NKA-induced Contraction Research Articles

Role of tachykinin and neurokinin receptors in the regulation of ovine omasal contractions

The present study investigated a role of tachykinins (TK) and neurokinin (NK) receptors (NK-R) in the non-cholinergic regulation of omasal contractions in sheep. Semiquantitative reverse transcription (RT)-PCR revealed that both preprotachykinin (PPT)-A and PPT-B mRNA were distributed in the omasal muscle layers and that NK-R type-1 (NK-1R) and type-2 (NK-2R) mRNA were largely expressed in the same tissues. Cumulative application of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) at 0.03–10μM induced tonic contractions of omasal muscle strips, and the contractile amplitude increased in order of NKB<SP≪NKA in longitudinal and circular muscle strips. Although cumulative application of NK-1R antagonist, L-732,138, at 1–100μM did not significantly inhibit SP- and NKA-induced contractions in both muscle layers, NK-2R antagonist GR159897 at 30–100μM significantly inhibited NKA-induced contraction of longitudinal muscles and showed tendency to inhibit that in circular muscles. Electric field stimulation (EFS)-induced contractions of omasal muscle strips, which were atropine-resistant, were significantly inhibited by GR159897 at 30 and 100μM in both muscle strips, and L-732,138 at 30 and 100μM in longitudinal muscles, though the inhibition in the latter was very weak. The results of the present study suggest that, though all mRNA coding TKs and NK-R subtypes were expressed in omasal muscle layer, NK-2R in both muscle layers and, to much lesser extent, NK-1R in longitudinal muscles are involved in the regulation of omasal contraction, and that NKA is presumably a primary non-cholinergic excitatory neurotransmitter released from motor neurons in the ovine omasum.

ROLE OF PLATELET ACTIVATING FACTOR INDUCED H2o2 PRODUCTION IN MOTOR DYSFUNCTION IN ULCERATIVE COLITIS

We have previously shown that sigmoid circular muscle cells from UC patients exhibit reduced contraction and Ca2+ signaling in response to the endogenous neurotransmitter neurokinin A (NKA) and that H2O2 is elevated in the UC circular muscle layer. The H2O2 scavenger catalase restored the decreased Ca2+ signal and cell shortening to normal levels, suggesting that H2O2 contributes to the motor dysfunction of UC. It is known that PAF levels are elevated in colonic mucosa from patients with ulcerative colitis. In the present study we examined the role of PAF in UC in causing H2O2 production and subsequent motor dysfunction. PAF levels were significantly elevated in sigmoid circular muscle from patients with ulcerative colitis, when compared to normal controls. Treatment of normal sigmoid circular smooth muscle cells with PAF (10-6M, 2 hours) significantly decreased NKA-induced contraction. Treatment of UC sigmoid muscle strips with the PAF antagonist CV3988 (10-6M) for 2 hours, however, did not improve the motor dysfunction, suggesting that other inflammatory mediators e.g. H2O2 and IL-1b may be present and affect muscle contraction, even after blockade of PAF. PAF 10-8M significantly increased hydrogen peroxide (H2O2) production in sigmoid smooth muscle cells in primary culture, and H2O2 production was abolished by the NADPH oxidase inhibitor apocynin, suggesting that NADPH oxidases mediate PAF-induced production of H2O2. PAF-induced H2O2 was significantly reduced by the MEK1 inhibitor PD98059, the P38 MAP kinase inhibitor SB203580 and the cytosolic phopholipase A2 inhibitor AACOCF3, suggesting that PAF-induced H2O2 production depends on activation of MAP kinases (ERK1/2, p38) and cytosolic phospholipase A2. We conclude that PAF may contribute to motor dysfunction of sigmoid colon in UC and that PAF-induced H2O2 production depends on activation of MAP kinases (ERK1/2, p38), cytosolic phospholipase A2 and NADPH oxidases. Supported by NIDDK R21 DK62775-01.

Ontogeny of excitatory and inhibitory control of gastrointestinal motility in the African clawed frog,Xenopus laevis

The transparent body wall of Xenopus laevis larvae during the first developmental stages allows in vivo studies of gastrointestinal tract activity. The purpose of this study was to chart the ontogeny of gut motility in Xenopus larvae and to identify the most important control systems during the first developmental stages. Coordinated descending contraction waves first occurred in the gut at Nieuwkoop and Faber stage 43 [0.8 +/- 0.1 contractions/min (cpm)] and increased to 4.9 +/- 0.1 cpm at stage 47. The cholinergic receptor agonist carbachol (5-10 microM) increased contraction frequency already at stage 43, as did neurokinin A (NKA, 0.3-1 microM). The muscarinic antagonist atropine (100 microM) first affected contraction frequency at stage 45, which coincides with the onset of feeding. The tachykinin antagonist MEN-10,376 (6 microM) blocked NKA-induced contractions but not spontaneous motility. Both sodium nitroprusside [nitric oxide (NO) donor, 1-10 microM] and vasoactive intestinal peptide (VIP, 0.1-1 microM) inhibited contractions from the earliest stage onward. Blocking NO synthesis using NG-nitro-L-arginine methyl ester (100 microM) had no effect per se, but antagonized VIP evoked inhibition at stage 47. We conclude that gastrointestinal motility is well developed in the Xenopus laevis larvae before the onset of feeding. Functional muscarinic and tachykinin receptors are present already at the onset of motility, whereas a cholinergic tone develops around the onset of feeding. No endogenous tachykinin tone was found. Functional VIP receptors mediate inhibition at the onset of motility. NO seems to mediate the VIP effect at later stages.

Depressed Contractile Responses to Neurokinin A in Idiopathic but not Neurogenic Overactive Human Detrusor Muscle

ObjectiveThe role of tachykinins such as neurokinin A in regulating bladder function is unclear, but NK2 receptors seem to mediate contraction in the human bladder and it has been suggested that these peptides may have a role in the pathophysiology of bladder dysfunction. The present study investigates neurokinin receptor-mediated contractility of detrusor muscle in the idiopathic overactive and neurogenic overactive bladder and investigates the neurokinin receptor subtypes involved. MethodsHuman bladder was obtained from patients undergoing cystectomy (normal) or clam cystoplasty (idiopathic overactive) and from patients with spinal injuries (neurogenic overactive). Strips of isolated detrusor muscle were mounted in physiological Krebs-bicarbonate solution and cumulative concentration-response curves to 1nM to 300μM neurokinin A (NKA) were obtained in the absence and presence of neurokinin receptor antagonists, either the NK2 receptor-selective antagonist SR 48968 or the NK3 receptor-selective antagonist SB 223412. ResultsNKA evoked concentration-dependent contraction of normal, idiopathic, and neurogenic overactive detrusor strips. In idiopathic overactive detrusor muscle, NKA-induced contraction was significantly reduced relative to normal detrusor (0.031±0.005mg/g, n=11 versus 0.193±0.039mg/g, n=7). Sensitivity to the peptide was also significantly (p<0.01) reduced in idiopathic overactive detrusor, with mean pEC50 values (concentration producing 50% maximal response) of 6.62±0.16 (n=11) compared to 7.47±0.19 (n=7) in normal detrusor. In contrast, NKA-induced responses of neurogenic overactive detrusor were similar to those in normal detrusor, with a mean maximum contraction of 0.199±0.036mg/g (n=10) and mean pEC50 value of 7.85±0.16 (n=10). NKA curves in all groups were shifted to the right by the NK2 receptor-selective antagonist SR 48968 with high affinity, pKB values being similar in normal, idiopathic, and neurogenic overactive detrusor (8.85+0.08, n=14; 8.97±0.13, n=12; 8.73±0.12, n=8, respectively). In contrast the NK3 receptor-selective antagonist SB 223412 had a minimal effect on NKA responses and affinity values were low (pKB 5.81±0.11, n=12 in normal; 5.75±0.08, n=12 in idiopathic overactive, and 5.77±0.13, n=11 in neurogenic overactive). ConclusionThese data indicate that NKA-induced responses are impaired in detrusor muscle from idiopathic overactive human bladder, but not in detrusor muscle from neurogenic overactive bladder. The NK2 receptor subtype appears to mediate NKA responses in the normal, idiopathic overactive, and neurogenic overactive detrusor. This is important evidence suggesting a difference between the bladder pathophysiology observed in idiopathic versus neurogenic overactive detrusor.

Hydrogen peroxide contributes to motor dysfunction in ulcerative colitis

Ulcerative colitis (UC) affects colonic motor function, but the mechanism responsible for this motor dysfunction is not well understood. We have shown that neurokinin A (NKA) may be an endogenous neurotransmitter mediating contraction of human sigmoid colonic circular muscle (HSCCM). To elucidate factors responsible for UC motor dysfunction, we examined the role of hydrogen peroxide (H(2)O(2)) in the decrease of NKA-induced response of HSCCM. As previously demonstrated, NKA-induced contraction or Ca(2+) increase of normal muscle cells is mediated by release of Ca(2+) from intracellular stores, because it was not affected by incubation in Ca(2+)-free medium (CFM) containing 200 microM BAPTA. In UC, however, CFM reduced both cell contraction and NKA-induced Ca(2+) increase, suggesting reduced Ca(2+) release from intracellular stores. In normal Ca(2+) medium, NKA and KCl caused normal Ca(2+) signal in UC cells but reduced cell shortening. The decreased Ca(2+) signal and contraction in response to NKA or thapsigargin were partly recovered in the presence of H(2)O(2) scavenger catalase, suggesting involvement of H(2)O(2) in UC-induced dysmotility. H(2)O(2) levels were higher in UC than in normal HSCCM, and enzymatically isolated UC muscle cells contained much higher levels of H(2)O(2) than normal cells, which were significantly reduced by catalase. H(2)O(2) treatment of normal cells in CFM reproduced the reduction of NKA-induced Ca(2+) release observed in UC cells. In addition, H(2)O(2) caused a measurable, direct release of Ca(2+) from intracellular stores. We conclude that H(2)O(2) may contribute to reduction of NKA-induced Ca(2+) release from intracellular Ca(2+) stores in UC and contribute to the observed colonic motor dysfunction.

Investigation of neurokinin-2 and -3 receptors in the human and pig bladder.

To investigate the role of neurokinin (NK)-2 and -3 receptors in mediating the contraction of detrusor muscle strips from human and pig, to determine whether the pig is a good model for the study of tachykinin receptors in the human bladder, as the biological actions of tachykinins, e.g. substance P and NKA are mediated via three distinct receptor subtypes, NK-1, -2 and -3. Strips of detrusor muscle were obtained from the bladder dome and neck of female pigs and from patients undergoing cystectomy. Cumulative concentration-response curves to NKA were obtained in the absence and presence of either the NK-2 receptor-selective antagonist SR48968 or the NK-3 receptor-selective antagonist SB223412. NKA produced concentration-dependent contractions in the human and pig detrusor muscle; the curves were shifted to the right by SR48968, with high affinity (pKB 8.9, 8.3 and 8.0 in the human, pig dome and pig neck, respectively), whereas SB223412 had a minimal effect (pKB 5.8, 5.8 and 6.3, respectively). These data confirm that the NK-2 receptor subtype mediates NKA-induced contraction of the human and pig detrusor muscle. The NK-3 receptor appears to have no role in detrusor contraction of either species. The results also provide evidence that the NK-2 receptor in human and pig are the same, and the latter may be an appropriate species to study tachykinin-induced contractions in human bladder.

Interleukin-1β (IL-1β) induced H2O2 production contributes to the motor dysfunction in human ulcerative colitis (UC)

We have shown that neurokmin-A (NKA) is an important excitatory neumtransmitter in normal human sig~mid colonic circular muscle and that NKA-induced contraction is mediated by release of calcium from intracelhdar stores. We have also shm~m that NKA induced contraction of sigmoid circular muscle and its intracellular Ca 2+ stores are reduced in ulcerative colitis and that H2Oa may contribute to the obsep,'ed changes in the response to NKA. In this study we examined the role of 1L-lf~ in the motor dysfunction in UC and in H202 production. IL-I~ content was significantly higher(ll.2+_4.7pgkng protein) m the circular muscle layer of UC patients than in nom~al citvular muscle (0.7 _+ O. lpg/mg protein). In circular muscle cells isolated by enzymatic digestion exposure to IL-I~ (100tdml, 23 h) significantly reduced NKA and thapsigargin-induced contraction. Because NKA and thapsigargin cause release of Ca ~ + from intracellular stores, the data suggest that 1L1 ~ may reduce intracellular Ca > stores. Tbe reduced contractions were partially reversed by the H20~ scavenger catalase (87u/ml, lhr), suggesnng that the motor dyshmction ts partially mediated by production of H~O> in fura 2 loaded cells, 20 mM caffeine, which selectively releases Ca ~ + from intracellular stores, caused a 630.+_ 62 nM increase in intracellular Ca ~ + in normal cells and 324+ 71riM in UC cells, confirming that intracellular Ca > stores are reduced in UC. 1L-II3 treatment similarly reduced the caffeine-induced Ca > signal (432 _+ 69 riM) and this reduction was partially restored by catalase (520+-78 riM), confirming that IL-l[3 reduces intraceflular Ca > stores by producing H~O_,. H20~ levels in circular smooth muscle of UC patients were higher (026 +-O.06nmd/mg protein) than in normal sigmoid (0.05-+ 0,01nmol/ng protein). In normal sigmoid circular muscle 1L-113 treatment (200u/ ml, 2hr) significantly increased the production of HaO~ (from O. 15 +0.02 to 0.3 -+ 0.05nmol/ mg protein). The increase was signihcantly mhihited by the MEK inhibitor PD98059 and by the cyIosohc phosphofipase A~ (cPLa,~) inhibitor AACOCF3, but not by' the p38 MAP kin_ase inhibitor SB203580 or by the NF-KB inhibitor D'rrolidine dithiocarbamate, suggesting that lL-I~-induced production of H~O~ depends on activation of ERK1/ERK2 MAP kinase and cPLA> We conclude that IL-l[3 can mimic the changes observed ni ulceratwe colitis and may contribute to the motor dysfunction in UC by producing H~O~. Supported by Litespan Research Funds.

SCH 206272: a potent, orally active tachykinin NK 1, NK 2, and NK 3 receptor antagonist

Experiments were performed to characterize the pharmacology of SCH 206272 [( R, R)-1′[5-[(3,5-dichlorobenzoyl)methylamino]-3-(3,4-dichlorophenyl)-4( Z)-(methoxyimino)pentyl]- N-methyl-2-oxo-[1,4′bipiperidine]-3-acetamide] as a potent and selective antagonist of tachykinin (NK) NK 1, NK 2, and NK 3 receptors. SCH 206272 inhibited binding at human tachykinin NK 1, NK 2, and NK 3 receptors ( K i =1.3, 0.4, and 0.3 nM, respectively) and antagonized [Ca 2+] i mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human tachykinin NK 1, NK 2, or NK 3 receptors. SCH 206272 inhibited relaxation of the human pulmonary artery (p K b=7.7±0.3) induced by the tachykinin NK 1 receptor agonist, [Met-O-Me] substance P and contraction of the human bronchus (p K b=8.2±0.3) induced by the tachykinin NK 2 receptor agonist, neurokinin A. In isolated guinea pig tissues, SCH 206272 inhibited substance P-induced enhancement of electrical field stimulated contractions of the vas deferens, (p K b=7.6±0.2), NKA-induced contraction of the bronchus (p K b=7.7±0.2), and senktide-induced contraction of the ileum. In vivo, oral SCH 206272 (0.1–10 mg/kg, p.o.) inhibited substance P-induced airway microvascular leakage and neurokinin A-induced bronchospasm in the guinea pig. In a canine in vivo model, SCH 206272 (0.1–3 mg/kg, p.o.) inhibited NK 1 and NK 2 activities induced by exogenous substance P and neurokinin A. Furthermore, in guinea pig models involving endogenously released tachykinins, SCH 206272 inhibited hyperventilation-induced bronchospasm, capsaicin-induced cough, and airway microvascular leakage induced by nebulized hypertonic saline. These data demonstrate that SCH 206272 is a potent, orally active tachykinin NK 1, NK 2, and NK 3 receptor antagonist. This compound may have beneficial effects in diseases thought to be mediated by tachykinins, such as cough, asthma, and chronic obstructive pulmonary disease.

Gq-linked NK2 receptors mediate neurally induced contraction of human sigmoid circular smooth muscle

Because tachykinins have been identified as neurotransmitters in the guinea pig colon and human ileum, we examined a possible role of tachykinin receptors and neurokinin (NK) A in neurally induced contraction of human sigmoid colon circular muscle. Muscle strips were stimulated electrically for 10 seconds. Single cells were isolated by enzymatic digestion and permeabilized by saponin. [(35)S]GTPgammaS binding was assayed with or without NKA for 5 minutes. Intracellular Ca(2+) was measured using Fura 2. In the presence of 100 micromol/L L-NNA, 100 micromol/L atropine did not affect electrical field stimulation (EFS)-induced contraction. A peptide NK(2)-receptor antagonist (NK-2ra) but not an NK(1) antagonist FK888 (1 micromol/L) eliminated EFS-induced contraction. NKA-induced contraction in muscle strips and single cells was virtually abolished by NK-2ra, but not by FK888. In permeabilized cells, contraction was blocked by Gq-protein antibodies, but not by other G-protein antibodies, suggesting that NKA activates Gq, which was confirmed by a [(35)S]GTPgammaS binding assay. NKA-induced contraction and increase in cytosolic Ca(2+) were abolished by depletion of intracellular Ca(2+) stores. Tachykinins may be the main excitatory neurotransmitters in human sigmoid circular muscle. NKA activates Gq-linked NK(2) receptors, which cause Ca(2+) release, followed by contraction.

ACTIVITY OF NONPEPTIDE TACHYKININ ANTAGONISTS ON NEUROKININ A INDUCED CONTRACTIONS IN DOG URINARY BLADDER

There is abundant evidence indicating that tachykinin (TK) containing sensory nerves (C-fibers) play an important role in neural regulation of reflex micturition in mammals. Desensitization of urinary bladder C-fibers with resiniferatoxin ameliorates bladder hyperreflexia and incontinence and supports a similar role for C-fibers in man. TK-induced contraction of isolated human urinary bladder smooth muscle appears to be exclusively neurokinin NK2-receptor mediated. Dog urinary bladder contractility to a series of TK-receptor agonists also suggests a predominance of NK2-receptor activation by tachykinins and indicates the dog may provide a useful model for the development of pharmacotherapy for bladder hyperreflexia. We evaluated the activity of the nonpeptide NK-receptor selective antagonists SR 48968 (NK2), SB 223412 (NK3) and CP 99994 (NK1) against neurokinin A (NKA)-induced contractions in isolated dog urinary bladder strips to determine the NK-receptor type which mediates contractile responses to NKA. The dual NK1 and NK2 antagonist MDL 103392 was also tested in this preparation. NKA-induced contractions (pD2 = 8.0) were dose dependently blocked by SR 48968 (pA2 = 8.2 +/- 0.2) while CP 99994 (1.0 microM) and SB 223412 (1.0 microM) were inactive. MDL 103392 was a weak functional antagonist (pKb = 6.1 +/- 0.1) of NKA. Relative activities of SR 48968, CP 99994 and SB 223412 confirm that the NK2-receptor is the mediator of NKA-induced contractions of dog urinary bladder smooth muscle.

Modulation by 5-HT1A receptors of the 5-HT2 receptor-mediated tachykinin-induced contraction of the rat trachea in vitro.

1. In the Fisher 344 rat, tachykinins have been shown to cause the release of 5-hydroxytryptamine (5-HT) from airway mast cells, which then causes direct smooth muscle activation as well as the release of acetylcholine from cholinergic nerves. The aim of the present study was to examine the modulatory effects of 5-HT receptors on the neurokinin A (NKA)-induced release of endogenous 5-HT and airway smooth muscle contraction in the isolated Fisher 344 rat trachea. 2. The selective 5-HT2 receptor antagonist ketanserin (0.1 microM) produced an almost complete inhibition of the contractions caused by NKA (n=4, P<0.0001, two-way ANOVA), and a significant rightward shift of the concentration-response curve to 5-HT (n=8, P<0.001, two-way ANOVA). 3. The partial agonist for 5-HT1A receptors, 8-OH-DPAT (1 microM), and the full agonist for 5-HT1 receptors, 5-CT (0.3 microM), potentiated the submaximal contractions induced by the 5-HT2 receptor agonist alpha-methyl-5-HT (0.1 microM) (n=4; P<0.005 and P<0.05, respectively). 8-OH-DPAT (1 microM), as well as the 5-HT1A receptor antagonists pMPPI, SDZ 216525 and NAN-190 (0.1 microM each), caused significant inhibition of the tracheal contractions induced both by NKA (10 nM-3 microM) and 5-HT (10 nM-10 microM) (n=4-10). This suggests that activation of 5-HT1A receptors potentiates the 5-HT2 receptor-mediated contractions. 4. SDZ 216525 (0.1 microM) significantly reduced the maximal contraction produced by 1 microM NKA (n=10, P< 0.001), without affecting the release of endogenous 5-HT. These data rule out the involvement of a 5-HT1A receptor-mediated positive feedback mechanism of the 5-HT release from mast cells. 5. Even in the presence of atropine (1 microM), 8-OH-DPAT (1 microM) further reduced the maximal NKA-induced contraction (n=4, P<0.0001), while the contractions of the rat isolated trachea induced by electrical field stimulation and the concentration-response curve to carbachol were unaffected by pMPPI (0.1 microM), SDZ 216525 (0.1 microM), NAN-190 (0.1 microM) and 8-OH-DPAT (1 microM) (n=4-6). These data demonstrate that the 5-HT1A receptor-mediated potentiation of contractile responses is not due to nonspecific inhibition of airway smooth muscle contraction or to modulation of postganglionic nerve activation. 6. The selective 5-HT1B/1D receptor antagonist GR 127935, the selective 5-HT3 receptor antagonist tropisetron and the selective 5-HT4 receptor antagonists SB 204070 and GR 113808 (0.1 microM each) had no effect on the concentration-response curve for NKA (n=6-10), ruling out the involvement of 5-HT1B/1D, 5-HT3 and 5-HT4 receptors. 7. The alpha-adrenoreceptor antagonist phentolamine (1 microM) had no effect on the 5-HT-induced contractions (n=4), ruling out the involvement of alpha-adrenoreceptors. 8. In conclusion, the tachykinin-induced contraction of the F334 rat isolated trachea is mediated by the stimulation of 5-HT2 receptors. Activation of 5-HT1A receptors located on airway smooth muscle potentiates the direct contractile effects of 5-HT2 receptor activation. The 5-HT1B/1D, 5-HT3 and 5-HT4 receptors are not involved in the NKA-induced contraction of rat airways.

Tachykinin antagonist FK224 inhibits neurokinin A-, substance P- and capsaicin-induced human bronchial contraction.

To determine the roles of endogenously released tachykinins (substance P [SP] and neurokinin A [NKA]) in the human bronchial tissues, we studied the effects of tachykinin antagonist FK224 on bronchial smooth muscle contraction induced by SP, NKA and capsaicin in an organ bath. FK224 (10(-6) M and 10(-5) M, respectively) significantly inhibited NKA-induced contraction and 10(-5) M FK224 shifted the dose-response curve to more than one log unit higher concentration. Because SP- and capsaicin-induced contractions were small, we pretreated the tissues with the neutral endopeptidase inhibitor phosphoramidon (10(-5) M), which inhibits degradation of exogenous tachykinins in order to potentiate the contractions. FK224 (10(-5) M) significantly inhibited SP-induced contraction and it shifted the dose-response curves to about one log unit higher concentration. FK224 (10(-5) M) also significantly inhibited capsaicin-induced contraction and it shifted the dose-response curves to more than one log unit higher concentration. In contrast, FK224 (10(-5) M) did not affect on acetylcholine-, histamine-, and leukotriene D4-induced contraction. These results suggest that FK224 is a tachykinin receptor antagonist in the human bronchial smooth muscle, and that capsaicin-induced contraction is due to endogenously released tachykinin-like substances in the human bronchus.

Ketamine and its isomers have equipotent relaxant effects on tracheal smooth muscle contracted by tachykinins

Recent studies indicate that not only inflammatory cells but also neural mechanisms by which tachykinins such as substance P (SP) and neurokinin A (NKA) are released from vagal afferent C-fiber contribute to asthma. Although ketamine (K) has been used in the anesthetic management of asthmatic patients, the mechanism by which K relaxes the airway smooth muscle is still uncertain, and no information exists on any differential effect of K and its isomers. We determined the spasmolytic effect of racemic [R(±)]K and its isomers S(+) K and R(-) K on SP and NKA-induced contraction of tracheal smooth muscle in guinea pigs. Strips of guinea pig trachea were mounted in an organ bath filled with Tyrode's solution at 37°C bubbled with 95% O2/5% CO2. Strip tension was measured isometrically with a force displacement transducer. Strip contraction was elicited with SP 10(-6) M or NKA 5×10(-7) M.R(±), R(-), or S(+) K (4.5-18.0×10(-4)M) was cumulatively administered into the bath. The calculated ED50 values (the concentration that relaxed the contraction by 50%) of R(±), R(-) and S(+) K were 7.6±0.5, 7.8±0.6, and 7.6±0.5 (10(-4)M), respectively, when the contraction was elicited with SP, and 8.0±1.0, 8.2±1.2, and 7.9±1.3 (10(-4)M), respectively, when NKA was used. We concluded that K and its isomers have equipotent spasmolytic effects on airway smooth muscle precontracted with tachykinins.

'Sensory-efferent' neural control of mucus secretion: characterization using tachykinin receptor antagonists in ferret trachea in vitro.

1. We characterized the tachykinin receptor(s) mediating 'sensory-efferent' neural control of release of 35SO4-labelled macromolecules (mucus) from ferret trachea in vitro in Ussing chambers using selective tachykinin antagonists. Secretion was induced by substance P (SP), neurokinin A (NKA), capsaicin, the NK1 tachykinin receptor agonist [Sar9, Met(O2)11]substance P ([Sar9]SP), or acetylcholine (ACh), or by electrical stimulation of nerves. Antagonists used were FK888 and L-668,169, selective for the NK1 receptor, SR 48968, selective for the NK2 receptor, and FK224, a dual antagonist at NK1 and NK2 receptors. The selectivity of FK888 and SR 48968 was examined on NKA-induced contraction of ferret tracheal smooth muscle in vitro. 2. SP (1 microM) increased mucus secretion by 695% above vehicle controls. FK888 (0.1 microM-30 microM) inhibited SP-induced secretion in a dose-dependent manner, with complete inhibition at 10 microM and an IC50 of 1 microM. L-668,169 (1 microM) also completely inhibited SP-induced secretion. 3. NKA (1 microM) significantly increased mucus secretion by 271% above baseline, a response which was completely inhibited by FK888 (10 microM) or L-668,169 (microM). Secretion induced by ACh (10 microM: 317% above baseline) was not inhibited by FK888 but was inhibited by atropine. Capsaicin (10 microM)-induced secretion (456% above vehicle controls) was significantly inhibited by FK888 and by L-668,169 (111% and 103% inhibition respectively). 4. Electrical stimulation (50 V, 10 Hz, 0.5 ms, 5 min) increased mucus output above baseline (increased by 12 to 26 fold), a response blocked by tetrodotoxin (0.1 microM). FK888 (10 microM) inhibited the increase in secretion due to electrical stimulation by 47%. Atropine, propranolol and phentolamine in combination(APP) inhibited the response to electrical stimulation by 48%. The remaining NANC response, i.e. in the presence of APP, was further reduced by 66% with FK888. FK224 (10 microM) inhibited neurally evoked secretion by 73%. SR 48968 (0.1 fLM) had no effect on electrically-stimulated or [Sar9]SP-induced secretion.5. NKA (10nM- 1O microM: in the presence of DMSO control vehicle) induced tracheal smooth muscle contraction in a concentration-dependent manner with a maximal contraction of 30% of the maximal response to ACh (10 mM) and an ECm of 0.3 JAM. SR 48968 (0.1 microM in DMSO) inhibited the NKA induced contraction whereas FK888 did not. Neither antagonist had any inhibitory effect on ACh induced contraction.6. We conclude that 'sensory-efferent' neurogenic mucus secretion in ferret trachea in vitro is mediated via tachykinin NK, receptors with no involvement of NK2 receptors. Potent and selective tachykinin antagonists may have therapeutic potential in bronchial diseases such as asthma and chronic bronchitis in which neurogenic mucus hypersecretion may be aetiologically important.

Neurokinin A-induced contraction of guinea-pig isolated trachea: potentiation by hepoxilins.

1. Hepoxilin A3 (8R and 8S isomers) (HxA3), hepoxilin A3-C (8R and 8S isomers) (HxA3-C) and trioxilin A3 (8S isomer) (TrXA3, the stable derivative of HxA3) were tested for their effects on helicoidal strips of guinea-pig isolated tracheae. 2. None of the compounds (10(-9)-10(-6) M) tested had a direct effect on resting tension of trachea. 3. HxA3 (8S) and HxA3-C (8R) (10(-8) M) produced a significant leftward shift of the log concentration-response curves to neurokinin A (NKA) (EC50 (nM), control = 29.0 +/- 2.8, HxA3 (8S) = 21.7 +/- 3.7, HxA3-C (8R) = 13.8 +/- 3.8, n = 6 for each). Also the maximal response to NKA was significantly increased when the tissues were exposed to these hepoxilins (% of the maximal response to NKA, control = 100, HxA3 (8S) = 114.5 +/- 5.3, HxA3-C (8R) = 139.0 +/- 6.2, n = 6 for each). The threshold concentrations for both hepoxilins was 10(-8) M and their effects were dose-related. 4. Stereochemical specificity was observed. The 8S-isomer of HxA3 was active in potentiating the NKA-induced contraction of the trachea while the 8R isomer was inactive. In contrast, the 8R isomer of HxA3-C was active while the 8S isomer was inactive. The trihydroxy metabolite of the active isomer of HxA3 (8S), i.e. TrXA3 (8S) (10(-6) M), was inactive in potentiating the NKA-induced contraction of the trachea. 5. It is concluded that hepoxilins sensitize the guinea-pig isolated trachea to the potent bronchoconstrictor, NKA.

Antiinflammatory and analgesic activity of a non-peptide substance P receptor antagonist

CP-96,345, a potent non-peptide antagonist of the substance P (SP) receptor, inhibited SP-, neurokinin A (NKA)- and neurokinin B-induced plasma extravasation in guinea pig dorsal skin. The inhibition was specific for the three tachykinins; CP-96,345 was not active against plasma leakage caused by histamine, bradykinin, platelet-activating factor or leukotriene D 4. CP-96,345 inhibited capsaicin-induced plasma extravasation in the ureter, an inflammatory response caused by neuropeptides released from afferent C-fibers. Thus, the NK 1 receptor appears to play a major role in vascular permeability increases induced by exogenous and endogenous tachykinins. In contrast, CP-96,345 was inactive against SP- and NKA-induced contraction of guinea pig ureter, suggesting that the smooth muscle contraction is not NK 1-mediated. CP-96,345 exhibited analgesic activity in acetic acid-induced abdominal stretching in mice, indicating for the first time that SP plays a critical role in this model. The results of these studies support a pathophysiological role of SP and the NK 1 receptor under acute neurogenic inflammatory conditions and in pain.

Postsynaptic potentiation of neurotransmission by neurokinin A in rat vas deferens.

Effects of neurokinin A (NKA) on sympathetic neurotransmission were studied in rat vas deferens. Although neither prazosin, an alpha 1-adrenoceptor blocker, nor alpha, beta-methylene adenosine triphosphate, a P2-purinoceptor blocker, inhibited the NKA-induced contractions in the epididymal site, high concentration of NKA-induced contractions in the prostatic site were slightly decreased by either of the two blockers. Treatment with guanethidine, which prevents the release of sympathetic transmitters from presynaptic nerve endings, also had no effect on NKA-induced contractions in either site. To investigate the effects of NKA on the adrenergic and purinergic neurotransmission in more detail, we measured transmitter release by using [3H]norepinephrine or [14C]adenosine. Neither spontaneous or nor evoked 3H efflux, indicating NE release, was affected by NKA in either site. NKA enhanced 14C efflux, indicating ATP release, evoked by electrical stimulation in the epididymal site, which may be originated from smooth muscle. In the prostatic site, contractions induced by electrical stimulation were enhanced in spite of no increase in 3H or 14C efflux. These results suggest that: 1) NKA has no effect on presynaptic nerve terminals in both sites, 2) NKA potentiates the effects of neurotransmitters in the prostatic site, and 3) NKA modulates the neurotransmissions.

Postsynaptic Potentiation of Neurotransmission by Neurokinin A in Rat Vas Deferens

Effects of neurokinin A (NKA) on sympathetic neurotransmission were studied in rat vas deferens. Although neither prazosin, an α1-adrenoceptor blocker, nor α,β-methylene adenosine triphosphate, a P2-purinoceptor blocker, inhibited the NKA-induced contractions in the epididymal site, high concentration of NKA-induced contractions in the prostatic site were slightly decreased by either of the two blockers. Treatment with guanethidine, which prevents the release of sympathetic transmitters from presynaptic nerve endings, also had no effect on NKA-induced contractions in either site. To investigate the effects of NKA on the adrenergic and purinergic neurotransmission in more detail, we measured transmitter release by using [3H]norepinephrine or [l4C]adenosine. Neither spontaneous or nor evoked 3H efflux, indicating NE release, was affected by NKA in either site. NKA enhanced 14C efflux, indicating ATP release, evoked by electrical stimulation in the epididymal site, which may be originated from smooth muscle. In the prostatic site, contractions induced by electrical stimulation were enhanced in spite of no increase in 3H or l4C efflux. These results suggest that: 1) NKA has no effect on presynaptic nerve terminals in both sites, 2) NKA potentiates the effects of neurotransmitters in the prostatic site, and 3) NKA modulates the neurotransmission.

Neurokinin A-induced guinea pig gallbladder contraction: Potential mechanism of action

The present study was performed to examine the mechanism of action of neurokinin A (NKA) on guinea pig gallbaldder smooth muscle. Muscle strips were prepared and mounted in 10 mL tissue bath containing Krebs' solution under 1 g tension. NKA induced a concentration-dependent increase in gallbladder muscle tension and reached a maximal response at 1 μM. The EC50 value was approximately 30nM. Preincubation of the muscle strips with neurotoxins, tetrodotoxin (1 μM), or omega-conotoxin (0.1 μM) had no effect on the NKA contractile response. NKA-induced gallbladder contractions were insensitive to cyclooxygenase inhibitors (5 μM) piroxicam and indomethacin. In contrast, the calcium channel blockers verapamil and diltiazem (0.1–1 μM) significantly blocked the contractile response to NKA. The intracellular calcium chelator BAPTA/AM had no significant effect on NKA activity. The removal of extracellular calcium, however, completely abolished the contractile response of NKA. These data suggest that NKA has a direct contractile effect on guinea pig gallbladder smooth muscle, which is independent of prostaglandin release. The primary source of calcium involved in mediating the NKA contractile response is the extracellular pool, suggesting that NKA might act via activation of L-type voltage-operated calcium channels to mediate its action. © 1992 Wiley-Liss, Inc.

Efficient Ca 2+ mobilization induced by neurokinin A in rat vas deferens

Although both neurokinin A (NKA) and norepinephrine (NE) induced similar maximal contractions in the epididymal and the prostatic site of vas deferens, NKA affected sensitivity more potently than did NE in both sites. The NKA-induced contractions were more strongly inhibited by nicardipine, a dihydropyridine Ca 2+ entry blocker, or by elimination of extracellular Ca 2+ (Ca 2+ o) in both sites. However, ryanodine, which interferes with the release of intracellular C 2+ (Ca 2+ i), abolished the contractions caused by NKA in the prostatic site whereas it had no effect in the epididymal site. These results suggest that NKA-induced contraction utilizes both Ca 2+ i and Ca 2+ o in the prostatic site but mobilizes only Ca 2+ o in the epididymal site. Ca 2+ i concentration ([Ca 2+] i)was measured directly with a Ca 2+ -sensitive fluorescent dye, fura-2. In the epididymal site NKA induced contractions with smaller increase in [Ca 2+] i compared to that necessary for NE-induccd contractions. These results suggest that NKA utilizes Ca 2+ more efficiently than does NE and plays a role as a neuromodulator in rat vas deferens.