Tachykinin NK1 Receptor Research Articles

Alteration in airway microvascular leakage induced by sensorineural stimulation in rats exposed to inhaled formaldehyde

Inhaled formaldehyde can rapidly produce microvascular leakage in the airway through stimulation of tachykinin NK1 receptors by tachykinins released from sensory nerves. Tachykinin NK1 receptors are known to be internalized in the cytoplasm after being stimulated, thus leading to transient attenuation of their action. We investigated time changes in airway microvascular leakage during formaldehyde inhalation for 45 min, and whether pre-inhalation of formaldehyde (5 ppm, 30 min) decreases the responses induced by subsequent inhaled formaldehyde (5 ppm, 15 min), intravenous capsaicin (75 μg/kg) and intravenous substance P (10 μg/kg) in rat airway. Evans blue dye content extravasated into the tissues was measured as an index of plasma leakage. Formaldehyde rapidly produced dye leakage in the airway, a response that ended within 15 min after the start of formaldehyde inhalation. Pre-inhalation of formaldehyde markedly decreased the responses induced by formaldehyde and capsaicin, but not substance P. However, dye leakage induced by formaldehyde was significantly enhanced by formaldehyde inhalation 20 h earlier. Our results suggest that tachyphylaxis in neurogenic airway microvascular leakage seen after formaldehyde inhalation may be due to impairment of tachykinin release from sensory nerves or decreases in tachykinins within sensory nerves. However, desensitization of tachykinin NK1 receptors was unlikely to be important in the tachyphylactic response.

Opioid-substance P chimeric peptides

ABSTRACT Chimerization of tachykinin and opioid pharmacophores offers a new avenue for analgesic development. The complexities of such design are illustrated by the analgesic efficacy (via different mechanisms) of chimeras that combine pharmacophores with opioid activity and substance P activity, as well as those that combine opioid agonist and substance P antagonist moieties. Although the interaction between substance P and opioid neural systems is more complex than a simple one-way inhibition, the relative balance of activities between tachykinin and opioid pharmacophores will generally determine the net effect of the chimeric molecule as pro-nociceptive, antinociceptive, or neutral. Intriguingly, endomorphins—μ-opioid receptor agonists with high intrinsic activity—may owe some of this high activity to weak but significant antagonist proper ties at tachykinin NK2 receptors, implying that these native peptides are endogenous chimeric opioid agonist–tachykinin antagonist compounds.

Expression of the nuclear transport protein importin ß-1 and its association with the neurokinin 3 receptor in the rat hypothalamus following acute hyperosmotic challenge

The tachykinin NK3 receptor (NK3R) is a G-protein coupled receptor that is activated, internalized, and trafficked to the nuclei of magnocellular neurons in the paraventricular nucleus of the hypothalamus (PVN) in response to acute hyperosmolarity. The lack of information on the nuclear import pathway raises concerns about the physiological role of nuclear NK3R. NK3R contains a nuclear localizing sequence (NLS) and this raises the possibility that importins are involved in transport of NK3R through the nuclear pore complex. The following experiments utilized: (1) co-immunoprecipitation to determine if NK3R is associated with importin ß-1 following activation in response to acute hyperosmolarity in vivo, and (2) immuno-neutralization of importin ß-1 in vitro to determine if nuclear transport of NK3R was blocked. Rats were given an i.v. injection of hypertonic saline (2 M) and 10 min after the infusion, the PVN was removed and homogenized. Importin ß-1 co-immunoprecipitated with the NK3R following treatment with 2 M NaCl, but not following isotonic saline treatment. Immuno-neutralization of importin ß-1 decreased the transport of NK3R into the nuclei in a time dependent fashion. The results indicate that in response to acute hyperosmotic challenge, NK3R associates with importin ß-1 which enables the nuclear transport of NK3R. This is the first in vivo study linking importin ß-1 and the nuclear transport of a G protein coupled receptor, the NK3R, in brain.

P.3.f.006 Deletion of CB2 cannabinoid receptor induces schizophrenia-related behaviours in mice

The tachykinin NK1 receptor was suggested to be involved in psychiatric disorders, but its antagonists have failed to be effective as antidepressants in clinical trials. Hemokinin-1 (HK-1), the newest tachykinin, is present in several brain regions and activates the NK1 receptor similarly to substance P (SP), but acts also through other mechanisms. Therefore, we investigated the roles of the Tac4 gene-derived HK-1 in comparison with SP and neurokinin A (NKA) encoded by the Tac1 gene, as well as the NK1 receptor in anxiety and depression-like behaviors in mice.Mice lacking SP/NKA, HK-1 or the NK1 receptor (Tac1−/−, Tac4−/−, Tacr1−/−, respectively) compared to C57Bl/6 wildtypes (WT), and treatment with the NK1 antagonist CP99994 were used in the experiments. Anxiety was evaluated in the light-dark box (LDB) and the elevated plus maze (EPM), locomotor activity in the open field (OFT) tests. Hedonic behavior was assessed in the sucrose preference test (SPT), depression-like behavior in the tail suspension (TST) and forced swim (FST) tests. FST-induced neuronal responsiveness was evaluated with Fos immunohistochemistry in several stress-related brain regions.In the LDB, Tac4−/− mice spent significantly less, while Tacr1−/− and CP99994-treated mice spent significantly more time in the lit compartment. In the EPM only Tac4−/− showed reduced time in the open arms, but no difference was observed in any other groups. In the OFT Tac4−/− mice showed significantly reduced, while Tac1−/− and Tacr1−/− animals increased motility than the WTs, but CP99994 had no effect. NK1−/− consumed markedly more, while Tac4−/− less sucrose solution compared to WTs. In the TST and FST, Tac4−/− mice showed significantly increased immobility. However, depression-like behavior was decreased both in cases of genetic deletion and pharmacological blockade of the NK1 receptor. FST-induced neuronal activation in different nuclei involved in behavioral and neuroendocrine stress responses was significantly reduced in the brain of Tac4 −/− mice.Our results provide the first evidence for an anxiolytic and anti-depressant-like actions of HK-1 through a presently unknown target-mediated mechanism. Identification of its receptor and/or signaling pathways might open new perspectives for anxiolytic and anti-depressant therapies.

BLOCKADE OF TACHYKININ NK-3 RECEPTOR REVERSES HYPERTENSION THROUGH A DOPAMINERGIC MECHANISM IN THE VENTRAL TEGMENTAL AREA OF SPONTANEOUSLY HYPERTENSIVE RATS: PP.29.161

Intracerebroventricularly (i.c.v.) injected tachykinin NK-3 receptor (R) antagonists normalized mean arterial blood pressure (MAP) in spontaneously hypertensive rats (SHR). This study was pursued to define the role played by NK-3R located on dopamine (DA) neurons of the ventral tegmental area (VTA) in the regulation of MAP in SHR. SHR (16 weeks) were implanted permanently with i.c.v. and/or VTA guide cannula. Experiments were conducted 24 h after catheterization of the abdominal aorta to measure MAP and heart rate (HR) in freely behaving rats. Cardiovascular responses to i.c.v. or VTA injected NK-3R agonist (senktide) and antagonists (SB222200 and R-820) were measured before and after systemic administration of selective DA antagonists for D-1R (SCH23390), D-2R (raclopride) or non-selective D-2R (haloperidol) and after destruction of the VTA with ibotenic acid. Senktide (10, 25, 65 and 100 pmol) elicited more prominent increases of MAP and HR when injected in the VTA than i.c.v. and this cardiovascular response was blocked by R-820, SCH23390 and haloperidol. I.c.v. or VTA injected SB222200 and R-820 (500 pmol) evoked anti-hypertension, which was blocked by raclopride. Destruction of the VTA prevented the pressor response to i.c.v. senktide and the anti-hypertension to i.c.v. R-820. VTA injected SB222200 prevented the pressor response to i.c.v. senktide, and vice versa, i.c.v. senktide prevented the anti-hypertension to VTA SB222200. Repeated i.c.v. injection of R-820, once a day for 5 days, caused a sustained anti-hypertensive effect in SHR. Tachykinin NK-3R in the VTA is implicated in the maintenance of hypertension by increasing midbrain DA transmission in SHR. Hence, these receptors may represent a therapeutic target in the treatment of arterial hypertension.

Effects of rat/mouse hemokinin-1, human hemokinin-1 and human hemokinin-1(4-11), mammalian tachykinin peptides, on rate and perfusion pressure in the isolated guinea pig heart

Rat/mouse hemokinin-1 (r/m HK-1), human hemokinin-1 (h HK-1) and human hemokinin-1(4-11) (h HK-1(4-11)) are members of the tachykinin family. In the present study, the coronary vascular activities and cardiac functions of r/m HK-1, h HK-1 and h HK-1(4-11) were investigated in isolated, spontaneously beating guinea pig hearts. Bolus injections of r/m HK-1 caused decrease in perfusion pressure indicative of coronary vasodilation, which was primarily due to the action on tachykinin NK1 receptors on vascular endothelial cells, causing the release of nitric oxide that relaxed the coronary vessels. H HK-1 caused biphasic perfusion pressure changes that were coronary vasodilation followed by coronary vasoconstriction. The mechanisms involved in the vasodilation induced by h HK-1 were similar to that of r/m HK-1 while the mechanisms for coronary vasoconstriction were mediated through the activation of tachykinin NK2 receptors on coronary sympathetic neurons to release catecholamines. H HK-1(4-11) only produced coronary vasoconstriction and the mechanisms involved in this effect were similar to that of h HK-1 in vasoconstriction. Moreover, r/m HK-1 and h HK-1 produced similar decreases in heart rate indicative of negative chronotropic responses and the decreases were mainly mediated through the activation of tachykinin NK1 receptors to release ACh acting on muscarinic receptors. H HK-1(4-11) also produced negative chronotropic response, which was mainly mediated through tachykinin NK2 receptors and muscarinic receptors. Our present results provide evidence that all of the three tachykinins could influence cardiac function and coronary vascular activity in the isolated guinea pig heart.

Development of Mechanisms Associated With Neurogenic-Mediated Skin Inflammation During the Growthof Rats

Neurogenic-mediated inflammation may be associated with several inflammatory skin diseases including atopic dermatitis. However, age-dependent differences in neurogenic-mediated skin responses are not fully understood. We compared skin plasma leakage in rats aged 2 and 8 wk, which was induced by topical capsaicin, topical formalin, and intracutaneous substance P, whose effects are mediated via tachykinin NK1 receptors. Evans blue dye extravasation served as an index of the increase in skin vascular permeability. Capsaicin, formalin, and substance P caused a skin response in a dose-dependent manner in both age groups. However, the skin response was much greater in adults than in pups. In addition, the localization of sensory C-fibers and tachykinin NK1 receptors in the skin was investigated by immunofluorescent staining with antisubstance P and antitachykinin NK1 receptor antibodies, respectively. Substance P-immunoreactive nerves were detected throughout the dermis and tachykinin NK1 receptors were mainly detected in blood vessel walls in the dermis in both age groups. However, they were more sparsely distributed in pups. In conclusion, the weak neurogenic-mediated skin inflammation in pups is probably because of immature neural mechanisms associated with skin inflammation such as reduced innervation of sensory C-fibers and low expression of tachykinin NK1 receptors.

Effect of the flavonoid galangin on urinary bladder rat contractility in-vitro.

Galangin is a flavanol with several biological activities. We have evaluated the effect of galangin on the contractile response elicited by electrical field stimulation (EFS) in the rat isolated urinary bladder. Galangin (10(-8)-10(-4) M) produced a concentration-dependent inhibition of the EFS contractile response without modifying the contractions produced by exogenous acetylcholine (10(-6) M). Blockade of adrenergic and cholinergic nerves with a combination of atropine (10(-6) M), phentolamine (10(-6) M) and propranolol (10(-6) M) or blockade of tachykinin NK1 and NK2 receptors with SR140333 (10(-7) M) and SR48968 (10(-6) M) did not modify the inhibitory effect of galangin. However, verapamil (10(-7) M) significantly reduced the inhibitory effect of galangin. It is concluded that the galangin inhibits EFS-induced contractions of the rat urinary bladder by acting on L-type calcium channels on presynaptic nerves.

A flexible approach to the design of new potent substance P receptor ligands.

The development of small-molecule antagonists of the substance-P-preferring tachykinin NK1 receptor offers an excellent opportunity to exploit these molecules as novel therapeutic agents in diverse pathologies such as depression, emesis or asthma. GR71251 has previously been identified as a potent and selective substance-P-receptor antagonist. We have therefore undertaken the synthesis of new pseudopeptidic analogues based on the C-terminal sequence of GR71251. The evaluation of binding affinities toward NK1 and NK2 receptors has enabled us to propose new selective NK1 ligands with high affinity. Structure-activity relationships showed that the Trp-OBzl(CF3)2 moiety is essential for NK1 affinity and that the introduction of building units such as spirolactam, lactam or proline, leading to a constrained peptide, increased selectivity for NK1 receptors. These compounds constitute a useful starting point for new substance P antagonists and represent an attractive lead series for further studies on the design of specific NK1 antagonists.

Influence of tachykinin NK2 receptors on intestinal sensitivity and motility in newborn rats

The effect of tachykinin neurokinin NK(2) receptors activation on intestinal propulsion and colorectal sensitivity was studied in 7-15 days old newborn rats. In a first set of experiments investigating the intestinal transit, the selective NK(2) receptor agonist, [betaAla(8)]NKA-(4-10) was used. It produced an increase of the small intestinal transit measured by charcoal test of 54%, that was inhibited in a dose-dependent manner by nepadutant ([N(4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparaginyl-L-aspartyl-L-tryptophyl-L-phenylalanyl-L-2,3-diaminopropionyl-L-leucyl]-C-4.2-N-3.5-lactam-C-1.6-N-2.1-lactam), a known selective NK(2) receptor antagonist, orally administered 2-48 h before the challenge with the NK(2) receptor agonist. Nepadutant did not affect the basal intestinal propulsion and showed a good oral bioavailability and long duration of action. In another set of experiments investigating visceral sensitivity, a fixed distension volume of a balloon inserted intrarectally in 14-15 days old newborns rats produced abdominal contractions (AC) that were increased after colonic application of acetic acid (50 microl, 0.5%). In this latter condition nepadutant, at 0.5 and 2.5 mg/kg p.o., significantly reduced the resulting AC. In control rats, untreated with acetic acid, nepadutant did not affect AC evoked by colorectal distension. These findings show for the first time two models to assess intestinal motility and visceral sensitivity in newborn rats and indicate nepadutant as a valuable tool to assess the role of NK(2) receptors in the intestinal propulsive and nociceptive activity in infants.

Compensatory mechanisms to maintain blood pressure in paraplegic rats: Implication of central tachykinin NK-1 and NK-3 receptors?

People with high level spinal cord injury (SCI) suffer from both hypotension and spontaneous hypertension due to loss of supraspinal control of spinal sympathetic outflow. Few reports have addressed whether any changes occur in central regulation of blood pressure (BP) and heart rat (HR) at the supraspinal level. Central tachykinin NK-1 and NK-3 receptors are located in many cardiovascular areas in the brain and are known to modulate BP and HR. This study examined the intracerebroventricular (i.c.v.) effects of the selective NK-1 receptor agonist [Sar(9), Met(O(2))(11)]SP (65pmol, n=6) and NK-3 receptor agonist senktide (650pmol, n=6) on mean arterial pressure (MAP) and HR before and after complete spinal cord transection at thoracic level 4 (T4). [Sar(9), Met(O(2))(11)]SP evoked increases in MAP and HR which were still present 4days after the T4 SCI. Further analysis using the beta(1)-adrenoceptor antagonist atenolol (10mgkg(-1)) revealed an increased contribution of HR in the MAP increase after SCI. For senktide, 2 and 5weeks after T4 SCI, the rise in MAP induced by senktide was significantly increased in magnitude and was similar to a normal response at 8weeks. These effects were accompanied by a bradycardia, which was still present and amplified at 8weeks. Our results reveal a transient potentiation of the senktide-mediated MAP effect and a greater contribution of the HR in MAP increase by [Sar(9), Met(O(2))(11)]SP in T4 transected rats. Although the significance of these changes remains to be established. This suggest a reorganization of supraspinal mechanisms regulating BP and HR after a high level SCI. Central NK-1 and NK-3 receptors might therefore contribute to the maintenance of MAP following high thoracic SCI.

MicroRNAs May Mediate the Down-Regulation of Neurokinin-1 Receptor in Chronic Bladder Pain Syndrome

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B(1) receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.

In vitro and in vivo comparison of two non-peptide tachykinin NK3 receptor antagonists: Improvements in efficacy achieved through enhanced brain penetration or altered pharmacological characteristics

Clinical evaluation of tachykinin NK(3) receptor antagonists has provided support for the therapeutic utility of this target in schizophrenia. However, these studies have not been entirely conclusive, possibly because of the pharmacokinetic limitations of these molecules. In the search for tachykinin NK(3) receptor antagonists with improved properties, we have discovered GSK172981 and GSK256471. Both compounds demonstrated high affinity for recombinant human (pK(i) values 7.7 and 8.9, respectively) and native guinea pig (pK(i) values 7.8 and 8.4, respectively) tachykinin NK(3) receptors. In vitro functional evaluations revealed GSK172981 to be a competitive antagonist (pA(2)=7.2) at cloned human tachykinin NK(3) receptor whereas GSK256471 diminished the neurokinin B-induced E(max) response, indicative of non-surmountable antagonist pharmacology (pA(2)=9.2). GSK172981 also exhibited a competitive profile in antagonizing neurokinin B-stimulated neuronal activity recorded from the guinea pig medial habenula slices (apparent pK(B)=8.1), whilst GSK256471 abolished the agonist-induced response. Central nervous system penetration by GSK172981 and GSK256471 was indicated by dose-dependent ex vivo tachykinin NK(3) receptor occupancy in medial prefrontal cortex (ED(50) values of 0.8 and 0.9 mg/kg, i.p., respectively) and the dose-dependent attenuation of agonist-induced "wet dog shake" behaviours in guinea pigs. Finally, in vivo microdialysis studies demonstrated that acute GSK172981 (30 mg/kg, i.p.) and GSK256471 (1mg/kg, i.p.) attenuated haloperidol-induced increases in extracellular dopamine in the guinea pig nucleus accumbens. Taken together, these in vitro and in vivo characterisations of the tachykinin NK(3) receptor antagonists GSK172981 and GSK256471 support their potential utility in the treatment of schizophrenia.

The effect of the tachykinin NK2 receptor antagonist MEN11420 (nepadutant) on neurokinin A-induced bronchoconstriction in asthmatics

We previously reported that the nonpeptide tachykinin NK(2) receptor antagonist SR48968 (saredutant) significantly inhibits neurokinin A-induced bronchoconstriction in patients with asthma. MEN11420 (nepadutant) is a bicyclic peptide tachykinin NK(2) receptor antagonist. The aim of the trial was to examine the effect of nepadutant on neurokinin A-induced bronchoconstriction in man. 12 patients with stable, mild to moderate asthma participated in a double-blind, placebo-controlled crossover trial and received, with intervals of 1 week, MEN11420 2 mg, MEN11420 8 mg and placebo (i.v.). Increasing concentrations of NKA (10(-9) to 10(-6) moles/ml) were inhaled immediately after (d1) and 24 hours after (d2) administration of treatment. On d1 both MEN11420 2 and 8 mg shifted the dose response curve for neurokinin A to the right (log PC(20) FEV(1) neurokinin A [moles/ml]; mean + or = or - SEM -6.38 + or - 0.26 after 2 mg, -6.11 + or - 0.23 after 8 mg, versus -6.95 + or - 0.27 after placebo]. On d2 MEN11420 had no effect on neurokinin A-induced bronchoconstriction. In conclusion, the tachykinin NK(2) receptor antagonist nepadutant significantly inhibits bronchoconstriction induced by neurokinin A in patients with asthma.

Role of the tachykinin NK1 receptor in a murine model of cigarette smoke-induced pulmonary inflammation

BackgroundThe tachykinins, substance P and neurokinin A, present in sensory nerves and inflammatory cells such as macrophages and dendritic cells, are considered as pro-inflammatory agents. Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and increased tachykinin levels are recovered from the airways of COPD patients. The aim of our study was to clarify the involvement of the tachykinin NK1 receptor, the preferential receptor for substance P, in cigarette smoke (CS)-induced pulmonary inflammation and emphysema in a mouse model of COPD.MethodsTachykinin NK1 receptor knockout (NK1-R-/-) mice and their wild type controls (all in a mixed 129/sv-C57BL/6 background) were subjected to sub acute (4 weeks) or chronic (24 weeks) exposure to air or CS. 24 hours after the last exposure, pulmonary inflammation and development of emphysema were evaluated.ResultsSub acute and chronic exposure to CS resulted in a substantial accumulation of inflammatory cells in the airways of both WT and NK1-R-/- mice. However, the CS-induced increase in macrophages and dendritic cells was significantly impaired in NK1-R-/- mice, compared to WT controls, and correlated with an attenuated release of MIP-3α/CCL20 and TGF-β1. Chronic exposure to CS resulted in development of pulmonary emphysema in WT mice. NK1-R-/- mice showed already enlarged airspaces upon air-exposure. Upon CS-exposure, the NK1-R-/- mice did not develop additional destruction of the lung parenchyma. Moreover, an impaired production of MMP-12 by alveolar macrophages upon CS-exposure was observed in these KO mice. In a pharmacological validation experiment using the NK1 receptor antagonist RP 67580, we confirmed the protective effect of absence of the NK1 receptor on CS-induced pulmonary inflammation.ConclusionThese data suggest that the tachykinin NK1 receptor is involved in the accumulation of macrophages and dendritic cells in the airways upon CS-exposure and in the development of smoking-induced emphysema. As both inflammation of the airways and parenchymal destruction are important characteristics of COPD, these findings may have implications in the future treatment of this devastating disease.

Current investigational drugs for major depression

Background: The World Health Organization (WHO) report has predicted that major depression will become a key cause of illness-induced disability by the year 2020, second only to ischemic heart diseases. Objectives/methods: Although a large number of antidepressant drugs (from monoamine oxidase inhibitors and tricyclic antidepressants to dual reuptake inhibitors) are available for treatment of the disease, approximately 30% of patients failed to respond to this therapy. Therefore, the search for newer or novel drug targets for the treatment of major depression continues. Some of these targets include dopamine, triple reuptake inhibition, L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway, sigma-1 receptors, neurosteroids, melatonin, glutamate, 5HT6, 5HT7 serotonin receptor antagonists, β-3 adrenoceptor antagonist, vasopressin V(Ib) receptor antagonists, NK2 tachykinin receptor antagonists, glucocorticoid receptor antagonists and corticotropin-releasing factor-1 receptor antagonists, as well as herbal antidepressant drugs. The present review attempts to discuss the status of some of these novel approaches and the drugs that are under investigation for the treatment of major depression. An attempt is also made to review the status of three indigenous plant-derived drugs, berberine, curcumin and rutin, as novel and safe future herbal antidepressants. Results/conclusion: There is an exciting future in the discovery of novel targets and target-specific agents for the management of major depression.

Electrical stimulation of the mucosa evokes slow EPSPs mediated by NK1 tachykinin receptors and by P2Y1 purinoceptors in different myenteric neurons

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300-900 ms) or slow (durations 1.3-9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2-4-disulfonic acid (30 microM, N = 3) or the specific P2Y(1) antagonist MRS 2179 (10 microM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 (N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y(1), S neurons).

Characterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3

Tachykinin NK receptors (NKRs) differ to a large degree among species with respect to their affinities for small molecule antagonists. The aims of the present study were to clone NKRs from gerbil (NK2R and NK3R) and dog (NK1R, NK2R and NK3R) in which the sequence was previously unknown and to investigate the potency of several NKR antagonists at all known human, dog, gerbil and rat NKRs.The NKR protein coding sequences were cloned and expressed in CHO cells. The inhibitory concentrations of selective and non-selective NKR antagonists were determined by inhibition of agonist-induced mobilization of intracellular Ca2+. Receptor homology models were constructed based on the rhodopsin crystal structure to investigate and identify the antagonist binding sites and interaction points in the transmembrane (TM) regions of the NKRs.Data collected using the cloned dog NK1R confirmed that the dog NK1R displays similar pharmacology as the human and the gerbil NK1R, but differs greatly from the mouse and the rat NK1R. Despite species-related amino acid (AA) differences located close to the antagonist binding pocket of the NK2R, they did not affect the potency of the antagonists ZD6021 and saredutant. Two AA differences located close to the antagonist binding site of NK3R likely influence the NK3R antagonist potency, explaining the 3–10-fold decrease in potency observed for the rat NK3R. For the first time, detailed pharmacological experiments in vitro with cloned NKRs demonstrate that not only human, but also dog and gerbil NKR displays similar antagonist pharmacology while rat diverges significantly with respect to NK1R and NK3R.

Cross talk between facilitatory adenosine A2A, Tachykinin NK1 and Muscarinic M3 receptors in the rat Mynteric plexus: on the role of Capsaicin-sensitive Tachykinergic neurons

Event Abstract Back to Event Cross talk between facilitatory adenosine A2A, Tachykinin NK1 and Muscarinic M3 receptors in the rat Mynteric plexus: on the role of Capsaicin-sensitive Tachykinergic neurons I. Silva1*, C. Vieira1, F. Ferreirinha1, M. Duarte-Araujo1 and P. Correia- De-Sa1 1 Instituto de Ciencias Biomedicas Abel Salazar - Universidade do Porto (ICBAS-UP), Laboratorio de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigacao Biomedica (UMIB), Portugal Gastrointestinal motility relies on the release of acetylcholine (ACh), purines and tachykinins (e.g. substance P) from excitatory enteric neurons. Tachykinins (TK) acting via NK1 and NK3 receptors facilitate cholinergic neurotransmission in the enteric nervous system. Recently, we showed that muscarinic M3 receptors facilitate ACh release indirectly by increasing adenosine outflow from stimulated myenteric neurons leading to the activation of excitatory A2A receptors (Vieira et al., 2009, Neurogastroenterol. Motil. in press). These findings prompted us to investigate the cross talk between facilitatory A2A, M3 and NK1 receptors in the rat myenteric plexus, focusing our attention on the role of tachykinergic nerve fibres which can be selectively eliminated (>95%) by neonatal capsaicin administration (CAP, 50 mg/Kg, SC).The muscarinic agonist, oxotremorine (Oxo, 300 μM) and the selective adenosine A2A agonist, CGS21680C (CGS, 3 nM), increased evoked (5 Hz, 200 pulses) [3H]-ACh release from myenteric neurons respectively by 34±4% (n=5) and 53±10% (n=5) in controls (C) but were devoid of effects in CAP animals. Selective activation of NK1 receptors with Sar9,Met(O2)11-Substance P (s,m-SP, 300 nM) restored CGS facilitation (46±17%, n=6) to control levels in CAP rats. Cross talk between A2A and NK1 receptors was evaluated using selective antagonists of NK1 (L732128, 20 nM) and A2A (ZM241385, 50 nM) receptors. Pretreatment with s,m-SP did not restore Oxo facilitation (3±7%, n=4) in CAP animals. Incubation of C with the M3 antagonist (J104129, 6 nM) attenuated CGS (3±5%, n=5)-induced facilitation of [3H]-ACh release, but (like in CAP rats) its effect was partially recovered (23±2%, n=3) in the presence of s,m-SP.Data suggest that facilitation of [3H]-ACh release by A2A receptors requires tonic activation of NK1 receptors by endogenously released TK from CAP-sensitive myenteric neurons. Confocal microscopy studies show that A2A receptors co-localize with cholinergic nerve fibres (labelled with VAChT), but not with CAP-sensitive neurons exhibiting substance P immunoreactivity. Data also indicate that endogenous release of TK (as well as adenosine, see Vieira et al., 2009) may be tightly controlled by M3 receptors. In support of this hypothesis, we showed that CAP-sensitive tachykinergic neurons might be an important source of purines in the rat myenteric plexus (see, Marques et al., 2009, this meeting).Work supported by FCT (FEDER funding, PTDC/CVT/74462/2006) Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Poster Presentation Topic: Neuronal Communication Citation: Silva I, Vieira C, Ferreirinha F, Duarte-Araujo M and Correia- De-Sa P (2009). Cross talk between facilitatory adenosine A2A, Tachykinin NK1 and Muscarinic M3 receptors in the rat Mynteric plexus: on the role of Capsaicin-sensitive Tachykinergic neurons. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.149 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Aug 2009; Published Online: 12 Aug 2009. * Correspondence: I. Silva, Instituto de Ciencias Biomedicas Abel Salazar - Universidade do Porto (ICBAS-UP), Laboratorio de Farmacologia e Neurobiologia, Unidade Multidisciplinar de Investigacao Biomedica (UMIB), Porto, Portugal, isabelsds@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa Google I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa Google Scholar I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa PubMed I. Silva C. Vieira F. Ferreirinha M. Duarte-Araujo P. Correia- De-Sa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Physiological evidence of a postsynaptic inhibition of the tail flick reflex by a cannabinoid receptor agonist

Current evidence indicates that cannabinoids are antinociceptive and this effect is in part mediated by spinal mechanisms. Anatomical studies have localized cannabinoid CB 1 receptors to pre- and postsynaptic sites within the spinal cord. However, behavioural tests have not clearly indicated the relative importance of each of these sites. In this study, the tail flick test was used as a model of acute pain in the rat to determine the site of action of WIN 55,212-2 (( R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate), a synthetic cannabinoid receptor agonist. WIN 55,212-2 (3 mg/kg, i.p.) increased the latency of tail withdrawal from a noxious radiant heat source, indicating it is antinociceptive in this model. Using the same paradigm, WIN 55,212-2 was then tested against the synaptically-induced nociceptive hypersensitivity in response to noxious thermal stimulation of the tail (hot water tail immersion). WIN 55,212-2 blocked this hypersensitivity, confirming a spinal site of action of the cannabinoid receptor agonist. Further, WIN 55,212-2 blocked the nociceptive hypersensitivity induced by intrathecal administration of substance P. As substance P acts on postsynaptic tachykinin NK1 receptors in the dorsal horn of the spinal cord, the data are interpreted to suggest that WIN 55,212-2 expressed its anti-hypersensitivity effects at least partially via a postsynaptic site of action; the data do not rule out a presynaptic site of action. This study suggests that cannabinoids may induce analgesia via a postsynaptic site of action in the spinal cord, as well as the possibility that they may interact with substance P signaling.