Differential Inhibitory Effects of CysLT1 Receptor Antagonists on P2Y6 Receptor-Mediated Signaling and Ion Transport in Human Bronchial Epithelia

BACKGROUND: Cysteinyl leukotriene (CysLT) is one of the proinflammatory mediators released by the bronchi during inflammation. CysLTs exert their biological effects via specific G-protein-coupled receptors. CysLT(1) receptor antagonists are available for clinical use for the treatment of asthma. Recently, crosstalk between CysLT(1) and P2Y(6) receptors has been delineated. P2Y receptors are expressed in apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Previous research suggests that CysLT(1) receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors. However, the detailed molecular mechanism underlying the inhibition remains unresolved. METHODOLOGY/PRINCIPAL FINDINGS: In this study, western blot analysis confirmed that both CysLT(1) and P2Y(6) receptors were expressed in the human bronchial epithelial cell line 16HBE14o-. All three CysLT(1) antagonists inhibited the uridine diphosphate (UDP)-evoked I(SC), but only montelukast inhibited the UDP-evoked [Ca(2+)](i) increase. In the presence of forskolin or 8-bromoadenosine 3'5' cyclic monophosphate (8-Br-cAMP), the UDP-induced I(SC) was potentiated but was reduced by pranlukast and zafirlukast but not montelukast. Pranlukast inhibited the UDP-evoked I(SC) potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2'-O-Me-cAMP), while montelukast and zafirlukast had no such effect. Pranlukast inhibited the real-time increase in cAMP changes activated by 8-CPT-2'-O-Me-cAMP as monitored by fluorescence resonance energy transfer imaging. Zafirlukast inhibited the UDP-induced I(SC) potentiated by N(6)-Phenyladenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-6-Phe-cAMP; a PKA activator) and UDP-activated PKA activity. CONCLUSIONS/SIGNIFICANCE: In summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT(1) receptor antagonists exert differential inhibitory effects on P2Y(6) receptor-coupled Ca(2+) signaling pathways and the potentiating effect on I(SC) mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia

Effects of CysLT₁ receptor antagonists on Epac activation and PKA activity.

<p>(A) The monochrome CFP and FRET images showing the cytosolic distribution of the fluorescent Epac probe in 16HBE14o- cells transfected with CFP-Epac-YFP. (B) Representative pseudocolor images of CFP/FRET emission ratios before and after the addition of 8-CPT-2′-<i>O</i>-Me-cAMP. (D) Real-time cAMP changes (normalized CFP/FRET emission ratio) recorded in cells stimulated with 50 µM 8-CPT-2′-<i>O</i>-Me-cAMP with or without 1 µM pranlukast shown in (B). The agents were added at time zero. (C) Summarized data showing the effect of CysLT₁ receptor antagonists on the CFP/FRET emission ratio. Each column represents the mean ± S.E. (<i>n</i> = 8–10). (*, <i>p</i><0.05, Student's <i>t</i>-test compared with control). (E) Confluent 16HBE14o- cells were treated with either vehicle alone (control), 100 µM UDP, or UDP with different CysLT₁ receptor antagonists (1 µM) for 5 min. PKA activity was measured as a function of fluorescence intensity. (F) Summarized data showing the relative fluorescence level as compared with the control level. Each column represents the mean ± S.E. (*, <i>p</i><0.05, <i>n</i> = 4, one-way ANOVA with Bonferroni post-hoc test).</p

Effect of CysLT₁ receptor antagonists, montelukast, pranlukast, and zafirlukast on UDP-evoked [Ca²⁺]_i responses.

<p>(A) Cells were stimulated with UDP in the absence (control) or presence of different concentrations of CysLT₁ receptor antagonists applied 10 min prior to the addition of UDP (100 µM). (B) The UDP-evoked [Ca²⁺]_i responses were inhibited by different concentrations of an IP₃ receptor antagonist, XeC, or a PLC inhibitor, U73122. Each column represents the mean ± S.E. (<i>n</i> = 4–6). (*, <i>p</i><0.05, one-way ANOVA with Bonferroni post-hoc test compared with control).</p

Concentration-dependent effect of UDP on <i>I_SC</i> and [Ca²⁺]_i.

<p>(A) Epithelia were initially bathed in normal K-H solution while changes in <i>I_SC</i> were recorded. A serosal-to-mucosal-directed Cl⁻ gradient was applied across the monolayers by changing the apical K-H solution to one with reduced Cl⁻ concentration to facilitate Cl⁻ secretion (not shown). Different concentrations of UDP were added to the apical side as indicated. Data presented are representative of at least four independent experiments. (B) UDP-evoked increases in [Ca²⁺]_i were monitored by a microfluorimetric technique using the calcium-sensitive fluorescent dye Fura-2. Cells grown on glass coverslips were initially superfused with normal K-H solution. The K-H solution was then changed to a nominally Ca²⁺-free K-H solution, and the cells were stimulated with different concentrations of UDP as indicated. Data presented are representative of at least four independent experiments. (C) Concentration-response relationship for the effect of UDP upon Δ<i>I_SC</i> in 16HBE14o- epithelia. The changes in <i>I_SC</i> for the first and second peaks were quantified and plotted against the concentration of UDP used. Each data point represents the mean ± S.E. (<i>n</i> = 4–5). (D) Concentration-response relationship for the effect of UDP upon changes in [Ca²⁺]_i represented by Δratio. Each data point represents the mean ± S.E. (<i>n</i> = 4–5). (E) Application of CysLT₁ receptor agonists (10 µM), LTC₄ or LTD₄, did not cause any discernible increase in <i>I_SC</i> and did not affect the subsequent 100-µM UDP-evoked <i>I_SC</i> responses. Data presented are representative of at least four independent experiments.</p

Effect of CysLT₁ receptor antagonists, montelukast, pranlukast, and zafirlukast, on UDP-evoked <i>I_SC</i> responses.

<p>Representative recordings of <i>I_SC</i> in response to the apical application of 100 µM UDP (shown by arrows) in the presence of montelukast (A). The CysLT₁ receptor antagonist was applied 10 min before the application of UDP (data not shown). Data are summarized for the inhibitory effects of montelukast (B) on the first and second peaks of <i>I_SC</i> increases in response to UDP. Similar experiments were performed using pranlukast (C and D) or zafirlukast (E and F). The control was the apical application of UDP alone in the absence of any CysLT₁ receptor antagonist. Each column represents the mean ± S.E. (<i>n</i> = 4–5). (*, <i>p</i><0.05, one-way ANOVA with Bonferroni post-hoc test compared with control).</p

Inhibition by CysLT₁ receptor antagonists of the potentiating effect of cAMP and Epac on UDP-evoked <i>I_SC</i>.

<p>Representative recordings of <i>I_SC</i> showing the response to the apical application of 100 µM UDP alone (A) or in combination with pretreatment of the epithelia with 1 µM forskolin (B), 8-Br-cAMP (C), 8-CPT-2′-<i>O</i>-Me-cAMP (D), or Sp-6-Phe-cAMP (E) 10 min prior to the application of UDP. (F) Summarized data showing the potentiating effect of cAMP or Epac on UDP-evoked <i>I_SC</i> could be inhibited by different CysLT₁ receptor antagonists. Control refers to the <i>I_SC</i> responses of the epithelia to UDP in the presence of various potentiating agents. Each column represents the mean ± S.E. (<i>n</i> = 4–6). (*, <i>p</i><0.05, one-way ANOVA with Bonferroni post-hoc test compared with control).</p

Immunolocalization of Epac 1 and Epac 2 in 16HBE14o- cells.

<p>With the use of specific antibodies, immunofluorescence staining of Epac 1 (A) and Epac 2 (C) were localized to the cytoplasm of 16HBE14o- cells, as indicated by the red colour. Under the conditions specified in the Methods section, the signal intensity of Epac 1 was stronger than that of Epac 2, which may suggest a higher and more uniform expression of Epac 1 in these cells. In the negative control (E), omission of the primary antibodies resulted in a complete loss of the immunofluorescence. The corresponding images taken in brightfield were shown in B, D and F.</p

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research