Novel Measuring System of ATP-Induced Transmembrane Potential Change of Nucleus Pulposus Cells

Low back pain is a serious concern in industrialized societies that affects millions of people around the world [1]. It can be attributed to several spine disorders; intervertebral disc (IVD) degeneration being one of the most common causes [2]. IVDs are the largest avascular tissue in the body and are composed of two differentiated anatomical structures: the nucleus pulposus (NP) and the annulus fibrosus (AF). The ability to withstand compressive loads due to weight and bending is provided by the swelling of the NP structure, while the ability to resist tensile forces during bending and twisting is provided by the AF fibers [3]. The biomechanical functions of NP and AF rely on their extracellular matrix (ECM) structure and composition. Previous studies have demonstrated that static and dynamic compressive loading alters ATP production, which may have an effect on ECM synthesis [2]. In addition, dynamic loading has shown an increase in ATP release from NP cells, which may contribute to endplate calcification and therefore to IVD degeneration [2, 4]. When tissue is damaged, ATP, which is found in millimolar concentrations in all cells, leaks or is released into the extracellular milieu [5, 6]. Extracellular ATP is a powerful signaling molecule that can regulate cell metabolism, survival, and growth [7]. However, IVD cell response to ATP has not been investigated. The receptors involved in transducing responses to ATP are found in many tissues throughout the body and are responsible for different kinds of intercellular communication. ATP receptor subtypes are ligand-gated ion channels (P2X) and G-protein coupled receptors (P2Y). P2X receptors show calcium permeability while P2Y receptors mediate calcium release from intracellular stores in response to ATP [6]. Direct ATP application to the cell has been reported to cause a change in membrane conductance in a variety of tissues [8]. The voltage sensitive dye di-8-ANEPPS allows for a noninvasive method of measuring fluorescence changes of the cell membrane, which are proportional to variations of the transmembrane potential [8]. Therefore, the objectives of this study were (1) to develop a novel transmembrane potential measuring system using di-8-ANEPPS dye and (2) to investigate the response of NP cells to ATP by measuring the change in transmembrane potential.