E-NTPDases in human airways : Regulation and relevance for chronic lung diseases Chronic obstructive lung diseases are characterized by the inability to prevent bacterial infection and a gradual loss of lung function caused by recurrent inflammatory responses . In the past decade , numerous studies have demonstrated the importance of nucleotide-mediated bacterial clearance . Their interaction with P2 receptors on airway epithelia provides a rapid ` on-and-off ' signal stimulating mucus secretion , cilia beating activity and surface hydration . On the other hand , abnormally high ATP levels resulting from damaged epithelia and bacterial lysis may cause lung edema and exacerbate inflammatory responses . Airway ATP concentrations are regulated by ecto nucleoside triphosphate diphosphohydrolases -LRB- E-NTPDases -RRB- which are expressed on the mucosal surface and catalyze the sequential dephosphorylation of nucleoside triphosphates to nucleoside monophosphates -LRB- ATP → ADP → AMP -RRB- . The common bacterial product , Pseudomonas aeruginosa lipopolysaccharide -LRB- LPS -RRB- , induces an acute reduction in azide-sensitive E-NTPDase activities , followed by a sustained increase in activity as well as NTPDase 1 and NTPDase 3 expression . Accordingly , chronic lung diseases , including cystic fibrosis -LRB- CF -RRB- and primary ciliary dyskinesia , are characterized by higher rates of nucleotide elimination , azide-sensitive E-NTPDase activities and expression . This review integrates the biphasic regulation of airway E-NTPDases with the function of purine signaling in lung diseases . During acute insults , a transient reduction in E-NTPDase activities may be beneficial to stimulate ATP-mediated bacterial clearance . In chronic lung diseases , elevating E-NTPDase activities may represent an attempt to prevent P2 receptor desensitization and nucleotide-mediated lung damage . Introduction Airway epithelia constitute an essential protective barrier against lung infection , coordinating luminal and interstitial responses to inhaled pathogens through signals -LRB- growth factors , cytokines and nucleotides -RRB- provided by epithelial , inflammatory and immune cells -LSB- 1 -RSB- . It is now widely accepted that extracellular nucleotides provide an elaborated cell communication system in mammalian tissues -LSB- 2 , 3 -RSB- including the airways -LSB- 4 , 5 -RSB- . Each signaling event constitutes a brief ` on-and-off ' switch mechanism allowing the target cells to perceive the subsequent signal . The major source of extracellular nucleotides in normal airways is the epithelium , releasing ATP under resting conditions and in response to mechanical or osmotic stress -LSB- 6 -RSB- . While basal ATP concentrations maintained under resting conditions are insufficient to activate surface receptors , the levels reached near the site of stimulated release initiate a variety of P2 receptor-mediated responses -LSB- 7 -RSB- . Two P2 receptor families have been identified : Fast-acting -LRB- ionotropic -RRB- P2X ligand-gated cation channels and slow-acting -LRB- metabotropic -RRB- G protein-coupled P2Y receptors -LSB- 8 -RSB- . Although ATP activates members of both families , P2X receptors generally respond to higher concentrations -LRB- EC50 = 1 -- 10 µM -RRB- than P2Y receptors -LRB- EC50 = 0.1 -- 1.0 µM -RRB- . Each signal is promptly terminated by surface conversion of ATP to adenosine -LSB- 9 , 10 -RSB- and dispersal into the interstitial fluid . Adenosine also initiates cellular responses through P1 -LRB- A1 , A2A , A2B or A3 -RRB- surface receptors -LSB- 11 -RSB- . In the airways , nucleotide - and adenosine-mediated communications are involved in wound healing -LSB- 4 -RSB- , bacterial clearance -LSB- 12 -RSB- and inflammatory responses -LSB- 1 , 3 , 13 , 14 -RSB- . Cell surface nucleotide concentrations are regulated by three families of ectonucleotidases in mammalian tissues : Ecto-nucleotide pyrophosphatase/phosphodiesterases -LRB- E-NPPs : ATP → AMP -RRB- , alkaline phosphatases -LRB- APs : ATP → AMP → AMP → adenosine -RRB- and ecto-nucleoside triphosphate diphosphohydrolases -LRB- E-NTPDases : ATP → AMP → AMP -RRB- -LSB- 15 -RSB- . This review describes the importance of regulating extracellular nucleotide concentrations for airway homeostasis and the impact of chronic lung diseases on E-NTPDases . Few studies have addressed the activity or expression of airway E-NTPDases . In this review , the available information is complemented by original data . Nucleotide-mediated bacterial clearance The importance of purine signaling in the airways is well illustrated in nucleotide-mediated mucociliary clearance -LRB- MCC -RRB- , which constitutes the first line of defense against bacterial infection -LSB- 16 -RSB- . Mucosal epithelial surfaces are protected by an airway surface liquid -LRB- ASL -RRB- layer containing a thin mucin sheath kept above the cilia by a periciliary liquid -LRB- PCL -RRB- layer -LRB- Fig. 1 -RRB- . Inhaled bacteria adhere to the mucin and are continuously displaced upward by coordinated cilia beating activity -LSB- 17 , 18 -RSB- . Comparative analysis of the bioelectric properties of airway epithelia from normal donors and CF patients revealed that PCL height is regulated by a delicate balance between the activities of the epithelial sodium channel -LRB- ENAC -RRB- and the cystic fibrosis transmembrane regulator -LRB- CFTR -RRB- -LSB- 19 -RSB- . Cystic fibrosis is characterized by mutations in the gene encoding CFTR -LSB- 16 -RSB- . The lack of ion secretion through CFTR is associated with Na + hyperabsorption by ENAC and water influx , resulting in the depletion of the PCL layer essential for cilia beating activity . The cilia become collapsed under a thick layer of mucus filled with bacteria and leukocytes , leading to severe inflammatory responses and progressive loss of lung function . Fig. 1Impact of chronic obstructive lung diseases on mucociliary clearance . The polarized epithelium is composed of columnar -LRB- ciliated or mucin-secreting -RRB- cells facing the lumen and basal cells facing the serosal compartment . Bacterial clearance in healthy lungs is maintained by coordinated cilia beating activity within the PCL layer , moving upward the overlying mucus and pathogens . In chronic obstructive lung diseases , cilia are collapsed under a thick layer of mucus containing bacteria and leukocytes . Extracellular nucleotides and nucleosides regulate CFTR and ENAC -LSB- 20 -RSB- . The basal activity of CFTR is maintained by adenosine -LRB- A2B -RRB- receptors , which induce G protein-coupled adenylate cyclase , followed by cAMP-dependent activation of type II protein kinase A -LSB- 5 -RSB- . In contrast , ATP stimulates Cl − secretion through Ca2 + - activated Cl − channels . This signaling pathway involves P2Y2 receptors , leading to G protein-coupled phospholipase C activation and cytosolic Ca2 + mobilization . Water efflux is also stimulated by P2Y2 receptor-mediated inhibition of Na + absorption by ENAC using Ca2 + - dependent signaling pathways -LSB- 21 -RSB- . Members of the P2X receptor family also support luminal Cl − / water efflux in human airway epithelia , as demonstrated in primary cultures and cell lines -LRB- Beas2B , 16HBE14o − , Calu-3 , -LRB- ΣCFTE-29o − -RRB- by Ussing chamber and patch-clamp experiments -LSB- 22 -RSB- . The receptors identified in airway epithelia by RT-PCR were P2X2 , P2X4 and P2X5 . These non-selective cation channels allow Ca2 + influx , which then stimulates Ca2 + - activated Cl − channels . Interestingly , P2X7 receptors are only detected in CF airway epithelia , primarily in nasal polyps -LSB- 22 -RSB- . Because they are known to induce apoptosis in response to high ATP concentrations -LSB- 23 -RSB- , P2X7 receptors may contribute to disease-associated epithelial damage . The central role of ASL nucleotides for MCC was further substantiated by the observation that P2Y2 receptor activation constitutes the most potent stimulus for cilia beating activity -LSB- 24 -- 26 -RSB- and mucin secretion -LSB- 27 , 28 -RSB- . In cultured CF airway epithelia , P2Y2 receptor activation restores normal PCL height and rotational mucus transport -LSB- 29 -RSB- . Based on these findings , aerosolized nucleotides were proposed for the treatment of chronic obstructive lung diseases -LSB- 16 -RSB- . However , clinical studies showed that patients with asthma or chronic obstructive pulmonary disease exposed to aerosolized metabolites of ATP -LRB- AMP and adenosine -RRB- experience coughing and bronchoconstriction -LSB- 30 , 31 -RSB- . Furthermore , aerosolized ATP -LRB- not adenosine -RRB- induced similar reactions from healthy subjects -LSB- 32 , 33 -RSB- . In developing nucleotide-mediated MCC therapies , the equally potent P2Y2 receptor agonist , UTP , was evaluated -LSB- 6 -RSB- . Airway clearance in sheep , measured with nebulized technetium-labeled albumin , was transiently increased by UTP -LSB- 34 -RSB- . Aerosolized UTP also improved MCC in patients with mild chronic bronchitis -LSB- 35 -RSB- , CF -LSB- 36 , 37 -RSB- and primary ciliary dyskinesia -LSB- 38 -RSB- . A major obstacle to the treatment of chronic lung diseases with aerosolized UTP is the rapid disappearance of therapeutic concentrations -LRB- 0.1 mM -RRB- from the ASL layer -LRB- < 5 min -RRB- -LSB- 10 , 29 -RSB- . Incidentally , the metabolically stable UTP analogs INS365 -LRB- diquafosol -RRB- and INS37217 -LRB- denufosol -RRB- developed by Inspire Pharmaceuticals Inc. -LSB- 39 -RSB- were reported to improve cough induced-sputum clearance in CF patients -LSB- 40 , 41 -RSB- . However , long-term treatments with these drugs may require dose optimization to minimize P2Y2 receptor desensitization -LSB- 42 -RSB- . P2 receptors and airway inflammation In vivo studies conducted on the injurious effects of mechanical ventilation demonstrated that endogenous ATP may reach ASL concentrations sufficiently high to trigger inflammatory responses and lung damage in the absence of infection -LSB- 43 -RSB- . The bronchoalveolar lavage fluid of rats subjected to positive-pressure mechanical ventilation contains significantly higher protein , ATP and cytokine -LRB- IL-6 , TNFα -RRB- levels than control animals . Instillation of an equivalent ATP concentration increased lung fluid volume , supporting the existence of P2 and/or P1 receptor-mediated lung edema -LSB- 43 -RSB- . Analysis of bronchoalveolar lavage fluid nucleotide content by etheno-derivatization revealed an increase in AMP + adenosine/ADP + ATP ratio -LSB- 44 -RSB- , suggesting that mechanical ventilation up-regulates airway ectonucleotidases . Real-time PCR on total lung tissue indicated that mechanical ventilation increases A2B , but decreases P2X7 , receptor expression . Rats subjected to both mechanical ventilation and positive end-expiratory pressure exhibited normal bronchoalveolar lavage composition and receptor expression . These findings suggest that patients subjected to positive end-expiratory pressure during large-volume ventilation may avoid ATP-mediated lung injuries . On the other hand , the fact that adenosine levels and A2B receptor expression were raised , whereas ATP levels and P2X7 receptor expression were decreased , by mechanical ventilation supports a dynamic role for ectonucleotidases in the regulation of purine signaling in the airways . The role of P2 receptors in innate defense against bacterial infection was investigated using mice deficient in P2Y1 , P2Y2 or both receptors -LSB- 45 -RSB- . All these mice exhibited lower survival and lower cytokine levels in lung homogenates than wild-type animals measured 24 h after intranasal instillation of P. aeruginosa . These results suggest a protective role for ATP-induced inflammation -LSB- 45 -RSB- . In human airways , P2Y2 receptor activation amplified inflammatory responses to bacterial infection . Primary cultures of bronchial epithelial cells exposed to UTP and sputum from CF patients exhibited an eight-fold increase in IL-8 secretion , compared to two-fold with UTP alone -LSB- 46 -RSB- . Furthermore , P2X7 receptors are known to support several processes relevant to inflammation , including LPS neutralization -LSB- 47 -RSB- , pathogen killing , nitric oxide production -LSB- 1 -RSB- and cytokine release from mast cells , leukocytes , dendritic cells and neurons -LSB- 3 , 14 -RSB- . In human blood , LPS-induced IL-1β release is increased 5-fold by P2X7 receptor activation -LSB- 48 -RSB- . Altogether , these studies entail that P2 receptor activation may not only induce innate immunity but also amplify pathogen-induced inflammatory responses in chronic infectious lung diseases . Too much of a good thing . . Excessive inflammatory response to recurrent bacterial infection is a major component in the pathogenesis of chronic lung diseases , irreversibly damaging the airways and leading to bronchiectasis and respiratory failure -LSB- 49 -- 51 -RSB- . With respect to purine signaling , nucleotide-mediated cell communications may be overwhelmed by massive ATP release from cell lysis of bacteria and damaged epithelia , as well as stimulated leukocytes and epithelia -LSB- 6 -RSB- . The sensitivity of certain P2 receptors -LRB- P2Y2 -LSB- 42 -RSB- , P2X1 and P2X3 -LSB- 52 -RSB- -RRB- to agonist-induced desensitization may reduce the efficiency of nucleotide-mediated MCC -LSB- 53 -RSB- . On the other hand , chronically elevated ATP may recruit signaling pathways normally dormant in healthy lungs . For instance , P2X7 receptor activation only induces cytokine release and cell death in response to high ATP concentrations encountered in damaged tissues -LSB- 23 -RSB- . On astrocytes , LPS-induced TNFα secretion is increased by P2Y receptor activation at low micromolar ATP , but reduced by P2X7 receptor activation at high ATP concentrations -LSB- 54 -RSB- . The authors propose a beneficial effect for the potentiation of TNFα secretion by P2Y receptors during mild or acute inflammation and a protective role for P2X7 receptors in damaged tissues or chronic infectious diseases . These studies suggest that P2X7 receptor-mediated responses may be tissue-specific and/or influenced by bacterial infections . In the respiratory system , P2Y2 receptors are expressed throughout airway epithelia -LSB- 39 -RSB- while P2X7 receptors have only been reported in nasal polyps of CF patients -LSB- 22 -RSB- . The impact of P2X and P2Y receptor activation on inflammatory responses mediated by airway epithelia under normal and pathological conditions remains to be investigated . E-NTPDases regulate airway ATP Several studies demonstrate the presence of ectonucleotidase activities on human airway epithelia -LSB- 9 , 10 , 55 -- 58 -RSB- . Time-course experiments conducted on polarized primary cultures of human bronchial epithelial cells showed that mucosal ATP is dephosphorylated into ADP , AMP and adenosine -LRB- Fig. 2a -RRB- . Experiments repeated with nasal , bronchial and bronchiolar epithelial cultures indicated that ATP metabolism accelerates toward the alveolar region -LRB- Fig. 2b -RRB- . Such gradient distribution was also reported for non-specific alkaline phosphatase -LRB- NS AP -RRB- in human airways -LSB- 9 -RSB- , an ectonucleotidase capable of sequentially dephosphorylating ATP to adenosine -LSB- 59 -RSB- . However , complete inhibition of NS AP with 10 mM levamisole -LSB- 9 -RSB- only reduced by 25 % total ectoATPase activities -LSB- 10 -RSB- . Other ATP-regulating ectonucleotidases were identified in mammalian tissues as members of the ENTPDase family -LSB- 15 , 60 -RSB- . They catalyze the hydrolysis of γ - and/or β-phosphate residues , resulting in the formation of nucleoside monophosphates . Their activities are Ca2 + and Mg2 + - dependent and insensitive to inhibitors of P - , F - and V-type ATPases or APs -LSB- 61 -RSB- . Four E-NTPDases are localized to cell surfaces : NTPDase 1 , 2 , 3 and 8 . Functional studies showed that NTPDase 1 -LSB- 62 -RSB- and NTPDase 3 -LSB- 63 -RSB- are inhibited by azide , whereas NTPDase 2 -LSB- 64 -RSB- and NTPDase 8 -LSB- 65 -RSB- are insensitive . The mRNA expression of NTPDase 1 , 2 and 3 in human airways was reported in total lung RNA -LSB- 66 -RSB- and cultured bronchial epithelial cells -LSB- 67 -RSB- . We now provide evidence that NTPDase 1 and NTPDase 3 are both expressed throughout human airways by real-time PCR on freshly excised epithelial cells -LRB- Fig. 3 -RRB- . Their expression distribution followed surface ectoATPase activities -LRB- Fig. 2b -RRB- , exhibiting higher mRNA levels in smaller airways -LRB- Fig. 3 -RRB- . The fact that 20 mM azide reduced total ectoATPase activities by 45 % on human bronchial epithelial cells -LSB- 67 -RSB- suggests that the high affinity -LRB- Km < 15 µM -RRB- -LSB- 68 -RSB- NTPDases 1 and/or 3 play major roles in the regulation of physiological ASL nucleotide concentrations . Fig. 2Ectonucleotidases regulate nucleotide concentrations on airway epithelial surfaces . a Polarized primary cultures of human bronchial epithelial cells were assayed with 100 µM ATP added to the mucosal surface , as we previously described -LSB- 10 -RSB- . Buffer sample analysis by high-pressure liquid chromatography -LRB- 10 -RRB- shows that exogenous ATP -LRB- • -RRB- is dephosphorylated into ADP -LRB- ▪ -RRB- , AMP -LRB- ≆ -RRB- and adenosine -LRB- ○ -RRB- . b Similar experiments repeated on primary cultures of human nasal , bronchial and bronchiolar epithelial cells show that the elimination rate of ATP increases toward alveoli -LRB- N = 3 , * , P < 0.05 ; Mann-Whitney test -RRB- . Fig. 3Expression level of NTPDase 1 and NTPDase 3 along human airways . Total RNA from excised epithelia was analyzed by real-time PCR using SYBR green assays and normalized to the expression level of the house-keeping gene , 18S , as we previously described -LSB- 89 -RSB- . The mRNA level of E-NTPDase 1 -LRB- □ -RRB- and NTPDase 3 -LRB- ▪ -RRB- increases toward alveoli -LRB- N = 4 , * , P < 0.05 ; Mann-Whitney test -RRB- . The physiological importance of azide-sensitive E-NTPDases for nucleotide-mediated MCC was recently established using an in vitro model of rhythmic breathing . Airway epithelia are continuously subjected to mechanical stress generated by breathing , coughing or chest movement . Since mechanical stress induces epithelial ATP release -LSB- 6 -RSB- , static culture conditions may underestimate the lungs capacity to regulate MCC in vivo . Rhythmic pressure changes mimicking normal tidal breathing were reproduced in vitro by a system applying cyclic compressive stress -LRB- CCS -RRB- to the mucosal surface of primary bronchial epithelial cultures -LSB- 69 -RSB- . Whereas CF cultures under static conditions exhibit a depleted PCL layer and mucostasis , CCS mimicking normal tidal breathing -LRB- 20 cmH2O ; 15 cycles/min -RRB- restored normal PCL height and mucus transport through ATP release and P2Y2 receptor activation . These results also suggest that purine signaling may provide an explanation for the beneficial effects of oscillatory therapeutic devices clinically used to stimulate sputum clearance -LSB- 70 -RSB- . Cyclic compressive stress also enhances MCC through a reduction in ASL nucleotide metabolism . We recently demonstrated that CCS decreases the rate of ATP hydrolysis on the mucosal surface of normal and CF bronchial epithelial cultures -LSB- 67 -RSB- . More importantly , CCS restored normal ectoATPase activities on CF epithelial surfaces . The inhibitory effect of CCS on ATP metabolism was abrogated by 20 mM azide , indicating that E-NTPDase 1 and/or E-NTPDase 3 are key components in the regulation P2 receptormediated MCC . Perhaps the ability of CF airway epithelia to provide adequate nucleotide-mediated MCC under conditions mimicking normal breathing may contribute to the relatively healthy state of young patients before the establishment of chronic infection -LSB- 71 -RSB- . Chronic lung diseases shorten the signals Recent studies demonstrate that chronic lung diseases accelerate all metabolic steps supporting the mucosal conversion of ATP to adenosine -LSB- 10 , 72 -RSB- . We identified the ectonucleotidases involved using primary bronchial epithelial cultures from healthy donors and patients with primary ciliary dyskinesia , CF or α1-antitrypsin deficiency . All airway diseases were characterized by a two - to four-fold increase in the elimination rate of ATP on the mucosal surface , without significant effect on the serosal surface -LRB- Fig. 4 -RRB- . Selective inhibitors of NS AP -LRB- 10 mM levamisole -RRB- , NTPDase 1 and NTPDase 3 -LRB- 20 mM azide -RRB- partially restored normal activity levels . However , the lower substrate affinities of airway NS AP -LRB- 36 µM and 717 µM -RRB- -LSB- 9 -RSB- suggest that NTPDase 1 and/or NTPDase 3 are responsible for the accelerated clearance of P2 receptor agonists in diseased airways . Fig. 4Chronic lung diseases enhance azide-sensitive E-NTPDases . Primary bronchial epithelial cultures from healthy donors -LRB- N -RRB- and patients diagnosed with primary ciliary dyskinesia -LRB- PCD -RRB- , CF or α1-antitrypsin deficiency -LRB- α AT -RRB- were assayed with bilateral 1 mM ATP in the absence -LRB- ▪ -RRB- or presence -LRB- □ -RRB- of 20 mM azide , as we previously described -LSB- 10 -RSB- . Analysis of buffer samples by high-pressure liquid chromatography showed that azide-sensitive E-NTPDases are concentrated on the mucosal surface under normal and pathological conditions -LRB- N = 4 , * , P < 0.05 ; Mann-Whitney test -RRB- . Pseudomonas aeruginosa is among the most frequently isolated pathogens from the airways of patients with gram-negative infections -LSB- 51 -RSB- . In patients with damaged airways from mechanical ventilation , trauma or viral infection , airway colonization by P. aeruginosa is often followed by pneumonia , sepsis and death . Much of the airway inflammation induced by these organisms is caused by released bacterial products adhering to the epithelial surface , such as LPS . The contribution of bacterial infection to the deregulation of E-NTPDase activities in lung diseases was examined using P. aeruginosa LPS . Primary cultures of human bronchial epithelial cells exposed 24 h on the mucosal surface to an optimum LPS concentration -LRB- 100 ng/ml -RRB- -LSB- 73 -RSB- exhibited a biphasic response over time -LRB- Fig. 5A -RRB- . Azide-sensitive E-NTPDases were transiently reduced over 8 h , without affecting mRNA expression . In contrast , prolonged exposures -LRB- 24 h -RRB- raised the activities two-fold above control levels and increased NTPDase 1 and NTPDase 3 expression by 4 and 10-fold -LRB- Fig. 5B -RRB- . These opposite effects suggest that two regulatory mechanisms may be involved in the acute and chronic effects of LPS on airway E-NTPDases . Fig. 5Impact of P. aeruginisa LPS on NTPDase 1 and NTPDase 3 . A The mucosal surface of primary bronchial epithelial cultures were exposed 0 , 1 , 8 or 24 h to 100 ng/ml LPS , then assayed for surface activity by high-pressure liquid chromatography , as we previously described -LSB- 10 -RSB- . The activities of azidesensitive mucosal E-NTPDases measured with 0.03 mM ATP were transiently reduced by LPS . B Quantification of their mRNA expression by real-time PCR using the house-keeping gene 18S , as we previously described -LRB- 89 -RRB- . LPS induced a delayed increase in the expression level of both E-NTPDases -LRB- N = 5 , * , P < 0.05 ; Mann-Whitney test -RRB- . Oxidative stress is one of the major causes of epithelial injury in chronic lung diseases -LSB- 74 -RSB- . Reactive oxygen and nitrogen species released from airway epithelia or activated leukocytes damage cell membranes by peroxidation of lipids , amino acids and carbohydrates . P aeruginosa LPS was reported to induce their release from airway epithelia -LSB- 75 -RSB- . Incidentally , intrahepatic cholestasis induced by intraperitoneal injection of LPS reduced total liver NTPDase 1 activity within 2 h -LSB- 76 -RSB- . In a rat model of glomerulopathy , short-term exposure -LRB- 1 h -RRB- to LPS inactivated endothelial NTPDase 1 , which was prevented by pre-treatment with the anti-oxidant superoxide dismutase -LSB- 77 -RSB- . Oxidative stress induced by membrane depolarization -LSB- 78 -RSB- or ischemia / reperfusion -LSB- 79 -RSB- also inhibited endothelial NTPDase 1 . Furthermore , short exposures -LRB- 4 h -RRB- to the pro-inflammatory cytokine , TNFα , inhibited endothelial NTPDase 1 , which was mimicked by hydrogen peroxide but prevented by superoxide dismutase -LSB- 80 -RSB- . Altogether , these studies suggest that oxidative stress may be responsible for the early and transient reduction in azide-sensitive E-NTPDase activities detected in LPS-treated airway epithelia . Since the mRNA expression of E-NTPDases is unaffected by acute oxidative stress -LSB- 80 -RSB- or LPS -LRB- Fig. 5B -RRB- , and the inactivation of NTPDase 1 by ischemia/reperfusion was prevented by inhibitors of lipid peroxidation -LSB- 79 -RSB- , the early loss of surface activity may involve local peroxidation of membrane lipids . The long-term effects of LPS on the azide-sensitive E-NTPDases have not been documented in mammalian tissues . On the other hand , a 24 h exposure to endotoxin stimulated NTPDase 2 on astrocytes -LSB- 81 -RSB- , as well as NS AP on endothelial , mesengial -LSB- 82 , 83 -RSB- and bronchial epithelial -LSB- 73 -RSB- cells . Additionally , the mRNA expression of NTPDase 1 in rat forebrain was up-regulated seven days after transient ischemia -LSB- 84 -RSB- . P. aeruginosa LPS -LSB- 85 , 86 -RSB- is well-known to influence gene expression through the activation of the transcription factors : Nuclear factor-kappa B and activator protein-1 -LSB- 87 -RSB- . Recent studies have shown that CFTR acts as a pattern recognition molecule for P. aeruginosa LPS -LSB- 88 -RSB- . Endocytosis of the complex triggers translocation of these transcription factors to the nucleus , which initiates innate immunity through the expression of numerous mediators recruiting and activating inflammatory cells . These signaling pathways could be involved in the up-regulation of NTPDase 1 and NTPDase 3 expression resulting from long-term exposures to P. aeruginosa LPS in human airways . Adaptation of purine signaling to airway diseases In past decade , the therapeutic potential of aerosolized nucleotides for the treatment of chronic obstructive lung diseases has motivated numerous studies on the importance of purine signaling in the airways . This elaborated cell communication system is involved in major defense mechanisms including bacterial clearance and inflammatory responses to bacterial infection . Each signaling event constitutes a brief ` on-and-off ' switch mechanism allowing the target cells to perceive the subsequent signal . Environmental changes are communicated to target cells in the form of released ATP , which binds surface receptors and triggers an appropriate response . The efficiency of this communication system depends on the cells capacity to rapidly eliminate these local bursts in extracellular ATP concentrations to prevent receptor desensitization . This review establishes the pivotal role of E-NTPDases in the regulation of airway defenses mediated by purine signaling , namely bacterial clearance -LRB- Fig. 6 -RRB- . Cyclic compressive stress , mimicking normal breathing or clinical oscillatory devices , stimulates P2 receptormediated MCC through ATP release and reduced azide-sensitive E-NTPDase activities . We also presented original data indicating that NTPDase 1 and 3 activities and expressions are up-regulated in chronic lung diseases by inflammatory mediators , including endotoxin . Interestingly , P. aeruginosa LPS generated a biphasic modulation of airway E-NTPDases which may reconcile previous conflicting reports based on exposure duration . Acute LPS exposures reduced ENTPDase activities , as observed during CCS , most likely to clear a pathogen through P2 receptor-mediated MCC . The signaling pathways activated by P. aeruginosa LPS and the lack of concomitant reduction in ENTPDase mRNA levels support a local inhibitory mechanism that may involve oxidative stress-mediated membrane damage . On the other hand , prolonged endotoxin exposures mimicked the impact of chronic lung diseases on NTPDases 1 and 3 , characterized by an up-regulation of both activities and mRNA expression . In chronically infected lungs , an increase in E-NTPDase activities may represent an attempt to prevent lung damage , excessive inflammation and P2 receptor desensitization by chronically elevated ATP concentrations . Fig. 6Purine signaling and nucleotide salvage pathways on human airway epithelia . In normal lungs , basal adenosine levels maintain adequate PCL height for mucus transport through A2B receptor-mediated CFTR activity . Mechanical stimulation of the epithelial surface by an irritant , a pathogen , mechanical ventilation or cyclic compressive stress induces ATP release and elevates ASL concentrations above activation threshold level for P2Y2 receptor activation . This autocrine signal transiently enhances basal MCC through cilia beating , mucin secretion and ion/water efflux into the lumen . The signal is rapidly terminated by NTPDase 1 and/or NTPDase 3 dephosphorylating excess ATP to AMP . Ecto 5 ′ - nucleotidase -LRB- ecto 5 ′ - NT ; CD73 -RRB- produces ASL adenosine from the resulting AMP , and excess adenosine is transported back to the cytosol . Chronic lung diseases burdened by considerable tissue damage recruits additional receptors -LRB- P2X4 , 5 , 7 -RRB- responding to higher ATP cocnentrations . The information provided in this review also exposes the complexity of the signaling interactions taking place between different classes of mediators , including bacterial products , cytokines and extracellular nucleotides , as combinations of mediators may induce different responses than when tested individually . Furthermore , communication networks in the airways may evolve with time during the establishment of a chronic infection , as new target cells -LRB- bacteria , leukocytes , lymphocytes -RRB- agglomerate on either side of the epithelial barrier . It is proposed that nucleotide-mediated airway functions defined in normal tissues or aseptic culture conditions may require reassessment under conditions mimicking chronic lung diseases before their therapeutic potential may be clearly established .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

bacterial clearance
endotoxin
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chrosobstructive lung diseases
diphosphohydrolase
purinergic signaling


Simultaneous

prescribing

of

atypical

antipsychotics

conventional

antipsychotics

and

anticholinergics

a

European

study

Objective

The

aim

of

this

study

was

to

investigate

to

what

extent

atypical

antipsychotics

conventional

antipsychotics

and

anticholinergics

are

prescribed

simultaneously

in

daily

clinical

practice

in

Europe

Introduction

All

antipsychotics

are

solely

registered

for

use

as

monotherapy

The

prescription

of

two

or

more

antipsychotics

simultaneously

also

called

antipsychotic

polypharmacy

has

no

empirical

foundation

-LSB-

-RSB-

However

in

the

past

antipsychotic

polypharmacy

with

two

conventional

-LRB-

early

generation

-RRB-

antipsychotics

was

frequently

practised

with

reported

frequencies

ranging

from

approximately

-LSB-

-RSB-

In

the

last

years

the

conventional

antipsychotics

are

increasingly

replaced

by

the

atypical

-LRB-

second

generation

-RRB-

antipsychotics

Few

studies

from

the

UK

and

the

US

have

focused

on

antipsychotic

polypharmacy

with

atypical

antipsychotics

and

reported

frequencies

ranging

from

to

-LSB-

-RSB-

We

carried

out

a

pharmaco-epidemiological

study

to

atypical

antipsychotic

polypharmacy

and

included

data

of

patients

of

several

psychiatric

hospitals

in

various

European

countries

Additionally

we

also

focused

on

the

co-prescription

of

anticholinergic

drugs

There

is

considerable

evidence

that

atypical

antipsychotics

cause

fewer

extrapyramidal

side

effects

than

conventional

antipsychotics

-LSB-

-RSB-

Therefore

there

should

be

less

need

for

anticholinergic

co-medication

to

be

prescribed

with

the

atypicals

This

European

study

was

conducted

to

investigate

to

what

extent

atypical

antipsychotics

conventional

antipsychotics

and

anticholinergics

are

being

prescribed

simultaneously

in

daily

clinical

practice

Method

The

study

was

carried

out

with

the

co-operation

of

members

of

the

European

Pharmacists

for

Psychiatry

and

Neurology

-LRB-

EPPN

-RRB-

an

organisation

of

hospital

pharmacists

working

in

psychiatry

and

neurology

All

participating

hospital

pharmacists

were

working

in

psychiatric

hospitals

Data

were

collected

between

November

and

October

In

that

period

the

hospital

pharmacists

screened

their

hospital

pharmacy

database

on

one

single

day

for

patients

who

had

been

using

an

atypical

antipsychotic

for

a

minimum

of

weeks

The

period

was

chosen

so

that

titration

to

the

atypical

antipsychotic

should

have

been

completed

No

restrictions

were

imposed

with

regard

to

age

and

diagnosis

The

following

data

were

collected

for

each

patient

included

age

sex

type

and

dose

of

the

atypical

-LRB-

s

-RRB-

and

if

prescribed

type

and

dose

of

conventional

antipsychotics

-LRB-

maximum

of

four

-RRB-

and

the

type

of

anticholinergics

-LRB-

maximum

of

four

-RRB-

As

needed

-LRB-

PRN

-RRB-

medication

was

considered

as

used

medication

The

doses

of

antipsychotic

depot-medications

were

recalculated

to

daily

oral

doses

-LSB-

-RSB-

All

data

were

checked

for

improbable

doses

duplication

of

patients

and

other

errors

Median

doses

of

the

antipsychotics

are

given

because

of

positively

skewed

distributions

The

frequencies

with

which

antipsychotic

polypharmacy

was

practised

and

anticholinergics

were

prescribed

are

calculated

for

the

total

group

of

patients

and

per

individual

atypical

antipsychotic

Conventional

antipsychotics

with

a

chlorpromazine

equivalent

dose

of

more

than

mg

-LRB-

compared

to

mg

chlorpromazine

-RRB-

are

considered

to

be

low

potency

drugs

-LSB-

-RSB-

The

frequencies

with

which

antipsychotic

polypharmacy

was

practised

are

calculated

with

and

without

the

low-potent

antipsychotics

Results

Hospital

pharmacists

from

six

West

European

countries

participated

Belgium

Denmark

France

Germany

The

Netherlands

and

Scotland

In

total

data

were

received

for

patients

Of

these

patients

-LRB-

-RRB-

were

excluded

for

the

following

reasons

patients

mentioned

twice

-LRB-

n

-RRB-

no

atypical

antipsychotic

prescribed

-LRB-

n

-RRB-

improbably

low

clozapine

doses

-LRB-

n

-RRB-

and

other

errors

-LRB-

n

-RRB-

Table

shows

details

of

the

participating

countries

and

patients

Most

patients

came

from

Belgium

-LRB-

-RRB-

followed

by

The

Netherlands

-LRB-

-RRB-

and

France

-LRB-

-RRB-

Table

of

hospitals

and

patients

included

in

the

participating

countries

with

the

mean

age

of

patients

and

the

percentages

of

male

patientsCountryNumber

of

hospitals

-LRB-

-RRB-

Number

of

patients

-LRB-

-RRB-

Mean

age

-LRB-

sd

-RRB-

of

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

The

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Table

shows

the

frequencies

with

which

the

atypical

antipsychotics

were

prescribed

the

median

dose

the

frequencies

with

which

of

antipsychotic

polypharmacy

-LRB-

with

and

without

the

low-potent

conventional

antipsychotics

-RRB-

was

practised

the

frequencies

with

which

anticholinergics

were

administrated

and

the

frequencies

of

no

co-prescription

of

antipsychotics

or

anticholinergics

Table

Details

of

the

atypical

antipsychotics

prescribed

their

frequencies

median

dose

the

frequencies

of

patients

prescribed

antipsychotic

polypharmacy

-LRB-

with

and

without

low-potent

antipsychotics

-RRB-

and

anticholinergics

the

frequencies

of

no

co-prescription

of

antipsychotics

and

anticholinergics

-LRB-

n

-RRB-

Atypical

antipsychoticNumber

of

patients

-LRB-

-RRB-

Median

dose

-LRB-

mg

-RRB-

Antipsychotic

polypharmacy

-LRB-

-RRB-

-LRB-

including

low-potent

antipsychotics

-RRB-

Antipsychotic

polypharmacy

-LRB-

-RRB-

-LRB-

excluding

low-potent

antipsychotics

-RRB-

Anticholinergic

prescription

-LRB-

-RRB-

No

co-prescription

of

antipsychotics

or

anticholinergics

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Two

-LRB-

-RRB-

-LRB-

-RRB-

See

Table

for

the

classification

of

the

low-potent

antipsychotics

Total

median

dose

not

calculated

By

definition

By

definition

Risperidone

was

prescribed

most

often

-LRB-

-RRB-

followed

by

clozapine

-LRB-

-RRB-

olanzapine

-LRB-

-RRB-

and

others

-LRB-

in

total

-RRB-

For

patients

-LRB-

-RRB-

atypical

antipsychotics

were

prescribed

the

combinations

used

most

were

clozapine/risperidone

-LRB-

n

-RRB-

olanzapine/risperidone

-LRB-

n

-RRB-

and

sulpiride/risperidone

-LRB-

n

-RRB-

In

our

population

of

patients

using

an

atypical

antipsychotic

polypharmacy

was

prescribed

in

of

cases

Of

patients

receiving

antipsychotic

polypharmacy

and

were

prescribed

two

three

four

and

five

or

more

antipsychotics

respectively

Table

shows

the

antipsychotics

prescribed

concurrently

with

the

atypicals

The

conventional

antipsychotics

prescribed

most

often

were

levomepromazine

haloperidol

prothipendyl

cyamemazine

and

zuclopentixol

When

the

low-potent

conventional

antipsychotics

are

not

included

in

the

analysis

of

the

patients

was

prescribed

antipsychotic

polypharmacy

Table

frequencies

and

median

doses

antipsychotics

prescribed

concurrently

for

patients

using

atypical

antipsychoticsAntipsychoticAntipsychotic

prescribed

concurrently

-LRB-

-RRB-

Median

dose

-LRB-

mg

-RRB-

Levomepromazine

Prothipendyl

Cyamemazine

Pipamperon

Thioridazine

Chlorpromazine

Chlorprothixeen

Other

conventional

Second

Considered

as

low-potent

antipsychotic

In

total

of

the

patients

were

prescribed

an

anticholinergic

drug

The

anticholinergics

prescribed

most

often

were

biperiden

-LRB-

-RRB-

procyclidine

-LRB-

-RRB-

trihexyphenidyl

-LRB-

-RRB-

and

tropatepine

-LRB-

-RRB-

In

total

of

the

patients

are

prescribed

the

atypical

antipsychotic

without

any

other

antipsychotic

or

anticholinergic

are

prescribed

an

atypical

plus

one

or

more

other

antipsychotics

are

given

an

atypical

plus

an

anticholinergic

and

an

atypical

plus

an

anticholinergic

and

one

or

more

other

antipsychotics

Discussion

This

European

study

clearly

shows

that

it

is

common

clinical

practice

to

prescribe

an

atypical

antipsychotic

and

one

or

more

other

antipsychotics

simultaneously

Low-potent

antipsychotics

may

be

added

to

an

atypical

antipsychotic

mainly

because

of

their

sedative

anxiolytic

and

anticholinergic

properties

rather

than

because

of

their

antipsychotic

properties

Even

when

the

low-potent

conventional

antipsychotics

are

not

included

in

the

analysis

we

found

that

antipsychotic

polypharmacy

was

prescribed

for

almost

a

quarter

of

the

patients

Additionally

we

did

not

expect

to

find

that

almost

a

third

of

the

patients

were

prescribed

an

anticholinergic

in

addition

to

the

atypical

drug

The

results

of

this

study

imply

that

more

than

half

-LRB-

-RRB-

of

the

patients

are

not

being

treated

in

the

way

according

to

the

psychiatric

handbooks

and

guidelines

The

atypical

is

frequently

combined

with

another

antipsychotic

and/or

an

anticholinergic

Several

studies

have

shown

that

antipsychotic

polypharmacy

with

two

conventional

antipsychotics

was

in

frequent

use

in

clinical

practice

-LSB-

-RSB-

Others

have

shown

that

fairly

large

numbers

of

patients

-LRB-

ranging

from

to

-RRB-

are

being

prescribed

a

therapy

that

combines

clozapine

with

a

conventional

antipsychotic

-LSB-

-RSB-

Recently

a

few

studies

have

focused

on

antipsychotic

polypharmacy

with

the

atypical

antipsychotics

with

reported

frequencies

ranging

from

to

-LSB-

-RSB-

There

could

be

several

reasons

to

explain

this

large

variation

in

co-prescribing

such

as

the

definition

of

antipsychotic

polypharmacy

whether

inpatients

or

outpatients

were

included

stage

of

the

disease

the

differences

in

prescription

patterns

between

countries

and

hospitals

and

different

use

of

guidelines

We

consider

several

hypotheses

for

the

relatively

high

rate

of

antipsychotic

polypharmacy

involving

atypicals

First

of

all

it

could

be

that

the

patients

with

an

inadequate

treatment

response

to

one

antipsychotic

are

treated

with

combinations

of

antipsychotics

including

the

atypical

antipsychotics

However

there

are

hardly

any

studies

that

show

that

antipsychotic

polypharmacy

is

effective

in

patients

who

do

not

respond

to

one

antipsychotic

-LSB-

-RSB-

Secondly

it

is

also

possible

that

antipsychotic

polypharmacy

is

continued

although

the

patient

shows

no

improvement

because

psychiatrists

are

hesitant

to

discontinue

any

medication

in

patients

with

persistent

psychotic

symptoms

Thirdly

it

could

be

that

in

some

patients

the

recommended

doses

of

the

atypical

antipsychotics

are

too

low

to

be

effective

and

that

adding

a

second

antipsychotic

is

in

fact

a

dose-increase

strategy

In

such

cases

however

a

higher

dose

of

one

particular

atypical

might

be

just

as

effective

Fourthly

when

switching

between

two

antipsychotics

it

is

common

to

titrate

the

first

drug

downwards

while

simultaneously

titrating

the

second

drug

upwards

If

the

patient

responds

halfway

through

the

titrating

process

the

clinician

might

decide

to

continue

both

antipsychotics

a

situation

referred

to

as

the

cross-titration

trap

-LSB-

-RSB-

Our

study

included

only

patients

who

had

been

using

an

atypical

antipsychotic

for

more

than

weeks

so

the

titrating

process

would

normally

have

been

completed

It

is

remarkable

that

the

potential

risks

of

antipsychotic

polypharmacy

have

received

limited

research

attention

-LSB-

-RSB-

However

Waddington

et

al.

-LSB-

-RSB-

found

that

antipsychotic

polypharmacy

is

associated

with

reduced

survival

among

patients

with

schizophrenia

Centorrino

et

al.

-LSB-

-RSB-

reports

that

the

risk

of

adverse

effects

is

higher

with

antipsychotic

polypharmacy

There

have

been

previous

reports

of

anticholinergics

being

added

to

atypical

antipsychotics

Of

patients

using

clozapine

-LSB-

-RSB-

and

-LSB-

-RSB-

were

also

prescribed

anticholinergics

Anticholinergics

were

added

to

of

patients

using

atypical

antipsychotics

-LSB-

-RSB-

These

numbers

are

in

line

with

our

finding

-LRB-

-RRB-

The

prescription

of

this

combined

therapy

could

be

attributed

to

the

use

of

a

second

almost

always

conventional

antipsychotic

-LRB-

-RRB-

which

might

induce

extrapyramidal

symptoms

But

nevertheless

of

patients

are

prescribed

an

atypical

antipsychotic

in

combination

with

an

anticholinergic

without

any

other

-LRB-

conventional

-RRB-

antipsychotics

Minzenberg

et

al.

-LSB-

-RSB-

studied

the

effects

of

anticholinergic

properties

of

psychiatric

medications

and

found

clinically

significant

effects

on

memory

and

complex

attention

The

frequencies

of

atypical

polypharmacy

in

the

separate

countries

range

between

-LRB-

Germany

-RRB-

and

-LRB-

Belgium

-RRB-

without

the

low-potent

antipsychotics

it

varies

between

-LRB-

Denmark

-RRB-

and

-LRB-

Belgium

-RRB-

However

these

numbers

should

be

appraised

cautiously

because

this

study

was

not

primary

set

up

to

detect

differences

between

the

various

countries

-LRB-

some

countries

are

under

represented

-RRB-

Furthermore

the

study

does

not

allow

to

draw

inferences

about

differences

between

the

various

atypicals

in

antipsychotic

polypharmacy

and

anticholinergic

use

These

differences

could

easily

be

biased

as

a

result

of

the

different

introduction

data

of

the

atypicals

in

the

diverse

countries

For

example

at

the

moment

of

data-collection

olanzapine

and

sertindole

had

been

introduced

very

recently

in

most

countries

quetiapine

was

available

only

in

Scotland

and

zotepine

was

used

only

in

a

clinical

trial

Nevertheless

the

similarity

between

the

frequencies

with

which

antipsychotic

polypharmacy

is

prescribed

for

the

various

atypicals

-LRB-

excluding

quetiapine

and

zotepine

on

account

of

their

low

frequencies

-RRB-

is

remarkable

Excluding

low-potent

antipsychotics

the

frequencies

range

from

to

The

study

however

has

its

limitations

First

of

all

the

population

included

is

not

a

random

sample

from

Western

European

countries

that

participated

Therefore

hypothetically

it

could

be

argued

that

the

results

were

biased

because

only

hospitals

practising

extremely

high

rates

of

antipsychotic

polypharmacy

were

included

However

it

is

unlikely

that

only

such

hospitals

were

selected

Secondly

we

estimate

on

the

basis

of

the

information

obtained

from

the

participating

hospitals

that

of

the

data

are

from

inpatients

The

symptomatology

of

inpatients

may

be

more

severe

than

that

of

outpatients

and

such

patients

may

be

prescribed

antipsychotic

polypharmacy

more

frequently

than

outpatients

-LSB-

-RSB-

Thirdly

we

did

regard

PRN-medication

as

used

medication

This

may

have

heightened

our

results

somewhat

but

can

not

explain

the

frequent

prescription

of

antipsychotic

polypharmacy

A

major

advantage

of

this

study

is

the

large

number

of

patients

included

and

the

differentiation

made

between

high

and

low-potent

antipsychotics

This

makes

extrapolation

more

feasible

Conclusion

It

is

common

practice

to

prescribe

a

combination

of

atypical

and

conventional

antipsychotics

Furthermore

the

use

of

atypical

antipsychotics

does

rule

out

the

use

of

anticholinergics

On

the

contrary

atypical

antipsychotics

are

often

prescribed

in

combination

with

anticholinergics

Apparently

monotherapy

involving

the

atypicals

is

not

considered

to

be

an

effective

therapy

for

a

substantial

number

of

patients

in

clinical

practice

Possible

conflicts

of

interest

P.N.

van

Harten

gives

lectures

on

symposia

and

congresses

that

are

sponsored

by

pharmaceutical

companies

O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

antipsychotic agents
drug therapy combination
drug use study
pharmaco-epidemiology


Oxidative Stress and the Induction of Cyclooxygenase Enzymes and Apoptosis in the Murine Placenta Placental oxidative stress has been implicated in many complications of human pregnancy , including preterm delivery and preeclampsia . It is now appreciated that reactive oxygen species can induce a spectrum of changes , ranging from homeostatic induction of enzymes to apoptotic cell death . Little is known regarding the occurrence of placental oxidative stress in other species . We investigated markers of oxidative stress in the labyrinthine -LRB- LZ -RRB- and junctional -LRB- JZ -RRB- zones of the murine placenta across gestational age , and correlated these with expression of the cyclooxygenase enzymes COX-1 and COX-2 , and apoptosis . We tested a causal link between the two by subjecting placental explants to hypoxia-reoxygenation -LRB- H/R -RRB- in vitro , a known stimulus for generation of oxidative stress . Western blotting demonstrated significant increases in the concentrations of hydroxynonenal -LRB- HNE -RRB- , COX-1 and COX-2 with gestational age . Dual-labelling demonstrated co-localisation of HNE , and COX-1 and COX-2 within the trophoblast of the LZ , and glycogen cells of the JZ . An apoptotic index based on TUNEL-positivity demonstrated an increase with gestational age , and dual-labelling showed co-localisation of TUNEL labelling with HNE and active caspase-3 within the trophoblast of the LZ . H/R significantly increased oxidative stress , induction of COX-1 and COX-2 , and the apoptotic index . Co-localisation demonstrated the increases in COX to be within the trophoblast of the LZ , and in particular the glycogen cells of the JZ . Apoptosis was restricted to the LZ . We speculate that the induction of COX enzymes is a physiological response to oxidative stress , and may play a role in initiating or augmenting parturition . Generation of oxidative stress may also play a role in influencing the growth trajectory of the placenta , and its component cell types . The mouse may provide an experimental genetic model in which to investigate these phenomena . 1 Introduction Oxidative stress of the placenta has been implicated in the pathogenesis of many complications of human pregnancy , including miscarriage , preeclampsia , and preterm labour -LSB- 1 -- 3 -RSB- . These complications all share the common predisposing feature of reduced trophoblast invasion and incomplete conversion of the uterine spiral arteries -LSB- 4,5 -RSB- . Therefore , it is generally held that malperfusion of the placenta leads to increased oxidative stress , resulting in placental dysfunction . In the case of preeclampsia it is thought that the stress induces the release of a cocktail of factors , including pro-inflammatory cytokines , anti-angiogenic factors and apoptotic debris -LSB- 6 -RSB- , into the maternal circulation that causes activation of the peripheral endothelial cells . Placental inflammatory lesions have also been associated with premature onset of labour -LSB- 7 -RSB- . By contrast , there are few data relating to placental oxidative stress available for other species . The mouse is potentially a powerful model in which to study the mechanisms of placental and obstetrical pathologies , and is the best-studied mammalian experimental genetic model system . The definitive murine placenta is a discoid haemochorial organ as in the human , and there are three anatomically and physiologically distinct regions : the labyrinth zone -LRB- LZ -RRB- , junctional zone -LRB- JZ -RRB- and decidua basalis -LRB- DB -RRB- -LSB- 8 -RSB- . The labyrinth zone is the principal area of exchange , and consists of a meshwork of maternal blood spaces lined by trophoblast . The trophoblast comprises three layers ; an outer layer -LRB- I -RRB- composed of cytotrophoblast cells , and two layers -LRB- II & III -RRB- of syncytiotrophoblast -LSB- 9 -RSB- . Deep to the syncytiotrophoblast are the fetal capillaries , embedded in a small amount of connective tissue . The junctional zone , interposed between the labyrinth and the decidua basalis , is of unknown function , but contains spongiotrophoblast cells and trophoblastic glycogen cells -LRB- GC -RRB- -LSB- 8 -RSB- . The decidua basalis is composed principally of maternal uterine tissues , and contains maternal arteries and veins that are continuous with the arterial and venous channels traversing the JZ . Glycogen cells migrate into the DB and surround the maternal arteries that supply the placenta late in gestation . The mechanisms of myometrial stimulation and activation appear to parallel those of the human -LSB- 10 -RSB- . For example , prostaglandins -LRB- PGs -RRB- are central components of labour in both humans and mice -LSB- 11 -RSB- . There are fundamental similarities in the changes occurring prior to labour , including an increase in myometrial contractile activity , increased coupling of myometrial cells through the formation of gap junctions , an increase in PG receptors in the myometrium , and enhanced sensitivity to oxytocin at term . There is also a rise in intracellular calcium resulting in an increase in myosin light chain phosphorylation . In this study we determined the degree of oxidative stress present in the murine placenta at different gestational ages in normal pregnancies . We also tested whether there was a temporal and spatial association between oxidative stress and two markers of placental function , one physiological and one pathological . These were the induction of cyclooxygenase -LRB- COX -RRB- enzymes and apoptosis respectively . Evidence from other systems points to potential links between oxidative stress and COX induction , resulting in the production of PGs -LSB- 12 -- 16 -RSB- . PGs are derived from arachidonic acid -LRB- AA -RRB- through the actions of the COX enzymes , which are rate-limiting . There are two known isoforms of COX , COX-1 and COX-2 , which share similar structures but differ in their function . COX-1 is believed to play a constitutive `` housekeeping '' role , whereas COX-2 is considered to be an inducible isoform . However , these contrasting roles have not been proven in the mouse -LSB- 17 -RSB- . In mice induction of COX-2 by administration of lipopolysaccharide leads to preterm labour , which can be blocked using the specific COX-2 inhibitor SC-236 -LSB- 18 -RSB- . Apoptosis has been described within the LZ of the normal mouse placenta , and is most frequent towards term and in post-mature placentas -LSB- 19 -RSB- . The stimulus for this is not known , but oxidative stress is a potent inducer of trophoblast apoptosis in the human placenta -LSB- 20 -RSB- . 2 Materials and methods 2.1 Tissue collection Placentas were collected from C57BL/6J inbred mice , and all experiments were carried out in accordance with the UK Government Home Office licensing procedures . Stages E14 , E16 , E18 , and E19 were studied , where E1 of gestation was the morning when a copulation plug was found . 2.2 Western blotting Placentas from three different animals at each gestational age were homogenized in ice-cold sample buffer , and centrifuged at 15,000 rpm . Protein concentration within the supernatant was determined using a colorimetric assay -LRB- Bio-Rad Bradford protein assay -RRB- . An equal amount of protein from each sample -LRB- 30 μg -RRB- was subjected to electrophoresis in a 10 % SDS-polyacrylamide gel under reducing conditions and transferred onto nitrocellulose membrane by a semidry transfer machine . Membranes were incubated in a blocking solution -LRB- TBS containing 5 % powdered non-fat milk and 0.01 % Tween-20 -RRB- at room temperature for 1 h , and then in the same solution containing primary antibody -LRB- Table 1 -RRB- at 4 °C overnight . After washes in TBS-T , membranes were incubated with the horseradish peroxidase secondary antibody at room temperature for 1 h . The immunoreactive bands were visualized by enhanced chemiluminescence with the ECL system -LRB- Amersham Biosciences -RRB- according to the manufacturer instructions . The levels of protein expression were quantified densitometrically and normalised against their respective expressions of β-actin . Prestained protein markers were used as molecular weight standards for each analysis . 2.3 Immunohistochemistry At each gestational age three placentas were fixed by immersion in 4 % paraformaldehyde overnight , dehydrated and embedded in paraffin wax . Sections -LRB- 6 μm thick -RRB- were dewaxed and immunostained according to our standard protocol -LSB- 21 -RSB- . When necessary , antigen retrieval was performed in a pressure cooker for 2 min in 0.01 M Citrate buffer at pH 6 . Primary antibodies -LRB- Table 1 -RRB- , diluted in goat serum , were applied overnight in a humidified chamber at 4 °C . Binding was detected using Vectastain Elite ABC kits -LRB- Vector Laboratories -RRB- and SigmaFast DAB -LRB- Sigma -RRB- , according to the manufacturers ' instructions . Sections were then lightly counterstained with haematoxylin . Negative controls were performed by omitting incubation with the primary antibody . For each antibody slides from all the gestational ages were prepared and immunostained in the same batch , ensuring identical conditions for comparisons . In order to localise expression of the antigens to specific cell types staining intensity was graded visually from 0 -- 3 , where 0 represented the negative control . Three people scored the sections independently , blinded to the gestational age or experimental protocol , and the mean value was taken . 2.4 Immunofluorescent dual-labelling Dual immunofluorescent labelling was performed in order to test for co-localisation of markers of oxidative stress and COX expression in individual cells . Sections were dewaxed , permeabilised in TBS containing Triton X-100 -LRB- 0.1 % -RRB- and Tween 20 -LRB- 0.1 % -RRB- -LRB- TBS-TT -RRB- for 30 -- 60 min , and blocked in 5 % bovine serum albumin for 20 min at room temperature . Primary antibodies diluted in TBS-TT were applied overnight at 4 °C . Negative control sections had primary antibodies omitted . After three 10-min washes in TBS-TT , sections were incubated for 1 h at room temperature with a mixture of fluorescent secondary antibodies , containing anti-goat Alexa 488 and anti-rabbit Alexa 568 -LRB- both used 1/100 ; from Molecular Probes -RRB- in TBS-TT . Sections were washed in TBS-TT as before and then twice in distilled water for 5 min and subsequently mounted in Vectashield mounting medium containing DAPI -LRB- Vector , UK -RRB- . Images were captured using a Leica confocal microscope -LRB- LeicaTCS-NT , Leica Instruments GmbH -RRB- . Co-localisation was identified in overlay images by a yellow signal caused by the combination of the green signal -LRB- COX-1 / COX-2 -RRB- and the red signal -LRB- HNE -RRB- . 2.5 TUNEL assay Staining was performed using In-Situ Cell Death Detection kit-Texas Red -LRB- Roche Applied Science -RRB- according to the manufacturer 's instructions . Conditions were adjusted so that the staining pattern matched morphological assessments of apoptosis based on examination of resin-embedded samples . Once the protocol had been optimised an apoptotic index was calculated as follows : 2.6 Dual-fluorescence TUNEL assay & Active Caspase-3 , and HNE co-localisation Staining was performed using In-Situ Cell Death Detection kit-Texas Red -LRB- Roche Applied Science -RRB- according to the manufacturer 's instructions , and using an Anti-Active Caspase-3 antibody -LRB- Promega -RRB- at 1:50 , and Anti-HNE as previously at 1:200 . Samples were also DAPI stained to intercalate the DNA and fluoresce cell nuclei . For negative controls , omission of primary antibody and TdT enzyme were performed separately . For positive controls , post-lactational mouse mammary glands were used . For confocal microscopy , all samples were analysed during one session to avoid bias , and multiple -LRB- usually 5 -RRB- fields of view per labyrinth were saved for further analysis . 2.7 Ex-vivo culture experiment In order to induce oxidative stress placental samples were subjected to hypoxia-reoxygenation -LRB- H/R -RRB- in vitro -LSB- 22 -RSB- . Placentas were taken from 3 animals at gestational age E18 , halved on an ice block and immersed in ice-cold PBS prior to culture . Three samples , each 5 -- 10 mm3 , from each placenta were incubated in culture medium -LRB- TCS large vessel endothelial cell basal medium -LRB- TCS CellWorks , Milton Keynes , UK -RRB- -RRB- , which had been maintained for several hours in the specified gas composition at 37 °C . Data for intraplacental oxygen concentrations are not yet available for the mouse , and so estimates were made on the basis of results for another small mammal , the guinea-pig . The umbilical venous pO2 towards term is 29.5 mmHg , which is close to that for fetal primates and ungulates -LSB- 23 -RSB- . Therefore , for normoxia a gas mix of 5 % O2 , 90 % N2 , 5 % CO2 was used , whereas for hypoxia it was 1 % O2 , 94 % N2 , 5 % CO2 . Gas composition was monitored and controlled throughout using Biospherix -LRB- Redfield , NY -RRB- Pro-Ox and Pro-CO2 probes . Control samples were cultured for 4 h in normoxia , whereas samples subjected to H/R experienced 1 h of hypoxia followed by 3 h of normoxia . Following culture the tissues were either frozen or fixed for Western blotting and immunohistochemistry respectively . 2.8 Statistical analyses Statistical significance was calculated by using one-way analysis of variance -LRB- ANOVA -RRB- . Differences between groups were assessed using Fisher 's Least Significant Difference Test . Statistical significance was assumed at a P-value < 0.05 . 3 Results 3.1 Markers of oxidative stress Hydroxynonenal -LRB- HNE -RRB- is a marker of lipid peroxidation . Western blotting revealed significant changes across gestational age -LRB- P = 0.003 -RRB- , with low concentrations of HNE at E14 , a significant increase at E16 , and a subsequent decline by E18 -LRB- Fig. 1a , b -RRB- . Scoring based on IHC revealed different intensities of immunoreactivity in the different cell populations , and different temporal patterns . At E14 immunoreactivity was low in all cell types -LRB- Fig. 1c , d -RRB- . At E16 there was moderate staining -LRB- scored 2 -RRB- in the syncytiotrophoblast of the LZ , which peaked -LRB- 3 -RRB- at E18 and declined to 1 by E19 -LRB- Fig. 1e -RRB- . The cytotrophoblast cells of the LZ stained uniformly at level 1 throughout , and no immunoreactivity was observed in the fetal endothelial cells . In the JZ strong staining was observed in the GCs at E16 -LRB- 2.5 -RRB- and E18 -LRB- 3 -RRB- -LRB- Fig. 1f -RRB- , and this was maintained at 2.5 at E19 . The spongiotrophoblast cells were less immunoreactive , scoring only 0.5 throughout gestation , and no immunoreactivity was observed in the giant cells or the decidua . Nitrotyrosine -LRB- NT -RRB- staining indicates the formation of the prooxidant peroxynitrite , and of an imbalance in the production of the superoxide ions and nitric oxide . Immunostaining revealed a similar pattern to HNE , with almost undetectable staining in early samples and significant increases from day 16 onwards -LRB- data not shown -RRB- . Therefore , oxidative stress increases during gestation in the murine placenta , specifically in syncytiotrophoblast and glycogen cells . 3.2 COX-1 Western blotting revealed significant changes in concentrations of COX-1 across gestational age -LRB- P = 0.013 -RRB- . There was an increase from E14 to E16 , followed by a significant decrease by E18 -LRB- Fig. 1a , b -RRB- . At E14 COX-1 immunoreactivity was not observed in any cell type , except for low intensity -LRB- level 1 -RRB- staining in the GCs . At E16 strong reactivity -LRB- 3 -RRB- was visible in the GCs , with lower intensity staining -LRB- 1 -RRB- being present in the spongiotrophoblast and cytotrophoblast cells -LRB- data not shown -RRB- . By E18 there was a general increase in staining , with many of the cytotrophoblast cells -LRB- 2 -RRB- reacting positively , along with the syncytiotrophoblast -LRB- 2 -RRB- , and all GCs -LRB- 3 -RRB- -LRB- Fig. 2a , b -RRB- . This pattern persisted at E19 , when the decidua -LRB- 2 -RRB- was also immunoreactive . By contrast , the spongiotrophoblast cells displayed low -LRB- 0.5 -RRB- immunoreactivity throughout gestation . Towards term COX-1 and HNE immunoreactivity were present within the same cells in both the LZ and JZ , as evidenced by the dual-labelling experiments -LRB- Fig. 2c , d -RRB- . 3.3 COX-2 Western blotting revealed a significant change in concentrations of COX-2 across gestation -LRB- P = 0.002 -RRB- , peaking at E19 -LRB- Fig. 1 -RRB- . IHC demonstrated that COX-2 expression is low -LRB- level 1 -RRB- within the syncytiotrophoblast , spongiotrophoblast and GCs at E14 . With advancing gestational age immunoreactivity increased , principally in the cytotrophoblast cells -LRB- 2 -RRB- , GCs -LRB- 2.5 -RRB- and spongiotrophoblast -LRB- 1 -RRB- at E18 and E19 -LRB- Fig. 3a , b -RRB- . The syncytiotrophoblast displayed low immunoreactivity -LRB- 0.5 -RRB- throughout gestation . Again there was strong co-localisation between immunoreactivity for COX-2 and HNE -LRB- Fig. 3c -RRB- . 3.4 Detection of apoptotic changes During apoptosis cytokeratins within trophoblast cells are cleaved by active-caspase 3 to yield a specific product detected by the M30 antibody -LSB- 24 -RSB- . The labyrinth showed low immunoreactivity for active-caspase 3 until E18 -LRB- Fig. 4b -RRB- , but thereafter it increased in intensity until E19 . The staining was localised to both the syncytiotrophoblast and individual cytotrophoblast cells . Immunoreactivity for M30 also increased with gestational age , and was most abundant at E18 , declining slightly at E19 -LRB- data not shown -RRB- . In order to estimate the number of apoptotic nuclear profiles TUNEL staining was performed and an apoptotic index calculated . TUNEL-positive nuclei were first observed at E16 when the apoptotic index was 2.7 % , and steadily increased in frequency until E19 , when the index reached 20.0 % -LRB- P < 0.001 -RRB- . There was a strong temporal and spatial association between TUNEL staining and immunoreactivity for HNE -LRB- Fig. 4a -RRB- and active caspase-3 -LRB- Fig. 4b -RRB- . At E16 a number of cells were caspase-positive but TUNEL-negative . Later , almost all TUNEL-positive nuclei were surrounded by cytoplasm immunoreactive for active caspase 3 . TUNEL-positive nuclei were rarely observed in the absence of caspase-positivity , when they were considered to be necrotic . To further confirm trophoblast cells were undergoing apoptosis , morphological evidence was sought by examining resin embedded samples . Cells displaying peripheral nuclear chromatin condensation , some in the classical crescent shape , were seen with increasing frequency towards E19 -LRB- Fig. 4c -RRB- . There was occasional evidence of apoptotic body formation , but other classical characteristics , such as plasma membrane blebbing , could not be resolved with light microscopy . The position of the nuclei suggested that the majority was within the syncytiotrophoblast , but the involvement of cytotrophoblast and endothelial cells could not be excluded at the light microscopic level . 3.5 Ex-vivo culture experiments To test the relationships between oxidative stress and the induction of COX enzymes and apoptosis experimentally , placental explants were subjected to hypoxia/reoxygenation -LRB- H/R -RRB- , a known stimulus for the generation of reactive oxygen species -LSB- 22 -RSB- . Western blotting confirmed increased concentrations of HNE and COX-1 in the explants following H/R compared to control samples frozen at the start of the experiment -LRB- time zero -RRB- or cultured under normoxic conditions . For COX-2 a significant increase was only observed compared to the time zero controls -LRB- Fig. 5a , b -RRB- . IHC localised the HNE to individual cytotrophoblast cells and to the syncytiotrophoblast layers of the labyrinth . In the JZ individual spongiotrophoblast cells were immunopositive , but the strongest staining was seen in the GCs within the JZ and decidua . Dual-labelling revealed strong co-localisation for COX-1 and COX-2 in the spongiotrophoblast and GCs in the JZ , and within the syncytiotrophoblast and cytotrophoblast cells in the labyrinth -LRB- Fig. 5c -RRB- . IHC revealed increased immunoreactivity for M30 within the trophoblast following H/R . The apoptotic index based on TUNEL labelling increased from 2.8 % and 1.6 % in the time zero and normoxic controls respectively , to 20.6 % following H/R -LRB- P = 0.006 -RRB- . The majority -LRB- 78 % -RRB- of TUNEL positive cells were also immunoreactive for HNE on dual-labelling -LRB- Fig. 5d -RRB- . From resin-embedded material it appeared that most of the cells with condensed peripheral chromatin following H/R were within the syncytiotrophoblast layers -LRB- data not shown -RRB- . From these data it can be concluded that there is a significant increase in oxidative stress following H/R , and that this is associated with increased expression of COX-1 and COX-2 expression in the LZ and JZ , and with increased apoptosis in the labyrinth . 4 Discussion These results indicate that oxidative stress increases with gestational age in the murine placenta during normal pregnancies , and that it may play a significant physiological role by inducing higher concentrations of the COX-1 and COX-2 enzymes , and hence increasing prostaglandin synthesis . Our data also indicate a close temporal and spatial association between oxidative stress and trophoblast apoptosis within the labyrinth . Trophoblast apoptosis has been linked to the pathophysiology of preeclampsia , and the mouse might provide a useful genetic model in which to elucidate the mechanisms underlying the shedding of apoptotic debris . We have recently proposed that placental oxidative stress arises through fluctuations in oxygenation -LSB- 3,25 -RSB- , and its development in the murine placenta towards term is consistent with this view . As fetal and placental oxygen extraction reach their peaks so any transient mismatch between maternal oxygen supply and feto-placental demand will lead to a dip in the intraplacental oxygen concentration . The fact that H/R in vitro is a powerful inducer of oxidative stress , and that the stress localises to the same tissues as in late gestation supports this hypothesis . It is now accepted that oxidative stress induces a spectrum of cellular changes , ranging from the physiologically homeostatic to the frankly pathologic . Induction of enzymes is towards the more physiological end of that spectrum , whereas apoptosis is towards the opposite extreme . Induction of the COX enzymes has been linked to oxidative stress through activation of the p38MAPK and the NF-κB family of transcription factors in other cell types -LSB- 26 -- 30 -RSB- . In our study there appeared to be strong co-localisation between the formation of HNE and expression of COX-1 and -2 to individual cytotrophoblast , spongiotrophoblast and glycogen cells , both in vivo and in vitro . However , there was an apparent discrepancy between the Western blot results and our IHC findings , for the former indicated a peak in oxidative stress and COX-1 at E16 while the staining intensity for HNE and COX-1 increased until E19 . This difference can be accounted for by the fact that the number of GCs declines by about half between E16 and E18 -LSB- 31 -RSB- , so reducing their contribution to the overall tissue homogenate . Expression of the COX enzymes appears to be mainly altered in placental rather than decidual tissues . Changes with gestational age were observed in the trophoblast populations within the LZ , but the greatest changes in immunoreactivity were seen within the GCs and the spongiotrophoblast cells in the JZ . Expression of COX-1 and COX-2 has recently been reported in the rat placenta , and that of COX-2 similarly increases with gestational age , particularly in the JZ -LSB- 32 -RSB- . The placental cells in this zone have direct access to maternal blood as they line the maternal venous sinuses . Therefore , it is possible that PGs synthesized in this zone in response to oxidative stress are released into the maternal circulation . It is also notable that the GCs migrating into the DB are strongly immunoreactive for COX , and so PGs synthesised by these cells could exert local paracrine effects in the decidua . As PGs have been linked with the induction of parturition -LSB- 10,11,33 -RSB- this raises the possibility that placental oxidative stress could play a role in initiating or augmenting uterine contractions should the mismatch in maternal blood supply and feto-placental demands become extreme . Trophoblast apoptosis increased late in gestation as assessed by IHC for M30 and TUNEL , and cell morphology . There was a close temporal and spatial co-localisation with oxidative stress , although on Western blotting evidence of oxidative stress appeared to decline in the last days of gestation . This may again reflect changes in the cell populations within the placenta and associated decidua . The apoptotic index based on TUNEL-positivity showed a continual increase until term , although M30 immunoreactivity suggested a decline after E19 . There is strong evidence that following initial cleavage , exposing the M30 epitope , cytokeratin 18 becomes further cleaved to smaller fragments -LSB- 24 -RSB- . In these late stages of apoptosis the M30 epitope may be lost , and so the antibody no longer detects all apoptotic trophoblast -LSB- 34 -RSB- . A causal link between oxidative stress and apoptosis was strengthened by the fact that apoptosis could be induced by H/R in vitro . As in the human placenta this was associated with increased concentrations of active caspase-3 -LSB- 20 -RSB- . The mitochondrial pathway was strongly implicated in the apoptotic process in the human , but further work is required to determine whether the same mechanism operates in the mouse . The increase in oxidative stress and apoptosis in the LZ with gestational age may explain some of the growth dynamics of the murine placenta . Overall placental volume increases with gestational age until E16 .5 and then plateaus , coinciding with the rise in oxidative stress . It is notable that within the LZ the volume and surface area of the trophoblast plateaus at E16 .5 whereas those of the fetal capillaries continue to increase until E18 .5 -LSB- 35 -RSB- . The fetal endothelial cells did not display immunoreactivity for HNE or nitrotyrosine , and did not appear to label with TUNEL , suggesting that oxidative stress and its consequences are lower in this cell type . The links between oxidative stress and apoptosis suggest that the mouse may provide a useful genetic model in which to investigate the relative roles of antioxidant enzymes and signalling pathways in regulating trophoblast apoptosis . Apoptosis increases towards term in normal human pregnancies -LSB- 36 -RSB- , but increased rates have been reported in preeclamptic placentas and in placentas from cases of intrauterine growth restriction -LSB- 37 -- 39 -RSB- . It is thought that microparticles of apoptotic , or aponecrotic , debris released from the apical surface of the syncytiotrophoblast into the maternal circulation result in an enhanced maternal inflammatory response in preeclampsia -LSB- 6 -RSB- . Whether apoptotic debris is shed from the murine labyrinth as in the human placenta towards the end of gestation remains to be determined .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

oxidative stress
apoptosis
murine placenta