Effect Of C-Type Natriuretic Peptide On Pacemaker Currents In Interstitial Cells Cajal From Murine Small Intestine

Considerable evidence suggests that interstitial cells of Cajal (ICC) are the pacemaker cell of slow wave in gastro intestinal. According to the position, ICC can be divided into myenteric ICC (ICC-MY) and intramuscular ICC (ICC-IM), which take the different functions in the generation, propagation of pacemaker currents and mediation of neurotransmition. C-type natriuretic peptide (CNP) is one of the members of the natriuretic peptides family. CNP distributes widely and has diverse biological functions. In our previous study, we have shown that CNP can inhibit the motility of gastric smooth muscle. However, the effect of CNP on pacemaker currents and its possible mechanism have not been clear.Cultured ICC was identified with C-Kit immunofluorescence antibody. Whole-cell, inside-out and on-cell mode patch clamp techniques were used to characterize pacemaker currents of ICC. Our experiments showed that almost all the cultured cells were immunopositive for C-Kit antibody protein. In whole-cell patch clamp mode, decreasing the intracellular Ca2+ concentrations by using 10mmol/L EGTA in pipette, the sustained inward holding currents were activated in ICC. W-7, a calmodulin inhibitor, potantiated the amplitude of spontaneous transient inward currents in ICC. There were some cells insensitve to intracellular low free calcium and W-7. In inside-out patch clamp mode, when the intracellular calcium from 1μmol/L decreased to 0.1μmol/L, the activities of pacemaker channel were significantly potantiated. In on cell mode, W-7 also significantly potantiated pacemaker conductance activities. By using immunocytochemistry technique to exhibit natriuretic peptide receptors (NPR) in ICC and the membrane currents were recorded by using whole-cell patch clamp technique. Our experiments showed that NPR-A and NPR-B were expressed in ICC from murine small intestine. Whole cell recordings further showed that the amplitude of pacemaker currents in intestinal small networks of ICC was 322±22pA and the frequency was 16.25±0.95Hz. CNP significantly suppressed the amplitude of pacemaker currents in a dose-dependent manner in networks ICC. The inhibited percentage of pacemaker currents by CNP were 76.05±1.51%, 38.24±3.74% and 18.33±2.86%, at the concentrations of 0.01μmol/L,0.1μmol/L and 1μmol/L, respectively. The frequencies of pacemaker currents can be inhibited by CNP from 16.25±0.95Hz to 13±0.9Hz, 12±0.8Hz and 3±0.2Hz, at the concentrations of 0.01μmol/L,0.1μmol/L and 1μmol/L, respectively. CNP significantly inhibited the enhanced effect of W-7, a calmodulin inhibitor, on the amplitude of pacemaker currents in single ICC. The inhibitory effect of CNP was mimicked by 8-Br-cGMP, a membrane permeable cGMP analogue, and potentiated by zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase. Under on-cell patch clamp, CNP and the analogue of cGMP had an inhibitory effect on the activity of pacemaker conductance. However, under inside-out patch clamp, the analogue of cGMP had no effect on the activity of pacemaker conductance. Under whole cell patch clamp, CNP and the analogue of cGMP had not an effect on the sustained inward currents activated by buffering intracellular Ca2+ with EGTA.In conclusion, our results suggest that ICC-MY spontaneously elicited inward transient currents and the currents carry by intracellular low calcium and W-7-sensitive nonselective cation channel. The expression of natriuretic peptide receptor in ICC are immunopositive, furthermore CNP inhibite the amplitude of pacemaker current in ICC. It is possible that CNP has a direct effect on pacemaker ICC. Our results also suggest that the inhibitory effect of CNP on pacemaker currents is mediated by cGMP dependent signal pathway, but the activities of pacemaker conductance can not be directly affected by cGMP which may exert its effect through modulating the intracellular concentration of Ca²⁺.