CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED₅₀ values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.     

Assessment of TRH as a potential MSH release stimulating factor in Xenopus laevis

This study considers the possible involvement of the tripeptide TRH (thyrotropin releasing hormone) in the physiological regulation of melanophore stimulating hormone (MSH) secretion from the pars intermedia of the toad, Xenopus laevis. TRH was shown to stimulate release of MSH from superfused neurointermediate lobes obtained from white-background adapted animals, but had no effect on secretion from lobes of black-background adapted animals. Immunohistochemical analysis revealed a rich TRH-containing neuronal network terminating in the neural lobe of the Xenopus pituitary. Plasma levels of TRH, determined with a specific radioimmunoassay, proved to be extremely high and no significant difference in this level could be found between white- and black-adapted animals. Plasma TRH probably originates from the skin, and our results show that its concentration is within the effective concentration range established for this peptide in stimulating MSH release from the pars intermedia. Therefore, while both our superfusion and immunohistochemical results argue favourably for a function of TRH in the regulation of MSH secretion, we conclude that, in any regulatory role, it would likely have to function within the pars intermedia at concentrations exceeding the high plasma values. While TRH could be involved in short-term activation of the secretory process in white-background adapted animals or in animals undergoing the initial stages of black background adaptation, our results indicate that this peptide may have no function in the maintenance of secretion from the pars intermedia of animals fully adapted to black background.     

Neuropeptide Y in the intermediate lobe of the frog pituitary acts as an alpha-MSH-release inhibiting factor

The presence of neuropeptide tyrosine (NPY) in the intermediate lobe of the frog pituitary was demonstrated using indirect immunofluorescence, the immunogold technique and a specific radioimmunoassay combined with high pressure liquid chromatography (HPLC). A high density of NPY-containing fibers, was found among the parenchymal cells of the intermediate lobe. These fibers originated from the ventral infundibular nucleus, travelled via the median eminence to the pars intermedia. At the electron microscopic level, NPY-like material was found exclusively in nerve fibers where the product of the immunoreaction was associated to dense-core vesicles. High concentrations of NPY-like peptide were found in neurointermediate lobe extracts. After Sephadex G-50 gel filtration the major peak of immunoreactive material appeared to co-elute with synthetic porcine NPY. Conversely, HPLC analysis revealed that the NPY-like peptide of the frog pituitary had a retention time shorter than the porcine NPY. The localization of NPY-like material in the pars intermedia suggested a possible role of NPY in the regulation of melanotropic cell secretion. In fact, graded concentrations of synthetic NPY induced a dose-dependent inhibition of alpha-melanotropin (alpha-MSH) release in vitro. The lack of effect of a dopaminergic antagonist on NPY-induced alpha-MSH release inhibition demonstrated that the local dopaminergic system could not account for the NPY action. These results indicate that NPY located in the hypothalamo-hypophyseal system of the frog may act as a melanotropin-release inhibiting factor.     

Adrenocorticotrophic and melanocyte-stimulating peptides in the human pituitary

The adrenocorticotrophic and melanocyte-stimulating peptides of the human pituitary were investigated by means of radioimmunoassay, bioassay and physicochemical procedures. Substantial amounts of adrenocorticotrophin and a peptide resembling beta-lipotrophin were identified in pituitary extracts, but alpha-melanocyte-stimulating hormone, beta-melanocyte-stimulating hormone and corticotrophin-like intermediate lobe peptide, which have been identified in the pars intermedia of pituitaries from other vertebrates, were not found. The absence of beta-melanocyte-stimulating hormone appears to contradict previous chemical and radioimmunological studies. Our results suggest, however, that it is not a natural pituitary peptide but an artefact formed by enzymic degradation of beta-lipotrophin during extraction.     

Immunocytochemical localization of vimentin in the posterior lobe of the cat, rabbit and rat pituitary glands

The presence and distribution of vimentin, a subunit of intermediate filaments, in the neural lobe and in the pars intermedia of the cat, rabbit and rat pituitary glands were investigated immunocytochemically. In the pars intermedia, our study revealed the presence of vimentin in glial-like cells located between glandular secretory cells of the three species and in the cells of the marginal layer of the cat and rat hypophyseal cleft. In the neural lobe of the cat and rabbit pituitary glands, there was a large amount of cell processes and immunoreactive pituicytes. In contrast, in the rat neural lobe, few pituicytes exhibited immunoreactivity, and these were located principally in the posterior region near the pituitary stalk. The significance of immunoreactive vimentin in these cells is discussed.     

Gamma-endorphin-like peptides in pituitary tissue: evidence for their existence in vivo

Beta and gamma endorphin-like peptides were measured by radioimmunoassay in whole pituitary. Boiling of acetic acid extracts prior to tissue disruption increased the concentration of both beta E- and gamma E-like peptides. The gamma E-like immunoreactivity from the neurointermediate lobe of the pituitary co-eluted with synthetic gamma E upon gel permeation chromatography. Immunoreactivity for beta E-like and gamma E-like peptides in the intermediate lobe of the pituitary was also shown by immunoperoxidase staining. The results suggest that gamma E-like peptides are present primarily in the pars intermedia in vivo and do not arise as artifacts of acid extraction of pituitary tissue.     

Interrelationships between pars intermedia and posterior pituitary with regard to corticotrophic and thyrotrophic partial function in rats

Functional interrelationships between the pars intermedia and posterior pituitary were proved under different experimental conditions. After stimulation of the thyrotropical axis of rats by an acute intraperitoneal application of 50 microgram TRH/rat the nuclear sizes of the thyroid follicular and the anterior pituitary thyrotropical cells increased according to a monophasic time curve with maximal amplitude at the time of 30 minutes. Interestingly, the nuclear sizes of the posterior pituitary cells were also enhanced. Under the same experimental conditions the nuclear areas of the cells of the external layer of the adrenal zona fasciculata decreased as did the nuclei of the pars intermedia cells (without regard to the cell type or localization of the cells in the intermediate lobe). Stimulation of the adrenocorticotropical axis by an acute injection of 0.2 ml isotonic saline solution/rat was followed by a time-dependent increase of nuclear sizes of the fasciculata cells and pars intermedia, whereas the nuclear volumes of the thyroid follicular cells, the anterior pituitary thyrotropical cells and the posterior pituitary cells decreased. Thus the functional state of the pars intermedia was in accordance with that of the adrenal cortex. Also the posterior pituitary cells responded to stimuli applied to the thyrotropical axis at the same degree as the thyrotropic organs themselves. Between the nuclear sizes of the pars intermedia and posterior pituitary we established the same inverse functional relationships as between the adrenal cortex and the thyroid gland.     

Immunoreactive alpha-melanotropin and beta-endorphin in the toad pars intermedia: dissociation in storage, secretion and subcellular localization

Storage, secretion and subcellular localization of immunoreactive α -melanotropin (α -MSH_i) and β -endorphin (β -END_i) were examined in the pars intermedia of toads adapted to a black or white background. During white adaptation, there was a selective increase in storage of α -MSH_i but not β -END_i. Subcellular fractionation and release studies suggest the presence of two pools of peptides in the toad pars intermedia, each containing different molar ratios of α -MSH_i and β -END_i and regulated differently in their release.     

The dopamine receptor in the intermediate lobe of the rat pituitary gland is negatively coupled to adenylate cyclase

The potency of a series of drugs to inhibit cyclic AMP accumulation in cells of the intermediate lobe of the rat pituitary gland in culture is typically dopaminergic. Dopaminergic antagonists reverse the inhibition of cyclic AMP levels according to their known pharmacological activity. The present data show that activation of the dopamine receptor in pars intermedia cells leads to inhibition of basal cyclic AMP accumulation and thus suggest that this receptor is negatively coupled to adenylate cyclase.     

Oxytocin-immunoreactive nerve fibers in the pars intermedia of the pituitary in the rabbit and hare

The pars intermedia of the pituitary in the rabbit and hare is abundantly innervated by axons reacting selectively with antibodies against oxytocin. These axons contain dense secretory vesicles about 140 nm in diameter, i.e., smaller than those in the neurosecretory axons of the neural lobe. No fiber elements staining for other peptides (vasopressin, somatostatin, substance P) were observed in the pars intermedia, except rare leu-enkephalin axons restricted to the rostral zone of the gland. Dopaminergic innervation appears to be completely absent from the intermediate lobe. This was shown by the lack of reaction with an antibody against tyrosine-hydroxylase, which did reveal a well-developed tubero-infundibular system of nerve fibers. Axons reacting with an antibody against serotonin were irregularly distributed in the pars intermedia. In the absence of dopaminergic axons, the extensive oxytocin-like innervation may play a major role in regulating the melanotrophic cell activity in the Leporidae.     

Cloning of corticotropin-releasing hormone (CRH) precursor cDNA and immunohistochemical detection of CRH peptide in the brain of the Japanese eel,paying special attention to gonadotropin-releasing hormone

The stress-related corticotropin-releasing hormone (CRH) was first identified by isolation of its cDNA from the brain of the Japanese eel Anguilla japonica. CRH cDNA encodes a signal peptide, a cryptic peptide and CRH (41 amino acids). The sequence homology to mammalian CRH is high. Next, the distribution of CRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined by immunohistochemistry. CRH-ir cell bodies were detected in several brain regions, e.g., nucleus preopticus pars magnocellularis, nucleus preopticus pars gigantocellularis and formatio reticularis superius. In the brain, CRH-ir fibers were distributed not only in the hypothalamus but also in various regions. Some CRH-ir fibers projected to adrenocorticotropic hormone (ACTH) cells in the rostral pars distalis of the pituitary and also the α-melanocyte-stimulating hormone (α-MSH) cells in the pars intermedia of the pituitary. Finally, the neuroanatomical relationship between the CRH neurons and gonadotropin-releasing hormone (GnRH) neurons was examined by dual-label immunohistochemistry. CRH-ir fibers were found to be in close contact with GnRH-ir cell bodies in the hypothalamus and in the midbrain tegmentum and GnRH-ir fibers were in close contact with CRH-ir cell bodies in the nucleus preopticus pars magnocellularis. These results suggest that CRH has some physiological functions other than the stimulation of ACTH and α-MSH secretion and that reciprocal connections may exist between the CRH neurons and GnRH neurons in the brain of the Japanese eel.     

Vascular Connections in the Pituitary of Anolis carolinensis with Special Reference to the Pars intermedia

The pituitary vascular system of Anolis carolinensis is similar to that of other lizards. Distinct portal vessels connect the primary plexus of the median eminence with the secondary plexus of the pars distalis. The primary plexus has some connections with the neural lobe. The neural lobe plexus is fed by separate arteries, and drains into the pars intermedia. The latter lobe receives its blood supply by this route. The pituitary is drained into the vena retrohypophysea. The findings are discussed in relation to existing theories regarding the neurovascular control of the pars intermedia.     

Intermediate filament expression in non-neoplastic pituitary cells.

Fifty-one non-neoplastic human pituitary glands, including examples with Crooke's hyalinization or amyloidosis, were examined by an immunoperoxidase method using antibodies to keratin, vimentin, neurofilaments (NFs), glial fibrillary acidic protein (GFAP), desmin, actin, S-100 protein and a variety of pituitary hormones. It was confirmed that most of the epithelial cells in the pituitary gland express keratin immunoreactivity. These cells included endocrine cells in the anterior lobe, endocrine cells and squamous metaplastic cells in the pars tuberalis, columnar and ciliated epithelia forming follicular structures and salivary-type epithelium in the pars intermedia, and anterior lobe cells infiltrating the posterior lobe. This study also demonstrated that keratin and NFs may be co-expressed in endocrine cells in the pituitary anterior lobe, that keratin, vimentin and GFAP may be co-expressed in the epithelial cells forming cyst-like follicle in the pars intermedia, and that vimentin and GFAP may be co-expressed in folliculo-stellate cells and pituicytes. In addition, the GFAP and S-100 protein-negative high columnar epithelium in the pars intermedia tended to be positive for adrenocorticotropic hormone and melanocyte stimulating hormone, while the low columnar epithelium with the co-expression of GFAP and S-100 protein was negative for pituitary hormones.     

An investigation of N-terminal pro-opiocortin peptides in the rat pituitary

Extracts of neurointermediate lobe (NIL) and anterior lobe (AL) of the rat pituitary, and material released from perfused rat pars distalis (PD) and pars intermedia (PI) cells were gel chromatographed and monitored using three antisera, each recognizing different regions of the non-corticotropin (ACTH)-lipotropin (LPH) portion of pro-opiocortin (POC). Two peaks (termed N-POC I) which emerged close to the elution position of rat beta-LPH were detected. The first peak was reduced significantly in the PI. Two smaller N-POC fragments which eluted near beta-endorphin were detected only in extracts and secretions of intermediate lobe tissue. One peak cross-reacted in the gamma 3-melanotropin (MSH) assay (N-POC III) whereas the other peak possessed amino (N)-terminal N-POC immunoreactivity (N-POC II). The results demonstrated differences in the distribution and nature of N-POC peptides released and extracted from the PD and PI of the rat pituitary, and suggest that the enzymic processing of N-POC is different in the two pituitary lobes.     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Endorphin in rat pituitary glands: its distribution within the gland, and age related changes in gland content in male and female rats

Endorphins were extracted with glacial acetic acid:acetone from freshly microdissected single lobes of rat pituitary and assayed for inhibition of ³H-etorphine stereospecific binding to rat brain opiate receptors. Highest tissue concentrations of endorphin were found in pars intermedia and pars distalis, with minimal activity observed in pars nervosa. In addition, mature female rats exhibited a significantly higher anterior lobe endorphin content than males, reflecting the larger gland weight, although no significant differences were found between neurointermediate lobe endorphin content of either sex. Pituitary endorphin content increased substantially with age, rising rapidly between the fifth and tenth week after birth. This elevation was still apparent at twenty-five weeks. These studies emphasize the importance of employing rats of closely similar age and weight in any experiments designed to examine pituitary endorphin function, and suggest that ontological development of pituitary endorphin may proceed in a manner similar to that of other pituitary hormones (e.g., MSH) derived from the same precursor peptide.     

Some functional aspects of canine corticotrophs

To examine the regulation and functional significance of canine pituitary pars intermedia corticotrophs, ACTH and cortisol responses to CRF were studied in healthy dogs before and after treatment with dexamethasone. In addition the effects of the dopamine agonist bromocriptine and the dopamine antagonist pimozide were investigated. In the latter two instances prolactin concentrations were also measured. Finally the pituitaries were studied immunocytochemically for ACTH and alpha-MSH. No response of ACTH or cortisol to bromocriptine was observed. Pimozide caused a slight rise in ACTH levels in some dogs. However, prolactin levels significantly decreased with bromocriptine and increased with pimozide. Injection of synthetic ovine CRF to dogs was followed by sharp increases in ACTH and cortisol values. These responses were obliterated by prior treatment with dexamethasone. In 1 of 4 dogs given dexamethasone before euthanasia, there were few pars distalis cells with ACTH(1-24) immunopositivity, although persistence of ACTH(1-24) reaction was noted within cells of the pars intermedia. The results indicate that none of the CRF-induced ACTH secretion in dogs is derived from pars intermedia corticotrophs. Dosages of bromocriptine and pimozide that clearly alter prolactin secretion do not consistently affect ACTH levels.     

Distribution of opioid peptides in the pituitary: a new hypothalamic-pars nervosa enkephalinergic pathway

The distribution of the endorphins, beta-endorphin and enkephalin (Met5-enkephalin and Leu5-enkephalin), was determined in the pars distalis, intermedia, and nervosa of the rat pituitary using both immunocytochemical and radioimmunological methods. Immunoreactive (ir) beta-endorphin was found in pars distalis and pars intermedia. On gel filtration of the pars distalis extracts, beta-endorphin immunoreactivity was eluted in three peaks corresponding to pro-opiocortin (5%), beta-lipotropin (75%), and beta-endorphin (20%). beta-Endorphin was the only component in the pars intermedia. Enkephalin was found in high amount in the pars nervosa. A new enkephalinergic hypothalamic-pars nervosa pathway was observed. Dehydration experiments on normal rats and analysis of the genetically polyuric Brattleboro rat suggest that this enkephalinergic pathway may modulate neurohypophyseal neurosecretion.     

Actions of PACAP and VIP on melanotrope cells of Xenopus laevis

The neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are implicated in the regulation of gene expression and hormone secretion in mammalian melanotrope cells and a mammalian pro-opiomelanocortin (POMC)-producing tumor cell line, but the physiological relevance of this regulation is elusive. The purpose of the present study was to establish if these peptides affect biosynthetic and secretory processes in a well-established physiological model for endocrine cell functioning, the pituitary melanotrope cells of the amphibian Xenopus laevis, which hormonally control the process of skin color adaptation to background illumination. We show that both PACAP and VIP are capable of stimulating the secretory process of the Xenopus melanotrope cell. As the peptides are equipotent, they may exert their actions via a VPAC receptor. Moreover, PACAP stimulated POMC biosynthesis and POMC gene expression. Strong anti-PACAP immunoreactivity was found in the pituitary pars nervosa (PN), suggesting that this neurohemal organ is a source of neurohormonal PACAP action on the melanotropes in the intermediate pituitary. We propose that the PACAP/VIP family of peptides has a physiological function in regulating Xenopus melanotrope cell activity during the process of skin color adaptation.