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C A Maggi

Publications and source records attributed to C A Maggi.

At least 91 records · Page 5Linked to original sources
PubMed1999

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.

Animals↗
PubMed1999

Tachykinin receptors are involved in the "local efferent" motor response to capsaicin in the guinea-pig small intestine and oesophagus.

The sensory neuron stimulant drug capsaicin stimulates primary afferent nerve endings in the guinea-pig small intestine, which in turn activate myenteric cholinergic neurons by an unknown mechanism. The tachykinins substance P and neurokinin A are present in primary afferent neurons. This study was performed to assess the possible involvement of endogenous tachykinins acting via neurokinin-1, neurokinin-2 and neurokinin-3 receptors in the contractile effect of capsaicin in the isolated guinea-pig ileum and oesophagus by using the receptor-specific antagonists GR 82334 (3 microM) for neurokinin-1 receptors, MEN 10627 (3 microM; ileum) or MEN 11420 (1 microM; oesophagus) for neurokinin-2 receptors and SR 142801 (0.1 microM) for neurokinin-3 receptors. In the ileum, the peak contraction evoked by capsaicin (2 microM) was not reduced when tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors were blocked separately, whereas an inhibition of neurokinin-3 receptors diminished the area under the curve of the capsaicin response. A combined blockade of neurokinin-1 and neurokinin-3 receptors significantly depressed the effect of capsaicin; the amplitude of the contractile response was 53.3+/-3.7% of the maximal longitudinal spasm in control preparations, whereas in the presence of GR 82334 plus SR 142801 it reached only 27.6+/-5% (P<0.001, Kruskal-Wallis test; n=9 and 10, respectively). Also, the area under the curve of the contractile response to capsaicin was more than 85% lower in the group of preparations treated with GR 82334 plus SR 142801 than in the control group (P<0.001). Including a neurokinin-2 blocker in the combination did not produce any further inhibition. A concomitant tachyphylaxis to substance P (natural neurokinin-1 receptor stimulant) and the neurokinin-3 receptor agonist senktide (5 and 1 microM, respectively) also reduced the contractile effect of capsaicin. In the oesophagus, capsaicin (1 microM) induced biphasic contractions which were strongly inhibited by atropine (1 microM) or capsaicin pretreatment (1 microM for 10 min). Here again, a blockade of tachykinin neurokinin-1, neurokinin-2 or neurokinin-3 receptors separately failed to inhibit the response to capsaicin, whereas a combined blockade of any two tachykinin receptors caused a partial inhibition. The reduction of the contractile effect of capsaicin was strongest when all three tachykinin receptors were blocked. In seven control preparations, peaks for the first and second phases of contraction reached 35.3+/-3.7% and 20+/-3.2% of maximal longitudinal spasm; the corresponding values in the presence of a combination of GR 82334, MEN 11420 and SR 142801 were 7.5+/-0.8% and 9.1+/-2.2%, respectively (n=6, P<0.001 and 0.05, respectively). Tetrodotoxin (0.5 microM) practically abolished the contractile effect of capsaicin in both tissues studied. It is concluded that an interplay of neuronal tachykinin neurokinin-1 and neurokinin-3 receptors (ileum) and neurokinin-1, neurokinin-2 and neurokinin-3 receptors (oesophagus) is involved in the contractile action of capsaicin, probably in mediating excitation of myenteric neurons by tachykinins released from primary afferents. In both tissues, there also seems to be a non-tachykininergic component of the capsaicin-induced contraction.

Animals↗
PubMed1999

Substance P activates responses correlated with tumour growth in human glioma cell lines bearing tachykinin NK1 receptors.

The neuropeptide substance P (SP), by stimulating tachykinin NK1 receptors (NK1R), triggers a number of biological responses in human glioma cells which are potentially relevant for tumour growth. First, radioligand binding studies demonstrated the presence of tachykinin NK1R on SNB-19, DBTRG-05 MG and U373 MG, but not on U138 MG and MOG-G-GCM human glioma cell lines. Second, application of SP or neurokinin A (NKA) to NK1R+ glioma cell lines increased the secretion of interleukin 6 (IL-6) and potentiated IL-6 secretion induced by IL-1beta. SP also up-regulated the release of transforming growth factor beta1 (TGF-beta1) by the U373 MG glioma cell line. Third, SP induced new DNA synthesis and enhanced the proliferation rate of NK1R+, but not of NK1R- glioma cell lines. Also, NKA stimulated the proliferation and cytokine secretion in NK1R+ glioma cell lines. All the stimulant effects of SP/NKA on NK1R+ glioma cell lines were completely blocked by a specific tachykinin NK1R antagonist, MEN 11467. These data support the potential use of tachykinin NK1R antagonist for controlling the proliferative rate of human gliomas.

Cell Division↗
PubMed1999

Tachykinin NK(1)receptor-mediated inhibitory responses in the guinea-pig small intestine.

We used in vivo, in vitro studies and immunohistochemistry to elucidate the mechanisms activated by tachykinin NK(1)receptors in evoking inhibitory motor response in the guinea-pig small intestine. In vivo, the selective NK(1)receptor agonist GR 73,632 produced a dose-dependent suppression of the distension-induced duodenal contractions, and a decrease of basal tone. These effects were reduced by pretreatment with the NK(1)receptor antagonist SR 140,333. In L-Nomega-nitro-L-arginine methylesther hydrochloride-pretreated animals, the suppressant effect of GR 73,632 on duodenal contractions was reduced, whereas the relaxation of the basal tone was unaffected. In vitro, GR 73,632 evoked a biphasic response consisting of a transient, tetrodotoxin-sensitive inhibitory effect followed by tetrodotoxin-resistant contractions. SR 140,333 blocked both inhibitory and excitatory motor responses induced by GR 73,632. NK(1)immunoreactivity was localized to myenteric and submucosal neurons and to interstitial cells of Cajal in the deep muscular plexus of the small intestine. NK(1)receptor-expressing neurons had Dogiel type I morphology and many of them were beta-nicotinamide adenine phosphate dinucleotide-diaphorase-positive, indicating they are inhibitory neurons. In conclusion, in the guinea-pig small intestine, NK(1)receptor stimulation evokes a myogenic excitatory motor response and a neurogenic inhibitory motor response that involves, at least in part, a nitrinergic pathway.

Adrenergic Agents↗
PubMed1999

MEN 11270, A novel selective constrained peptide antagonist with high affinity at the human B2 kinin receptor.

We investigated the pharmacological profile of MEN 11270, or H-D-Arg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10 alpha), a conformationally constrained derivative of the B2 kinin receptor antagonist Icatibant. MEN 11270 bound with high-affinity to the B2 kinin receptor constitutively expressed by WI38 human fibroblasts, inhibiting 3H-bradykinin (BK) with a pKi value of 10.3 +/- 0.08 (n = 5). The rank order of affinity of several peptide and nonpeptide antagonists was also assessed: Icatibant (pKi = 10.6) approximately MEN 11270 (pKi = 10.3) approximately B9430 (pKi = 10.0) > B9858 (pKi = 8.0) > FR173657 (pKi = 7.6) > WIN64338 (pKi = 7.2) > Lys-[des-Arg9, Leu8]-BK (pKi < 6) > [des-Arg9,Leu8]-BK (pKi < 5). MEN 11270 showed a low affinity in inhibiting 3H-Lys-[des-Arg9]-BK binding at the human B1 kinin receptor constitutively expressed by the same cells (pKi 6.0 +/- 0.33; n = 3). MEN 11270 showed no binding affinity (pIC50 < 5.5) at 29 different receptors and ion channels. In the human umbilical vein contraction assay, MEN 11270, shifted the concentration-response curve to BK to the right in a concentration-dependent manner (pA2 8.14 +/- 0.22, n = 7). The Schild plot was linear (slope 0.95 +/- 0.11), consistent with a competitive antagonism. In the same bioassay, MEN 11270 (10 microM) did not affect the concentration-response curve to the B1 agonist Lys-[des-Arg9]-BK nor the contractile responses elicited by noradrenaline or serotonin. These findings indicate MEN 11270 as an antagonist at the human B2 kinin receptor, with potency and selectivity comparable to those of the linear peptide antagonist, supporting the hypothesis that a constrained C-terminal beta-turn conformation preserves a high affinity for the interaction of Icatibant with the B2 kinin receptor.

Adult↗
PubMed1999

Relevance of aromatic residues in transmembrane segments V to VII for binding of peptide and nonpeptide antagonists to the human tachykinin NK(2) receptor.

We used membranes from Chinese hamster ovary cells stably transfected with the human tachykinin NK(2) receptor, either wild-type or mutated, at four aromatic residues (His(198), Tyr(266), Phe(270), Tyr(289)) located in transmembrane segments V to VII, to assess the role of these residues in the binding of natural tachykinins and peptide and nonpeptide antagonists. Three radioligands, the agonist [(125)I]neurokinin A (NKA), the peptide antagonist [(3)H]MEN 11420, and the nonpeptide antagonist [(3)H]SR 48968 bound to the wild-type receptor with high affinity (K(d) = 2.4 nM, 0.3 nM, and 4.0 nM, respectively). Four of the six mutant receptors tested retained high affinity for at least one of the radioligands. H(198)A mutation abrogated the binding of NKA but not that of MEN 11420 or SR 48968 (K(d) = 4.8 and 11.5 nM, respectively); Y(266)F mutation abrogated the binding of MEN 11420 but not that of NKA or SR 48968 (K(d) = 2.8 nM and 1.2 nM, respectively); F(270)A mutation abrogated the binding of both NKA and MEN 11420 but not that of SR 48968 (K(d) = 1.6 nM); Y(289)F mutation abrogated the binding of SR 48968 but not that of NKA and MEN 11420 (K(d) = 2.0 and 2.9 nM, respectively). Y(266)A and Y(289)A mutations abrogated the binding of all radioligands. Among the unlabeled antagonists, the affinity of the nonpeptide GR 159897, at variance with SR 48968, resulted heavily compromised by H(198)A and Y(266)F mutations; the peptide antagonists R396 and MEN 10376 essentially followed the binding profile of NKA, but R396 showed markedly increased affinity for the Y(289)F mutant receptor. Taken together, these results indicate that different, partially overlapping sets of sites may be involved in the binding of agonists and diverse antagonists to the human tachykinin NK(2) receptor.

Animals↗
PubMed1998

Characterization of the antibronchoconstrictor activity of MEN 11420, a tachykinin NK2 receptor antagonist, in guinea-pigs.

We have investigated the antibronchoconstrictor activity of a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist, MEN 11420 c¿[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta++ +)¿, as compared to MEN 10627 c[(Met-Asp-Trp-Phe-Dpr-Leu)c(2beta-5beta)] and to the nonpeptide antagonist SR 48968 ((S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2-3,4-dichlorophenyl)bu tyl] benzamide. In the guinea-pig isolated bronchus MEN 11420 (pK(B) 8.40+/-0.07) and MEN 10627 (pK(B) 8.67+/-0.09) competitively antagonized the contraction induced by the tachykinin NK2 receptor agonist, [betaAla8]neurokinin A-(4-10). SR 48968 showed an apparent pK(B) of 9.57+/-0.2. The atropine-resistant response to electrical stimulation was reduced in a concentration-dependent manner by MEN 11420, MEN 10627 and SR 48968. In urethane-anaesthetized guinea-pigs, MEN 11420 produced a dose-dependent inhibition of bronchoconstriction induced by [betaAla8]neurokinin A-(4-10). Comparable inhibitory effects were observed after i.v. administration of SR 48968 and MEN 10627. Bilateral electrical stimulation of the vagi (20 Hz for 20 s) induced a bronchoconstriction that was dose-dependently inhibited by i.v. MEN 11420, SR 48968 and MEN 10627. MEN 11420 was also effective in inhibiting the capsaicin (20 nmol/kg i.v.)-induced bronchoconstriction. MEN 11420 (1.1 micromol/kg i.v.) showed a longer plasma half-life and a greater area under the plasma concentration-time curve value (AUC) than those of MEN 10627. These findings indicate that MEN 11420 is a potent and selective antagonist of the tachykinin NK2 receptor in guinea-pig airways with a long duration of action.

Amino Acid Sequence↗
PubMed1998

Characterization of [3H]MEN 11420, a novel glycosylated peptide antagonist radioligand of the tachykinin NK2 receptor.

[3H]MEN 11420, a radiolabeled glycosylated peptide antagonist of the tachykinin NK2 receptor, has been investigated in ligand-receptor binding assays using membranes of CHO cells transfected with the human tachykinin NK2 receptor. [3H]MEN 11420 bound to a single class of high affinity binding sites: its binding was inhibited by natural tachykinins (potency ranking: NKA >> SP > or = NKB), as well as by peptide (MEN 11420 > MEN 10376 >> R 396) and nonpeptide (SR 48968 > GR 159897) selective NK2 receptor antagonists. These data indicate that [3H]MEN 11420 is a potent radioligand for the human tachykinin NK2 receptor that may represent a useful tool for studying ligand-receptor interactions at the molecular level.

Animals↗
PubMed1998

Receptor binding profile of Otilonium bromide.

The interaction of Otilonium bromide (OB) with binding sites for 63 different receptors and ion channels in appropriate preparations has been investigated. Experiments were also performed in rat colon, the preferred tissue for OB 'in vivo' uptake after oral administration. Among the receptors investigated OB binds with sub microM affinity to muscarinic M1, M2, M4, M5 and PAF receptors and with microM affinity to the diltiazem binding site on L type Ca2+ channels. In the rat colon OB shows competitive interaction with the verapamil binding site on L type Ca2+ channels and with muscarinic M2 receptors with IC50 of 1020 and 1220 nM, respectively. These findings provide a molecular rationale to explain the spasmolytic action exerted by OB on intestinal smooth muscle. In particular, a combination of antimuscarinic and Ca2+ channel blocker properties seems to best account for the action of this compound.

Animals↗
PubMed1998

Evidence for the involvement of multiple mechanisms in the excitatory action of bradykinin in the circular muscle of guinea-pig colon.

We have investigated, by using the sucrose gap technique, the mechanisms of the excitatory action of bradykinin in the circular muscle of the guinea-pig proximal colon. In the presence of atropine (1 microM) and S-ketoprofen (3 microM), the application of bradykinin (1 microM for 20 s) produced complex changes in membrane potential and muscle tension. The prevailing response was a small hyperpolarization followed by a slowly developing depolarization and a tonic contraction. The selective B2 receptor antagonist, HOE 140 (0.3 microM) blocked the responses to bradykinin (1 microM) while tetrodotoxin (0.3 microM) had no affect. The selective B1 receptor agonist, [des-Arg9]bradykinin (1 microM) did not affect the electrical or mechanical activities of the circular muscle. Apamin (0.1 microM) blocked the transient hyperpolarization and potentiated the bradykinin-induced depolarization and contraction. In the presence of apamin, nifedipine (1 microM) blocked spikes (when present) and the phasic contraction while leaving the tonic contraction unaffected. The excitatory action of bradykinin was further investigated in the presence of atropine (1 microM), S-ketoprofen (3 microM), apamin (0.1 microM) and nifedipine (1 microM). The depolarization but not the contraction induced by bradykinin was reduced by about 30% in low-Na+ (25 mM) but not in low Cl- (9.7 mM) Krebs solution. The depolarization and contraction evoked by bradykinin were reduced (by about 30 and 75%, respectively) in Ca2+-free (2 min) Krebs solution. The blocker of the sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (CPA, 10 microM) reduced the nifedipine-resistant depolarization and contraction induced by bradykinin by about 40 and 60%, respectively. The inhibitor of receptor-operated cation channels, SKF 96365 (50 microM) reduced the nifedipine-resistant bradykinin-induced depolarization and contraction by about 40 and 30%, respectively, whereas the inhibitor of Ca2+-dependent chloride channels, niflumic acid (100 microM) was without effect. The inhibitory effect of SKF 96365 (50 microM) and CPA (10 microM) was additive: in the presence of both drugs the bradykinin-induced depolarization and contraction were reduced by about 70-80%. The protein kinase C inhibitor, GF 109203x (10 microM) did not affect the nifedipine-resistant bradykinin-induced depolarization and contraction. At a concentration of 30 microM, GF 109203x reduced the bradykinin-induced contraction by about 50% while leaving the bradykinin-induced depolarization unaffected. The KCl (40 mM)-induced contraction was significantly reduced (by about 30%) by GF 109203x (30 microM). The present findings indicate that, in the presence of apamin and nifedipine, the bradykinin-induced contraction of circular muscle of the guinea-pig colon is due to the influx of extracellular Ca2+ via non-selective cation channels and, in part, to the release of Ca2+ from a loosely bound internal store. Intracellular Ca2+ facilitates the bradykinin-induced depolarization, a response which does not involve a protein kinase C-dependent mechanism.

Animals↗
PubMed1998

Depolarization evoked co-release of tachykinins from enteric nerves in the guinea-pig proximal colon.

The aim of this study was to assess at which extent an even co-release of the tachykinins, substance P (SP) and neurokinin A (NKA), occurs from enteric neurons/nerves of the guinea-pig proximal colon during graded depolarization. In this preparation, a sharply diverging NK1/NK2 receptor pattern of tachykininergic co-transmission has been observed in physiological studies. The experiments were performed in capsaicin-pretreated (10 microM for 15 min) mucosa-free smooth muscle of guinea-pig proximal colon, to exclude the mucosa and the peripheral endings of primary afferent nerves as possible sources of released tachykinins. The content of extractable tachykinins was measured as SP- and NKA-like immunoreactivities (-LI) by radioimmunoassay. Chromatographic characterization of aqueous acetic acid extracts showed one peak of SP-LI corresponding to authentic SP, whereas there were multiple peaks of NKA-LI, the major one co-eluting with authentic NKA. An increased outflow of both SP- and NKA-LI was evenly produced in a concentration-dependent manner when the preparations were superfused with a high potassium (K) medium in which NaCl had been replaced with equimolar amounts (20-100 mM) of KCl. The high K-evoked release of SP- and NKA-LI was dependent upon the presence of extracellular calcium and was inhibited by about 50% in the presence of the N-type voltage-dependent calcium channel blocker, omega-conotoxin GVIA (0.1 microM). Omega-conotoxin MVIIC (1 microM), a non-selective blocker of N-, P- and Q-type voltage-dependent calcium channels, likewise produced about 40% inhibition of evoked release of both peptides. No evidence for a role of L-type channels in tachykinin release was obtained, since the addition of nifedipine (1 microM) or Bay K8644 (1 microM) did not significantly affect the response to high K. Neither NK1 receptor agonist (septide, 0.1 microM) or antagonist (SR 140333, 10 nM) nor NK2 receptor agonists ([betaAla8]NKA(4-10) and GR 64349, 0.1 microM each) or antagonist (SR 48968, 10 nM) did affect the high K-evoked release of tachykinins. We conclude that SP and NKA are evenly co-released in response to graded depolarization of enteric nerves in the guinea-pig colon. Therefore, the specialization of tachykininergic transmission observed in functional studies does not originate at the prejunctional level. The co-release of tachykinins involves the influx of extracellular calcium via N-type but not L-type calcium channels. No evidence for the presence of NK1 or NK2 autoreceptors affecting tachykinin release from enteric neurons was obtained.

Animals↗
PubMed1998

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, pD2 7.40, Emax 71% of the response to 80 mM KCl) and the synthetic tachykinin NK2 receptor selective agonist [betaAla8]NKA(4-10) (pD2 7.48, Emax 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 [betaAla8]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 micromol/kg i.v.) produced a phasic contraction of the hamster bladder in vivo that was abolished by hexamethonium (110 micromol/kg i.v.) or by surgical ablation of pelvic ganglia. In vivo [betaAla8]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 [betaAla8]NKA(4-10) was comparable to that observed after administration of hexamethonium. Moreover, hexamethonium did not affect the contractile responses to [betaAla8]NKA(4-10) in ganglionectomized animals. MEN 10627 and MEN 11420 produced a dose-dependent and long-lasting inhibition of the contractile response to [betaAla8]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 microl/min) produced repetitive micturition cycles which were abolished by hexamethonium (110 micromol/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.

Animals↗
PubMed1998

Independent coupling of the human tachykinin NK2 receptor to phospholipases C and A2 in transfected Chinese hamster ovary cells.

The human tachykinin NK2 receptor stably expressed in Chinese hamster ovary cells (CHO-hNK2R cells) was characterized by studying the effect of neurokinin A (NKA), the preferred natural ligand, and that of other agonists and antagonists in both binding experiments and functional assays. Competition experiments using [125I]NKA showed that CHO-hNK2R cells express binding sites which have high affinity for NKA (Ki=3.4+/-0.9 nM), GR 64349 (Ki=12+/-3 nM) and [betaAla8]NKA(4-10) (Ki=21+/-8 nM) and for the antagonists MEN 10627 (Ki=0.55+/-0.2 nM), and MEN 11420 (Ki=2.4+/-0.8 nM). In contrast, the tachykinin NK1 and NK3 receptor agonists [Sar9,Met(O2)11]SP and senktide, respectively, were recognized with low affinity (Ki>10 microM). NKA (EC50=68+/-18 nM) induced a rapid and concentration-dependent increase in the intracellular level of inositoltrisphosphate (IP3). The concentration-response curve to GR 64349 (EC50=155+/-14 nM) was close to that of NKA, whereas [betaAla8]NKA(4-10) (EC50=445+/-78 nM) and SP (EC50=3197+/-669 nM) were 7- and 50-fold less potent, respectively. In addition, NKA stimulated the release of arachidonic acid and the production of prostaglandin E2 (PGE2) in a concentration-dependent manner. Also in this assay, NKA was found to be more potent than the other agonists tested (the EC50 values were 3+/-0.3, 9+/-3, 7.8+/-0.9 and 217+/-37 nM for NKA, GR 64349, [betaAla8]NKA(4-10) and SP, respectively). MEN 10627 and MEN 11420 were potent and competitive antagonists in blocking NKA-induced IP3 formation and PGE2 release: MEN 10627 and MEN 11420 displayed comparable potencies in blocking the two functional responses initiated by occupancy of the NK2 receptor by NKA. Pretreatment of the cells with pertussis toxin (500 ng/ml for 18 h) did not significantly modify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE2 release by about 35%. The phospholipase C inhibitor U-73122 (10 microM) prevented the NKA-induced formation of IP3 but did not affect PGE2 release. Conversely, the phospholipase A2 inhibitor quinacrine (100 microM) blocked the release of arachidonic acid and PGE2 without affecting the NKA-stimulated formation of IP3. Chelation of extracellular calcium with 3 mM EGTA inhibited the NKA-induced PGE2 release by 81% but was without effect on basal and NKA-stimulated IP3 production. The calcium channel blockers verapamil (10 microM) and omega-conotoxin GVIA (0.1 microM) did not modify the basal PGE2 production and had no significant effect on the response to tachykinins while the blocker of non-selective cation channels, SKF-96365 (10 microM), inhibited the response to NKA by about 74%. SKF-96365 did not affect the basal or the NKA-induced IP3 formation. In conclusion, our data demonstrate that the human tachykinin NK2 receptor expressed in CHO cells displays binding affinity and functional properties which are those of a native NK2 receptor. No pharmacological evidence for heterogeneity of the human NK2 receptor was obtained in this study. Our findings indicate that the human tachykinin NK2 receptor is independently coupled to both PLC and PLA2 signaling pathways. Activation of the PLA2 pathway may be linked to the opening of a voltage-independent cation channel which activates a Ca2+-dependent PLA2.

Animals↗
PubMed1998

The tachykinin NK1 receptor. Part II: Distribution and pathophysiological roles.

The tachykinin NK1 receptor is widely distributed in both the central and peripheral nervous system. In the CNS, NK1 receptors have been implicated in various behavioural responses and in regulating neuronal survival and degeneration. Moreover, central NK1 receptors regulate cardiovascular and respiratory function and are involved in activating the emetic reflex. At the spinal cord level, NK1 receptors are activated during the synaptic transmission, especially in response to noxious stimuli applied at the receptive field of primary afferent neurons. Both neurophysiological and behavioural evidences support a role of spinal NK1 receptors in pain transmission. Spinal NK1 receptors also modulate autonomic reflexes, including the micturition reflex. In the peripheral nervous system, tachykinin NK1 receptors are widely expressed in the respiratory, genitourinary and gastrointestinal tracts and are also expressed by several types of inflammatory and immune cells. In the cardiovascular system, NK1 receptors mediate endothelium-dependent vasodilation and plasma protein extravasation. At respiratory level, NK1 receptors mediate neurogenic inflammation which is especially evident upon exposure of the airways to irritants. In the carotid body, NK1 receptors mediate the ventilatory response to hypoxia. In the gastrointestinal system, NK1 receptors mediate smooth muscle contraction, regulate water and ion secretion and mediate neuro-neuronal communication. In the genitourinary tract, NK1 receptors are widely distributed in the renal pelvis, ureter, urinary bladder and urethra and mediate smooth muscle contraction and inflammation in response to noxious stimuli. Based on the knowledge of distribution and pathophysiological roles of NK1 receptors, it has been anticipated that NK1 receptor antagonists may have several therapeutic applications at central and peripheral level. At central level, it is speculated that NK1 receptor antagonists could be used to produce analgesia, as antiemetics and for treatment of certain forms of urinary incontinence due to detrusor hyperreflexia. In the peripheral nervous system, tachykinin NK1 receptor antagonists could be used in several inflammatory diseases including arthritis, inflammatory bowel diseases and cystitis. Several potent tachykinin NK1 receptor antagonists are now under evaluation in the clinical setting, and more information on their usefulness in treatment of human diseases will be available in the next few years.

Animals↗
PubMed1998

Dissociation of dorsal root ganglion neurons into afferent and efferent-like neurons.

Although the textbook function of dorsal root ganglion neurons is to signal information to the brain, a group of C-fibre and some A delta-fibre afferents have the capacity to release peptide transmitters from their peripheral terminations and thereby to regulate vascular and other tissue activities. Depending on the species under study, vasoactive dorsal root ganglion neurons in the skin belong to heat nociceptors or a subclass of polymodal nociceptors, and it has been a matter of long-standing discussion how the local effector role of dorsal root ganglion neurons is related to their afferent function. The observation that local vascular regulation may take place independently of nociception has commonly been explained by a number of transductional differences between peripheral peptide release and afferent nerve activity. However, the view that it is only one population of dorsal root ganglion neurons that can work in either mode is increasingly challenged, and after consideration of the available information we hypothesize that some dorsal root ganglion neurons are specialized in controlling peripheral effector mechanisms but have no role in sensation. These local effector neurons represent a novel class of autonomic neurons that lack a preganglionic input, but operate in an efferent-like mode after stimulation by internal or external stimuli.

Afferent Pathways↗
PubMed1998

Pharmacological evidence for the existence of multiple P2 receptors in the circular muscle of guinea-pig colon.

1. By using the sucrose gap technique, we have investigated the effect of the metabolically stable P2Y receptor agonist, adenosine 5'-O-2-thiodiphosphate (ADPbetaS), on the membrane potential and tension in the circular muscle of the guinea-pig proximal colon. All experiments were performed in the presence of atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM), nifedipine (1 microM), L-nitroarginine (L-NOARG, 100 microM) and of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (0.1 microM) and GR 94800 (0.1 microM), respectively. 2. ADPbetaS (100 microM for 15 s) evoked a tetrodotoxin- (1 microM) resistant hyperpolarization and contraction of the smooth muscle. In the presence of apamin (0.1 microM), the ADPbetaS-induced hyperpolarization was converted to depolarization and the contraction was potentiated while tetraethylammonium (TEA, 10 mM) did not affect significantly the response to ADPbetaS. The combined application of apamin and TEA reproduced the effect observed with apamin alone. 3. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acids (PPADS, 30 microM) slightly but significantly increased the ADPbetaS-induced hyperpolarization, while the contraction evoked by ADPbetaS was reduced by about 80%. Suramin (100 microM) did not affect the ADPbetaS-induced hyperpolarization but totally blocked the ADPbetaS-induced contraction. In the presence of suramin (100 microM), a small relaxation of the circular muscle was observed upon application of ADPbetaS. 4. The contraction and hyperpolarization evoked by ADPbetaS were abolished in Ca2+-free Krebs solution. The blocker of sarcoplasmic reticulum Ca2+ pump, cyclopiazonic acid (10 microM) reduced contraction and hyperpolarization induced by ADPbetaS by about 60 and 50%, respectively. 5. A comparison of our present and previous findings enables to conclude that at least 3 types of P2 receptors are present on the smooth muscle of the guinea-pig colon, as follows: (1) inhibitory P2 receptors, producing an apamin-sensitive hyperpolarization, which are activated by alpha,beta-methylene ATP (alpha,beta-meATP) and by endogenously released purines, sensitive to suramin and PPADS; (2) inhibitory P2 receptors, producing an apamin-sensitive hyperpolarization, which are activated by ADPbetaS and are resistant to suramin and PPADS; (3) excitatory P2 receptors, producing contraction, which are activated by ADPbetaS and are sensitive to suramin and PPADS. The data also support the idea of the existence of a restricted pool of specialized junctional P2 receptors producing the apamin-sensitive NANC inhibitory junction potential in response to endogenous ligand(s).

Adenosine Diphosphate↗
PubMed1998

MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist.

1. The pharmacological profile was studied of MEN 11420, or cyclo[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dap-Leu]cyclo(2beta-5beta )], a glycosylated derivative of the potent, selective, conformationally-constrained tachykinin NK2 receptor antagonist MEN 10627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2beta-5beta)). 2. MEN 11420 competitively bound with high affinity to the human NK2 receptor stably transfected in CHO cells, displacing radiolabelled [125I]-neurokinin A and [3H]-SR 48968 with Ki values of 2.5+/-0.7 nM (n = 6) and 2.6+/-0.4 nM (n = 3), respectively. 3. MEN 11420 showed negligible binding affinity (pIC50 < 6) at 50 different receptors (including tachykinin NK1 and NK3 receptors) and ion channels. 4. In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2 receptor-mediated contractions (pK(B) = 8.6+/-0.07, n = 10, and 9.0+/-0.04, n = 12; Schild plot slope = -1.06 (95% c.l. = -1.3; -0.8) and -1.17 (95% c.l. = -1.3; -1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2 receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pK(B) of 10.2+/-0.14, n = 9, and 9.8+/-0.15, n = 9, was calculated in the hamster trachea and mouse urinary bladder, respectively. 5. MEN 11420 showed low affinity (pK(B) < 6) at guinea-pig and rat tachykinin NK1 (guinea-pig ileum and rat urinary bladder) and NK3 (guinea-pig ileum and rat portal vein) receptors. On the whole, the affinities (potency and selectivity) showed by MEN 11420 for different tachykinin receptors, measured either in binding or in functional bioassays, were similar to those shown by the parent compound, MEN 10627. 6. The in vivo antagonism of the contractions produced by [betaAla8]neurokinin A(4-10) (1 nmol kg(-1)) was observed after intravenous (dose range: 1-10 nmol kg(-1)), intranasal (3-10 nmol kg(-1)), intrarectal (30-100 nmol kg(-1)) and intraduodenal (100-300 nmol kg(-1)) administration of MEN 11420. MEN 11420 was more potent (about 10 fold) and longer lasting than its parent compound MEN 10627, possibly due to a greater metabolic stability. 7. A dose of MEN 11420 (100 nmol kg(-1), i.v.), that produced potent and long lasting inhibition of the contraction of the rat urinary bladder induced by challenge with the NK2 selective receptor agonist [betaAla8]neurokinin A(4-10) (10-300 nmol kg(-1)), was without effect on the responses produced by the NK1 receptor selective agonist [Sar9]substance P sulphone (1-10 nmol kg(-1)). 8. These findings indicate that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist. The introduction of a sugar moiety did not produce major changes in the affinity profile of this antagonist as compared to MEN 10627, but markedly improved its in vivo potency and duration of action. With these characteristics, MEN 11420 is a suitable candidate for studying the pathophysiological significance of tachykinin NK2 receptors in humans.

Animals↗