Expression of monocarboxylate transporters in rat ocular tissues

The aim of the present study was to determine the distribution of monocarboxylate transporter (MCT) subtypes 1-4 in the various structures of the rat eye by using a combination of conventional and real-time RT-PCR, immunoblotting, and immunohistochemistry. Retinal samples expressed mRNAs encoding all four MCTs. MCT1 immunoreactivity was observed in photoreceptor inner segments, Müller cells, retinal capillaries, and the two plexiform layers. MCT2 labeling was concentrated in the inner and outer plexiform layers. MCT4 immunolabeling was present only in the inner retina, particularly in putative Müller cells, and the plexiform layers. No MCT3 labeling could be observed. The retinal pigment epithelium (RPE)/choroid expressed high levels of MCT1 and MCT3 mRNAs but lower levels of MCT2 and MCT4 mRNAs. MCT1 was localized to the apical and MCT3 to the basal membrane of the RPE, whereas MCT2 staining was faint. Although MCT1-MCT4 mRNAs were all detectable in iris and ciliary body samples, only MCT1 and MCT2 proteins were expressed. These were present in the iris epithelium and the nonpigmented epithelium of the ciliary processes. MCT4 was localized to the smooth muscle lining of large vessels in the iris-ciliary body and choroid. In the cornea, MCT1 and MCT2 mRNAs and proteins were detectable in the epithelium and endothelium, whereas evidence was found for the presence of MCT4 and, to a lesser extent, MCT1 in the lens epithelium. The unique distribution of MCT subtypes in the eye is indicative of the pivotal role that these transporters play in the maintenance of ocular function. retina; eye; immunohistochemistry; polymerase chain reaction     

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.     

Effects of the extracellular matrix on fetal choroid plexus epithelial cells: changes in morphology and multicellular organization do not affect gene expression.

We have developed a primary culture system for fetal mouse choroid plexus epithelial cells which maintains their differentiated phenotype. When grown on a reconstituted basement membrane substrate (Matrigel) epithelial cells formed aggregates which became embedded in the matrix and developed into characteristic and highly reproducible multicellular vesicular structures. These vesicles consisted of a squamous layer of epithelial cells with extensive attachment to the matrix substrate, surrounding a fluid-filled lumen. Electron microscopy showed that cells comprising these vesicles had a high degree of membrane specialization and polarized morphology which in many respects mimicked the in vivo morphology. Biochemical analyses demonstrated that under these culture conditions the tissue-specific pattern of gene expression of fetal choroid plexus epithelium was maintained. After 6 days in culture these cells contained approximately the same amount of transthyretin mRNA as the 12.5-day choroid plexus in vivo, and the level of total RNA per cell, which is proportional to the protein synthetic capability of the cells, was also maintained. The pattern of protein secretion was also very similar to that generated by fetal mouse choroid plexus cells in vivo. In contrast choroid plexus epithelial cells attached poorly to collagen I gels. Heterogeneous aggregates were formed in which cell-cell interactions were more extensive than cell-substrate interactions, and in no cases was a central lumen observed. Cells on the surface of large aggregates showed some evidence of membrane polarization, while the majority of cells in the cultures exhibited little evidence of polarized morphology. Despite the striking difference in morphology and multicellular organization these cells still expressed high levels of transthyretin mRNA and maintained the same pattern of protein synthesis as cells cultured on Matrigel. These results indicate that the basement membrane is important for the organization of choroid plexus epithelial cells into a functional epithelium in vitro and thus presumably the maintenance of the integrity of the blood-brain barrier in vivo. In contrast to several other epithelial systems which have been studied, the type of extracellular matrix does not appear to directly influence tissue-specific gene expression by choroid plexus epithelial cells. Thus the level of gene expression is not dependent on the cytoarchitecture and multicellular organization of this cell type.     

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.     

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.     

Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.     

Molecular expression of SLC4-derived Na+-dependent anion transporters in selected human tissues

NaHCO(3) transporters are involved in maintenance of intracellular pH and transepithelial HCO(3)(-) movement in many rodent tissues. To establish the human relevance of the many investigations on rodents, this study aimed to map these transporters and a related polypeptide, NaBC1 [solute carrier 4 (SLC4)A11], to several human tissues by using PCR on reverse transcribed human mRNA and immunoperoxidase histochemistry. The mRNA encoding the electroneutral Na(+):HCO(3)(-) cotransporter (NBCe1; SLC4A4), was expressed in renal cortex, renal medulla, stomach, duodenum, jejunum, ileum, colon, pancreas, choroid plexus, cerebellum, cerebrum, and hippocampus. NBCe2 (SLC4A5) and NBCn1 (SLC4A7) mRNAs were mainly found in kidney and brain tissues, as was mRNA encoding the Na(+)-dependent anion exchangers NCBE (SLC4A10) and NDCBE1 (SLC4A8). In addition to previous findings, NBCn1 protein was localized to human renal medullary thick ascending limbs and duodenal epithelial villus cells and NBCe2 protein to renal collecting ducts. Finally, the message encoding NaBC1 was found in kidney, stomach, duodenum, pancreas, and brain, and the corresponding protein in the anterior and posterior corneal epithelia, renal corpuscules, proximal tubules, collecting ducts, pancreatic ducts, and the choroid plexus epithelium. In conclusion, the selected human tissues display distinct expression patterns of HCO(3)(-) transporters, which closely resemble that of rodent tissues.     

Patterning the optic neuroepithelium by FGF signaling and Ras activation.

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.     

Cellular expression of monocarboxylate transporters in the female reproductive organ of mice: implications for the genital lactate shuttle

The present study examined the cellular localization of monocarboxylate transporters (MCTs), glucose transporters (GLUTs), and some glycolysis-related molecules in the murine female genital tract to demonstrate existence of lactate/pyruvate-dependent energy systems. MCT1, a major MCT subtype, was localized selectively in the ovarian granulosa, oviductal-ciliated cells, and vaginal epithelium; all localizations were associated with intense expressions of glycolytic enzymes. MCT1 was localized in the cell membrane of granulosa cells, including fine processes extending from cumulus cells toward oocytes. The cumulus cells and oocytes showed intense signals for lactate dehydrogenase (LDH)-A and -B, respectively. The basolateral membrane of oviductal-ciliated cells expressed MCT4 as well as MCT1, while adjacent non-ciliated cells contained an intense immunoreactivity for aldolase-C, a glycolytic enzyme. The expression of GLUTs in the ovary was generally weak with an intense expression of GLUT1 only in some vascular endothelia. The oviductal epithelium expressed GLUT1 and GLUT3, respectively, in the basolateral and apical membrane of non-ciliated cells. In the vagina, the basal layers of epithelium were immunolabeled for MCT1 with the entire length of cell membrane, and expressed abundantly both GLUT1 and LDH-A. The findings correspond well with the rich existence of lactate in the genital fluids and strongly suggest the active transport of lactate/pyruvate in the female reproductive tract, which provides favorable conditions for oocytes, sperms, and zygotes.     

24S-hydroxycholesterol induces cholesterol release from choroid plexus epithelial cells in an apical- and apoE isoform-dependent manner concomitantly with the induction of ABCA1 and ABCG1 expression

The release of cholesterol from choroid plexus epithelial cells (CPE) plays an important role in cholesterol homeostasis in the CSF. The purpose of this study was to clarify the molecules involved in cholesterol release in CPE and the regulation mechanisms of the cholesterol release by the liver X receptor (LXR) using a conditionally immortalized CPE line (TR-CSFB3). The mRNA expression of LXRalpha, LXRbeta and their target genes, ATP-binding cassette transporter (ABC)A1, ABCG1, ABCG4 and ABCG5, were detected in rat choroid plexus. ABCA1 and ABCG1 protein were detected in the plasma membrane of TR-CSFB3 cells. Following treatment with 24S-hydroxycholesterol, an endogenous LXR ligand, the expression of ABCA1 and ABCG1 were induced in TR-CSFB3 cells. Moreover, apolipoprotein (apo)AI- and high-density lipoprotein (HDL)-mediated cholesterol release to the apical side of TR-CSFB3 cells was facilitated by this treatment, whereas that to the basal side was not affected. Following 24S-hydroxycholesterol treatment, apoE3-dependent cholesterol release from TR-CSFB3 cells was enhanced more than the apoE4-dependent release. These results suggest that LXR activation facilitates cholesterol release into the CSF from CPE through the functional induction of ABCA1 and ABCG1. The difference between apoE3 and apoE4 suggests that the cholesterol release from CPE is related to the development of neurodegenerative diseases.