Changes And Mechanisms In Gap-Junction Permeability Mediated By Connexin 43 In Silica-induced Pneumocytes

Gap junctions are transmembrane channe1s composed of tWohemichanneIs from contiguous ceIIs that permit the transfer of ionsand small molecules directly betWeen the ceIls. These hemichan-ne1s are composed of hexameric connexons, which are attached tothe connexons in the p1asma membranes of neighboring ceIls.Connexons are consisted of transmembrane proteins called con-nexins. The connexins are a fami1y of gapdunction proteins (rangedbetWeen molecular weights of 26 and 70 kDa), that are highIy evo-Iutionari1y conserved in the transmembrane and eXtracellular region,but they differ in their CytopIasmic domain. There are approximate1ya dozen corresponding high1y conserved genes coding for theseconnexins. Connexin 45, 43, 40,37 and 32 mRNAs and proteinsoccur in a wide variety of fetaI and adult tissues, with particularabundance in lung. HoweveF, in the remode1ling process during fi-brogenesis, the predominant one is connexin 43.Gap junctions pIay a role in both the rapid propagation of e1ectricalsignaIs and the synchronization of metabolic activity. They haveaIso been implicated in the reguIation of ceII proliferation, differen-tiation and deveIopment. Many cancer ceIls, which Iack groWthCQntroI and the ability to terminally differentiate, have been associ-ated with deficient-or defeCtive gap junCtional intercel1uIar commu-nication (GJIC). The downregulation of GJIC can occur at the levelsof connexin expression and transport, gap junCtion formation, andgapdunction function by endogenous factof, incIuding growth fac-tors and reactive oxygen l nitrogen species (ROS l RNS), as welIas by exogenous factors such as poIlutions and naturaI toxins, et al.IntraceIIular second messages aItered by these endogenous andexogenous faCtors, such as changes in intraceIlular caIcium, cAMPpH, and gIutathione leveIs, have been Iinked to the regulation ofgap junctions.Silicosis is a chronic Iung disease, which is caused by inhaIation ofsiIica-containing dusts, and Iung fibrosis is a prominent histoIogicfeature of both experimental and human siIicosis. The mechanismsproducing fibrosis in siIicosis are incompIetely understood and yetto be eIucidated, but an interaction betWeen alveoIar macrophages(AMs), fibrobIasts and epitheIial ceIls, couId be thought to be a keyprocess. As a resuIt of phagoCytosis, the macrophages are knownto reIease a variety of active mediators that stimuIate or facilitatefibroblast proliferation, incIuding the various cytokines, such aspIateIet derived groWth factor (PDGF), insuIin--Iike groWth factor1(IGF-1), tumor necrosis factor (TNF), and reactive oxygen l nitro-gen species (ROS l RNS). lt is possible, therefore, that the media-tors released by siIica-exposed AMs, cy'tokines and ROS, exert theeffeCts on fibroblasts to aIter their intracelIuIar second messages,and then fUrther to downreguIate the GJlC function. In the studyreported here, we evaluated this hypothesis iD vHro by anaIysis us-ing nuorescence-recovery- afterphotobleaching (FRAP), en-zyme-linked-immunosorbent assay (ELlSA), and immunofIuores-cent histochemistry to determine to what leveI and at what extentsilica-exposed AMs supernatants disrupted GJIC mediated byconnexin 43 in a lung fibroblast ceII Iine CHL (Chinese hamsterlung fibrobIast), and epitheliaI celI Iine CCL-64 (a normaI mink lungepitheIiaI ceII).Part1.Establishment of an In-WtIO SiIicotic Test System.The puImonary alveolar macrophage (AMs) or PMA-primed THP-1(a monocyte-Iike ceIl Iine with properties of the pulmonary aIveoIarmacrophage) supernatants was prepared by incubating the ceIIs inthe serum1ree RPMI1640 with a series of concentrations of siIicondioxide for 24 hours (AMs) or 36 hours (PMA-primed THP-1). CHLand CCL-64 celIs were re-incubated with the prepared supernatants fOr 24 hours. After incubation, the proliferation of CHL andCCL-64 celIs were measured using MTT assay. ln the range of 0,50, 100, 20...