The cellular site of action of angiotensin.

The site of action and the distribution of angiotensin II have been studied in the mouse. A comparison of the ratios of angiotensin-(14)C and inulin-(3)H at the time of the pressor effect reveals an extracellular pattern of distribution. Morphological studies were made using angiotensin coupled to exogenous enzymes which can be demonstrated histochemically. Coupling of angiotensin to horseradish peroxidase or cytochrome c, with glutaraldehyde or difluorodinitrodiphenylsulfone (FNPS) as the coupling agent, does not alter the pattern of its vasopressor response or that of its inactivation; nor are differences present between angiotensin and the angiotensin-enzyme complexes in the stimulation of in vitro tissue preparations. Dissociation of the complexes was shown not to occur in vitro, but the possibility of a serum factor splitting the complexes immediately after intravenous injection cannot be excluded. Since these complexes are localized on the endothelium and not on the smooth muscle at the time of maximum hypertension, the endothelium is proposed as the site of action for angiotensin.

Two morphologically distinct blood-brain barriers preventing entry of cytochrome c into cerebrospinal fluid.

After intravenous injection, cytochrome c does not enter the cerebrospinal fluid. In most areas of the brain, the marker is prevented from leaving cerebral vessels by the capillary endothelium. In the choroid plexus, the marker passes freely out of capillaries into the extracellular space. However, it does not traverse tight junctions between epithelial cells and is rapidly incorporated into mnembrane-bound vesicles within the cell cytoplasm. Thereafter, cytochrome c is apparently removed by lysosomal degradation. These data suggest that there are at least two morphologically distinct blood-brain barriers to cytochrome c and that pinocytosis may be a mechanism for intracellular degradation rather than transcellular transport.

( 3 H)lysergic acid diethylamide: cellular autoradiographic localization in rat brain.

Intravenous administration of [(3)H]lysergic acid diethylamide(LSD) to rats resulted in accumulation of the drug in the brain within 15 minutes. Autoradiographic methods were used to differentiate free and bound [(3)H]LSD in brain tissue. Free [(3)H]LSD was generally distributed in the pituitary and pineal glands, cerebellum, hippocampus,and choroid plexus. Bound [(3)H]LSD was localized in neurons of the cortex, caudate nucleus, midbrain, and medulla,as well as in choroid plexus epithelium.

An immunofluorescence microscopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein within the adult rat brain.

A collection of antibodies specific to different intermediate filament proteins were applied to frozen sections of adult rat brains. The relative distribution of these proteins was then studied using double label immunofluorescence microscopy. Antibodies specific to each of the neurofilament "triplet" proteins (of approximate molecular weight 68 K, 145 K and 200 K) stained exclusively neuronal structures. The distribution of these three antigens was in general identical, except that certain neurofilament populations such as those in the dendrites and cell bodies of pyramidal cells of the hippocampus and cerebral cortex, contained relatively little if any 200 K protein. Some neurone populations, such as the granule cells of the cerebellar cortex, could not be visualized by neurofilament antibodies, indicating that neurofilaments may not be essential for function of all neurones in vitro. Antibodies to GFA and vimentin stained an entirely different population of processes, none of which stained with any of the neurofilament antibodies. Vimentin antibody stained sheath material around the brain, a monolayer of ependymal cell bodies lining the ventricles, fibrous material associated within the choroid plexus, the walls of blood vessels and capillaries, and the processes of cells in certain regions. GFA antibody stained a second layer of sheath material under the vimentin layer, and numerous processes visible throughout the brain. Some specific populations of GFA-positive processes proved to stain also with vimentin. These included the processes of Golgi "epithelial" cells (Bergmann glial fibres), those of certain astrocytes in bundles of myelinated fibers. In addition, some processes apparently derived from ependymal cells proved to stain for both vimentin and GFA, whilst other could only be reliably visualized by vimentin alone. These results are discussed in terms of the previously described morphological characteristics of the various cell types of the brain.

Distribution of insulin-like growth factor II receptor immunoreactivity in rat tissues.

Antibodies specific for the insulin-like growth factor II (IGF-II) receptor were used to study its distribution in a number of rat tissues and cell lines in order to determine which cells might be responsive to local or circulating IGF-II. In cultured 18-54,SF and B104 neuroblastoma cells, plasma membrane and cytoplasmic staining corresponding to Golgi apparatus could be seen, consistent with the glycoprotein nature of this receptor. Antibody binding was also seen in the central nervous system, confined primarily to the choroid plexus, and the vascular and ependymal elements. Some staining was seen in the parenchyma of the brain, in addition to binding around nerve sheaths and axon bundles. There were high levels of immunoreactivity in all three lobes of the pituitary, including vascular and cellular elements. In liver, highest levels of immunoreactivity occurred in the sinusoidal cells. In lung, IGF-II receptor immunostaining was seen in the alveoli and around the bronchioles. Staining in kidney was observed in glomeruli, tubules, and Bowman's capsules. Lower levels of immunostaining were seen in skeletal muscle, located primarily around the muscle sheaths. Localization of IGF-II receptor to cells of known function in different tissues will help elucidate the role of this ligand-receptor system in regulating growth and metabolism.

Catecholamines and serotonin in the area postrema of normal and sodium-depleted dogs.

Sodium depletion, a maneuver that is accompanied by a 14-fold elevation of plasma renin activity (PRA), alters the norepinephrine concentration of the canine area postrema (AP), a circumventricular organ of the 4th ventricle known to be sensitive to circulating angiotensin II. The norepinephrine concentration of the AP after 3 weeks of sodium depletion decreased by 43%, whereas the concentration of epinephrine and dopamine and the activity of phenylethanolamine-N-methyltransferase (PNMT) did not change. In the pyramidal tract (PT) and choroid plexus (CP) catecholamines were present in significantly lower amounts than in the AP; their concentrations were unaffected by sodium depletion in the PT, but in the CP the norepinephrine concentration was reduced. Serotonin was present in the AP but its concentration was unaltered by sodium depletion. These findings provide evidence that sodium depletion produced an alteration in the concentration of norepinephrine of the area postrema without any change in the concentration of epinephrine, dopamine or serotonin.

Localization of specific binding sites for atrial natriuretic factor in peripheral tissues of the guinea pig, rat, and human.

Specific, high affinity atrial natriuretic factor (ANF) binding sites were identified and localized by autoradiographic techniques in peripheral tissues of the guinea pig, rat, and human. In the guinea pig kidney, high concentrations of ANF binding sites were located in the glomerular apparatus, outer medulla, and small renal arteries. Other peripheral tissues containing ANF binding sites included the zona glomerulosa of the adrenal cortex, the smooth muscle layer of the aorta and gallbladder, the lung parenchyma, the posterior lobe of the pituitary, the ciliary body of the eye, and the leptomeninges and choroid plexus of the brain. The distribution of ANF binding sites in the rat and human kidney was nearly identical to those seen in the guinea pig kidney; high concentrations were present in the glomerular apparatus, outer medulla, and small renal arteries. These results are consistent with earlier physiological and pharmacological studies that suggested that ANF plays a functional role in the regulation of extracellular fluid volume and blood pressure. There appears to be little species variation in the location and concentration of renal ANF binding sites, suggesting that, at least in the kidney, the results in experimental animals are relevant to the actions of ANF in humans. The finding that ANF binding sites were stable and present in high concentrations in human postmortem kidneys further suggests that these tissues may be amenable to testing for the involvement of ANF receptor dysfunction in diseases such as hypertension and congestive heart failure.

Angiotensinogen levels in the brain and cerebrospinal fluid of the genetically hypertensive rat.

The present experiments were designed to document changes in the regional distribution of angiotensinogen in the rat brain with the development of hypertension in spontaneously hypertensive rats (SHR) relative to age-matched normotensive Wistar-Kyoto rats (WKY). Levels of angiotensinogen were measured in discrete brain nuclei and cerebrospinal fluid from rats at 4, 7, and 16 weeks of age and in cerebrospinal fluid obtained by cisternal puncture at 7 and 16 weeks. Age-dependent changes in angiotensinogen were found, with levels higher in both strains at 4 weeks of age compared with 7 or 16 weeks. In contrast, plasma levels of angiotensinogen were essentially the inverse of the brain levels, low at 4 weeks and higher at 7 and 16 weeks. Levels in a number of regions adjacent to the rostral third ventricle from the 4-week-old SHR (prehypertensive phase) were significantly elevated relative to the WKY (p less than 0.05), while levels in the amygdala and posterior hypothalamus were significantly lower in the SHR (p less than 0.05). In 7-week-old rats (evolving phase), levels in nine brain regions were significantly elevated in the SHR relative to the WKY and included the nucleus tractus solitarii (p less than 0.01). Unlike the prehypertensive and evolving phases, in 16-week-old rats (maintenance phase) only two brain areas, the nucleus of the diagonal band and the lateral hypothalamus, had significantly elevated levels in the SHR (p less than 0.05). Cerebrospinal fluid levels of angiotensinogen did not correlate well with brain levels of angiotensinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in the cerebrospinal fluid outflow glycosaminoglycans.

The glycosaminoglycan distribution patterns of the cerebrospinal fluid (CSF) outflow pathway, dura mater and cerebral cortex of young New Zealand red rabbits and 1-, 3- and 12-week-old C-57 mice were identified by analyses of the glycosaminoglycan moieties and by the use of zone electrophoresis. The glycosaminoglycans were identified by specific degradation procedures, i.e., hyaluronate lyase, chondroitin ABC lyase, endo-beta-D-galactosidase and nitrous acid treatment. The CSF outflow pathway and dura mater glycosaminoglycan components were primarily hyaluronic acid and chondroitin sulfate-dermatan sulfate, whereas the cerebral cortex glycosaminoglycan components were hyaluronic acid, chondroitin sulfate-dermatan sulfate, keratan sulfate and heparan sulfate. The glycosaminoglycan components of the dura mater and cerebral cortex decreased and those of the CSF outflow pathway increased as a function of age. These results demonstrate the feasibility of analyses of the CSF outflow pathway glycosaminoglycan components and suggest that topographical changes in the glycosaminoglycan distribution profiles may contribute to the pattern of cerebrospinal fluid outflow.

The use of polyethyleneimine for demonstration of anionic sites in basement membranes and collagen fibrils.

Strong cationic polyethyleneimine (PEI) is used to trace anionic sites in lung, choroid plexus, glomeruli, blood vessels and also on collagen fibrils. In the mentioned tissues, PEI is found in the basement membranes with a spacing identical to that found on collagen fibrils. It is assumed that the acid endgroups of tropocollagen molecules are responsible for the binding of PEI.

Localization of human prealbumin in choroid plexus epithelium.

Cerebrospinal fluid (CSF) is generally considered to be derived from plasma through a combined process of ultrafiltration and secretion by the choroid plexus. However, the mechanisms ultimately responsible for achieving the final protein composition of CSF are uncertain. Some proteins, in particular prealbumin, are present in quantities not easily explained by usual physicochemical considerations. To investigate the possibility of de novo synthesis by choroid epithelium, we have examined human choroid plexus an ependyma for the presence of prealbumin. Using the peroxidase-antiperoxidase method at the light and electron microscopic level, as well as immunofluorescence, we localized prealbumin in choroid epithelial cytoplasm on the endoplasmic reticulum and in association with the Golgi apparatus. Ependymal cells and stroma did not reveal immunocytochemical labeling. These findings indicate that the choroid plexus epithelium contributes to the final CSF composition by de novo protein synthesis.

Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets.

We examined transthyretin immunoreactivity (TTR-IR) in human and porcine liver, choroid plexus, and pancreatic islets with both polyclonal and monoclonal antibodies to TTR. The specificity of the immunoreactions and the effects of various fixatives were tested in immunohistochemical and dot-blot systems. B-5 fixative (mercuric chloride and sodium acetate in formalin) was the best immunopreservative. In both species, the TTR-IR in choroid plexus epithelial cells was strong and was much greater than that in hepatocytes. Glucagon cells in pancreatic islets were also strongly TTR immunoreactive. Insulin cells were slightly TTR immunoreactive in human but strongly so in porcine pancreas. The finding of TTR-IR in normal islets explains the presence of TTR-IR in human endocrine pancreatic tumors, notably glucagonomas and malignant insulinomas.

Histaminergic cells in the choroid plexus of rat.

The choroid plexus of the rat was examined immunocytochemically using both an antibody directed towards a histamine thyroglobulin conjugate as well as to histidine decarboxylase. Histamine- as well as histidine decarboxylase-immunoreactive cells were found within or in the vicinity of the plexus epithelium, with processes extending to the neighbouring epithelial cells or surrounding tissue, suggesting a specific function for histamine on the choroid plexus.

Third ventricle choroid plexus function and its response to acute perturbations in plasma chemistry.

The homeostatic role of the third ventricle choroid plexus (3VCP) in the maintenance of CSF electrolytes was investigated by quantifying alterations in CP epithelial ion concentrations induced by chemical perturbations of plasma in adult Sprague-Dawley rats. Significant regional differences (third vs fourth (4VCP) and lateral ventricle CP (LVCP] were found in epithelial content of Na+ and K+, with respect to baseline levels as well as alterations caused by 5-60 min of systemic metabolic acidosis. 3VCP, which comprises ca. 10% of total choroidal tissue, has a water content, extracellular fluid volume and vascularity comparable to 4VCP and LVCP; yet 3VCP is characterized by relatively high and low values for cellular [Na+] (68 mM) and [K+] (118 mM). Compared to time-matched controls, acute metabolic acidosis (i.p. NH4Cl) effected a response, i.e. increases [K+] and decreases [Na+], in 3VCP that was less than in 4VCP, and substantially smaller than in LVCP. The onset and duration of induced electrolyte changes were qualitatively similar among the 3 plexus regions. Although systemic acidosis severely altered CP electrolyte concentrations, it did not compromise CSF homeostasis of [K+] and [Na+]. The function of 3VCP is discussed in terms of secretory capacity, embryological origin, and innervation. Overall, the findings indicate that transport/permeability phenomena which mediate transmembrane distribution of Na+ and K+ in 3VCP differ quantitatively from other regions of the blood-CSF barrier.

A morphological and chemical study of calcification of the choroid plexus.

Human choroid plexus was submitted to low temperature ashing (LTA) in order to isolate the calcification. The ashing residue was then subjected to morphological, chemical and structural studies using technics such as scanning electron microscopy, flame and flameless atomic absorption spectrometry, infrared spectrometry and X-ray diffraction. Morphologically, the calcification consisted of wound-up fibers forming granules with a diameter of 0.05 to 0.15 mm. The concretions were identified as cristalline Ca3(PO4)2 and hydroxylapatite. The content of trace elements was high, but within the limits found in other biological apatites. In some cases, however, the Fe content exceeded these limit values.

The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site.

The kinetic and pharmacological characteristics of the binding of [3H]5-HT (serotonin), [3H]8-OH-DPAT (8-OH-2-di-n-propylaminotetraline), [3H]LSD, [3H]ketanserin and [3H]mesulergine to membranes from frontal cortex, hippocampus and choroid plexus of pig brain were studied. The binding of these ligands to frontal cortex and hippocampus demonstrated the presence of 5-HT1 and 5-HT2 sites in both tissues, although hippocampus was richer in 5-HT1 (subtype 5-HT1A) sites. [3H]5-HT, [3H]mesulergine and [3H]LSD labeled the pig choroid plexus with high affinity. The pharmacological profiles of [3H]5-HT and [3H]mesulergine binding to this tissue were closely comparable. Ligands reported as selective for 5-HT1A, 5-HT1B or 5-HT2 subtypes did not show high affinity for these binding sites. Therefore, these 5-HT binding sites in pig choroid plexus could be named 5-HT1C. Other drugs with a high affinity for these sites were methysergide and mianserine. In pig frontal cortex, [3H]5-HT labeled the different subtypes of 5-HT1 sites. In contrast, [3H]mesulergine bound in pig frontal cortex to a small population of sites with pharmacological properties similar to those of the choroid plexus 5-HT1C sites. Possible physiological functions in which these sites might be involved are discussed.

Immunofluorescent localization of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase in the choroid plexus.

An immunofluorescent technique has demonstrated that tissue-bound pools of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase are localized within the cytoplasm of the epithelial cells of the choroid plexus. In all other cell types and regions of the central nervous system previously examined, however, these molecules have shown contrasting immunofluorescent localization. These results suggest that the interaction in the choroid plexus may be related to the specialized physiological functions of the neuroglial epithelial cell.

High levels of messenger RNA for transthyretin (prealbumin) in human choroid plexus.

We have investigated the expression of the gene for transthyretin (prealbumin) in the human choroid plexus. RNA was isolated from the human choroid plexus, fractionated by electrophoresis in agarose gel and transferred onto a nitrocellulose filter membrane. Transthyretin messenger RNA (mRNA) was identified by hybridization to radioactive complementary DNA for rat transthyretin. The level of transthyretin mRNA in the human choroid plexus was found to be at least 40 times higher than in human liver, suggesting very active synthesis of transthyretin in the choroid plexus.

The influence of the choroid plexus on the concentration of prealbumin in CSF.

Prealbumin and albumin concentrations were measured in homogenates of choroid plexus, ventricular and lumbar CSF, and in serum. The passive permeability-independent prealbumin concentration was estimated by subtracting from the total concentration that portion which enters the plexus and CSF by a passive molecular sieve effect, assuming that the diffusion of albumin and prealbumin is similar. The passive permeability-independent prealbumin concentration was highest in homogenates of the choroid plexus (mean = 56 micrograms/g). It was almost 3 times that in ventricular CSF (mean = 18 micrograms/ml). The passive permeability-independent concentration decreased only slightly during passage from the ventricular to the lumbar CSF space (mean = 16 micrograms/ml). These data suggest that much of the prealbumin in CSF enters the ventricular CSF via the choroid plexus by a mechanism different from that responsible for the transit of albumin.

Transferrin gene expression in choroid plexus of the adult rat brain.

Transferrin immunoreactivity and transferrin messenger RNA (mRNA) were recently found to be present in oligodendrocytes of the adult rat brain by using immunohistochemistry and in situ hybridization procedure. The present study demonstrates, in the same way, that epithelial cells of the choroid plexus also contain transferrin together with transferrin mRNA. Choroid plexus of the lateral and the third ventricle are rich in transferrin mRNA, while choroid plexus of the fourth ventricle contain few if any transferrin mRNA. These results demonstrate that epithelial cells of the choroid plexus as well as oligodendrocytes express the transferrin gene in the adult rat brain.