Paul J. Linser

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.

Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut.

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH ≈ 3 units⋅mm−1) in which an extremely alkaline (pH ≈ 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7–8). A plasma membrane H+ V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate/carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 ± 5.64 and 50.8 ± 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 ± 7.84, than in the lumen, 3.51 ± 2.58, whereas bicarbonate was higher in the lumen, 58.1 ± 7.34, than the hemolymph, 3.96 ± 2.89. Time-lapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), at 10−4 M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by ≈65% and 85% respectively. Self-referencing chloride-selective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H+ V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl−/HCO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.

Müller cell differentiation in the zebrafish neural retina: Evidence of distinct early and late stages in cell maturation

The vertebrate neural retina is mainly composed of cells of neuroectodermal origin. The primary cell types found in all vertebrate retinas are several categories of neurons and the archetypical retina glial cell the Müller cell. Although the neurons and the single glial cell type of the retina are specialized for very distinct functions, they all have a common developmental origin within the tissue. How the distinctions between cell types, in particular between neurons and glia, arise during embryonic development remains a central issue in neurobiology. In this report, we examine the genesis of Müller glial cells during zebrafish (Danio rerio) eye development. Particular emphasis is placed on the expression of the Müller cell maturation markers carbonic anhydrase and glutamine synthetase. In addition, we report that the HNK‐1 monoclonal antibody, which identifies a particular glycoconjugate frequently found on cell surface recognition molecules, also identifies zebrafish retina Müller cells early in development. The expression patterns of these three markers clearly show that the Müller cells mature in stages: HNK‐1 labeling and glutamine synthetase arise earlier than carbonic anhydrase expression. In addition, the embryonic zebrafish neural retina is characterized by the presence of amoeboid, carbonic anhydrase–positive microglial cells even before the genesis of retinal neuroectodermal glia. The stepwise maturation of the glia is likely to be indicative of an overall retinal maturational program in which cell differentiation and the expression of certain phenotype‐defining gene products may be separately regulated. J. Comp. Neurol. 429:530–540, 2001.

Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan, Anopheles gambiae.

SUMMARY We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes (Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned, nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

Cationic pathway of pH regulation in larvae of Anopheles gambiae.

SUMMARY Anopheles gambiae larvae (Diptera: Culicidae) live in freshwater with low Na+ concentrations yet they use Na+ for alkalinization of the alimentary canal, for electrophoretic amino acid uptake and for nerve function. The metabolic pathway by which larvae accomplish these functions has anionic and cationic components that interact and allow the larva to conserve Na+ while excreting H+ and HCO3–. The anionic pathway consists of a metabolic CO2 diffusion process, carbonic anhydrase and Cl–/HCO3– exchangers; it provides weak HCO3– and weaker CO32– anions to the lumen. The cationic pathway consists of H+ V-ATPases and Na+/H+ antiporters (NHAs), Na+/K+ P-ATPases and Na+/H+ exchangers (NHEs) along with several (Na+ or K+):amino acid+/– symporters, a.k.a. nutrient amino acid transporters (NATs). This paper considers the cationic pathway, which provides the strong Na+ or K+ cations that alkalinize the lumen in anterior midgut then removes them and restores a lower pH in posterior midgut. A key member of the cationic pathway is a Na+/H+ antiporter, which was cloned recently from Anopheles gambiae larvae, localized strategically in plasma membranes of the alimentary canal and named AgNHA1 based upon its phylogeny. A phylogenetic comparison of all cloned NHAs and NHEs revealed that AgNHA1 is the first metazoan NHA to be cloned and localized and that it is in the same clade as electrophoretic prokaryotic NHAs that are driven by the electrogenic H+ F-ATPase. Like prokaryotic NHAs, AgNHA1 is thought to be electrophoretic and to be driven by the electrogenic H+ V-ATPase. Both AgNHA1 and alkalophilic bacterial NHAs face highly alkaline environments; to alkalinize the larva mosquito midgut lumen, AgNHA1, like the bacterial NHAs, would have to move nH+ inwardly and Na+ outwardly. Perhaps the alkaline environment that led to the evolution of electrophoretic prokaryotic NHAs also led to the evolution of an electrophoretic AgNHA1 in mosquito larvae. In support of this hypothesis, antibodies to both AgNHA1 and H+ V-ATPase label the same membranes in An. gambiae larvae. The localization of H+ V-ATPase together with (Na+ or K+):amino acid+/– symporter, AgNAT8, on the same apical membrane in posterior midgut cells constitutes the functional equivalent of an NHE that lowers the pH in the posterior midgut lumen. All NATs characterized to date are Na+ or K+ symporters so the deduction is likely to have wide application. The deduced colocalization of H+ V-ATPase, AgNHA1 and AgNAT8, on this membrane forms a pathway for local cycling of H+ and Na+ in posterior midgut. The local H+ cycle would prevent unchecked acidification of the lumen while the local Na+ cycle would regulate pH and support Na+:amino acid+/– symport. Meanwhile, a long-range Na+ cycle first transfers Na+ from the blood to gastric caeca and anterior midgut lumen where it initiates alkalinization and then returns Na+ from the rectal lumen to the blood, where it prevents loss of Na+ during H+ and HCO3– excretion. The localization of H+ V-ATPase and Na+/K+-ATPase in An. gambiae larvae parallels that reported for Aedes aegypti larvae. The deduced colocalization of the two ATPases along with NHA and NAT in the alimentary canal constitutes a cationic pathway for Na+-conserving midgut alkalinization and de-alkalinization which has never been reported before.

Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

Abstract: The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell‐cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P‐40‐solubilized retina membranes had two components as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), a 45.5‐kDa doublet and a 69‐kDa polypeptide. Immunoreactive bands of 46‐50 kDa were recognized following SDS‐PAGE of detergent‐solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N‐glycosidase F (EC 3.2.2.18) converted the 45.5–50‐kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N‐Glycosidase Ftreatment also converted the 69‐kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32‐kDa species. Treatment with endo‐β‐N‐acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo‐β‐N‐acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5‐kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O‐glycosidase (endo‐α‐N‐acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for α2, 6‐linked sialic acid) was confined to the higher molecular mass species of the 45.5‐kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue‐specific posttranslational modifications, particularly differences in sialylation of antigen‐bearing polypeptides.

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 marked dependent on neuronal-glial associations.

Isolation and characterization of a carbonic anhydrase homologue from the zebrafish (Danio rerio).

Abstract. We have isolated a 29,000-Da carbonic anhydrase (CA) protein from the zebrafish, Danio rerio, sequenced two peptide fragments, and tentatively identified it as a high-activity CA by inhibition kinetics. We have also characterized a 1,537-bp message whose deduced sequence of 260 amino acids matches that of the isolated protein. This CA is clearly an α-CA based on the similarity of its sequence to that of other members of the α-CA gene family. A phylogenetic analysis suggested CAH-Z diverged after the branching of the CA-V and CA-VII genes and prior to the duplications that generated the CA-I, CA-II, and CA-III genes of amniotes. This marks the first characterization of the mRNA and its protein product from the CA gene of a teleost.

Carbonic anhydrase in the adult mosquito midgut

SUMMARY We have previously demonstrated the involvement of carbonic anhydrase (CA) in larval mosquito midgut physiology. In this study, we used Hanssons histochemistry to examine the distribution of the enzyme in the midgut of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex nigripalpus, Ochlerotatus taeniorhynchus, Anopheles albimanus and Anopheles quadrimaculatus adult mosquitoes. Additionally, we quantitated CA content in the anterior and posterior midgut of adult males and females from these species using the 18O isotope exchange method coupled to mass spectrometry. We also tested the effect of CA inhibitors such as methazolamide and acetazolamide in the alkalization of the midgut in females from these species. Our results indicate that CA is present in the midgut of adults from the species studied and that it appears to be preferentially associated with the posterior midgut in both males and females. CA inhibitors appear to have a profound effect on midgut pH indicating that this enzyme might play a key role in the maintenance of this pH.

Glutamine synthetase is a glial-specific marker in the olfactory regions of the lobster (Panulirus argus) nervous system.

Glutamine synthetase (GS) has been qualified as a very specific marker of astroglial‐type neuroglia in vertebrate neural tissues. In this paper we have begun to examine the possibility that glial localization of GS could be a ubiquitous characteristic of complex nervous systems. To this end we have used immunohistochemistry to localize GS‐like immunoreactivity in the olfactory regions of the complex nervous system of the arthropod, the spiny lobster Panulirus argus. We describe a novel method for affinity isolation of antibodies from crude serum. Using this approach we purified GS‐specific antibodies to chick retina GS and used these to analyze the lobster brain and the primary olfactory organ. Western blots showed that the lobster brain contains an immunoreactive peptide with nearly the same molecular mass as that of chick retina GS. Northern blot analyses of mRNA and enzymatic activity assays also confirm that the lobster brain produces GS. Immunohistochemical staining of sectioned lobster olfactory lobes and sensory sensilla showed strong reactivity in specific cells. Comparison of the GS immunostaining pattern with that for FMRFamide, a well characterized marker of neurons in invertebrate neural tissues, it became clear that GS is indeed glial‐specific in lobster neural tissues as it is in vertebrates. These results suggest that the compartmentalization of GS in non‐neuronal cells is either an early step in neural evolution or is an obligate and fundamental characteristic of complex neural systems composed of both neurons and neuroglia. GLIA 20:275–283, 1997.