Proteomic changes in the membrane fraction of SH-SY5Y neuroblastoma cells induced by fentanyl treatment

For many years, treatment of SH-SY5Y neuroblastoma cells with opioids has been used as an in vitro model to reflect biochemical changes observed during the in vivo development of opioid tolerance. However, the bulk of these studies have focused on specific target molecules in whole cell extracts. To gain a better understanding of global changes induced in membrane proteins after opioid treatment, we used a proteomic approach to study the adaptations induced by 12 hours of fentanyl treatment in this cell line. Proteins were extracted from control and fentanyl treated cells using a two-stage differential solubility process, first removing more soluble proteins ("cytoplasmic" fraction) followed by detergent extraction of less soluble proteins ("membrane" fraction). The less-soluble fraction then was analyzed using 2-dimensional gel electrophoresis (2-DE). Analysis of these gels revealed numerous alterations in protein abundance induced by fentanyl treatment. The proteins with the most significantly changed expression levels were identified using MALDI-TOF (matrix-assisted desorption/ionization-time of flight) based peptide mass fingerprinting. We identified several proteins that had previously been thought to be regulated by opioids, as well as many proteins that had not previously been implicated in opioid-mediated cell signaling. The identified proteins can be divided into groups that are involved in diverse cellular functions, such as metabolic maintenance, signal transduction, cytoskeletal dynamics, RNA editing, and protein processing. These results demonstrate that opioids have multiple and diverse cellular effects. Further application of this novel approach should enable the detection of additional molecules and novel signaling mechanisms that mediate both the desirable main effects and unwanted side effects of opioids.