α 1G T‐type Ca 2+ channel plays a dual role in control of pulmonary microvascular endothelial cell P‐selectin surface expression

Signaling pathways directing alveolar endothelial cell activation are of pivotal importance in controlling lung inflammation as lung alveolar capillaries constitute the major site for neutrophil trafficking. We had previously established that the expression of an α 1G T‐type Ca 2+ channel, the only voltage‐gated Ca 2+ channel identified in lung endothelium, is restricted to the alveolar capillary network. Notably, the α 1G ‐mediated Ca 2+ entry is pro‐inflammatory as it specifically leads to P‐selectin surface expression, which enables intra‐capillary neutrophil sequestration. More recently we observed that, in pulmonary microvascular endothelial cells (PMVECs), the α 1G directly interacts with endothelial‐type nitric oxide (NO) synthase (NOS3) and blockade of the α 1G abolishes agonist‐stimulated NO generation. These observations suggest that the NOS3/NO signaling cascade, known to inhibit endothelial exocytosis, is also activated downstream of α 1G activation. The present study tested whether the α 1G ‐mediated Ca 2+ entry activates NO generation which in turns leads to inhibition of α 1G ‐dependent P‐selectin surface expression. In PMVECs over‐expressing rat α 1G and NOS3 loaded with an NO‐sensitive fluorophore, DAF‐FM, real‐time fluorescence imaging was utilized to assess the NO production in response to high external [K + ] (40 mmol/L) stimulation that is to depolarize the endothelial cell membrane potential from the resting level to a voltage at which the T‐type “window current,” i.e. , a constant α 1G ‐mediated Ca 2+ entry, occurs. Depolarization of plasma membrane via high [K + ] elicited a sustained NO production that was nearly abolished by pharmacologic NOS blockade as well as T‐type channel blockade with L‐NAME (1 mmol/L) and mibefradil (5 μmol/L), respectively. Next, in PMVECs stably transduced with a GFP tagged P‐selectin, fluorescence microscopy was employed to examine the real‐time dynamics of the P‐selectin in response to plasma membrane depolarization. Indeed, depolarization provoked rigorous P‐selectin translocation from cytosol to the plasma membrane leading to P‐selectin surface expression. While such depolarization‐stimulated P‐selectin surface expression was nearly abolished by T‐type channel blockade (mibefradil, 5 μmol/L), it was significantly potentiated by NOS blockade (L‐NAME, 1 mmol/L). Taken together, these findings revealed that the NO signaling cascade is a specific physiological target of the Ca 2+ signal initiated by α 1G activation and that the α 1G ‐dependent NO generation subsequently limits the α 1G ‐mediated P‐selectin surface expression, as in a negative feedback mechanism. In this regard, α 1G serves a dual role in control of endothelial cell pro‐inflammatory phenotype. Support or Funding Information HL‐066299