Background and Aims:
We investigated the impact of altered intracellular calcium levels and G-protein-coupled receptor (GPCRs) signaling inhibition on migration and NETosis of polymorphonuclear leukocytes (PMNs) under influence of bupivacaine and ropivacaine.

Methods:
PMNs were isolated from whole blood of healthy volunteers by centrifugation. In vitro µSlide chemotaxis assays were conducted, where PMNs migrated along a formyl-methionyl-leucyl-phenylalanine (fMLP) chemotactic gradient through a type I collagen matrix, tracked over 6 h using fluorescence microscopy. Bupivacaine and ropivacaine were added, along with the calcium chelator BAPTA AM, GPCR inhibitor gallein and phospholipase C (PLC) inhibitor U-73122.

Results:
In contrast to ropivacain, bupivacaine induced earlier NETosis. Both local anesthetics caused an earlier cessation of PMN migration. Chelation of intracellular calcium demonstrated a concentration-dependent effect on migration. The addition of Gallein and U-73122 resulted in earlier NETosis and an increase in maximum intracellular calcium concentration.

Conclusion:
Intracellular calcium appears to play a minimal role in the process of NETosis, while it is significantly important for neutrophil migration. Inhibition of the Gβγ subunit using gallein and PLC using U-73122 led to an earlier onset of NETosis and an increase in the maximum intracellular calcium. An additional effect of ropivacaine on the GPCR signaling pathway was not detectable.