Nerve growth factor-like activity in the rat pituitary intermediate lobe

Nerve growth factor (NGF)-like immunoreactivity was demonstrated in the rat pituitary intermediate lobe by indirect immunofluorescence method using antisera specific to beta-NGF isolated from adult male mouse submaxillary salivary gland. Co-culture of frozen or fresh intermediate lobes with newborn rat superior cervical ganglion resulted in marked fiber growth from the ganglion, which was totally inhibited by NGF antiserum, suggesting the presence in situ and secretion in vitro of biologically active pituitary NGF. Pituitary stalk transection caused decrease in both the NGF immunoreactivity and biological activity. These findings suggest that pituitary NGF level is under neural regulation.     

Patch-clamp analysis of voltage-gated currents in intermediate lobe cells from rat pituitary thin slices

Ionic currents of hypophyseal intermediate lobe cells were studied using a thin-slice preparation of the rat pituitary in conjunction with conventional and perforated whole-cell patch-clamp recording techniques. A majority (89%) of the cells studied generated Na⁺, Ca²⁺ and K⁺ currents upon depolarizing voltage steps and responded to bath application of -aminobutyric acid (GABA; 20–50 M) with inward currents (in symmetrical chloride, holding potential –80 mV). A small percentage of cells (11%) did not display inward membrane currents upon depolarization and was unresponsive to GABA. In the first type of cells, Ca²⁺ and K⁺ currents were further studied in isolation. Ca²⁺ tail currents showed a biphasic time course upon repolarization, with time constants and amplitudes of 2.07±0.29 ms, 123±22 pA (for the slowly deactivating component) and 0.14±0.06 ms, 437±33 pA (for the fast-deactivating component; means±SD of n=4 cells). Slowly and fast-deactivating conductances were half-maximally activated at around –10 mV and +10 mV respectively. Depolarizing voltage steps elicited two types of K⁺ current, which were separated using a prepulse protocol. A fast-activating, transient component showed half-maximal steadystate inactivation between –65 mV and –45 mV depending on the divalent cation composition of the external solution. Its decay was fitted by single-exponential functions with time constants of 36±11 ms and 3.9±0.9 ms at –20 mV and +40 mV respectively (mean±SD; n=4 cells). Whereas the peak current amplitudes of the transient K⁺ current component remained stable, the amplitude of the second, delayed component increased progressively throughout the course of whole-cell experiments. In cells recorded with the perforated whole-cell technique, bath application of dopamine (10 nM–1 M) induced large hyperpolarizations from a spontaneous membrane potential of –40 mV, but did not consistently affect the amplitude of the voltage-gated K⁺ conductances. These data are compared to previous studies using other preparations of the intermediate lobe, and differences are discussed, thus helping to extend our knowledge of electrical excitability of hypophyseal cells.     

Ontogenesis of the rabbit pituitary intermediate lobe

Summary The development of the intermediate lobe of the rabbit pituitary was investigated by light and electron microscopy and by using immunocytological techniques. The first immunoreactive melanotrophic cells were detected at the fetal day 17 in the dorsal zone of Rathke's pouch epithelium facing the neural lobe; this coincided ultrastructurally with the appearance in this area of a few cells exhibiting secretory vesicles and granular condensations in the Golgi saccules. The differentiation of the gland probably required an infundibular inductive effect. Secretory cells increased in number following a dorsoventral gradient during the next fetal and neonatal stages until postnatal day 20, the stage at which the intermediate lobe exhibited its definitive organization. The gland innervation occurred during the first days after birth. The advent of these oxytocin- and neurophysin-immunoreactive fibres coincided with an obvious stimulation of the synthetic activity of the melanotrophic cells. The possible neurotrophic effect of these cells on their innervating system remains to be established.This article is dedicated to the memory of Georgette Haller     

A study of intermediate lobe differentiation in the human pituitary gland

The immunohistochemical demonstration of neurofilament (NF) polypeptide was used to identify nerves in a series of 17 pituitary adenomas. NF-positive fibres were present in two out of five corticotroph adenomas sited deep in the anterior lobe, in one out of five sited in the intermediate zone and in two out of seven non-corticotroph adenomas. Such nerve fibres were often seen in relation to blood vessels. The distribution of alpha-MSH immunoreactive cells was examined in 25 normal pituitaries and in 23 cases of Cushing's disease. Such cells were scattered throughout the normal gland and there was no increase in numbers in pregnancy. alpha-MSH was demonstrated in 18 corticotroph adenomas in Cushing's disease. There was no correlation with the site of the tumour or the presence of nerve fibres. alpha-MSH cells were distributed normally in the para-adenomatous gland. Crooke's hyaline change and alpha-MSH coexisted in some corticotrophs. These findings support the concept that 'intermediate lobe' function, as found in animals, has no discrete anatomical location in man.     

Ultrastructure of the pituitary intermediate lobe in aging rats

The pituitary intermediate lobe, a source of pro-opiomelanocortin (POMC) peptides, was examined with light and electron microscopic techniques in Sprague-Dawley rats aged 5-18 months and Zucker rats aged 18 months. Cysts were common in the intermediate lobe in the Zucker animals, a finding also noted in human pituitary glands by other investigations. The nuclear envelopes were often indented in cells of aging rats, while those of young animals were generally smooth. Lipid droplets and lysosomes, rarely seen in tissue from young animals, were frequently observed in endocrine cells of older rats. Most cells had an abundance of secretory granules, suggestive of enhanced storage of peptides in the cytoplasm. Nerve terminals which were present among endocrine cells contained myelin figures in some of the old rats, and may indicate degenerative changes, while other terminals appeared normal. These morphologic findings suggest that the aging phenomena in intermediate lobe tissue have characteristics in common with nervous tissue, and may also reflect a diminished inhibitory neuroregulation.     

Non-secretory neuronal elements in the bovine pituitary intermediate lobe

The bovine pituitary intermediate lobe was studied by light microscopy after staining with a modified Bodian procedure. The study revealed a number of dissimilar intermediate lobe elements characteristic of mature and immature cells found in the adult and embryonic central nervous system and ganglia. In this study the elements are referred to as non-secretory neuronal elements. The elements, forming part of the parenchyma, have multiple processes which distribute themselves throughout the lobe. The typical secretory intermediate lobe cells have only a slight affinity for the Bodian stain. The intermediate lobe basophilic staining secretory cells and the neuronal elements maintain their general differences in appearance and possibly their function. Since the intermediate lobe in bovine is rather avascular and neuronal elements are usually associated with blood vessels, these elements may be involved in certain mechanisms yet unknown. It is possible that the elements provide common precursor proteins that reach the secretory intermediate lobe cells through their dendritic branches. In addition, the non-secretory neuronal elements may be directly involved in the spike potentials recorded from the lobe as well as in stimulus-secretion coupling. This study does not treat the function of the neural elements at this time, but directs attention strictly to their presence in the intermediate lobe of the bovine pituitary gland.     

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1–4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10^–8 M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I>Br>Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23–31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR 42641, 1 M) blocked activity evoked by GABA (1–10 M), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 M, and were practically abolished at Ca_i 1 M. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Ca_i reached 1 M.     

GABAA and GABAB receptor-mediated inhibition of release of 5-hydroxytryptamine in the intermediate lobe of the rat pituitary gland

Isolated neurointermediate lobes or neural lobes of the rat pituitary gland attached to the pituitary stalk were incubated in vitro and the spontaneous or electrically (pituitary stalk stimulation, 5 Hz, 1,500 pulses) evoked release of 5-hydroxytryptamine was determined. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe was increased fivefold in the presence of the dopamine receptor antagonist (-)-sulpiride (1 microM). The evoked release of 5-hydroxytryptamine from the isolated neural lobe was not altered by (-)-sulpiride. The evoked release of 5-hydroxytryptamine from the isolated neural lobe in the presence of (-)-sulpiride was less than 5% of that from the combined neurointermediate lobe showing that most of the 5-hydroxytryptamine released from the combined neurointermediate lobe originated in the intermediate lobe. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe in the presence of (-)-sulpiride showed a diurnal variation. It was three to five times higher between 9.30 and 14.00 h than between 8.30 and 9.30 h or between 14.00 and 16.00 h. The 5-hydroxytryptamine tissue content at the end of the incubation experiments also showed similar variations which were, however, less pronounced. The evoked release of 5-hydroxytryptamine from the neurointermediate lobe, in the presence of (-)-sulpiride, was reduced by the preferential GABAA receptor agonist muscimol or the selective GABAB receptor agonist (-)-baclofen in a concentration-dependent manner. Bicuculline, a selective GABAA receptor antagonist inhibited the effect of muscimol, but not that of (-)-baclofen. Bicuculline alone did not affect the release of 5-hydroxytryptamine from the gland. It is concluded that the release of 5-hydroxytryptamine in the intermediate lobe is influenced by both dopaminergic and GABAergic mechanisms.     

Release of endogenous 5-hydroxytryptamine from the intermediate lobe of the rat pituitary gland is inhibited by endogenous dopamine

The release of endogenous 5-hydroxytryptamine (5-HT) from the in vitro incubated neurointermediate lobe (NIL) of the rat hypophysis was measured by HPLC with electrochemical detection. 5-HT release evoked by electrical stimulation of the pituitary stalk from the NIL, but not from the isolated neural lobe (NL) was enhanced in the presence of the dopamine receptor antagonist sulpiride (1 microM). The opiate receptor antagonist naloxone (1 microM) had no effect on the evoked release of 5-HT from the NIL or NL. In conclusion, the release of endogenous 5-HT from the intermediate lobe is under inhibitory control of endogenous dopamine.     

Release of endogenous 5-hydroxytryptamine from the neural and the intermediate lobe of the rat pituitary gland evoked by electrical stimulation of the pituitary stalk

A calcium-dependent release of 5-hydroxytryptamine from the neural and intermediate lobe of the rat pituitary gland has been demonstrated following electrical stimulation of the pituitary stalk with stimulation parameters thought to evoke propagated action potentials. The 5-hydroxytryptamine release from the intermediate lobe was double that from the neural lobe. The mass of the intermediate lobe of the rat is about 80% of that of the neural lobe [Holzbauer, Racké, Mann, Cooper, Cohen, Krause and Sharman (1984) J. Neural Transm. 59, 91-104]. The relatively high overflow of 5-hydroxytryptamine from the intermediate lobe agrees with immunohistochemical studies in which a larger number of 5-hydroxytryptamine fibres were seen in the intermediate lobe than in the neural lobe. The present results have demonstrated that the rat hypophysis contains neuronal 5-hydroxytryptamine. They also suggest that this amine may act as a neurotransmitter substance in the neural and intermediate lobe.     

Presence of arginine-vasopressin in bovine pituitary intraglandular colloid of intermediate lobe origin

In order to determine whether pituitary intraglandular colloid might contain enough hormonal material to be considered a transport medium for intermediate lobe peptides, arginine-vasopressin was assayed in three pools of colloid collected from nearly 100 steers. Colloid samples taken from the pituitary glands of freshly slaughtered cattle were pooled, freed of extraneous tissue, and lyophilized. Three such pools were further extracted on octadecylsilyl-silica and assayed. The radioimmunoassay showed that arginine-vasopressin was indeed present in the bovine intraglandular colloid at levels ranging from 0.18 to 6.95 ng/mg dry weight. These results indicate that the intraglandular colloid contains a sufficient amount of arginine-vasopressin to be considered as a transport medium for this and other intermediate lobe peptides.     

Hypothalamic deafferentation in the rat appears to discriminate between the anterior lobe and intermediate lobe response to stress

Anterolateral deafferentation of the mediobasal hypothalamus prevented the increase of the plasma corticosterone concentration induced by ether, histamine and electric footshock. Hypothalamus deafferentation also prevented the ether stress-induced increase of the plasma levels of ACTH and beta-endorphin immunoreactivity (ACTHi, beta-ENDi). Infusion of isoproterenol evoked an increase of the plasma levels of corticosterone, ACTHi, beta-ENDi and alpha-MSH immunoreactivity (alpha-MSHi) in sham-operated rats. In rats with a deafferented hypothalamus, the responses of plasma corticosterone and ACTHi to isoproterenol were blocked but the responses of plasma beta-ENDi and alpha-MSHi remained intact. We conclude that circulating beta-ENDi after exposure to ether is of anterior lobe origin while circulating beta-ENDi after infusion of isoproterenol is of intermediate lobe origin.     

Ultrastructural morphometry of the rat pituitary intermediate zone after stimulation and suppression

The intermediate zone of the rat pituitary was studied after stimulation for 3 weeks with the adrenostatic drugs metyrapone and aminogfutethimide and after suppression for 3 weeks with the glucocorticoid 6α-methylprednisolone using morphometry by the point-counting method on electron micrographs. After metyrapone, intermediate cells showed a significant (P <.05) increase in volume percentages of the smooth endoplasmic reticulum, the Golgi apparatus, and the lucent immature secretory granules, as well as a significant decrease in the lucent secretory granules, the vacuoles, the lysosomes, and the cell membranes in comparison with the untreated controls. After aminoglutethimide, the volume percentages of the nuclei, the smooth endoplasmic reticulum, and the dense and lucent immature secretory granules were significantly increased in comparison with the controls, whereas the unorganized cytoplasm, the lucent secretory granules, the vacuoles, and the lysosomes were significantly decreased. The morphological changes after both adrenostatic drugs clearly indicate the pathophysiologically expected increase of secretory activity. After 6α-methyfprednisolone, the rough endoplasmic reticulum, the vacuoles, and the lysosomes significantly decreased in comparison with the controls, and the smooth endoplasmic reticulum, the Golgi fields, and the immature secretory granules were increased. These changes cannot be interpreted without certain reservations as the first alterations indicate decreased activity, whereas the increased Golgi fields and immature secretory granules are usually signs of increased secretory activity, which cannot be expected after glucocorticoid treatment. The possible causes are discussed.     

Biosynthesis of pro-opiomelanocortin is increased in the rat intermediate pituitary following denervation

In order to study the effect of pituitary intermediate lobe denervation on pro-opiomelanocortin (POMC) biosynthesis and processing, radioactive amino acids were incorporated in vitro into whole neurointermediate lobe (NIL) explants obtained from hypothalamic lesioned rats and control animals. The lesion in the basal hypothalamus removed the neural input to the intermediate pituitary and cut the neurohypophyseal neurons. One week after the lesion, approximately a 3-fold increase in the rate of synthesis of POMC peptides was found in the NIL. The content of POMC peptides was decreased. The results imply that denervation increases the rate of POMC synthesis and release, without altering the pattern of proteolytic processing.     

Modulation of expression of intermediate filaments during the development of established rat liver cell lines

A number of clear epithelial-like cell lines were established from the liver of fetal and neonatal rats. The intermediate filaments of these cells were investigated using polyclonal prekeratin antisera, monoclonal antibodies against a cytokeratin subfamily and vimentin by means of the indirect immunofluorescence technique and SDS-PAGE analysis. Changes in the expression of cytokeratin filaments were found during the evolution of permanent cell lines. Cells positively stained for cytokeratins could be seen near to other cells which were negative. In early passages (up to the 40th) nearly all cells were strongly stained by the different keratin antibodies. During the following subcultivation the pattern of staining considerably changed. In FRL and NP-RL cell lines keratin-negative cells could already be observed in the early passages, rapidly increasing in the later passages. Compared to this, vimentin-staining of all cells remained constant in its morphological expression. The keratin filaments were seen in thick fiber bundles arranged particularly in the perinuclear ring as well as in finer networks throughout the cytoplasm. Every cell in the established lines showed their very individual staining pattern. The vimentin filaments extended to the whole cytoplasm up to the cell margin. Our observations demonstrate the variability of the system of keratin filaments in established epithelial-like liver cells under cultural conditions.     

Global transcriptome analysis of Borrelia burgdorferi during association with human neuroglial cells

As adherence and entry of a pathogen into a host cell are key components to an infection, identifying the molecular mechanisms responsible for cellular association will provide a better understanding of a microbe's pathogenesis. We previously established an in vitro model for Borrelia burgdorferi infection of human neuroglial cells. To expand on our earlier study, we performed B. burgdorferi whole-genome expression analysis following a 20-hour infection of human neuroglial cells to identify borrelial genes that were differentially regulated during host-cell association compared with cultured Borrelia in cell-free medium. This study identifies several regulated genes, the products of which may be important mediators of cellular pathogenesis.     

Changes in fine structure of pericytes and novel desmin-immunopositive perivascular cells during postnatal development in rat anterior pituitary gland

Pericytes are perivascular cells associated with capillaries. We previously demonstrated that pericytes, identified by desmin immunohistochemistry, produce type I and III collagens in the anterior pituitary gland of adult rats. In addition, we recently used desmin immunoelectron microscopy to characterize a novel type of perivascular cell, dubbed a desmin-immunopositive perivascular cell, in the anterior pituitary. These two types of perivascular cells differ in fine structure. The present study attempted to characterize the morphological features of pituitary pericytes and novel desmin-immunopositive perivascular cells during postnatal development, in particular their role in collagen synthesis. Desmin immunostaining revealed numerous perivascular cells at postnatal day 5 (P5) and P10. Transmission electron microscopy showed differences in the fine structure of the two cell types, starting at P5. Pericytes had well-developed rough endoplasmic reticulum and Golgi apparatus at P5 and P10. The novel desmin-immunopositive perivascular cells exhibited dilated cisternae of rough endoplasmic reticulum at P5–P30. In addition, during early postnatal development in the gland, a number of type I and III collagen-expressing cells were observed, as were high expression levels of these collagen mRNAs. We conclude that pituitary pericytes and novel desmin-immunopositive perivascular cells contain well-developed cell organelles and that they actively synthesize collagens during the early postnatal period.