Analysis of sigma receptor (sigmaR1) expression in retinal ganglion cells cultured under hyperglycemic conditions and in diabetic mice

The type 1 sigma receptor (sigmaR1) is a nonopiate and nonphencyclidine binding site that has numerous pharmacological and physiological functions. In some studies, agonists for sigmaR1 have been shown to afford neuroprotection against overstimulation of the NMDA receptor. sigmaR1 expression has been demonstrated recently in retinal ganglion cells (RGC). RGCs undergo apoptosis early in diabetic retinopathy via NMDA receptor overstimulation. In the present study we asked whether RGCs cultured under hyperglycemic conditions and RGCs of diabetic mice continue to express sigmaR1. RGCs were cultured 48 h in RPMI medium containing either 45 mM glucose or 11 mM glucose plus 34 mM mannitol (osmolar control). C57BL/6 mice were made diabetic using streptozotocin. The retina was dissected from normal and streptozotocin-induced diabetic mice 3, 6 and 12 weeks post-onset of diabetes. sigmaR1 was analyzed in cells using semiquantitative RT-PCR and in tissues by semiquantitative RT-PCR, in situ hybridization, Western blot analysis and immunolocalization. The RT-PCR analysis of cultured RGCs showed that sigmaR1 mRNA is expressed under hyperglycemic conditions at levels similar to control cells. Similarly, analysis of retinas of diabetic mice showed no difference in levels of mRNA encoding sigmaR1 compared to retinas of control mice. In situ hybridization analysis showed that expression patterns of sigmaR1 mRNA in the ganglion cell layer were similar between diabetic and control mice. Western blot analysis suggested that levels of sigmaR1 in retina were similar between diabetic and control retinas. Immunohistochemical analysis of sigmaR1 showed a similar pattern of sigmaR1 protein expression between control and diabetic retina. These studies demonstrate that sigmaR1 is expressed under hyperglycemic conditions in vitro and in vivo.     

Transcellular transfer of folate across the retinal pigment epithelium

The differential polarized distribution of the folate receptor alpha (FR alpha) in the basal membrane and reduced-folate transporter (RFT-1) in the apical membrane was demonstrated previously in the retinal pigment epithelium (RPE). Based on this, we hypothesized that folate would enter the RPE via FR alpha and exit the cell via RFT-1. To test this, we performed in vitro transport assays using ARPE-19 cells cultured on permeable supports. The cells were grown for 4 weeks and electron microscopic analysis indicated that the cells have the phenotypic features of normal RPE cells including apical microvillous processes and junctional complexes. Measurement of transepithelial resistance showed that the resistance increases in the cells as they differentiate over several weeks. Transport assays showed that ARPE-19 cells transport folate in a basal-to-apical direction, but do not transport this vitamin significantly in the apical-to basal-direction. This was not a diffusional process, as the paracellular markers inulin and sucrose were transferred across the cell monolayer at a much lower level. The presence of FR alpha in the basal membrane was demonstrable by folate binding and that of RFT-1 in the apical membrane by blockade of folate transport by RFT-1-specific antibody. This study represents the first in vitro demonstration of transcellular transfer of folate across RPE and suggests that folate is transported from the choriocapillaris to the adjacent photoreceptor cells in vivo by the concerted action of FR alpha in the basal membrane and RFT-1 in the apical membrane.     

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of β-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.     

High affinity electrophilic and photoactivatable covalent endocannabinoid probes for the CB1 receptor

We have designed and synthesized the first two high affinity covalent anandamide probes for the CB1 receptor by introducing either an electrophilic isothiocyanato or a photoactivatable azido group at the terminal carbon of the arachidonic acid moiety. The headgroup of these anandamide analogues was optimized by using a cyclopropylamide substituent to impart optimal CB1 affinity. Both 20-isothiocyanato-eicosa-5,8,11,14-tetraenoic acid cyclopropylamide (1, AM3677) and 20-azido-eicosa-5,8,11,14-tetraenoic acid cyclopropylamide (2, AM3661) exhibited high selectivities for the CB1 receptor with K(i) values of 1.3 and 0.9 nM, respectively. Using suitable experimental conditions, both ligands were shown to covalently label the CB1 receptor with high efficiency. These two covalent probes for the endocannabinoid CB1 binding site open the door for exploring the ligand binding motifs involved in the activation of the CB1 receptor by its endogenous ligand, anandamide.     

Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2)

Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrate-evoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2.Amino (and imino) acid transport via rPAT2 was pH-dependent, Na(+)-independent and electrogenic. At extracellular pH 5.5 (in Na(+)-free conditions) proline uptake was saturable (Km 172+/-41 muM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter.PAT2 preferred substrates are L-alpha-amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4- or 5-membered heterocyclic amino and imino acids such as 2-azetidine-carboxylate, proline and cycloserine, where both D- and L-enantiomers are transported.The major restrictions on transport are side chain size (the ethyl group of alpha-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH(2) groups, as in beta-alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O-methyl esters show either reduced transport (alanine-O-methyl ester) or are excluded.The structural characteristics that determine the substrate specificity of rPAT2 have been identified. This information should prove valuable in the design of selective substrates/inhibitors for PAT1 and PAT2.     

Carnitine content and expression of mitochondrial beta-oxidation enzymes in placentas of wild-type (OCTN2(+/+)) and OCTN2 Null (OCTN2(-/-)) Mice

Placenta requires energy to support its rapid growth, maturation, and transport function. Fatty acids are used as energy substrates in placenta, but little is known about the role played by carnitine in this process. We have investigated the role of carnitine in the expression of the enzymes involved in fatty acid beta-oxidation in placenta of OCTN2(-/-) mice with defective carnitine transporter (OCTN2). Heterozygous (OCTN2(+/-)) female mice were mated with heterozygous (OCTN2(+/-)) male mice. Pregnant mice were killed and fetuses and placentas were collected. Carnitine was measured using HPLC and tandem mass spectrometry. Immunohistochemistry was used to detect enzyme expression. Enzyme activities were measured spectrophotometrically. The fetal and placental weights were similar among the three genotypes (OCTN2(+/+), OCTN2(+/-), and OCTN2(-/-)). The levels of carnitine were markedly reduced (<20%) in homozygous OCTN2(-/-) null fetuses and placentas compared with wild-type OCTN2(+/+) controls. However, carnitine concentration in placenta was 2- to 7-fold higher than in the fetus in all three genotypes. Immunohistochemistry revealed that beta-oxidation enzymes are expressed in trophoblast cells. Catalytic activities of these enzymes were present at comparable levels in wild-type (OCTN2(+/+)) and homozygous (OCTN2(-/-)) mouse placentas, with the exception of SCHAD, for which activity was significantly higher in OCTN2(-/-) placentas than in OCTN2(+/+) placentas. These data show that placental OCTN2 is obligatory for accumulation of carnitine in placenta and fetus, that fatty acid beta-oxidation enzymes are expressed in placenta, and that reduced carnitine levels up-regulate the expression of SCHAD in placenta.     

The sigma receptor ligand (+)-pentazocine prevents apoptotic retinal ganglion cell death induced in vitro by homocysteine and glutamate

Recent studies demonstrated that the excitotoxic amino acid homocysteine induces apoptotic death of retinal ganglion cells in vivo. In the present study, an in vitro rat retinal ganglion cell (RGC-5), culture system was used to analyze the toxicity of acute exposure to high levels of homocysteine, the mechanism of homocysteine-induced toxicity, and the usefulness of type 1 sigma receptor (sigmaR1) ligands as neuroprotectants. When cultured RGC-5 cells were subjected to treatment with 1 mM D,L-homocysteine, a significant increase in cell death was detected by terminal dUTP nick end labeling (TUNEL) analysis and analysis of activated caspase. When cells were treated with homocysteine- or glutamate in the presence of MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) receptor, the cell death was inhibited significantly. In contrast, NBQX, an antagonist of the AMPA/Kainate receptor, and nifedipine, a calcium channel blocker, did not prevent the homocysteine- or glutamate-induced cell death. Semiquantitative RT-PCR and immunocytochemical analysis demonstrated that RGC-5 cells were exposed to homocysteine or glutamate express type 1 sigma receptor at levels similar to control cells. Treatment of RGC-5 cells with 3 or 10 microM concentrations of the sigmaR1-specific ligand (+)-pentazocine inhibited significantly the apoptotic cell death induced by homocysteine or glutamate. The results suggest that homocysteine is toxic to ganglion cells in vitro, that the toxicity is mediated via NMDA receptor activation, and that the sigmaR1-specific ligand (+)-pentazocine can block the RGC-5 cell death induced by homocysteine and glutamate.     

Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines

PURPOSE: Efflux pumps such as P-glycoprotein (P-gp; MDR1) are believed to be a major barrier to drug delivery. The purpose of this work was to determine whether cornea and corneal epithelial cells expresses the functionally active P-gp efflux pump. METHOD: Cultured rabbit primary corneal epithelial cells (rPCECs) and a corneal cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) were selected as the model. Rhodamine-123 (Rho-123), a P-gp substrate, was used as a P-gp probe. To confirm gene expression, RT-PCR was performed with appropriate pairs of primers for rabbit and human MDR1. Subcloning, sequencing, and protein sequence determination were performed to confirm P-gp. RESULTS: Permeability of [(3)H] cyclosporin A (CsA) across SIRC cells was found at 1.74 x 10(-6) cm/s in the apical-to-basolateral and 5.1 x 10(-6) cm/s in the basolateral-to-apical directions. Uptake of Rho-123 across both SIRC cells and rPCECs was time and temperature dependent. Rho-123 uptake in SIRC cells was 14.4 picomoles/mg protein and in the presence of CsA (10 micro M) was 70.8 picomoles/mg protein at 3 hours. Uptake in rPCECs was the highest at 3 hours. Western blot analysis indicated a 170-kDa band confirming the presence of P-gp. Human cornea was also checked for the presence of P-gp. RT-PCR data indicated one single band, which was subcloned and sequenced to confirm the presence of P-gp. The protein sequence deduced from the fragment product indicated more than 89% homology with human MDR1. CONCLUSIONS: Functional and molecular characterization showed the existence of P-gp in human cornea, rabbit cornea, and a rabbit corneal cell line. This knowledge of the existence of P-gp will help in development of better ocular drug delivery strategies.     

Inhibition of system A amino acid transport activity by ethanol in BeWo choriocarcinoma cells

OBJECTIVE: Our purpose was to investigate the influence of ethanol on system A amino acid transporter in BeWo cells. STUDY DESIGN: BeWo cells were cultured in the absence or presence of ethanol. The function of system A was monitored by the transport of alpha-(methylamino)isobutyric acid. Messenger RNA levels for system A were assessed by Northern analysis. RESULTS: Treatment of BeWo cells with ethanol reduced the activity of system A. The effect was dose and treatment time dependent. The decrease in system A activity was 38% +/- 3% at 0.75% ethanol with a 16-hour treatment time. The activities of several other transporters tested were not affected. The effect on system A activity was associated with a decrease in the maximal velocity of the transport system without affecting the substrate affinity. Ethanol did not alter the messenger RNA levels for system A. CONCLUSION: Exposure of BeWo cells to ethanol significantly reduces the function of system A. This finding has potential implications that may be relevant to the pathogenesis of the fetal alcohol syndrome.