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M Criscuoli

Publications and source records attributed to M Criscuoli.

At least 19 recordsLinked to original sources
PubMed2003

Pharmacological profile of MEN 11066, a novel potent and selective aromatase inhibitor.

MEN 11066 is a new non-steroidal compound which potently inhibits human placenta (K(i)=0.5 nM) and rat ovarian (K(i)=0.2 nM) aromatase in vitro. In vivo, a single oral dose of 0.3 mgkg(-1) significantly decreased uterus weight in immature rats after stimulation of uterus growth by androstenedione. MEN 11066 reduced in a dose-dependent manner plasma estradiol levels in adult female rats treated with pregnant mare serum gonadotropin (PMSG). After 2 weeks of repeated daily treatment in adult rats, a significant decrease in uterine weight was observed together with a 65% decrease in plasma estradiol, whereas plasma levels of testosterone, progesterone, aldosterone, corticosterone, cholesterol, LH and FSH were not affected. The lack of any effect by MEN 11066 on adrenal steroids was confirmed by the unchanged plasma corticosterone and aldosterone levels in immature rats and also in adult rats when the repeated treatment with MEN 11066 (15 days) was followed by the administration of a synthetic ACTH analogue. No change in 11beta-hydroxylase or 21-hydroxylase activities was produced in vitro by the addition of 10 microM MEN 11066. Fifteen-day treatment with MEN 11066 did not produce changes in several rat hepatic enzymatic activities involved in the metabolism of xenobiotics. These results demonstrated that MEN 11066 is a potent inhibitor of aromatase which does not interfere with the cytochrome P450 involved in the synthesis of other steroids or in the metabolism of xenobiotics.

Administration, Oral↗
PubMed2001

Effect of a tachykinin NK(2) receptor antagonist, nepadutant, on cardiovascular and gastrointestinal function in rats and dogs.

The effect of the tachykinin NK(2) receptor antagonist, nepadutant (MEN 11420 or (c[[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta)])) was assessed on cardiovascular function (unanaesthetized rats and anaesthetized dogs) and gastrointestinal motor activity (fasted unanaesthetized dogs). The selective tachykinin NK(2) receptor agonist, [betaAla(8)]neurokinin A (4-10), up to 100 nmol/kg, i.v., did not produce changes on mean blood pressure or heart rate in unanaesthetized rats. Nepadutant did not affect blood pressure and heart rate up to 10 micromol/kg, whereas saredutant (SR 48968 or ((S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl] benzamide), a nonpeptide antagonist, produced a transient reduction of mean blood pressure and heart rate. Nepadutant up to 20 micromol/kg, i.v. neither caused changes of cardiovascular and respiratory parameters in anaesthetized dogs nor induced any changes in left ventricular systolic pressure, left ventricular dP/dt or of electrocardiogram (lead II) waveforms. Intravenous administration of neurokinin A (9 nmol/kg) in unanaesthetized dogs stimulated gastrointestinal motility for 20-25 min. Nepadutant at 0.1 micromol/kg suppressed the stimulant effects of neurokinin A but, up to a dose of 10 micromol/kg, did not produce significant changes in the basal migrating motor complexes. We conclude that tachykinin NK(2) receptors do not participate in the physiologic regulation of resting cardiovascular and respiratory functions and that they do not regulate the fasted pattern of gastrointestinal motility. The cardiovascular changes induced by the nonpeptide tachykinin NK(2) receptor antagonist, saredutant, likely arise from nonspecific effects unrelated to tachykinin NK(2) receptor blockade.

Animals↗
PubMed2001

Nepadutant pharmacokinetics and dose-effect relationships as tachykinin NK2 receptor antagonist are altered by intestinal inflammation in rodent models.

Tachykinin NK2 receptor antagonists could reduce motility and symptoms during gastrointestinal diseases characterized by local inflammation such as diarrhea or colitis; however, how these conditions change pharmacodynamic and pharmacokinetic characteristics of NK2 receptor antagonists is unknown. We investigated the effect of the peptide NK2 receptor antagonist nepadutant on spontaneous intestinal motility or [betaAla8]NKA(4-10)-induced colonic and bladder contractions in rodent models of intestinal inflammation (enteritis induced by castor oil and rectocolitis induced by local instillation of acetic acid in rats, enteritis induced by bacterial toxins in mice). In the castor oil model, the oral/intraduodenal bioavailability of nepadutant was also determined. The intrarectal (i.r.) administration of nepadutant (100 nmol/kg) did not reduce [betaAla8]NKA(4-10) (10 nmol/kg i.v.)-induced colonic and bladder contractions in normal animals, but the same dose of nepadutant produced an inhibitory effect in the two organs following rectocolitis; in contrast, nepadutant is equieffective by the intravenous route in normal and colitic animals. In this model, nepadutant (100 nmol/kg i.r. or i.v.) decreased spontaneous colonic hypermotility, without affecting motility in controls. The intraduodenal administration of nepadutant (30 nmol/kg), which was ineffective on [betaAla8]NKA(4-10) (10 nmol/kg i.v.)-induced colonic and bladder contractions in control animals, abolished bladder contractions in castor oil-pretreated animals. In this latter group, the oral and intraduodenal bioavailability of nepadutant showed a 7- to 9-fold increase with respect to controls. Oral administration of nepadutant, in nanomolar or subnanomolar dosage, reduced diarrhea induced by bacterial toxins in mice. It is concluded that intestinal inflammation increases nepadutant absorption in the intestine, enhancing its activity. These results suggest that a drug with a limited oral bioavailability could be used for treating gastrointestinal diseases associated with a local inflammation.

Acetates↗
PubMed2000

Effect of the aromatase inhibitor, MEN 11066, on growth of two different MCF-7 sublines.

The racemate compound MEN 11066 (1-[(benzofuran-2-yl)(4'-cyanophenyl)methyl]-1H-1,2,4-triazole) and its enantiomers, (+)-MEN 11623 and (-)-MEN 11622, showed potent and selective aromatase activity on human placental microsomes. In addition, to better evaluate their potency as anticancer drugs, the compounds were assayed on testosterone-induced cell proliferation to measure their ability in inhibiting oestrogen-dependent tumour growth. Two different sublines originated from the human breast carcinoma MCF-7 were used. One, named MCF-7(tumour aromatase) (TA), that had maintained its intrinsic aromatase activity, was more sensitive to estradiol or testosterone-induced growth than the second subline named MCF-7(human placental aromatase) (hPA). The latter had been transfected with the human placental aromatase cDNA, after recognizing that the parental cells had aromatase activity reduced to undetectable levels. The MEN compounds completely reverted the testosterone-induced proliferation in both MCF-7(TA) and MCF-7(hPA) cells, while they did not affect the estradiol-triggered proliferation as a proof of their specificity for aromatase enzyme. Interestingly, MCF-7(TA) cells were more susceptible to the effects of aromatase inhibitors than the MCF-7(hPA) cell. These data suggest the efficacy of aromatase inhibitors in breast cancer when the growth dependency from oestrogen is high and a relatively low aromatase activity may be extremely important for tumour development.

Androstenedione↗
PubMed2000

Pharmacological evaluation of the role of cyclooxygenase isoenzymes on the micturition reflex following experimental cystitis in rats.

Prostanoids, generated from cyclooxygenase (COX) isoenzymes, play a role in the physiological function of the lower urinary tract and are important mediators of inflammatory hyperalgesia. The present work evaluates the effects of the COX-1/COX-2 inhibitor dexketoprofen as well as of a selective COX-2 inhibitor, NS-398, on urodynamic function following endotoxin (LPS) or cyclophosphamide (CYP)-induced inflammation of the urinary bladder. The application of arachidonic acid (330 microgram rat(-1)) onto the serosal surface of the urinary bladder in control rats elicited bladder contractions which could be blocked in a dose-dependent manner by dexketoprofen (0.1 - 3 mg kg(-1), i.v.) but not by NS-398 (0.2 - 6 mg kg(-1), i.v. ). Dexketoprofen (3 mg kg(-1), i.v.) decreased the micturition frequency and increased the pressure threshold for triggering the micturition either when administered within 15 min or 3 h following surgery in control animals. NS-398 (6 mg kg(-1), i.v.) decreased the micturition frequency and increased the pressure threshold when administered 3 h but not 15 min following surgery. Administration of LPS (2 mg kg(-1), i.v., 90 - 120 min) increased both the micturition frequency and the pressure threshold for triggering the micturition reflex. Changes in urodynamic parameters induced by LPS were prevented by doses of either dexketoprofen (1 mg kg(-1), i.v.) or NS-398 (2 mg kg(-1), i.v.) which were ineffective in control animals. Pretreatment with CYP (150 mg kg(-1), i.p., 48 h) increased the micturition frequency, pressure threshold, and the minimal intravesical pressure but decreased the mean amplitude of micturition contractions. In CYP-treated rats, dexketoprofen (1 mg kg(-1), i.v.) or NS-398 (2 mg kg(-1), i.v.) blocked the CYP-induced urodynamic changes with exception of the micturition contraction amplitude. These results indicate that COX-1 may be involved in modulating the threshold for activating the micturition reflex in the normal rats and also demonstrates that inhibition of COX-2 prevents or reverses the urodynamic changes associated with bladder inflammation induced either by surgery, LPS or CYP treatments.

Animals↗
PubMed2000

Multiple sites of action in the inhibitory effect of nociceptin on the micturition reflex.

PURPOSE: Nociceptin, the endogenous peptide ligand for the opioid receptor-like1 (ORL1) receptors, exerts a naloxone-resistant suppressant effect on micturition reflex after intravenous administration. This work aims to elucidate the mechanism and the site of action of the inhibitory effect of nociceptin on the micturition reflex. MATERIALS AND METHODS: The bladder of urethane-anesthetized rats was cannulated through the dome (cystometries) or the urethra in isovolumetric conditions (distension-induced reflex contractions, DIRCs). In this latter model, the effect of the application of nociceptin onto the serosal surface of the urinary bladder was determined. The effect of intravenous, intrathecal and intracerebroventricular administration of nociceptin on ongoing cystometries at two different infusion rates (50 and 250 microL/min.) was assessed. The effect of the intravenous administration of nociceptin on cystometries was also studied in capsaicin-pretreated animals. RESULTS: When cystometric recordings were obtained at a low infusion-rate (50 microL/min.), the intravenous administration of nociceptin (10 to 100 nmol./kg.) induced a dose-dependent reduction in the micturition frequency associated to an increase of the pressure threshold for activating the micturition reflex, whereas the amplitude of micturition contractions was unaffected. These effects faded within 60 minutes. The intracerebroventricular administration of nociceptin (0.3 nmol./rat) produced urodynamic changes similar to those observed after the intravenous route and, in addition, also reduced the amplitude of micturition contractions. The intrathecal administration of nociceptin up to 1 nmol./rat was ineffective. Capsaicin pretreatment (164 micromol./kg., s.c. 5 to 6 days before) significantly reduced the micturition frequency as compared with controls. In capsaicin pretreated animals intravenous nociceptin was ineffective. When cystometries were recorded at a high infusion-rate (250 microL/min.) either intravenous (100 nmol./kg.), i.t. (1 nmol./rat) nociceptin or capsaicin pretreatment had no effect. In contrast, intracerebroventricular nociceptin (0.3 and 1 nmol./rat) inhibited the micturition reflex by reducing both the frequency and the amplitude of micturition contractions: these effect were not modified by naloxone (0.5 micromol./kg., i.v.). The topical application of nociceptin (5 and 50 nmol./rat) caused a dose-dependent inhibition of DIRCs. CONCLUSION: Nociceptin inhibits the micturition reflex at a peripheral and at a supraspinal site. The effects observed after the intravenous administration of nociceptin indicate that the functional integrity of capsaicin-sensitive bladder afferents is required for exerting its inhibitory activity at the peripheral level. In contrast, the supraspinal effect of nociceptin involves both the afferent and the efferent pathways of the micturition reflex, possibly through a direct effect on ORL1 receptors located in the pontine micturition center.

Animals↗
PubMed2000

A distribution study with (14)C-otilonium bromide in the rat: evidence for selective tropism for large intestine after oral administration.

The aim of this study was to determine the plasma levels and the tissue distribution of otilonium bromide, measured as total radioactivity, after oral administration of 2 mg/kg of (14)C-labeled drug to rats. Radioactivity levels were very low in the plasma (ranging from 2.7 ng Eq/ml at 1.5 h to 0.6 ng Eq/ml at 24 h) as compared with those found in the gastrointestinal (GI) tract, indicating negligible systemic otilonium bromide absorption. Results from both quantitative radioluminography of whole body tissue distribution and radioassay of dissected parts of the GI tract carried out with liquid scintillation counting clearly demonstrate the presence of radioactive compounds in the walls of the GI tract at all sacrifice times. In the other tissues and organs examined, radioactivity was only found in trace amounts in the liver. The presence of radioactivity in the GI walls reflected the transit kinetics of drug-enriched contents. The radioactivity in large intestine walls was measurable at otilonium bromide concentrations in the range of micromole equivalents/kg, from 4 to 8 h after drug administration. Total body radioactivity recovery was 95, 101, 24, and 9% at 1.5, 4, 8, and 24 h, respectively. In conclusion, orally administered (14)C-otilonium bromide is poorly absorbed systemically, as indicated by the very low plasma radioactivity levels, but it is able to effectively penetrate into the large intestine walls, a recognized target for drugs oriented toward irritable bowel syndrome therapy.

Administration, Oral↗
PubMed1999

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.

Animals↗
PubMed1999

Kinin B1 receptor-mediated motor responses in normal or inflamed rat urinary bladder in vivo.

The rat urinary bladder is one of the few in vivo preparations in which kinin B1 receptor-mediated contractile responses have been described, but the nature (local or reflex) of these responses has not been characterized. We have investigated the motor effects of i.v. or topical (onto the bladder serosa) administration of the selective kinin B1 receptor agonist [des-Arg9]-bradykinin ([des-Arg9]-BK) in the normal or inflamed (cyclophosphamide-induced) urinary bladder in urethane-anaesthetized rats. In both normal and inflamed bladders [des-Arg9]-BK produced a tonic contraction of low amplitude (< 15 mmHg) with phasic contractions of high amplitude (> or = 15 mmHg) superimposed (micturition reflex contractions). In inflamed bladders, the response to [des-Arg9]-BK was more prominent than in controls. Similar observations were made after the topical administration of [des-Arg9]-BK. In order to evaluate any time-dependency in the expression of B1 receptor-mediated bladder responses, [des-Arg9]-BK was administered in separate groups of control animals at 30 and 240 min after the completion of surgical procedures required for set-up of the preparation: no bladder contraction was detected at 30 min whereas both local and reflex contractions could be elicited by [des-Arg9]-BK at 240 min after the set up. In ganglionectomized rats, the response to [des-Arg9]-BK or the selective tachykinin NK2 receptor agonist [betaAla8]NKA(4-10) was evaluated at 30 and 240 min after the set up in inflamed or in control animals. The response to [des-Arg9]-BK was greater after inflammation although a time-dependent increase was evident in both groups; in contrast, the response to [betaAla8]NKA(4-10) was similar in both groups and remained constant over the observation period. After induction of inflammation, the tonic contraction induced by [des-Arg9]-BK in ganglionectomized rats was dose-dependently reduced by the kinin B1 receptor antagonist [desArg10]Hoe 140. The contractile response (number of micturition reflex contractions) induced by [des-Arg9]-BK in normal rats with intact pelvic nerves at 240 min from the set up was not changed after the administration of the selective B2 receptor antagonist Hoe 140. These results indicate that stimulation of bladder kinin B1 receptors evokes a local, tonic-type contraction with reflex contractions superimposed in both normal and inflamed bladders, but in the latter situation the motor responses are magnified.

Administration, Topical↗
PubMed1999

Effect of dexamethasone on cyclophosphamide-induced cystitis in rats: lack of relation with bradykinin B1 receptor-mediated motor responses.

We investigated the role of bradykinin B receptors in inducing urinary bladder contraction and maintaining bladder compliance in anaesthetized rats following cyclophosphamide-induced bladder inflammation and the influence of dexamethasone treatment on these responses. In the group treated with cyclophosphamide the amplitude of the contraction induced by the selective bradykinin B1 receptor agonist des-Arg9-bradykinin was larger than that in controls and dexamethasone prevented the up-regulation of this response induced by inflammation. The specific binding of [3H]des-Arg10-kallidin to bladder membranes was only detected in cyclophosphamide-treated rats: this binding was prevented by dexamethasone pretreatment. The bladder contraction induced by des-Arg9-bradykinin in cyclophosphamide-treated rats was antagonized by the bradykinin B1 receptor antagonist des-Arg9-D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (des-Arg10-Hoe 140). Cyclophosphamide treatment increased the bladder weight and dexamethasone reversed this effect. Bladder compliance was decreased in the bladder inflammation group and this effect was partially reversed by dexamethasone pretreatment. Neither des-Arg10-Hoe 140 nor the combined administration of des-Arg10Hoe 140 and the selective bradykinin B2 receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (Hoe 140) affected bladder compliance, thus excluding a role of kinins in the maintenance of bladder tone during inflammation. These results indicate that: (1) dexamethasone pretreatment ameliorates cyclophosphamide-induced bladder inflammation: (2) dexamethasone pretreatment prevents cyclophosphamide-induced up-regulation of bradykinin B receptors; (3) kinins do not contribute to the increased vesical tone during inflammation.

Animals↗
PubMed1999

Capsaicin pretreatment does not alter rat urinary bladder motor responses induced by a kinin B1 receptor agonist after endotoxin treatment.

The kinin B1 receptor is generally expressed after inflammation or tissue injury. Kinin B1 receptor stimulation induces excitatory motor responses in the urinary bladder and, in this preparation, the effect of many excitatory transmitters involves the stimulation of capsaicin-sensitive afferent nerves. In this study we have investigated the effect of capsaicin pretreatment on the bladder contractions induced by [Sar0, D-Phe8, des-Arg9]bradykinin (SDABK), a kinin B1 receptor agonist, by inducing the expression of B1 receptors via the intravesical administration of a bacterial endotoxin (LPS, 1 mg/ml) in urethane-anaesthetized rats. Three and half hours after LPS, the bladder was filled with saline until the micturition reflex was evoked, then 0.15 ml of saline was withdrawn, in order to avoid spontaneous reflex contractions. In LPS-pretreated rats the threshold volume for micturition was lower than in the control group (248 +/- 44 vs. 534 +/- 112 microl). After capsaicin pretreatment the bladder capacity was increased in both control and LPS-treated groups and the LPS-induced hyperreflexia was abolished (threshold volumes: 901 +/- 96 vs. 837 +/- 120 microl, respectively). The administration of SDABK (30 nmol/kg i.v., 4 h after LPS or saline application) produced a local, low amplitude tonic contraction (< 15 mmHg) or a tonic contraction with superimposed high amplitude (> or = 15 mmHg) reflex contractions but no effect of LPS or capsaicin pretreatment was observed in the incidence of these responses. The amplitude of the local response was increased by LPS treatment (1.4 +/- 0.3 vs. 4.0 +/- 0.7 mmHg) but capsaicin pretreatment did not modify this effect (2.3 +/- 0.4 vs. 4.3 +/- 0.6 mmHg). Likewise, the number of reflex contractions induced by SDABK was increased after LPS treatment (1.1 +/- 0.4 vs. 2.7 +/- 0.5) irrespective of capsaicin pretreatment (1.3 +/- 0.4 vs. 2.8 +/- 0.6). These results indicate that: (1) topical application of LPS induces a bladder hyperreflexia that is sensitive to capsaicin pretreatment; (2) B1 receptor-mediated motor responses (either reflex or local) are enhanced after LPS treatment; (3) capsaicin pretreatment does not modify B1 receptor-mediated motor response (either reflex or local).

Administration, Intravesical↗
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

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

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↗