Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at −80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current- voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.

Sodium-potassium-chloride co-transporter (NKCC) is a membrane bound channel protein that plays a prominent role in a variety of epithelial absorptive, secretory processes and a direct role in cell volume regulation, in which NKCC transports sodium, potassium, and chloride ions across the cell membrane. It has been known that prostaglandin E2 (PGE2) induces an acute cell lysis of specific hemocyte type, oenocytoid, to release prophenoloxidase into the plasma and ouabain (a specific sodium pump inhibitor) inhibits the oenocytoid cell lysis resulting in preventing phenoloxidase activation. However, it is not clear how the intracellular signaling pathway leads to oenocytoid cell lysis in response to PGE2. This study was designed to analyze functional role of NKCC in the cell lysis to release prophenoloxidase. A gene structure of NKCC was derived from cDNA library of Spodoptera exigua hemocyte, NKCC was expressed in all developmental stages and tissues. A real time quantitative RT-PCR showed that bacterial challenge significantly induced its expression. Specific inhibitors of NKCC, bumetanide and chlorothiazide, clearly prevented the cell lysis in a dose dependent manner. When RNA interference using double stranded RNA (dsRNA) specific to NKCC suppressed its expression, the oenocytoid lysis and PO activation was significantly inhibited in response to PGE2. It also reduced nodule formation to bacterial challenge. These results indicate that NKCC is associated with oenocytoid cell lysis probably by increasing cell volume through inward transport of ions in response to PGE2.