0 2 ) /cm-10 A (y -20 t i s en -30 d t ren -40 r u C-50 -60 100 2 ) 50 m /cA 0 ( y is t -50 n e d tne-100 r r u C-150 -200 ATP UTP UDP 10 min Amiloride 100 M 3 min meATP BzATP Genistein Amiloride 100 M 78 Clinical and Experimental Otorhinolaryngology Vol. 1, No. 2: 75-79, June 2008 A A 100 50 2 ) /cm 0 A t(y -50 i s n de-100 t n e ru-150 r C -200 50 2 )cm 0 / A ( y isn t -50 e d t n e r u-100 r C -150 Fig. 3. Effects of ATP, UTP, UDP, and adenosine on the Isc. (A) A representative trace of 5 experiments. UDP acting on P2Y6 receptors was less potent than UTP on P2Y2 receptors. UDP was preincubated with hexokinase in the presence of glucose to eliminate small amount of UTP cont- amination. (B) Effect of adenosine in the presence of amiloride on the Isc. Adenosine (Ad) also activated chloride secretion, which was inhibited by a nonspecific blocker (8-SPT). Ad: Adenosine; 8-SPT: 8-(p-Sulfophenyl) theophylline. Fig. 4. Effects of P2X agonists, genistein (A), and nitric oxide donors (B) in the presence of amiloride on the Isc. None of these agents had an effect on the nasal polyps in situ. (A) αβmeATP, BzATP (100 M), and genistein (20 M) were used. (B) L-arginine (L-arg, 1 mM), sodium nitroprus- side (SNP, 1 mM), and ATP (100 M) were used. Alpha-beta-meATP: Alpha-beta-methyleneadenosine 5’-triphosphate; BzATP: 2’- & 3’-O-(4-benzoyl-benzoyl) adenosine 5’-triphosphate. activator (12), had no effect on the Isc (from -14.1±2.5 to -14.4 ±2.9 μA/cm2, n=6, Fig. 4A). Perfusion of L-arginine (1 mM, sub- strate for nitric oxide synthase, n=5) and sodium nitroprusside (1 mM, nitric oxide donor, n=7) showed no response in the absence (-101.7±20.7 μA/cm2 to -95.8±22.3 μA/cm2, -122.1 ±19.6 μA/cm2 to -118.8±20.5 μA/cm2, respectively) and pres- ence (from -19.8±4.6 to -18.9±4.3 μA/cm2, -18.8±4.6 to -18.9 ±4.3 μA/cm2, respectively, Fig. 4B) of amiloride. DISCUSSION The Isc measured in polyps largely represents the current from the ciliated epithelial cells because the superficial epithelium of the polyp is characterized by ~80% ciliated cells (13). The block- ade of Na+ absorption by amiloride is consistent with a previ- ous report involving nasal polyps in situ (14). In the presence of amiloride, DIDS had little effect on the Isc, but DPC abolished the Isc. Although DIDS and DPC are non-specific anion channel blockers, current pharmacology indicates that DIDS does not act on the CFTR chloride channel, even at high concentrations (15), and DPC induces a voltage-dependent blockade of CFTR when used at micromolar concentrations (16). Therefore, after the addition of amiloride the Isc is likely to be dominated by CFTR-mediated chloride conductance. ATP strongly stimulated DPC-sensitive chloride secretion and its potency was similar to UTP. This is consistent with the activation of the human P2Y2 receptor (17) because ATP acts as a competitive antagonist on the human P2Y4 receptor (18). The EC50 of ATP and UTP mea- sured in this study was 5.3 and 7.3 μM, respectively, which was similar to the EC50 reported in cultured cells in terms of anion transport (ATP, 1.0 μM) (19) and ciliary beat frequency (UTP, B B 10 min Amiloride 100 M 8-SPT 100 M L-arg SNP ATP 4 min Amiloride 100 M