76 Clinical and Experimental Otorhinolaryngology Vol. 1, No. 2: 75-79, June 2008 MATERIALS AND METHODS The polyps were freshly obtained from patients with chronic sinusitis during endoscopic sinus surgery and were immediately delivered to the laboratory in cold physiologic saline. No local anesthetic agents were introduced before extirpating the polyps. The submucosa of the polyp was removed under a stereomicro- scope and the tissue was divided into several small pieces. The tissue was folded with the apical membrane of the surface ciliat- ed epithelium facing outward and transferred into a recording chamber. Our Institutional Committee approved the use of these tissues with informed consent. The vibrating probe is a technique for measuring extracellu- lar electrical currents that are steady or slowly changing. The diameter of the vibrating probe tip is about 20 μm and allows the detection of voltages in the low nanovolt range; vibration between two positions within the line of current flow yields voltages that correspond to current flow through resistive phys- iologic saline. The technique has been described in detail else- where (10). Briefly, the short circuit current (Isc) was monitored by vibrating a platinum-iridium wire microelectrode that was insulated with parlene-C (Micro Electrodes, Gaithersburg, MD, USA) and which had been coated with platinum black on the exposed tip. The vibration was about 20 μm along both a hori- zontal (X) and vertical (Z) axis. The X-axis was perpendicular to the face of the epithelium. The probe was positioned 30-40 μm from the apical surface of the epithelium with computer-con- trolled, stepper-motor manipulators (Applicable Electronics, Forestdale, MA, USA) and specialized probe software (ASET, version 2.0, Science Wares, East Falmouth, MA, USA). The bath references were 26-gauge platinum-black electrodes. Calibration was performed in physiologic saline (see below) using a glass microelectrode (tip, < 1 μm OD) filled with 3 M KCl as a point source of the current. The frequencies of vibration were in the range of 300-400 Hz and were well-separated for the two orthogo- nal directions. The signals from the oscillators driving the probe were also fed to a dual channel phase-sensitive detector. The signal of the X and Z detectors were connected to a 16 bit ana- log to digital converter (CIO-DAS1602/16, ComputerBoards, Mansfield, MA, USA) in a Pentium IV computer. The sampling interval was 0.6s, which is the minimum in this software. The electrode was positioned such that Isc showed a maximum X value and a minimum Z value; data are expressed as the vector length of the current density and plotted with Origin software, version 6.1 (OriginLab Software, Northampton, MA, USA). The output from the vibrating probe depends not only on the spe- cific short circuit current of the epithelium, but also on the posi- tion of the probe from the surface of the tissue and the exact geometry of each tissue sample. The current density reported here refers to the flux at the position of the probe and repre- sents only a fraction of the current crossing the epithelium. No changes in the relative position of the probe due to swelling or shrinking of the tissue during experimental treatments were observed. In all experiments, both sides of the polyp were perfused with physiologic saline containing (in mM) 130 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 5 glucose, and 10 HEPES (pH 7.4). Adenosine 5’-tri- phosphate (ATP), uridine 5’-triphosphate (UTP), 2’- and 3’-O- (4-benzoyl-benzoyl)adenosine 5’-triphosphate (BzATP), α,β- methyleneadenosine 5’-triphosphate αβmeATP), 4,4 ′-diisothio- cyanatostilbene-2,2 ′-disulfonic acid disodium salt hydrate (DIDS), L-arginine, sodium nitroprusside (SNP), and 8-(p-sulfophenyl) theophylline (8-SPT) were directly dissolved in physiologic saline just before use. Uridine 5 ’-diphosphate (UDP) was pre-incubat- ed for 1.5 h with hexokinase (1 unit/mL) and glucose (5 mM) because the commercial preparations of UDP may be supplied with a minor component of UTP (11). Amiloride, N-phenylan- thranilic acid (DPC), genistein, and ouabain were dissolved in DMSO and then diluted to 0.1% DMSO in the control solution prior to application. DMSO at this concentration had no effect on the Isc. Each tissue was mounted in a perfusion chamber on the stage of an inverted microscope and continuously perfused at 37℃ at an exchange rate of 0.7 times/sec. Ouabain (10 μM), an inhibitor of Na+, K+-ATPase, was tested to estimate the time for the per- fusate to migrate to the basolateral membrane of the surface epithelium. Application of ouabain decreased the Isc from 8.6± 2.5 min (n=15), which indicates that the ion channel modula- tors used here acted in a short time on the apical membrane of surface epithelium. For analysis, the peak Isc was chosen. Data are expressed as the mean±S.E.M. (n=number of tissues) of the Isc. An increase or decrease in Isc were considered significant at a level of P<0.05. A paired t-test was used. RESULTS The Isc measured in physiologic saline was -124.0±17.0 μA/cm2 (n=33). Perfusion of amiloride (100 μM), an epithelial sodium channel (ENaC) blocker, significantly decreased the Isc by 78.7± 3.1% (to -18.8±2.6 μA/cm2, n=33). In the presence of amiloride, DIDS (1.5 mM) slightly decreased the Isc by 11.1±2.5% (n=17) and subsequent application of DPC (0.5 mM) significantly dec- reased the Isc by 88.0±2.6% (n=17, Fig. 1A). In the presence of amiloride, 100 μM ATP significantly increased the Isc by 460.1 ±171.8% (n=7), which was inhibited by DPC (92.1±1.8%), but not by DIDS (17.9±6.7%, Fig. 1B). ATP and UTP increased the Isc in a dose-dependent manner (Fig. 2). The EC50 of ATP and UTP was 5.3±0.3 (n=5) and 7.7 ±0.8 μM (n=5), respectively. UDP, a P2Y6 receptor agonist, also increased the Isc to 44.5±8.9% of the UTP response (n=5, Fig. 3A). Adenosine, a purinergic receptor type 1 (P1) receptor agonist, increased the Isc by 340.1±167.9% (from -21.4±6.6