Carbonic anhydrase IV on brain capillary endothelial cells: a marker associated with the blood-brain barrier.

Carbonic anhydrase (CA) activity plays an important role in controlling cerebrospinal fluid production and also influences neuroexcitation and susceptibility to seizures. Until recently, CA II was the only CA demonstrated in brain. Its distribution is limited to the epithelial cells of the choroid plexus and to the myelin-forming cells, the oligodendrocytes. In this report, we present immunoblots, using an antibody raised to CA IV from rat lung, that show that CA IV is also present in rat and mouse brain. Results of immunohistochemistry and immunoelectron microscopy on sections from rat and mouse brain are presented that show the distribution of CA IV to be quite distinct from that of CA II. CA IV is expressed on and is limited to the luminal surface of endothelial cells of cerebral capillaries. These results establish CA IV as a cytochemical marker associated with the blood-brain barrier and suggest an important role for CA IV in CO2 and HCO3- homeostasis in brain.

Characterization of two chromaffin cell populations isolated from bovine adrenal medulla.

Cultures of chromaffin cells isolated from the bovine adrenal medulla have been extremely useful for investigating secretory mechanisms, but such cultures used up to the present time represent mixed populations of adrenergic and noradrenergic cells. This report describes how, with slight modifications to standard procedures, two separate chromaffin cell populations may be separated from bovine adrenal medullae. These two cell fractions have been characterized by biochemical, immunocytochemical, and morphological techniques as enriched populations of adrenergic or noradrenergic cells, respectively. The adrenergic cell-enriched fraction consists of greater than 90% adrenergic cells, whereas the noradrenergic cell-enriched fraction contains greater than 60% noradrenergic cells. We also demonstrate that these cells may be cultured with their secretory machinery intact: analysis of secreted catecholamines from nicotine- or high K+ concentration-stimulated cells cultured from each fraction confirms that adrenaline is the major catecholamine secreted by one fraction, whereas noradrenaline is mainly secreted by the other.

Neurofilament expression in bovine chromaffin cells.

Neurofilament (NF) expression was examined in adult bovine adrenal chromaffin cells by immunocytochemistry using a series of monoclonal antibodies directed against either nonphosphorylated or phosphorylated epitopes of the heavy NF subunit. In situ, this NF subunit was not detected in chromaffin cells. However, chromaffin cells grown in primary culture under standard conditions contained NF proteins, but only in a nonphosphorylated state. Phosphorylation of NFs could be induced under culture conditions favouring the development of a neuronal phenotype--40% of the cells developed neurites within a week while NF phosphorylation occurred later. Phosphorylated NFs were restricted to neurites, unlike nonphosphorylated NFs which were observed in both perikarya and neurites.

Expression of the neural cell adhesion molecule NCAM in endocrine cells.

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.

Effect of cell surface trypsinization on ethanolamine base exchange enzymatic activity of astrocyte primary cultures and derived spontaneously transformed cell lines.

Trypsinization of neonatal rat astrocyte primary cultures (normal cells) inhibited the activity of ethanolamine base exchange enzyme (EBEE) by 80%, whereas ethanolamine phosphotransferase (EPT) and choline base exchange (CBEE) enzymatic activities were not affected; subcellular fractionation demonstrated that trypsin treatment affected the intracellular EBEE activity. During trypsinization the enzyme was not taken up by cultured astrocytes but the cell surface was affected. In contrast, the same treatment did not alter EPT, CBEE and EBEE activities of spontaneously transformed cell lines derived from the primary cultures. However, treatment of the transformed cells with db-cAMP prior to trypsin, restored the pattern found in the primary culture, i.e. only EBEE activity was affected. These data suggest that a relationship exists between cell surface organization and intracellular EBEE activity in a culture system which possesses the property to control its own cell division or/and differentiation.

Induction of neurofilament phosphorylation in cultured chromaffin cells.

The distribution, structural organization and state of phosphorylation of neurofilaments have been examined in chromaffin cells from adult bovine adrenal medulla cultured under various conditions using a series of monoclonal antibodies directed against phosphorylated and nonphosphorylated epitopes of the 200,000 mol. wt subunit. Nonphosphorylated neurofilament epitopes were detected immunocytochemically to varying extents in chromaffin cells maintained under standard culture conditions for up to 3 weeks. Staining was usually limited to a perinuclear region from which fine filaments sometimes appeared to radiate around the nucleus. In marked contrast, none of the antibodies directed against phosphorylated neurofilament epitopes stained these structures. When cells were cultured under conditions favouring neurite outgrowth, in conditioned medium derived from intermediate lobe cultures, there was a more extensive expression of the nonphosphorylated neurofilament epitopes. In addition, phosphorylation of neurofilaments was induced. The phosphorylated neurofilament epitopes were restricted to the neurite, whereas the nonphosphorylated neurofilament epitopes were localized in both neurite extensions and perikarya. These results demonstrate that conditioned medium from intermediate lobe cells of the hypophysis not only provokes neurite outgrowth from chromaffin cells, but also supports neuronal maturation as demonstrated by the phosphorylation of neurofilaments in neurites.

Expression of neurofilament proteins by Purkinje cells: ultrastructural immunolocalization with monoclonal antibodies.

Purkinje cell bodies in rodent cerebellum have been shown to express neurofilament protein epitopes but neurofilaments are rarely seen in these perikarya by classical morphological approaches. In an attempt to solve this enigma the ultrastructural distribution of two neurofilament epitopes was studied by immunoelectron microscopy with two monoclonal antibodies (Mabs) of divergent specificity: one, Mab 04-7 recognized a phosphorylated epitope, the other, Mab 02-135 a non-phosphorylated epitope. Longitudinal filamentous elements were heavily labeled in basket cell axons and afferent nerve fibers with both Mabs. While Mab 04-7 was unreactive with Purkinje cells, the immunoperoxidase reaction product with Mab 02-135 was distributed in the form of patches with no filamentous substructure throughout the cytoplasm of these cells. The data complement the results of other immunocytochemical studies showing the presence of all 3 neurofilament constituent proteins in Purkinje cell bodies, and lead to the conclusion that in these perikarya the majority of neurofilament proteins are not assembled in the form of neurofilaments.

Endocrine cells share expression of N-CAM with neurones.

The expression of the neural cell adhesion molecule, N-CAM, was examined in the anterior lobe of rat hypophysis by immunocytochemistry at light and electron microscope levels. In addition, N-CAM antigenic determinants present in adrenal medulla, anterior hypophysis and PC12 cells were compared by immunoblotting with those found in cerebellum. All secretory cells in the anterior hypophysis were found to be N-CAM positive on their surfaces, but not all of the three polypeptide determinants typical of cerebellum were present in the endocrine tissues or cell line tested. In addition, a new N-CAM determinant of 49 kDa not present in cerebellum was found in adrenal medulla and hypophysis, although it was absent from PC12 cells. The possible implications of these data are discussed.

Anti-alpha-fodrin inhibits secretion from permeabilized chromaffin cells.

Chromaffin cells release catecholamine- and peptide-containing granules by exocytosis, by a mechanism involving movement of secretory granules towards the cell membrane, their apposition to it and the fusion of the granule membrane with the plasma membrane. One of the two subunits of membrane-associated brain spectrin, alpha-fodrin is an actin-binding protein which is found at the periphery of chromaffin cells and may be involved in secretion. Because cultured chromaffin cells can be permeabilized with detergents, giving pores large enough to permit the entry of immunoglobulin molecules, we used permeabilized cells to test the effect of specific antibodies on secretory mechanisms. Incubation of permeabilized cells with polyclonal immunoaffinity-purified monospecific anti-alpha-fodrin antibody or its Fab fragments did not modify basal release but did specifically inhibit Ca2+-induced catecholamine release by exocytosis. Our observations indicate that fodrin and the cytoskeleton participate in the release mechanism.

Alpha-fodrin in the adrenal gland: localization by immunoelectron microscopy.

Localization of fodrin, the brain equivalent of spectrin (a protein constituent of the erythrocyte membrane cytoskeleton), was investigated at the ultrastructural level in rat adrenal gland. By use of an affinity purified antibody directed against the alpha-fodrin subunit, all chromaffin cells, cortical cells, nerve fibers, and their surrounding Schwann cells were found to be labeled close to the cytoplasmic side of their plasma membranes. The labeling appeared more intense for chromaffin cells, and secretory granules and mitochondria were frequently found to be associated with the zone containing alpha-fodrin in these cells. The immunostained zone was estimated to extend 230 +/- 70 nm into the cytoplasm. This localization is discussed in terms of what is known of the properties of spectrin, and possible roles of the molecule in the chromaffin cell are suggested.

Secretory cell actin-binding proteins: identification of a gelsolin-like protein in chromaffin cells.

Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.

Surface expression of neural cell adhesion molecule in cultured bovine paraneurones: immunogold and immunoperoxidase methods compared.

The D2 neural cell adhesion molecule immunolabelling patterns of cultured bovine chromaffin cells are compared at the ultrastructural level, using the indirect immunohistochemical method with the secondary antibody labelled with either 17 or 5 nm gold particles or horseradish peroxidase (HRP). With HRP-conjugated antibody an apparently continuous surface staining was produced, while with 17 nm gold particles relatively large areas of membrane remained unlabelled; 5 nm particles gave a pattern more closely resembling the localization obtained with HRP. These differences are explained by differences in steric hindrance inherent to each method.

Cellular development and myelin production in primary cultures of embryonic mouse brain.

The development of cell cultures from embryonic mouse cerebral hemispheres has been followed in detail for periods up to 40 days in culture using a variety of approaches. Functionally well differentiated neurons (shown by receptor binding studies, immunocytochemistry and morphological examination) were found to be abundant early in culture and to form cell contacts with oligodendrocytes characterized both immunocytochemically and morphologically. Myelin-like membranes with the periodicity of classical myelin elaborated by oligodendrocytes were detected only after 30 days in culture when neurones were no longer present. These results are discussed with regard to possible mechanisms of initiation of myelin synthesis.

A developmentally modified neurone-specific marker in rodent cerebellum.

Out of several monoclonal antibodies secreted by hybridomas resulting from the fusion of a mouse myeloma cell line with spleen cells from mice immunized with cerebellar membranes from 12 day old rats, one, called 11.9, produced an unusual immunolabelling pattern when tested on sections of rat cerebellum. The cerebellar distribution of the antigenic sites recognized by this antibody using an immunoperoxidase technique at the optical and ultrastructural levels is described in detail in this report. The immunoreaction product was found in the adult rat to be associated with the microtubules and the zone immediately beneath the plasma membrane of parallel fibres. In young animals the density of immunostaining appears to be higher than in the adult, and the staining is detectable in addition in the perikaryal cytoplasm of granule cells. Biochemical studies using the Western immunoblot technique demonstrate that the antigens consist of two polypeptides of molecular weights 120 and 185 kD. The possible relation of the antigens to cytoskeletal structures is discussed and the labelling pattern is compared with that produced by other known monoclonal antibodies.

Ultrastructural immunocytochemical demonstration of D2-protein in adrenal medulla.

The ultrastructural localization of the glycoprotein D2 in rat adrenal gland was investigated using immunohistochemical methods, and D2 localization in cultures of adult bovine chromaffin cells was studied by immunofluorescence. D2 was found to be situated on nerve fibers passing through the adrenal cortex and in the medulla zone, and also on the surface of all chromaffin cells. In addition, it was strongly expressed on the surface of glial (Schwann) cells. Cortical cells were unreactive to the antiserum. In cultures, all adrenalin and noradrenalin [dopamine-beta-hydroxylase (DBH)-positive] cells were surface labelled for D2. A less frequent second cell type was recognized in vitro which was DBH negative but D2 positive. Such cells were presumed to be Schwann cells. These data are discussed in terms of the developmental origin of the cells and with regard to the putative functional rôle of D2 in cell adhesion phenomena.

Arguments in favour of endocytosis of glycoprotein components of the membranes of parallel fibers by Purkinje cells during the development of the rat cerebellum.

Chloroquine (a drug known to induce a dysfunction of lysosomes) was used to study the behavior of Concanavalin A binding glycoproteins located on the axolemma of parallel fibers in young rat cerebella, and abundant on these membranes at a period preceding synaptogenesis with the dendrites of Purkinje cells. Chloroquine induces in Purkinje cells a large accumulation of grains consisting of membrane whorls in lysosomes. These grains stain for Concanavalin A, and do not stain either for a mitochondrial marker (aspartate aminotransferase mitochondrial isoenzyme) or for a marker of the Purkinje cell internal membrane (PSG). It is suggested that the material accumulating in the Purkinje cells under the effect of chloroquine comes from the parallel fibers. Together with the observation that alpha-D-mannosidase (involved in the degradation of these glycoproteins) is exclusively located inside Purkinje cells, these results provide a firm indication that this material enters the Purkinje cells through pinocytosis. The absence of ATPase activity (ATPase is a glycoprotein plasma membrane marker highly concentrated on parallel fibers) within these grains suggested that not all the components of these membranes are pinocytosed, but that the process is specific for certain molecules. These results are compatible with the ultrastructural observations of others, and support the arguments in favour of the pinocytosis phenomenon being one of the first steps of synapse formation. The observed specificity of pinocytosis for certain molecules suggests that a receptor-mediated recognition of some glycans of glycoproteins is the preliminary event in the establishment of synapses.

A monoclonal antibody recognizing subpopulations of neurones in mouse brain.

A monoclonal antibody, designated C138, was raised against a particulate fraction from young postnatal mouse cerebellum. In sections from adult mouse brain and cerebellum, this antibody stained a sub-population of neurons. Cell bodies of large neurones and fibre tracts were negative in all brain regions examined, whereas neuropil was heavily labelled. Immune-electron microscopy confirmed the specificity of the antibody for certain types of neurones and indicated that the antigen may be associated with microtubular structures. Immunofluorescence studies on dissociated cerebellar cultures showed that the antigen was expressed inside all tetanus toxin-positive neurones but not by non-neuronal cells.