Hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat urinary bladder

In the rat isolated urinary bladder, NaHS (30 microm-3 mm) and capsaicin (10 nm-3 microm) produced concentration-dependent contractile responses (pEC(50)=3.5+/-0.02 and 7.1+/-0.02, respectively) undergoing dramatic tachyphylaxis. In preparations in which sensory nerves were rendered desensitized (defunctionalized) by high-capsaicin (10 microm for 15 min) pretreatment, neither capsaicin itself nor NaHS produced any motor effect. NaHS-induced contractile effects were totally prevented by the simultaneous incubation with tachykinin NK(1) (GR 82334; 10 microm) and NK(2) (nepadutant; 0.3 microm) receptor-selective antagonists. Tetrodotoxin (1 microm) only partially reduced the response to NaHS. These results provide pharmacological evidence that H(2)S stimulates capsaicin-sensitive primary afferent nerve terminals, from which tachykinins are released to produce the observed contraction by activating NK(1) and NK(2) receptors. While the molecular site of action of H(2)S remains to be investigated, our discovery may have important physiological significance since H(2)S concentrations capable of stimulating sensory nerves overlap those occurring in mammalian tissues under normal conditions.     

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.     

Evidence that tachykinin NK2 receptors modulate resting tone in the rat isolated small intestine.

1. In the progress of experiments aimed at evaluating the role of tachykinins as enteric nonadrenergic noncholinergic (NANC) transmitters, we noted that certain tachykinin receptor antagonists produce a relaxation of circular muscle strips in the rat small intestine. This study aimed to assess the nature of this response and to determine the receptor type involved. The majority of the experiments were performed in capsaicin- (10 microM for 15 min) pretreated mucosa-free circular muscle strips from the rat small intestine, in the presence of atropine (1 microM), guanethidine (3 microM) and indomethacin (10 microM). 2. Under isometric recording of mechanical activity, the tachykinin NK1 receptor antagonist SR 140,333 (0.1 microM) had no effect on resting tone or spontaneous activity in duodenal or ileal circular muscle strips. The NK2 receptor antagonists, MEN 10,627 (0.1 microM) and GR 94,800 (0.1 microM) produced, after a delay of 10-15 min, a relaxation which averaged 61 +/- 3 and 57 +/- 6% (n = 6 and 4, respectively) of the maximal response (Emax) to isoprenaline (1 microM). The effect of maximal concentrations of MEN 10,627 and GR 94,800 when applied together was non-additive. The relaxant effect of MEN 10,627 (0.1 microM) was similar in the absence and presence of apamin (0.3 microM) and L-nitroarginine (100 microM). 3. Under isotonic recording of mechanical activity, MEN 10,627 (10 nM-1 microM) produced a concentration- and time-related relaxation of duodenal strips. The maximal relaxation averaged 72 +/- 4 and 69 +/- 4% (n = 5 each) of Emax to isoprenaline (1 microM) and was achieved 15-20 or 20-30 min after application of 1.0 or 0.1 microM MEN 10,627, respectively. 4. Duodenal strips were relaxed by other NK2 receptor selective antagonists (values in parentheses are % of Emax to isoprenaline at the given concentration of antagonist) GR 94,800 (69 +/- 3% at 1 microM, n = 4), SR 48,968 (60 +/- 3% at 1 microM, n = 4) and MDL 29,913 (66 +/- 4% at 1 microM, n = 4). SR 48,965 (1 microM), the inactive enantiomer of SR 48,968, was without effect. The NK1 receptor selective antagonists, SR 140,333 (0.1 microM), FK 888 (10 microM) RP 67,580 (1 microM) and GR 82,334 (10 microM) were also without effect (n = 4-5). 5. A cocktail of peptidase inhibitors, thiorphan, bestatin and captopril (1 microM each) had no significant effect on tone or spontaneous activity of duodenal strips. In the presence of peptidase inhibitors, MEN 10,627 (1 microM) produced a relaxation of duodenal strips (72 +/- 6% of Emax to isoprenaline, n = 5), whilst GR 82,334 (10 microM, n = 6) had no significant effect. 6. The relaxant response to MEN 10,627 was preserved in mucosa-free strips not pre-exposed to capsaicin. Tetrodotoxin (1 microM), saxitoxin (1 microM), hexamethonium (100 microM) and omega-conotoxin (0.1 microM) had no significant effect on the resting tone of duodenal strips nor did they affect the relaxation to MEN 10,627. L-Nitroarginine (100 microM) increased the tone of the strips but did not affect the response to MEN 10,627. Nifedipine (1 microM) relaxed the strips by 62 +/- 4% (n = 4), but in its presence a small relaxant effect to MEN 10,627 (26 +/- 5%, n = 4) was still evident. 7. Under isotonic recording of mechanical activity along the longitudinal axis, MEN 10,627 (1 microM) produced a slowly developing relaxation (39 +/- 3% of Emax to isoprenaline; n = 6) of whole segments of rat duodenum. When similar experiments were performed on whole segments of rat proximal colon MEN 10,627 had no effect. 8. The present findings document the observation that tachykinin NK2 receptors contribute to the maintenance of resting tone of the rat isolated small intestine. We found no evidence to suggest that this effect follows the blockade of the contractile effect of spontaneously released endogenous tachykinins. The present findings raise the possibility that constitutively active NK2 receptors account for the relaxant effect produced by NK2 receptor ant     

Neurokinin-induced changes in pial artery diameter in the anaesthetized guinea-pig.

1. The effects of selective neurokinin agents on pial artery diameter, measured with an on-line image analyser, have been studied in anaesthetized guinea-pigs in order to characterize the neurokinin receptors present on pial arteries. 2. Perivascular injection of either substance P (0.01-1 microM) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01-1 microM) and GR73632 (0.1 microM), increased pial artery diameter. 3. In contrast, the selective NK2 receptor agonist, GR64349 (1 microM), produced a small vasoconstriction while the NK3 receptor-selective agonist, senktide (1 microM) was inactive. 4. Co-administration of GR82334 (1 microM), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 microM) but not that caused by calcitonin gene-related peptide (CGRP, 0.01 microM). 5. The results are consistent with an involvement of NK1 receptors in the neurokinin-induced increase in guinea-pig pial artery diameter.     

Mechanisms of the cutaneous vasodilator response to local external pressure application in rats: involvement of CGRP, neurokinins, prostaglandins and NO

1. Local pressure-induced vasodilation (PIV) is a neural vasodilator response to non-nociceptive externally applied pressure in the skin, previously described in humans. We first determined whether PIV exists in rats and depends on capsaicin-sensitive fibres as it does in humans. We then examined the mediators involved in the efferent pathway of PIV. 2. Cutaneous blood flow was measured by laser Doppler flowmetry during 11.1 Pa s(-1) increases in local applied pressure in anaesthetized rats. The involvement of capsaicin-sensitive fibres in PIV was tested in rats treated neonatally with capsaicin. To antagonize CGRP, neurokinin-1, -2, or -3 receptors, different groups of rats were treated with CGRP(8 - 37), SR140333, SR48968 or SR142801, respectively. Prostaglandins involvement was tested with indomethacin treatment. To inhibit nitric oxide synthase (NOS) activity or specific neuronal NOS, rats were treated with N(G)-nitro-L-arginine or 7-nitroindazole, respectively. 3. PIV was found in rats, as in humans. PIV was abolished by neonatal treatment with capsaicin and by administration of CGRP(8 - 37) but remained unchanged with SR140333, SR48968 and SR142801 treatments. Prostaglandin inhibition resulted in a significant decrease in PIV. Inhibition of NOS abolished PIV, whereas inhibition of neuronal NOS caused a diminution of PIV. 4. These data suggest that PIV depends on capsaicin-sensitive fibres in rats, as in humans. It appears that CGRP plays a major role in the PIV, whereas neurokinins have no role. Furthermore, PIV involves a contribution from prostaglandins and depends on endothelial NO, whereas neuronal NO has a smaller role.     

Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon.

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of ATP, but not of VIP/PACAP or nitric oxide, in the excitatory effect of capsaicin in the small intestine

The contractile effect of capsaicin in the guinea-pig small intestine involves an activation of enteric cholinergic neurons. Our present data show that the P(2) purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) significantly reduces the contractile response to capsaicin (2 microM) in the presence, but not in the absence, of the tachykinin receptor antagonists [O-Pro(9), (Spiro-gamma-lactam)Leu(10), Trp(11)]physalaemin (1-11) (GR 82334; 3 microM) and (S)-(N)-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidine-4-yl)-N -methylacetamide (SR 142804: 100 nM) (for blocking tachykinin NK1 and NK3 receptors, respectively). PPADS (30 microM) fails to influence submaximal cholinergic contractions evoked by cholecystokinin octapeptide (CCK-8; 2-3 nM) or senktide (1 nM), or the direct smooth muscle-contracting effect of histamine (100-200 nM). A higher concentration (300 microM) of PPADS is also without effect against the stimulatory action of cholecystokinin octapeptide. This means that PPADS can probably be safely used as a purinoceptor antagonist in intestinal preparations. The putative pituitary adenylate cyclase activating peptide (PACAP) receptor antagonist PACAP-(6-38) (3 microM) significantly reduces the contractile effect of PACAP-(1-38) (10 nM) and abolishes that of vasoactive intestinal polypeptide (VIP; 10 nM). PACAP-(6-38) (3 microM) fails to influence the effect of capsaicin (2 microM) both in the absence and in the presence of tachykinin receptor antagonists. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM) also fails to inhibit the capsaicin-induced motor response. We conclude that an endogenous ligand of PPADS-sensitive P(2) purinoceptors (possibly ATP), but not a VIP/PACAP-like peptide or NO, is involved in the nontachykininergic activation of cholinergic neurons in the course of the capsaicin-induced contraction.     

Radioligand binding and functional characterisation of tachykinin receptors in chicken small intestine

Tachykinin receptors in chicken intestine were studied using radioligand binding and functional techniques. Mechanisms of tachykinin-induced contraction were also investigated. Binding of [125I]Bolton-Hunter substance P ([125I]BH-SP) to chicken ileal membranes was rapid, saturable, of high affinity and to a single population of binding sites with Kd 0.72 nM and Bmax 0.48 fmol/ wet weight tissue. The rank order of agonists competing for [125I]BH-SP binding sites was [Sar9]SP > [Arg3]SP (natural tachykinin in chickens) > SP > [Pro9]SP > or = NKA > eledoisin > [Sar9,Met(O2)11]SP > [Lys5,MeLeu9,Nle10]-NKA(4-10) > senktide, suggesting similarities to the mammalian NK1 receptor. The NK1 receptor antagonist CP 99994, and NK2 receptor antagonist SR 48968 were weak competitors while spantide, RP 67580, GR 82334, GR 94800 and MEN 11420 were ineffective. The radioligand [125I]NKA showed no specific binding to ileal membranes. The potency order of most tachykinins in contacting isolated ileal longitudinal segments was in good agreement with that obtained from competition binding studies. Contractions to [Arg3]SP, NKA and senktide were greatly reduced by tetrodotoxin, suggesting that neurally-mediated responses were primarily involved. [Arg3]SP and NKA acted mainly by increasing release of acetylcholine, prostaglandins and probably tachykinins. Responses to [Arg3]SP were virtually abolished by nifedipine but were unaffected by NK1 receptor antagonists. Senktide-induced contraction was inhibited by the NK3 receptor antagonist, SR 142801, but was unaffected by atropine or L-NAME. The study provides evidence for a tachykinin receptor with similarities to the NK1 receptor in the chicken small intestine. In addition, senktide may act on a receptor similar to the mammalian NK3 receptor.     

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-induced contraction of the guinea-pig isolated oesophageal mucosa is mediated by NK(2) receptors

1. The tachykinin receptor present in the guinea-pig oesophageal mucosa that mediates contractile responses of the muscularis mucosae has been characterized, using functional in vitro experiments. 2. The NK(1) receptor-selective agonist, [Sar(9)(O(2))Met(11)]SP and the NK(3) receptor-selective agonists, [MePhe(7)]-NKB and senktide, produced no response at submicromolar concentrations. The NK(2) receptor-selective agonists, [Nle(10)]-NKA(4 - 10), and GR 64,349 produced concentration-dependent contractile effects with pD(2) values of 8.20+/-0.16 and 8.30+/-0.15, respectively. 3. The concentration-response curve to the non-selective agonist, NKA (pD(2)=8.13+/-0.04) was shifted significantly rightwards only by the NK(2) receptor-selective antagonist, GR 159,897 and was unaffected by the NK(1) receptor-selective antagonist, SR 140,333 and the NK(3) receptor-selective antagonist, SB 222,200. 4. The NK(2) receptor-selective antagonist, GR 159,897, exhibited an apparent competitive antagonism against the NK(2) receptor-selective agonist, GR 64,349 (apparent pK(B) value=9.29+/-0.16) and against the non-selective agonist, NKA (apparent pK(B) value=8.71+/-0.19). 5. The NK(2) receptor-selective antagonist, SR 48,968 exhibited a non-competitive antagonism against the NK(2) receptor-selective agonist, [Nle(10)]-NKA(4 - 10). The pK(B) value was 10.84+/-0.19.6. It is concluded that the guinea-pig isolated oesophageal mucosa is a useful preparation for studying the effects of NK(2) receptor-selective agonists and antagonists as the contractile responses to various tachykinins are mediated solely by NK(2) receptors.     

Nitric oxide is involved in the relaxant effect of capsaicin in the human sigmoid colon circular muscle

The relaxant effect of capsaicin (300 nM) has been studied on mucosa-free circular strips of the human sigmoid colon in vitro. The response of precontracted preparations to capsaicin (sub-maximal relaxation) was reduced by over 50% by the nitric oxide synthase inhibitor N(G)-nitro- L-arginine (L-NOARG; 20 microM or 100 microM) or by the guanylate cyclase inhibitor 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), but not by tetrodotoxin (1 microM) or the P(2) purinoceptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS; 50 microM). L-NOARG or ODQ caused moderate contraction of the circular muscle, indicating a tonic "nitrergic" control. Anandamide (1-100 microM), an endogenous cannabinoid and capsaicin VR(1) receptor stimulant, failed to either mimic or modify the response to capsaicin (300 nM).It is proposed that capsaicin causes the release of smooth muscle relaxant substance(s) from afferent nerve endings in the gut wall, in a tetrodotoxin-resistant manner. Nitric oxide (possibly released from capsaicin-sensitive afferents) plays an important role in the capsaicin-evoked response. No evidence has been found for an involvement of PPADS-sensitive P(2) purinoceptors in the response to capsaicin or for a stimulation or inhibition of capsaicin-sensitive receptors by anandamide in the human sigmoid colon.     

Lack of effect of sumatriptan and UK-14,304 on capsaicin-induced relaxation of guinea-pig isolated basilar artery.

1. The objectives of this study were to assess the effects of sensory neuropeptide antagonists and presynaptically acting receptor agonists on capsaicin-induced relaxations of guinea-pig isolated basilar artery (GPBA). 2. Capsaicin, human alpha-calcitonin gene-related peptide (CGRP) and substance P (SP) caused concentration-related relaxations of GPBA which had been pre-contracted with prostaglandin F2 alpha (PGF2 alpha). Responses to capsaicin were not modified by the peptidase inhibitors, phosphoramidon (1 microM) and bestatin (100 microM). 3. The relaxant responses to capsaicin were blocked in a selective manner by ruthenium red (3 microM) and by the CGRP antagonist, CGRP8-37 (1 microM). CGRP8-37 also selectively inhibited the relaxant effects of CGRP. 4. The selective NK1 receptor antagonist, GR82334 (10 microM), inhibited SP-induced relaxations but had little effect on capsaicin-induced relaxations. 5. The 5-HT1 receptor agonist, sumatriptan, produced small contractions of GPBA under conditions of resting tone. In the presence of PGF2 alpha, sumatriptan had no further contractile effect. Sumatriptan (0.3 and 3 microM) did not modify capsaicin-induced relaxations of GPBA. 6. The alpha 2-adrenoceptor agonist, UK-14,304 (0.1 microM), had no effect on basal or PGF2 alpha-induced tone. UK-14,304 did not modify capsaicin-induced relaxations. 7. These results suggest that capsaicin causes relaxation of GPBA via a release of CGRP. This process is amenable to blockade by CGRP8-37 and ruthenium red, but not to modulation by either sumatriptan or UK-14,304.     

Functional characterization of tachykinin NK1 receptors in the mouse uterus

1. Contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of oestrogen-treated mice. 2. In the presence of thiorphan (3 microM), captopril (10 microM), and bestatin (10 microM), substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) produced concentration-related contractions of uterine preparations. The order of potency was SP > or =NKA>NKB. 3. Neither atropine (0.1 microM) nor l-NOLA (100 microM), nor indomethacin (10 microM) alone or in combination with either ranitidine (10 microM) or mepyramine (10 microM), affected responses to SP. These findings indicate that SP actions are not mediated or modulated through the release of acetylcholine, nitric oxide, prostanoids or histamine. 4. In the presence of peptidase inhibitors, the tachykinin NK(1) receptor-selective agonist [Sar(9)Met(O(2))(11)]SP, produced a concentration-dependent contractile effect. The tachykinin NK(2) and NK(3) receptor-selective agonists [Lys(5)MeLeu(9)Nle(10)]NKA(4-10) and [MePhe(7)]NKB were relatively inactive. The potencies of SP analogues in which Glu replaced Gln(5) and/or Gln(6) were similar to that of SP. 5. The tachykinin NK(1) receptor-selective antagonist, SR140333 (10 nM), alone or combined with the tachykinin NK(2) receptor-selective antagonist, SR48968 (10 nM), shifted log concentration curves to SP, NKA and NKB to the right. SR140333 (10 nM) reduced the effect of [Sar(9)Met(O(2))(11)]SP. SR48968 did not affect responses to SP or [Sar(9)Met(O(2))(11)]SP, but reduced the effect of higher concentrations of NKA and shifted the log concentration-response curve to NKB to the right. The tachykinin NK(3) receptor-selective antagonist, SR 142801 (0.3 microM), had little effect on responses to SP and NKB. 6. We conclude that the tachykinin NK(1) receptor mediates contractile effects of SP, NKA and NKB and [Sar(9)Met(O(2))(11)]SP in myometrium from the oestrogen-primed mouse. The tachykinin NK(2) receptor may also participate in the responses to NKA and NKB.     

Modulation of acetylcholine release in the guinea-pig trachea by the nitric oxide donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP)

The effects of the nitric oxide (NO) donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) and the NO synthase inhibitor L-N(G)-nitroarginine (L-NOARG) on the electrically evoked [(3)H]-acetylcholine release were studied in an epithelium-free preparation of guinea-pig trachea that had been preincubated with [(3)H]-choline. SNAP (100 and 300 microM) caused small but significant increases of the electrically evoked [(3)H]-acetylcholine release (121+/-4% and 124+/-10% of control). Resting outflow of [(3)H]-ACh was not affected by SNAP. The increase by SNAP was abolished by the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 1 microM). The facilitatory effect of SNAP (100 and 300 microM) was reversed into inhibition of release (to 74+/-4% and to 78+/-2%) after pretreatment of the trachea with capsaicin (3 microM). ODQ prevented the inhibition. Capsaicin pretreatment alone did not significantly alter the release of [(3)H]-acetylcholine. A significant inhibition by SNAP (100 microM) of [(3)H]-acetylcholine release (78+/-3%) was also seen in the presence of the NK(2) receptor antagonist SR 48968 (30 nM). L-NOARG (10 and 100 microM) significantly enhanced the electrically-evoked smooth muscle contractions, but caused no significant increases of the evoked release from capsaicin pretreated trachea strips. This might indicate that the inhibitory effect of endogenous NO on acetylcholine release is too small to be detected by overflow studies. It is concluded that NO has dual effects on the evoked acetylcholine release. NO enhances release in the absence of modifying drugs, but NO inhibits acetylcholine release after blockade of the NK(2) receptor or after sensory nerve depletion with capsaicin. This suggests that NO and endogenous tachykinins act in series to produce an increase in acetylcholine release.     

Defects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells

A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK2 receptor to yield a tyrosine/phenylalanine (Tyr/Phe) substitution. Chinese hamster ovary cells stably transfected with the wild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [betaAla8]NKA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinity as compared to NKA. In a functional assay (production of inositol-1,4,5-trisphosphate, IP3), NKA, GR 64349, and [betaAla8]INKA(4-10) stimulated IP3 accumulation via the wild-type and mutant receptors with similar potencies. On the other hand, NKB and SP exhibited a dramatic reduction in their agonist efficacies at the mutant receptor, NKB acting as a partial agonist (maximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicated that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized the agonist effect of NKA. The antagonist action of SP at the mutant NK2 receptor cannot be ascribed to receptor internalization. The Tyr/Phe replacement at position 289 markedly reduced the binding affinity and antagonist potency of the non-peptide ligand, SR 48968, without affecting the binding affinity and antagonist potency of the bicyclic peptide antagonist MEN 11420. The results indicate that the hydroxyl radical function of Tyr289 in transmembrane segment 7 of the human NK2 receptor is, directly or indirectly, involved in stimulus transduction when the NK2 receptor is occupied by NKB or SP, but not when using NKA or NK2 receptor-selective agonists.     

Respiratory action of capsaicin microinjected into the nucleus of the solitary tract: involvement of vanilloid and tachykinin receptors.

1. The respiratory response to microinjection of capsaicin into the commissural nucleus of the solitary tract (cNTS) of urethane-anaesthetized rats was investigated in the absence and presence of the competitive vanilloid (capsaicin) antagonist, capsazepine, and selective tachykinin NK1, NK2 and NK3 antagonists (RP 67580, SR 48968 and SR 142801, respectively). 2. Microinjection of capsaicin reduced respiratory frequency but not tidal volume (VT), leading to an overall reduction in minute ventilation (VE). The effect was dose-dependent between 0.5 and 2 nmol capsaicin. Doses greater than 2 nmol produced apnoea. Tachyphylaxis was observed following repeated injection of capsaicin (1 nmol, 30 min apart). 3. Capsazepine (1 nmol) had no effect on frequency or VT when injected alone but completely blocked the respiratory response to capsaicin (1 nmol). 4. RP 67580 (1 but not 5 nmol) alone depressed frequency and VT slightly. Moreover, RP 67580 appeared to potentiate the bradypnoeic effect of capsaicin. In contrast, SR 48968 and SR 142801 (1 and 5 nmol) alone had no significant effect on respiration. However, both agents significantly attenuated the reduction in frequency produced by capsaicin. 5. In conclusion, microinjection of capsaicin into the cNTS decreases overall ventilation, primarily by reducing frequency. The action of capsaicin appears from the data to be mediated by vanilloid receptors since it is blocked by the competitive vanilloid antagonist capsazepine and is subject to tachyphylaxis. However, since NK2 (SR 48968) and NK3 (SR 142801) receptor antagonists block the actions of capsaicin, we propose that capsaicin acts also by releasing tachykinins from central afferent terminals in the cNTS.     

Vanilloid receptors on capsaicin-sensitive sensory nerves mediate relaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arteries

In the present study, the vasodilator actions of methanandamide and capsaicin in the rat isolated mesenteric arterial bed and small mesenteric arterial segments were investigated. Methanandamide elicited concentration-dependent relaxations of preconstricted mesenteric arterial beds (pEC(50)=6.0+/-0.1, E(max)=87+/-3%) and arterial segments (pEC(50)=6.4+/-0.1, E(max)=93+/-3%). In arterial beds, in vitro capsaicin pre-treatment blocked vasorelaxation to 1 and 3 microM methanandamide, and reduced to 12+/-7% vasorelaxation to 10 microM methanandamide. Methanandamide failed to relax arterial segments pre-treated in vitro with capsaicin. In arterial beds from rats treated as neonates with capsaicin to cause destruction of primary afferent nerves, methanandamide at 1 and 3 microM did not evoke vasorelaxation, and relaxation at 10 microM methanandamide was reduced to 26+/-4%. Ruthenium red (0.1 microM), an inhibitor of vanilloid responses, attenuated vasorelaxation to methanandamide in arterial beds (pEC(50)=5.6+/-0.1, E(max)=89+/-1%). Ruthenium red at 1 microM abolished the response to 1 microM methanandamide, and greatly attenuated relaxation at 3 and 10 microM methanandamide in arterial beds. In arterial segments, ruthenium red (0.15 microM) blocked vasorelaxation to methanandamide, but not to CGRP. In arterial segments, the vanilloid receptor antagonist capsazepine (1 microM) inhibited, and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8 - 37) (3 microM) abolished, methanandamide-induced relaxations. CGRP(8 - 37), but not capsazepine, attenuated significantly relaxation to exogenous CGRP. These data show that capsaicin and ruthenium red attenuate vasorelaxation to methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments. In addition, CGRP(8 - 37) and capsazepine antagonize responses to methanandamide in mesenteric arterial segments. In conclusion, vanilloid receptors on capsaicin-sensitive sensory nerves play an important role in the vasorelaxant action of methanandamide in the rat isolated mesenteric arterial bed and small mesenteric arterial segments.     

Calcitonin gene-related peptide facilitates serotonin release from guinea-pig colonic mucosa via myenteric neurons and tachykinin NK2/NK3 receptors

The ability of calcitonin gene-related peptide (CGRP), to alter the outflow of 5-hydroxytryptamine (5-HT) from the guinea-pig proximal colon, was evaluated using three different isolated preparations: whole colon, mucosa-free muscle layer and submucosa/mucosa preparations. In the presence of the monoamine oxidase A inhibitor, clorgyline, CGRP elicited a concentration-dependent increase in 5-HT outflow from the whole colon, but not from mucosa-free muscle layer preparations. The CGRP-evoked 5-HT outflow was sensitive to tetrodotoxin (TTX) or hexamethonium, but was not detectable in submucosa/mucosa preparations. HCGRP8-37 (3 microM) inhibited the submaximal effect of CGRP on the 5-HT outflow. [Cys(ACM)2,7]hCGRP had a slight stimulant influence on the 5-HT outflow. The selective NK2 and NK3 receptor antagonists, SR48968 or SR142801, respectively, prevented the enhancing effect of CGRP. By contrast, a selective NK1 receptor antagonist L703606, failed to block the effect of CGRP. The enhancing effect of CGRP was mimicked by the NK2 receptor agonist [beta-Ala8]-neurokinin A (NKA)4-10 and the NK3 receptor agonist senktide. The effect of [beta-Ala8]-NKA4-10 on the 5-HT outflow was unaffected by TTX, while the effect of senktide was prevented by TTX, hexamethonium or SR48968. The present data also demonstrated a synergistic action of the NK2 and NK3 receptor agonists on the CGRP-evoked 5-HT outflow. We concluded that CGRP facilitates 5-HT release from the guinea-pig colonic mucosa through an action on myenteric neurons and that this effect is mediated by endogenously released tachykinins, acting via tachykinin NK2/NK3 receptors in cascade.British Journal of Pharmacology (2004) 141, 385-390. doi:10.1038/sj.bjp.0705624     

Pharmacological profile of the novel mammalian tachykinin, hemokinin 1.

1. The effects of the novel mammalian tachykinin, hemokinin 1 (HEK-1), have been investigated by radioligand binding and functional in vitro and in vivo experiments. 2. Similar to SP (K(i)=0.13 nM), HEK-1 inhibited in a concentration-dependent manner and with high affinity [(3)H]-substance P (SP) binding to human NK(1) receptor (K(i)=0.175 nM) while its affinity for [(125)I]-neurokinin A (NKA) binding at human NK(2) receptor was markedly lower (K(i)=560 nM). 3. In isolated bioassays HEK-1 was a full agonist at tachykinin NK(1), NK(2) and NK(3) receptors. In the rat urinary bladder (RUB) HEK-1 was about 3 fold less potent than SP. In the rabbit pulmonary artery (RPA) HEK-1 and in the guinea-pig ileum (GPI), HEK-1 was about 500 fold less potent than NKA and NKB, respectively. 4. The responses to HEK-1 were antagonized by GR 82334 in RUB (pK(B)=5.6+/-0.07), by nepadutant in RPA (pK(B)=8.6+/-0.04) and by SR 142801 in GPI (pK(B)=9.0+/-0.2) with apparent affinities comparable to that measured against tachykinin NK(1), NK(2) and NK(3) receptor-selective agonists, respectively. 5. Intravenous HEK-1 produced dose-related decrease of blood pressure in anaesthetized guinea-pigs (ED(50)=0.1 nmol kg(-1)) and salivary secretion in anaesthetized rats (ED(50)=6 nmol kg(-1)) with potencies similar to that of SP. All these effects were blocked by the selective tachykinin NK(1) receptor antagonist, SR 140333. 6. We conclude that HEK-1 is a full agonist at tachykinin NK(1), NK(2) and NK(3) receptors, possesses a remarkable selectivity for NK(1) as compared to NK(2) or NK(3) receptors and acts in vivo experiments with potency similar to that of SP.     

Tachykinin NK1 and NK2 receptors mediate inhibitory vs excitatory motor responses in human isolated corpus cavernosum and spongiosum

Motor effects produced by tachykinins were studied in human isolated corpus spongiosum and cavernosum. In quiescent preparations neurokinin A caused potent contractions (pD(2)=8.3 - 7.9 respectively) prevented by the NK(2) receptor-selective antagonist nepadutant, whereas [Sar(9)]SP sulfone and senktide (NK(1) and NK(3) receptor-selective agonists) produced no effect or spare contractions. In KCl-precontracted corpus spongiosum septide (pD(2)=7.1) and [Sar(9)]SP sulfone (pD(2)=7.7) produced tetrodotoxin-resistant relaxations, abolished by the tachykinin NK(1) receptor-selective antagonist SR 140333. [Sar(9)]SP sulfone (1 microM) produced similar relaxations in precontracted corpus cavernosum. Electrical field stimulation (EFS) elicited tetrodotoxin-sensitive relaxations, which were additive to those produced by [Sar(9)]SP sulfone. N(omega)-nitro-L-arginine (L-NOARG) totally prevented both [Sar(9)]SP sulfone- and EFS-induced relaxations. These results show that tachykinin NK(1) and NK(2) receptors mediate opposite motor effects in human penile tissues, suggesting a possible modulatory role of tachykinins on smooth muscle tone in these organs.