Immunohistochemical demonstration of the carbonic anhydrase isoenzymes I and II in pancreatic tumours

Summary The location of carbonic anhydrase (CA) isoenzymes I, II and VI in normal and neoplastic pancreatic tissue was studied using polyclonal antisera and the immunoperoxidase technique. Samples were obtained from patients with well-differentiated (n = 4), moderately differentiated (n = 1) and poorly differentiated (n = 4) ductal adenocarcinomas, cystadenocarcinoma (n = 2), adenosquamous carcinoma (n = 1), acinar adenocarcinoma (n = 1), gastrinoma (n = 3), insulinoma (n = 3) and glucagonoma (n = 1). The control specimens were from a patient with traumatic laceration of the pancreas. The normal and malignant endocrine tissue showed intense positive staining for CA I localized in the cells expressing glucagon. In the exocrine pancreatic tissue, CA II was detected in the normal and neoplastic ductal epithelium. No specific staining was detected with anti-CA VI serum in either normal or malignant tissue.     

Characteristics of tumour vessels in cytological squash smears of astrocytic tumours

S. Sato, Y. Sato, K. Marutsuka, H. Takeshima and Y. Asada Characteristics of tumour vessels in cytological squash smears of astrocytic tumours Objective: Smear preparations are useful tools from which to diagnose brain tumours intraoperatively. Although vascular proliferation is histologically a key feature of high‐grade astrocytoma, the characteristics of tumour vessels in smear preparations have not been determined. Methods: We examined the density and morphological parameters (area, width, nuclear layer and branches of vessel wall) of tumour vessels in squash smears of 43 primary astrocytomas (grade II diffuse astrocytomas, n = 9; grade III anaplastic astrocytomas, n = 13; grade IV glioblastomas, n = 21) and normal brain tissues (n = 11). Results: Vessel density and all morphological parameters were significantly higher in grade IV than in the other grades of tumours and in normal brain tissue. Vessel area, width and nuclear layer were greater in grade III than in normal brain tissue. The sensitivity and specificity of these vessel parameters for astrocytomas were 75–100% and 82–100%, respectively. Conclusions: Tumour vessel evaluations from squash smears provide useful information for the intraoperative diagnosis and grading of astrocytic tumours.     

Choroid plexus and paraphysis in lower vertebrates

Cytoarchitecture of the choroid plexus of the third ventricle and the paraphysis was investigated in some lower vertebrates to compare the histologic characteristics of these organs. Both epithelia are similar in appearance in the same class. Minor microscopic variations exist in the different classes of vertebrates, but do not provide a fundamental distinction between the two organs. The epithelia, moreover, have similar staining properties, contain mucicarmine- and PAS-reactive materials, and are derived from a common neuroepithelium. Tubules are identified in the choroid plexus and in the paraphysis; all are similarly formed by simple folding of epithelium on the surface into the stroma. The paraphyses in all vertebrates studied contain villi similar to those seen in the choroid plexus. Cilia are identified in both choroidal and paraphyseal epithelia, and are not an indication of degree of epithelial differentiation. Many types of epithelium are noted in both organs during histologic differentiation as well as in the mature stage. Functionally, the choroid plexus is active in both secretion and absorption. Accumulation of particulate material within the epithelial cytoplasm may indicate phagocytic as well as absorptive activity of cells. Based on a common neuroepithelial origin and similar histochemical properties, we conclude that the paraphysis is a modified choroid plexus. The velum transversum is an arbitrary boundary between diencephalon and telencephalon, and is itself formed of choroid plexus. The medial telencephalic ventricle is the rostral portion of the third ventricle. All neuroepithelial infoldings at the rostral end of the diencephalic roof including the velum transversum are intraventricular choroid plexuses; the neuroepithelial outpouchings in this region are the extraventricular choroid plexuses (paraphysis) of the diencephalon.     

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus

Abstract: The serotonin 5-HT_2C receptor (formerly designated the 5-HT_1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5-HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five-fold basal level, with an EC₅₀ of 11 nM. This response was not inhibited by muscarinic or β-adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent K_i values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin-stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin-stimulated cGMP formation was calcium, phospholipase A₂, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A₂ or lipoxygenase inhibitors p-bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5-HT_2C receptors trigger cGMP formation in a calcium-sensitive manner.     

Expression of carbonic anhydrases II, IV, VII, VIII and XII in rat brain after kainic acid induced status epilepticus

Carbonic anhydrases (CAs) are important enzymes in the central nervous system (CNS), where they participate in regulating cerebrospinal fluid (CSF) secretion, blood-brain barrier and glial cell function. Using RT-PCR we found CA XII mRNA in rat and mouse brain. Cloning of rat CA XII revealed 94% homology with the mouse CA XII. To map the putative functional roles of different CAs, we studied the expression and localization of CA II, CA IV, CA VII, CA-related protein (CA-RP) VIII and CA XII mRNAs in rat brain after kainic acid induced epileptic seizures using Northern blot analysis and in situ hybridization. The expression of CA IV, CA VII and CA-RP VIII was somewhat similar: they were expressed in the cortex, hippocampus and midbrain structures and their expression did not change after the kainic acid treatment. The expression of CA II was concentrated in the white matter structures, which is in line with the preferential expression of CA II in the oligodendrocytes. High levels of CA II mRNA were also detected in the choroid plexus. Surprisingly, CA II was induced 3-12 h after seizures in the vulnerable CA1 region. CA XII was expressed in dentate granule cells, cortex and choroid plexus. Kainic acid stimulated CA XII expression throughout the cortical layer I. The observed hippocampal induction of CA II may indicate a pro-apoptotic and/or epileptogenic role of CA II after prolonged seizures. The physiological significance of the observed cortical induction of CA XII remains obscure. Cytosolic CA II is known to participate in CSF secretion, and the high expression of CA XII in the choroid plexus suggests an analogous role for this membrane-bound isozyme.     

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n?=?24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n?=?16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.     

NBCe2 exhibits a 3 HCO3(-):1 Na+ stoichiometry in mouse choroid plexus epithelial cells

The transport stoichiometry of the electrogenic sodium-bicarbonate cotransporter (SLC4A5 or NBCe2) in mouse choroid plexus was examined. Whole-cell recording methods measured the currents carried by the NBCe2, using experimental solutions determined to minimise the contributions of the other ion conductances present. Increases in outward current were observed when 21.2 mM was added to the bath solution in the presence of Na⁺, but not N-methyl-d-glucamine. This -induced current was completely abolished by 500 μM 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid. The reversal potential for the -induced current was −95.1 ± 7.1 mV (n = 11), a value which corresponds to a NBCe2 transport stoichiometry of 3 with 1 Na⁺. The NBCe2, with this stoichiometry, will mediate the efflux of and Na⁺ from the cell into the cerebrospinal fluid at the apical membrane of the choroid plexus.     

In situ localization of transthyretin-mRNA in the adult human liver,choroid plexus and pancreatic islets and in endocrine tumours of the pancreas and gut

Summary In situ hybridization with ³⁵S-labeled single stranded RNA probes was used on sections from formaldehyde-fixed and paraffin-embedded tissue specimens to provide semiquantitative data on the occurrence of transthyretin(TTR)-mRNA in human liver, choroid plexus and pancreatic islets as well as in 15 endocrine tumours of the pancreas and gut. A monoclonal antibody to TTR was used for immunocytochemical identification of the protein in consecutive sections.The amount of TTR-mRNA in hepatocytes was found to be much less than that in epithelial cells of the choroid plexus. Glucagon cells of the pancreatic islets were also specifically labeled and the level of TTR-mRNA in these cells was intermediate between that of hepatocytes and choroid plexus epithelial cells. Four glucagonomas, one malignant insulinoma and two midgut carcinoids were shown to contain TTR-mRNA. The in situ labeled cells were also found to be TTR immunoreactive. These findings present the first conclusive evidence for TTR synthesis in pancreatic islets and in endocrine tumours. They also establish that the high serum concentration of TTR found in some patients with endocrine tumours (notably glucagonomas) is most likely due to tumour production of TTR.     

Carbonic anhydrase inhibitors. Interaction of the antitumor sulfamate EMD 486019 with twelve mammalian carbonic anhydrase isoforms: Kinetic and X-ray crystallographic studies

The new antitumor sulfamate EMD 486019 was investigated for its interaction with twelve catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I – XIV. Similarly to 667-Coumate, a structurally related compound in phase II clinical trials as steroid sulfatase/CA inhibitor with potent antitumor properties, EMD 486019 acts as a strong inhibitor of isozymes CA II, VB, VII, IX, XII, and XIV (K_Is in the range of 13–19 nM) being less effective against other isozymes (K_Is in the range of 66–3600 nM against hCA I, IV, VA, VI, and mCA XIII, respectively). The complete inhibition profile of 667-Coumate against these mammalian CAs is also reported here for the first time. Comparing the X-ray crystal structures of the two adducts of CA II with EMD 486019 and 667-Coumate, distinct orientations of the bound sulfamates within the enzyme cavity were observed, which account for their distinct inhibition profiles. CA II/IX potent inhibitors belonging to the sulfamate class are thus valuable clinical candidates with potential for development as antitumor agents with a multifactorial mechanism of action.     

The Vascularization of the Anuran Brain Diencephalon and Choroid Plexus: A scanning electron microscopical study of vascular corrosion casts

Albrecht, U., Lametschwandtner, A., Adam, H. 1979. The vascularization of the anuran brain. Diencephalon and choroid plexus. A scanning electron microscopical study of vascular corrosion casts. (Department of Zoology, University of Saulzburg, Austria.) — Acta zool. (Stockh.) 61(4): 203–220. The vascularization of the diencephalon (with choroid plexus of the third ventricle, epithalamus and pineal region, thalamus and hypothalamus) of the toad, Bufo bufo (L.) has been studied by means of scanning electron microscopy of vascular corrosion casts. To localize angioarchitectonic patterns of distinct diencephalic regions the authors refer to critical point dried specimens and to histological sections. In the choroid plexus a supply via one choroid artery, which arises from the posterior telencephalic artery, was found. Its strict dichotomous branching is pointed out. In generally a similar vascular pattern like that in the choroid plexus of the fourth ventricle has to be reported. Furthermore the epithalamic region with the epiphysial area was under investigation. No special angioarchitecture of the epiphysis was found. There are also no prominent vascular connections with thalamic or hypothalamic regions. The thalamic region is supplied by branches of the posterior telencephalic artery as well as by branches of the preoptic artery. Epithalamic and thalamic regions are drained via the posterior diencephalic vein. Special attention was also paid to the preoptic, the chiasmatic and the retrochiasmatic area. No special vascular connections, however, were found.     

High Activities of Glutamine Transaminase K (Dichlorovinylcysteine β-Lyase) and ω-Amidase in the Choroid Plexus of Rat Brain

Abstract: Certain halogenated hydrocarbons, e.g., dichlo-roacetylene, are nephrotoxic to experimental animals and neurotoxic to humans; cysteine-S-conjugate β-lyases may play a role in the nephrotoxicity. We now show that with dichlorovinylcysteine as substrate the only detectable cysteine-S-conjugate β-lyase in rat brain homogenates is identical to glutamine transaminase K. The predominant (mitochondrial) form of glutamine transaminase K in rat brain was shown to be immunologically distinct from the predominant (cytosolic) form of the enzyme in rat kidney. Glutamine transaminase K and ω-amidase (constituents of the glutaminase II pathway) activities were shown to be widespread throughout the rat brain. However, the highest specific activities of these enzymes were found in the choroid plexus. The high activity of glutamine transaminase K in choroid plexus was also demonstrated by means of an immunohistochemical staining procedure. Glutamine transaminase K has a broad specificity toward amino acid and α-keto acid substrates. The ω-amidase also has a broad specificity; presumably, however, the natural substrates are α-ketoglutaramate and α-ketosuccinamate, the α-keto acid analogues of glutamine and aspara-gine, respectively. The high activities of both glutamine transaminase K and ω-amidase in the choroid plexus suggest that the two enzymes are linked metabolically and perhaps are coordinately expressed in that organ. The data suggest that the natural substrate of glutamine transaminase K in rat brain is indeed glutamine and that the metabolism of glutamine through the glutaminase II pathway (i.e., l -glutamine and α-keto acid α-ketoglutarate and l -amino acid + ammonia) is an important function of the choroid plexus. Moreover, the present findings also suggest that any explanation of the neurotoxicity of halogenated xenobiotics must take into account the role of glutamine transaminase K and its presence in the choroid plexus.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VITRO STUDIES

Abstract— The transport into and release of tritium labeled vitamin B₆ ([³H]B₆) from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated [³H]B₆ by an energy dependent uptake system when [³H]pyridoxine (PIN) was added to the incubation medium. Most of the [³H] within the tissues was phosphorylated [³H]B₆. In each tissue, the nonphosphorylated vitamers inhibited the uptake of [³H]PIN from the medium significantly more than the phosphorylated vitamers. The concentrations of the nonphosphorylated B₆ vitamers necessary to inhibit brain and choroid plexus uptake of [³H]PIN from the medium by 50% were approx 0.4 μm and 5–10μm respectively after a 30 min incubation. Both brain slices and choroid plexus readily released (46 and 56% respectively in 30 min) previously accumulated [³H]B₆ into artificial CSF. However, brain slices released only nonphosphorylated [³H]B₆, whereas the choroid plexus released predominantly phosphorylated [³H]B₆. Addition of unlabeled PIN to the release media significantly increased the percentage of [³H]B₆ released by both brain slices and choroid plexus. The results of these in vitro studies provide evidence that: (1) both brain slices and chloroid plexus possess specific uptake and release mechanisms for B₆, and (2) these mechanisms tend to regulate intracellular B₆ levels. These studies also suggest that the choroid plexus serves as a locus for the transfer of B₆ from blood to CSF and is the source of most of the phosphorylated B₆ in CSF.     

Expression of the β-subunit isoforms of the Na,K-ATpase in rat embryo tissues,inner ear and choroid plexus

Summary— We report evidence of the apical localization of the two Na, K-ATPase β-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na, K-ATPase β1 and β2 subunits. These polyclonal rabbit antisera were raised against truncated β-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the β1 isoform and five bands in the β2 iso-form immunoblots. In E15 rat embryos, the β1 isoform was detected in brain, heart and kidney and the β2 isoform only in brain. While β-subunit mRNA expression (Watts AG, Sanchéz-Watts G, Emanuel JR, Levenson R 1991 Proc Natl Acad Sci USA 88, 7425–7429), and immunoblotting and enzymatic activity have been determined (Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A 1993 J Biol Chem 268, 8019–8025), very little is known about the specific localization of each β-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the β1 and β2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus     

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

 Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks. Accepted: 7 January 1998     

Carbonic anhydrase isoenzymes in nine troops of Kenya baboons, Papio cynocephalus (Linnaeus 1766)

The distributions of alleles at the carbonic anhydrase I (CA I = CA B) and carbonic anhydrase II (CA II = CA C) loci in nine troops of Papio cynocephalus were determined. Two alleles were found at the CA I locus, and three at the CA II locus; the frequencies were: CA I^a = 0.856; CA I^b = 0.144; CA II^a = 0.784; CA II^b = 0.209; CA II^c = 0.007. Results of tests for Hardy-Weinberg equilibrium, homogeneity tests, and calculations of migration rates were used in support of the interpretation that migration and genetic drift may affect the distribution of alleles at the CA I locus and that selection is the process responsible for the distribution of alleles at the CA II locus.     

The role of nucleotide pyrophosphatase in Mullerian duct regression

Mullerian inhibiting substance (MIS), a glycoprotein from the fetal testis causing regression of the embryonic Mullerian duct, can be inhibited in vitro in the presence of Mn²⁺ by a wide range of nucleotides including GTP, NAD, ATP, AMP, and several nonhydrolyzable synthetic ATP analogs. Extracellular nucleotide pyrophosphatase (NPPase), an enzyme able to hydrolyze the wide variety of the nucleotides and analogs found to inhibit Mullerian duct regression, was studied by histochemical staining (H. Sierakowska and D. Shugar (1963) to determine if NPPase localized in or around the Mullerian duct during regression. Frozen sections of urogenital ridges from to rat fetuses (n = 77) were incubated with a-naphthyl thymidine-5′-phosphate (naphthyl TMP) and Fast Red TR. Nucleotide pyrophosphatase hydrolyzes naphthyl TMP, releasing naphthol, which then reacts with Fast Red to produce color at the enzyme site. Nucleotide hydrolysis was detected around regressing male (n = 16) Mullerian duct cells at days of gestation, but no hydrolysis was detected around female (n = 17) Mullerian duct cells at any stage. Controls (n = 24) incubated without substrate did not stain. Addition of exogenous ATP (n = 20) to the histochemical incubation medium inhibited nucleotide hydrolysis on male Mullerian ducts, suggesting that this staining is specific for pyrophosphatase activity. Results in vivo were confirmed in vitro by incubating day female rat urogenital ridges with MIS for 72 hr prior to histochemical staining. The addition of testosterone to MIS was obligatory to detect staining in vitro (n = 10). The localized NPPase activity around the regressing Mullerian duct suggests that NPPase may appear as a consequence of duct regression and may act to control the degree of membrane phosphorylation by degrading excess trinucleotides.     

Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides

A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO₂ hydration reaction were measured. With a k_cat/K_m of 1.1?×?10⁸ M^?1 s^?1, and a k_cat of 1.3?×?10⁶ s^?1, clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K_Is in the range of 1.9–3460?nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.     

Epithelial differentiation in gliomas,meningiomas and choroid plexus papillomas

The immunohistological findings using antibodies to different intermediate filaments (glial fibrillary acidic protein, vimentin and two types of cytokeratin) and epithelial membrane antigen are described in 89 gliomas, 19 meningiomas and 8 choroid plexus papillomas (CPPs) from adult patients. All the patients had total or subtotal surgical excision of their tumours with clinical follow up for between 3 and 7 years. The immunohistological results were correlated with the histological features and patient survival. Tumours other than low grade astrocytomas, oligodendrogliomas and anaplastic ependymomas expressed one or more epithelial markers. This immunohistological evidence of epithelial differentiation in the absence of histological epithelial features in gliomas confirms that the two are not necessarily correlated. It is concluded that the expression of epithelial markers in some intradural tumours may reflect aberrant differentiation related to the degree of anaplasia in poorly differentiated astrocytomas and glioblastomas. All the patients with anaplastic epithelial marker-positive gliomas died within 1 year, whereas only 68% of patients with marker-negative tumours died within the follow-up period. In ependymomas and meningiomas, the expression of epithelial markers may reflect their histogenesis, while in malignant CPPs such expression could denote either their aberrant differentiation or histogenetic derivation.