Functional evidence for NO‐synthase activation by substance P through a mechanism not involving classical tachykinin receptors in guinea‐pig ileum in vitro

1 This study tested the hypothesis that a nitric oxide synthase (NOS) was activated in guinea‐pig ileum in vitro in response to substance P (SP), and attempted to characterize the tachykinin receptor involved in this activation by the use of selective receptor agonists and antagonists. 2 Strips of guinea‐pig ileum (8 × 2 mm) were superfused (Krebs, 37°C, 2 ml min −1 ) with: (i) tachykinin receptor agonists: SP, GR 73,632 (NK 1 ), GR 64,349 (NK 2 ), senktide (NK 3 ), and neuropeptide (NP)γ; (ii) tachykinin receptor antagonists: CP 99,994 (NK 1 ), SR 48,968 (NK 2 ), SR 142,801 (NK 3 ); (iii) nerve‐related agents: carbachol (CCh), atropine, tetrodotoxin (TTX), hexamethonium; (iv) NOS inhibitors: N/>ω‐nitro‐L‐arginine‐methyl‐ester (L‐NAME), N ω ‐monomethyl‐L‐arginine (L‐NMMA) and aminoguanidine (AG); (v) NO‐related agents, L‐arginine (L‐Arg), D‐arginine (D‐Arg), sodium nitroprusside (NaNP) and methaemoglobin. Muscle contractility was recorded isometrically and quantified as integrated area of activity. 3 SP, tachykinin receptor agonists and NP γ (10 pM to 10 μ m ), produced concentration‐dependent contractions of ileal strips, with EC 50 s in the nanomolar range, and maximal responses ( E max ) attained at 0.1 μ m for SP and 1 μ m for the other agonists. The E max response to SP equalled that to KCl (60 mM) taken as a 100% control (99.3% [93.0–105.7]; mean and 95% CI; n = 12); a comparable E max contraction was obtained with the other tachykinin receptor agonists (1 μ m ) as well as with CCh (1 μ m ). 4 Under baseline conditions, L‐NAME (1 μ m ), L‐NMMA (1 μ m ) and AG (1 μ m ), failed to contract the muscle strip. In contrast, when superfused for 3 min, 10 min after SP (0.1 μ m ), they induced a transient contraction of the strip (e.g. for 1 μ m L‐NAME: 50 to 70 s duration; amplitude 73 ± 12%, n = 24). 5 The NOS inhibitor‐induced contractile response was not obtained after KCl (60 mM), GR 73,632, GR 64,349, senktide or CCh (all up to 1 μ m ). In contrast, this contractile response was obtained after NP γ (1 μ m ). 6 Blockade of tachykinin NK 1 , NK 2 and NK 3 receptors by continuous superfusion of CP 99,994, SR 48,968 and SR 142,801 (1 μ m ) respectively, starting 5 min before SP, did not modify the response to L‐NAME, superfused 10 min after SP (0.1 μ m ). The contractile response to L‐NAME (1 μ m ) was blocked by atropine (1 μ m ), superfused either before or after SP. In contrast, it persisted after TTX or hexamethonium (1 μ m ) superfused in the same conditions. 7 The amplitude of NOS inhibitor‐induced contraction (1 μ m ) was dependent on the concentration of priming SP (1 pM to 1 μ m ). In contrast, the contractile response to NOS inhibitors (1 nM to 10 μ m ) of the ileum strip primed with SP (0.1 μ m ) was not concentration‐related. 8 L‐NAME‐induced contraction was prevented by continuous superfusion of L‐Arg (1 μ m ), but not D‐Arg (1 μ m ). In addition, the NO donor, sodium nitroprusside (1 μ m ) and the NO scavenger, methaemoglobin (10 μg ml −1 ), both prevented the contractile response to L‐NAME. 9 In summary, SP and to a lesser extent NP γ , exert a permissive action allowing contractile stimulating effects of L‐NAME, L‐NMMA and AG, in guinea‐pig ileum in vitro , by a mechanism which apparently does not involve tachykinin NK 1 , NK 2 and NK 3 receptors. This action is likely to result from the activation of a NO‐synthase by SP in the vicinity of intestinal myocytes. Thus, L‐NAME, L‐NMMA or AG, by blocking this SP‐induced NO production, unveiled a smooth muscle contraction which involves a cholinoceptor (atropine‐sensitive) mechanism.