Mechanical Stretch Activates TRPV4 and Hemichannel Responses in the Nonpigmented Ciliary Epithelium.

Previously, we reported a mechanosensitive ion channel, TRPV4, along with functional connexin hemichannels on the basolateral surface of the ocular nonpigmented ciliary epithelium (NPE). In the lens, TRPV4-mediated hemichannel opening is part of a feedback loop that senses and respond to swelling. The present study was undertaken to test the hypothesis that TRPV4 and hemichannels in the NPE respond to a mechanical stimulus. Porcine NPE cells were cultured on flexible membranes to study effects of cyclic stretch and ATP release was determined by a luciferase assay. The uptake of propidium iodide (PI) was measured as an indicator of hemichannel opening. NPE cells subjected to cyclic stretch for 1-10 min (10%, 0.5 Hz) displayed a significant increase in ATP release into the bathing medium. In studies where PI was added to the bathing medium, the same stretch stimulus increased cell PI uptake. The ATP release and PI uptake responses to stretch both were prevented by a TRPV4 antagonist, HC067047 (10 µM), and a connexin mimetic peptide, Gap 27 (200µm). In the absence of a stretch stimulus, qualitatively similar ATP release and PI uptake responses were observed in cells exposed to the TRPV4 agonist GSK1016790A (10 nM), and Gap 27 prevented the responses. Cells subjected to an osmotic swelling stimulus (hypoosmotic medium: 200 mOsm) also displayed a significant increase in ATP release and PI uptake and the responses were abolished by TRPV4 inhibition. The findings point to TRPV4-dependent connexin hemichannel opening in response to mechanical stimulus. The TRPV4-hemichannel mechanism may act as a mechanosensor that facilitates the release of ATP and possibly other autocrine or paracrine signaling molecules that influence fluid (aqueous humor) secretion by the NPE.

Activation of Piezo1 Increases Na,K-ATPase-Mediated Ion Transport in Mouse Lens.

Lens ion homeostasis depends on Na,K-ATPase and NKCC1. TRPV4 and TRPV1 channels, which are mechanosensitive, play important roles in mechanisms that regulate the activity of these transporters. Here, we examined another mechanosensitive channel, piezo1, which is also expressed in the lens. The purpose of the study was to examine piezo1 function. Recognizing that activation of TRPV4 and TRPV1 causes changes in lens ion transport mechanisms, we carried out studies to determine whether piezo1 activation changes either Na,K-ATPase-mediated or NKCC1-mediated ion transport. We also examined channel function of piezo1 by measuring calcium entry. Rb uptake was measured as an index of inwardly directed potassium transport by intact mouse lenses. Intracellular calcium concentration was measured in Fura-2 loaded cells by a ratiometric imaging technique. Piezo1 immunolocalization was most evident in the lens epithelium. Potassium (Rb) uptake was increased in intact lenses as well as in cultured lens epithelium exposed to Yoda1, a piezo1 agonist. The majority of Rb uptake is Na,K-ATPase-dependent, although there also is a significant NKCC-dependent component. In the presence of ouabain, an Na,K-ATPase inhibitor, Yoda1 did not increase Rb uptake. In contrast, Yoda1 increased Rb uptake to a similar degree in the presence or absence of 1 µM bumetanide, an NKCC inhibitor. The Rb uptake response to Yoda1 was inhibited by the selective piezo1 antagonist GsMTx4, and also by the nonselective antagonists ruthenium red and gadolinium. In parallel studies, Yoda1 was observed to increase cytoplasmic calcium concentration in cells loaded with Fura-2. The calcium response to Yoda1 was abolished by gadolinium or ruthenium red. The calcium and Rb uptake responses to Yoda1 were absent in calcium-free bathing solution, consistent with calcium entry when piezo1 is activated. Taken together, these findings point to stimulation of Na,K-ATPase, but not NKCC, when piezo1 is activated. Na,K-ATPase is the principal mechanism responsible for ion and water homeostasis in the lens. The functional role of lens piezo1 is a topic for further study.

Terbutaline, forskolin and cAMP reduce secretion of aqueous humour in the isolated bovine eye.

In order to elucidate involvement of cyclic AMP and intracellular Ca2+,[Ca2+]i, in the modulation of aqueous humour formation (AHF), we studied the effects of terbutaline, forskolin and 8-Br-cAMP in the isolated bovine eye. We also studied the interaction of cAMP on calcium signaling in cultured ciliary epithelial (CE) cells. Drug effects on AHF were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the fura-2 fluorescence ratio technique. Terbutaline (100 nmol-100 M), forskolin (30 nM-100 M) or 8-Br-cAMP (100 nM- 10 µM), administered in the arterial perfusate produced significant reductions in AHF. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT-5720). ATP (100 M) caused a rapid, transient (peak) increase in [Ca2+]i followed by a sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8-Br-cAMP significantly reduced the peak calcium response to ATP. The sustained plateau phase of the response, on the other hand, was augmented by each of the agents. KT-5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. These data indicate: (a) that AHF in the bovine eye can be manipulated through cyclic AMP, operating via protein kinase A, (b) that protein kinase A can affect [Ca2+]i homeostasis, (c) that calcium release from the intracellular store, not the entry, affects AHF, and (d) that interaction of [Ca2+]i with cAMP plays a role in modulating AH secretion.

Correcting deregulated Fxyd1 expression rescues deficits in neuronal arborization and potassium homeostasis in MeCP2 deficient male mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the MECP2 gene. In the absence of MeCP2, expression of FXYD domain-containing transport regulator 1 (FXYD1) is deregulated in the frontal cortex (FC) of mice and humans. Because Fxyd1 is a membrane protein that controls cell excitability by modulating Na+, K+-ATPase activity (NKA), an excess of Fxyd1 may reduce NKA activity and contribute to the neuronal phenotype of Mecp2 deficient (KO) mice. To determine if Fxyd1 can rescue these RTT deficits, we studied the male progeny of Fxyd1 null males bred to heterozygous Mecp2 female mice. Maximal NKA enzymatic activity was not altered by the loss of MeCP2, but it increased in mice lacking one Fxyd1 allele, suggesting that NKA activity is under Fxyd1 inhibitory control. Deletion of one Fxyd1 allele also prevented the increased extracellular potassium (K+) accumulation observed in cerebro-cortical neurons from Mecp2 KO animals in response to the NKA inhibitor ouabain, and rescued the loss of dendritic arborization observed in FC neurons of Mecp2 KO mice. These effects were gene-dose dependent, because the absence of Fxyd1 failed to rescue the MeCP2-dependent deficits, and mimicked the effect of MeCP2 deficiency in wild-type animals. These results indicate that excess of Fxyd1 in the absence of MeCP2 results in deregulation of endogenous K+ conductances functionally associated with NKA and leads to stunted neuronal growth.

TRPV1-dependent ERK1/2 activation in porcine lens epithelium.

Recently we determined that the Transient Receptor Potential Vanilloid 4 ion channel (TRPV4) has a crucial signaling role in a pathway that regulates various aspects of lens epithelium function. Here, we report on a different TRPV channel, TRPV1, in porcine lens. The presence of TRPV1 in the lens was evident from RT-PCR studies and Western blot analysis of MAPK signaling pathway activation caused by the TRPV1 agonist capsaicin. TRPV1 mRNA was detected in the epithelium of porcine as well as human lens. Transient ERK1/2 and p38 MAPK phosphorylation was detected within 1 min in the epithelium isolated from intact porcine lenses exposed to capsaicin (100 nM), a selective TRPV1 agonist, and the response was significantly inhibited by A889245 (1.0 µM), a TRPV1 antagonist. A similar ERK 1/2 and p38 response in the epithelium, also inhibitable by A889245, was evident in lenses treated with hyperosmotic solution (350 vs 300 mOsm). Lenses pre-treated with either the cytosolic Ca2+ chelator BAPTA-AM or the PKC inhibitor sotrastaurin (1.0 µM) had a diminished ERK1/2 activation response to capsaicin and hyperosmotic solution. Taken together the findings support the notion that TRPV1 functions as a plasma membrane ion channel that, when activated, permits the entry of extracellular calcium into the lens epithelium, leading to activation of PKC, ERK1/2 and p38 MAPK. It is significant that the findings confirm earlier proposals that hyperosmotic stress is linked to TRPV1 channel activation in the mouse lens. Further studies are ongoing to determine what functional changes are triggered by the TRPV1-linked signaling pathways and how they might relate to lens volume homeostasis.

Body mass-specific Na+-K+-ATPase activity in the medullary thick ascending limb: implications for species-dependent urine concentrating mechanisms.

In general, the mammalian whole body mass-specific metabolic rate correlates positively with maximal urine concentration (Umax) irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass-specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na+-K+-ATPase activity and Na+-K+-ATPase α1-gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na+-K+-ATPase activity (~70%) compared with two rat strains (Sprague-Dawley and Munich-Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na+-K+-ATPase α1-protein (~4- to 6-fold) and mRNA (~13-fold) and higher TAL mitochondrial volume density (~20%) occur in the kangaroo rat compared with both rat strains. Rat TAL Na+-K+-ATPase α1-protein expression is relatively unaffected by body hydration status or, shown previously, by dietary Na+, arguing against confounding effects from two unavoidably dissimilar diets: grain-based diet without water (kangaroo rat) or grain-based diet with water (rat). We conclude that higher TAL Na+-K+-ATPase activity contributes to relationships between whole body mass-specific metabolic rate and high Umax. More vigorous TAL Na+-K+-ATPase activity in kangaroo rat than rat may contribute to its steeper Na+ and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

A Role for Calcium-Activated Adenylate Cyclase and Protein Kinase A in the Lens Src Family Kinase and Na,K-ATPase Response to Hyposmotic Stress.

Purpose: Na,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response. Methods: Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na,K-ATPase activity. Results: An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na,K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na,K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium. Conclusions: Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na,K-ATPase activity.

Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K-ATPase in Nonpigmented Ciliary Epithelium.

Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2-fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2, and p38 activation, all of which were prevented by a pI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates pI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. J. Cell. Physiol. 232: 1489-1500, 2017. © 2016 Wiley Periodicals, Inc.

A renal-like organic anion transport system in the ciliary epithelium of the bovine and human eye.

The purpose of this study was to determine the direction of organic anion (OA) transport across the ciliary body and the transport proteins that may contribute. Transport of several OAs across the bovine ciliary body was examined using ciliary body sections mounted in Ussing chambers and a perfused eye preparation. Microarray, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to examine OA transporter expression in human ocular tissues. Microarray analysis showed that many OA transporters common to other barrier epithelia are expressed in ocular tissues. mRNA (RT-PCR) and protein (immunoblotting) for OAT1, OAT3, NaDC3, and MRP4 were detected in extracts of the human ciliary body from several donors. OAT1 and OAT3 localized to basolateral membranes of nonpigmented epithelial cells and MRP4 to basolateral membranes of pigmented cells in the human eye. Para-aminohippurate (PAH) and estrone-3-sulfate transport across the bovine ciliary body in the Ussing chambers was greater in the aqueous humor-to-blood direction than in the blood-to-aqueous humor direction, and active. There was little net directional movement of cidofovir. Probenecid (0.1 mM) or novobiocin (0.1 mM) added to the aqueous humor side of the tissue, or MK571 (5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid; 0.1 mM) added to the blood side significantly reduced net active PAH transport. The rate of 6-carboxyfluorescein elimination from the aqueous humor of the perfused eye was reduced 80% when novobiocin (0.1 mM) was present in the aqueous humor. These data indicate that the ciliary body expresses a variety of OA transporters, including those common to the kidney. They are likely involved in clearing potentially harmful endobiotic and xenobiotic OAs from the eye.

Connexins form functional hemichannels in porcine ciliary epithelium.

The expression of connexins in the ciliary epithelium is consistent with gap junctions between the pigmented (PE) and nonpigmented ciliary epithelium (NPE) that form when connexon hemichannels from adjacent cells pair to form a channel. Here we present evidence that suggests undocked connexons may form functional hemichannels that permit exchange of substances between NPE and the aqueous humor. Intact porcine eyes were perfused via the ciliary artery and propidium iodide (PI) (MW 668) was added to the aqueous humor compartment as a tracer. After calcium-free solution containing PI was introduced into the aqueous humor compartment for 30 min, fluorescence microscopy revealed PI in the NPE cell layer. PI entry into the NPE was inhibited by calcium and by the connexin antagonist 18α-glycyrrhetinic acid (18-AGA). Studies also were carried out with cultured porcine NPE. Under normal conditions, little PI entered the cultured cells but calcium-free medium stimulated PI accumulation and the entry was inhibited by 18-AGA. In cells loaded with calcein (MW 622), calcium-free solution stimulated calcein exit. 18-AGA partially suppressed calcein exit in calcium-free medium. Connexin 43 and connexin 50 proteins were detected by western blot analysis in both native and cultured NPE. In the intact eye, immunolocalization studies revealed connexin 50 at the basolateral, aqueous humor-facing, margin of the NPE. In contrast, connexin 43 was observed at the junction of the PE and NPE layer and on the basolateral membrane of PE. The results point to functional hemichannels at the NPE basolateral surface. It is feasible that hemichannels might contribute to the transfer of substances between the ciliary epithelium cytoplasm and aqueous humor.

Nitric oxide regulation of Na, K-ATPase activity in ocular ciliary epithelium involves Src family kinase.

The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ~40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ; 10 µM) and PKG (KT5823; 1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation.

Nonpigmented ciliary epithelial cells respond to acetazolamide by a soluble adenylyl cyclase mechanism.

PURPOSE: The nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI). METHODS: Cyclic AMP was measured by radioimmunoassay in primary cultured porcine NPE. The pH-sensitive dye BCECF was used to examine cytoplasmic pH regulation. Subcellular protein translocation was examined by Western blot. RESULTS: A transient cAMP increase, detectable within minutes of acetazolamide treatment, was prevented by KH7, a specific sAC inhibitor. Following 10-minute exposure to acetazolamide, the abundance of H(+)-ATPase B1 subunit and sAC was doubled in a plasma membrane-rich fraction, suggesting subcellular translocation. Similar evidence of H(+)-ATPase translocation was observed in NPE exposed to 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Consistent with increased capacity for proton export, acetazolamide increased the rate of pH recovery from acidification. KH7 and bafilomycin A1, an inhibitor of H(+)-ATPase, both prevented the stimulatory effect of acetazolamide on pH recovery. In a parallel study, H(+)-ATPase abundance was found to be higher in the plasma membrane of HEK293 cells that overexpress sAC compared to the normal HEK293 cells. HEK cells that overexpress sAC and had higher H(+)-ATPase abundance displayed a faster rate of pH recovery and greater sensitivity to KH7. CONCLUSIONS: Acetazolamide increases cAMP in a response that involves activation of sAC. Subcellular translocation of H(+)-ATPase and an increase in the capacity for proton export by acetazolamide-treated NPE cells is a cAMP-dependent response.

Defects in synapse structure and function precede motor neuron degeneration in Drosophila models of FUS-related ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. We show here that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by >80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS.

DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism.

The anion transport inhibitor DIDS is known to reduce aqueous humor secretion but questions remain about anion dependence of the effect. In some tissues, DIDS is reported to cause Na-K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na-K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for (86)Rb uptake measurements or homogenized to measure Na-K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive (86)Rb uptake, activated Src family kinase (SFK), and caused a reduction of Na-K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na-K-ATPase activity inhibition, (86)Rb uptake suppression, and SFK activation were observed when a similar reduction of pHi was imposed by low-pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction, but U0126 did not prevent SFK activation or the Na-K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na-K-ATPase activity by a mechanism that hinges on SFK activation associated with a reduction of cytoplasmic pH.

TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity.

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells.

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.

A novel mechanism of acid and bile acid-induced DNA damage involving Na+/H+ exchanger: implication for Barrett's oesophagus.

OBJECTIVE: Barrett's oesophagus is a premalignant disease associated with oesophageal adenocarcinoma. The major goal of this study was to determine the mechanism responsible for bile acid-induced alteration in intracellular pH (pH(i)) and its effect on DNA damage in cells derived from normal oesophagus (HET1A) or Barrett's oesophagus (CP-A). DESIGN: Cells were exposed to bile acid cocktail (BA) and/or acid in the presence/absence of inhibitors of nitric oxide synthase (NOS) or sodium-hydrogen exchanger (NHE). Nitric oxide (NO), pH(i) and DNA damage were measured by fluorescent imaging and comet assay. Expression of NHE1 and NOS in cultured cells and biopsies from Barrett's oesophagus or normal squamous epithelium was determined by RT-PCR, immunoblotting or immunohistochemistry. RESULTS: A dose dependent decrease in pH(i) was observed in CP-A cells exposed to BA. This effect of BA is the consequence of NOS activation and increased NO production, which leads to NHE inhibition. Exposure of oesophageal cells to acid in combination with BA synergistically decreased pH(i). The decrease was more pronounced in CP-A cells and resulted in >2-fold increase in DNA damage compared to acid treatment alone. Examination of biopsies and cell lines revealed robust expression of NHE1 in Barrett's oesophagus but an absence of NHE1 in normal epithelium. CONCLUSIONS: The results of this study identify a new mechanism of bile acid-induced DNA damage. We found that bile acids present in the refluxate activate immediately all three isoforms of NOS, which leads to an increased NO production and NHE inhibition. This consequently results in increased intracellular acidification and DNA damage, which may lead to mutations and cancer progression. Therefore, we propose that in addition to gastric reflux, bile reflux should be controlled in patients with Barrett's oesophagus.

New therapeutic and biomarker discovery for peripheral diabetic neuropathy: PARP inhibitor, nitrotyrosine, and tumor necrosis factor-{alpha}.

This study evaluated poly(ADP-ribose) polymerase (PARP) inhibition as a new therapeutic approach for peripheral diabetic neuropathy using clinically relevant animal model and endpoints, and nitrotyrosine (NT), TNF-alpha, and nitrite/nitrate as potential biomarkers of the disease. Control and streptozotocin-diabetic rats were maintained with or without treatment with orally active PARP inhibitor 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one (GPI-15,427), 30 mg kg(-1) d(-1), for 10 wk after first 2 wk without treatment. Therapeutic efficacy was evaluated by poly(ADP-ribosyl)ated protein expression (Western blot analysis), motor and sensory nerve conduction velocities, and tibial nerve morphometry. Sciatic nerve and spinal cord NT, TNF-alpha, and nitrite/nitrate concentrations were measured by ELISA. NT localization in peripheral nervous system was evaluated by double-label fluorescent immunohistochemistry. A PARP inhibitor treatment counteracted diabetes-induced motor and sensory nerve conduction slowing, axonal atrophy of large myelinated fibers, and increase in sciatic nerve and spinal cord NT and TNF-alpha concentrations. Sciatic nerve NT and TNF-alpha concentrations inversely correlated with motor and sensory nerve conduction velocities and myelin thickness, whereas nitrite/nitrate concentrations were indistinguishable between control and diabetic groups. NT accumulation was identified in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia. The findings identify PARP as a compelling drug target for prevention and treatment of both functional and structural manifestations of peripheral diabetic neuropathy and provide rationale for detailed evaluation of NT and TNF-alpha as potential biomarkers of its presence, severity, and progression.

Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior.

An understanding of sleep requires the identification of distinct cellular circuits that mediate the action of specific sleep:wake-regulating molecules, but such analysis has been very limited. We identify here a circuit that underlies the wake-promoting effects of octopamine in Drosophila. Using MARCM, we identified the ASM cells in the medial protocerebrum as the wake-promoting octopaminergic cells. We then blocked octopamine signaling in random areas of the fly brain and mapped the postsynaptic effect to insulin-secreting neurons of the pars intercerebralis (PI). These PI neurons show altered potassium channel function as well as an increase in cAMP in response to octopamine, and genetic manipulation of their electrical excitability alters sleep:wake behavior. Effects of octopamine on sleep:wake are mediated by the cAMP-dependent isoform of the OAMB receptor. These studies define the cellular and molecular basis of octopamine action and suggest that the PI is a sleep:wake-regulating neuroendocrine structure like the mammalian hypothalamus.

Responses of sodium-hydrogen exchange to nitric oxide in porcine cultured nonpigmented ciliary epithelium.

PURPOSE: To better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity. METHODS: Cytoplasmic pH (pH(i)) was measured in cultured porcine NPE loaded with BCECF (2',7'-bis(2-carboxyl)-5(6)-carboxyfluorescein-acetoxyethyl ester). Na-H exchanger (NHE) was examined by immunolocalization. RESULTS: In cells acidified by 5 minutes of exposure to 20 mM ammonium chloride, pH(i) recovery was partially inhibited by sodium nitroprusside (SNP), an NO donor, and l-arginine, the endogenous substrate for NO synthase. SNP and dimethyl amiloride (DMA), an NHE inhibitor, inhibited pH(i) recovery to a similar degree. In bicarbonate-free buffer SNP+DMA elicited no additional change in pH(i) recovery beyond that elicited by DMA alone. This suggests that SNP causes NHE inhibition. the SNP's effect on pH(i) recovery was mimicked by 8-pCPT-cGMP but suppressed by ODQ and H-8. Ouabain alone reduced pH(i) recovery, but SNP+ouabain caused significant further reduction. Immunolocalization studies revealed NHE1 and -4 in native and cultured NPE. CONCLUSIONS: NHE1 and -4 are expressed at the NPE basolateral margin. The findings suggest the NHE is inhibited by NO which acts via a cGMP and protein kinase G signaling pathway. The NHE response does not appear to be the consequence of NO-induced Na,K-ATPase inhibition. Because NO synthases are expressed in porcine NPE, NO could act as an autocrine regulator of NHE activity. Although NHE inhibitors are known to lower intraocular pressure (IOP), further studies are needed to understand whether changes in NHE activity contribute to the IOP-lowering effect of NO donors.