Agents that chelate intracellular Ca²⁺, increase intracellular cAMP levels, or inhibit MLCK did not inhibit EC stiffening response induced by adherent neutrophils.

Twenty-four-hour TNF-α–treated ECs were pretreated with the agent or its control vehicle for 30 minutes, and the EC stiffness before or 2, 6, 10, and 15 minutes after neutrophil adherence was evaluated as before. (A) Pretreatment with 50 μM BAPTA/am, an intracellular Ca²⁺ chelator. (B) Pretreatment with 1 μM forskolin along with 0.5 mM IBMX to increase intracellular cAMP levels. (C) Pretreatment with 10 μM ML-7, an MLCK inhibitor. Data are expressed as mean ± SEM (n = 4). ●, control vehicle; ○, agent.