The role of cholecystokinin (CCK) in the effect of dietary lipid on proximal gastrointestinal function and satiety is controversial. Recent work suggests that fatty acid chain length may be a determining factor. We investigated the mechanism by which long- and short-chain fatty acids activate jejunal afferent nerves in rats. Whole mesenteric afferent nerve discharge was recorded in anaesthetized male Wistar rats during luminal perfusion of saline, sodium oleate, and sodium butyrate (both 10 mM). Both fatty acids evoked characteristic afferent nerve responses, distinct from the mechanical response to saline, that were abolished in rats following chronic subdiaphragmatic vagotomy. The effect of oleate was abolished by the CCK-A receptor antagonist Devazepide (0.5 mg/kg), whereas the effect of butyrate persisted despite pretreatment with either Devazepide or a combination of the calcium channel inhibitors nifedipine (1 mg/kg) and the omega-conotoxins GVIA and SVIB (each 25 microg/kg). In summary, long- and short-chain fatty acids activate intestinal vagal afferents by different mechanisms; oleate acts via a CCK-mediated mechanism and butyrate appears to have a direct effect on afferent terminals.
1. This study was performed to elucidate the actions of 5_hydroxytryptamine (5_HT) on mesenteric afferent discharge and to determine the receptor-mechanisms responsible for these effects. The activity of mesenteric afferents innervating the mid-jejunum of urethaneanaesthetized rats was recorded with extracellular microelectrodes. The discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software. 2. The intravenous injection of 5_HT produced a complex pattern of afferent activation with two distinct components which could be distinguished both in terms of the response characteristics and the receptors involved. Initially, in 64% of nerve bundles, there was a brief (2·0 ± 0·1 s) but intense activation of afferent discharge with peak afferent firing increasing with incremental doses of 5_HT. The discharge frequency in seventeen single units from these bundles during the initial response to 10 ìg 5_HT was 13·0 ± 1·8 impulses s¢ from a baseline discharge of 1·0 ± 0·1 impulses s¢. 3. This initial response was mimicked by the 5_HT× receptor agonist, 2-methyl-5_HT, whereas 5_methoxytryptamine (5_MEOT, 10-100 ìg) had no comparable effect. Similarly, the initial 5_HT response was completely abolished by the 5_HT× receptor antagonist, granisetron (0·5 mg kg¢). 4. 5_HT also evoked, in approximately 35% of nerve bundles, a delayed response that single unit analysis showed to be mediated by an entirely different population of afferents from those activated during the initial response. This secondary response to 5_HT was characterized by a more prolonged (> 30 s) but less intense period of afferent activity which was coincident with an increase in intrajejunal pressure, and was mimicked by 5_MEOT (10-100 ìg). 5. The secondary response to 5_HT and the response to 5_MEOT were significantly attenuated by the 5_HT2A receptor antagonist, ketanserin (0·5 mg kg¢), which had no effect on the initial response. 6. The initial response to 5_HT was unaffected by the L-type calcium channel inhibitor nifedipine (1 mg kg¢) or the N-type calcium channel inhibitor ù_conotoxin GVIA (25 ìg kg¢). However, the secondary response to 5_HT was significantly reduced after treatment with nifedipine. 7. These results demonstrate that 5_HT activates different populations of afferent fibres innervating the rat jejunum. One population of afferents is activated directly via stimulation of 5_HT× receptors, while another population responds to 5_HT with a time course consistent with secondary activation of mechanosensitive afferents following 5_HT2A-mediated contractile activity.
and {Department of Biomedical Sciences, University of She eld, Western Bank, She eld S10 2TN1 The e ects of anandamide on K + currents and membrane potential have been examined in freshlyisolated smooth muscle cells from rat hepatic artery and the results compared with the e ects of this arachidonic acid derivative on tension and membrane potential changes in segments of whole artery. 2 In the presence of 0.3 mM L-NOARG and 10 mM indomethacin, anandamide (0.1 ± 100 mM) and endothelium-derived hyperpolarizing factor (EDHF; liberated by acetylcholine, 0.01 ± 10 mM) each relaxed endothelium-intact segments of hepatic artery precontracted with phenylephrine. These e ects of anandamide, but not those of EDHF, were antagonized by the cannabinoid receptor antagonist, SR141716A (3 mM).3 The relaxant e ects of anandamide were una ected by a toxin combination (apamin plus charybdotoxin, each 0.3 mM) which abolishes EDHF relaxations and were essentially unchanged in endothelium-denuded arteries. The relaxant e ects of anandamide in endothelium-intact arteries were signi®cantly reduced in a physiological salt solution containing 30 mM KCl and abolished when the K + concentration was raised to 60 mM. 4 Anandamide (10 mM), acetylcholine (1 mM, via release of EDHF) and levcromakalim (10 mM) each markedly hyperpolarized the membrane potential of the smooth muscle cells of endothelium-intact arteries. However, when the endothelium was removed, the hyperpolarizing e ects of both anandamide (10 mM) and acetylcholine were essentially abolished whereas those of levcromakalim (10 mM) were una ected. 5 Under voltage-clamp conditions, anandamide (10 mM) abolished spontaneous transient outward currents (STOCs) in freshly-isolated single hepatic artery cells held at 0 mV but had no e ect on the holding current at this potential. In current-clamp mode, the spontaneous hyperpolarizing potentials which corresponded to the STOCs were abolished with no signi®cant change in basal membrane potential. 6 Anandamide (10 mM) abolished the iberiotoxin-sensitive K + current (I BK(Ca) ) produced by ca eine and the corresponding hyperpolarizations generated by this xanthine derivative in current-clamp mode. In contrast, anandamide had no e ect on I BK(Ca) generated on exposure to NS1619 (30 mM). 7 It was concluded that anandamide is not EDHF in the rat hepatic artery. Anandamide-induced hyperpolarization is exerted indirectly and requires the presence of the endothelium. Anandamide also acts on the smooth muscle cells to inhibit processes which require functional intracellular calcium stores. This direct action seems more important than membrane hyperpolarization in relaxing phenylephrinecontracted vessels.
BACKGROUND AND PURPOSENaV1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Nav1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACHThe inhibition of Nav1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTSPF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50: 331 nM) and in recombinantly expressed h Nav1.8 channels (IC50: 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav1.5 channels (IC50: ∼10 μM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50: ∼10-18 μM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONSUsing PF-01247324, we have confirmed a role for Nav1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.
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