The effect of C-type natriuretic peptide on delayed rectifier potassium currents in gastric antral circular myocytes of the guinea-pig

C-type natriuretic peptides (CNP) play an inhibitory role in smooth muscle motility of the gastrointestinal tract, but the effect of CNP on delayed rectifier potassium currents is still unclear. This study was designed to investigate the effect of CNP on delayed rectifier potassium currents and its mechanism by using conventional whole-cell patch-clamp technique in guinea-pig gastric myocytes isolated by collagenase. CNP significantly inhibited delayed rectifier potassium currents [I(K (V))] in dose-dependent manner, and CNP inhibited the peak current elicited by depolarized step pulse to 86.1+/-1.6 % (n=7, P<0.05), 78.4+/-2.6 % (n=10, P<0.01) and 67.7+/-2.3 % (n=14, P<0.01), at concentrations of 0.01 micromol/l, 0.1 micromol/l and 1 micromol/l, respectively, at +60 mV. When the cells were preincubated with 0.1 micromol/l LY83583, a guanylate cyclase inhibitor, the 1 ?micromol/l CNP-induced inhibition of I(K (V)) was significantly impaired but when the cells were preincubated with 0.1 micromol/l zaprinast, a cGMP-sensitive phosphodiesterase inhibitor, the 0.01 micromol/l CNP-induced inhibition of I(K (V)) was significantly potentiated. 8-Br-cGMP, a membrane permeable cGMP analogue mimicked inhibitory effect of CNP on I(K (V)). CNP-induced inhibition of I(K (V)) was completely blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). The results suggest that CNP inhibits the delayed rectifier potassium currents via cGMP-PKG signal pathway in the gastric antral circular myocytes of the guinea-pig.

TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs+ > K+ > Na+, similar to that in the murine stomach. With 0.2 mM GTPγS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-Gq/11 antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La3+ and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.

ATP-sensitive K+ channels composed of Kir6.1 and SUR2B subunits in guinea pig gastric myocytes

This study was designed to identify the single-channel properties and molecular entity of ATP-sensitive K+ (KATP) channels in guinea pig gastric myocytes with patch-clamp recording and RT-PCR. Pinacidil and diazoxide activated KATP currents in a glibenclamide-sensitive manner. The open probability of channels was enhanced by the application of 10 μM pinacidil from 0.085 ± 0.04 to 0.20 ± 0.05 ( n = 7) and was completely blocked by 10 μM glibenclamide. Single-channel conductance was 37.3 ± 2.5 pS ( n = 4) between −80 and −20 mV in symmetrical K+ gradient conditions. In inside-out mode, KATP channels showed no spontaneous openings and were activated by the application of nucleotide diphosphates to the cytoplasmic side. These single-channel properties are similar to those of the nucleotide diphosphate-dependent K+ channels in vascular smooth muscle, which are composed of Kir6.1 and sulfonylurea receptor (SUR)2B. RT-PCR demonstrated the presence of Kir6.1, Kir6.2, and SUR2B in guinea pig stomach smooth muscle cells. These results suggest that KATP channels in smooth muscle cells of the guinea pig stomach are composed of Kir6.1 and SUR2B.