RESUMO
Recent data have shown a unique relationship between Ca2+ signaling in macrophages through L-type channels and the outcome of C. pneumoniae infection of such cells. The present investigation seeks to provide insights into the manner in which macrophage L-type Ca2+ channel operation affects major outer membrane protein (MOMP) and heat shock protein-60 (HSP-60) mRNA gene expression (factors associated with Chlamydia chronicity), and the possible effect of this on antibiotic susceptibility. Intracellular calcium ([Ca2+]i) chelation using varying doses of 1,2-bis (o-aminophenoxy) ethane-N,N,N'N'--tetra acetic acid (acetoxymethyl) ester (BAPTA-AM) induced an increase in MOMP and a decrease in HSP-60 mRNA gene expression. L-type Ca2+ channel antagonists produced an identical but enhanced effect. Since these findings associate specialized Ca2+ channels to Chlamydia chronicity, it was important to determine Ca2+ channel effect on the usual antibiotic refractory form of C. pneumoniae in macrophages. Inhibition of macrophage L-type Ca2+ channel operation improved C. pneumoniae antibiotic susceptibility assessed by decreased inclusion counts or down-regulated MOMP and HSP-60 mRNA gene expression. These findings provide molecular insights into how specialized Ca2+ channels influence Chlamydia chronic course in macrophages and demonstrates a role for L-type Ca2+ channel inhibitors in enhanced C. pneumoniae susceptibility to antibiotic therapy.
Assuntos
Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/metabolismo , Canais de Cálcio Tipo L/metabolismo , Chaperonina 60/metabolismo , Chlamydophila pneumoniae/metabolismo , Ácido Egtázico/análogos & derivados , Macrófagos/microbiologia , Animais , Antibacterianos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio , Linhagem Celular , Chaperonina 60/genética , Quelantes/farmacologia , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/genética , Ácido Egtázico/farmacologia , Expressão Gênica , Macrófagos/metabolismo , Camundongos , Nifedipino/antagonistas & inibidores , Nimodipina/antagonistas & inibidores , RNA Mensageiro/metabolismoRESUMO
Macrophage immune capability depends on their efficient redox potential expressed in the effective release of reactive oxygen species (ROS) and nitric oxide. In this study the effect of the activation of a specialized Ca(2+) channel on macrophage redox function during Chlamydia pneumoniae infection was explored. C. pneumoniae exhibited a profound and sustained Ca(2+) influx capacity, with evidence of activity attributable to their lipopolysaccharide (cLPS) content. Also the organism showed an additional Ca(2+) influx signal in macrophages exposed to thapsigargin, and there was evidence for the operation of a single ion channel of the L type as demonstrated by the effect of L-type channel antagonists (methoxyverapamil and nimodipine) despite exposure to Ca(2+)-rich medium. C. pneumoniae or cLPS induced intracellular ROS and NO generation in a manner consistent with dependence on intracellular calcium. L-type Ca(2+) channel blocking significantly prompted C. pneumoniae inclusion formation. These findings suggest that Ca(2+) influx signal and redox function in C. pneumoniae-infected macrophages depend on L-type Ca(2+) channel activation.
