RESUMEN
The widespread presence of the Na-K-2Cl (NKCC) cotransporter protein suggests that chronic administration of inhibitors may result in adverse effects. Inhibition of the NKCC cotransporter by loop diuretics is felt to underlie the diuretic and the pulmonary smooth muscle relaxant effects of this drug class. However, the fundamental regulation of salt and water movement by this cotransporter suggests that it may also mediate cell volume changes occurring during cell cycle progression. Thus we hypothesized that NKCC cotransporter inhibition by loop diuretics would decrease cellular proliferation. Normal human bronchial smooth muscle cells (BSMC) showed a significant concentration-dependent decrease in cell counts after 7 days of exposure to both bumetanide (n=5-10) and furosemide (n=6-16) compared with controls. Proliferation was similarly inhibited in normal human lung fibroblasts (n=5-9). To determine whether this was due to loss of cells, we performed apoptosis assays on BSMC. Both annexin V-propidium iodide staining (n=5-10) and single cell gel electrophoresis assays (n=4) were negative for necrosis and apoptosis in BSMC exposed to 10 microM bumetanide. Subsequent analysis of the cell cycle by flow cytometry showed that bumetanide-exposed BSMC were delayed in G1 phase compared with controls (n=4-8). This is the first evidence for loop diuretic inhibition of airway smooth muscle cell proliferation. NKCC cotransporter inhibition impeded G1-S phase transition without facilitating cell death. Thus although inhibition by loop diuretics relaxes airway smooth muscle, the NKCC cotransporter may have a more important role in cell proliferation regulation.
Asunto(s)
Bronquios/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Apoptosis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Bumetanida/farmacología , Recuento de Células , División Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Diuréticos/farmacología , Fibroblastos/efectos de los fármacos , Furosemida/farmacología , Fase G1/efectos de los fármacos , Humanos , Miocitos del Músculo Liso/efectos de los fármacos , Fase S/efectos de los fármacos , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
Inhibition of the Na-K-2Cl (NKCC) cotransporter by loop diuretics is associated with airway relaxation, but there has been no direct evidence for the expression of this protein in airway smooth muscle. Thus we hypothesized that a NKCC cotransporter is present and functional in airway smooth muscle cells. Monoclonal and polyclonal antibodies were used first to demonstrate the presence of a NKCC cotransporter protein in isolated human fetal trachea and normal human bronchial smooth muscle cells (BSMC) by Western blotting. The cotransporter protein was then localized by immunohistochemical staining to airway smooth muscle cells in culture and in situ. The localization was confirmed by indirect immunofluorescence and laser confocal microscopy in the BSMC. Cotransporter function in BSMC was also confirmed in vitro by bumetanide-mediated inhibition of rubidium uptake. Our present findings thus document the presence of a functional NKCC cotransporter in human airway smooth muscle, providing a basis for defining the role of this ion cotransporter in airway smooth muscle function.