Carbonic anhydrase-C Research Articles

Peripapillary glial cells in the chick retina: A special glial cell type expressing astrocyte, radial glia, neuron, and oligodendrocyte markers throughout development

Peripapillary glial cells of the chick are a special type of glia, not only because of their position, forming a boundary between the retina on one side and the optic nerve head (ONH) and the pecten on the other, but also because although they have the same orientation and similar shape as the retinal Müller cell (a type of radial glia) and express common markers for these cells and astrocytes, they do not express glutamine synthetase (GS) or carbonic anhydrase C (CA-C), enzymes intensely expressed by Müller cells and astrocytes. In this study, we present further molecular characterization of these cells, using immunohistochemistry techniques. We show that peripapillary glial cells express a novel neuron antigen, 3BA8, that in the adult retina is located only in one neuron type (the amacrine cell) and in the inner plexiform layer (IPL). They also express an antigen specific to myelin and oligodendrocytes, MOSP, and a glial antigen, 3CB2, expressed by radial glia and astrocytes throughout the CNS. The study of the developmental expression of these three antigens in the peripapillary glial cell territory shows different spatiotemporal labeling patterns: 3CB2 and 3BA8 are expressed much earlier (embryonic days E3 and E5, respectively) than MOSP (E12), and during a developmental window (E6-E10) 3BA8 labels the peripapillary glial cells intensely and does not label the ONH or the optic nerve (ON), which are labeled later. The expression of 3CB2 is much more intense in the peripapillary glial cells than in Müller cells from early stages of development up to E16, and the expression of MOSP starts earlier in the peripapillary glial cells than in the Müller cells and is maintained with much higher intensity in the peripapillary glial cells throughout development. These findings show that Müller and peripapillary glial cells follow independent courses of differentiation, which together with the fact that the peripapillary glial cells express molecules typical of neurons, oligodendrocytes, radial glia, and astrocytes are evidence that peripapillary glial cells are a unique type of glia in the CNS.

Human Papillomavirus-16 E6/E7 Transfected Retinal Cell Line Expresses the Müller Cell Phenotype

The introduction of viral transforming genes into mammalian cells has been used in establishing cultures of unlimited lifespan. Although Müller cells, the predominant glial cells in the mammalian retina, have been isolated using a variety of techniques, most of these cultures have limited capacity for cell division and are often contaminated by other cell types especially astrocytes, endothelial cells and microglial cells. We have established pure cultures of retinal cells which express Müller cell characteristics and exhibit unlimited growth in vitro. We now report the techniques involved in the propagation and characterization of these cultures. Mixed retinal cultures isolated from dystrophic rat retinas were infected with defective retroviruses coding for human papillomavirus (HPV) type 16 E6 and E7 proteins. The disabled viral constructs also contained the neomycin gene allowing selection of the cultures using Geneticin, a neomycin analogue. Pure cultures were then obtained from Geneticin-selected populations by limiting end-dilution techniques. The expression of the HPV-16 E6/E7 genes in the transfected cell line was established using an HPV-16 E6/E7 PCR product to probe Northern blots. Cloned cells were found to be highly reactive for Müller cell markers including S-100, carbonic anhydrase-C, cellular retinaldehyde binding protein, and glial fibrillary acidic protein but not for glutamine synthetase. Ultrastructural studies showed stacks of cells with long elaborate processes, short microvilli, coated pits, cytoplasmic filaments, abundant perinuclear rough endoplasmic reticulum, and smooth endoplasmic reticulum extending to the cell processes. Growth patterns of late passage cells (>50 passages) showed a lag phase of 48 hr followed by exponential growth extending past visual confluence at day 5. Since the cultures have undergone more than 240 population doublings, they can be characterized as a continuous cell line with unlimited lifespan. The HPV-16 E6/E7 transfected Müller cell line may prove useful in studies requiring abundant and pure cultures of Müller cells.

Mullerian glia in dystrophic rodent retinas: an immimocytochemical analysis

Mullerian glia in retinas of Royal College of Surgeons (RCS) dystrophic rats and retinal degeneration (rd/rd) mice undergo biochemical and morphological alterations concomitant with photoreceptor loss. To follow the fate of Mullerian glia in these degenerating retinas, two Muller cell-specific markers, carbonic anhydrase-C (CAC) and cellular retinaldehyde-binding protein (CRALBP), were examined by light microscopic immunocytochemistry. In retinas of 1-to 12-month-old RCS dystrophic rats, cell bodies in the inner nuclear layer and radial processes were immunostained for CAC, but appeared to diminish with age. In addition, a material in the region of the retinal pigment epithelium (RPE), representing expansion of Muller cell processes into the subretinal space, was immunolabelled for CAC in retinas of 2-month-old and older RCS rats. The CAC-immunoreactive Muller cells seen in retinas of 12-month-old RCS rats were disorganized, as significant photoreceptor degeneration had occurred by this time. In retinas of 6-week-old RCS rats, Muller cells and their processes were immunolabelled for CRALBP, which spanned from the nerve fiber layer (NFL) through the outer nuclear layer. The density of this immunostaining increased, especially in the subretinal space, with advancing age in RCS rats, seen most prominently in retinas of 9-month-old RCS rats and decreased by 12 months. In retinas of rd/rd mice beginning by day 14, minimal CAC-and CRALBP-immunoreactive material was observed in the subretinal space. By 6 weeks, when a majority of the photoreceptors had degenerated, the CAC-staining pattern appeared significantly reduced and patchy. This study showed that Muller cells in degenerating retinas of RCS rats and rdJ.rd mice ultimately exhibited decreased immunolabelling for CAC and CRALBP at the more advanced stages of retinopathy, which coincided with the loss of photoreceptors. This is in contrast to the progressive increase in glial fibrillary acid protein (GFAP), an intermediate filament protein, throughout the retinal dystrophy in both animal models.

Reactive cell proliferation and microglia following injury to the rat brain

The non-astrocytic cells which proliferate in the rat brain after the induction of an area of necrosis have been characterized and counted by means of combined in vivo bromodeoxyuridine (BrdU) administration and immunohistochemical demonstration of glial fibrillary acid protein (GFAP), vimentin, Ricinus communis agglutinin 120 (RCA-1), Griffonia simplicifolia B4 isolectin (GSI-B4), keratan sulphate (KS), carbonic anhydrase C (CA.C), transferrin (TF) and ferritin. Two days after the injury, 7.5% of the proliferating cells were GFAP-positive reactive astrocytes, 5.7% were RCA-1-positive cells and 17.4% were GSI-B4-positive cells. Lectin-binding cells had the microscopic and ultrastructural aspects of microglia; they proliferated around the needle track and in the corpus callosum. Microglia represented a large fraction of the proliferating cells. Evidence is presented for the origin of at least a proportion of perilesional astrocytes and microglia from the periventricular matrix, and of microglia from blood precursors. Other non-proliferating microglia cells transiently appeared in the normal brain around the wound, in agreement with the existence of two different microglia cell populations reacting with different modalities to an area of necrosis.

RPE conditioned medium stimulates photoreceptor cell survival, neurite outgrowth and differentiation in vitro

In the present study we have investigated retinal pigment epithelium-photoreceptor cell interactions in vitro, and their contributions to photoreceptor cell survival and differentiation. Preparations enriched for intact photoreceptor cells from neonatal rat retina were grown in either serum-free medium supplemented with RPE-conditioned medium (RPE-CM) or in serum-free medium alone. A variety of substrate conditions were tested for the best neurite outgrowth. Cultures were monitored for 7 days by light and electron microscopy, as well as by opsin, vimentin and carbonic anhydrase-C immunocytochemistry. RPE-CM was found to stimulate both proliferation of flat cells and photoreceptor differentiation. The number of photoreceptors bearing neurites and their neurite length measurements showed significant differences between the RPE-CM group and the control group within 20 hr in culture. Elimination of contaminating flat cells by the addition of an antimitotic drug prevented photoreceptor cell morphological maturation; however, these cells survived as round cell bodies without processes for at least 10 days in the presence of RPE-CM and expressed opsin during this period. Conditioned medium from the flat-cell monolayers did not support photoreceptor differentiation or their survival. However, the presence of flat cells was a requisite to achieve any neurite outgrowth even in the presence of RPE-CM. In the absence of RPE-CM, neither photoreceptors nor flat cells survived or proliferated. Heat and trypsin treatment of the RPE-CM abolished all its growth-supporting activities which indicates its proteinaceous nature. This represents the first time in vitro that an RPE-derived factor(s) has been shown to be responsible for photoreceptor cell survival and differentiation.

IMMUNOCYTOCHEMICAL STUDY OF AN EYE WITH PROLIFERATIVE VITREORETINOPATHY AND RETINAL TACKS

After an eye-wall resection for a choroidal melanoma, a 32-year-old woman had subsequent retinal detachment with proliferative vitreoretinopathy (PVR), and an unsuccessful attempt at repair with retinal tacks. Gross and light-microscopic examination of the globe revealed a total retinal detachment with extensive preretinal and subretinal membranes. The membranes surrounded the tack heads and extended in taut bands to form a tractional detachment of the pars plana. The membranes contained glial and nonglial cells. The glial cells immunolabeled for glial fibrillary acidic protein (GFAP), carbonic anhydrase-C (CA-C), vimentin, and glutamine synthetase (GS), thus suggesting that they were Müller's cells. While the tacks did not seem to cause PVR, in this case they may have provided an anchoring point from which membranes were able to exert traction on the retina and pars plana.

The human chromophobe cell renal carcinoma: Its probable relation to intercalated cells of the collecting duct

In the present study we have examined ten cases of the chromophobe type renal cell carcinoma. This type of tumor is distinguished from the other carcinomas of the kidney with light cytoplasm (formerly called "hypernephroid") by (a) a positive Hale's iron colloid stain of the cytoplasm, (b) the occurrence of numerous invaginated vesicles within the cytoplasm that resemble the invaginated vesicles of intercalated cells of the collecting duct system, and (c) a positive immunoreaction of both the plasma membrane and the cytoplasm with antibodies to the epithelial membrane antigen (EMA) and carbonic anhydrase C (CAC), respectively. Unlike oncocytomas, which also express CAC and EMA, the chromophobe renal cell carcinoma does not express the erythrocyte anion exchanger band 3. These findings strongly indicate that chromophobe renal cell carcinomas as well as oncocytomas of the kidney are histogenetically related to the two populations of intercalated cells of the collecting duct system. Thus, both tumors represent examples of renal tumors which disprove the broadly accepted hypothesis that all epithelial tumors of the kidney are histogenetically related to the proximal tubule.

Gliogenesis in the embryonic avian optic tectum: neuronal-glial interactions influence astroglial phenotype maturation

We have analyzed the development of neuroglial cells in the chick optic tectum under 3 sets of developmental conditions to assess the role of heterotypic cell-cell interactions in gliogenesis. Immunochemical and biochemical methods were employed to measure and localize the expression of the glial markers glutamine synthetase, glial fibrillary acidic protein, S-100 protein, carbonic anhydrase-C and myelin basic protein as functions of development in situ and in aggregation and monolayer cultures of dissociated embryonic tissue. The results showed that certain astroglial cells can be recognized as early as day 9 of development in situ. Oligodendroglial development manifests several days later and the full range of glial subtypes are not evident until nearly the time of hatching. Culture of 7-day embryonic tectum cells either in aggregates or in monolayer cultures failed to yield definitive oligodendroglia. Fibrous astroglia, as defined by glutamine synthetase and glial fibrillary acidic protein, developed well in both culturing systems. However, as previously noted in the embryonic neural retina system, glutamine synthetase expression was markedly dependent on neuronal-glial associations.

Multiple marker analysis in the avian optic tectum reveals three classes of neuroglia and carbonic anhydrase-containing neurons

The distribution and biochemical diversity of neuroglial cells in the avian optic tectum were analyzed using immunohistochemical techniques. Polyclonal and monoclonal antibodies were employed to define the distributions and localizations of four proteins that are considered to be glial-specific in the mammalian central nervous system: glutamine synthetase, carbonic anhydrase-C, glial fibrillary acidic protein, and S-100 protein. The results showed the presence of at least three distinguishable populations of glia: glutamine synthetase/glial fibrillary acidic protein-positive fibrous astrocytes, glutamine synthetase/carbonic anhydrase-C-positive protoplasmic astrocytes, and S-100-positive oligodendroglia. In addition, a distinct population of neurons was observed that showed very intense immunostaining for the presence of carbonic anhydrase-C. The results suggest that the segregation of biochemical functions in the major categories of brain neuroglial cells (oligodendroglia and astroglia) varies significantly between vertebrate species. This observation raises the possibility of using comparative studies to refine our understanding of the roles that glial cells play in nervous tissue.

1-Naphthol basic dye (1-NBD)

The usefulness of 1-naphthol as substrate for horseradish peroxidase (HRP) in immunohistochemistry was studied using the peroxidase-antiperoxidase (PAP) and avidin-biotin-complex (ABC) methods in the demonstration of glial fibrillary acidic protein (GFAP), vimentin, carbonic anhydrase C (CA.C), and factor VIII-related antigen (FVIII/RAg) in central nervous tissue and cerebral tumors. In the presence of ammonium carbonate, 1-naphthol is oxidized by HRP and hydrogen peroxide, producing a fine gray-violet precipitate. The oxidation product of 1-naphthol proved capable of binding a great number of basic dyes. For each stain the final reaction product had a characteristic color that was different from the spontaneous color of the dye and from the color displayed by nuclei. The final color obtained with this procedure was alcohol resistant and could be mounted in solvent-based mounting media. The results obtained with the 1-naphthol basic dye (1-NBD) method were compared with those obtained using the diaminobenzidine (DAB) reaction in the demonstration of GFAP-positive astrocytes. The DAB reaction produced a more intense staining but also a coarser precipitate than the 1-NBD reaction. The 1-NBD procedure showed more morphological detail of fine structures and did not obscure nuclei and mitosis. The very low toxicity of 1-naphthol compared with DAB (a suspected carcinogen) is an important advantage of the 1-NBD method, as is its high specificity and sensitivity.

Comparative characterization of monoclonal antibodies to carbonic anhydrase

Monoclonal antibodies (Mabs) were generated to avian carbonic anhydrase-C and characterized; their reactivity with human, murine, bovine, chicken and fish erythrocyte carbonic anhydrase-C, and with human carbonic anhydrase-B was investigated by ELISA and electroblot techniques. Reactivity of the Mabs with native and SDS-denatured carbonic anhydrase was compared. Mabs that recognize antigenic determinants shared by all the carbonic anhydrases examined were identified. The results demonstrate the potential usefulness of these particular probes for investigating various aspects of function, evolution, development and regulation of this important, but not well understood group of enzymes.

Cellular compartmentalization of carbonic anhydrase-C and glutamine synthetase in developing and mature mouse neural retina

Using immunohistochemical methods, we have determined the cellular localization of the enzymes, glutamine synthetase (GS) and carbonic anhydrase-C (CA-C), in mouse neural retina during development and in the mature tissue. GS is always confined exclusively to the Müller glial cells; it is first detectable in these cells post-natally on about day 12, i.e. shortly before the eyes open. Also CA-C in the mature retina is localized in the Müller cells but, in addition, it is found in certain amacrine neurons as well. CA-C is first detectable in the retina already several days before birth; at that time it is found in most of the cells, with the exception of the emerging ganglion cells. However, with advancing differentiation, CA-C becomes progressively restricted to Müller cells and to a sub-category of amacrine neurons, and persists only in these cells in the mature retina. The present results extend our previous studies on these enzymes in the avian retina; they demonstrate that also in mammalian retina, different temporal and cellular patterns of GS and CA-C expression and localization earmark distinct phases of structural and functional differentiation of the retina. The striking developmental changes in the cellular localization of CA-C, and the finding of this enzyme in certain amacrine neurons as well as in Müller cells, raise questions about the role of CA-C in the retina, and about mechanisms regulating its expression in specific cell types.

Hormonal induction of glutamine synthetase in cultures of embryonic retina cells: Requirement for neuron-glia contact interactions

Cortisol induces glutamine synthetase (GS) in gliocytes of chick embryo neural retina. Using adherent cultures of retina cells we have demonstrated that responsiveness of the gliocytes to GS induction by the hormone requires contact with neurons. GS is not inducible in high-density cultures depleted of neurons and consisting only of gliocytes. In neuron-containing cultures, induced GS was detected immunohistochemically only in those gliocytes that were closely juxtaposed with clusters of neurons. Unlike the induction of GS, the expression of carbonic anhydrase-C (which does not require cortisol) persisted in these glia cells also in the absence of neurons. The nature and role of glia-neuron interactions in the hormonal induction of GS are briefly discussed.

Chapter 6 Developmental and Experimental Changes in Retinal Glia Cells: Cell Interactions and Control of Phenotype Expression and Stability

This chapter is concerned with the differentiation of neural retina cells. It focuses mainly on the glia cells, and on the role of contact interactions with neurons in the control of their phenotypic characteristics. The main purpose is to bring together findings and interpretations within a framework of a unifying working hypothesis. Studies have been concerned mainly with the characteristics of glia cells in the neural retina (NR) of the chick embryo. There is growing awareness of the importance of glia cells in the development and function of the NR, prompting increased interest in their properties. Among the key functions of glia cells is the regulation of the neuronal extracellular milieu because of their ability to take up and metabolize various transmitters and other products released from nerve terminals and also because they contribute to nerve cells and their immediate microenvironment metabolic and physical conditions essential for normal activity. The chapter reviews published and ongoing work on the developmental programs of two “markers” of definitive glia cells in the NR, the enzymes carbonic anhydrase-C and glutamine synthetase. It describes experiments on modification of definitive glia cells into lens-like cells and proposes that the cell surface and cell interactions play a key role in the expression and stability of Muller glia phenotype.

Physiological significance of fluid secretion in the testis and blood-testis barrier.

Physiological significance of fluid secretion in the testis and blood-testis barrier.G M Waites, and R T GladwellG M WaitesSearch for more papers by this author, and R T GladwellSearch for more papers by this authorPublished Online:01 Apr 1982https://doi.org/10.1152/physrev.1982.62.2.624MoreSectionsPDF (8 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByIn Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated VirusesStem Cell Reports, Vol. 10, No. 5Anatomy and Physiology of the Male Reproductive System and Potential Targets of Toxicants ☆Effects of levamisole on hyaluronidase activity and sperm characteristics in ramsTheriogenology, Vol. 62, No. 1-2Metastatic testicular rhabdomyosarcoma—a report of two casesJournal of Pediatric Surgery, Vol. 38, No. 8Paracrine control of spermatogenesisTrends in Endocrinology & Metabolism, Vol. 5, No. 3Theobromine toxicity on sertoli cells and comparison with cocoa extract in male ratsToxicology Letters, Vol. 70, No. 2Human sertoli cells in vitro: Morphological features and androgen-binding protein secretionThe Journal of Steroid Biochemistry and Molecular Biology, Vol. 43, No. 5The stimulatory effect of albumin on luteinizing hormone-stimulated Leydig cell steroid production depends on its fatty acid content and correlates with conformational changesMolecular and Cellular Endocrinology, Vol. 82, No. 1Direct evaluation of acidification by rat testis and epididymis: role of carbonic anhydraseC. R. Caflisch, and T. D. DuBose1 January 1990 | American Journal of Physiology-Endocrinology and Metabolism, Vol. 258, No. 1Regulation of transferrin secretion in Sertoli cells of the calf testisAnimal Reproduction Science, Vol. 20, No. 1The albumin fraction of rat testicular fluid stimulates steroid production by isolated Leydig cellsMolecular and Cellular Endocrinology, Vol. 59, No. 3Time course of the tri-o-cresyl phosphate-induced testicular lesion in F-344 rats: Enzymatic, hormonal, and sperm parameter studiesToxicology and Applied Pharmacology, Vol. 89, No. 1Paracrine regulation of testicular functionJournal of Steroid Biochemistry, Vol. 27, No. 1-3Androgen binding protein levels and FSH binding to testicular membranes in vitamin A deficient rats and during subsequent replenishment with vitamin AJournal of Steroid Biochemistry, Vol. 25, No. 4The effect of hemicastration on sperm reserves of cockerels on a restricted dietary intake, sperm reserves and deposition of sperm in different strains of cockerelsAnimal Reproduction Science, Vol. 9, No. 3Ethane dimethane sulphonate (EDS) specifically inhibits LH stimulated steroidogenesis in Leydig cells isolated from mature rats but not in cells from immature ratsMolecular and Cellular Endocrinology, Vol. 42, No. 2Androgen stimulation of sertoli cell function is enhanced by peritubular cellsMolecular and Cellular Endocrinology, Vol. 40, No. 2-3The effect of 2-methoxyethanol and methoxyacetic acid on sertoli cell lactate production and protein synthesis in vitroToxicology and Applied Pharmacology, Vol. 76, No. 1Intratesticular secretion of a factor(s) with major stimulatory effects on Leydig cell testosterone secretion in vitroMolecular and Cellular Endocrinology, Vol. 37, No. 2 More from this issue > Volume 62Issue 2April 1982Pages 624-71 Copyright & PermissionsCopyright © 1982 by American Physiological Societyhttps://doi.org/10.1152/physrev.1982.62.2.624PubMed6803269History Published online 1 April 1982 Published in print 1 April 1982 Metrics

An improved isolation procedure for carbonic anhydrase C from red cell lysate.

An effective isolation of carbonic anhydrase C (CA-C) from red cell lysate was described. The lysate dialyzed against 0.0175 M phosphate buffer pH 6.3 was applied onto a CM-Sephadex column equilibrated with the same buffer. The elution was performed with the starting buffer and 0.1 M dibasic potassium phosphate containing 0.14 M NaCl. The hemoglobin fraction eluted with the second eluant was applied onto a DEAE-cellulose column and eluted with 0.2 M glycine containing 0.01 percent KCN, resulting in complete isolation of CA-C at high recovery rate. For the preparation in a large scale, the hemoglobin fraction prepared from CM-Sephadex semi-batch-type chromatography was treated with cold ethanol and chloroform. The purity of these preparations was confirmed by polyacrylamide gel disc electrophoresis and immunoelectrophoresis.

Determination of carbonic anhydrase C and β2-microglobulin by radioimmunoassay in urine of heavy-metal-exposed subjects and patients with renal tubular acidosis

Carbonic anhydrase C (CA-C) was measured by the radioimmunoassay in urine specimens from normal individuals, residents in mercury-polluted area, cadmium-exposed workers, lead-exposed workers, and patients with primary or secondary renal tubular acidosis. None of the urine in the normal subjects demonstrated CA-C levels above 40 μg/g creatinine. Some of the cadmium-exposed workers, residents in mercury-polluted area, and patients with renal tubular acidosis excreted large amounts of CA-C that were 10–250 times higher than the normal. Urinary β 2-microglobulin (BMG) was also determined in relation to the CA-C levels. Most of the subjects exposed to heavy metals had high levels of urine CA-C or BMG or both. Some individuals had high levels of urine CA-C although BMG levels were within normal values. These findings seem to suggest that the mechanisms of urinary excretion of these two proteins were different and the quantitative determination of urinary CA-C by radioimmunoassay appears to be also a useful and sensitive test for detecting the renal tubular disorders in environmental exposure of heavy metals or those in renal tubular acidosis.

Carbonic anhydrase isoenzymes in infants with respiratory distress syndrome.

Blood obtained from umbilical cords of 106 preterm infants was analyzed for carbonic anhydrase (CA) activity. Carbonic anhydrase isoenzymes were measured in red blood cells from five adults, eight full-term newborn infants, and 16 premature infants. Carbonic anhydrase activity increased progressively with gestation. Infants with respiratory distress syndrome (RDS) had lower cord blood CA activity than infants without RDS. Carbonic anhydrase B (CA-B), carbonic anhydrase C (CA-C), and total carbonic anhydrase protein (CA-B + CA-C) were highest in adults, then full-term newborns, premature newborns without RDS, and infants with RDS. The ratio of CA-B/CA-C was the same for all newborn groups and lower than the adult ratio. All enzyme activity in newborns could be accounted for by CA-B and CA-C. No inhibitors or abnormal enzymes were present. Importance of findings to RDS pathogenesis is discussed.