Depletion of the lipid raft constituents, sphingomyelin and ganglioside, decreases serotonin binding at human 5-HT7(a) receptors in HeLa cells

Aim: The localization and function of several G protein‐coupled receptors, including β‐adrenergic receptors and NK 1 receptors, are regulated via lipid rafts in the plasma membrane. These domains are enriched in cholesterol, gangliosides and sphingolipids, and play an important role in regulating signal transduction in most cell types. Serotonin (5‐hydroxytryptamine, 5‐HT), acting via 14 different receptors, regulates as diverse effects as mood, metabolism and smooth muscle contraction. 5‐HT₇ receptors are involved in the regulation of depression, circadian rhythms, thermoregulation and vasodilatation. Ligand binding and signalling via the 5‐HT₇ receptor are regulated by membranous cholesterol. Here we investigated the role of sphingomyelin and gangliosides on binding of 5‐HT to 5‐HT₇ receptors to further examine the role of lipid raft constituents on 5‐HT₇ receptor function. Methods: HeLa cells stably transfected with the human 5‐HT₇ receptor were treated with Fumonisin B₁ or (±)‐threo‐1‐Phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) to reduce sphingomyelin or ganglioside levels, respectively. The effects of these treatments were investigated by the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl‐tetrazolium bromide (MTT) viability assay, cholesterol analysis and [³H]5‐HT binding studies on intact cells. Results: Treatments with 20 μm Fumonisin B₁ for 24 h or with 10 μm PDMP for 48 h had no effects of total levels if 5‐HT₇ receptors, but caused significant decreases in maximum [³H]5‐HT binding to 5‐HT₇ receptors. The effects were cholesterol‐independent as levels of cholesterol remained unaffected by either treatment. Conclusion: These data demonstrate a role for sphingomyelin and gangliosides in regulating binding of [³H]5‐HT to 5‐HT₇ receptors. These observations further strengthen that actions of 5‐HT via 5‐HT₇ receptors are dependent upon lipid raft integrity.     

Role of 5‐HT2A, 5‐HT4 and 5‐HT7 receptors in the antigen‐induced airway hyperresponsiveness in guinea‐pigs

Background A possible role of 5‐hydroxytryptamine (5‐HT) in the origin of antigen‐induced airway hyperresponsiveness (AI‐AHR) has been scarcely investigated. Objective To explore the participation of different 5‐HT receptors in the development of AI‐AHR in guinea‐pigs. Methods Lung resistance was measured in anaesthetized guinea‐pigs sensitized to ovalbumin (OVA). Dose–response curves to intravenous (i.v.) acetylcholine (ACh) were performed before and 1 h after antigenic challenge and expressed as the 200% provocative dose (PD₂₀₀). Organ bath experiments, confocal microscopy and RT‐PCR were additionally used. The 5‐HT content in lung homogenates was measured by HPLC. Results Antigenic challenge significantly decreased PD₂₀₀, indicating the development of AI‐AHR. This hyperresponsiveness was abolished by a combination of methiothepin (5‐HT₁/5‐HT₂/5‐HT₅/5‐HT₆/5‐HT₇ receptors antagonist) and tropisetron (5‐HT₃/5‐HT₄ antagonist). Other 5‐HT receptor antagonists showed three different patterns of response. Firstly, WAY100135 (5‐HT_1A antagonist) and ondansetron (5‐HT₃ antagonist) did not modify the AI‐AHR. Secondly, SB269970 (5‐HT₇ antagonist), GR113808 (5‐HT₄ antagonist), tropisetron or methiothepin abolished the AI‐AHR. Thirdly, ketanserin (5‐HT_2A antagonist) produced airway hyporresponsiveness. Animals with bilateral vagotomy did not develop AI‐AHR. Experiments in tracheal rings showed that pre‐incubation with LP44 or cisapride (agonists of 5‐HT₇ and 5‐HT₄ receptors, respectively) induced a significant increase of the cholinergic contractile response to the electrical field stimulation. In sensitized lung parenchyma strips, ketanserin diminished the contractile responses to ACh. Sensitization was associated with a ninefold increase in the 5‐HT content of lung homogenates. Confocal microscopy showed that sensitization enhanced the immunolabelling and co‐localization of nicotinic receptor and 5‐HT in airway epithelium, probably located in pulmonary neuroendocrine cells (PNECs). RT‐PCR demonstrated that neither sensitization nor antigen challenge modified the 5‐HT_2A receptor mRNA levels. Conclusions Our results suggested that 5‐HT was involved in the development of AI‐AHR to ACh in guinea‐pigs. Specifically, 5‐HT_2A, 5‐HT₄ and 5‐HT₇ receptors seem to be particularly involved in this phenomenon. Participation of 5‐HT might probably be favoured by the enhancement of the PNECs 5‐HT content observed after sensitization. Cite this as: P. Segura, M. H. Vargas, G. Córdoba‐Rodríguez, J. Chávez, J. L. Arreola, P. Campos‐Bedolla, V. Ruiz, L. M. García‐Hernández, C. Méndez and L. M. Montaño, Clinical & Experimental Allergy, 2010 (40) 327– 338.     

Tachykinins are involved in local reflex modulation of vagally mediated striated muscle contractions in the rat esophagus via tachykinin NK1 receptors

The objective of the present study was to investigate the hypothesis of the presence of a local neural reflex modulating the vagally mediated contractions of striated muscle in the rat esophagus and to determine the possible involvement of tachykinins in such a local neural reflex. Electrical stimulation of the vagus nerve evoked twitch contractile responses that were abolished by d-tubocurarine (5 microM). Capsaicin (1-100 microM) inhibited the vagally mediated twitch contractions o f the normal rat esophageal preparations concentration-dependently but not those of the neonatally capsaicin-treated ones. NG-nitro-L-arginine methyl ester (100 microM), a nitric oxide synthase inhibitor, blocked the inhibitory effect of capsaicin and exogenous application of a nitric oxide donor (1 mM) inhibited the vagally mediated twitch contractions. Capsaicin suppressed acetylcholine release from the normal rat esophageal segments evoked by vagus nerve stimulation but not that from the neonatally capsaicin-treated ones. A selective tachykinin NK1 receptor antagonist (0.1 or 1 microM) attenuated the inhibitory effect of capsaicin. However, antagonists of tachykinin NK2, tachykinin NK3 and calcitonin gene-related peptide receptors (1 microM) did not have any effect. A tachykinin NK1 receptor agonist (1 or 5 microM) inhibited the vagally mediated twitch contractions, which was prevented by NG-nitro-L-arginine methyl ester (100 microM). These data suggest that the rat esophagus might have a local neural reflex inhibiting the vagally mediated striated muscle motility, which consists of capsaicin-sensitive sensory neurons and myenteric nitrergic neurons, and that tachykinins might be involved in the neural reflex through tachykinin NK1 receptors.     

Lack of an association between 5‐HT1A receptor gene structural polymorphisms and suicide victims

A serotonergic dysfunction in the brain has been reported to be involved in suicidal behavior independently of the presence of a specific psychiatric disorder. Serotonin 1A (5‐HT_1A) receptors are known to be located on serotonergic nerve terminals and to be involved in the presynaptic regulation of serotonin release. Genetic factors partly explain the risks for suicide, and a suicide completion group is thought to be more uniform than a suicide attempt group. To explore the hypothesis that the 5‐HT_1A receptor‐induced serotonergic dysfunction is implicated genetically in suicide, we focused on the structural polymorphisms, Pro16Leu and Gly272Asp, of the 5‐HT_1A receptor gene, and examined the association between suicide victims who completed suicide and these two polymorphisms. In both polymorphisms, we found no significant difference in genotype distribution or allele frequencies between suicide victims and controls. These findings suggest that neither of these two polymorphisms is associated with suicide victims and it is unlikely that the 5‐HT_1A receptor gene is implicated in the susceptibility to suicide. © 2002 Wiley‐Liss, Inc.     

犬和大鼠回肠壁丛内5-羟色胺能神经元免疫组织化学观察

目的　观察犬和大鼠回肠壁丛内 5 羟色胺能神经元。方法　应用 5 羟色胺 (5 HT)抗体的免疫组织化学改良法对正常小狗 (5只 )回肠切片标本、正常大鼠 (8只 )和 5 羟色胺酸前处理大鼠 (4只 )回肠外纵肌全层铺片标本内含 5 HT免疫反应性 (5 HT IR)神经元进行了观察研究。结果　正常大鼠回壁内神经节 (丛 )内可见少数 5 HT IR核周体 ,及肠肌丛周围和节间束中含有丰富的 5 HT IR纤维。 5 羟色胺酸 (5 HTP)处理后大鼠与正常鼠相比较 ,回肠壁丛内 5 HT IR胞体和带膨体纤维的可见数稍多 ,及免疫反应性增强。正常狗远端内 5 HT IR神经元胞体和纤维非常丰富 5 HT IR基础丛内有 1～ 4个 5 HT IR神经元胞体。结论　本研究对犬和大鼠肠内 5 HT能神经元的存在提供了直接的形态学证据 ,肠 5 HT能神经元与胃肠运动的调节功能及其可能的受体机制有关     

Tachykinin receptors are involved in the "local efferent" motor response to capsaicin in the guinea-pig small intestine and oesophagus

The sensory neuron stimulant drug capsaicin stimulates primary afferent nerve endings in the guinea-pig small intestine, which in turn activate myenteric cholinergic neurons by an unknown mechanism. The tachykinins substance P and neurokinin A are present in primary afferent neurons. This study was performed to assess the possible involvement of endogenous tachykinins acting via neurokinin-1, neurokinin-2 and neurokinin-3 receptors in the contractile effect of capsaicin in the isolated guinea-pig ileum and oesophagus by using the receptor-specific antagonists GR 82334 (3 microM) for neurokinin-1 receptors, MEN 10627 (3 microM; ileum) or MEN 11420 (1 microM; oesophagus) for neurokinin-2 receptors and SR 142801 (0.1 microM) for neurokinin-3 receptors. In the ileum, the peak contraction evoked by capsaicin (2 microM) was not reduced when tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors were blocked separately, whereas an inhibition of neurokinin-3 receptors diminished the area under the curve of the capsaicin response. A combined blockade of neurokinin-1 and neurokinin-3 receptors significantly depressed the effect of capsaicin; the amplitude of the contractile response was 53.3+/-3.7% of the maximal longitudinal spasm in control preparations, whereas in the presence of GR 82334 plus SR 142801 it reached only 27.6+/-5% (P<0.001, Kruskal-Wallis test; n=9 and 10, respectively). Also, the area under the curve of the contractile response to capsaicin was more than 85% lower in the group of preparations treated with GR 82334 plus SR 142801 than in the control group (P<0.001). Including a neurokinin-2 blocker in the combination did not produce any further inhibition. A concomitant tachyphylaxis to substance P (natural neurokinin-1 receptor stimulant) and the neurokinin-3 receptor agonist senktide (5 and 1 microM, respectively) also reduced the contractile effect of capsaicin. In the oesophagus, capsaicin (1 microM) induced biphasic contractions which were strongly inhibited by atropine (1 microM) or capsaicin pretreatment (1 microM for 10 min). Here again, a blockade of tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors separately failed to inhibit the response to capsaicin, whereas a combined blockade of any two tachykinin receptors caused a partial inhibition. The reduction of the contractile effect of capsaicin was strongest when all three tachykinin receptors were blocked. In seven control preparations, peaks for the first and second phases of contraction reached 35.3+/-3.7% and 20+/-3.2% of maximal longitudinal spasm; the corresponding values in the presence of a combination of GR 82334, MEN 11420 and SR 142801 were 7.5+/-0.8% and 9.1+/-2.2%, respectively (n=6, P<0.001 and 0.05, respectively). Tetrodotoxin (0.5 microM) practically abolished the contractile effect of capsaicin in both tissues studied. It is concluded that an interplay of neuronal tachykinin neurokinin-1 and neurokinin-3 receptors (ileum) and neurokinin-1, neurokinin-2 and neurokinin-3 receptors (oesophagus) is involved in the contractile action of capsaicin, probably in mediating excitation of myenteric neurons by tachykinins released from primary afferents. In both tissues, there also seems to be a non-tachykininergic component of the capsaicin-induced contraction.     

Serotonin in the rabbit ileum: localization,uptake, and effect on motility

Repeated experiments to localise serotonin in the myenteric plexus of rabbit ileum failed. After preincubation in serotonin (10(-5) M), an extensive varicose fibre system was detected by immunocytochemical methods. Stained fibres left the myenteric plexus and ran to the muscle layers. Labelled cell bodies could not be found, even after pretreatment with colchicine or pargyline. Application of reserpine (10(-5) M) and fluoxetine (10(-5) M) prevented serotonin uptake. Antisera against tryptophan hydroxylase revealed a rich fibre system, including those processes that entered the tertiary plexus. These fibres were able to accumulate serotonin, but again the cell bodies could not be detected. Serotonin caused concentration-dependent contraction in the longitudinal muscle layer of the rabbit ileum. Pretreatment with tetrodotoxin strongly reduced the effect of serotonin. Preapplication of atropine caused a slight decrease of response evoked by serotonin. Combined administration of tetrodotoxin and atropine significantly reduced the responses to serotonin, but did not abolish them. At the same time, agonists of 5-HT(2) and 5-HT(4) receptors caused concentration-dependent contractions. Our studies show that: 1). Without pretreatment, serotonin cannot be detected in the myenteric plexus of rabbit ileum. 2). An extensive uptake system works in this plexus. If released from myenteric nerve fibres, serotonin may evoke contractions in indirect and direct ways. 3). There may be an extrinsic serotoninergic innervation from the mesenteric ganglia. 4). Serotonin exerts its effect through 5-HT(2) and 5-HT(4) receptors on smooth muscle cells and nerve elements.     

Additive hypothermic effects of the 5‐HT1A receptor agonist 8‐OH‐DPAT and the dopamine D2/3 receptor agonist 7‐OH‐DPAT in the rat

The objective of this study was to examine possible interactions between serotonergic and dopaminergic agents lowering core temperature via stimulation of 5‐HT_1A and dopamine (DA) D₂ receptors, respectively. The effects of the 5‐HT_1A receptor agonist (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin HBr (8‐OH‐DPAT) and the DA D_2/3 receptor agonist 7‐OH‐DPAT on core temperature was monitored in adult male Wistar rats, approximately 300 g body weight. The temperature probe was connected to a PC‐assisted temperature instrument, and an automated printer device was activated when the temperature reading had stabilized (±0.1 °C) for 10 s. As expected, 7‐OH‐DPAT [0.5 and 2.0 μmol kg^–1 subcutaneous (s.c.)] as well as 8‐OH‐DPAT (0.15–2.4 μmol kg^–1 s.c.), produced a dose‐dependent hypothermia. When combined, there were additive effects of the two compounds, although the effects of 7‐OH‐DPAT were attenuated by 8‐OH‐DPAT at the higher doses (0.6–2.4 μmol kg^–1), in all probability because of emerging DA D₂ receptor blocking properties of the latter compound.     

Neurokinin-1 and -3 receptor blockade inhibits slow excitatory synaptic transmission in myenteric neurons and reveals slow inhibitory input

Recent studies have shown that tachykinins mediate slow synaptic transmission to myenteric AH (afterhyperpolarising) neurons via neurokinin-3 receptors (NK(3)R). This study investigated a similar role for neurokinin-1 receptors (NK(1)R) and compared the effect of selective receptor antagonists on non-cholinergic slow excitatory post-synaptic potentials (EPSPs) recorded in myenteric AH neurons of the guinea-pig ileum. Slow EPSPs evoked by electrical stimulation of circumferentially oriented presynaptic nerves were mimicked by application of senktide, an NK(3)R agonist. [Sar(9),Met(O(2))(11)]-substance P, an NK(1)R agonist, depolarised a smaller number of neurons. SR142801, a selective NK(3)R antagonist (100 nM), inhibited slow EPSPs and responses to senktide, but had no effect on depolarisations evoked by forskolin, an activator of adenylate cyclase. SR140333, a selective NK(1)R antagonist, inhibited slow EPSPs in a subset of neurons and blocked responses to [Sar(9),Met(O(2))(11)]-substance P, but not to senktide or forskolin. Slow EPSPs that were predominantly mediated by NK(1)R had significantly shorter latencies than those due to activation of NK(3)R. After blockade of slow EPSPs, slow hyperpolarizing responses to presynaptic nerve stimulation were revealed in one-third of neurons. These events, which were associated with a decrease in input resistance and blocked by tetrodotoxin, were equated with slow inhibitory postsynaptic potentials. They were abolished by the 5-hydroxytryptamine(1A) receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine (NAN-190), but unaffected by phentolamine, an alpha-adrenoceptor antagonist. In conclusion, these results provide the first direct evidence that NK(1)R mediate some slow excitatory synaptic input to myenteric AH neurons, and suggest that NK(1)R and NK(3)R activate distinct signal transduction pathways. These results also demonstrate that slow inhibitory synaptic transmission, which may be mediated by 5-hydroxytryptamine, is more prevalent in the myenteric plexus than previously indicated.     

5-HT1A, but not 5-HT2 and 5-HT7, receptors in the nucleus raphe magnus modulate hypoxia-induced hyperpnoea

Aim: In the present study, we assessed the role of 5‐hydroxytryptamine (5‐HT) receptors (5‐HT_1A, 5‐HT₂ and 5‐HT₇) in the nucleus raphe magnus (NRM) on the ventilatory and thermoregulatory responses to hypoxia. Methods: To this end, pulmonary ventilation (V_E) and body temperature (T_b) of male Wistar rats were measured in conscious rats, before and after a 0.1 μL microinjection of WAY‐100635 (5‐HT_1A receptor antagonist, 3 μg 0.1μL^?1, 56 mm ), ketanserin (5‐HT₂ receptor antagonist, 2 μg 0.1μL^?1, 36 mm ) and SB269970 (5‐HT₇ receptor antagonist, 4 μg 0.1 μL^?1, 103 mm ) into the NRM, followed by 60 min of severe hypoxia exposure (7% O₂). Results: Intra‐NMR microinjection of vehicle (control rats) or 5‐HT antagonists did not affect V_E or T_b during normoxic conditions. Exposure of rats to 7% O₂ evoked a typical hypoxia‐induced anapyrexia after vehicle microinjections, which was not affected by microinjection of WAY‐100635, SB269970 or ketanserin. The hypoxia‐induced hyperpnoea was not affected by SB269970 and ketanserin intra‐NMR. However, the treatment with WAY‐100635 intra‐NRM attenuated the hypoxia‐induced hyperpnoea. Conclusion: These data suggest that 5‐HT acting on 5‐HT_1A receptors in the NRM increases the hypoxic ventilatory response.     

Serotonin receptors contribute to the promnesic effects of P. olacoides (Marapuama)

Nootropic, antioxidant, and neuroprotective properties have been shown in a standardized ethanol extract of Ptychopetalum olacoides (POEE), a medicinal plant traditionally used by the Amazonian elderly population. It has been revealed that POEE mechanisms of action include anticholinesterase effects, and involve β-adrenergic and dopamine D₁ receptors. The purpose of this study was to verify the role of serotonin receptors in the promnesic effects of this standardized extract. The step-down task in mice and selective serotonin antagonists were used. The study reveals that POEE promnesic effects on short-term (acquisition, consolidation and retrieval) and long-term (retrieval) declarative aversive memories are increased by 5HT_2A (but not 5HT_1A) serotonin antagonists (spiperone and pindolol, respectively). The observed synergism between POEE and spiperone can be interpreted as the combined effects of two subeffective doses of two 5HT antagonists, or the known synergism between an acetylcholinesterase inhibitor (POEE) and a 5HT antagonist. In conclusion it is suggested that 5HT_2A serotonin receptors are relevant for the promnesic effects of this extract, adding to its multiple mechanisms of action.     

Antipsychotic medications,glutamate, and cell death: A hidden,but common medication side effect?

We hypothesize the interaction between antipsychotic medications and regulation of extracellular glutamate which has gone largely unnoticed in the medical community has significant clinical importance. Typical antipsychotic medications such as haloperidol elevate extracellular glutamate because they exert antagonist effects on dopamine D₂ and serotonin 5HT_1A receptors. In contrast, serotonin 5HT_2A receptor antagonists inhibit glutamate release. Glutamate is potentially excitotoxic through effects on ionotropic receptor channels and may exert synergistic effects with other neurotoxic pathways. In contrast to typical antipsychotic drugs, pharmacological properties of atypical antipsychotic medications at dopamine D₂, serotonin 5HT_1A and 5HT_2A receptors limit extracellular glutamate and may theoretically be neuroprotective in certain clinical settings. In this review we discuss three common clinical settings in which typical antipsychotic medications may potentiate neurotoxicity by elevating extracellular glutamate. The most common clinical setting, hypoglycemia during combined use of antipsychotic medications and insulin, presents a theoretical risk for 35 million diabetic patients worldwide using antipsychotic medications. Antipsychotic medication treatment during hypoxic episodes in the intensive care unit and following traumatic brain injury are two other common clinical settings in which this interaction poses theoretical risk. Further study is needed to test hypothesized risk mechanisms, and determine clinical and epidemiological consequences of these exposures.     

NK1, NK2 and NK3 tachykinin receptor localization and tachykinin distribution in the ileum of the mouse

Tachykinin receptors NK1r, NK2r and NK3r bind tachykinins with different affinities and share pharmacological and molecular differences among animal species. NK1r, NK2r, NK3r and tachykinin (SP/NKA) distribution was studied by immunohistochemistry in the ileum of mouse since no data are available for this species. The results were then compared to those obtained in the rat and guinea pig either by us or by others to ascertain interspecies similarities and/or differences. NK1r- and NK3r-immunoreactivity (IR) were detected in neurons and NK1r-IR in the interstitial cells of Cajal at the deep muscular plexus. At variance with rat and guinea pig, NK1r-IR was also found in the myoid cells of the villi, while NK2r-IR was never detected in nerve varicosities. This latter datum suggests that the NK2r does not play a presynaptic role in the mouse. Unexpectedly, a high NK2r-IR and the presence of NK3r-IR were observed at the inner portion of the circular muscle layer in the mouse as well as in the rat and guinea pig, demonstrating a subregional distribution of these receptors. Tachykinin distribution did not show noticeable species-related differences. The present findings show species-related differences in the tachykinin receptor distribution that might be related to a different tachykinin control of intestinal motility.     

Relationship between postsynaptic NK1 receptor distribution and nerve terminals innervating myenteric neurons in the guinea‐pig ileum

The amounts of neurokinin 1 (NK₁) receptor immunolabelling on the membranes of myenteric cell bodies at appositions with tachykinin‐immunoreactive nerve terminals, other nerve terminals, and glial cells were compared at the ultrastructural level using pre‐embedding, double‐label immunocytochemistry. NK₁ receptor immunoreactivity was revealed using silver‐intensified, 1 nm gold, and tachykinin‐immunoreactive nerve terminals were revealed using diaminobenzidine. The density of NK₁ receptor immunolabelling (silver particles per length of cell membrane) on the membrane at appositions with tachykinin‐immunoreactive nerve terminals was not significantly different from that at appositions with other (nonimmunoreactive) nerve terminals or with glial cells. Synaptic specializations (“active zones”) were present at a small proportion of the appositions between NK₁ receptor‐immunoreactive cell bodies and tachykinin‐immunoreactive or other nerve terminals. The density of NK₁ receptor immunolabelling at synaptic specializations was lower than that at regions of appositions where no synaptic specializations were present. The presence of NK₁ receptor on the cell surface in areas not directly apposed to tachykinin‐containing nerve terminals suggests that tachykinins that diffuse away from their site of release may still exert an action via NK₁ receptors. Although NK₁ receptors do not appear to be targetted to particular sites on the surfaces of myenteric nerve cell bodies and proximal dendrites, they are reduced in density at regions of the membrane‐forming synaptic specializations. Anat Rec 263:248–254, 2001. © 2001 Wiley‐Liss, Inc.     

Neurokinin-1 receptor activation induces reactive oxygen species and epithelial damage in allergic airway inflammation

Background An induction of reactive oxygen species (ROS) is characteristic for inflammation but the exact pathways have not been identified for allergic airway diseases so far. Objective The aim of this study was to characterize the role of the tachykinin NK‐1 receptor on ROS production during allergen challenge and subsequent inflammation and remodelling. Methods Precision‐cut lung slices of ovalbumin (OVA)‐sensitized mice were cultivated and ROS‐generation in response to OVA challenge (10 μg/mL) was examined by the 2′,7′‐dichloroflourescein‐diacetate method. Long‐term ROS effects on epithelial proliferation were investigated by 5‐bromo‐2′‐deoxyuridine incorporation (72 h). In vivo, the results were validated in OVA‐sensitized animals which were treated intra‐nasally with either placebo, the tachykinin neurokinin 1 (NK‐1) receptor antagonist SR 140333 or the anti‐oxidant N‐acetylcystein (NAC) before allergen challenge. Inflammatory infiltration and remodelling were assessed 48 h after allergen challenge. Results ROS generation was increased by 3.7‐fold, which was inhibited by SR 140333. [Sar⁹,Met¹¹(O₂)]‐Substance P (5 nm ) caused a tachykinin NK‐1 receptor‐dependent fourfold increase in ROS generation. Epithelial proliferation was decreased by 68% by incubation with [Sar⁹,Met¹¹(O₂)]‐SP over 72 h. In‐vivo, treatment with SR 140333 and NAC reduced epithelial damage (91.4% and 76.8% vs. placebo, respectively, P<0.01) and goblet cell hyperplasia (67.4% and 50.1% vs. placebo, respectively, P<0.05), and decreased inflammatory cell influx (65.3% and 45.3% vs. placebo, respectively, P<0.01). Conclusion Allergen challenge induces ROS in a tachykinin NK‐1 receptor‐dependent manner. Inhibition of the tachykinin NK‐1 receptor reduces epithelial damage and subsequent remodelling in vivo. Therefore, patients may possibly benefit from treatment regime that includes radical scavengers or tachykinin NK‐1 receptor antagonists.     

Regulation of the human serotonin transporter mediated by long-term action of serotonin in Caco-2 cells

Aim: The aim of this study was to determine the effect of long‐term serotonin (5‐hydroxytryptamine, 5‐HT) treatment on the human serotonin transporter (hSERT) function and its expression. Methods: This study was carried out in the enterocyte‐like cell line Caco‐2. These cells constitutively express the hSERT and have been shown to be an excellent model for the study of this protein. We measured serotonin transport, levels of mRNA expression and of the SERT protein after treating the cells with serotonin. Results: Serotonin treatment diminished hSERT activity in a concentration and period‐dependent way by increasing the K_t value and reducing V_max. This inhibition was reversible and was not mediated by either the action of 5‐HT₂, 5‐HT₃ or 5‐HT₄ receptors, or by the intracellular second messengers, protein kinase C and cAMP. 5‐HT did not seem to affect either the mRNA level of the SERT or the protein transporter measured in either the membrane or the cell lysate. The 5‐HT treatment effect was additive to the inhibitory effect of treatment with a low concentration of citalopram and fluoxetine. Nevertheless, 5‐HT did not increase the inhibition yielded by treatment with high concentration citalopram. Conclusion: The chronic increase in serotonin in the extracellular medium diminishes the function of the SERT. This effect seems to be due to an effect on the transporter molecule itself in the membrane, without altering protein synthesis, intracellular traffic, or its availability.     

Contractile responses of rat duodenum caused by transmural nerve stimulation: interaction between tachykininergic and cholinergic mechanisms

We have studied the importance of tachykinins and acetylcholine for motor stimulation of the rat duodenum in vitro. Contractions induced by transmural nerve stimulation and tachykinin receptor agonists selective for NK1, NK2 and NK3 receptors were used in combination with a neurokinin (NK)2 receptor antagonist, MEN 10,627, and atropine as a muscarinic receptor antagonist. Transmural nerve stimulation in the range 0.5–32 Hz caused frequency-dependent contractions. MEN 10,627 (10^-8, 10^-7 and 10^-6m ) dose-dependently reduced the contractile frequency–response curve (P<0.01–0.001). Addition of atropine (10^-6m ) completely inhibited the response to transmural nerve stimulation (P<0.001). As control, atropine alone reduced this response only by about 65%. Of the tachykinin analogues, [β-Ala⁸]-neurokinin A(4-10) selective for NK2 receptors caused concentration-dependent contractions with high potency (pD₂ 8.01) and high efficacy, while substance P methyl ester acting on NK1 receptors had lower potency (pD₂ 7.94) and low efficacy, and senktide acting on NK3 receptors had a low potency (pD₂ 7.52) but high efficacy. With increasing concentrations of MEN 10,627 the response to [β-Ala⁸]-neurokinin A(4-10) was markedly reduced (P<0.01), while responses to substance P methyl ester and senktide were only slightly affected. Our results indicate that the physiological contractile responses of the rat duodenum are co-mediated by acetylcholine and tachykinins, for which NK2 receptors seem to be most important.     

Important roles of tachykinins in the development of allergic nasal hyperresponsiveness in guinea-pigs

Background Although it has been suggested that the use of tachykinin receptor antagonists might prove to be an effective treatment for allergic rhinitis (AR), they are not used clinically. Therefore, we decided to examine the effects of tachykinin receptor antagonists on AR symptoms in an appropriate experimental model. Objective To evaluate newly developed tachykinin receptor antagonists in a Japanese cedar pollen‐induced AR model and to determine their effect on allergen‐induced sneezing, nasal blockage, and nasal hyperresponsiveness (NHR). Methods Sensitized guinea‐pigs were challenged by forced inhalation of pollen once every week. Sneezing and nasal blockage were observed after pollen challenges. NHR (nasal blockage) to an intranasal application of leukotriene D₄ was assessed 2 days after an antigen challenge. We also evaluated whether intranasal dosing with a tachykinin causes NHR. NK₁ and NK₂ receptor antagonists were administered before an intranasal treatment with antigen or tachykinin. Amounts of tachykinins present in nasal cavity lavage fluid were measured by an enzyme immunoassay. Results Although an NK₁ and NK₂ receptor dual antagonist showed no effect on pollen‐induced sneezing and biphasic nasal blockage, it did completely suppress the development of NHR. Experiments using specific NK₁ or NK₂ receptor antagonists revealed that NK₂ receptor activation was preferentially involved in the development of hyperresponsiveness. Increases in the levels of substance P (SP) and neurokinin A (NKA) in the nasal tissue were noted 20 min–1 h after the challenge. Intranasal instillation of either SP or NKA‐induced NHR, which was almost completely inhibited by NK₂ receptor antagonists and partially inhibited by NK₁ receptor antagonists. Conclusions SP and NKA, which are released early after the challenge, mediate the development of NHR by preferentially activating NK₂ receptors. Therefore, NK₂ receptor antagonists might prove to be effective treatment of AR.     

Novel and previously reported single‐nucleotide polymorphisms in the human 5‐HT1B receptor gene: No association with cocaine or alcohol abuse or dependence

Evidence from animal self‐administration and human genetics studies suggests that the serotonin_1B (5‐HT_1B) receptor may be involved in modulating responses to cocaine or alcohol. We hypothesize that polymorphisms, including single‐nucleotide polymorphisms (SNPs), in the human 5‐HT_1B receptor gene, may be associated with individual differences in vulnerability to cocaine or alcohol abuse or dependence. A total of 210 subjects were studied, including individuals with a primary diagnosis (DSM‐IV criteria) of cocaine abuse or dependence, alcohol abuse or dependence, and controls with no history of previous or current illicit drug or alcohol abuse or dependence. Genomic DNA samples were isolated from each individual. For 157 of the subjects, polymerase chain reaction (PCR) was used to amplify the entire coding region of the 5‐HT_1B receptor gene as well as parts of the 5′ and 3′ untranslated regions. PCR products were sequenced in forward and reverse directions on an automated sequencer. Amplified DNA from an additional 53 subjects was sequenced in the 5′ untranslated region to gain additional data on the frequency of one identified SNP. Seven polymorphisms were identified: one novel SNP in the 5′ untranslated region (UTR) of the gene (A‐161T); one SNP not reported in any published scientific communication (but found to be recorded in GenBank) in the 3′ UTR (A1180G); two novel dinucleotide deletions at positions ? 184/? 183 and ? 182/? 181; and three previously identified SNPs (T‐261G, C129T, G861C). Data were stratified by ethnicity and pooled Relative Risk was calculated for combined alcohol abuse and dependence cases and controls, and also for combined cocaine abuse and dependence cases and controls. No significant differences between cases and controls were found. © 2001 Wiley‐Liss, Inc.