Substance P (SP) nerve terminals innervate the intermediolateral cell column (IML) of the thoracic spinal cord, where SP coexists with serotonin (5-HT), neurokinin A (NKA) and thyrotropin-releasing hormone (TRH). Neither the depolarization-induced release of SP nor the presence of other neurochemicals in the regulation of SP release has been directly studied in this system. In the present study, basal and K+-stimulated release of SP from the microdissected intermediate area (including the IML, intercalated nucleus and central autonomic nucleus) of the rat thoracic spinal cord, and the regulation of SP release by presynaptic autoreceptors and by coexisting neurochemicals (5-HT, NKA and TRH) were studied using an in vitro superfusion system. Potassium evoked a concentration-and extracellular Ca2+-dependent release of SP. In rats pretreated with the serotoninergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), both SP content and the absolute amount of SP released were decreased. However, the fraction of the remaining tissue content of SP released by K+ depolarization was not changed subsequent to 5,7-DHT treatment. Moreover, 5-HT, 5-HTlB agonists (CGS-12066B and RU 24969) and a 5-HT3 agonist (2-methyl-5-HT) did not alter the K+-evoked release of SP. These data demonstrate that SP is released from the intermediate area of the rat thoracic spinal cord and some of the SP released comes from serotoninergic nerve terminals. Although 5-HT coexists with SP in the IML, neither endogenous 5-HT nor 5-HT receptor ligands appear to regulate the release of SP. Other colocalized neuropeptides (NKA and TRH) are not involved in the regulation of SP release because neither NKA, a NK2 agonist (GR 64349) nor a TRH analog (MK-771) changed the K+-evoked release of SP.
A neurokinin-1 (NK,) antagonist (GR 82334) dose-dependently (10-9-10-7 M) increased the K+-stimulated release of SP. These data suggest the presence of presynaptic inhibitory NK, autoreceptors. Whereas, NK, agonists, and [Sar9, Met (02)11]SP ( 10-8-10-6 M)], increased the basal and K+-stimulated release of SP, the excitatory effects of GR 73632 were not blocked by the NK1 antagonist. Moreover, GR 73632 increased the emus of SP to a greater extent in the absence of peptidase inhibitors. Thus, the effect of NK, agonists on the release of SP may be related to an inhibition of peptide degradation rather than activation of NK, autoreceptors.