Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca(2+)-dependent MAPK pathways. Since the role of Ca(2+) influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca(2+) (Ca(V)) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The Ca(V) channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of Ca(V) channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (−6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of Ca(V) channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K(+) (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca(2+)-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.