates do not reside only in the content of secretory organelles, since in all systems so far investigated they have also been found in the limiting membrane, where at least some of them are oriented according to a specific geometry.

Somatomammotrophic cells in GH-secreting and PRL-secreting human pituitary adenomas

A morphological study has been carried out on 20 GH-secreting adenomas removed from acromegalic normoprolactinemic patients, on 29 PRL-secreting adenomas removed from hyperprolactinemic patients without signs of acromegaly and on one normal human anterior pituitary gland collected at autopsy. The protein A-gold immunoelectron microscopic technique has been utilized in order to verify the presence of mixed cells producing both GH and PRL (somatomammotropins) in these pituitary tissues. In the normal pituitary a considerable number of somatomammotropins (15–20%) was found, thus supporting the idea that these cells are normal components of the human anterior pituitary gland. In 10 GH-secreting adenomas and in 10 PRL-secreting adenomas somatomammotrophs were present in a variable number (from 4 to 20% of the whole cell population in GH adenomas and from 1 to 47% in PRL tumors). It can be concluded therefore that these cells, largely present in all GH/PRL-secreting adenomas, can also be found in GH-secreting and PRL-secreting tumors without clinical evidence of a mixed secretion. Adenomatous somatomammotrophs displayed ultrastructural features of adenomatous somatotrophs and mammotrophs (prominent Golgi complexes, abundant rough endoplasmic reticulum, irregular nuclei). The size and the number of granules were variable. In some cells GH and PRL were stored in distinct secretory granules, in others in mixed granules or both in mixed and distinct granules, thus suggesting that in adenomatous somatomammotrophs the efficiency of the mechanisms of sorting of the two hormones varies from one cell to another. The fact that somatomammotrophs have been found in normal pituitaries and were lacking in some tumors would suggest that in tumors these cells might originate from their normal cellular counterparts and not be a consequence of the tumoral process.

Combined effects of vasoactive intestinal peptide and dopamine on adenylate cyclase in prolactin-secreting cells

VIP stimulates adenylate cyclase activity of male and female rat anterior pituitaries and human prolactinomas, while dopamine inhibits the enzyme activity of female rat pituitaries and prolactinomas. A dopamine inhibited cyclase can be detected also in male rats provided the enzyme activity is increased by VIP. The analysis of the dose-response curves for one agent (VIP or dopamine) in the absence or in the presence of the other indicates that the two agents exhibit a different pattern of interaction in the different systems. In fact, in female rat pituitaries and in human prolactinomas, the curves for dopamine +/- VIP and for VIP +/- dopamine were parallel, indicating that the two agents exherted their effects independently from one another. On the contrary, in male rat pituitaries, the curves were definitively non parallel, that is, the inhibitory effect of dopamine was greatly amplified by VIP. In no case was the apparent affinity (EC50) of one agent modified by the presence of the other. It is concluded that two different modes of interaction between stimulatory and inhibitory neurohormones might exist at the level of adenylate cyclase from anterior pituitary cells.