- The hypothesis that substance P (SP) elicits both direct and indirect responses on the canine proximal colon was tested using two different in vitro preparations. The mucosa contained the muscularis mucosa and the attendant submucosal plexuses whereas the epithelium, being devoid of both, was functionally "nerve-free." Dose-dependent stimulation was noted on both preparations, increases in peak current (microamperes per squared centimeter) as well as charge transfers (millicoulombs per squared centimeter) were monitored. Tetrodotoxin significantly reduced mucosal responses whereas atropine and the H1 antagonists mepyramine and diphenhydramine had only marginal effects. None of these agents affected the responses of the epithelium to SP. Indomethacin significantly reduced responses in both preparations. Removal of Na+ or Cl- or the use of C- channel blockers (9-anthracene carboxylic acid and N-phenylanthranilic acid) produced a significant reduction of SP responses across the epithelium. Thus, SP has both direct and indirect affects on the colon; activation of the cyclooxygenase pathway could be significant.