Paradoxical behavior of neuromedin U in isolated smooth muscle cells and intact tissue.

Neuromedin U (NmU) is a neuropeptide showing high levels of structural conservation across different species. Since its discovery in 1985, NmU has been implicated in numerous physiological roles, including smooth muscle contraction, energy homeostasis, stress, intestinal ion transport, pronociception, and circadian rhythm. Two G-protein-coupled receptors have been identified for NmU and cloned from humans, rats, and mice. Recombinantly expressed NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins, and NmU binds essentially irreversibly, preventing signaling to repetitive applications of NmU. However, it is unclear whether these properties reflect those of endogenously expressed NmU receptors or how these properties influence the functional consequences of NmU receptor signaling. Here, we have explored the signaling by rat NmU receptors expressed endogenously in cultured rat colonic smooth muscle cells and explore the functional consequence of this signaling by investigating the NmU-mediated contraction of ex vivo rat colonic smooth muscle preparations. We demonstrate that endogenous rat NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins. Furthermore, we show complex patterns of Ca(2+) signaling, including oscillations, and provide evidence of essentially irreversible binding of NmU to smooth muscle cells. Challenge of either circular or longitudinal rat isolated colonic smooth muscle preparations with NmU resulted in robust contractions. Stimulation was direct, and paradoxically, repetitive applications of NmU mediated repetitive contractions with no evidence of desensitization, highlighting a major discrepancy in the behavior of NmU in single cells and in intact tissues. The reason for this discrepancy is presently unknown.

The prokinetic-like activity of ghrelin in rat isolated stomach is mediated via cholinergic and tachykininergic motor neurones.

Ghrelin increases electrically evoked, neuronally mediated contractions of rat isolated forestomach, a prokinetic-like activity. Since the nerve type sensitive to ghrelin is unclear, we examined the activity of ghrelin in the presence of antagonists at receptors for the main gastric motor neurotransmitters. Electrical field stimulation (EFS; 5 Hz, 0.5 ms, +/-50 V, 30 s every 3 min) of circular muscle preparations evoked tetrodotoxin 1 microM-sensitive responses, consisting of a small initial contraction followed by a further contraction or more usually, by muscle relaxation. Termination of EFS evoked a large rapidly developing after-contraction. Atropine 1 microM prevented contractions during EFS, increased any relaxations and prolonged the after-contractions. Nomega-Nitro-L-arginine-methyl-ester-hydrochloride (L-NAME) 0.3 mM prevented relaxations during EFS, changing the triphasic response into a monophasic contraction. The tachykinin NK1 and tachykinin NK2 receptor antagonists N-acetyl-L-tryptophan-3,5-bistrifluoromethyl-benzyl-ester (L-732,138 1 microM) and Cyclo[Gln-Trp-Phe-Gly-Leu-CH2N(CH3)-Leu] (MDL-29,913 1 microM) each reduced EFS-evoked relaxations; the latter also reduced the after-contractions. The tachykinin NK3 receptor antagonist (-)-(S)-N-(alpha-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB-235375, 0.1 microM) had no effects. The combination of tachykinin NK(1,2,3) receptor antagonists reduced the after-contractions and abolished relaxations during EFS, replacing this with a contraction. In control tissues, ghrelin 1 microM increased EFS-induced contractions and tended to reduce any relaxations. In the presence of atropine 1 microM, L-NAME 0.3 mM or the tachykinin receptor antagonists (as above), ghrelin 1 microM increased any EFS-induced contraction but in the presence of atropine had no effects on EFS-evoked relaxations. We conclude that EFS evokes responses mediated by acetylcholine, nitric oxide and tachykinins. Ghrelin facilitates both cholinergic and tachykininergic excitatory pathways, consistent with activity within the enteric nervous system and possibly the vagus nerve.

Prokineticin-2, motilin, ghrelin and metoclopramide: prokinetic utility in mouse stomach and colon.

The ability of agents described as gastrointestinal prokinetics (prokineticin-2, [Nle(13)]-motilin, ghrelin), to modulate nerve-mediated contractions of mouse isolated stomach and colon was determined and compared with the prokinetic and 5-HT(4) receptor agonist, metoclopramide. Circular muscle preparations were electrically field-stimulated (EFS) to evoke cholinergically mediated contractions. Metoclopramide 10-100 microM facilitated EFS-evoked contractions in forestomach (n = 5-11, P < 0.05); 1 mM inhibited. Metoclopramide had no effects in colon, apart from 100 microM which reduced contractions. Prokineticin-2 0.001 nM-0.1 microM (n = 3-7) or [Nle(13)]-motilin 0.1 nM-1 microM (n = 4-8) had no effects in forestomach or colon. Ghrelin 0.01-1 microM facilitated EFS-evoked contractions in forestomach (n = 5-7, P < 0.05) but not in colon (n = 5-8). We conclude that ghrelin and metoclopramide facilitate excitatory nerve activity because neither affected inhibitory responses to EFS in the presence of atropine, or contractions to carbachol. Further, prokineticin-2 and [Nle(13)]-motilin are unlikely to exert gastric prokinetic activity in this species, the inactivity of the latter being consistent with an absence of the motilin receptor in rodents.