Beta-endorphin in pituitary intraglandular colloid of intermediate lobe origin.

Radioimmunoassay (RIA) detected the presence of beta-endorphin in the intraglandular colloid (IGC) of bovine pituitary intermediate lobe origin. The amount of beta-endorphin recovered in each of twelve samples ranged from 0.15 to 218.30 pmol/mg protein. A second group of assays [amino acid analysis, high performance liquid chromatography (HPLC) and mass spectral analysis] confirmed the RIA findings in another series of colloid samples. Approximately 75 pmol was collected from eight pooled glands. beta-Endorphin is an addition to the list of peptide hormones (e.g., methionine-enkephalin, adrenocorticotropin, arginine-vasopressin, alpha-melanocyte-stimulating hormone, beta-lipotropin and somatostatin) previously discovered in IGC by this laboratory.

Intraglandular colloid induced nuclear proliferation of murine thymic cells as determined by flow cytometry.

The ability of intraglandular colloid, the holocrine secretion of cells of the marginal half of the bovine pituitary intermediate lobe, to enhance or suppress the proliferation of immature thymic cells of CH3/6JH mice, 4 to 6 weeks of age, was studied. The mice were given subcutaneous injections of colloid for 3 consecutive days at a dose of 20 mg/kg body weight in 0.2 ml of saline. The analysis of propidium iodide stained thymic cell preparations by means of flow cytometry showed that there was a significant increase in the percentage of cells in the S phase (proliferative phase) of the cell cycle of mice injected with colloid (33%) compared with saline injected (15%) and non-injected controls (7%). These findings are the first to suggest that various combinations and concentrations of intraglandular colloid neuropeptides play a role in the immune system and shed light on additional functions of the intermediate lobe.

Somatostatin-like immunoreactivity in pituitary intraglandular colloid of intermediate lobe origin.

The objective of this study was to determine the presence of somatostatin-like immunoreactivity (SSIR) in bovine pituitary intraglandular colloid (IGC) of intermediate lobe (IL) origin. Three colloid samples, sufficient to perform the assays, were taken from freshly slaughtered steers, freed of extraneous tissue and lyophilized. The pools were extracted with acetone and petroleum either and the extract radioimmunoassayed for the presence of SSIR. SSIR was present in two of the three samples assayed. We conclude SS is one of the many peptides found in pituitary IGC of IL origin.

Light microscopic study of the hypophyseal angioarchitecture in the rabbit.

This study describes the hypophyseal angioarchitecture found in 79 adult New Zealand white rabbits. The pituitary glands and attached hypothalami were removed and carefully processed following routine histological methods, and the vascular organization was studied by light microscopy. Whole mounts of the pituitary median eminence complex were prepared and studied with a binocular dissecting microscope employing transmitted and epi-illumination. Arterial blood was found to be directed primarily to the neurohypophysis by the superior hypophyseal artery (SHA) and the inferior hypophyseal artery (IHA). A direct arterial blood supply was found to the adenohypophysis, but was limited solely to the pars intermedia by branches of the anterior hypophyseal artery (AHA) and the IHA. Capillaries of the pars intermedia were subdivided into an intermediate and a superficial plexus. The superficial plexus was situated between the intermediate plexus and the capillaries of the infundibular process. Capillaries of the superficial plexus did not form anastomoses between themselves, but ramified into the intermediate plexus to form a dense network of anastomosing capillaries that were continuous with capillaries of the pars distalis. A direct arterial blood supply was found only to the superficial plexus.

Beta-lipotropin-like immunoreactivity in intraglandular colloid from pituitary intermediate lobe cells.

The aim of this paper is the identification of beta-lipotropin (beta/LPH) as a peptide present in intraglandular colloid (the holocrine secretion of cells in the marginal half of the bovine pituitary intermediate lobe). beta/LPH, although not an opioid peptide itself, contains the peptide beta-endorphin. The methodology used allowed detection of beta/LPH when present in the samples in sufficient amounts.

Adrenocorticotropic hormone (ACTH) in pituitary colloid and extrapituitary tissues.

The level of ACTH in bovine pituitary intraglandular colloid (IGC/ACTH), the holocrine secretion of the marginal half of the intermediate lobe (IL) cells, was found to correlate with the concentration of this peptide in blood plasma (BP/ACTH), cerebrospinal fluid (CSF/ACTH), the paraventricular nuclei (PVN/ACTH) and the arcuate nuclei (AN/ACTH). The ACTH content in all sites was measured by radioimmunoassay (RIA). Although the IL is virtually avascular, the intraglandular lumen housing the IGC communicates nonetheless with the venous cavernous sinuses as well as the subarachnoid cerebral spinal fluid (CSF). Therefore, we content that the IGC serves as a transport medium whereby IL materials gain access to these extrapituitary sites.

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.

Immunoglobulin IgG detected by ELISA in bovine pituitary intraglandular colloid, a non-vascular holocrine secretion of intermediate lobe cells.

The purpose of this study was to determine the presence of immunoglobulin G in bovine pituitary intraglandular colloid (IGC). Intraglandular colloid, housed in the intraglandular lumen (residual lumen), is the holocrine secretion of the marginal layers of intermediate lobe (IL) cells. Immunoglobulin G was detected and quantitated in bovine IGC using an Enzyme Linked Immunosorbent Assay (ELISA). This study establishes the presence of immunoglobulin G in the intraglandular colloid at levels that are of the order of 10 micrograms per mg of colloid. The existence of this molecule in material scrupulously prepared to exclude the possibility of serum contamination in this normally avascular compartment is a unique phenomenon.

Presence of alpha-melanocyte-stimulating hormone in bovine pituitary intraglandular colloid of intermediate lobe origin.

Alpha-melanocyte-stimulating hormone (alpha-MSH), one of several peptide hormones originating in the intermediate lobe of the pituitary as proopiomelanocortin, was discovered in bovine pituitary intraglandular colloid by using radioimmunoassay. The quantity of alpha-MSH varied from 5 to 368 micrograms/mg protein in the three pools. The importance of this finding is discussed in light of the possibility that the colloid is a transport medium for alpha-MSH and other intermediate lobe hormones.

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.

Pituitary intermediate lobe cysts: their formation and contribution to intraglandular (residual lumen) colloid.

3 types of bovine pituitary intermediate lobe cysts referred to in the present study as type 1, 2 and 3, normally occur in the deep zone of the intermediate lobe. The deep zone of the intermediate lobe, like the entire lobe, is composed entirely of basophilic cells which make contact and invade the posterior lobe. The deep zone abuts the marginal zone which faces the intraglandular lumen (residual lumen) between the anterior and intermediate lobe. The marginal zone is readily distinguished from the deep zone since cysts do not form there and the cells in the marginal zone continually break down into a colloid-like material that sloughs into the intraglandular lumen. The major characteristics of the type 1 cyst is the constant addition of intermediate lobe cells to its peripheral ring and the presence of an isolated clump of unknown material in the colloid. The type 2 cyst is characterized by the continual disintegration of its peripheral cells which are cast into the cavity forming its contents. The type 3 cyst is characterized by a peripheral ring made up of a single layer of basophilic intermediate lobe cells as well as a limiting membrane which surrounds the cavity of the cyst. Intermediate lobe cysts may be a reservoir of active intermediate lobe materials. It is widely accepted that intermediate lobe cells synthesize a family of peptide hormones and a recent study shows that intraglandular colloid housed in the intraglandular lumen (residual lumen) contains intermediate lobe peptide hormones ACTH, alpha MSH and beta LPH.

Presence of met-enkephalin in bovine pituitary intraglandular colloid of intermediate lobe origin.

Met-enkephalin, and opioid peptide present in the intermediate lobe of the pituitary gland, is herein demonstrated, for the first time, in bovine pituitary intraglandular colloid. Colloid samples were taken from the glands of freshly slaughtered cattle, pooled, lyophilized, extracted on octadecylsilyl-silica and assessed by radioimmunoassay. Met-enkephalin was indeed present in this colloid extract at levels varying from 0.40 to 0.85 nanograms per milligram dry weight. Locating an intermediate lobe peptide in intraglandular colloid is a significant finding since this implies that colloid, housed in the intraglandular lumen, may serve as a transport medium. Intermediate lobe peptides which, because the intermediate lobe is avascular, are otherwise denied the vascular transport routes available to the anterior and posterior lobe hormones.

ACTH in bovine pituitary intraglandular colloid, the holocrine secretion of intermediate lobe cells.

The presence of bovine pituitary intermediate lobe peptides in intraglandular colloid, the holocrine secretion of intermediate lobe cells, is explored by ELISA. Intraglandular colloid collected immediately after sacrificing the animal, is placed in phosphate buffered saline, pH 7.6. This material is homogenized, centrifuged to remove extraneous tissue, lyophilized and stored at -20 degrees C. ACTH in intraglandular colloid is measured by competitive ELISA. Human ACTH (1-24) is used in the preparation of the solid phase antigen and as the standard for competition. The antibody is rabbit anti-human ACTH (1-24), and the alkaline phosphatase conjugate is goat anti-rabbit IgG with p-nitrophenyl phosphate as substrate. It is concluded that ACTH is present in bovine pituitary intraglandular colloid of intermediate lobe origin and that the colloid may serve as a transport medium for intermediate lobe materials.

Transport and anatomic distribution of a high molecular weight fraction of bovine pituitary intraglandular colloid of intermediate lobe origin.

Bovine pituitary intraglandular colloid (IGC) of intermediate lobe (IL) origin, was separated into seven primary fractions (F1-F7) on a 2.6 X 70 cm column of dextran gel G-100 superfine (Pharmacia). The molecular weight of F1-F3 was much greater than 1000 000; F4, 63 000; F5, 34 000 and F6-F7 less than 4000. The first fraction (F1) was used in this study and was labelled with (125I)Na via a modification of the chloramine-T method. Male Wistar rats were divided into 5 experimental and 5 control groups. The animals were fed Purina Lab Chow and Lugol's iodine was mixed with the drinking water to minimize any uptake by the thyroid gland of 125I liberated by metabolism. Under anesthesia, the animals in the experimental group were administered via the right internal jugular vein, 0.15 ml of (125I)F1 and those in the control were administered with 0.15 ml of free (125I)NA. Animals were killed with sodium pentobarbital at 2, 4, 6, 12 and 24 h periods. There are 5 replications for each of the 5 treatments (time periods) in every experiment including the control. Twenty-one tissues were retrieved and the kidney, spleen and thyroid were the only tissues that had retained the (125I)F1 at the 24 h recording period. Autoradiographic labelling was detected in hematoxylin-eosin stained sections of these tissues. Although it is difficult to make an exact determination as to whether the labelled fraction recovered is the same as the one injected, the kidney, spleen and thyroid continued to be significant tissues after several repeated experiments. The autoradiographic picture was the same each time the experiment was repeated. These results tend to indicate that IGC of IL origin is involved in the transport of IL materials.

Changes in the spontaneous action potentials from the whole, isolated, intact bovine pituitary gland following an injection of various bovine pituitary intraglandular colloid fractions.

Bovine pituitary intraglandular colloid (IGC) of intermediate lobe (IL) origin with accessions from the anterior lobe (AL), can modify the spontaneous action potentials (SAP) from AL, IL and posterior lobe (PL) cells. It was discovered that intraglandular colloid contains peptides ACTH, alpha-MSH, and beta-LPH when subjected to a series of radioimmunoassays. These peptides are thought, in part, to be responsible for altering the SAP.

Bovine pituitary intraglandular colloid fraction F5 localized in the rat endocrine pancreas.

Bovine pituitary intraglandular colloid thought to be a waste product, is the holocrine secretion of intermediate lobe cells. It is housed in the intraglandular lumen (residual lumen) and is extruded into the venous circulation of the cavernous sinuses via clefts in the capsule of the gland aligned with the intraglandular lumen. Intraglandular colloid, fraction F5 (mol.wt 34,000), radiolabeled with (125I)Na and injected (0.15 ml) into the right internal jugular vein of male Wistar rats, accumulated in the endocrine pancreas. Autoradiographs showed that the material had specifically localized in the capillary network of the endocrine pancreas. Since the intermediate lobe is poorly vascularized, intraglandular colloid is considered to be the transport medium for intermediate lobe materials.

Spontaneous action potentials recorded from the isolated, whole, encapsulated bovine pituitary gland: changes induced by certain physiological agents.

Extracellular spontaneous action potentials (SAP) were recorded from each lobe of the whole, isolated, encapsulated bovine pituitary gland bathed in Ringer's solution and maintained at 37 degrees C. All recordings were made with the gland in a shielded cage. Subdermal electrodes inserted into the various lobes of the gland were attached to a polygraph outside of the cage. Agents: dopamine (DA), norepinephrine (NE), gamma-aminobutyric acid (GABA) and serotonin (5HT) known to induce changes in the frequency and amplitude of the SAP in dissociated pituitary cells were used in this study. Although there are some questions about recording from dissociated pituitary cells in culture, in general, using these agents, there were no major differences in the SAP recorded from either dissociated cells in culture or cells in the whole intact bovine pituitary gland. Since pituitary cells and cells in nervous tissue share a common parentage, a comparative study of the SAP recorded from these cells show that the potentials recorded from cells in nervous tissue, e.g. ganglia, are inconsistent whereas those from pituitary cells show a consistent pattern of activity.

Electrical activity from cells in the whole, isolated, intact bovine pituitary gland. I. Changes induced by injection of hypothalamic, pineal and intraglandular colloid material.

Spontaneous electrical activity can be recorded from all lobes of the whole, isolated, intact bovine pituitary gland with subdermal electrodes. The potentials can be modified by injecting either crude hypothalamic, pineal gland or intraglandular colloid material. The pituitary cells yield grossly similar responses to these materials, and it is thought that the materials act through distinctly separate receptors.

Electrical activity from cells in the whole, isolated, intact bovine pituitary gland. II. Changes induced by injection of pentobarbital and morphine.

Spontaneous electrical activity can be recorded from certain pockets of cells in the whole, intact, isolated bovine pituitary gland. Other pockets of cells are either just noticeably active or electrically silent. An infusion of either pentobarbital or morphine enhanced spontaneous electrical activity and induced activity in electrically silent cells. An application of these agents resulted in an unprecedented excitatory response.

Antigenic determinants of pituitary intermediate lobe: accessions to CSF.

In bovine, the intermediate lobe is highly active and the holocrine secretion of its marginal cell layer forms a colloid-like substance housed in the intra-glandular cleft (residual lumen). Immunodiffusion of fractions of intraglandular colloid shows determinants sharing antigenic specificity with GH, FSH, CSF and serum immunoglobulins. The fractions tested by PAGE show determinants common to intraglandular colloid, CSF and serum albumin. The intraglandular cleft housing intermediate lobe colloid communicates by well defined channels passing through the intermediate lobe with the subarachnoid space, and with the cavernous sinuses by well defined venous channels. The venous channels are provided with well developed valve-like structures preventing venous blood from entering the cleft. These results question the relationship of the blood-brain barrier to the transport of pituitary substances. It is known that some pituitary peptide hormones (e.g. GH, ACTH, etc.) cannot cross the blood-brain barrier and their concentration in the CSF may not be due to an impairment of the barrier. We contend that intraglandular colloid of intermediate lobe origin is a part of major pituitary transport system and, fluctuating hormones and immunoglobulins in the CSF may be due to direct contributions from the pituitary.