Abstract
Voltage-gated sodium ion channels (VGSCs) are transmembrane proteins found on excitable cells, such as neurons, cardiac, and muscle cells. These channels are important for electrical signaling and development of action potential required for pain stimulus and motor functions. Previous studies have suggested that non-excitable cells, such as immune macrophages, may express VGSCs. This study is focused on the evaluation of two VGSCs modulators, brevetoxin-2 and bupivacaine hydrochloride, on the inflammatory responses from RAW 264.7 macrophage cells. Brevetoxins are a class of neurotoxins produced by Karenia brevis, a marine algal dinoflagellate, that functions by activating voltage-gated sodium ion channels (VGSCs). Oppositely, bupivacaine hydrochloride is a long-acting local analgesic that functions by blocking VGSCs, leading to the suppression of action potentials in excitable tissue. The aims of this study were carried out by treatment of the RAW 264.7 cell line with various concentrations of each modulator to assess the major inflammatory responses of macrophages using a WST-1 assay, crystal violet staining, zymosan uptake assay, wound healing assay, and nitrite quantification followed by ELISA kits. Whole-cell patch clamp protocols were also conducted to determine if VGSCs are present on the plasma membrane of RAW 264.7 cells. This study found that concentrations greater than 333 µM of bupivacaine hydrochloride inhibited cellular proliferation with toxicity starting at 1,000 µM. All tested concentrations of brevetoxin-2 treatment showed no change in proliferation or toxicity. In regard to the macrophage’s inflammatory cytokine responses, lipopolysaccharide (LPS)-stimulated nitric oxide production was enhanced with bupivacaine hydrochloride at 0.1 µM and 1.0 µM and with brevetoxin-2 at 20 ng/mL and 200 ng/mL. TNF-α production was inhibited at all tested concentrations of bupivacaine hydrochloride treatment with LPS-stimulation, while brevetoxin-2 treatment at 2,000 ng/mL resulted in enhanced cytokine production on its own. Unlike bupivacaine hydrochloride’s TNF-α production, a dose-dependent trend was seen with increased TNF-α levels without the need for LPS simulation. Both bupivacaine hydrochloride and brevetoxin-2 showed to inhibit LPS stimulated IL-1β production at 100 µM and 20 ng/mL, respectively. LPS-induced IL-6 production was inhibited with bupivacaine hydrochloride at 10 µM and 100 µM, while treatment with brevetoxin-2 at 200 ng/mL showed significant enhancement in LPS-induced IL-6 cytokine production. RAW 264.7 macrophage’s phagocytic activity was not affected by either modulator’s treatment in cell culture. Cell migration for wound repair increased with bupivacaine hydrochloride at 100 µM with 58% of the wound filled in after 48 hours. Treatment with brevetoxin-2 at 20 ng/mL and 200 ng/mL also showed enhanced migration when cells were induced with LPS, for a total wound closure of 48% and 46%. Lastly, RAW 264.7 cells showed no induction of antiviral activity or interferon production under the treatment of either compound against vesicular stomatitis virus (VSV) infections. Also, neither compound was able to provide protection against VSV through virus neutralization as the cells remained susceptible to virus-induced lysis. Overall, the differential effects of bupivacaine hydrochloride and brevetoxin-2 on the various inflammatory responses produced by macrophages may be caused by these compounds interacting with VGSCs. However, whole-cell patch clamp electrophysiology studies showed that the tested RAW 264.7 cells do not express VGSCs on their plasma membrane. The voltage clamp protocols were used to evoke ion channel activation and inactivation; although, sodium ion currents were not detected from these murine macrophages. In summary, the assays presented in this study increased our understanding of brevetoxin-2 and bupivacaine hydrochloride, while also demonstrating their influences on macrophage immune functions. Results showed that bupivacaine hydrochloride and brevetoxin-2 do influence some inflammatory responses of the RAW 264.7 macrophage cells. Although, since VGSCs were not detected on the plasma membrane of these cells, we concluded that bupivacaine hydrochloride’s and brevetoxin-2’s mechanism of action does not involve modulation of transmembrane VGSCs.