Hyposmotic stress causes ATP release and stimulates Na,K-ATPase activity in porcine lens

Purinergic receptors in lens epithelium suggest lens function can be altered by chemical signals from aqueous humor or the lens itself. Here we show release of ATP by intact porcine lenses exposed to hyposmotic solution (200 mOsm). 18α-glycyrrhetinic acid (AGA) added together with probenecid eliminated the ATP increase. N-ethylmaleimide (200 μM), an exocytotic inhibitor, had no significant effect on ATP increase. Lenses exposed to hyposmotic solution displayed a ~400% increase of propidium iodide (PI) entry into the epithelium. The increased ability of PI (MW 668) to enter the epithelium suggests possible opening of connexin and/or pannexin hemichannels. This is consistent with detection of connexin 43, connexin 50, and pannexin 1 in the epithelium and the ability of AGA + probenecid to prevent ATP release. Na,K-ATPase activity doubled in the epithelium of lenses exposed to hyposmotic solution. The increase of Na,K-ATPase activity did not occur when apyrase was used to prevent extracellular ATP accumulation or when AGA + probenecid prevented ATP release. The increase of Na,K-ATPase activity was inhibited by the purinergic P2 antagonist reactive blue-2 and pertussis toxin, a G-protein inhibitor, but not by the P2X antagonist PPADS. Hyposmotic solution activated Src family kinase (SFK) in the epithelium, judged by Western blot. The SFK inhibitor PP2 abolished both SFK activation and the Na,K-ATPase activity increase. In summary, hyposmotic shock-induced ATP release is sufficient to activate a purinergic receptor- and SFK-dependent mechanism that stimulates Na,K-ATPase activity. The responses might signify an autoregulatory loop initiated by mechanical stress or osmotic swelling.     

Light-induced ATP release from the lens

The recent discovery of the photoreceptor melanopsin in lens epithelial cells has opened the possibility of modulating this protein by light stimulation. Experiments carried out on New Zealand white rabbits have demonstrated that the release of ATP from the lens to the aqueous humor can be reduced either when a yellow filter or a melanopsin antagonist is used. Compared to control (1.10?±?0.15 μM ATP), the application of a yellow filter (λ465–480) reduced ATP in the aqueous humor 70%, while the melanopsin antagonist AA92593 reduced the presence of ATP 63% (n?=?5), an effect which was also obtained with the PLC inhibitor U73122. These results indicate that when melanopsin is blocked either by the lack of light, a filter, or an antagonist, the extracellular presence of ATP is significantly reduced. This discovery may be relevant, on the one hand, because many ocular physiological processes are controlled by ATP and, on the other hand, because it is possible to stimulate ATP release with just light and without using any added substance.     

The effect of endothelin-1 on Src-family tyrosine kinases and Na,K-ATPase activity in porcine lens epithelium

Previous studies show Src family kinase (SFK) activation is involved in a response that stimulates Na,K-ATPase. Here, we tested whether SFK activation is involved in the Na,K-ATPase response to endothelin-1 (ET-1). Intact porcine lenses were exposed to 100 nM ET-1 for 5-30 min. Then, the epithelium was removed and used for Na,K-ATPase activity measurement and Western blot analysis of SFK activation. Na,K-ATPase activity was reduced by ～30% in lenses exposed to ET-1 for 15 min. The response was abolished by the SFK inhibitor PP2 or the ET receptor antagonist, PD145065. Activation of a ～61 kDa SFK was evident from an increase in Y416 phosphorylation, which reached a maximum at 15 min ET-1 treatment, and a decrease in Y527 phosphorylation. PP2 prevented SFK activation. Since Fyn, Src, Hck, and Yes may contribute to the observed 61 kDa band, these SFKs were isolated by immunoprecipitation and analyzed. Based on Y416 phosphorylation, ET-1 appeared to activate Fyn, while Src and Hck were inhibited and Yes was unaltered. ET-1 requires SFK activation to cause Na,K-ATPase inhibition. ET-1 elicits a different pattern of SFK activation from that reported earlier for purinergic agonists that stimulate Na,K-ATPase activity and activate Src. In the ET-1 response Src is inhibited and Fyn is activated. The findings suggest SFK phosphorylation is involved in a regulatory mechanism for Na,K-ATPase. Knowing this may help us understand drug actions on Na,K-ATPase. Faulty regulation of Na,K-ATPase in the lens could contribute to cataract formation since an abnormal sodium content is associated with lens opacification.     

Axonal neuropathy-associated TRPV4 regulates neurotrophic factor-derived axonal growth

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.     

Basal cells express functional TRPV4 channels in the mouse nasal epithelium

Basal cells in the nasal epithelium (olfactory and airway epithelia) are stem/progenitor cells that are capable of dividing, renewing and differentiating into specialized cells. These stem cells can sense their biophysical microenvironment, but the underlying mechanism of this process remains unknown. Here, we demonstrate the prominent expression of the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca²⁺-permeable channel that is known to act as a sensor for hypo-osmotic and mechanical stresses, in the basal cells of the mouse nasal epithelium. TRPV4 mRNA was expressed in the basal portions of the prenatal mouse nasal epithelium, and this expression continued into adult mice. The TRPV4 protein was also detected in the basal layers of the nasal epithelium in wild-type but not in TRPV4-knockout (TRPV4-KO) mice. The TRPV4-positive immunoreactions largely overlapped with those of keratin 14 (K14), a marker of basal cells, in the airway epithelium, and they partially overlapped with those of K14 in the olfactory epithelium. Ca²⁺ imaging analysis revealed that hypo-osmotic stimulation and 4α-phorbol 12,13 didecanoate (4α-PDD), both of which are TRPV4 agonists, caused an increase in the cytosolic Ca²⁺ concentration in a subset of primary epithelial cells cultured from the upper parts of the nasal epithelium of the wild-type mice. This response was barely noticeable in cells from similar parts of the epithelium in TRPV4-KO mice. Finally, there was no significant difference in BrdU-labeled proliferation between the olfactory epithelia of wild-type and TRPV4-KO mice under normal conditions. Thus, TRPV4 channels are functionally expressed in basal cells throughout the nasal epithelium and may act as sensors for the development and injury-induced regeneration of basal stem cells.     

The influence of Lyn kinase on Na,K-ATPase in porcine lens epithelium

Na,K-ATPase is essential for the regulation of cytoplasmic Na⁺ and K⁺ levels in lens cells. Studies on the intact lens suggest activation of tyrosine kinases may inhibit Na,K-ATPase function. Here, we tested the influence of Lyn kinase, a Src-family member, on tyrosine phosphorylation and Na,K-ATPase activity in membrane material isolated from porcine lens epithelium. Western blot studies indicated the expression of Lyn in lens cells. When membrane material was incubated in ATP-containing solution containing partially purified Lyn kinase, Na,K-ATPase activity was reduced by 38%. Lyn caused tyrosine phosphorylation of multiple protein bands. Immunoprecipitation and Western blot analysis showed Lyn treatment causes an increase in density of a 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase ₁ polypeptide. Incubation with protein tyrosine phosphatase 1B (PTP-1B) reversed the Lyn-dependent tyrosine phosphorylation increase and the change of Na,K-ATPase activity. The results suggest that Lyn kinase treatment of a lens epithelium membrane preparation is able to bring about partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase ₁ catalytic subunit. lens; Na,K-ATPase; tyrosine phosphorylation; Lyn     

HSF4 regulates DLAD expression and promotes lens de-nucleation

HSF4 mutations lead to both congenital and age-related cataract. The purpose of this study was to explore the mechanism of cataract formation caused by HSF4 mutations. The degradation of nuclear DNA is essential for the lens fiber differentiation. DNase 2β (DLAD) is highly expressed in lens cells, and mice with deficiencies in the DLAD gene develop nuclear cataracts. In this study, we found that HSF4 promoted the expression and DNase activity of DLAD by directly binding to the DLAD promoter. In contrast, HSF4 cataract causative mutations failed to bind to the DLAD promoter, abrogating the expression and DNase activity of DLAD. These results were confirmed by HSF4 knockdown in zebrafish, which led to incomplete de-nucleation of the lens and decreased expression and activity of DLAD. Together, our results suggest that HSF4 exerts its function on lens differentiation via positive regulation of DLAD expression and activity, thus facilitating de-nucleation of lens fiber cells. Our demonstration that HSF4 cataract causative mutations abrogate the induction of DLAD expression reveals a novel molecular mechanism regarding how HSF4 mutations cause cataractogenesis.     

Rho signaling regulates pannexin 1-mediated ATP release from airway epithelia

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.     

OS-9 regulates the transit and polyubiquitination of TRPV4 in the endoplasmic reticulum

Transient receptor potential (TRP) proteins constitute a family of cation-permeable channels that are formed by homo- or heteromeric assembly of four subunits. Despite recent progress in the identification of protein domains required for the formation of tetramers, the mechanisms governing TRP channel assembly, and biogenesis in general, remain largely elusive. In particular, little is known about the involvement of regulatory proteins in these processes. Here we report that OS-9, a ubiquitously expressed endoplasmic reticulum (ER)-associated protein, interacts with the cytosolic N-terminal tail of TRPV4. Using a combination of co-expression and knockdown approaches we have found that OS-9 impedes the release of TRPV4 from the ER and reduces its amount at the plasma membrane. Consistent with these in vitro findings, OS-9 protected zebrafish embryos against the detrimental effects of TRPV4 expression in vivo. A detailed analysis of the underlying mechanisms revealed that OS-9 preferably binds TRPV4 monomers and other ER-localized, immature variants of TRPV4 and attenuates their polyubiquitination. Thus, OS-9 regulates the secretory transport of TRPV4 and appears to protect TRPV4 subunits from the precocious ubiquitination and ER-associated degradation. Our data suggest that OS-9 functions as an auxiliary protein for TRPV4 maturation.     

Constitutive ERK MAPK activity regulates macrophage ATP production and mitochondrial integrity

A unique feature of human alveolar macrophages is their prolonged survival in the face of a stressful environment. We have shown previously that the ERK MAPK is constitutively active in these cells and is important in prolonging cell survival. This study examines the role of the ERK pathway in maintaining mitochondrial energy production. The data demonstrate that ATP levels in alveolar macrophages depend on intact mitochondria and optimal functioning of the electron transport chain. Significant levels of MEK and ERK localize to the mitochondria and inhibition of ERK activity induces an early and profound depletion in cellular ATP coincident with a loss of mitochondrial transmembrane potential. The effect of ERK suppression on ATP levels was specific, since it did not occur with PI3K/Akt, p38, or JNK suppression. ERK inhibition led to cytosolic release of mitochondrial proteins and caspase activation. Both ERK inhibition and mitochondrial blockers induced loss of plasma membrane permeability and cell death. The cell death induced by ERK inhibition had hallmarks of both apoptotic (caspase activation) and necrotic (ATP loss) cell death. By blocking ERK inhibition-induced reactive oxygen species, caspase activation was prevented, although necrotic pathways continued to induce cell death. This suggests that mitochondrial dysfunction caused by ERK inhibition generates both apoptotic and necrotic cell death-inducing pathways. As a composite, these data demonstrate a novel mitochondrial role for ERK in maintaining mitochondrial membrane potential and ATP production in human alveolar macrophages.     

IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells

To investigate the mechanisms of eosinophil recruitment in allergic airway inflammation, we examined the effects of interleukin (IL)-4, a Th2-type cytokine, on eotaxin and monocyte chemoattractant protein-4 (MCP-4) expression in human peripheral blood mononuclear cells (PBMCs; n = 10), in human lower airway mononuclear cells (n = 5), in the human lung epithelial cell lines A549 and BEAS-2B, and in human cultured airway epithelial cells. IL-4 inhibited eotaxin and MCP-4 mRNA expression induced by IL-1 beta and tumor necrosis factor-alpha in PBMCs but did not significantly inhibit expression in epithelial cells. Eotaxin and MCP-4 mRNA expression was not significantly induced by proinflammatory cytokines in lower airway mononuclear cells. IL-1 beta-induced eotaxin and MCP-4 protein production was also inhibited by IL-4 in PBMCs, whereas IL-4 enhanced eotaxin protein production in A549 cells. In contrast, dexamethasone inhibited eotaxin and MCP-4 expression in both PBMCs and epithelial cells. The divergent effects of IL-4 on eotaxin and MCP-4 expression between PBMCs and epithelial cells may create chemokine concentration gradients between the subepithelial layer and the capillary spaces that may promote the recruitment of eosinophils to the airway in Th2-type responses.     

Mechanotransduction via TRPV4 regulates inflammation and differentiation in fetal mouse distal lung epithelial cells

Background

Mechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury.

Methods

Expression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively.

Results

TRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4.

Conclusion

These studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.     

Characterization of Na-K-ATPase in dog tracheal epithelium: enzymatic and ion transport measurements

The dog tracheal epithelium actively secretes Cl and absorbs Na. The possible dependency of this electrolyte transport on a Mg-dependent, Na-K-activated adenosine triphosphatase (Na-K-ATPase, EC 3.6.1.3) was examined. The characteristics of this enzyme system were investigated using homogenates of tracheal epithelium. The electrical properties and ion fluxes of this epithelium were determined in tissues mounted in Ussing chambers. Addition of Na and K produced an approximate 50% activation of basal Mg-ATPase activity. The apparent Km values for ATP, Na, K, and Mg were 0.4, 12.7, 1.9, and 1.6 mM, respectively. The total specific ATPase activity was 8.1 +/- 0.4 and that of the Mg-ATPase 4.3 +/- 0.1 mumol Pi. mg protein -1.h-1. Addition of ouabain (1 muM) or omission of K from the submucosal bathing solution reduced potential difference (PD) and short-circuit current (SCC) significantly. Relatively low concentrations (0.1 mM or less) of ethacrynic acid, furosemide, or 2,4-dinitrophenol (2,4-DNP) depressed SCC and PD significantly, i.e., at concentrations that were without effect on the Na-K-ATPase activity. Ethacrynic acid inhibited Cl secretion, whereas 2,4-DNP lowered both Na and Cl transport. These data demonstrate that 1) the tracheal mucosa of dogs contains a Na-K-ATPase at relatively high specific activity, 2) this enzyme is likely contained in the basal aspect of this membrane, 3) it appears to be essential for maintenance of Cl secretion, and 4) Cl secretion can be reduced (by ethacrynic acid, furosemide, and 2,4-DNP) without Na-K-ATPase inhibition.