NK2 receptor-mediated detrusor muscle contraction involves Gq/11-dependent activation of voltage-dependent Ca2+ channels and the RhoA-Rho kinase pathway.

Tachykinins (TKs) are involved in both the physiological regulation of urinary bladder functions and development of overactive bladder syndrome. The aim of the present study was to investigate the signal transduction pathways of TKs in the detrusor muscle to provide potential pharmacological targets for the treatment of bladder dysfunctions related to enhanced TK production. Contraction force, intracellular Ca2+ concentration, and RhoA activity were measured in the mouse urinary bladder smooth muscle (UBSM). TKs and the NK2 receptor (NK2R)-specific agonist [ß-Ala8]-NKA(4-10) evoked contraction, which was inhibited by the NKR2 antagonist MEN10376. In Gαq/11-deficient mice, [ß-Ala8]-NKA(4-10)-induced contraction and the intracellular Ca2+ concentration increase were abolished. Although Gq/11 proteins are linked principally to phospholipase Cß and inositol trisphosphate-mediated Ca2+ release from intracellular stores, we found that phospholipase Cß inhibition and sarcoplasmic reticulum Ca2+ depletion failed to have any effect on contraction induced by [ß-Ala8]-NKA(4-10). In contrast, lack of extracellular Ca2+ or blockade of voltage-dependent Ca2+ channels (VDCCs) suppressed contraction. Furthermore, [ß-Ala8]-NKA(4-10) increased RhoA activity in the UBSM in a Gq/11-dependent manner and inhibition of Rho kinase with Y-27632 decreased contraction force, whereas the combination of Y-27632 with either VDCC blockade or depletion of extracellular Ca2+ resulted in complete inhibition of [ß-Ala8]-NKA(4-10)-induced contractions. In summary, our results indicate that NK2Rs are linked exclusively to Gq/11 proteins in the UBSM and that the intracellular signaling involves the simultaneous activation of VDCC and the RhoA-Rho kinase pathway. These findings may help to identify potential therapeutic targets of bladder dysfunctions related to upregulation of TKs.

NK2 and NK1 receptor-mediated effects of NKA and analogs on colon, bladder, and arterial pressure in anesthetized dogs.

Tachykinin NK2 receptor (NK2R) agonists have potential to alleviate clinical conditions associated with bladder and gastrointestinal under activity. The effects of agonists with differing selectivity for NK2R over NK1Rs on colorectal, bladder, and cardiovascular function were examined in anesthetized dogs. Intravenous (IV) administration of NKA, LMN-NKA ([Lys5,MeLeu9,Nle10]-NKA(4-10)), and [ß-Ala8]-NKA(4-10) caused a dose-related increase in colorectal pressure (up to 98 mmHg) that was blocked by pretreatment with the NK2R antagonist GR 159897 (1 mg/kg), and hypotension (decrease in mean arterial pressure of ~40 mmHg) that was blocked by the NK1R antagonist CP-99,994 (1 mg/kg). Despite the greater in vitro selectivity of LMN-NKA and [ß-Ala8]-NKA(4-10) for NK2R over NK1Rs compared with NKA, all 3 agonists increased colorectal pressure and caused hypotension within a similar dose range when administered as a bolus (0.1-300 µg/kg IV), or even as a slow IV infusion over 5 min (NKA; 0.02-0.6 µg/kg/min). In contrast, subcutaneous (SC) administration of LMN-NKA (3-10 µg/kg) increased colorectal pressure (up to 50 mmHg) and elicited micturition (≧ 85% voiding efficiency) without causing hypotension. NK2R agonists can produce rapid-onset, short-duration, colorectal contractions, and efficient voiding of urine without hypotension after SC administration, indicating that routes of administration that avoid the high plasma concentrations associated with IV dosing improve the separation between desired and unwanted pharmacodynamic effects. The potent hypotensive effect of NKA in dogs was unexpected based on published studies in humans in which IV infusion of NKA did not affect blood pressure at doses that increased gastrointestinal motility.

Ablation of Tacr2 in mice leads to gastric emptying disturbance.

BACKGROUND: Tacr2 is one of the G protein-coupled receptors(GPCRs) that mediate the biological actions of tachykinins. It is abundantly expressed in the gastrointestinal (GI) system and is thought to play an important role in GI motility, secretion, and visceral sensitivity. Previously, the physiological and pathophysiological functions of Tacr2 were mainly studied using Tacr2 selective agonists or antagonists. Here, we seek to investigate the effect of Tacr2 disruption in mice to provide further insights. METHODS: The Tacr2 knockout mice were generated by homologous recombination and the phenotypic changes of the Tacr2-null mice were analyzed and compared with their wild type (wt) littermates. KEY RESULTS: Increased food retention was detected in Tacr2-/- mice. The stomach of Tacr2-/- mice had thinner muscularis externa and less neurons in the myenteric plexus. The stomach and small intestine exhibited longer duration of electrical field stimulation (EFS)-induced inhibition in the gastric fundus and decreased frequency of migrating motor complex (MMC), respectively. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were significantly up-regulated due to Tarc2 deficiency, contributing to enhanced nitric oxide (NO) signaling in the stomach of Tacr2-/- mice. Intraperitoneal application of 7-nitroindazole (7-NI) to Tacr2-/- mice effectively relieved the gastric emptying disturbance. Moreover, Creb and NF-κB signalings were involved in the regulation of these physiological changes initiated by Tacr2 deficiency. CONCLUSIONS & INFERENCES: Tacr2 negatively regulated the expression of nNOS and VIP both in vivo and in vitro. Its ablation in mice elevated the expression of nNOS and VIP, enhanced NO signaling and changed the Creb and NF-κB signalings, finally leading to the gastric emptying disturbance of Tacr2-/- mice.

Impaired colonic motility and reduction in tachykinin signalling in the aged mouse.

Ageing is associated with an increased incidence of constipation in humans. The contribution that the ageing process makes to this condition is unclear. The aim of this study was to determine the effects of age on faecal output and colonic motility in male C57BL/6J mice and to determine the role that altered tachykinin signalling plays in this process. Total faecal output recorded over a 24h period decreased with age due to a reduction in the number of pellets produced and their water content. These changes occurred in the absence of any significant change in food and water intake. There was an increase in the amount of faecal matter stored in the isolated colon with age which caused a proportional increase in colonic length. Analysis of colonic motility using an artificial pellet demonstrated that pellets moved in a stepwise fashion through the colon. There was an age-related increase in pellet transit time due to decreases in the step distance, velocity, and frequency of stepwise movements. These changes were reversed using the neurokinin 2 (NK2) receptor agonist neurokinin A. Addition of the NK2receptor antagonist GR159897 significantly increased transit time in the young animals by decreasing step distance, velocity and frequency, but was without effect in the aged colon. In summary, the ageing C57BL/6J mouse shows an impaired motility phenotype. These effects appear, at least in part, to be due to an attenuation of tachykinin signalling via NK2 receptors.

Peristalsis and propulsion of colonic content can occur after blockade of major neuroneuronal and neuromuscular transmitters in isolated guinea pig colon.

We recently identified hexamethonium-resistant peristalsis in the guinea pig colon. We showed that, following acute blockade of nicotinic receptors, peristalsis recovers, leading to normal propagation velocities of fecal pellets along the colon. This raises the fundamental question: what mechanisms underlie hexamethonium-resistant peristalsis? We investigated whether blockade of the major receptors that underlie excitatory neuromuscular transmission is required for hexamethonium-resistant peristalsis. Video imaging of colonic wall movements was used to make spatiotemporal maps and determine the velocity of peristalsis. Propagation of artificial fecal pellets in the guinea pig distal colon was studied in hexamethonium, atropine, ω-conotoxin (GVIA), ibodutant (MEN-15596), and TTX. Hexamethonium and ibodutant alone did not retard peristalsis. In contrast, ω-conotoxin abolished peristalsis in some preparations and reduced the velocity of propagation in all remaining specimens. Peristalsis could still occur in some animals in the presence of hexamethonium + atropine + ibodutant + ω-conotoxin. Peristalsis never occurred in the presence of TTX. The major finding of the current study is the unexpected observation that peristalsis can occur after blockade of the major excitatory neuroneuronal and neuromuscular transmitters. Also, the colon retained an intrinsic polarity in the presence of these antagonists and was only able to expel pellets in an aboral direction. The nature of the mechanism(s)/neurotransmitter(s) that generate(s) peristalsis and facilitate(s) natural fecal pellet propulsion, after blockade of major excitatory neurotransmitters, at the neuroneuronal and neuromuscular junction remains to be identified.

Characterization of ibodutant at NK(2) receptor in human colon.

We have characterized the pharmacological profile of the nonpeptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) through radioligand binding and contractility assays in the human colon smooth muscle. The antagonist affinity of ibodutant was evaluated through concentration-dependent inhibition curves at the [(125)I]NKA specific binding by using membranes prepared from human colon smooth muscle. In this assay the affinity of ibodutant (pKi 9.9) was compared to that of other two selective NK2 receptor antagonists, nepadutant (pKi 8.4) and saredutant (pKi 9.2). The antagonist potency of ibodutant was evaluated towards the [ßAla(8)]NKA(4-10)-mediated contractions of human colon smooth muscle strips. In this assay ibodutant (3, 10, 30 and 100 nM) induced a concentration-dependent rightward shift of the [ßAla(8)]NKA(4-10) concentration-response curves without depressing the maximal contractile effect. The analysis of the curves yielded a Schild-plot linear regression with a slope not different from unity (1.02), thus indicating a surmountable antagonist behavior. The calculated apparent antagonist potency as pKB value was 9.1. No sex related differences were observed in NK2 receptor pharmacology for [ßAla(8)]NKA(4-10) or ibodutant in colonic strips obtained from male or female patients. Reversibility experiments of tachykinin NK2 receptor blockade indicated that the inhibition of the agonist-induced contractions in preparations pre-exposed to ibodutant, and afterwards subjected to repeated washing cycles remained almost constant showing no sign of recovery during the 3h observation period. Overall, the present study indicates ibodutant as a potent tachykinin NK2 receptor antagonist in the human colon tissue, also endowed with a persistent duration of action.

Role of tachykinins and neurokinin receptor subtypes in the regulation of motility of the forestomach and abomasum in conscious sheep.

The present study was planned to evaluate role of tachykinins (TKs) and neurokinin (NK) receptors in the regulation of gastric motility in sheep. We examined the effects of intravenous (i.v.) injection of neurokinin A (NKA) and substance P (SP) on motility of the rumen, omasum, and abomasum in conscious sheep and the effects of NK receptor blockade on the effect of TKs using NK-1 receptor antagonist L-732,138 and NK-2 receptor antagonist SR48968. Moreover, the effect of NK receptor blockade on omasal cyclic contractions was examined. Intravenous injection of NKA and SP induced tonic contraction of rumen, omasum, and abomasum, and the contractile effect of NKA was more potent than that of SP in all the gastric regions. Although the effect of SP was not inhibited by L-732,138, the effect of NKA was significantly inhibited by SR48968. However, single infusion of SR48968 and L-732,138 did not alter cyclic electromyographic activity and basal intraluminal pressure in the omasum. These results imply that NKA and NK-2 receptors play a primary role in non-cholinergic regulation of ovine gastric motility, though NK-2 and NK-1 receptors seem unlikely to be involved in the physiological regulation of omasal cyclic contractions.

Antagonist profile of ibodutant at the tachykinin NK(2) receptor in guinea pig isolated bronchi.

In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK(2) receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK(2) receptor agonist [ßAla8]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300 nM) induced a concentration-dependent rightward shift of the [ßAla8]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65-1.25), thus, indicating a surmountable behavior. The calculated apparent antagonist potency as pK(B) value was 8.31 ± 0.05. Ibodutant (0.3-100 nM) produced a concentration-dependent inhibition of the nonadrenergic-noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100 nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95 ± 4% inhibition), the calculated IC(50) value was 2.98 nM (95% c.l. 1.73-5.16 nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100 nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrates that ibodutant is a potent NK(2) receptor antagonist in guinea pig airways.

Establishment and validation of a rabbit model for in vivo pharmacodynamic screening of tachykinin NK2 antagonists.

We attempted to establish and validate an in vivo pharmacodynamic (PD) rabbit model to screen tachykinin NK(2) receptor (NK(2)-R) antagonists using pharmacological and pharmacokinetic (PK)/PD analyses. Under urethane anesthesia, changes in intracolonic pressure associated with intravenous (i.v.) administration of a selective NK(2)-R agonist, ßAla(8)-neurokinin A(4-10) (ßA-NKA), was monitored as a PD marker. The analgesic effects of NK(2)-R antagonists were evaluated by monitoring visceromotor response (VMR) to colorectal distension in a rabbit model of visceral hypersensitivity induced by intracolonic treatment of acetic acid. Intravenous administration of ßA-NKA induced transient colonic contractions dose-dependently, which were inhibited by the selective NK(2)-R antagonists in dose- and/or plasma concentration-dependent manners. The correlation between PD inhibition and plasma concentration normalized with the corresponding in vitro binding affinity was relatively high (r(2) = 0.61). Furthermore, the minimum effective doses on the VMR and ID(50) values calculated in the PD model were highly correlated (r(2) = 0.74). In conclusion, we newly established and validated a rabbit model of agonist-induced colonic contractions as a screening tool for NK(2)-R antagonists. In a drug discovery process, this PD model could enhance the therapeutic candidate selection for irritable bowel syndrome, pharmacologically connecting in vitro affinity for NK(2)-R with in vivo therapeutic efficacy.

Hemokinin-1 stimulates prostaglandin E2 production in human colon through activation of cyclooxygenase-2 and inhibition of 15-hydroxyprostaglandin dehydrogenase.

Hemokinin-1 (HK-1) is a newly identified tachykinin, originating from the immune system rather than neurons, and may participate in the immune and inflammatory response. In colonic mucosa of patients with inflammatory bowel disease (IBD), up-regulation of the TAC4 gene encoding HK-1 and increased production of prostaglandin E2 (PGE2) occur. Our aim was to examine the mechanistic link between human HK-1 and PGE2 production in normal human colon. Exogenous HK-1 (0.1 µM) for 4 h evoked an increased PGE2 release from colonic mucosal and muscle explants by 10- and 3.5-fold, respectively, compared with unstimulated time controls. The HK-1-stimulated PGE2 release was inhibited by the tachykinin receptor antagonists (S)1-2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl-4-phenyl-l azonia-bicyclo[2.2.2]octane (SR140333) [neurokinin-1 (NK1)] and N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide (SR48968) [neurokinin-2 (NK2)] and was also inhibited by the cyclooxygenase (COX)-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide) (NS-398) but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560). A parallel study with substance P showed similar results. Molecular studies with HK-1-treated explants demonstrated a stimulatory effect on COX-2 expression at both transcription and protein levels. It is noteworthy that this was coupled with HK-1-induced down-regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) mRNA and protein expression. Immunoreactivity for 15-PGDH occurred on inflammatory cells, epithelial cells, platelets, and ganglia. This finding provides an additional mechanism for HK-1-evoked PGE2 increase, in which HK-1 may interfere with the downstream metabolism of PGE2 by suppressing 15-PGDH expression. In conclusion, our results uncover a novel inflammatory role for HK-1, which signals via NK1 and NK2 receptors to regulate PGE2 release from human colonic tissue, and may further explain a pathological role for HK-1 in IBD when abnormal levels of PGE2 occur.

Bidirectional regulation of human colonic smooth muscle contractility by tachykinin NK(2) receptors.

In this study, we attempted to clarify the mechanism of tachykinin-induced motor response in isolated smooth muscle preparations of the human colon. Fresh specimens of normal colon were obtained from patients suffering from colonic cancer. Using mucosa-free smooth muscle strips, smooth muscle tension with circular direction was monitored isometrically. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) produced marked contraction. All of these contractions were inhibited by saredutant, a selective NK(2)-R antagonist, but not by CP122721, a selective NK(1)-R antagonist or talnetant, a selective NK(3)-R antagonist. ßAla(8)-NKA(4-10) induced concentration-dependent contraction similar to NKA, but Sar(9)-Met(11)-SP and Met-Phe(7)-NKB did not cause marked contraction. Colonic contraction induced by ßAla(8)-NKA(4-10) was completely blocked by saredutant, but not by atropine. Tetrodotoxin or N(G)-nitro-L-arginine methyl ester pretreatment significantly enhanced ßAla(8)-NKA(4-10)-induced contraction. Immunohistochemical analysis showed that the NK(2)-R was expressed on the smooth muscle layers and myenteric plexus where it was also co-expressed with neuronal nitric oxide synthase in the myenteric plexus. These results suggest that the NK(2)-R is a major contributor to tachykinin-induced smooth muscle contraction in human colon and that the NK(2)-R-mediated response consists of an excitatory component via direct action on the smooth muscle and an inhibitory component possibly via nitric oxide neurons.

Three days after a single exposure to ozone, the mechanism of airway hyperreactivity is dependent on substance P and nerve growth factor.

Ozone causes persistent airway hyperreactivity in humans and animals. One day after ozone exposure, airway hyperreactivity is mediated by release of eosinophil major basic protein that inhibits neuronal M(2) muscarinic receptors, resulting in increased acetylcholine release and increased smooth muscle contraction in guinea pigs. Three days after ozone, IL-1ß, not eosinophils, mediates ozone-induced airway hyperreactivity, but the mechanism at this time point is largely unknown. IL-1ß increases NGF and the tachykinin substance P, both of which are involved in neural plasticity. These experiments were designed to test whether there is a role for NGF and tachykinins in sustained airway hyperreactivity following a single ozone exposure. Guinea pigs were exposed to filtered air or ozone (2 parts per million, 4 h). In anesthetized and vagotomized animals, ozone potentiated vagally mediated airway hyperreactivity 24 h later, an effect that was sustained over 3 days. Pretreatment with antibody to NGF completely prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone and significantly reduced the number of substance P-positive airway nerve bundles. Three days after ozone, NK(1) and NK(2) receptor antagonists also blocked this sustained hyperreactivity. Although the effect of inhibiting NK(2) receptors was independent of ozone, the NK(1) receptor antagonist selectively blocked vagal hyperreactivity 3 days after ozone. These data confirm mechanisms of ozone-induced airway hyperreactivity change over time and demonstrate 3 days after ozone that there is an NGF-mediated role for substance P, or another NK(1) receptor agonist, that enhances acetylcholine release and was not present 1 day after ozone.

Propofol preferentially relaxes neurokinin receptor-2-induced airway smooth muscle contraction in guinea pig trachea.

BACKGROUND: Propofol is the anesthetic of choice for patients with reactive airway disease and is thought to reduce intubation- or irritant-induced bronchoconstriction by decreasing the cholinergic component of vagal nerve activation. However, additional neurotransmitters, including neurokinins, play a role in irritant-induced bronchoconstriction. We questioned the mechanistic assumption that the clinically recognized protective effect of propofol against irritant-induced bronchoconstriction during intubation was due to attenuation of airway cholinergic reflexes. METHODS: Muscle force was continuously recorded from isolated guinea pig tracheal rings in organ baths. Rings were subjected to exogenous contractile agonists (acetylcholine, histamine, endothelin-1, substance P, acetyl-substance P, and neurokinin A) or to electrical field stimulation (EFS) to differentiate cholinergic or nonadrenergic, noncholinergic nerve-mediated contraction with or without cumulatively increasing concentrations of propofol, thiopental, etomidate, or ketamine. RESULTS: Propofol did not attenuate the cholinergic component of EFS-induced contraction at clinically relevant concentrations. In contrast, propofol relaxed nonadrenergic, noncholinergic-mediated EFS contraction at concentrations within the clinical range (20-100 mum, n = 9; P < 0.05), and propofol was more potent against an exogenous selective neurokinin-2 receptor versus neurokinin-1 receptor agonist contraction (n = 6, P < 0.001). CONCLUSIONS: Propofol, at clinically relevant concentrations, relaxes airway smooth muscle contracted by nonadrenergic, noncholinergic-mediated EFS and exogenous neurokinins but not contractions elicited by the cholinergic component of EFS. These findings suggest that the mechanism of protective effects of propofol against irritant-induced bronchoconstriction involves attenuation of tachykinins released from nonadrenergic, noncholinergic nerves acting at neurokinin-2 receptors on airway smooth muscle.

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.

Further investigation into the mechanism of tachykinin NK(2) receptor-triggered serotonin release from guinea-pig proximal colon.

The effects of the monoamine oxidase A (MAO-A) inhibitor clorgyline, the L-type calcium-channel blocker nicardipine, the syntaxin inhibitor botulinum toxin type C, and the potent thiol-oxidant phenylarsine oxide (PAO) on the selective tachykinin NK(2)-receptor agonist [beta-Ala(8)]-neurokinin A(4-10) [betaAla-NKA-(4-10)]-evoked 5-hydroxytryptamine (5-HT) outflow from colonic enterochromaffin (EC) cells was investigated in vitro using isolated guinea-pig proximal colon. The betaAla-NKA-(4-10)-evoked outflow of 5-HT from clorgyline-treated colonic strips was markedly higher than that from clorgyline-untreated colonic strips. The betaAla-NKA-(4-10)-evoked 5-HT outflow from the clorgyline-treated colonic strips was sensitive to nicardipine or botulinum toxin type C. Moreover, PAO concentration-dependently suppressed the betaAla-NKA-(4-10)-evoked 5-HT outflow from the clorgyline-treated colonic strips. The suppressant action of PAO was reversed by the reducing agent dithiothrietol, but was not blocked by the protein tyrosine kinase inhibitor genistein. These results suggest that the tachykinin NK(2) receptor-triggered 5-HT release from guinea-pig colonic EC cells is mediated by syntaxin-related exocytosis mechanisms and that colonic mucosa MAO-A activity has the important function of modulating the tachykinin NK(2) receptor-triggered 5-HT release. It also appears that PAO-mediated sulfhydryl oxidation plays a role in modulating the tachykinin NK(2) receptor-triggered 5-HT release through a mechanism independent of inhibition of protein tyrosine phosphatase activity.

Neurokinin receptors in recurrent airway obstruction: a comparative study of affected and unaffected horses.

The purpose of the study was to compare in vitro airway responses to neurokinin A & B (NKA and NKB) and expression of NK-2 receptors in airways of horses affected and unaffected with recurrent airway obstruction (RAO). Neurokinin-A, an inflammatory mediator belonging to the tachykinin family of neuropeptides, causes bronchoconstriction by binding to NK-2 receptors. Neurokinin-B is a lesser-known neuropeptide that acts on NK-3 receptors. Horses were placed into RAO-affected and RAO-unaffected groups based on their history, clinical scoring, and pulmonary function testing. Lung tissue from each lobe was collected for immunohistochemical staining for NK-2 receptors. Cumulative concentration-response relationships were determined on bronchial rings (4-mm wide) collected and prepared from the right diaphragmatic lung lobe to graded concentrations (half log molar concentrations 10(-7)M to 10(-4)M) of NKA and NKB. The results showed that NKA caused significantly greater contraction than NKB in both groups. In RAO-affected horses, both agents produced significantly greater bronchial contractions than those in the RAO-unaffected horses. Immunohistochemical staining showed that the overall NK-2 receptor distribution was significantly increased in bronchial epithelium and smooth muscles of bronchi and pulmonary vessels of RAO-affected than RAO-unaffected horses. The findings indicate that NK-2 receptors are up-regulated in RAO, suggesting that NK-2 receptor antagonists may have some therapeutic value in controlling the progression of airway hyperreactivity in horses affected with RAO.

Involvement of the neurokinin-2 receptor in airway smooth muscle stretch-activated contractions assessed in perfused intact bovine bronchial segments.

The airway response to deep inspirations (DIs) in asthmatics has been shown to be ineffective in producing bronchodilation and can even cause bronchoconstriction. However, the manner by which a DI is able to cause bronchoconstriction remains ambiguous. We sought to investigate the pathway involved in this stretch-activated contraction and whether this contraction is intrinsic to airway smooth muscle (ASM). In brief, intact bovine bronchial segments were dissected, and side branches were ligated and then mounted horizontally in an organ bath. Intraluminal pressure was measured under isovolumic conditions. Instantaneously opening and then closing the tap on a column of fluid 5 to 30 cm high evoked a sudden increase in intraluminal pressure (equivalent to the height of the column of fluid) followed by a stress relaxation response of the ASM. When tissues were stimulated with carbachol (10(-8) M) or serotonin (10(-7) M) for 10 min, and the consequent agonist-evoked pressure response was dissipated manually, the response to the same transmural stretch was accompanied by a slowly developing and prolonged increase in intraluminal pressure. This stretch-activated response was significantly diminished by the stretch-activated cation channel blocker gadolinium (10(-3) M), the L-type Ca2+ channel blockers nifedipine (2 x 10(-6) M), diltiazem (10(-5) M), and verapamil (10(-5) M), the sensory neurotoxin capsaicin (10(-5) M), and the neurokinin (NK)(2) receptor antagonists MEN 10376 ([Tyr(I),d-Trp(6,8,9),Lys(10)]-NKA(4-10)) (10(-5) M) and SR48968 (N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide) (3 x 10(-6) M). These results show the ability of isolated airways to exhibit stretch-activated contractions and suggest a role for stretch-activated cation channels, sensory afferent neurons, the neurotransmitter NKA, and L-type Ca(2+) channels in these isolated airway responses.

Effects of ischemia on tachykinin-containing nerves and neurokinin receptors in the rabbit bladder.

OBJECTIVES: Our previous studies showed marked changes in efferent nerve structure and reactivity in the ischemic bladder. The goal of this study was to examine the effects of bladder ischemia on tachykinin (TK) containing sensory nerves and neurokinin receptors (NKR) in a rabbit model. METHODS: We recorded bladder blood flow and spontaneous contractions in treated animals at week 8 after the induction of iliac arteries atherosclerosis and in age-matched controls. Bladder tissues were processed for studies of isometric smooth muscle tension in the organ bath, NK2R gene expression using quantitative real-time polymerase chain reaction (PCR), immunohistochemical staining of TK containing nerves and epithelial TK expression, and transmission electron microscopy. RESULTS: Atherosclerosis-induced ischemia significantly increased the frequency of spontaneous bladder contractions in vivo. Electrical field stimulation (EFS)-induced smooth muscle contractions were significantly greater in the ischemic tissues. Inhibition of NK1R diminished contractions to low-frequency EFS in control tissues while having no significant effect on the ischemic tissues. In contrast, NK2R inhibition significantly decreased contractions to both low- and high-frequency EFS in the ischemic tissues. Inhibition of NK3R had no significant effect on EFS-induced contractions. Real-time PCR showed a significant increase in NK2R gene expression in the ischemic bladder. The number of TK immunopositive nerves and epithelial TK immunoreactivity were significantly greater in the ischemic bladder. These alterations were associated with marked ultrastructural reactions to bladder ischemia. CONCLUSIONS: Alterations of NK2R reactivity and gene expression, increased number of TK immunopositive nerves, and greater epithelial TK immunoreactivity may imply activated bladder afferents to signal ischemic insult.

Role of tachykinin NK(1) and NK(2) receptors in colonic sensitivity and stress-induced defecation in gerbils.

The pharmacology of tachykinin NK receptors varies greatly among species. The aim of the present study was to assess the role of NK(1) and NK(2) receptors in mediating colorectal distension-evoked nociception and psychological stress-induced defecation in gerbils, a species with human-like NK receptor pharmacology. The effects of the selective NK(1) and NK(2) receptor antagonists, aprepitant and saredutant, on acute (1 h) restraint stress-evoked defecation and plasma adenocorticotropin (ACTH) levels in gerbils were assessed. The effects of antagonists alone or in combination on colorectal distension-evoked visceral pain in conscious gerbils were evaluated using the visceromotor response as a surrogate marker of pain. Restraint stress increased fecal pellet output 2-3-fold and plasma ACTH levels 9-fold. Aprepitant inhibited the defecatory and endocrine responses to stress by 50%, while saredutant completely normalized the same parameters. Visceral pain responses during colorectal distension were attenuated by both compounds, but aprepitant (19+/-6% inhibition, P<0.01) was slightly more effective than saredutant (10+/-9% inhibition, P<0.05). A combination of both compounds resulted in an additive effect (30+/-10% inhibition, P<0.01). The results demonstrate that NK(1) and NK(2) receptors are involved in stress-related colonic motor alterations and visceral pain responses in gerbils and that combined antagonism provides enhanced inhibition of visceral pain responses. This suggests that for therapeutic use in for instance functional gastrointestinal disorders, dual NK(1)/NK(2) receptor antagonists may provide better clinical outcome than selective compounds.