Identification of SK-951, a novel benzofuran derivative, as an agonist to 5-HT4 receptors

The pharmacological profile of SK-951 ((-)4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl) ethyl]-5-chloro-2,3-dihydro-2-methylbenzo[b]furan-7-carboxamide hemifumarate) was identified in relation to serotonin 5-HT3 and 5-HT4 receptors by the receptor binding assay and functional studies. The receptor binding assay showed that SK-951 bound to the 5-HT3 receptor with a high affinity, to the 5-HT4 receptor with relatively higher affinity and to the muscarinic M2 receptor with a low affinity, but not to dopamine D1 and D2 and serotonin 5-HT1 and 5-HT2 and muscarinic M1 and M3 receptors. SK-951 caused relaxations of tunica muscularis mucosae preparations from rat esophagus which were precontracted with carbachol, and the effects were antagonized by GR113808, a selective 5-HT4 antagonist. In the longitudinal muscle with myenteric plexus (LMMP) preparations from guinea pig ileum, SK-951 enhanced the electrically-stimulated contraction of preparations in which the 5-HT1, 5-HT2 and 5-HT3 receptors were blocked, and it enhanced the electrically-stimulated release of [3H]acetylcholine (ACh). These effects of SK-951 were antagonized by GR113808. SK-951 inhibited the 5-HT3 receptor-mediated contractions. These results indicate that SK-951 possesses properties of an agonist for the 5-HT4 receptor and an antagonist for the 5-HT3 receptor. Thus, SK-951 is a new and potent 5-HT4-receptor agonist and causes contractions of guinea pig ileum mediated by enhancement of ACh release via the 5-HT4 receptor.     

Effects of SK-951, a benzofuran derivative, as a prokinetic agent in rats and dogs

The gastrokinetic activity of SK-951 ((-)4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro-2,3-dihy dro-2-methylbenzo[b]furan-7-carboxamide hemifumarate), a benzofuran derivative with 5-hydroxytryptamine (5-HT)4-receptor agonist activity, was studied in rats and dogs. The effects of SK-951 were also investigated in a model of vagotomy-induced gastroparesis in comparison with cisapride. In rats, both SK-951 and cisapride enhanced gastric emptying of liquids (phenol red) at a dose of 1-100 mg/kg, p.o. Gastric emptying of liquid (acetaminophen) in fasted beagle dogs was enhanced significantly by SK-951 (1.0 mg/kg, i.v.), whereas the effect of cisapride (0.2-1.0 mg/kg, i.v.) was not statically significant. Similar results were found when radiopaque markers were given with standard meal to dogs with vagotomy-induced gastroparesis. The delayed gastric emptying of radiopaque markers by vagotomy was reversed by SK-951 (1.0 mg/kg, i.v.), whereas cisapride showed no effect at doses from 0.1 to 1.0 mg/kg, i.v. These results indicated that oral and intravenous administration of SK-951 accelerates gastric emptying of both liquids and solids in animal models. Thus, SK-951 may be a highly potent and useful prokinetic agent in comparison to cisapride.     

Investigation into the 5-hydroxytryptophan-evoked luminal 5-hydroxytryptamine release from the guinea pig colon

The effect of an endogenous 5-hydroxytryptamine (5-HT) precursor, 5-hydroxytryptophan (5-HTP), on the luminal outflow of 5-HT was examined using the luminally perfused isolated colon of the guinea pig, a model that would facilitate the pharmacological analysis of luminal 5-HT release from enterochromaffin cells (EC cells). 5-HTP (1-10 microM) concentration-dependently caused an increase of the luminal outflow of 5-HT. Either tetrodotoxin (0.3 microM) or atropine (0.2 microM) did not affect the 5-HTP-evoked increase in luminal 5-HT outflow, while the L-type calcium channel blocker, nicardipine (1 microM) or diltiazem (1 microM) reduced the 5-HTP-evoked 5-HT outflow by 47% and 61%, respectively. SB203186 (1 microM), a 5-HT4-receptor antagonist, enhanced the 5-HTP-evoked 5-HT outflow, while ramosetron (1 microM), a 5-HT3-receptor antagonist reduced the stimulating effect of 5-HTP by 66%. Ketanserin (0.1 microM), a 5-HT2A-receptor antagonist did not modify the stimulatory effect of 5-HTP. It is concluded that in the guinea pig colon, 5-HTP facilitates the luminal 5-HT release from EC cells, with no involvement of neuronal mechanisms and a non-neuronal cholinergic system. Furthermore, non-neuronal 5-HT3 and 5-HT4 receptors appear to contribute to the regulation of the luminal 5-HT release evoked by 5-HTP. This new bioassay of the guinea pig colon allows the pharmacological characterization of uncomplicated luminal 5-HT release from EC cells.     

Cholinergic modulation of the release of 5-hydroxytryptamine from the guinea pig ileum

Summary Isolated segements of the guinea pig ileum were vascularly perfused and the release of 5-HT and its metabolite 5-HIAA into the portal venous effluent determined by HPLC with electrochemical detection. Test substances were applied via the arterial perfusion medium. Oxotremorine inhibited concentration-dependently the release of 5-HT and 5-HIAA (by 47% at 1 mol/l). Scopolamine (0.1 mol/1) did not affect the release of 5-HT and 5-HIAA, but antagonized the effect of oxotremorine. In the presence of TTX (1 mol/1) oxotremorine (1 pmol/1) increased the release of 5-HT by 150% and that of 5-HIAA by 220%. This increase was completely blocked by scopolamine. Hexamethonium (100 pmol/1) and TTX (1 pmol/1) reduced the release of 5-HT by 32 and 40%, respectively. DMPP (10 pmol/1) increased the release of 5-HT by 57%, and this effect was prevented by hexamethonium. Neither DMPP nor hexamethonium significantly affected the release of 5-HIAA. The enhancing effect of DMPP on 5-HT release was increased and prolonged in the presence of TTX or scopolamine. Nicotine (1, 10 or 30 mol/l) alone did not cause a consistent increase in the release of 5-HT. However, in the presence of scopolamine nicotine increased the release of 5-HT by 57%. In conclusion, the release of intestinal 5-HT is facilitated via muscarine and nicotine receptors located on the enterochromaffin cells. Indirect evidence suggests that the release of 5-HT is additionally modulated by an as yet unknown inhibitory neurotransmitter released by muscarine receptor activation.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium - 5-HT 5-hydroxytryptamine - 5-HIAA 5-hydroxyindoleacetic acid - TTX tetrodotoxin Send offprint requests to H. S. at the above address     

In vivo assessment of acceleration of motor activity associated with acetylcholine release via 5-hydroxytryptamine4 receptor in dog intestine

Effect of mosapride, a benzamide, on the motor activity associated with the release of endogenous acetylcholine (ACh) from enteric neurons was examined in the ileum of anesthetized dogs using an in vivo microdialysis method and compared with the effect of 5-hydroxytryptamine (5-HT). Intraarterial administration of 5-HT accelerated intestinal motor activity and increased the concentration of dialysate ACh, and the responses were inhibited by SB204070, a specific 5-HT4-receptor antagonist, but were apparently not affected by methiothepin, ketanserin and granisetron. Intraarterial administration of mosapride, a prokinetic benzamide, accelerated intestinal motor activity and the concentration of dialysate ACh increased. The effects of mosapride were antagonized by SB204070. Specific [125I]SB207710 binding was observed in the myenteric and submucosal plexuses and muscle layers of dog ileum by in vitro receptor autoradiography. High densities of [125I]SB207710 binding sites were detected in the myenteric and submucosal plexuses. Mosapride as well as SB204070 inhibited [125I]SB207710 binding. Thus, in the whole body of dogs, 5-HT and mosapride accelerated the intestinal motor activity due to the increases in ACh release mediated by stimulation of the 5-HT4 receptor.     

Presynaptic regulation of the release of acetylcholine by 5-hydroxytryptamine.

1 The effect of 5-hydroxytryptamine (5-HT) on the release of acetylcholine (ACh) from bullfrog sympathetic preganglionic nerve terminals and frog sciatic nerve terminals was studied with intra-cellular microelectrodes. 2 The change in transmitter release was measured from the mean quantal content calculated by the variance method from evoked fast e.p.s.ps or e.p.ps in low Ca2+-high Mg2+ Ringer solution. 3 5-HT facilitated the release of ACh in low concentrations and depressed it in relatively high concentrations at both preganglionic and motor nerve terminals. 4 These results suggest the possibility that 5-HT may play a role in regulating cholinergic transmission in general.     

5-Hydroxytryptamine receptors,especially the 5-HT4 receptor,in guinea pig urinary bladder

The function of 5-hydroxytryptamine (5-HT) receptors, especially the 5-HT4 receptor, in the urinary bladder were examined in preparations isolated from the guinea pig by in vitro receptor autoradiography and determinations of mechanical activity and acetylcholine (ACh) release. Specific [125I]SB207710 binding sites were detected evenly throughout the urinary bladder. 5-HT (3 x 10(-8)-10(-4) M) caused contractions of strips of the urinary bladder, in a concentration dependent manner. Ketanserin antagonized the 5-HT-induced contractions, while granisetron and SB204070 antagonized the contractions induced by high concentrations of 5-HT. Atropine inhibited the contractions induced by high concentrations of 5-HT. Ketanserin prevented the 5-HT-induced contractions in the presence of atropine, but granisetron and SB204070 did not affect the contractions under such a condition. 5-HT enhanced the electrically-stimulated (5 Hz, 0.5 ms) outflow of [3H]acetylcholine from strips preloaded with [3H]choline, and the enhancement was antagonized by granisetron and SB204070. Thus, the contractile response to 5-HT was mediated by activations of 5-HT2, 5-HT3 and 5-HT4 receptors. The 5-HT2 receptor may be a property of high affinity to 5-HT and located on the smooth muscle cells. The 5-HT4 as well as 5-HT3 receptor may be a property of low affinity to 5-HT and located on the cholinergic neurons.     

Regional difference in correlation of 5-HT4 receptor distribution with cholinergic transmission in the guinea pig stomach.

Localization and function of 5-HT4 receptors in the stomach were examined in mucosa-free preparations of antrum, corpus and fundus from guinea pig stomach by determination of acetylcholine release and in vitro receptor autoradiography. Specific [125I]SB207710, (1-n-butyl-4-piperidinyl) methyl-8-amino-7-iodo-1,4-benzodioxane-5-carboxylate, binding sites were detected in 3 regions of the stomach. High densities of binding were observed in the myenteric plexus of antrum and corpus, but not fundus. In mucosa-free preparations treated with 5-HT1, 5-HT2 and 5-HT3 receptor antagonists, 5-HT (10(-8)-10(-6) M) potentiated the electrically stimulated (0.5 Hz, 1 ms) outflow of [3H]acetylcholine from antrum and corpus strips preloaded with [3H]choline, but not from fundus strips, and the potentiation was antagonized by SB204070, (1-n-butyl-4-piperidinyl) methyl-8-amino-7-chloro-1,4-benzodioxane-5-carboxylate. Thus, 5-HT4 receptors are located on myenteric cholinergic neurons in the antrum and corpus of guinea pig stomach and their activation evokes the release of acetylcholine.     

Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.     

Descending release of acetylcholine from the locally distended guinea pig ileum.

The effects of local distension of the intestinal wall on the release of acetylcholine (ACh) from the adjacent non-distended part were studied with the segment os isolated guinea pig ileum. Local distension of the intestinal wall induced the increased release of ACh in the distended part and in its anal side but not in its oral side. Such aboral release of ACh by local distension was abolished by tetrodotoxin or atropine in the concentrations which did not block the release in the distended part. When hexamethonium was applied exclusively to the distending part, significant increase of ACh release was observed in both the regions oral to and anal to the distended part. It is suggested that distension stimuli applied to the myenteric plexus are transmitted aborally along the network of the Auerbach's plexus to the anal direction. The release of ACh from the intestine by nicotine or DMPP differed from the occurring during local distension in that the release was localized to the part of the intestine to which the drug was applied.     

Effect of amantadine on cerebral acetylcholine release and content in the guinea pig

The effect of amantadine on cortical acetylcholine release and on cerebral acetyl-choline content in guinea pigs was examined in comparison with amphetamine. Both drugs stimulated gross behaviour and increased cortical acetylcholine release in conscious freely-moving animals. The effects of amantadine were still evident in guinea pigs pretreated with reserpine or α-methyl-p-tyrosine, but were abolished by the combined inhibition of catecholamine synthesis and granular uptake. On the contrary, α-methyl-p-tyrosine pretreatment alone, prevented amphetamine effects both on behaviour and on acetylcholine release. Amantadine increased acetylcholine content in the caudate nucleus, α-methyl-p-tyrosine showed the opposite effect, while amphetamine was ineffective.The discussion is based on the hypothesis that the activation of the cerebral adrenergic neurons is associated with a cholinergic counter-action. The conclusion is reached that the role of dopamine in determining this counter-action is predominant.     

Dual agonist-antagonist effects of 5-hydroxytryptamine (5-HT) in the guinea pig ileum: evidence for a selective receptor desensitization effect

The application of 5-HT to strips of whole ileum or the longitudinal muscle-myenteric plexus preparation from guinea pigs caused dose-dependent muscle contractions that were followed by relaxation to baseline tension without washing off the drug (fade). The contractile effect of a subsequent addition of 5-HT, 4 min after a priming dose of 5-HT, was markedly reduced. Increasing priming doses of 5-HT caused proportional increases in the 5-HT Emax50, up to a priming dose that completely abolished the contractile effects caused by further additions of 5-HT. The auto-blockade of the 5-HT responses was selective to drugs acting on serotonergic receptors. 5-HT did not antagonize the effects of acetylcholine, dimethylphenylpiperazinium, nicotine, histamine, prostaglandin E2, substance P or angiotensin II. N-methyl-5-HT was the most potent analogue in mimicking the effects of 5-HT to produce auto-blockade and fade of the contractile responses. Other structural analogues of 5-HT that shared the 5-HT blocking effect were 5-methoxytryptamine, 5,6-dihydroxytryptamine, and N,N-dimethyltryptamine although these compounds were considerably less potent than 5-HT as blockers or as agonists. Results suggest that fade and the auto-blockade are part of a common effect and are discussed in relation to a model of drug-induced selective receptor desensitization.     

Modification of 5-hydroxytryptophan-evoked 5-hydroxytryptamine formation of guinea pig colonic mucosa by reactive oxygen species

We studied whether reactive oxygen species (ROS) generated by normal colonic mucosa affect 5-hydroxytryptophan (5-HTP)-evoked 5-HT formation (measured as the sum of 5-HT plus 5-hydroxyindole acetic acid (5-HIAA) accumulation) of guinea pig's isolated colonic mucosa. Catalase (3000-6000 U/ml), a hydrogen peroxide (H2O2) scavenger or diphenylene iodonium (DPI, 10-100 microM), an NADPH oxidase inhibitor, concentration-dependently caused an increase of the sum of 5-HT plus 5-HIAA accumulation in the presence of 5-HTP (10 microM), but these drugs did not significantly affect the 5-HT-metabolite in the colonic mucosa measured as the ratio of 5-HIAA/5-HT. Exogenously applied H2O2 (10-100 microM) concentration-dependently inhibited the sum of 5-HT plus 5-HIAA accumulation. In contrast, neither superoxide dismutase (SOD, 100-300 U/ml), superoxide anion scavenger, nor dimetyl sulfoxide (1-5%, DMSO), a hydroxyl radical scavenger affected the sum of 5-HT plus 5-HIAA accumulation. Moreover, mucosa ROS generation was estimated using the chemiluminescence technique. SOD (100-300 U/ml), catalase (3000-6000 U/ml) or DPI (10-100 microM), concentration-dependently reduced luminol-enhanced chemiluminescence signal from the colonic mucosa, while allopurinol (10-100 microM), a xanthine oxidase inhibitor, did not affect the chemiluminescence signal. These results suggest that ROS is formed through an NADPH oxidase system in the guinea pig colonic mucosa, where it exerts a modulatory effect on mucosal 5-HT formation upon addition of 5-HTP. Thus, ROS formation from normal colonic mucosa could be considered to contribute to the control of 5-HT production in mucosa enterochromaffin cells.     

Metitepine distinguishes two receptors mediating inhibition of [3H]-5-hydroxytryptamine release in guinea pig hippocampus

Summary Inhibition of [³H]-5-hydroxytryptamine ([³H]-5-HT) release from guinea pig brain slices via activation of the terminal 5-HT autoreceptor has previously been characterised as a model of 5-HT_1D receptor activation, based on the rank potencies of a range of agonists, and the potent antagonism of the inhibitory effects of 5-HT by metitepine. The present study uses this model, in slices of the guinea pig hippocampus, to examine the antagonist potency of metitepine against the 5-HT receptor agonists sumatriptan, 5-carboxamidotryptamine (5-CT) and 5-HT Addition of metitepine to the perfusion buffer (30, 300 and 1000 nmol/l) significantly shifted the concentration-response curve to 5-HT, producing a Schild slope of 1.1, and a pA₂ value of 7.6. However, the ability of metitepine to antagonise the effects of sumatriptan or 5-CT in this model was less marked. A clear-cut shift in the concentration-response curve to sumatriptan was only achieved at1000 nmol/l metitepine (apparent pA₂ = 6.7),and this was similar to the ability of metitepine to attenuate the effects of 5-CT (apparent pA₂ 7.0 at 300 nmol/l and 6.7 at 1000 nmol/l). These findings suggest heterogeneity in the receptor mediating inhibition of [³H]-5-HT release in guinea pig hippocampus.Send offprint requests to L. O. Wilkinson at the above address     

Iontophoretic release of acetylcholine, noradrenaline, 5-hydroxytryptamine and D-lysergic acid diethylamide from micropipettes

1. The in vitro iontophoretic release of tritium-labelled acetylcholine and 5-hydroxytryptamine from large and small micropipettes and noradrenaline and D-lysergic acid diethylamide from small micropipettes was determined by liquid scintillation counting.2. The release was directly proportional to the electrical charge passed in the range normally used in the iontophoretic study of these compounds. The transport numbers obtained for the large micropipettes were approximately double those with the small micropipettes. A very low transport number was found for D-lysergic acid diethylamide.3. The spontaneous leakage was small and did not vary appreciably with time.4. The iontophoretic release of acetylcholine in vitro agreed with the in vitro measurements.5. The brain-stem tissue concentration of D-lysergic acid diethylamide after intravenous injection into intact and decerebrate cats was determined.     

LY393558, a 5-hydroxytryptamine reuptake inhibitor and 5-HT(1B/1D) receptor antagonist: effects on extracellular levels of 5-hydroxytryptamine in the guinea pig and rat.

The stimulation of terminal 5-HT(1B/1D) autoreceptors limits the effects of selective serotonin reuptake inhibitors on extracellular levels of 5-hydroxytryptamine (5-HT, serotonin) in vivo. Microdialysis studies show that acute oral administration of LY393558-a 5-HT reuptake inhibitor and antagonist at both the human 5-HT(1B) and 5-HT(1D) receptor-in the dose range 1-20 mg/kg, increases extracellular levels of 5-HT in both the guinea pig hypothalamus and rat frontal cortex. In both species, the levels of 5-HT that were attained were higher than following an acute, maximally effective dose of fluoxetine (20 mg/kg orally), reaching approximately 1500% in the guinea pig hypothalamus and 700% in the rat frontal cortex. In both species, the response to LY393558 (10 mg/kg p.o.) was impulse dependent, being absent in the presence of tetrodotoxin delivered at 1 microM via the microdialysis probe. The sensitivity to tetrodotoxin contrasted with the effects seen with DL-fenfluramine. Studies in rats showed that the microdialysate 5-HT concentration achieved in the frontal cortex after an acute challenge with LY393558 (5 mg/kg p.o.) was significantly greater than following a chronic regime of fluoxetine treatment (10 mg/kg/day orally for 21 days). Moreover, in rats chronically treated with LY393558 (5 mg/kg/day orally for 21 days), the mean basal concentration, 24 h after the final pretreatment dose, was of the same magnitude as that following chronic fluoxetine. However, in contrast to the response seen in fluoxetine-pretreated animals, a challenge dose of LY393558 still elicited a further increase in extracellular 5-HT in LY393558-pretreated animals. LY393558 is a potent 5-HT reuptake inhibitor and 5-HT(1B/1D) receptor antagonist. Microdialysis studies show that acute oral administration increases extracellular levels of 5-HT, by an impulse-dependent mechanism, above those produced by a maximally effective dose of fluoxetine, and in rats to levels only achieved following chronic fluoxetine treatment. Its neurochemical profile in vivo suggests that it may be a more effective antidepressant with the potential for producing an earlier onset of clinical activity than selective serotonin reuptake inhibitors.     

Pharmacological characterization of a novel 5-HT4 receptor agonist, TS-951, in vitro.

The pharmacological effect of a novel selective 5-HT4 receptor agonist, TS-951 (N-[endo-8-(3-hydroxypropyl)-8-azabicyclo[3.2.1]oct-3-yl]-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamide) was investigated in vitro. TS-951 potently inhibited specific [3H]GR113808 binding both in guinea-pig striatum and in mouse brain. The affinity of TS-951 for the 5-HT4 receptor was higher than those of other agonists, 5-HT, cisapride, mosapride and renzapride. On the longitudinal muscle of the guinea-pig ileum, TS-951 caused a concentration-dependent increase in the amplitude of electrically induced submaximal twitch contractions. On the longitudinal muscle of the guinea-pig distal colon, TS-951 also caused concentration-dependent contractions. TS-951 is a high-affinity, selective and potent 5-HT4 receptor agonist. This compound therefore can be considered as a useful pharmacological tool for investigating 5-HT4 receptor-mediated events.     

Facilitation of acetylcholine release in rat frontal cortex by indeloxazine hydrochloride: involvement of endogenous serotonin and 5-HT4 receptors

Effects of indeloxazine hydrochloride, an inhibitor of serotonin (5-HT) and norepinephrine (NE) reuptake with a facilitatory effect on 5-HT release, on acetylcholine (ACh) output in frontal cortex of conscious rats were characterized using an in vivo microdialysis technique. Systemic administration of indeloxazine (3 and 10 mg/kg, i.p.) increased ACh and 5-HT output in a dose-dependent manner. Depletion of endogenous monoamines by reserpine and of 5-HT by p-chlorophenylalanine, but not that of catecholamines by α-methyl-p-tyrosine, significantly attenuated the facilitatory effect of indeloxazine on ACh release. When applied locally by reverse dialysis, indeloxazine (10 and 30 μM) and the selective 5-HT reuptake inhibitor citalopram (10 μM), but not the NE reuptake inhibitor maprotiline (30 μM), increased cortical ACh output. Indeloxazine (10 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT₄ receptor antagonists RS23597 (50 μM) and GR113803 (1 μM), while the 5-HT_1A antagonist WAY-100135 (100 μM), 5-HT_1A/1B/β-adrenoceptor antagonist (–)propranolol (150 μM), 5-HT_2A/2C antagonist ritanserin (10 μM) and 5-HT₃ antagonist ondansetron (10 μM) failed to significantly modify this effect. Neither depletion of monoamines nor treatment with serotonergic antagonists significantly changed the basal ACh level, indicating that endogenous monoamines do not tonically activate ACh release. These results suggest that indeloxazine-induced facilitation of ACh release in rat frontal cortex is mediated by endogenous 5-HT and involves at least in part cortical 5-HT₄ receptors. Received: 22 May 1997 / Accepted: 26 August 1997     

Direct evidence for a release of acetylcholine from the myenteric plexus of guinea pig small intestine by substance P

Myenteric plexus-longitudinal muscle strips from guinea pig small intestine were labeled with [3H]choline while under continuous field stimulation. Release of newly synthesized [3H]acetylcholine was studied in the presence of substance P afterwards. Substance P evoked release of [3H]acetylcholine in a dose-related fashion. Both tetrodotoxin and [D-Pro2,D-Phe7,D-Trp9]substance P, a synthetic antagonist of substance P, completely blocked this release and provided evidence for a cholinergically mediated mechanism of substance P on enteric neurons.     

Palmitoylation of the 5-hydroxytryptamine4a receptor regulates receptor phosphorylation, desensitization, and beta-arrestin-mediated endocytosis

The mouse 5-hydroxytryptamine4a (5-HT4a) receptor is an unusual member of the G protein-coupled receptor superfamily because it possesses two separate carboxyl-terminal palmitoylation sites, which may allow the receptor to adopt different conformations in an agonist-dependent manner (J Biol Chem 277:2534-2546, 2002). By targeted mutation of the proximal (Cys-328/329) or distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT4a receptor undergoes rapid (t(1/2) approximately 2 min) and dose-dependent (EC50 approximately 180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase. Overexpression of GRK2 significantly reduced the receptor-promoted cAMP formation. The Cys328/329-Ser mutant, which is constitutively active in the absence of ligand, exhibited enhanced receptor phosphorylation under both basal and agonist-stimulated conditions and was more effectively desensitized and internalized via a beta-arrestin-2 mediated pathway compared with the wild-type 5-HT4a. In contrast, G protein activation, phosphorylation, desensitization, and internalization of the other palmitoylation-deficient receptor mutants were affected differently. These findings suggest that palmitoylation plays an important role in modulating 5-HT4a receptor functions and that G protein activation, phosphorylation, desensitization, and internalization depend on the different receptor conformations.