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 Ca²⁺-dependent MAPK pathways. Since the role of Ca²⁺ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca²⁺ (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 Ca²⁺-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.