Ophthalmic Nonsteroidal Anti-Inflammatory Drugs as a Therapy for Corneal Dystrophies Caused by SLC4A11 Mutation.

Purpose: SLC4A11 is a plasma membrane protein of corneal endothelial cells. Some mutations of the SLC4A11 gene result in SLC4A11 protein misfolding and failure to mature to the plasma membrane. This gives rise to some cases of Fuchs' endothelial corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED). We screened ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs) for their ability to correct SLC4A11 folding defects. Methods: Five ophthalmic NSAIDs were tested for their therapeutic potential in some genetic corneal dystrophy patients. HEK293 cells expressing CHED and FECD-causing SLC4A11 mutants were grown on 96-well dishes in the absence or presence of NSAIDs. Ability of NSAIDs to correct mutant SLC4A11 cell-surface trafficking was assessed with a bioluminescence resonance energy transfer (BRET) assay and by confocal microscopy. The ability of mutant SLC4A11-expressing cells to mediate water flux (SLC4A11 mediates water flux across the corneal endothelial cell basolateral membrane as part of the endothelial water pump) was measured upon treatment with ophthalmic NSAIDs. Results: BRET-assays revealed significant rescue of SLC4A11 mutants to the cell surface by 4 of 5 NSAIDs tested. The NSAIDs, diclofenac and nepafenac, were effective in moving endoplasmic reticulum-retained missense mutant SLC4A11 to the cell surface, as measured by confocal immunofluorescence. Among intracellular-retained SLC4A11 mutants, 20 of 30 had significant restoration of cell surface abundance upon treatment with diclofenac. Diclofenac restored mutant SLC4A11 water flux activity to the level of wild-type SLC4A11 in some cases. Conclusions: These results encourage testing diclofenac eye drops as a treatment for corneal dystrophy in patients whose disease is caused by some SLC4A11 missense mutations.

Cortical distal nephron Cl(-) transport in volume homeostasis and blood pressure regulation.

Renal intercalated cells mediate the secretion or absorption of Cl(-) and OH(-)/H(+) equivalents in the connecting segment (CNT) and cortical collecting duct (CCD). In so doing, they regulate acid-base balance, vascular volume, and blood pressure. Cl(-) absorption is either electrogenic and amiloride-sensitive or electroneutral and thiazide-sensitive. However, which Cl(-) transporter(s) are targeted by these diuretics is debated. While epithelial Na(+) channel (ENaC) does not transport Cl(-), it modulates Cl(-) transport probably by generating a lumen-negative voltage, which drives Cl(-) flux across tight junctions. In addition, recent evidence indicates that ENaC inhibition increases electrogenic Cl(-) secretion via a type A intercalated cells. During ENaC blockade, Cl(-) is taken up across the basolateral membrane through the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and then secreted across the apical membrane through a conductive pathway (a Cl(-) channel or an electrogenic exchanger). The mechanism of this apical Cl(-) secretion is unresolved. In contrast, thiazide diuretics inhibit electroneutral Cl(-) absorption mediated by a Na(+)-dependent Cl(-)/HCO3(-) exchanger. The relative contribution of the thiazide and the amiloride-sensitive components of Cl(-) absorption varies between studies and probably depends on the treatment model employed. Cl(-) absorption increases markedly with angiotensin and aldosterone administration, largely by upregulating the Na(+)-independent Cl(-)/HCO3(-) exchanger pendrin. In the absence of pendrin [Slc26a4((-/-)) or pendrin null mice], aldosterone-stimulated Cl(-) absorption is significantly reduced, which attenuates the pressor response to this steroid hormone. Pendrin also modulates aldosterone-induced changes in ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function, at least in part, by altering luminal HCO3(-). This review summarizes mechanisms of Cl(-) transport in CNT and CCD and how these transporters contribute to the regulation of extracellular volume and blood pressure.

Capsaicinoids regulate airway anion transporters through Rho kinase- and cyclic AMP-dependent mechanisms.

To investigate the effects of capsaicinoids on airway anion transporters, we recorded and analyzed transepithelial currents in human airway epithelial Calu-3 cells. Application of capsaicin (100 µM) attenuated vectorial anion transport, estimated as short-circuit currents (I(SC)), before and after stimulation by forskolin (10 µM) with concomitant reduction of cytosolic cyclic AMP (cAMP) levels. The capsaicin-induced inhibition of I(SC) was also observed in the response to 8-bromo-cAMP (1 mM, a cell-permeable cAMP analog) and 3-isobutyl-1-methylxanthine (1 mM, an inhibitor of phosphodiesterases). The capsaicin-induced inhibition of I(SC) was attributed to suppression of bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters)-sensitive components, which reflect anion uptake via basolateral cAMP-dependent anion transporters. In contrast, capsaicin potentiated apical Cl(-) conductance, which reflects conductivity through the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl(-) channel. All these paradoxical effects of capsaicin were mimicked by capsazepine. Forskolin application also increased phosphorylated myosin phosphatase target subunit 1, and the phosphorylation was prevented by capsaicin and capsazepine, suggesting that these capsaicinoids assume aspects of Rho kinase inhibitors. We also found that the increments in apical Cl(-) conductance were caused by conventional Rho kinase inhibitors, Y-27632 (20 µM) and HA-1077 (20 µM), with selective inhibition of basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1. Collectively, capsaicinoids inhibit cAMP-mediated anion transport through down-regulation of basolateral anion uptake, paradoxically accompanied by up-regulation of apical cystic fibrosis transmembrane conductance regulator-mediated anion conductance. The latter is mediated by inhibition of Rho-kinase, which is believed to interact with actin cytoskeleton.

High-affinity nitrate uptake by rice (Oryza sativa) coleoptiles.

Nitrate uptake by rice coleoptiles was evaluated using ¹5N-nitrate in relation to the expression of high-affinity nitrate uptake-related genes, OsNRT2s (OsNRT2.1-2.4) and OsNAR2s (OsNAR2.1 and 2.2). Apparent nitrate uptake by coleoptiles was about one-sixth of that by hydroponically cultured seedling roots. Real-time RT-PCR analysis revealed that OsNRT2.1, a root-specific key gene of inducible high-affinity transport system for nitrate, was most strongly induced in coleoptiles following nitrate supply initiation, while other OsNRT2s and OsNAR2s showed modest induction. These results suggest that rice coleoptiles may have high-affinity transport systems for nitrate similar to roots, and can be model organs for nutrient uptake by submerged plant shoots.

Functional expression and microdomain localization of prestin in cultured cells.

INTRODUCTION: Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. METHODS: We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. RESULTS & DISCUSSION: Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. CONCLUSION: Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.

Identification and characterization of a nitrate transporter gene in Neurospora crassa.

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.

Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells.

Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl(-)- and K(+)-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of approximately 30-60 s, and its activity persists for at least 8-12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21(rho), or phosphatidylinositol 3-kinase activity and was insensitive to Cl(-) channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca(2+) by using A-23187- or Ca(2+)-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.

Properties of the vertebrate skeletal muscle tubular system as a sealed compartment.

Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t-) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t-system upon mechanical skinning, the time course of dye loss from the sealed t-system in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t-system. In contrast to intact fibres, which have a t-system open to the outside, the sealed t-system of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t-system of rat-skinned fibres was infrequent. Application and removal of 200-400 mM glycerol to the sealed t-system did not produce any obvious changes in its morphology. The dyes fluo-3, fura-2 and Oregon green 488 were lost from the sealed t-system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo-3 and fura-2 loss from the sealed t-system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t-system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad-skinned fibres and lack of vesicle formation in rat-skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres.

Independent induction of two blue light-dependent monovalent anion transport systems in the plasma membrane of Monoraphidium braunii.

In the plasma membrane of the green alga Monoraphidium braunii there are at least two monovalent anion transport systems. One of them is specific for bicarbonate. This transport system is activated by blue light and its induction is triggered by a decrease in the external CO2 concentration. The second transport system is responsible for nitrate uptake at least. This transport system is also activated by blue light and its induction occurs when there is no ammonium in the external medium. Both transport systems are synthesized independently. Hence, when M. braunii cells grow with nitrate as the only nitrogen source under high CO2, they have a nitrate transport system but lack a bicarbonate transporter. Conversely, cells grown with ammonium under low CO2, have a bicarbonate transport system but lack a nitrate transporter. Both transport systems are induced in cells irradiated with white light in the absence of a carbon source, suggesting that there may be precursors in the plasma membrane that only need the synthesis and assembly of some component(s) to become fully active. The induction of nitrate and nitrite reductases, however, only takes place when a carbon source is supplied to the cells.

Effects of nitrate pulses on BnNRT1 and BnNRT2 genes: mRNA levels and nitrate influx rates in relation to the duration of N deprivation in Brassica napus L.

A de-repression mechanism based on the disappearance of 'signals' down-regulating N transporter activity has been proposed in the literature to explain the transient increase of NO(3)(-) uptake by the roots following N deprivation in higher plants. This hypothesis was investigated at the physiological and molecular levels by measuring NO(3)(-) influx into roots of Brassica napus L. grown under low or high external concentrations of KNO(3) following N deprivation. Parallel measurements were made of endogenous NO(3)(-), amino acid concentrations and abundance of mRNA for BnNRT1 and BnNRT2, genes encoding nitrate-inducible transport proteins. The effect of NO(3)(-) pulsing on NO(3)(-) transport components in N-deprived plants was also investigated by measuring influx of high- and low-affinity transport system (HATS and LATS) and assaying mRNA levels. Influx of NO(3)(-) via HATS and LATS, and transcript levels of BnNRT2 and BnNRT1 decreased with the duration of N deprivation. The results suggested that the absence of de-repression of NO(3)(-) influx and BnNRT2 gene expression following N starvation was related to a high amino acid status. Pulsing with NO(3)(-) induced a large increase in BnNRT2 mRNA level, but a comparatively small increase in NO(3)(-) influx via HATS. The level of BnNRT1 mRNA also increased, but there was no effect on LATS uptake activity. The absence of a strict correlation between the NO(3)(-) transport activity and the mRNA BnNRT1 and BnNRT2 levels is discussed in terms of possible post-transcriptional regulation by the amino acids.

Experimental therapeutics with a new 10-deazaaminopterin in human mesothelioma: further improving efficacy through structural design, pharmacologic modulation at the level of MRP ATPases, and combined therapy with platinums.

Studies described here sought to evaluate the therapeutic potential of a new 10-deazaaminopterin analogue, 10-propargyl-10-deazaaminopterin (PDX), alone and in combination with platinum compounds in the treatment of human pleural mesothelioma. In vitro studies documented 25-30-fold and 3-fold, respectively, greater cytotoxic potency of PDX compared with methotrexate and another 10-deazaaminopterin, edatrexate, against VAMT-1 and JMN cell lines derived from human mesothelioma. These tumor cell lines were also inhibited by platinum compounds. Cisplatin (CDDP) was somewhat more inhibitory than oxaloplatin and >1 log order in magnitude more inhibitory than carboplatin (CBCDA). Against the JMN tumor xenografted in nude mice, whereas methotrexate and, more so, edatrexate, were potently growth inhibitory, only PDX brought about substantial regression. By comparison, CDDP and CBCDA, but not oxaloplatin were markedly growth inhibitory to this same tumor in vivo. This high level of therapeutic activity of PDX could be additionally enhanced by coadministration of probenecid, an inhibitor of canicular multispecific organic anion transporter/multidrug resistance-related protein (MRP)-like ATPases, which increased the number of complete regressions by >-3 fold. Canicular multispecific organic anion transporter/MRP genes, primarily 1, 3, 4, 5, and 7, were in fact expressed in these human mesothelioma cell lines as determined by real-time reverse transcription-PCR. These same MRP genes, including, to a lesser extent, MRP-4, were also expressed in pleural mesotheliomas derived from patients as shown by the same methodology. When combined with CDDP or CBCDA, PDX achieved 2-fold greater overall regression of the JMN tumor with a 3-4-fold increase in complete regressions, although some attenuation of dosages of each were required in the combination. These results strongly suggest that PDX has significant potential in the treatment of human pleural mesothelioma, particularly when coadministered with probenecid or combined with platinum compounds.

Effect of S20787, a novel Cl--HCO3- exchange inhibitor, on intracellular pH regulation in guinea pig ventricular myocytes.

S20787 has recently been proposed to be a selective Cl--HCO3- anion exchange (AE) inhibitor in rat cardiomyocytes. The AE transporter mediates sarcolemmal acid influx but is only one part of the cardiac cell's dual acid loading mechanism, the other part being a sarcolemmal Cl--OH- exchanger (CHE). We have therefore (1) investigated the differential effects of S20787 on the AE and CHE transporters in isolated guinea pig ventricular myocytes and (2) re-examined the influence of the drug on other sarcolemmal acid transporters by monitoring its effect on intracellular pH (pH(i)) recovery from alkali or acid loads. The pH(i) was measured using microspectrofluorimetry (carboxy-SNARF-1). The results indicate that CHE activity was unaffected by the drug (1-20 microM), whereas up to 78% of AE activity was blocked (K(i) = 3.9 microM). Thus, S20787 targets only the AE component of the dual acid influx system. Activities of other acid-transporting carriers, such as Na+-H+ exchange, Na+-HCO3- co-transport and the monocarboxylic acid transporter, were unaffected by the drug. The inhibitory efficacy of S20787 for AE in guinea pig cardiomyocytes appears to be considerably higher (approximately 78%) than proposed previously for rat cardiomyocytes (50%). This is most likely because, in both cells, a significant fraction (20-30%) of acid influx is mediated through the S20787-insensitive CHE transporter. Previous studies made no allowance for the CHE component, which would result in an underestimation. S20787 is thus a highly selective AE inhibitor which may be useful as an experimental tool and a potential cardiac protective agent in the heart.