Contractile effects and intracellular Ca2+ signalling induced by motilin and erythromycin in the circular smooth muscle of human colon

Motilin has excitatory effects on the colon of the rabbit and the dog, but little is known of its effect on the human colon. The aim of this study was to investigate the effects induced by motilin and erythromycin A (EMA) on muscle strips and on single cells from primary cultures from human colon. Isotonic contraction was recorded in circular muscle strips from macroscopically normal resection specimens of patients operated on for colonic neoplasm. Agonist-induced intracellular Ca2+ ([Ca2+](i)) signalling was studied in primary cultures of colonic smooth-muscle cells using the ratiometric Ca2+ indicator Indo 1, on a laser-scanning confocal epifluorescence microscope. In circular muscle strips, norleucine(13)-porcine motilin ([Nle(13)]-pm)and EMA induced tonic contractions with an EC50 of 92 +/- 21 nmol L-1 and 31 +/- 16 mu mol L-1, respectively. The maximal contraction was 21 +/- 4% (motilin) and 33 +/- 12% (EMA) of the response to 10(-4) mol L-1 acetylcholine (ACh). The motilin antagonist OHM-11526 (10(-5.5) mol L-1) abolished the effects of both [Nle(13)]-pm and EMA. Neither tetrodotoxin (10(-5.5) mol L-1), L-nitro-D-arginine methyl ester (L-NAME) (10(-3.5) mol L-1) nor guanethidine (10(-5) mol L-1) interfered with the effects of [Nle(13)]-pm or EMA. [Nle(13)]-pm (10(-11)-10(-6) mol L-1) induced rises of [Ca2+](i) in cultured colonic myocytes. At 10(-6) mol L-1, 94% of the cells responded, and half of the cells responded at 1.4 nmol L-1 [Nle(13)]-pm. 81% (35/43) and 95% (75/79) responded to EMA (10(-6) mol L-1) and acetylcholine (ACh, 10(-4) mol L-1), respectively. The motilin antagonist GM-109 inhibited motilin- and EMA-induced [Ca2+](i) rises. In the absence of extracellular Ca2+, only 13% (7/52) of the cells responded to [Nle(13)]-pm (10(-6) mol L-1) vs. 90% (47/52) to ACh (10(-4) mol L-1). Motilin and EMA have direct excitatory effects on circular smooth muscle from the human colon and these effects are mediated via a smooth-muscle motilin receptor. These findings suggest that motilin may regulate colonic motility and that motilides may have therapeutic potential for the treatment of colonic hypomotility.