Depolarization evoked co-release of tachykinins from enteric nerves in the guinea-pig proximal colon

Abstract

The aim of this study was to assess at which extent an even co-release of the tachykinins, substance P (SP) and neurokinin A (NKA), occurs from enteric neurons/nerves of the guinea-pig proximal colon during graded depolarization. In this preparation, a sharply diverging NK₁/NK₂ receptor pattern of tachykininergic co-transmission has been observed in physiological studies. The experiments were performed in capsaicin-pretreated (10μM for 15min) mucosa-free smooth muscle of guinea-pig proximal colon, to exclude the mucosa and the peripheral endings of primary afferent nerves as possible sources of released tachykinins. The content of extractable tachykinins was measured as SP- and NKA-like immunoreactivities (-LI) by radioimmunoassay. Chromatographic characterization of aqueous acetic acid extracts showed one peak of SP-LI corresponding to authentic SP, whereas there were multiple peaks of NKA-LI, the major one co-eluting with authentic NKA. An increased outflow of both SP- and NKA-LI was evenly produced in a concentration-dependent manner when the preparations were superfused with a high potassium (K) medium in which NaCl had been replaced with equimolar amounts (20–100mM) of KCl. The high K-evoked release of SP- and NKA-LI was dependent upon the presence of extracellular calcium and was inhibited by about 50% in the presence of the N-type voltage-dependent calcium channel blocker, ω-conotoxin GVIA (0.1μM). ω-Conotoxin MVIIC (1μM), a non-selective blocker of N-, P- and Q-type voltage-dependent calcium channels, likewise produced about 40% inhibition of evoked release of both peptides. No evidence for a role of L-type channels in tachykinin release was obtained, since the addition of nifedipine (1μM) or Bay K8644 (1μM) did not significantly affect the response to high K. Neither NK₁ receptor agonist (septide, 0.1μM) or antagonist(SR 140333, 10nM) nor NK₂ receptor agonists ([βAla⁸]NKA(4-10) and GR 64349, 0.1μM each) or antagonist (SR 48968, 10nM) did affect the high K-evoked release of tachykinins. We conclude that SP and NKA are evenly co-released in response to graded depolarization of enteric nerves in the guinea-pig colon. Therefore, the specialization of tachykininergic transmission observed in functional studies does not originate at the prejunctional level. The co-release of tachykinins involves the influx of extracellular calcium via N-type but not L-type calcium channels. No evidence for the presence of NK₁ or NK₂ autoreceptors affecting tachykinin release from enteric neurons was obtained.