inositol-1-4-5-trisphosphate and Cystic-Fibrosis

Secretory leukoprotease inhibitor (SLPI) is an anti-inflammatory protein present in respiratory secretions. Whilst epithelial cell SLPI is extensively studied, neutrophil associated SLPI is poorly characterised. Neutrophil function including chemotaxis and degranulation of proteolytic enzymes involves changes in cytosolic calcium (Ca(2+)) levels which is mediated by production of inositol 1,4,5-triphosphate (IP3) in response to G-protein-coupled receptor (GPCR) stimuli. The aim of this study was to investigate the intracellular function of SLPI and the mechanism-based modulation of neutrophil function by this antiprotease. Neutrophils were isolated from healthy controls (n = 10), individuals with cystic fibrosis (CF) (n = 5) or chronic obstructive pulmonary disease (COPD) (n = 5). Recombinant human SLPI significantly inhibited fMet-Leu-Phe (fMLP) and interleukin(IL)-8 induced neutrophil chemotaxis (P < 0.05) and decreased degranulation of matrix metalloprotease-9 (MMP-9), hCAP-18, and myeloperoxidase (MPO) (P < 0.05). The mechanism of inhibition involved modulation of cytosolic IP3 production and downstream Ca(2+) flux. The described attenuation of Ca(2+) flux was overcome by inclusion of exogenous IP3 in electropermeabilized cells. Inhibition of IP3 generation and Ca(2+) flux by SLPI may represent a novel anti-inflammatory mechanism, thus strengthening the attractiveness of SLPI as a potential therapeutic molecule in inflammatory airway disease associated with excessive neutrophil influx including CF, non-CF bronchiectasis, and COPD.

Topics: Adult; Anti-Inflammatory Agents; Calcium; Cell Degranulation; Chemotaxis; Cystic Fibrosis; Cytoskeleton; Cytosol; Female; Humans; Immunologic Factors; Inositol 1,4,5-Trisphosphate; Intracellular Space; Male; Models, Biological; Neutrophil Activation; Neutrophils; Oxidation-Reduction; Pulmonary Disease, Chronic Obstructive; Recombinant Proteins; Secretory Leukocyte Peptidase Inhibitor

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca(2+) stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y(2)-R activation-dependent Ca(2+)(i) responses, which reproduced the increased Ca(2+)(i) signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca(2+)(i) signals elicited by apical P2Y

Topics: Adult; Blotting, Western; Bronchi; Calcium; Calcium Channels; Calreticulin; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Epithelium; Homozygote; Humans; Inflammation; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ions; Lung; Microscopy, Electron; Middle Aged; Phenotype; Protein Folding; Pseudomonas aeruginosa; Receptors, Bradykinin; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Signal Transduction; Time Factors; Type C Phospholipases; Up-Regulation; Uridine Triphosphate

We studied the intracellular free Ca2+ concentration ([Ca2+]i) response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine (100 microM; duration approximately 10 min) biphasically increased [Ca2+]i, with a rapid initial peak (30-45 s) followed by a smaller second peak that lasted for several minutes before returning to baseline. Neither peak specifically depended on Ca2+ influx. Exposure to bradykinin (10 microM) elicited a single peak that lasted 3-3.5 min before returning to baseline. Bradykinin increased intracellular inositol 1,4,5-trisphosphate (IP3), which peaked and returned to baseline within 150 s. Histamine also increased IP3 monophasically, but the peak was brief (< 20 s). Both phases of the Ca2+ response to histamine exhibited similar responsiveness to histamine concentration and sensitivity to antagonists. Cimetidine or thioperamide (1 mM) had no effect on the second peak. Pyrilamine blocked the second peak at concentrations similar to those required to block the initial peak. Activation of the second peak was observed at a threshold concentration of 1 microM comparable with the threshold of the initial peak. Neither adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, nor cyclic ADP (cADP)-ribose altered the second phase of the histamine response.

Topics: Bradykinin; Calcium; Cell Line, Transformed; Cystic Fibrosis; Epithelium; Histamine; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Nasal Mucosa; Second Messenger Systems; Time Factors; Trachea

Neutrophils from cystic fibrosis (CF) patients have been shown previously to be defective in their response (beta-glucuronidase exocytosis, NADPH oxidase activation) to the chemotactic peptide FMLP. In this work, we attempted to identify the defective step in this response. We showed that stimulated CF and control neutrophils do not differ in the formation of inositol phosphates. On the other hand, direct stimulation of protein kinase C with phorbol myristate acetate (PMA) revealed a subnormal stimulation of beta-glucuronidase exocytosis in CF neutrophils. Furthermore, retroinhibition exerted by PMA-activated protein kinase C on stimulated inositol phosphates or on beta-glucuronidase exocytosis was marginal or absent in CF neutrophils, whereas it was significant in the case of control neutrophils. Our observations suggest that the CFTR gene is expressed in neutrophils and is involved in protein kinase C-mediated actions.

Topics: Cystic Fibrosis; Diglycerides; Enzyme Activation; Exocytosis; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Kinase C; Signal Transduction; Sodium Fluoride; Tetradecanoylphorbol Acetate