Role of tachykinin NK2 receptors in normal and altered rectal sensitivity in rats

Irritable bowel syndrome is characterized by visceral hyperalgesia commonly associated with stress and inflammatory processes. We investigated the role of tachykinin NK2 receptors in the ability of trinitrobenzenesulphonic acid (TNBS) and stress to enhance the sensitivity of the rat rectum to distension using a selective tachykinin NK2 receptor antagonist (MEN 11420). Rats were fitted with electrodes implanted in the striated muscles of the abdomen. Rectal distension (RD) was performed with a balloon inflated by steps of 0.4 ml from 0 to 1.6 ml. Five groups were submitted to RD performed 3 days before and after intrarectal instillation of TNBS. Fifteen minutes before RD, rats were treated with saline or MEN 11420 (5 - 100 microg kg(-1) i.v.). Two other groups, submitted to 2 h restraint or sham stress sessions were randomly treated i.v. with saline or MEN 11420 (10 - 200 microg kg(-1)) prior to RD applied 20 min later. The basal response to RD was characterized by a significant increase in the number of abdominal contractions. This response occurred with a threshold volume of 0.8 ml and was dose-dependently reduced by MEN 11420 (5 - 100 microg kg(-1) i.v.). Rectal inflammation lowered the volume of distension producing abdominal contractions to 0.4 ml (allodynia). This effect was either reduced or suppressed by MEN 11420. A similar allodynia was observed after a stress session and this effect was reduced (49%) or suppressed by MEN 11420 at 200 and 100 microg kg(-1), respectively. Tachykinin NK2 receptors are involved in rectal hypersensitivity associated with inflammation and stress. British Journal of Pharmacology (2000) 129, 193 - 199     

NK1 receptor-mediated mechanisms regulate colonic hypersensitivity in the guinea pig

Neurokinin-1 (NK(1)) receptors activated by substance P (SP) are involved in the processing of nociceptive information and are a potential target for therapy of visceral pain. We have evaluated the role of NK(1) receptors using a selective antagonist of NK(1) receptors in two animal models of colorectal hypersensitivity. The behavioral response to colorectal distension was assessed in freely moving guinea pigs by recording visceromotor reflex contractions of the abdominal musculature. Colonic hypersensitivity was induced by intracolonic administration of a chemical irritant (0.6% of acetic acid), or by acute partial restraint stress. Sensitization was characterized by an exaggerated visceromotor response to a low level of colorectal distension (10 mm Hg). In both models of colonic hypersensitivity, oral administration of TAK-637 (0.1-10 mg/kg) normalized visceromotor responses. The intracerebroventricular (10 microg/kg) or intrathecal (10 microg/kg) administration of TAK-637 inhibited colonic hypersensitivity, suggesting an interaction with central NK(1) receptors. In contrast, TAK-637 had no effect on visceromotor responses to colorectal distension at 40 mm Hg in guinea pigs with normosensitive (nonsensitized) colons. In conclusion, central NK(1) receptors play a significant role in colonic hypersensitivity induced by visceral afferent nerve sensitization from gastrointestinal origin or acute psychosomatic stress, but not in the perception of colorectal distension in animals with normosensitive colons.     

Involvement of endogenous tachykinins and CGRP in the motor responses produced by capsaicin in the guinea-pig common bile duct

In functional experiments, we have investigated the effect exerted by neurotransmitters released from capsaicin-sensitive primary afferent nerve terminals in the isolated guinea-pig common bile duct. In resting preparations, capsaicin (0.1 microM) produced a quick contraction (45.1+/-4% of KCl 80mM) which was abolished by either atropine (1 microM) or tetrodotoxin (0.5 microM). The tachykinin receptor-selective antagonists GR 82334 (NK1 receptor-selective; 3 microM), MEN 11420 (NK2 receptor-selective; 1 microM) and SR 142801 (NK3 receptor-selective; 0.1 microM) administered separately failed to reduce the capsaicin-evoked contraction, whereas any combination of the three antagonists was effective: GR 82334 plus MEN 11420, 36+/-7% reduction; GR 82334 plus SR 142801, 48+/-4% reduction; MEN 11420 plus SR 142801, 55+/-3% reduction; GR 82334 plus MEN 11420 plus SR 142801, 57+/-5% reduction. Neither the CGRP1 receptor antagonist h-CGRP (8-37) (1.5 microM) nor the P2X purinoceptor antagonist PPADS (50 microM) affected the contractile response to capsaicin. The effect of capsaicin (0.1 microM) was abolished by pretreatment with capsaicin itself (10 microM for 15 min). Human calcitonin gene-related peptide (h-CGRP; 0.1 microM) mimicked the effect of capsaicin on resting preparations (contractile response =28% of KCl 80 mM). In preparations precontracted with a submaximal concentration of KCl (24 mM), and in the presence of atropine (1 microM), GR 82334 (3 microM) and MEN 11420 (3 microM), capsaicin (1 microM) produced a tetrodotoxin-insensitive long-lasting relaxation (45+/-3% reduction of tone, at 4min from administration), which was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM). h-CGRP (10-50 nM) produced a similar sustained relaxation of precontracted preparations (59+/-4% reduction of tone). h-CGRP (8-37) (1.5 microM) almost completely reversed the relaxations produced by both capsaicin and h-CGRP. Application of electrical field stimulation (EFS: trains of stimuli of 10Hz; 0.25ms pulse width; supramaximal voltage; for 60s) to precontracted preparations produced a sustained, tetrodotoxin (1 microM)-sensitive relaxation (32+/-4% reduction of tone). L-NOARG (100 microM) greatly reduced (69+/-5% inhibition) the EFS-elicited relaxation. A complete reversal of the relaxant response to EFS into a contraction was obtained by administering L-NOARG to preparations in which a functional blockade of capsaicin-sensitive primary afferent neurons had been achieved by incubating the tissue with capsaicin (10 microM) for 15 min. At immunohistochemistry, tachykinin- and CGRP-immunoreactivities (TK-IR/CGRP-IR) were detected in varicose nerve fibers throughout the common bile duct, while TK-IR cell bodies were observed in the terminal portion (ampulla) only. In vivo pretreatment with capsaicin (50 mg/kg; 6-7 days before) decreased the number of CGRP-IR nerves, whereas the TK-IR neural network was apparently unchanged. In conclusion, our data provide functional evidence for the presence of capsaicin-sensitive primary afferent nerve endings in the guinea-pig terminal biliary tract, whose stimulation by capsaicin or EFS produces the release of tachykinins and CGRP. In addition, morphological evidence is provided that the bulk of TK-IR material in the biliary tract is contained in intrinsic neuronal elements, while CGRP in this tissue is of extrinsic origin only. Tachykinins, probably released in small amounts by capsaicin, act by activating receptors of the NK1, NK2 and NK3 type, most probably located on intrinsic cholinergic neurons, which in turn release ACh to produce the final excitatory motor response. The contractile response to capsaicin obtained in the presence of the three tachykinin receptor antagonists could be due to the co-released CGRP and/or to other unknown neurotransmitters. CGRP produces either indirect excitatory or direct inhibitory responses by stimulation of CGRP2 and CGRP1 receptors, respectively.     

MEN 11420, a potent and selective tachykinin NK2 receptor antagonist in the guinea-pig and human colon

We have characterized the action of the novel, water-soluble, tachykinin NK₂ receptor antagonist MEN 11420 ([Asn(2-AcNH-β-D-Glc)-Asp-Trp-Phe-Dap-Leu] c(2β–5β)) on the circular muscle of the guinea-pig and human colon in vitro and on the guinea-pig colon in vivo. In organ bath experiments on guinea-pig colon MEN 11420 produced a concentration-dependent rightward shift of the concentration-response curve to the NK₂ receptor selective agonist, [βAla⁸]neurokinin A (NKA) (4–10) with a pK_B value of 8.1. Up to 1 μM MEN 11420 had no effect on the concentration-response curve to methacholine, to the NK₁ receptor selective agonist, [Sar⁹]substance P (SP) sulfone, to the NK₃ receptor selective agonist, senktide, or on the response to exogenous SP. The response to exogenous NKA was inhibited, although the shift of the concentration-response curve to NKA produced by MEN 11420 at 1 μM (dose ratio 5.3) was much smaller than that produced against [βAla⁸]NKA (4–10) (dose ratio 102), presumably because NKA also stimulates NK₁ receptors at relatively low concentrations. In sucrose gap, MEN 11420 concentration-dependently inhibited both depolarization (IC₅₀ 0.34 μM) and contraction (IC₅₀ = 0.32 μM) produced by [βAla⁸]NKA (4–10) (0.3 μM for 10 s) in the guinea-pig colon without affecting the corresponding responses produced by [Sar⁹]SP sulfone. When similar experiments were performed in the circular muscle of the human colon MEN 11420 concentration-dependently inhibited both depolarization and contraction induced by [βAla⁸]NKA(4– 10) with IC_50s of 99 and 75 nM, respectively. MEN 11420 (1 μM) had no effect on the nonadrenergic noncholinergic (NANC) depolarization and contraction produced by a short period of electrical field stimulation (EFS, 10 Hz for 1 s) in the guinea-pig colon and selectively inhibited the sustained component of depolarization produced during a prolonged period of EFS (3 Hz for 3 min), without affecting the concomitant depolarization. Nifedipine (1 μM) eliminated the NANC contraction to a short period of EFS and the phasic contraction in response to a prolonged period of EFS. MEN 11420 (1 μM) abolished the nifedipine-resistant NANC contraction produced by prolonged period of electrical field stimulation (EFS, 3 Hz for 3 min). All electrical and mechanical NANC responses to EFS which were resistant to MEN 11420, either in the absence or presence of nifedipine, were abolished by the subsequent application of the NK₁ receptor antagonist, SR 140333 (1 μM). Up to 3 μM, MEN 11420 had no significant effect on the cholinergic excitatory junction potential or the NANC inhibitory junction potential evoked by single pulse EFS, nor did it affect membrane conductance. In urethane-anaesthetized guinea-pigs MEN 11420 (10– 100 nmol/kg i.v.) produced a dose-dependent and long lasting (> 3 h) inhibition of the contractile response (15 ± 2 mmHg) of the proximal colon induced by [βAla⁸]NKA (4–10) (3 nmol/kg i.v.). MEN 11420 (300 nmol/kg i.v.) did not affect the contraction produced by [Sar⁹]SP sulfone. MEN 11420 (300 nmol/kg) produced a limited (Emax about 40% inhibition) and transient (recovery within 60 min) inhibition of the atropine- and hexamethonium-sensitive phasic contractions of the proximal colon induced by threshold distension of a colonic balloon. On the other hand, MEN 11420 (10–300 nmol/kg i.v.) produced a dose-dependent complete and prolonged (> 2 h from administration) inhibition of the atropine-resistant and hexamethonium-sensitive phasic contraction induced by suprathreshold distension of the colonic balloon. We conclude that MEN 11420 is a potent and selective tachykinin NK₂ receptor antagonist devoid of significant inhibitory activity toward excitatory transmission mediated via tachykinin NK₁ or muscarinic receptors. The present findings indicate that SP and NKA are likely involved in the preferential activation of NK₁ and NK₂ receptors during tachykininergic transmission, although NKA can act as an effective NK₁ receptor ligand. Received: 24 March 1997 / Accepted: 9 June 1997     

A novel tachykinin NK2 receptor antagonist prevents motility-stimulating effects of neurokinin A in small intestine

1. MEN 11420 (nepadutant) is a potent, selective and competitive antagonist of tachykinin NK2 receptors. 2. The objective of the present study was to assess the capability of the drug to antagonize the stimulatory effects of neurokinin A (NKA) on gastrointestinal motility, as well as to change the fasting migrating motor complex (MMC). 3. Thirty-four male volunteers were randomized to treatment with either placebo or MEN 11420 in a double-blinded manner. Effects of MEN 11420 (8 mg intravenously) were evaluated as changes in phases I, II and III of MMC, as well as contraction frequency, amplitude and motility index during baseline conditions and during stimulation of motility using NKA (25 pmol kg(-1) min(-1) intravenously). 4. NKA preceded by placebo increased the fraction of time occupied by phase II, increased contraction frequency, amplitude and motility index. 5. MEN 11420 effectively antagonized the motility-stimulating effects of NKA. MEN 11420 reduced the phase II-stimulating effect of NKA. In addition, the stimulatory effect of NKA on contraction frequency and amplitude, as well as motility index were inhibited by MEN 11420. MEN 11420 did not affect the characteristics of MMC during saline infusion. 6. Plasma levels of MEN 11420 peaked during the first hour after infusion and decreased to less than half during the first 2 h. 7. In conclusion, intravenous MEN 11420 effectively inhibited NKA-stimulated, but not basal gastrointestinal motility, and was well tolerated by all subjects.     

Tachykinin NK2 receptors in the hamster urinary bladder: in vitro and in vivo characterization

We have characterized the contractile responses produced by stimulation of the tachykinin NK₂ receptor in the hamster urinary bladder in vitro and in vivo. In isolated bladder strips, neurokinin A (NKA, pD ₂ 7.40, E_max 71% of the response to 80 mM KCl) and the synthetic tachykinin NK₂ receptor selective agonist [βAla⁸]NKA(4–10) (pD ₂ 7.48, E_max 77% of the response to KCl) both induced a concentration-dependent contraction, whereas the tachykinin NK₁ and NK₃ receptor selective agonists, [Sar⁹]substance P sulfone and senktide, respectively, produced a negligible contractile effect. The bicyclic peptide antagonists MEN 11420 and MEN 10627 behaved as competitive antagonists of the response to [βAla⁸]NKA(4–10) with apparent pK _B values of 9.3 and 9.7, respectively. Comparable apparent pK _B values were estimated against NKA (pK _B 9.2 and 9.4 for MEN 11420 and MEN 10627, respectively). Under isovolumetric recording of the intravesical pressure, the nicotinic receptor agonist DMPP (0.6 μmol/kg i.v.) produced a phasic contraction of the hamster bladder in vivo that was abolished by hexamethonium (110 μmol/kg i.v.) or by surgical ablation of pelvic ganglia. In vivo [βAla⁸]NKA(4–10) (10 nmol/kg i.v.) induced a tonic-type sustained bladder contraction with superimposed high frequency and small amplitude (<12 mmHg) phasic contractions and, in about 70% of cases examined, a few high amplitude (>20 mmHg) phasic contractions. Hexamethonium abolished the high amplitude phasic contractions, indicating their reflex origin. In animals subjected to the ablation of pelvic ganglia, the urinary bladder response to [βAla⁸]NKA(4–10) was comparable to that observed after administration of hexamethonium. Moreover, hexamethonium did not affect the contractile responses to [βAla⁸]NKA(4–10) in ganglionectomized animals. MEN 10627 and MEN 11420 produced a dose-dependent and long-lasting inhibition of the contractile response to [βAla⁸]NKA(4–10): the least effective doses of the two antagonists were 30 and 3 nmol/kg i.v. for MEN 10627 and MEN 11420, respectively. An almost complete and long-lasting inhibition of the response to the agonist was produced at doses of 10 and 100 nmol/kg i.v. of MEN 11420 and MEN 10627. In urethane-anaesthetized hamsters the non-stop intravesical infusion of saline (50 μl/min) produced repetitive micturition cycles which were abolished by hexamethonium (110 μmol/kg i.v.) or by surgical removal of the pelvic ganglia. MEN 11420 (100 nmol/kg) had no significant effect on the volume-evoked micturition reflex in anaesthetized hamsters. In conclusion, the hamster urinary bladder is a suitable preparation for studying the action of tachykinin NK₂ receptor antagonists in vivo: in this species, the stimulation of tachykinin NK₂ receptors induces bladder contractions. Blockade of tachykinin NK₂ receptors does not appreciably modify the volume-evoked micturition reflex in this species. Received: 22 April 1998 / Accepted: 12 June 1998     

Characterization of the [125I]-neurokinin A binding site in the circular muscle of human colon.

Neurokinin A (NKA) is a potent contractile agonist of human colon circular muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [125I]-NKA has been used to characterize binding sites in this tissue, using tachykinin agonists and antagonists. 125INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [125I]-NKA was saturable (K(D) 0.47+/-0.05 nM), of high capacity (Bmax 2.1+/-0.1 fmol mg(-1) wet weight tissue) and reversible (kinetically derived K(D) 0.36+/-0.07 nM). The rank order of agonists competing for the [125I]-NKA binding site was neuropeptide gamma (NPgamma) > or = NKA > or = [Lys5, MeLeu9,Nle10]NKA (4-10) (NK2 agonist) > substance P (SP) > neurokinin B (NKB) > or = [Pro9]SP (NK1 agonist) > senktide (NK3 agonist), indicating binding to an NK2 site. The nonpeptide selective NK2 antagonist SR48968 showed higher affinity for the [125I]-NKA site than selective peptide NK2 antagonists. The rank order of potency for NK2 antagonists was SR48968 > or = MEN11420 > GR94800 > or = MEN10627 > MEN10376 > or = R396. The NK1 antagonist SR140333 was a weak competitor. The competition curve for SP could be resolved into two sites. When experiments were repeated in the presence of SR140333 (0.1 microM), the curve for SP became monophasic and showed a significant shift to the right, whereas curves to NKA and NKB were unaffected. In conclusion, binding of the radioligand [125I]-NKA to membranes from circular muscle is predominantly to the NK2 receptor. There may be a small component of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscle is unclear.     

Tachykinin agonists modulate cholinergic neurotransmission at guinea-pig intracardiac Ganglia

Effects of substance P (SP) and selective tachykinin agonists on neurotransmission at guinea-pig intracardiac ganglia were studied in vitro. Voltage responses of neurons to superfused tachykinins and nerve stimulation were measured using intracellular microelectrodes. Predominant effects of SP (1 microM) were to cause slow depolarization and enable synaptic transmission at low intensities of nerve stimulation. Augmented response to nerve stimulation occurred with 29 of 40 intracardiac neurons (approx. 73%). SP inhibited synaptic transmission at 23% of intracardiac neurons but also caused slow depolarization. Activation of NK(3) receptors with 100 nM [MePhe(7)]neurokinin B caused slow depolarization, enhanced the response of many intracardiac neurons to low intensity nerve stimulation or local application of acetylcholine, and triggered action potentials independent of other stimuli in 6 of 42 neurons. The NK(1) agonist [Sar(9),Met(O(2))(11)]SP had similar actions but was less effective and did not trigger action potentials independently. Neither selective agonist inhibited cholinergic neurotransmission. We conclude that SP can function as a positive or negative neuromodulator at intracardiac ganglion cells, which could be either efferent neurons or interneurons. Potentiation occurs primarily through NK(3) receptors and may enable neuronal responses with less preganglionic nerve activity. Inhibition of neurotransmission by SP is most likely explained by the known blocking action of this peptide at ganglionic nicotine receptors.     

MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist

The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2相似文献   

Intraplantar injection of anandamide inhibits mechanically-evoked responses of spinal neurones via activation of CB2 receptors in anaesthetised rats

Anti-nociceptive effects of the endocannabinoid anandamide are well established. Anandamide has, however, also been shown to activate pro-nociceptive vanilloid 1 (VR1) receptors present on primary afferent nociceptors. The aim of the present study was to determine the effect of intraplantar injection of anandamide on dorsal spinal neuronal responses in control rats and rats with hindpaw carrageenan-induced inflammation. Effects of intraplantar administration of anandamide (50 microg in 50 microl) on peripheral mechanically-evoked responses of spinal neurones were studied in halothane-anaesthetised rats in vivo. Responses of spinal neurones to mechanical punctate stimulation (von Frey filaments, 8-80 g) of the peripheral receptive field were similar in non-inflamed rats and rats with hindpaw carrageenan-induced inflammation. Intraplantar injection of anandamide, but not vehicle, significantly (P<0.05) inhibited innocuous and noxious mechanically-evoked responses of spinal neurones in rats with hindpaw inflammation, but not in non-inflamed rats. Co-administration of the cannabinoid (2) (CB(2)) receptor antagonist, SR144528 (10 microg in 50 microl), but not the cannabinoid (1) (CB(1)) receptor antagonist, SR141716A (10 microg in 50 microl), significantly blocked inhibitory effects of anandamide on peripheral evoked neuronal responses in rats with hindpaw inflammation. This study demonstrates inhibitory effects of exogenous anandamide on mechanically-evoked responses under inflammatory conditions in vivo, which are mediated by peripheral CB(2) receptors.     

In vivo effects of the NMDA receptor agonist tetrazolyl-glycine related to the function of small-diameter primary afferents

The NMDA receptor agonist tetrazolyl-glycine (TG; 100 μg kg^–1, i.v.) caused a depressor reflex in anaesthetized rats. The NMDA receptor antagonist MK-801 (300 μg kg^–1, i.v.) inhibited this depressor reflex, but not that induced by pentylenetetrazol (PTZ; 100 mg kg^–1, i.v.), indicating a selective effect of TG on NMDA receptors in vivo. Capsaicin pretreatment, which excludes the function of small-diameter primary afferent fibres, caused only a reduction of the TG-induced depressor reflex, suggesting a reduction of NMDA receptors. The absence of effects of TG and PTZ on the blood pressure in pithed rats excluded any peripheral vascular actions of TG and PTZ. The depressor reflex evoked by afferent nerve stimulation was also inhibited by MK-801 (300 μg kg^–1, i.v.), but not by the tachykinin antagonist L-742694 (10 mg kg^–1, i.v.), confirming the essential role of glutamate in the neurotransmission of signals at central terminals of small-diameter afferent neurons. Plasma protein extravasation in the rat hind paw, induced by neurotransmitters released at peripheral terminals of small diameter afferent neurons by antidromic nerve stimulation, was not influenced by MK-801, indicating that glutamate is either not released or has no effect there. It is concluded that the NMDA agonist TG is a valuable tool to study the functions of primary afferents in vivo. Received: 8 February 1999 / Accepted: 12 April 1999 / Published online: 21 June 1999     

Nerve injury alters the effects of interleukin-6 on nociceptive transmission in peripheral afferents

Interleukin-6 (IL-6) is markedly upregulated in the peripheral and central nervous systems following nerve injury; however, the functional effects of this are unclear. This study investigates the effect of peripheral interleukin-6 on nociceptive transmission in naive and neuropathic states. Using an in vitro rat skin-nerve preparation, 50 ng interleukin-6 inhibited responses of single nociceptive fibers to noxious heat. A 20-ng sample of interleukin-6 only inhibited heat responses in the presence of soluble interleukin-6 receptors. To examine in vivo effects of peripheral interleukin-6, extracellular recordings from dorsal horn neurons were made in anaesthetised naive, sham-operated and neuropathic (spinal nerve ligated) rats. Peripheral interleukin-6 (40-100 ng) markedly inhibited all naturally evoked neuronal responses in naive rats, yet only neuronal responses to heat in neuropathic rats. Behaviourally, intraplantar administration of interleukin-6 (0.01-1 microg) elicited ipsilateral thermal hypoalgesia in naive rats. Thus, interleukin-6 inhibits normal peripheral nociceptive transmission, yet such anti-nociceptive effects are attenuated following nerve injury in a modality-specific manner.     

The involvement of tachykinin NK2 and NK3 receptors in central sensitization of a spinal withdrawal reflex in the decerebrated, spinalized rabbit

Repetitive electrical stimulation of the sural nerve at a strength sufficient to excite C-fibres results in prolonged (> 20 min) increases in the reflex responses of gastrocnemius motoneurones evoked by stimulation of myelinated axons in the sural nerve. We have tested the susceptibility of this effect to blockade of tachykinin NK2 and NK1 receptors. In the control state, iterative stimulation of sural nerve C fibres increased gastrocnemius reflexes to a peak of between 200 and 400% of pre-stimulus levels, an effect that recovered to baseline values over 23-30 min. Pre-treatment with selective antagonists for NK2 (SR 48968, 1 mg/kg i.v.) or NK3 (SR 142801, 0.1 and 1 mg/kg i.v.) receptors failed to alter the peak facilitation resulting from sural nerve stimulation. However, both drugs significantly reduced the duration of enhancement of reflexes, to 18 min after SR 48968 and to 5 min after SR 142801. The inactive enantiomers of these compounds (SR 48965 and SR 142806, both 1 mg/kg i.v.) did not reduce facilitation of reflexes after sural nerve stimulation. We conclude that activation of tachykinin NK3 receptors is a major factor in maintaining central sensitization of the sural-gastrocnemius reflex after electrical stimulation of sural nerve C-fibres. The effects of SR 48968 were surprisingly weak and may have resulted from an effect of this compound at NK3 receptors.     

Effects of selective tachykinin receptor antagonists on capsaicin- and tachykinin-induced bronchospasm in anaesthetized guinea-pigs.

Bronchospasm induced by i.v. injection of equieffective doses of acetylcholine, capsaicin or selective tachykinin receptor agonists ([Sar9]SP sulfone or [beta-Ala8]neurokinin A (NKA-4-10)) (for NK1 and NK2 receptors, respectively) was studied in anaesthetized guinea-pigs. The NK1 and NK2 receptor antagonists, (+/-)-CP96,345 (3 mumol/kg i.v.) and MEN 10,376 (3 mumol/kg i.v.), selectively abolished the bronchoconstriction induced by the respective agonist, showing that both NK1 and NK2 receptors mediate bronchoconstriction in guinea-pig airways and that they are activated independently. Capsaicin-induced bronchospasm was inhibited by atropine (1.5 mumol/kg i.v.) and MEN 10,376 (3 mumol/kg i.v.), but unaffected by (+/-)-CP96,345 (3 mumol/kg i.v.). Hexamethonium (79 mumol/kg i.v.), propranolol (17 mumol/kg i.v.) and physostigmine (0.9 mumol/kg i.v.) enhanced the airway constriction induced by acetylcholine, capsaicin, [Sar9]SP sulfone or [beta-Ala8]NKA-(4-10) while guanethidine (67 mumol/kg s.c. for two days) increased only bronchoconstriction induced by capsaicin or the selective NK2 receptor agonist. In hexamethonium-treated animals, MEN 10,376 still abolished the increase in insufflation pressure induced by [beta-Ala8]NKA-(4-10) and reduced the increase elicited by capsaicin. In summary, in anaesthetized guinea pig i.v. capsaicin-induced bronchospasm through activation of postjunctional NK2 (but not NK1) receptors along with activation of cholinergic pathways. This motor response is moderated by the simultaneous stimulation of a sympathetic bronchodilating mechanism(s), possibly through activation of NK2 receptors localized in sympathetic ganglia.     

Effects of tachykinin receptor agonists and antagonists on the guinea-pig isolated oesophagus

1. Vagal nerve stimulation of the guinea-pig isolated oesophagus produced a triphasic tetrodotoxin (TTX)-sensitive contractile response. The third phase, which was resistant to ganglion blocking drugs, was selectively abolished by capsaicin, suggesting the involvement of one or more neuropeptides released from afferent neurons. Receptors on cholinergic neurons were subsequently activated because the response was atropine sensitive. Contractile responses resulting from exogenous substance P were abolished by atropine and TTX and enhanced by physostigmine. These findings suggest that the third phase may be mediated by the action of a substance P-like neuropeptide released from sensory nerve endings that subsequently activated cholinergic neurons. 2. The tachykinin receptors in the body of the guinea-pig oesophagus were characterized by determining the relative agonist potencies of natural tachykinins as well as tachykinin receptor-selective analogues. Antagonist affinities were also determined. The results indicated the presence of both NK2 and NK3 receptors. In addition, the effects of a cocktail of peptidase inhibitors (captopril, thiorphan and amastatin) on responses to various tachykinins and synthetic analogues were determined. The results indicate that one or more peptidases are present in this preparation. 3. Experiments using various tachykinin receptor antagonists were performed to determine whether the activation of tachykinin receptors played a role in the mediation of the third phase of the response to vagal nerve stimulation. While this response was unaffected by NK1 and NK2 receptor-selective antagonists, it was only partially inhibited (23%) by the NK3 receptor antagonist SR 142801. Thus, in the guinea-pig oesophagus, it appears that NK3 receptors play only a minor role in mediating a contractile response when afferent neurons are excited by vagal nerve stimulation.     

Oral administration of MA-2029, a novel selective and competitive motilin receptor antagonist, inhibits motilin-induced intestinal contractions and visceral pain in rabbits

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA2 value of 9.17+/-0.01 (n=5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 microM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The pKi of MA-2029 was 8.58+/-0.04 in the rabbit colon homogenate (n=4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 microg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 microg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.     

Tachykinin receptors mediating responses to sensory nerve stimulation and exogenous tachykinins and analogues in the rabbit isolated iris sphincter.

1. We have used selective tachykinin receptor agonists and antagonists to investigate the nature of the receptors mediating responses to endogenous and exogenous tachykinins in the rabbit iris sphincter preparation in vitro. 2. The NK1-selective agonist, substance P methyl ester, induced contraction with a pD2 of 9.16 indicating the presence of NK1 receptors. In confirmation, the NK1-selective antagonist, GR82334, competitively antagonized responses to substance P methyl ester with high affinity (pKB 7.46). 3. NK3 receptors also mediate contraction since NK3-selective agonists exhibited high potency, e.g. the pD2 of [Me-Phe7]-neurokinin B was 9.67, and their responses were not inhibited by GR82334 (10 microM). 4. NK2 receptor activation does not seem to contribute to contraction since the NK2-selective agonist [beta-Ala8]-neurokinin A(4-10) had relatively low potency (pD2 6.43), and the NK2-selective antagonists MEN10207 (1 microM) and L-659,877 (10 microM) were inactive or had low affinity, respectively. 5. GR82334 (1 microM) significantly inhibited responses to electrical field-stimulation of non-adrenergic non-cholinergic sensory nerves (3, 10 and 30 Hz), and caused a rightward shift of the log concentration-response curve to bradykinin (lateral shift ca. 1000 fold). Higher concentrations of GR82334 (10 microM) significantly attenuated responses to capsaicin (1-60 microM) whilst completely abolishing responses to field-stimulation (3, 10 and 30 Hz) and bradykinin (1 nM- 3 microM). 6. In conclusion, NK1 and NK3 receptor activation results in contraction of the rabbit iris sphincter. The contractile response following sensory nerve stimulation by bradykinin, capsaicin and electrical field stimulation results from NK1 receptor activation.     

Effects of CP-99, 994, a tachykinin NK(1) receptor antagonist, on abdominal afferent vagal activity in ferrets: evidence for involvement of NK(1) and 5-HT(3) receptors.

The effect of CP-99, 994, a tachykinin NK(1) receptor antagonist, on abdominal vagal afferent nerve activity in the ferret was investigated. Substance P (1 microg/kg, i.v.) increased vagal afferent activity by 449.0+/-51.9% and this was reduced to 145.9+/-5.7% (p<0.01) by pre-treatment with CP-99, 994 (1 mg/kg, i.v.), and to 149.5+/-1.5% (p<0.001) by granisetron (1 mg/kg, i.v.), a 5-HT(3) receptor antagonist. In addition, the increase in vagal nerve activity induced by 5-HT (25 microg/kg, i.v., 552.0+/-57.0% increase from pre-injection level) was significantly reduced (401.3+/-10.6% increase from pre-injection level, p<0.05) by CP-99, 994 (100 microg/kg, i.v.). These results provide evidence for an involvement of peripheral NK(1) and 5-HT(3) receptors in substance P-induced vagal afferent activation. While the functional consequences (if any) of such peripheral effects were not investigated, they could contribute either directly (e.g. by blockade of receptors on vagal afferents) or indirectly (e.g. modulation of 5-HT release or reduction of local inflammatory response) to the antiemetic effects of CP-99, 994 against cisplatin and other emetic agents acting primarily via the vagus.     

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.     

Role of opioid receptors in neurogenic dural vasodilation and sensitization of trigeminal neurones in anaesthetized rats.

Migraine headache is thought to be caused by a distension of meningeal blood vessels, the activation of trigeminal sensory neurones and the the development of a central sensitization within the trigeminal nucleus caudalis (TNC). It has been proposed that clinically effective 5-HT(1B/1D) agonists act peripherally to inhibit the release of calcitonin gene-related peptide (CGRP) and neurogenic dural vasodilation, and to attenuate nociceptive neurotransmission within the TNC. Since opioids are also effective anti-migraine agents the present studies investigated the role of opioids within the trigemino-vascular system in anaesthetised rats. Electrical stimulation of the dura mater evoked neurogenic dural vasodilation which was significantly inhibited by morphine (1 mg kg(-1)) the selective mu-opioid agonist DAGO (10 microg kg(-1)) and the mixed agonist/antagonist butorphanol (1 mg kg(-1)) but not by the kappa- and delta-opioid agonists (+/-) U50488H (100 microg kg(-1)) and DPDPE (1 mg kg(-1)). Morphine had no effect on CGRP-evoked dural vasodilation. In electrophysiological studies morphine (1 - 10 mg kg(-1)) significantly attenuated brainstem neuronal activity in response to electrical stimulation of the dura by 65% at 10 mg kg(-1). Morphine (3 mg kg(-1)) also inhibited the TNC neuronal sensitization following CGRP-evoked dilation. The present studies have demonstrated that opioids block the nociceptive neurotransmission within the trigeminal nucleus caudalis and in addition inhibit neurogenic dural vasodilation via an action on mu-opioid receptors located on trigeminal sensory fibres innervating dural blood vessels. These peripheral and central actions are similar to those of the 'triptan' 5-HT(1B/1D) agonists and could account for the anti-migraine actions of opioids.