Na-K-2Cl cotransporter inhibition impairs human lung cellular proliferation

Abstract

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.

Enhanced insulin secretion and improved glucose tolerance in mice with homozygous inactivation of the Na+K+2Cl− co-transporter 1

“…Although the mechanisms involved in proliferation of b-cells lacking NKCC1 remain unknown, our findings are in contrast to the direct positive linear relationship that exists between b-cell mass and body weight (Montanya et al 2000, Herbach et al 2011 and to the known negative impact that pharmacological inhibition of NKCC1 or depletion of [Cl K ] i have on normal cell growth and proliferation (O'Brien et al 1988, Panet et al 1994, 2002, Lang et al 1998, Iwamoto et al 2004, Shiozaki et al 2006. Indeed, NKCC1 KO mice are half the weight of NKCC1…”

Acquisition of Cisplatin-resistance in Malignant Mesothelioma Cells Abrogates Na^+,K^+,2Cl^-;-cotransport Activity and Cisplatin-induced Early Membrane Blebbing

“…Bumetanide has a higher affinity for activated NKCC1 than for basal NKCC1 [36]. In the early G1 phase of the cell cycle, NKCC1 is activated, and inhibition impairs cell proliferation [37]. Conversely, over-expression of the NKCC1 gene causes cell proliferation [38].…”

Neurogenic Role of the Depolarizing Chloride Gradient Revealed by Global Overexpression of KCC2 from the Onset of Development

“…Chloride cotransporters have been involved previously in disruptions of the cell cycle, because NKCC1 blockade with bumetanide in bronchial smooth muscles cell cultures inhibited the G 1 -S phase transition without facilitating apoptosis (Iwamoto et al, 2004). It is also possible that progenitors started committing to a neuronal fate by expressing ion channels and establishing a resting potential, but without expressing voltage-gated conductances or other postdifferentiation features such as neural-specific transcription factors or neurites.…”