Expression of Transient Receptor Potential (TRP) channels of the M-family in cultured human neuroendocrine tumor (NET) cells

Abstract

Background: Human NET cells express numerous of ion channel types. So far, voltage-dependend Ca2+ channels (all subtypes), sodium-, and potassium channels were described. Recently, chloride channels were detected (unpublished observation). In addition to these selective ion channels, non-selective TRP channels were also registered. These channels could be activated by active or passive store depletion or artificially by extracellular stimulation such as G-protein coupled receptor activation. Generally, channel activation will induce an increase of intracellular free Ca2+ concentration ([Ca2+]i). However, the physiological role of these ion channels in NET cells is absolutely unclear. Aim: Study of the electrophysiological properties of TRP channels by patch-clamp technique and fura-2 measurements in order to characterize the relevance of TRPs in the NE system more closely. Methods: Permanent NET BON cells were used as a representative cell model for NET diseases and were cultured according to established methods. In parallel, freshly isolated primary NET cells from several patients were cultured and also investigated. The TRP channel activities were measured by patch-clamp technique. [Ca2+]i was registered with the fluorescent dye fura-2. Furthermore, real-time PCR was used. Results: TRP channel activity of the M8 subtype (TRPM8) could be detected using the cooling agents menthol or icilin. As a result, Ca2+ influx was at higher levels than without activation of these channels. Furthermore, Ca2+ influx could also be registered after induction of oxidative stress by hydrogen peroxide (1 mM H2O2). This effect could be due to activation of the M7 subtype (TRPM7). Importantly, nickel-sensitive TRP-channel activity could additionally be detected in BON cells by induction of capacitative Ca2+ entry (CCE) after passive store depletion with 10µM cyclopiazonic acid (CPA). In the presence of 1 mM NiCl2, the CCE amplitude (set to 100%) decreased by 60±4% (recovery occurred to 78±3%) (±SEM; all n=3). Conclusion: For the first time, TRP channel expression of the M-family in NET cells could be demonstrated by several methods. TRPs could have an important role in the NE system. Specific intracellular TRP channel modulators (e.g. bioactive lipids) could exist. However, further investigation about expression and function of TRPs in NET cells are necessary.