Rat Choroid Plexus Epithelium Research Articles

Identification and properties of a novel variant of NBC4 (Na+/HCO3− co-transporter 4) that is predominantly expressed in the choroid plexus

Secretion of HCO(3)- at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO(3)- permeability has been observed and suggested to be the major pathway for HCO(3)- transport across the apical membrane. Recently, it was found that NBC (Na(+)/HCO(3)- co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate co-transporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO(3)- transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO(3)- and contributes to cerebrospinal fluid production.

Expression of junctional proteins in choroid plexus epithelial cell lines: a comparative study

BackgroundThere is an increasing interest in using choroid plexus (CP) epithelial cell lines to study the properties of the blood-cerebrospinal fluid barrier (BCSFB). Currently, there are three major CP-derived cell lines available. Z310 and TR-CSFB3, two immortalized cell lines carrying the simian virus 40 large T-antigen gene, were derived from rat CP epithelium, whereas the CPC-2 cell line was derived from human CP carcinoma. Although these cell lines have previously been used in various functional studies, the expression of adherens junction (AJ) and tight junction (TJ) proteins in these epithelial cells has not been systematically studied. Accordingly, in the present study, we sought to characterize the expression of these junctional proteins in these three cell lines.MethodsThe cells were grown in six-well cell culture plates. Reverse-transcriptase polymerase chain reaction, Western blotting, and immunocytochemistry were used to characterize the expression of AJ and TJ proteins in the CP cell lines.ResultsZ310 and TR-CSFB3 cells expressed a TJ protein, occludin, and its cytosolic binding partner, zonula occludens 1, as well as an AJ protein, E-cadherin, and β-catenin, a cytoplasmic protein that interacts with E-cadherin. However, the expression of occludin and E-cadherin in TR-CSFB3 cells at both the mRNA and protein level was weaker than that found in Z301 cells. The immunocytochemical analysis also demonstrated that the staining pattern for these junctional proteins in TR-CSFB3 cells was discontinuous and the staining intensity was weaker than that observed in Z310 cells. The message for claudin 1 and claudin 2 was expressed at low levels in TR-CSFB3 cells and these cells were weakly immunopositive for claudin 1. In comparison, the message for these TJ proteins could not be detected in Z310 cells. CPC-2 cells expressed occludin, which was localized to areas of cell-cell contact, but the staining pattern for this TJ protein was found to be variable and irregular. Although CPC-2 cells expressed mRNA for claudin 1, claudin 2, and claudin 11, only claudin 1 was expressed at the protein level and it was localized to the nuclei rather than to areas of cell-cell contact. An AJ protein, E-cadherin, was also found to be mislocalized in CPC-2 cells, even though its cytosolic binding partner, β-catenin, was restricted to areas of cell-cell contact, as in normal CP.ConclusionThe three CP cell lines analyzed in this study vary considerably with regard to the expression of AJ and TJ proteins, which is likely reflected by different barrier properties of these in vitro models of BCSFB.

Na+-dependent HCO3− uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na+ and HCO3- transport in the formation of cerebrospinal fluid. Two Na+-dependent HCO3- transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601-C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pHi) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH2-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pHi in choroidal cells increased from 7.03 +/- 0.02 to 7.38 +/- 0.02 (n=41) after addition of CO2/HCO3- into the bath solution. This increase was Na+ dependent and inhibited by the Cl- and HCO3- transport inhibitor DIDS (200 muM). This suggests the presence of Na+-dependent, partially DIDS-sensitive HCO3- uptake. The pHi recovery after acid loading revealed an initial Na+ and HCO3- -dependent net base flux of 0.828 +/- 0.116 mM/s (n = 8). The initial flux in the presence of CO2/HCO3- was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na+- and HCO3- -dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na+-driven Cl- bicarbonate exchanger, and NBCe2 in this tissue.

Fatty acid amide hydrolase in brain ventricular epithelium: mutually exclusive patterns of expression in mouse and rat

Fatty acid amides and fatty acid ethanolamides are novel signalling molecules exemplified by the sleep-inducing lipid oleamide and the endocannabinoid anandamide, respectively. These substances are inactivated by fatty acid amide hydrolase (FAAH), an enzyme that is expressed by neurons and non-neuronal cells in the brain. In the rat, FAAH-immunoreactivity has been detected in epithelial cells of the choroid plexus and, in accordance with this finding, here we report FAAH mRNA expression in rat choroid plexus epithelium using in situ hybridisation methods. Surprisingly, a comparative analysis of mouse brain did not reveal FAAH mRNA expression or FAAH-immunoreactivity in the choroid plexus of this species. FAAH-immunoreactivity was, however, detected in non-choroidal ventricular ependymal cells in the mouse brain and the specificity of this immunostaining was confirmed by analysis of FAAH-knockout mice. FAAH-immunoreactivity was detected in ependymal cells throughout the ventricles of the mouse brain but with regional variation in the intensity of immunostaining. Intriguingly, in rat brain, although FAAH expression is observed in choroid plexus epithelial cells, little or no FAAH-immunoreactivity is present in the ventricular ependyma. Thus, there are mutually exclusive patterns of FAAH expression in the ventricular epithelium of rat and mouse brain. Our observations provide the basis for an experimental analysis that exploits differences in FAAH expression in rat and mouse to investigate FAAH function in ventricular epithelial cells and, in particular, the role of FAAH in regulating the sleep-inducing agent oleamide in cerebrospinal fluid.

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na +/K +-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.

The presence of arginine vasopressin and its mRNA in rat choroid plexus epithelium.

Arginine vasopressin (AVP) plays an important role in the regulation of secretory function and hemodynamics of choroid plexus, the primary site of cerebrospinal fluid (CSF) production. In the present study, localization of AVP and its transcripts in choroid plexus of adult male Sprague-Dawley rats was studied by immunohistochemistry and in situ hybridization histochemistry, respectively. For immunohistochemical analysis, AVP-specific polyclonal rabbit antibody was employed. Plasmid, pGrVP, containing a 232-bp fragment of rat AVP cDNA encoding the C-terminus of proAVP, was used as a probe to detect AVP mRNA. AVP-immunoreactive product was predominantly localized close to the apical (CSF-facing) membrane of choroidal epithelium while AVP transcripts were distributed throughout the cytoplasm of the cells. Our findings indicate that AVP is synthesized in choroid plexus epithelium, which suggests autocrine and/or paracrine actions of this peptide in choroidal tissue.

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia.

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)-ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal-lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)-dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B-cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity in the immunolocalization of cytokeratin specific monoclonal antibodies in the rat eye: evaluation of unusual epithelial tissue entities

The immunocytochemical localization of cytokeratin and vimentin in rat eye tissues was investigated using a panel of 39 monoclonal antibodies specific for single or multiple of cytokeratin polypeptides and one polyclonal anti CK20 antiserum. The retinal and the ciliary body pigment epithelial only expressed cytokeratins 8 and 18, whereas the fetal retinal pigment epithelium and focally the adult epithelium, in the transition zone of retina and ciliary body, exhibited a reactivity for cytokeratin 19. In contrast, the non-pigmented ciliary epithelium was positive for vimentin only. In the rat conjunctiva distributed goblet cell clusters were selectively stained with cytokeratin 7, 8, 18 and 19 specific monoclonal antibodies. Among them a group of cytokeratin 8 and 18 specific monoclonal antibodies which stained the goblet cells as well as cytokeratin 8 and 18 positive internal controls did not react with either the cytokeratin 8 and 18 positive neuroectodermal cells of the rat eye nor the rat choroid plexus epithelium. This indicates differences in the phenotype e.g. conformational epitope changes, of neuroectodermal derived and other cytokeratins. The corneal and conjunctival epithelium showed a more complex distribution of squamous epithelium type cytokeratins. The limbal region as a transient zone connecting both epithelia exhibited a changing cytokeratin pattern. In general, the study emphasized the necessity to work with an enlarged antibody panel to avoid misleading results in the immunolocalization of cytokeratins.

Arginine vasopressin causes morphological changes suggestive of fluid transport in rat choroid plexus epithelium.

The experiments described herein use an in vitro preparation of choroid plexus to demonstrate that it is a vasopressin-responsive organ by morphologic criteria. Choroid plexus from rats was incubated for one hour in graded concentrations of arginine vasopressin (AVP). Within physiologic range of molar concentration, incubation in vasopressin induced a decrease in basal and lateral spaces in choroid plexus epithelial cells as well as an increase in number of dark cells. The number of cells with basal spaces decreased significantly from 82.7 +/- 9.2 in control tissue to 19 +/- 18 in tissue incubated in 10(-12) M AVP; similarly, the number with lateral cellular spaces decreased from 20 +/- 8.8 to 7.6 +/- 2.2 cells in 10(-10) M AVP. Dark cells increased in number from 3.8 +/- 2.6 in control conditions to 49 +/- 4 with 10(-9) M vasopressin. These data suggest important effects of arginine vasopressin in cerebrospinal fluid (CSF) on choroid plexus, compatible with enhanced fluid transport across choroid epithelial cells.

Intracellular pH.

Intracellular pH.

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.

Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell.

Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na+, K+ -ATPase (Masuzawa et al. 1980).

Differential effects of chloroquine and of several other amphiphilic cationic drugs upon rat choroid plexus

Several cationic amphiphilic drugs, each of which is known to induce generalized lipidosis in rats, were compared with respect to their cytological effects on rat choroid plexus epithelium. Chloroquine induced large cytoplasmic vacuoles, whereas the other drugs (quinacrine, 4,4'-diethylaminoethoxyhexestrol, chlorphentermine, iprindole, 1-chloro-amitriptyline, clomipramine) caused formation of lamellated or crystalloid inclusions as usually seen in drug-induced lipidosis. The ultrastructure of the chloroquine-induced vacuoles suggested storage of water-soluble materials (polar lipids and/or non-lipid materials) in addition to non-water-soluble polar lipids.

Horseradish peroxidase uptake into the rat choroid plexus epithelium, with special reference to the lysosomal system

Uptake of horseradish peroxidase (HRP), lysosome formation, and acid phosphatase (AcPhase) activity in the rat choroid plexus epithelium were studied. HRP was administered intravenously or intraventricularly for various times, and the choroid plexuses were fixed by ventriculo—cisternal perfusion of aldehydes. AcPhase reaction was performed both on control material and on choroid plexuses from the HRP experiments. The uptake of HRP from the cerebrospinal fluid (csf) side of the epithelium was, in general, more pronounced than that from the blood side. HRP became micropinocytozed and sequestered in a well-developed lysosomal apparatus situated in the apical half of the epithelial cells. The main pathway for endocytozed HRP was from micropinocytic vesicles to large endocytic vacuoles which may appear as multivesicular bodies. These eventually developed into dense bodies or dense multivesicular bodies, representing secondary lysosomes. HRP labeling was also seen in elongated tubular structures, which may be chains of fused micropinocytic vesicles or smooth endoplasmatic reticulum cisterns. AcPhase activity was located in distinct Golgi cisterns within the Golgi complexes. Small Golgi vesicles (primary lysosomes), some (dark) multivesicular bodies, and most dense bodies were also AcPhase positive. The AcPhase activity of HRP-exposed choroid plexus epithelium and of control specimens was largely the same. However, the number of AcPhase-positive dense bodies appeared to increase after prolonged administration of a large dose of HRP into the csf. The results provide evidence that the choroid plexus epithelium is able to remove protein from the ventricular system.

Microperoxidase uptake into the rat choroid plexus epithelium

Uptake, transport, and diffusion of the heme-peptide tracer microperoxidase (MP; MW 1900) in the rat choroid plexus were studied. MP was perfused ventriculo—cisternally or intravenously. The plexuses were fixed by ventriculo—cisternal perfusion of aldehydes, and tissue sections were incubated in a diaminobenzidine—H 2 O 2 medium containing imidazole at pH 8.8. After intravenous administration MP was found in the connective tissue of the plexus. Basement membranes were heavily labeled, while intercellular spaces between epithelial cells exhibited only a little MP. Some micropinocytic uptake of MP into vacuoles and lysosomes of the epithelial cells occurred. After ventriculo-cisternal perfusion of MP, it was micropinocytozed and incorporated into numerous vacuoles, multivesicular bodies, and dense bodies. Labeling of elongated cisterns (ER cisterns, or cisterns formed by fusion of endocytic vesicles) also occurred. Apical tight junctions between choroid plexus epithelial cells were never penetrated by MP. The intercellular spaces between epithelial cells, the connective tissue between epithelium and endothelium, and the blood vessels were, in general, devoid of MP, although diffusion of MP between ependymal cells into the extracellular space of the neuropil close to the base of the plexus was pronounced, as evaluated from the density of reaction product. No MP could be located in the kidney tubules of rats exposed ventriculo—cisternally to MP. The observations indicate that the choroid plexus is involved in absorption of molecules—also smaller than proteins—from the cerebrospinal fluid (csf). No significant diffusion of MP via ependyma and neuropil (the “functional leak”) into the plexus occurred, and no support of a transepithelial vesicular csf-to-blood transport of MP was obtained. This discrepancy in relation to previous horseradish peroxidase experiments is discussed.