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.     

Salient characteristics of bovine pituitary intraglandular colloid: a transport medium for determinants to pituitary hormones and immunoglobulins

Bovine hypophyseal intraglandular colloid, the holocrine secretion of marginal intermediate lobe cells, contains high molecular weight (MW 45 000 to 158 000) protein fractions and low molecular weight (MW below 25 000) peptide fractions. The fractions display immunoreactive determinants similar to those in pituitary hormones, cerebrospinal fluid (CSF), serum IgG and albumin. Immunoelectrophoresis shows that components in high molecular weight fractions distribute themselves in three distinct bands (gamma, beta, alpha), while those in low molecular weight fractions distribute themselves in two bands (gamma and alpha). Physiochemical characteristics, i.e., sedimentation rate, percentage of hexose, total CHO and 12C as well as the content of cystine are presented. It is concluded that pituitary colloid should not be dismissed as a waste product of cellular degeneration, since there are strong suggestions that it serves as a transport medium for certain pituitary 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.     

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.     

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.     

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.     

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.     

Effect of anatomical fine structure on the dispersion of solutes in the spinal subarachnoid space

The dispersion of a solute bolus is calculated for cerebrospinal fluid undergoing oscillatory flow in the subarachnoid space of the spine. The fine structure of the subarachnoid space (nerves and trabeculae) enhances both longitudinal and transverse dispersions five to ten times over a simple model with an open annular space. Overall, dispersion is >10(3) times simple molecular diffusion. The result of enhanced dispersion is rapid spread and dilution of the bolus, effectively stirred by fluid movement around the fine structure.     

Spinal cord central canal of the German shepherd dog: Morphological,histological, and ultrastructural considerations

This study deals with some macroscopical, microscopical, and ultrastructural aspects of the spinal cord central canal of the German shepherd dog. The caudal end of the spinal cord is constituted by the conus medullaris, which may extend to the first sacral vertebra, the terminal ventricle, and the filum terminale. The latter structure is considered as internum (second to third sacral vertebrae) or externum (fifth caudal vertebra), according to its relation to the dura mater. Occasionally, there is a second anchorage which is close to the level of the sixth caudal vertebra. The central canal is surrounded by a ciliated ependymal epithelium, which differs depending upon the levels. The most caudal part of the filum terminale bears a columnar ciliated ependymal epithelium surrounded by two layers of glia and pia mater, which separate the central canal from the subarachnoid space. Microfil injections show a communication between the cavity and the subarachnoid space, as the plastic is able to pass through the ependymal epithelium. At the level of the terminal ventricle there are real separations of the ependymal epithelium, which seem to connect the lumen of the spinal canal with the subarachnoid space. These structures probably constitute one of the drainage pathways of the cerebrospinal fluid. The diameter of the central canal is related to the age of the animal. However, even in very old animals the spinal cord central canal reaches the tip of the filum terminale and remains patent until death. At the ultrastructural level the ependymal cells present villi, located on cytoplasmic projections, cilia, dense mitochondria, and oval nuclei. © 1995 Wiley-Liss, Inc.     

Meningeal Carcinomatosis

Meningeal carcinomatosis without gross tumour in the substance of the brain or spinal cord has been reported rarely. Two cases observed at the Victoria General Hospital, Halifax, presented a bizarre clinical picture consisting of signs of meningeal irritation without fever, and psychotic behaviour. Examination of the cerebrospinal fluid revealed low sugar concentration and increased pressure, protein and cells. In one case these cells were readily identified as malignant on stained smears. At autopsy the surfaces of the cerebral hemispheres, cerebellum and brain stem were covered by an opalescent film and on section the subarachnoid space was densely packed with malignant cells. Both primary tumours were adenocarcinomas, one originating in the gallbladder and one in the rectum. The diagnosis of meningeal carcinomatosis must be considered in patients presenting with profound mental changes and meningeal irritation without fever. Diagnosis may be confirmed by cytological examination of the cerebrospinal fluid. The primary tumour is most commonly an adenocarcinoma. There is no satisfactory treatment available.     

Ultrastructural study of the uptake of peroxidase by the rat median eminence

An active role of the ependymal cells (tanycytes) of the median eminence in the transport of hypothalamic hormones has been recently suggested. In order to investigate the fate of material present in the cerebrospinal fluid, a protein tracer, horse-radish peroxidase (HRP) was injected into the left lateral ventricle of rats. Two minutes after the injection, HRP had largely diffused between tanycytes and hypendymal cells. As soon as 5 min after the injection, HRP had completely penetrated all the layers of the median eminence. A few labelled vesicles and lysosomes were occasionally seen in ependymal and glial cells. At longer time intervals (20 min, 1 and 4 hrs), a reaction was observed in the lumen of fenestrated capillaries of the pituitary portal plexus. In many nerve endings of the external zone, vesicles and lysosomes were seen to contain HRP. An interesting observation was the localization of HRP between nerve endings and cells in both the pars nervosa and the pars intermedia of the pituitary gland. No reaction was recorded in the anterior pituitary and the kidney. Seventeen hours after the injection, the extracellular space was free of reaction but a few positive intracellular structure were still found. These results clearly indicate that some material from the third ventricle can rapidly diffuse between cells and axons of the median eminence to reach the fenestrated capillaries of the pituitary portal plexus and the posterior pituitary without involving an active transport by tanycytes.     

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.     

Circulation of marker substances in the cerebrospinal fluid of an amphibian,Rana pipiens

Summary Solutions of fluorescein-labelled dextran or Evans blue-albumin were infused into the lateral cerebral ventricle of Rana pipiens. The subsequent distribution in the cerebrospinal fluid (CSF) was investigated between 2 and 24 h after infusion by freezing and examination of the cut blocks of the head and vertebral column of the stage of a freezing microtome. These marker substances move out of the ventricles into the subarachnoid space at the caudal end of the fourth ventricle and spread rapidly along the subarachnoid space of the spinal cord. The spreading of marker substances is slower into the brain subarachnoid space. When the marker is infused into the subarachnoid space of the forebrain, it becomes distributed throughout the subarachnoid space of the brain and spinal cord but not in the ventricles.Partial clearance of markers from the ventricles takes place within 5 h and total clearance within 8 h. Clearance from the brain and cord subarachnoid space is somewhat slower and can only be detected in experiments lasting 10 h or more. Absorption of the markers from the CSF occurs via the intervertebral foramina of the spinal cord. Fluorescence microscopy of sections of the cord show that the fluorescence leaves the subarachnoid space at the point where the spinal nerves traverse the arachnoid membrane.     

Bovine Pituitary Membrane Glycoproteins Contain β-N-Acetylgalactosaminylated N-Linked Sugar Chains

Abstract: Bovine pituitary glycoprotein hormones contain unique N-linked sugar chains with GalNAcβ1 → 4GlcNAc structure in their outer chain moieties. In the present study, whether bovine pituitary membrane glycoproteins contain the sugar chains with the disaccharide structure was investigated. Western blot analysis of the membrane glycoproteins using Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with β- N -acetylgalactosamine residue(s), showed that most protein bands detected with Coomassie Brilliant Blue staining bind to WFA. However, no WFA binding was observed for the bands after treatment of the blotted filter with jack bean β- N -acetylhexosaminidase or N -Glycanase. The WFA-positive bands were also detected in membrane glycoprotein samples from bovine cerebrum, cerebellum, and medulla oblongata, although their expression levels were low. Structural analysis of the oligosaccharides released by hydrazinolysis from the pituitary membrane glycoproteins by serial lectin column chromatography and sequential exoglycosidase digestion revealed that the major oligosaccharides, which bound to a WFA-agarose column, are of biantennary complex type with one and two GalNAcβ1 → 4GlcNAc groups in their outer chain moieties. These results indicate that the β- N -acetylgalactosaminylation is not unique to the glycoprotein hormones but occurs to most bovine pituitary glycoproteins.     

Transventricular and transpial absorption of cerebrospinal fluid into cerebral microvessels

It is generally accepted that volume of cerebrospinal fluid (CSF) is secreted in brain ventricles and flows to subarachnoid space to be absorbed into dural venous sinuses or/and into lymphatics via perineural sheats of cranial nerves. Since 99% of CSF volume is water, in experiments on cats 3H-water was slowly infused into lateral ventricle and found that it does not flow to subarachnoid space but that it is rapidly absorbed transventricularly into periventricular capillaries. When 3H-water was infused in cortical subarachnoid space, it was absorbed locally into cerebral capillaries via pia mater. On the contrary, when macromolecule 3H-inulin is applied in CSF it is very slowly eliminated in bloodstream, and, with time, is carried by systolic-diastolic pulsations and mixing of CSF bidirectionally along CSF system. Thus, CSF volume (water) is absorbed rapidly into adjacent cerebral capillaries while inulin is distributed bidirectionally due to its long residence time in CSF Previously, the macromolecules have been used to study CSF volume hydrodynamics and with this misconception of CSF physiology arose.     

Interaction of putative vasopressin receptors in rat brain and bovine pituitary gland with a vasopressin anti-idiotype antibody as revealed by immunoblotting

Vasopressin (AVP)-binding proteins were obtained from rat brain and the anterior and posterior lobes of bovine pituitary glands by (a) preparation of crude membranes, (b) solubilization of membrane proteins, (c) passage through an affinity column containing immobilized AVP, and (d) elution from the column with excess AVP. Gel electrophoresis revealed protein bands of 55 and 62 kilodaltons in rat brain, bovine posterior lobe and, to a far lesser extent, in the anterior lobe, which were similar to those previously identified in rat brain to be associated with AVP binding. Immunoblotting demonstrated that the 55 kilodalton bands of rat brain and bovine pituitary gland were selectively immunoreactive with an AVP anti-idiotype antibody. In addition, immunoreactivity occurred with a 62 kilodalton component of rat brain.     

Expression of certain proteins in the subfornical organ and cerebrospinal fluid of spontaneously hypertensive rats

The objective of this study was to analyze the proteins in the cerebrospinal fluid of spontaneously hypertensive rats and to study their possible role in the relationship between hydrocephalus, arterial hypertension and variations in the subfornical organ. Brains and cerebrospinal fluid from control Wistar-Kyoto rats and spontaneously hypertensive rats sacrificed with chloral hydrate were used. Cerebrospinal fluid and extract of subfornical organ were processed by protein electrophoresis. Antisera against protein bands of 141, 117 and 48 kDa and Concanavalin A were used for immunohistochemical and western blot study of the subfornical organ, adjacent circumventricular structures and cerebrospinal fluid. Ventricular dilation in the spontaneously hypertensive rats and the presence of quite a lot of protein bands in the cerebrospinal fluid of the hypertensive rats, which were either not observed or scarcely present in the cerebrospinal fluid of the Wistar-Kyoto rats, were confirmed. The subfornical organ, third ventricle ependyma and choroideus plexus showed immunoreactive material for antibodies against 141kDa, 117 and 48 kDa proteins band (anti-B1, anti-B2 and anti-B3). The larger amount of the immunoreactive material was found in the subfornical organ of the spontaneously hypertensive rat. Our results and the alterations observed by other authors in the subfornical organ in hydrocephalic and hypertensive rats support the possibility that this circumventricular organ, some proteins of the cerebrospinal fluid and ventricular dilation could be connected with the physiopathology of this type of hypertension.     

Hypoglycosylation of a brain glycoprotein ({beta}-trace protein) in CDG syndromes due to phosphomannomutase deficiency and N-acetylglucosaminyl-transferase II deficiency

Human β-trace protein is a major intrathecally synthesizedpolypeptide constituent of human cerebrospinal fluid. We havepreviously shown that this protein is almost quantitativelymodified with biantennary complex-type N-linked oli-gosaccharideswhich show "brain-type" glycosylation characteristics (Hoffmann,A.et al, J. Neurochenu, 63, pp. 2185-2191,1994). In the presentstudy human β-trace protein from the cerebrospinal fluid(CSF) of patients with carbohydrate-deficient glycoprotein syndrome(CDGS) due to phospho-mannomutase (PMM) deficiency and N-acetyl-glucosami-nyltransferaseII (GlcNAc-T II) deficiency as well as from control individualswas studied by Western blot analysis. The protein from pooledCSFs was purified by immunoaffinity chromatography. The proteinfrom the five patients with CDGS PMM deficiency showed threeprotein bands upon SDS-PAGE analysis corresponding to the di-,mono-, and unglycosylated polypeptide forms. Carbohydrate structuralanalysis of the enzymatically liberated N-glycans was performedapplying mapping by HPAEC-PAD, methylation analysis as wellas MALD/TOF-MS. Essentially identical oli-gosaccharide structureswere detected in β-TP from type I patients and controladult pooled CSF. The β-trace protein from two patientswith GlcNAc-T II deficiency showed a single di-N-glycosylatedprotein band with a significantly lower molecular weight thanthe di-glycosylated polypeptide from control patients and theβ-trace protein from pooled adult CSF. β-TP from GlcNAc-TII deficiency patients shared only three oligosaccharides outof the 13 observed in β-TP from controls or patients withPMM deficiency. The major oligosaccharide structures of theglycoprotein from patients with GlcNAc-T n deficiency were foundto be monoanten-nary asialo- or monosialylated lactosamine-typechains with proximal fucose and bisecting GlcNAc. "brain-type" N-glycosylation carbohydrate deficiency glycoprotein syndrome human β-trace protein phosphomannomutase deficiency GlcNAc-transferase II deficiency human cerebrospinal fluid     

Mass spectrometric analysis of neuropeptidergic systems in the human pituitary and cerebrospinal fluid

Neuropeptidergic systems have been studied in human tissues and fluids, which include the pituitary and lumbar cerebrospinal fluid, respectively. This paper reviews the qualitative and quantitative mass spectrometric analytical data obtained from three areas of study. Methionine enkephalin (ME) and β-endorphin (BE) were quantified in the human pituitary by liquid secondary ion mass spectrometry (LSI MS)–tandem mass spectrometry. Corresponding stable isotope-incorporated synthetic peptide internal standards were used. Proenkephalin A and proopiomelanocortin produce ME and BE, respectively. The analysis of neuropeptides in macroadenomas demonstrated a decrease in both of those neuropeptidergic systems relative to controls. An analysis of prolactin-secreting microadenomas showed an increase in the proenkephalin A system. Mass spectrometry was also used to detect opioid peptide-containing proteins in the pituitary. Enzymes that process the precursors of proenkephalin A and tachykinin (substance P) neuropeptides were studied in human lumbar cerebrospinal fluid. Electrospray ionization mass spectrometry was used to characterize the molecular mass of each peptide product.     

Effects of fluid structure interaction in a three dimensional model of the spinal subarachnoid space

It is unknown whether spinal cord motion has a significant effect on cerebrospinal fluid (CSF) pressure and therefore the importance of including fluid structure interaction (FSI) in computational fluid dynamics models (CFD) of the spinal subarachnoid space (SAS) is unclear. This study aims to determine the effects of FSI on CSF pressure and spinal cord motion in a normal and in a stenosis model of the SAS. A three-dimensional patient specific model of the SAS and spinal cord were constructed from MR anatomical images and CSF flow rate measurements obtained from a healthy human being. The area of SAS at spinal level T4 was constricted by 20% to represent the stenosis model. FSI simulations in both models were performed by running ANSYS CFX and ANSYS Mechanical in tandem. Results from this study show that the effect of FSI on CSF pressure is only about 1% in both the normal and stenosis models and therefore show that FSI has a negligible effect on CSF pressure.