Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

BMES High School Student Chapter Member

HS Poster

Poster 98 - Investigating the Relationship Between Nav 1.8 and ASICs in PC-12 Cells: Implications for Pain Signaling and Drug Efficacy

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: Pain signaling in neurons involves complex interactions between various ion channels and receptors. Acid-Sensing Ion Channels (ASICs) are sensory ligand-gated channels which are responsible for initiating a nociception signal that respond to extracellular acidification and other stimuli to mediate pain. This signal often triggers calcium signaling pathways, which further amplify and sustain the pain response. Voltage-gated sodium channels, such as NaV 1.8, are responsible for receiving and propagating the signal initiated by sensory channels such as ASICs. Our study hypothesizes that NaV 1.8 is crucial for nociceptive pain signaling, which results from tissue damage and inflammation, in Pheochromocytoma (PC-12) cells, which model sensory neuron behavior. Specifically, we examine the efficacy of HBW-4600, a NaV 1.8 inhibitor, in reducing acid-induced signaling in a pH of 5.6—a known activator of ASICs. In our experiment, we use dimethyl sulfoxide (DMSO) as the solution for the NaV 1.8 inhibitor, ensuring proper solubility and delivery of the inhibitor to the cells. This research is crucial as it provides insights into the mechanisms of pain signaling and the efficacy of non-opioid pain treatments.

Materials and

Methods: Four dishes of Pheochromocytoma cells (PC-12) were calcium stained by Fluo-4 dye and analyzed in different states, with 20-30 cells per dish being measured for calcium intensity. Each dish experienced a change of condition, and their calcium intensity was recorded by a Zeiss 40x objective microscope in 3 second intervals for 15 minutes before and after the change. The change of condition was done by media replacement, and HCL acid was used to change the pH. Dish 1 served to test the effect of dimethyl sulfoxide (DMSO) on the cell’s calcium response by comparing the cell before and after DMSO was added. Dish 2 was used as a control to observe the cell’s response to a pH of 5.6. A different concentration of the HBW-4600 was added to Dishes 3 and 4 under the pH 5.6 environment to observe its effect on pain reduction, with 100nM in Dish 3 and 10nM in Dish 4. Dish 2-4 was first observed with a media of pH 7.4 containing DMSO (pre stimuli) for 15 minutes, then changed to the respective conditions. We utilized FIJI ImageJ to analyze the calcium intensity by measuring the mean gray values of the images.

Results, Conclusions, and Discussions: Our results strongly support our hypothesis and demonstrate the efficacy of the Nav 1.8 inhibitor. During this process, we encountered some unavoidable errors: photobleaching reduced fluorescence signals over time and the PC-12 cells employed in this study have mechanisms to maintain homeostasis, which activate pumps and transporters on the cell membrane to eject foreign substances such as the Fluo-4 dye. Both complications resulted in slight alterations in the measured calcium intensity. In summary, our study shows that acid-induced signaling can be suppressed through medically inhibiting NaV 1.8 function. This implies that nociceptive pain induced by lesions and tissue damage—or other causes for localized acidosis—can be reduced by limiting the function of NaV 1.8. However, the data from PC-12 cells cannot give us conclusive results, as these cells are not nociceptors. Therefore, a statement regarding a suppression of acid-induced pain cannot be fully justified until we pursue further experimentation of dorsal root ganglia. Additionally, we will work with other receptors such as TRPV1 to test other stimuli.

Acknowledgements (Optional): This work was supported by a generous gift from Beckman Laser Institute Inc. to LS. The high school students (*) participated in the UCSD IEM OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering and Dr. Lizhu Chen from CorDx Inc. for their donations

References (Optional):