Substance P modulates localized calcium transients and membrane current responses in murine colonic myocytes

1 Neurokinins contribute to the neural regulation of gastrointestinal (GI) smooth muscles. We studied responses of murine colonic smooth muscle cells to substance P (SP) and NK1 and NK2 agonists using confocal microscopy and the patch clamp technique. 2 Colonic myocytes generated localized Ca2+ transients that were coupled to spontaneous transient outward currents (STOCs). SP (10(-10) M) increased Ca2+ transients and STOCs. Higher concentrations of SP (10(-6) M) increased basal Ca2+ and inhibited Ca2+ transients and STOCs. 3 Effects of SP were due to increased Ca2+ entry via L-type Ca2+ channels, and were mediated by protein kinase C (PKC). Nifedipine (10(-6) M) and the PKC inhibitor, GF 109203X (10(-6) M) reduced L-type Ca2+ current and blocked the effects of SP. 4 SP responses depended upon parallel stimulation of NK1 and NK2 receptors. NK1 agonist ([Sar(9),Met(O-2)(11)]-substance P; SSP) and NK2 agonists (neurokinin A (NKA) or GR-64349) did not mimic the effects of SP alone, but NK1 and NK2 agonists were effective when added in combination (10(-10)-10(-6) M). Consistent with this, either an NK1-specific antagonist (GR-82334; 10(-7) M) or an NK2-specific antagonist (MEN 10,627; 10(-7) M) blocked responses to Sp (10(-6) M). 5 Ryanodine (10(-5) M) blocked the increase in Ca2+ transients and STOCs in response to SP (10(-10) M). 6 Our findings show that low concentrations of SP, via PKC-dependent enhancement of L-type Ca2+ current and recruitment of ryanodine receptors, stimulate Ca2+ transients. At higher concentrations of Sp (10-6 m), basal Ca2+ increases and spontaneous Ca2+ transients and STOCs are inhibited.