Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.     

P2Y receptor regulation of sodium transport in human mammary epithelial cells

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors. RT-PCR experiments also revealed expression of A(2b) adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (I(sc)) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na(+) channel (ENaC) alpha-, beta-, and gamma-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na(+) transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPgammaS. Basolateral stimulation with UTP produced a rapid but transient increase in I(sc) that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in I(sc) and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca(2+) mobilization evoked by P2Y receptor agonists stimulates Na(+) absorption by increasing the activity of Ca(2+)-activated K(+) channels located in the basolateral membrane.     

nPKCepsilon, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y(2) purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP(3)), diacylglycerol, Ca(2+) and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKCalpha, nPKCdelta, nPKCepsilon, and nPKCeta; of these, only nPKCdelta translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKCalpha, nPKCdelta, and nPKCeta had the same levels of ATPgammaS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKCepsilon (14.6 and 23.5%, for ATPgammaS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKCepsilon exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPgammaS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y(2)-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKCdelta knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKCepsilon KO mice relative to its WT littermates. We conclude that nPKCepsilon is the effector isoform downstream of P2Y(2)-R activation in the goblet cell secretory response. The translocation of nPKCdelta observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology.     

Erythromycin increases bactericidal activity of surface liquid in human airway epithelial cells

Macrolide antibiotics have clinical benefits in patients with diffuse panbronchiolitis and in patients with cystic fibrosis. Although many mechanisms have been proposed, the precise mechanisms are still uncertain. We examined the effects of erythromycin on bactericidal activity of airway surface liquid secreted by cultured human tracheal epithelial cells. Airway surface liquid was collected by washing the surface of human tracheal epithelial cells with a sodium solution (40 meq/l). Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were incubated with airway surface liquid, and the number of surviving bacteria was examined. The number of bacteria in airway surface liquid from the cells cultured in medium alone was significantly lower than that in the sodium solution. Furthermore, the number of bacteria in airway surface liquid from the cells treated with erythromycin was significantly lower than that in airway surface liquid from the cells treated with solvent alone. The production of mRNA and protein of human beta-defensin-1 and human beta-defensin-2 was significantly increased by erythromycin. Bactericidal activity of airway surface liquid was observed at low concentrations (40 meq/l) of sodium but not at higher concentrations (> or =80 meq/l). Airway surface liquid did not contain significant amounts of antibiotics supplemented in the culture medium. Erythromycin at the levels in airway surface liquid and in culture medium did not inhibit bacterial growth. These results suggest that erythromycin may increase bactericidal activity of airway surface liquid in human airway epithelial cells through human beta-defensins production and reduce susceptibility of the airway to bacterial infection.     

Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells

Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.     

ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca(2+)](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.     

nPKC{varepsilon}, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y₂ purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP₃), diacylglycerol, Ca²⁺ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC, nPKC, nPKC, and nPKC; of these, only nPKC translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149–L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC, nPKC, and nPKC had the same levels of ATPS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC (14.6 and 23.5%, for ATPS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y₂-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC KO mice relative to its WT littermates. We conclude that nPKC is the effector isoform downstream of P2Y₂-R activation in the goblet cell secretory response. The translocation of nPKC observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung     

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.     

Bradykinin increases IL-8 generation in airway epithelial cells via COX-2-derived prostanoids

Interleukin (IL)-8, the C-X-C chemokine, is a potent neutrophil chemoattractant that has been implicated in a number of inflammatory airway diseases such as cystic fibrosis. Here we tested the hypothesis that bradykinin, an inflammatory mediator and chloride secretagogue, would increase IL-8 generation in airway epithelial cells through autocrine generation of endogenous prostanoids. Bradykinin increased IL-8 generation in both a non-cystic fibrosis (A549) and cystic fibrosis epithelial cell line (CFTE29) that was inhibited by the nonselective cyclooxygenase (COX) inhibitor indomethacin and the COX-2 selective inhibitor NS-398. COX-2 was the only isoform of COX expressed in both cell lines. Furthermore, the COX substrate arachidonic acid and exogenous prostaglandin E(2) both increased IL-8 release in A549 cells. These results suggest that bradykinin may contribute to neutrophilic inflammation in the airway by generation of IL-8 from airway epithelial cells. The dependence of this response on endogenous production of prostanoids by COX-2 suggests that selective COX-2 inhibitors may have a role in the treatment of airway diseases characterized by neutrophilic inflammation such as cystic fibrosis or chronic obstructive pulmonary disease.     

Isoproterenol suppresses cytokine-induced RANTES secretion in human lung epithelial cells through the inhibition of c-jun N-terminal kinase pathway

It has been reported that beta2-agonists may potentially exert some anti-inflammatory action in addition to bronchodilation that may contribute to their beneficial effects on asthma control. Bronchial epithelial cells are well known to respond to a range of stimuli by producing various biologically active mediators that can influence airway inflammation. RANTES (regulated on activation, normal T cells expressed and secreted) plays an important role in the pathophysiology of airway inflammation of asthmatics through its chemotactic activity for eosinophils. In this study, the authors investigated whether cytokine-induced RANTES release from BEAS-2B human bronchial epithelial cells could be modulated by beta-agonist isoproterenol (ISO). The possible involvement of c-jun N-terminal kinase (JNK) pathway was also studied. Combination of tumor necrosis factor-alpha and interleukin-1beta (cytokine mix) increased RANTES release from BEAS-2B cells and stimulated JNK activity. Similar to JNK inhibitor SP600125, ISO inhibited not only the production of RANTES but also the activation of JNK pathway in cytokine mix-stimulated BEAS-2B cells. The effect of ISO was mediated by the beta2-adrenoceptor, since it was blocked by ICI 118,551, a selective beta2-receptor antagonist, but not by atenolol, a selective beta1-receptor antagonist. Adenylyl cyclase activator forskolin reproduced the effects of ISO. Isoproterenol was found to inhibit the release of RANTES from the human bronchial epithelial cells, at least in part, through the inhibition of JNK signaling pathway.     

Functional evidence for involvement of P2 purinoceptors in the ATP stimulation of phosphatidylcholine secretion in type II alveolar epithelial cells

There is evidence that phosphatidylcholine secretion in type II pneumocytes is stimulated by adenosine and adenine nucleotides and that the effect of adenosine is mediated by the A2 subtype of the P1 purinoceptor. To determine if the effect of ATP is also mediated by the same receptor following its catabolism to adenosine or by the P2 purinoceptor we compared the effects of adenosine and ATP. Adenosine and terbutaline stimulated phosphatidylcholine secretion approx. 2-fold, while ATP stimulated it by more than 3-fold, essentially to the same extent as the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate. The stimulatory effect of adenosine but not of ATP was abolished by adenosine deaminase. The effect of ATP was markedly diminished by the P2 desensitizing agent alpha,beta-methylene ATP, but only slightly by the P1 antagonist 8-phenyltheophylline. Adenosine increased the cAMP content of type II cells while ATP had little effect. The effects of ATP and terbutaline were additive while those of adenosine and terbutaline were not. These data show that ATP and adenosine stimulate phosphatidylcholine secretion via different mechanisms. Therefore, the effect of ATP is not mediated via catabolism to adenosine. Metabolically resistant analogs of ATP also stimulated secretion in a concentration-dependent manner although none were as potent as ATP. The order of potency was ATP greater than beta,gamma-methylene ATP = 2-methylthio ATP = 2-deoxy ATP greater than or equal to 8-bromo ATP greater than alpha,beta-methylene ATP. The facts that ATP analogs also stimulate secretion and that the effect of ATP was antagonized by alpha,beta-methylene ATP suggest that the stimulatory effect of ATP is mediated by the P2 purinoceptor.     

Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon

Eight human G protein-coupled P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (I_sc) or Ca²⁺ mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y₁, P2Y₁₁, P2Y₁₂, and P2Y₁₄ receptors reside at the basolateral membrane, P2Y₂, P2Y₄, and P2Y₆ receptors are expressed at the apical membrane, and the P2Y₁₃ receptor is unsorted. Biotinylation studies and I_sc measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the G_q-coupled P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁) in lung and colonic epithelial cells (16HBE14o– and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells. Madin-Darby canine kidney; 16HBE14o–; Caco-2; confocal microscopy; polarized targeting     

Crosstalk between platelet-derived growth factor-induced Nox4 activation and MUC8 gene overexpression in human airway epithelial cells

Reactive oxygen species (ROS) contribute to chronic airway inflammation, and NADPH oxidase (Nox) is an important source of ROS. However, little is known of the role that ROS play in chronic upper respiratory tract inflammation. We investigated the mechanism of ROS generation and its association with mucin gene overexpression in the nasal epithelium. The level of platelet-derived growth factor (PDGF) expression was increased in sinusitis mucosa, and high-level PDGF expression induced intracellular ROS, followed by MUC8 gene overexpression in normal human nasal epithelial cells. Knockdown of Nox4 expression with Nox4 siRNA decreased PDGF-induced intracellular ROS and MUC8 expression. Infection with an adenovirus containing Nox4 cDNA resulted in Nox4 overexpression and increased intracellular levels of ROS and MUC8 expression. PDGF and Nox4 overexpression are essential components of intracellular ROS generation and may contribute to chronic inflammation in the nasal epithelium through induction of MUC8 overexpression.     

Extracellular ATP induces P2X7-dependent nicotinamide phosphoribosyltransferase release in LPS-activated human monocytes

Nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in NAD biosynthesis, has recently been identified as a novel mediator of innate immunity. In the present study, we report that treatment of LPS-primed monocytes with ATP greatly enhanced the secretion of NAMPT in a time- and concentration-dependent manner without displaying any cytotoxic effect. NAMPT release was suppressed by pretreatment with the P2X(7) receptor (P2X(7)R) inhibitors oxidized ATP (oxATP) and KN-62, indicating the engagement of P2X(7)Rs. Furthermore, P2X(7)R was found to be involved in mediating cell permeability caused by the addition of ATP. To define a role of endogenous ATP in NAMPT secretion, LPS-primed monocytes were incubated in the presence of oxATP and KN-62, as well as the ATP-hydrolyzing enzymes apyrase and hexokinase. With the exception of oxATP, neither substance led to a decrease in NAMPT release, suggesting that autocrine/paracrine ATP is unlikely to be responsible for the LPS-induced release of NAMPT. In conclusion, the enhanced release of NAMPT by extracellular ATP described here indicates the requirement of a second stimulus for the efficient secretion of NAMPT. This mode of secretion, which also applies to IL-1β, might represent a general mechanism for the release of leaderless secretory proteins at locally restricted sites.     

The role of P2Y14 and other P2Y receptors in degranulation of human LAD2 mast cells

Mast cell degranulation affects many conditions, e.g., asthma and urticaria. We explored the potential role of the P2Y₁₄ receptor (P2Y₁₄R) and other P2Y subtypes in degranulation of human LAD2 mast cells. All eight P2YRs were expressed at variable levels in LAD2 cells (quantitative real-time RT-PCR). Gene expression levels of ADP receptors, P2Y₁R, P2Y₁₂R, and P2Y₁₃R, were similar, and P2Y₁₁R and P2Y₄R were highly expressed at 5.8- and 3.8-fold of P2Y₁R, respectively. Least expressed P2Y₂R was 40-fold lower than P2Y₁R, and P2Y₆R and P2Y₁₄R were ≤50 % of P2Y₁R. None of the native P2YR agonists alone induced β-hexosaminidase (β-Hex) release, but some nucleotides significantly enhanced β-Hex release induced by C3a or antigen, with a rank efficacy order of ATP > UDPG ≥ ADP >> UDP, UTP. Although P2Y₁₁R and P2Y₄R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y₄R agonist UTP nor P2Y₁₁R agonists ATPγS and NF546 had a substantial effect. P2Y₁R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y₁R potency, and the effect of P2Y₆R agonist 3-phenacyl-UDP was negligible. The enhancement by ADP and ATP appears mediated via multiple receptors. Both UDPG and a synthetic agonist of the P2Y₁₄R, MRS2690, enhanced C3a-induced β-Hex release, which was inhibited by a P2Y₁₄R antagonist, specific P2Y₁₄R siRNA and pertussis toxin, suggesting a role of P2Y₁₄R activation in promoting human mast cell degranulation.     

Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells

Tear hyperosmolarity is a key event in dry eye. In this work, we analyzed whether hyperosmolar challenge induces ATP release on the ocular surface. Moreover, as extracellular ATP can activate P2X7 receptor, the changes in P2X7 protein levels and its involvement in pathological process triggered by hypertonic treatment were also examined. High-performance liquid chromatography analysis revealed that ATP levels significantly increased in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge as well as in dry eye patients as compared to control subjects. A significant reduction in cell viability was detected after hyperosmolar treatment, indicating that the rise in ATP release was mainly due to cell lysis/death. Additionally, vesicular nucleotide transporter was identified in both cell lines and their protein expression was upregulated in hypertonic media. P2X7 receptor truncated form together with the full-length form was identified in both cell lines, and experiments using specific antagonist and agonist for P2X7 indicated that this receptor did not mediate cell death induced by hyperosmolar stress. In conclusion, hyperosmotic stress induces ATP release. Extracellular ATP can activate P2X7 receptor leading to cytotoxicity in many cells/tissues; however, this does not occur in human corneal and conjunctival epithelial cells. In these cells, the presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior on the ocular surface.     

Voltage- and [ATP]-dependent Gating of the P2X2 ATP Receptor Channel

P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X₂ channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this “activation” phase quantitatively. We characterized the conductance–voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel''s transition between a closed and an open state, we showed that the gating of P2X₂ is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X₂ even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate “gating.” We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X₂-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].     

IL13 activates autophagy to regulate secretion in airway epithelial cells

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.