Anterior pituitary cell networks.

Both endocrine and non-endocrine cells of the pituitary gland are organized into structural and functional networks which are formed during embryonic development but which may be modified throughout life. Structural mapping of the various endocrine cell types has highlighted the existence of distinct network motifs and relationships with the vasculature which may relate to temporal differences in their output. Functional characterization of the network activity of growth hormone and prolactin cells has revealed a role for cell organization in gene regulation, the plasticity of pituitary hormone output and remarkably the ability to memorize altered demand. As such, the description of these endocrine cell networks alters the concept of the pituitary from a gland which simply responds to external regulation to that of an oscillator which may memorize information and constantly adapt its coordinated networks' responses to the flow of hypothalamic inputs.

Functional importance of blood flow dynamics and partial oxygen pressure in the anterior pituitary.

The pulsatile release of hormone is obligatory for the control of a range of important body homeostatic functions. To generate these pulses, endocrine organs have developed finely regulated mechanisms to modulate blood flow both to meet the metabolic demand associated with intense endocrine cell activity and to ensure the temporally precise uptake of secreted hormone into the bloodstream. With a particular focus on the pituitary gland as a model system, we review here the importance of the interplay between blood flow regulation and oxygen tensions in the functioning of endocrine systems, and the known regulatory signals involved in the modification of flow patterns under both normal physiological and pathological conditions.

Effect of estrogen on the blood supply of pituitary autografts in rats.

Estrogens are known to cause pituitary enlargement and lactotroph proliferation. They also modulate pituitary angiogenesis and induce tumor formation. Pituitary grafts, due to the loss of hypothalamic dopamine, also show lactotroph hyperplasia. We investigated the role of estrogen on rat pituitary autograft vascularization by light and transmission electron microscopy, and assessed prolactin (PRL) blood levels, microvessel density (MVD) and cell proliferation using the BrdU labeling index. All adenohypophysial cell types were identified by immunohistochemistry (streptavidin-biotin-peroxidase complex method). The proangiogenic factors, vascular endothelial growth factor (VEGF), its receptor Flk-1, and hypoxia inducible factor-1alpha (HIF-1alpha) were similarly demonstrated. The prevalence of lactotrophs, as well as more intense staining for VEGF, Flk-1 and HIF-1alpha, was noted in those grafts exposed to estrogen, mainly in the area surrounding the central necrotic core. Immunostaining showed Flk-1 expression increased in endothelial cells of the estrogen-exposed grafts as compared with those unexposed. In contrast to the grafts not exposed to estrogen, in the estrogen-exposed grafts, only fenestrated endothelium could be demonstrated, suggesting that estrogen induces fenestration of newly formed capillaries. There was an increase in blood PRL levels in the estrogen-treated groups as compared with controls. Both MVD and BrdU labeling indices were higher in grafts exposed to estrogen, especially after 4 weeks. Our results suggest that estrogen administration not only enhances the expression of proangiogenic factors in the pituitary grafts but also induces their expression at earlier stages, leading to rapid neoformation of purely fenestrated capillaries.

Pituitary-brain vascular relations: a new paradigm.

Vascular casts of the pituitary gland have demonstrated a paucity of veins extending from the adenohypophysis to the systemic circulation and have suggested that some adenohypophyseal venous blood returns to the neurohypophysis. The neurohypophyseal capillary bed may function as a vascular switch and in this article a series of 14 questions are proposed regarding the vascular dynamics of the pituitary. Together these questions raise the larger question, namely, whether pituitary hormones are transported directly to the brain to modify brain function?

Immunohistochemical expression of nestin in adenohypophysial vessels during development of pituitary infarction.

OBJECTIVES: The aim of this work was to investigate the immunohistochemical expression of nestin, a member of the intermediate filament family, in adenohypophysial vasculature during development and progression of pituitary infarction. METHODS: Forty-five nontumorous adenohypophyses and 34 pituitary adenomas of various types, all exhibiting acute or healing infarcts, were examined immunohistochemically using the streptavidin-biotin-peroxidase complex method. RESULTS: In both adenohypophyses and pituitary adenomas without infarction, nestin was expressed in only a few capillaries and endothelial cells. In acute infarcts without a vascular response, no nestin was demonstrable within necrotic capillaries (50 cases). In organizing infarcts, newly formed vessels spreading into necrotic zones showed nestin expression in all capillaries and practically every endothelial cell (25 cases). In the hypocellular, fibrotic scar phase, only a few vessels (4) were apparent, and immunoreactivity was focal and mild. CONCLUSIONS: Nestin is strongly expressed in newly formed capillaries and is downregulated when infarcts transform to fibrous tissue. Nestin expression may provide valuable insight into the process of pituitary angiogenesis.

Isolation and characterisation of CD9-positive pituitary adult stem/progenitor cells in rats.

S100ß protein and SOX2-double positive (S100ß/SOX2-positive) cells have been suggested to be adult pituitary stem/progenitor cells exhibiting plasticity and multipotency. The aim of the present study was to isolate S100ß/SOX2-positive cells from the adult anterior lobes of rats using a specific antibody against a novel membrane marker and to study their characteristics in vitro. We found that cluster of differentiation (CD) 9 is expressed in the majority of adult rat S100ß/SOX2-positive cells, and we succeeded in isolating CD9-positive cells using an anti-CD9 antibody with a pluriBead-cascade cell isolation system. Cultivation of these cells showed their capacity to differentiate into endothelial cells via bone morphogenetic protein signalling. By using the anterior lobes of prolactinoma model rats, the localisation of CD9-positive cells was confirmed in the tumour-induced neovascularisation region. Thus, the present study provides novel insights into adult pituitary stem/progenitor cells involved in the vascularisation of the anterior lobe.

Inhibition of estrogen-induced pituitary tumor growth and angiogenesis in Fischer 344 rats by the matrix metalloproteinase inhibitor batimastat.

The development of estrogen-induced pituitary prolactinoma in Fischer 344 (F344) rats is associated with enhanced neovascularization. Based on the significance of matrix metalloproteinases (MMPs) for tumor growth and angiogenesis, we have studied the effect of batimastat (BB-94), a synthetic MMPs inhibitor (MMPI) on the progression of prolactin-secreting pituitary adenoma in rats. Pituitary tumors were induced in male F344 rats by s.c. implantation of Silastic tubes containing diethylstilbestrol (DES). The effects of chronic treatment with BB-94 (30 mg/kg b.w.) on pituitary weight, cell proliferation, apoptosis and vascular density were evaluated. We have stated that chronic treatment with batimastat caused a significant reduction in the pituitary weight. Batimastat has been found to decrease cell proliferation evaluated by a number of PCNA-positive stained cell nuclei. A marked increase in the apoptotic index within the pituitary was observed in the study group. Moreover, the density of microvessels identified by CD31 was reduced in the group treated with BB-94. The results of our study provide evidence for an inhibitory effect of batimastat, a synthetic MMPI, on the growth and angiogenesis in an experimental model of human prolactinoma. The ability of BB-94 to suppress established pituitary tumor growth suggests a possible application of MMPIs in the treatment of pituitary adenomas.

Transforming growth factor alpha in arterioles: cell surface processing of its precursor by elastases.

Analysis of the transforming growth factor alpha (TGF alpha) cDNA predicts that the mature TGF alpha polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGF alpha to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGF alpha, NR6 cells were transfected with the rat TGF alpha cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGF alpha precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGF alpha polypeptide. The released TGF alpha was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGF alpha but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic alpha 1-chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGF alpha precursor from the fixed cells. The location of TGF alpha in the arterioles and ability of PMN leukocytic elastase to process the membrane-bound TGF alpha precursor suggests a novel role for this elastase at the wound site.

Combined CNS and pituitary involvement as a primary manifestation of Wegener granulomatosis.

Wegener granulomatosis (WG) is a systemic vasculitis of small and medium vessels. It predominantly affects the upper and/or lower respiratory airway and kidneys. Its pathogenesis is not fully understood. WG relatively frequently affects the nervous system (in 30-50% according to the different studies). Most frequently, it manifests as necrotizing vasculitis that leads to the peripheral neuropathies or to the cranial nerves palsy. Impairment of the central nervous system (CNS) is less frequent and occurs in 2-8% of patients. Three major pathogenetic mechanisms were described: CNS vasculitis, spreading of granulomas from the adjacent anatomical areas (paranasal cavities, orbit etc.), and new formation of granulomas in brain tissue. This case report describes patients in whom WG manifested in the form of localized skin involvement and combined CNS involvement that included pituitary gland. Atypical presentation of WG impedes and slows down the process of diagnosis and emphasizes the need for collaboration between medical specialists.

Anterior pituitary vasodilation after hemorrhage in the dog.

A miniature thermoelectric probe was used to record continuously tissue blood flow in the anterior pituitary gland of anesthetized dogs. The output of the flow probe is linear in the range of tissue blood flow encountered in the pituitary. Probe placement required minimal tissue trauma, and zero blood flow was recorded at the end of each experiment. Little or no change in flow followed hemorrhage of 10 ml/kg body weight. However, hemorrhages of 20 or 30 ml/kg were followed by a biphasic deviation from control level, with an initial transient decrease in flow, followed by recovery to control at about 19 min and a rise above control to 90% of maximum at about 32 min. Hypothalamohypophysial vascular resistance was found to decrease significantly following hemorrhages of 20 or 30 ml/kg, and this was shown to be independent of resistance changes in the femoral vascular bed. It is concluded that anterior pituitary blood flow is well maintained following moderate hemorrhage as a result of intrinsic vasodilation in the hypothalamo-hypophysial vascular bed.

Correlation of spatial differences in concentrations of prolactin and growth hormone cells with vascular pattern in the female mouse adenohypophysis.

Adult female mice of the DDY/S strain were used to study the distribution of PRL or GH cells and the vasculature of the anterior pituitary lobe. Electron microscopy was used to quantify PRL or GH cells in horizontal sections. Most parenchymal cells were either PRL or GH cells, and both types of cells were present in all regions. The densities of PRL cells in the rostral and caudal areas were significantly greater than that of GH cells. The density of GH cells was greater in the anterolateral wings. Thus, the spatial differences in concentrations of PRL and GH cells were reversed. The vasculature was studied with scanning electron microscopy of vascular casts and with stereoscopy of pituitary glands injected with India ink. The adenohypophysis was supplied by long and short portal vessels. The long portal vessels originated from the primary capillary plexus on the median eminence and the upper portion of the pituitary stalk, and they supplied rostral regions of the adenohypophysis. Most of the short portal vessels connected caudal areas of the anterior lobe with the posterior lobe, crossing the surface of the intermediate lobe. The blood in the short portal vessels may flow from the posterior lobe toward the anterior lobe. Thus, within the rostral and caudal areas, which are supplied by long and short portal vessels, respectively, PRL cells predominated; the anterolateral wings where GH cells predominated were far from these regions. These data suggest that the anatomical pattern of the blood supply may account in part for the spatial distribution of PRL and GH cells.

Can the pituitary secrete directly to the brain? (Affirmative anatomical evidence).

Vascular casts of 10 rhesus monkey pituitary glands and three vascular casts of the rhesus monkey cavernous sinus were examined by scanning electron microscopy. A continuous neurohypophyseal capillary bed was found uniting the infundibulum, infundibular stem, and infundibular process. The neurophypophysis was supplied by three groups of arteries: superior hypophyseal, middle hypophyseal, and inferior hypophyseal. Numerous anastomoses were found between individual arteries, and some hypophyseal arteries formed anastomotic links between different portions of the circle of Willis. Veins located at the caudal pole of the infundibular process, capillaries linking the infundibulum to the hypothalamus, and portal vessels extending from the infundibulum to the adenohypophysis provided efferent vascular pathways from the neurohypophysis. The adenohypophysis received no direct arterial supply; its entire afferent vascular supply was provided by portal vessels. Lateral hypophyseal veins were not found; small adenohypophyseal veins joined larger neurohypophyseal veins to form confluent pituitary veins which extended to the cavernous sinus. The capacity of the venous connections draining the adenohypophysis directly to the cavernous sinus appeared small when compared to that of of the long portal vessels supplying the adenohypophysis. However, many of the short portal vessels interposed between the adenohypophysis and the infundibular stem and process were well arranged to function as alternative efferent routes from the adenohypophysis. The limited potential for venous drainage directly to the cavernous sinus suggests that blood leaves the adenohypophysis by other routes; blood carried via long portal vessels from the infundibulum to the adenohypophysis may return to the neurohypophyseal capillary bed via short portal vessels. This anatomical study suggests that hypothalamic and adenohypophyseal secretions are conveyed to the capillary bed of the neurohypohysis. These secretions may leave the neurohypophysis via any of seven potential routes: one efferent route is directed to the adenohypophysis, another route is directed to the systemic circulation, but five of the potential efferent routes are directed toward the brain.

Hemorrhage-induced secretion of corticotropin-releasing factor-like immunoreactivity into the rat hypophysial portal circulation and its inhibition by glucocorticoids.

A paradigm for reliably stimulating ACTH secretion in urethane-anesthetized male rats has been used to examine hypothalamic secretion of corticotropin-releasing factor-like immunoreactivity (CRF-LI) into the hypophysial portal circulation. Hemorrhage of 15% estimated blood volume evoked a maximal 4.6-fold elevation in circulating ACTH levels from an initial level of 178.4 +/- 51.2 (+/-se) to 814.7 +/- 184.6 pg ml-1. The cumulative amount of ACTH secreted in response to hemorrhage was 10-fold greater than the cumulative amount of ACTH secreted by nonhemorrhaged rats (unweighted cumulative effect over all time points). In another experiment from a similarly hemorrhaged group, the hypophysial portal plasma CRF-LI concentration rose 2-fold from an initial level of 429.7 +/- 34.2 to 839.3 +/- 170.4 pg ml-1. Pretreatment with dexamethasone (100 microgram/kg BW, im) had no effect on initial levels of either CRF-LI or ACTH. The hemorrhage-induced elevations of both CRF-LI and ACTH were abolished in dexamethasone-treated rats. The secretory rate of CRF-LI was calculated to be 1.61 +/- 0.7 pg min-1 in nonhemorrhaged animals. Reversible pharmacological hyperpolarization of the paraventricular nuclei by stereotaxically microinjected procaine (15 micrograms/100 nl) reduced portal plasma CRF-LI and peripheral plasma ACTH to undetectable levels. These observations led to the following conclusions: 1) CRF-LI is an important hypothalamic regulator of adenohypophysial ACTH secretion, 2) CRF-LI in the hypophysial portal circulation is derived from CRF-LI-containing neurons within the paraventricular nuclei, and 3) glucocorticoid negative feedback effects can be exerted at the central level.

Effect of hypothalamic deafferentation on hypophysial and other endocrine gland blood flows.

The effects of partial or complete deafferentation of the medical basal hypothalamus (MBH) on blood flow through endocrine glands was evaluated using a modification of Sapirstein's indicator fractionation method. Adult female Wistar rats were distributed into three groups consisting of normal control animals (n = 12), animals in which rostral deafferentation (Rd) of the MBH was performed (n = 11), and animals in which complete deafferentation (Cd) of the MBH was done (n = 6). Three weeks after the surgical procedures, Cd reduced adenohypophysial weight by 30% and raised its blood flow levels by 80% compared to those values in the Rd and control groups. In contrast to the anterior pituitary, both Rd and Cd led to weight reduction of 22% in posterior lobes, and Cd was also accompanied by a 50% net blood flow decrease. No statistically significant differences were noted in pineal gland weights or decreases in blood perfusion rates in adrenal and thyroid glands. There were no body weight changes in experimental animals compared to values in the control groups. These results suggest that there may be a loss of vasomotor tone in the primary vascular beds of the adenohypophysial portal systems after deafferentation which elicits the 80% increase in adenohypophysial blood flow. This increased circulation may play a role in pituitary function(s) and should be included along with diminished neuroendocrine inputs in interpretations of results obtained after deafferentation procedures.

Morphological analysis of growth hormone release from rat somatotrophs into blood vessels by immunogold electron microscopy.

The ultrastructure of GH release from the somatotrophs of the rat anterior pituitary was examined in vivo by immunogold electron microscopy. Under pentobarbital anesthesia, injection of GH-releasing factor clearly induced an increase in both plasma GH content and the number of exocytotic GH-immunopositive granules in the cells. The exocytotic events occurred from a part of the plasma membrane facing endocrine cells, including other somatotrophs, and other portions facing folliculo-stellate cells or the walls of blood vessels. When released from the plasma membrane, the GH-immunopositive secretory granules sometimes appeared to aggregate with each other and showed an irregular shape surrounded by a single unit membrane. The exocytotic secretory granules were released into the extracellular space, and then flowed into the sinusoids as irregularly shaped GH-immunopositive electron-dense masses. After reaching the vascular space via the intercellular spaces between the endothelial cells, the contents of each mass became diffusely dispersed into the blood stream, with concomitant disappearance of immunopositivity. The present study thus revealed the morphological aspects of the process of GH secretion from somatotrophs into the blood vessels.

Inhibition of estrogen-induced anterior pituitary enlargement and arteriogenesis by bromocriptine in Fischer 344 rats.

The interrelationship between dopamine (DA) regulation and changes in the blood supply of the anterior pituitary lobe (AP) in the etiology of estradiol (E2)-induced proliferation of pituitary cells was studied in Fischer 344 rats. Rats were implanted with E2-filled or empty Silastic capsules for 21 days alone or in conjunction with pellets of the potent DA agonist bromocriptine (CB-154). Changes in vascularization of the AP, median eminence DA content, and responsiveness to DA of cultured AP cells were measured. Development of a direct arterial blood supply was assessed by the injection of 15-microns microspheres that can only reach the AP by newly formed arteries (arteriogenesis). APs were enzymatically dispersed and cultured for 3 days before challenges with increasing concentrations of DA for 3 h. DA content was measured by radioenzymatic assay, and serum PRL was determined by RIA. E2 treatment increased the weight of the pituitary gland, serum PRL levels, and the number of microspheres in the AP 4.5-, 173-, and 142-fold, respectively, over control values. Median eminence DA content was decreased 71% by E2 treatment, while the ability of DA to suppress PRL secretion in vitro decreased from a maximum of 70% to 40% with no change in the ED50. Simultaneous treatment with CB-154 dramatically decreased the effect of E2 on arteriogenesis, pituitary weight, serum PRL levels, and median eminence DA content. Blockade of E2-induced AP enlargement by increased dopaminergic stimulation was closely correlated with inhibition of arteriogenesis, which further suggests an important role for vascular changes in lactotroph proliferation.

Morphological evidence for the presence of arteries in human prolactinomas.

This study was undertaken to clarify the vascular anatomy of human prolactinomas and specifically to determine whether arteries were present. Sixteen prolactinomas were studied by electron microscopy. The presurgical diagnosis of the tumors as prolactinomas was based on the findings of hyperprolactinemia and radiographic abnormalities, and was confirmed by the electron microscopic features of the specimens as well as by immunocytochemical staining for PRL. In addition to the presence of fenestrated endothelial cells, which are characteristic of the normal capillaries of the anterior pituitary, 13 of the 16 prolactinomas contained arteries. These arteries ranged from well formed vessels with multiple layers of smooth muscle cells to abnormal terminal arterioles, i.e. vessels with fenestrated endothelium surrounded by a variable number of smooth muscle cells. Arteries were not found in anterior pituitaries from 8 patients with no pituitary disease. In the prolactinomas, smooth muscle cells also were found, either isolated in the pericapillary connective tissue space or in small cords some distance from the vessel lumen. The results suggest that vascular changes, including arteriogenesis, occur in prolactinomas (and possibly other types of pituitary tumor). The arteries entering the anterior pituitary directly could be congenital or develop during formation of the tumor. An arterial blood supply to a region of the anterior pituitary could result in the escape of that area from hypothalamic regulation, since systemic blood contains negligible levels of hypothalamic hormones. In the case of PRL-secreting cells, which are tonically inhibited by the hypothalamic hormone dopamine, this would result in hypertrophy, hyperplasia, and possibly tumorigenesis.

Calcitonin-like immunoreactivity in the pituitary vascular bed of man.

The calcitonin-like immunoreactivity (CT-like immunoreactivity) was measured in blood aspirated from the vascular bed of the anterior pituitary gland during transsphenoidal surgery in 33 patients with PRL-producing microadenomas, 2 patients with Cushing's disease, and 1 patient with metastatic breast cancer with a normal pituitary gland. The mean level of CT-like immunoreactivity in the pituitary vascular bed was 2-3 times higher than in peripheral blood (2.4 +/- 0.9 ng/ml vs. 0.69 +/- 0.19 ng/ml), and the difference was highly significant (P less than 0.001). However, the serum ACTH, hGH, TSH, PRL, and FSH in the pituitary vascular bed was 1000 times or higher than that found in the peripheral blood. The serum CT-like immunoreactivity levels in the pituitary bed in the two patients with Cushing's disease were similar to that found in other patients. Our investigations indicate: 1) CT-like immunoreactivity in man is higher in the blood obtained from the pituitary vascular bed than that found in the peripheral blood; 2) the serum CT-like immunoreactivity level in the pituitary vascular bed is much less than ACTH or the other hormones secreted by the pituitary gland; 3) there is no correlation between CT-like immunoreactivity and ACTH levels.

The effect of angiotensin II receptor antagonists on diethylstilbestrol-induced vascular changes in the rat anterior pituitary gland: a quantitative evaluation.

The effects if diethylstilbestrol (DES) and of angiotensin II (Ang II) receptor antagonists, such as losartan (selective AT1 receptor antagonist) or PD 123319 (selective AT2 receptor antagonist) on the anterior pituitary microvasculature were studied by means of computer-assisted image analysis. The vascularization was visualized using Selye's method modified by Poely et al. (1964). It was found that DES induced a sharp increase in vessel area, mean vessel diameter and perimeter, whereas mean vessel number was reduced. These DES-induced changes were inhibited by simultaneous administration of losartan. On the other hand, PD 123319 was less effective. These findings suggest an involvement of Ang II, acting mainly via AT1 receptors, in the mechanism of estrogen-induced vascular changes in the rat anterior pituitary gland.

The effect of somatostatin analog octreotide on diethylstilbestrol-induced prolactin secretion, cell proliferation and vascular changes in the rat anterior pituitary gland.

The effects of diethylstilbestrol (DES) and of long-acting somatostatin analog, octreotide (SMS) on the rat anterior pituitary microvasculature have been studied by means of computer-assisted image analysis. Additionally, the effects of DES and SMS on prolactin secretion and anterior pituitary cell proliferation have been studied, as well. The vascularization was visualized using Selye's method modified by Poely et al. (1964). The prolactin serum levels were estimated by radio-immunoassay. The proliferation indices were assessed using bromodeoxyuridine incorporation assay. As expected, it was found that DES sharply increased serum prolactin levels and enhanced cell proliferation in the anterior pituitary gland. DES also induced changes in parameters of vascularization. Simultaneous treatment of rats with SMS inhibited the DES-induced elevation of prolactin levels and pituitary cell proliferation. It also suppressed some but not all DES-induced changes in the anterior pituitary vascularization. These data suggest that the angio-inhibitory activity of SMS might be involved in its anti-tumor action on pituitary adenomas, but not as a sole or principal mechanism.