Measurement of ATP-Induced Membrane Potential Changes in IVD Cells

Extracellular adenosine-5′-triphosphate (ATP) triggers biological responses in a wide variety of cells and tissues and activates signaling cascades that affect cell membrane potential and excitability. It has been demonstrated that compressive loading promotes ATP production and release by intervertebral disc (IVD) cells, while a high level of extracellular ATP accumulates in the nucleus pulposus (NP) of the IVD. In this study, a noninvasive system was developed to measure ATP-induced changes in the membrane potential of porcine IVD cells using the potential sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS).The responses of NP and annulus fibrosus cells to ATP were examined in monolayer and 3-dimensional cultures. It was found that the pattern and magnitude of membrane potential change in IVD cells induced by extracellular ATP depended on cell type, culture condition, and ATP dose. In addition, gene expression of P2X₄ purinergic receptor was found in both cell types. Inhibition of the ATP-induced response by pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonate, a non-competitive inhibitor of P2 receptors, suggests that ATP may modulate the biological activities of IVD cells via P2 purinergic receptors.