Blockade of nociceptive sensory afferent activity of the rat knee joint by the bradykinin B2 receptor antagonist fasitibant.

OBJECTIVE: The aim of this study was to determine in intact and inflamed knee joints of the rat, the effect of the bradykinin (BK) B2 receptor antagonist fasitibant (MEN16132) on nociceptor mechanosensitivity and hyperalgesia. METHODS: Joint afferent sensory fibers of the medial articular nerve of anesthetized animals were electrophysiologically recorded, measuring nerve impulse activity evoked by passive innocuous and noxious movements of the joint, in intact and kaolin and carrageenan-injected joints. Knee joints of rats were also acutely inflamed by intra-articular injection of carrageenan alone. Long term duration of fasitibant antinociceptive effects were behaviorally evaluated using the incapacitance test. RESULTS: BK (100 µM) injected into the saphenous artery, induced excitation and sensitization of multi- and single unit recordings. Fasitibant (300 µM) injected prior to BK, reduced its excitatory effects as well as the overall increase of movement-evoked activity resulting from repeated injections of BK. Fasitibant did not affect movement-evoked activity of sensory fibers of intact, non-inflamed knee joints. Intra-articular fasitibant (100 µg/knee) significantly reduced the carrageenan-induced inflammatory hyperalgesia measured with the incapacitance test up to four days after treatment. This antinociceptive effect was not obtained with systemic endovenous injection of the drug. CONCLUSIONS: Fasitibant prevents B2 receptor-mediated activation and sensitization of peripheral joint afferents and the ensuing inflammatory hyperalgesia, and may be a useful, novel drug for arthritis pain treatment.

Synovial fluid levels of bradykinin correlate with biochemical markers for cartilage degradation and inflammation in knee osteoarthritis.

OBJECTIVE: To determine the content of bradykinin (BK) and markers of cartilage degradation and inflammation in the synovial fluid (SF) of patients with knee osteoarthritis (OA), and to evaluate correlations with biomarkers or clinical parameters. METHODS: SFs were obtained from 30 patients with knee OA. Levels of basal and generated BK, cartilage oligomeric matrix protein (COMP), interleukin (IL) 1, IL-6, IL-8 and matrix metalloprotease (MMP) 1, MMP-3, MMP-13 and sulfated glycosaminoglycans (GAGs) were measured by enzyme-linked immunosorbent assay (ELISA) or colorimetric assays. RESULTS: The mean concentration of basal BK (in the presence of peptidase and protease inhibitors to avoid degradation and de novo formation of BK) was 422 pg/ml (95% confidence interval, CI, 281-563) whereas that of in vitro generated BK (in the presence of peptidase inhibitors SFs were incubated 60 min at 37°C to measure the potential capability to generate BK) was 3427 pg/ml (2591-4264). The content of MMP-13, IL-1α, and IL-1ß was under assay sensitivity. Basal BK levels positively correlated (Spearman's rank correlation) with GAGs (40 µg/ml, 26-54, r = 0.4834, P = 0.0308) and IL-6 (553 pg/ml, 171-935, r = 0.3946, P = 0.0377) similarly to the generated BK (GAGs, r = 0.4563, P = 0.0431; IL-6, r = 0.5605, P = 0.0019). Statistical analysis of basal BK and biomarkers was significant (P = 0.0483). When applying a stepwise logistic regression analysis considering biomarkers together with clinical parameters, results indicated that K/L radiographic OA grade and COMP improved the model (P = 0.0032). CONCLUSION: The presence of BK in the knee OA SF and its correlations with cartilage degradation and inflammation markers of OA support its participation in OA pathology.

Thermal and electrochemical decomposition of lithium peroxide in non-catalyzed carbon cathodes for Li-air batteries.

The decomposition of lithium peroxide during the charging process of lithium-air batteries is investigated. A novel preparation method for electrodes in the discharged state, i.e., prefilled with Li2O2 using polyethylene oxide as a binder, is presented. The composition and reactivity of Li2O2-prefilled electrodes are examined by thermal analysis coupled with on-line mass spectrometry. Voltage profiles and gas evolution during the charging process of Li2O2-prefilled electrodes in battery cells are correlated with the thermal decomposition process of Li2O2 and its impact on other electrode compounds. It is found that both thermal Li2O2 decomposition and the electrochemical decomposition of Li2O2 during charging enhance the oxidation of the electrolyte, the binder, and/or carbon, which is suggested to be due to the formation of "nascent" oxygen during Li2O2 decomposition into O2 and Li2O (thermally) or into O2 and lithium ions (electrochemically).

Characterization of kinin receptors in human cultured detrusor smooth muscle cells.

BACKGROUND AND PURPOSE: Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor. EXPERIMENTAL APPROACH: Human detrusor cells from biopsies were isolated and maintained in culture. B(1) and B(2) kinin receptors were characterized by means of radioligand and functional experiments (PI accumulation and PGE(2) release). KEY RESULTS: [(3)H]-[desArg(9)]-Lys-BK and [(3)H]-BK saturation studies indicated receptor density (B(max)) and K (d) values of 19 or 113 fmol mg(-1), and 0.16 or 0.11 nM for the B(1) or B(2) receptors, respectively. Inhibition binding studies indicated the selectivity of the B(1) receptor antagonist [desArg(9)Leu(8)]-Lys-BK and of the B(2) receptor antagonists Icatibant and MEN16132. [DesArg(9)]-Lys-BK and BK induced PI accumulation with an EC(50) of 1.6 and 1.4 nM and different maximal responses (E(max) of [desArg(9)]-Lys-BK was 10% of BK). BK also induced prostaglandin E(2) release (EC(50) 2.3 nM), whereas no response was detected with the B(1) receptor agonist. The incubation of detrusor smooth muscle cells with interleukin 1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1)) induced a time-dependent increase in radioligand-specific binding, which was greater for the B(1) than for the B(2) receptor. CONCLUSIONS AND IMPLICATIONS: Human detrusor smooth muscle cells in culture retain kinin receptors, and represent a suitable model to investigate the mechanisms and changes that occur under chronic inflammatory conditions.

Comparative antagonist pharmacology at the native mouse bradykinin B2 receptor: radioligand binding and smooth muscle contractility studies.

BACKGROUND AND PURPOSE: The aim was to characterize the recently discovered non-peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists. EXPERIMENTAL APPROACH: [3H]-BK binding experiments used mouse lung and ileum tissue membranes and antagonist potency was measured in the isolated ileum contractility assay. KEY RESULTS: Two BK binding sites resulted from saturation and homologous competition experiments. A role for the B1 receptor was excluded because of the poor affinity of B1 receptor ligands (pIC50<5). MEN16132, and the other reference antagonists, inhibited only one portion of BK specific binding, and the rank order of potency was (pIC50): Icatibant (lung 10.7; ileum 10.2)=MEN11270 (lung 10.4; ileum 9.9)=MEN16132 (lung 10.5; ileum 9.9).>LF16-0687 (lung 8.9; ileum 8.8)>FR173657 (lung 8.6; ileum 8.2). BK homologous curves performed with lung membranes after treatment with the antagonist MEN16132 or Icatibant (10 nM) displayed only the low affinity site. The functional antagonism by MEN16132 (pA2 9.4) and Icatibant (pA2 9.1), towards BK (control EC50 6.1 nM) induced ileum contractions, was concentration-dependent and surmountable, but the Schild plot slope was less than unity. CONCLUSIONS AND IMPLICATIONS: In mouse tissue, radiolabelled BK recognizes two binding sites and B2 receptor antagonists can compete only for the higher affinity one. The pharmacological profile of the novel non-peptide antagonist MEN16132 indicates that it exhibits subnanomolar affinity and potency for the mouse B2 receptor and is suitable for further characterization in in vivo pathophysiological models.

Draft Genome Sequence of Clostridium difficile Belonging to Ribotype 018 and Sequence Type 17.

Clostridium difficile, belonging to ribotype 018 (RT018), is one of the most prevalent genotypes circulating in hospital settings in Italy. Here, we report the draft genome of C. difficile CD8-15 belonging to RT018, isolated from a patient with fatal C. difficile-associated infection.

Long-term survival of patients with critical limb ischemia treated with iloprost: response rate and predictive criteria. A retrospective analysis of 102 patients.

OBJECTIVE: Critical limb ischemia (CLI) patients have poor long-term prognosis. We showed that iloprost improves outcomes (major amputation and survival) up a 5-year follow-up, but it is not known if in this length of time the survival curves, of clinical responders and non-responders, differ. PATIENTS AND METHODS: A retrospective study enrolling 102 consecutive patients between 2004-2008, with clinical and instrumental (ultrasound, angiography, transcutaneous tensiometry of oxygen TcpO2 and carbon dioxide TcpCO2 in the affected and contralateral limbs) diagnosis of critical ischemia. All patients received the best medical therapy. Iloprost was administered (0.5-2 ng/kg/min 6 hours/day for 2-4 weeks) in all patients initially considered unsuitable for revascularization, repeating it regularly in time every six-twelve months in the case of positive response. The minimum expected follow-up was 4 years. RESULTS: 71.5% of patients were treated with iloprost and the responder rate was 71.2%. Most of the patients were regularly retreated with repeated cycles. Initial median supine TcpCO2 in symptomatic limb was higher in untreated patients than those treated (58 vs. 49 mmHg; p < 0.05) and in non-responders compared to responders (60 vs. 49 mmHg; p < 0.05). TcpCO2 directly and significantly correlated with the highest risk of mortality and seems to represent a new accurate prognostic criterion of unfavourable short and long-term response to prostanoid. In iloprost group, major amputations were significantly reduced. Revascularization was significantly higher in non-responders (57.1% vs. 11.5%; p < 0.05). There was a significantly higher prevalence of subsequent myocardial infarction in the non-iloprost group (27.6% vs. 9.6%; p < 0.05). The survival rate of non-responders was higher than untreated up until the second year (76.2% vs. 62%; p < 0.05). At 4 years we found higher survival in patients treated with iloprost (64.3% vs. 41% in untreated; p < 0.05) and in responders (75% vs. 38.1% in non-responders; p < 0.05). CONCLUSIONS: Our results confirm the favourable role of iloprost on the long-term outcome in patients with CLI. In particular, the maximum benefit is obtained in responder patients treated with multiple cycles of infusion.

Long-term clinical outcomes in critical limb ischemia--A retrospective study of 181 patients.

OBJECTIVE: Critical limb ischemia (CLI) is the most severe manifestation of the peripheral arterial disease. To date, several prognostic factors have been identified but the data of long-term follow-up in real life setting are scarce. The aim of our study is to describe a large group of CLI patients and identify possible prognostic factors, in a long-term follow-up. PATIENTS AND METHODS: Case-control, retrospective study. 181 consecutive CLI patients with a minimum follow-up of 5 years were included in the study. RESULTS: Overall mortality was 15%, 24%, and 43% at 1, 2, and 5 years, respectively. Among known risk factors, only arterial hypertension was significantly correlated with survival rate; no differences were found between diabetics and non-diabetics. Patients treated with intravenous iloprost (46%), compared to untreated patients, showed a better (p < 0.0001) long-term outcome in terms of major amputation (6% vs. 21%), subsequent vascular surgery (4% vs. 32%) and survival rates (69% vs. 47%), at 5-year follow-up. Major amputations were significantly correlated with lower median forefoot transcutaneous values of O2 (0/3 mmHg, p < 0.001) and higher median values of CO2 (83/53 mmHg, p < 0.0001) in supine/dependent position, respectively. CONCLUSIONS: Our results confirm the poor prognosis of CLI patients in a very long-term follow-up and the severe metabolic damage caused by ischemia. A favourable role of iloprost was observed, in agreement with previous evidence in the literature.

Gender-related differential effect of tachykinin NK2 receptor-mediated visceral hyperalgesia in guinea pig colon.

BACKGROUND AND PURPOSE: The tachykinin NK2 receptor antagonist ibodutant is under Phase III clinical investigation to treat female patients with irritable bowel syndrome. The aim of this study was to investigate the NK2 receptor-related gender specificity in a model of colitis. EXPERIMENTAL APPROACH: Colitis was induced by rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 0.5 mL, 30 mg·mL(-1) in 30% ethanol) in female and male guinea pigs. Electromyographic recording of the responses to colorectal distension (CRD) was made 3 days later. Ibodutant (0.33 , 0.65, 1.9 and 6.5 mg·kg(-1) ) was given s.c., 30 min before CRD. Release of neurokinin A and substance P from isolated mucosal and smooth muscle tissues following treatment with KCl (80 mM) or capsaicin (10 µM) was measured by EIA. Plasma pharmacokinetics of ibodutant following a single s.c. administration (0.73 or 2.1 mg·kg(-1) ) were measured over 24 h. KEY RESULTS: Ibodutant did not affect abdominal contractions in control animals. After TNBS-induced colitis, ibodutant prevented the increased visceral hypersensitivity to CRD in females, at lower doses than in males. Ibodutant pharmacokinetics did not differ between females and males. Tachykinins release was greater in smooth muscle than in mucosal samples. Capsaicin-stimulated release of tachykinins from inflamed mucosal samples from females was significantly lower than in males. CONCLUSIONS AND IMPLICATIONS: Ibodutant prevented abdominal nociception in a model of visceral hypersensitivity in guinea pigs with a greater efficacy in females than in males. Our results highlight a gender-related difference in colonic visceral hypersensitivity and mucosal nerve activation.

Iloprost treatment reduces TNF-alpha production and TNF-RII expression in critical limb ischemia patients without affecting IL6.

Iloprost, a stable prostacyclin analogue, regulates expression of genes that are involved in inflammation and in cell growth and inhibits the in vitro production of cytokines. We evaluated the effect of an in vivo weekly iloprost treatment on TNF-alpha and IL6 monocyte production (evaluated by ELISA), on monocyte apoptosis (Annexin V/uptake of propidium iodide by flow cytometry) and on peripheral blood mononuclear cell (PBMC) TNF-alpha receptors (TNF-RI and TNF-RII) mRNA expression (RT-PCR) in 14 atherosclerotic critical limb ischemia patients. PBMC were stimulated with LPS for 24h. TNF-alpha production was significantly reduced by iloprost whereas IL6 production was not affected. Iloprost did not accelerate monocyte apoptosis. TNF-RI mRNA expression was not modified by iloprost, whereas TNF-RII mRNA expression was significantly reduced. Our data show that iloprost may have anti-inflammatory effects in addition to the well-known vasodilatatory and anti-aggregant ones.

Short-term and long-term effects of one-week treatment with intravenous iloprost in critical limb ischemia patients (Leriche-Fontaine stage III and IV).

AIM: Iloprost, usually administered through intravenous infusion for 6 hours per day for at least 21 days, is the main medical treatment for critical limb ischemia in patients unsuitable for surgical or endovascular approach. We evaluated the tolerance and the short-term and long-term effects of a single 1-week treatment in critical limb ischemia patients. METHODS: Twenty-nine patients in Leriche-Fontaine III and IV stage were treated with iloprost infusions for 16 hours per day for 7 days, achieving a maximal dose of 1.5 ng/kg/min. Tolerance and clinical assessment after treatment discontinuation and after 1 and 6 months were recorded; clinical evaluation (rest pain, trophic lesions), ankle/brachial pressure index (ABPI) and treadmill exercise test were performed before, immediately after treatment and after 1 and 6 months. RESULTS: No discontinuation of treatment occurred because of intolerance to iloprost. At the end of the treatment 69% of patients were responders, 55.2% at 1 month, 37.9% after 6 months. ABPI and treadmill maximum walking distance were improved by the treatment at every timepoint. After 6 months 10.3% mortality and 3.4% major amputation rates were recorded. There was a higher percentage of non-responders amongst women vs men, in diabetic patients vs non diabetic and in stage IV patients vs stage III. CONCLUSIONS: One-week treatment with iloprost is safe and effective in both Leriche-Fontaine stage III and IV patients. Clinical effects are persistent over time, often lasting up to the 6th month, similarly to the commonly used 28-day treatment, with clear implications in terms of patient's compliance and medical cost containment.

Extracorporeal blood oxygenation and ozonation (EBOO): a controlled trial in patients with peripheral artery disease.

BACKGROUND: Since 1990 our group has been using extracorporeal circulation to ozonate blood by an original method, known as extracorporeal blood oxygenation and ozonation (EBOO), with the aim of amplifying the results observed with ozone autohemotherapy. OBJECTIVE: To verify the hypothesis that EBOO improves the skin lesions typical of peripheral artery disease (PAD) patients. METHODS: Twenty-eight patients with PAD were randomized to receive EBOO or intravenous prostacyclin in a controlled clinical trial. The primary efficacy parameters were regression of skin lesions and pain,and improvement in quality of life and vascularisation. RESULTS: Patients treated with EBOO showed highly significant regression of skin lesions with respect to patients treated with prostacyclin. Other parameters that were significantly different in the two groups of patients were pain,pruritus, heavy legs and well-being. No significant differences in vascularisation of the lower limbs before and after treatment were found in either group. No side effects or complications were recorded during the 210 EBOO treatments. CONCLUSION: EBOO was much more effective than prostacyclin for treating skin lesions in PAD patients and also had a positive effect on patient general condition without any apparent change in arterial circulation. This suggests other mechanisms of action of EBOO.

Agonist activity at the kinin B1 receptor: structural requirements of the central tetrapeptide.

A series of analogues of desArg(9)-Lys-bradykinin (BK), Lys-Arg-X-Ac(n)c-X-Ser-Pro-Phe, in which the spacer X-Ac(n)c-X replaces the central tetrapeptide Pro-Pro-Gly-Phe of BK, have been synthesized and functionally characterized at the B1 receptor. The 1-aminocycloalkane-1-carboxylic acids (Ac(6)c, Ac(7)c, Ac(8)c, Ac(9)c, Ac(12)c) were incorporated to impart conformational constraint and probe the importance of the hydrophobicity of the residue in the central position. The linker is varied in length (X = Gly, betaAla, gammaAbu) to examine the optimal distance between the biologically important residues at the N- and C-termini. The biological assays indicate that the optimal length is obtained with X = Gly, with reduced activities for the longer linkers. Although the size of the central cyclic amino acid does not significantly alter the biological activity, the hydrophobic residue Ac(n)c which may tether the peptide in the membrane environment is required (Lys-Arg-Gly-Gly-Gly-Ser-Pro-Phe is inactive). Two of the analogues, Lys-Arg-Gly-Ac(7)c-Gly-Ser-Pro-Phe and Lys-Arg-gammaAbu-Ac(7)c-gammaAbu-Ser-Pro-Phe, have been structurally characterized in the presence of a zwitterionic lipid environment by high-resolution NMR. Both compounds have similar structural features, differing greatest in the distance between the termini (9 and 15 A for the Gly- and gammaAbu-containing analogues, respectively). The correlation of the smaller distance with activity at the B1 receptor is in complete accord with the results from our previous examination of Lys-Arg-NH-(CH(2))(11)-CO-Ser-Pro-Phe. With the results from this series of compounds we are beginning to define some of the molecular descriptors important for activity at the B1 BK receptor.

A new class of pseudopeptide antagonists of the kinin B1 receptor containing alkyl spacers.

Four previously reported kinin receptor peptide antagonists, including the B1 receptor-selective peptides desArg10-HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-OH) and B-9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH), have been modified by replacement of the central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue of desArg10-HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-D-Arg-Arg-NH-(CH2)10-CO-Ser-D-Tic-Oic-OH], was found to be slightly more potent than the unmodified peptide (pA2 = 7.1) as a kinin B1 receptor antagonist in the rat ileum longitudinal smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin B1 receptor (rat ileum) and antagonist activity at the kinin B2 receptor (guinea pig ileum longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg10-HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an improved pharmacological profile in terms of both potency and selectivity, and it represents a good template for the development of new peptidomimetic kinin B1 receptor antagonists. We also report an attempt to investigate the conformational role of the flexible, linear spacer of MEN 11575 and to design more constrained analogues, possibly locked in the bioactive conformation, using semirigid spacers based on Calpha-tetrasubstituted alpha-amino acids of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc).

Involvement of capsaicin-sensitive nerves in the bronchomotor effects of arachidonic acid and melittin: a possible role for lipoxin A4.

1. Functional studies have been performed to evaluate the potential involvement of capsaicin-sensitive nerves in the bronchomotor responses evoked by lipid mediators produced from the metabolic breakdown of arachidonic acid (AA) in the guinea-pig bronchus. 2. In the presence of indomethacin, the exogenous administration of AA (0.01-1 mM) produced a concentration-dependent contractile response in guinea-pig isolated bronchial rings. AA-induced contractions were augmented by epithelium-removal and by thiorphan (10 microM), an inhibitor of tachykinin breakdown. A sustained downward and rightward displacement of the complete concentration-response curve to AA was observed after in vitro capsaicin desensitization. 3. BWA4C (1 microM), a selective inhibitor of 5-lipoxygenase, shifted the AA concentration-response curve to the right. In the presence of this inhibitor, capsaicin desensitization did not have any further inhibitory action. 4. A potent, concentration-dependent and capsaicin-sensitive bronchoconstrictor effect was also observed with the polypeptide, melittin (10 nM-1 microM), an activator of phospholipase A2, which therefore should generate endogenous AA. 5. In vitro capsaicin-desensitization produced a significant reduction of the bronchomotor responses evoked by lipoxin A4 (1-6 microM), but not of those elicited by other lipoxygenases products such as leukotriene D4 (1-100 nM) or by 15-hydroxyeicosatetraenoic acid (15-HETE, 1-6 microM). 6. These findings indicate that lipoxin A4 but not leukotriene D4 or 15-HETE, might be one of the lipoxygenase mediators of excitatory effects of AA on capsaicin-sensitive sensory nerves.

Evidence for a capsaicin-sensitive, tachykinin-mediated, component in the NANC contraction of the rat urinary bladder to nerve stimulation.

1. In the presence of atropine (1 microM) and guanethidine (3 microM), electrical field stimulation (EFS) of the rat isolated urinary bladder for 30 s induced a frequency-dependent (1-30 Hz) nonadrenergic non-cholinergic (NANC) triphasic contraction characterized by a peak response (within 10 s from onset of stimulation), a late response (determined as the tension developed at the end of the stimulation period) and a prolonged post-stimulus 'off' response. The latter peaked at 2-6 min from the end of the stimulation period. At 10 Hz, the amplitude of the three responses averaged 89 +/- 6, 76 +/- 6 and 18 +/- 3% of the response to 40 mM KCl, respectively. Tetrodotoxin (1 microM) abolished all contractile responses to EFS. 2. In capsaicin-pretreated bladder strips (10 microM for 15 min) the amplitude of the peak response to EFS (1-30 Hz for 30 s) was unchanged, the 'late' response to EFS was significantly reduced as compared to controls, and the post-stimulus response was absent, being replaced by a transient relaxation. 3. When varying train duration from 1 to 120 s at a frequency of 10 Hz, the differences between control and capsaicin-treated strips became evident for periods of stimulation > 10 s. 4. The tachykinin NK1 receptor antagonist, SR 140,333 (0.1-1 microM) had no effect on the peak response to EFS (10 Hz for 30 s) while it decreased significantly the late response at both concentrations tested (16 +/- 3 and 33 +/- 3% inhibition). At 1 micro M, SR 140,333 also significantly reduced (29 +/- 9% inhibition)the peak of the post-stimulus contraction. The tachykinin NK2 receptor antagonist, MEN 10,627(0. 1-1 9 MicroM) had no significant effect on the peak response to EFS (10 Hz for 30 s), and decreased the late response at 1 MicroM only (32 +/- 4% inhibition). MEN 10,627 inhibited the post-stimulus response at both concentrations tested and almost abolished it at 1 MicroM.5. The combined administration of SR 140,333 and MEN 10,627 (1 MicroM each) produced a small reduction(22 +/- 3% inhibition) of the peak response to EFS, a marked reduction (48 +/- 3% inhibition) of the late response and the abolition of the post-stimulus response which was replaced by a post-stimulus relaxation as observed in capsaicin-pretreated strips.6. SR 140,333 (0.1 and 1.0 MicroM) produced a large rightward shift in the concentration-response curve tothe NKI receptor agonist, [Sar9]substance P sulphone (apparent pKB 8.97 +/- 0.14), without affecting the response to the NK2 receptor-selective agonist, [Beta Ala8]neurokinin A (4-10). MEN 10,627 (0.1 and 1 MicroM)produced a large rightward shift of the concentration-response curve to [Beta Ala8]neurokinin A (4-10)(apparent pKB 8.95 +/- 0.16) without affecting the response to [Sarl substance P sulphone. SR 140,333 and MEN 10,627 (1.0 MicroM each) did not affect the contraction produced by exogenous ATP (1 mM).7. These findings provide evidence that the NANC contraction of the rat isolated urinary bladder to transmural nerve stimulation has two components, which are sharply differentiated by blockade of the efferent function of sensory nerves following in vitro capsaicin administration. The first component,probably mediated by endogenous ATP, is fully activated during short periods of nerve activity (< 10 s)and does not involve capsaicin-sensitive nerve afferents. The second component, which is capsaicin sensitive and tachykinin-mediated, is evident as a late 'on' response during nerve stimulation and as a post-stimulus 'off response for periods of stimulation >lOs. Activation of both NK1 and NK2receptors contributes to the capsaicin-sensitive responses.

Tachykinin NK1 receptor subtypes in the rat urinary bladder.

1. Following the recent proposal that the selective agonist septide, ([pGlu6,Pro9]SP(6-11)), acts on a novel tachykinin receptor distinct from the 'classical' NK1 receptor, the aim of the study was to investigate the possible heterogeneity of tachykinin NK1 receptors in the rat urinary bladder. 2. The synthetic tachykinin receptor agonists, septide (pD2 7.87) and [Sar9]substance P (SP) sulphone (pD2 7.64) produced concentration-dependent contractions of the rat isolated urinary bladder. 3. The NK1 receptor antagonists GR82,334, (+/-)-CP96,345, and RP67,580 competitively antagonized (slopes of Schild plot not significantly different from unity) the response to septide with the rank order of potency (pKB values in parentheses): RP 67,580 (7.57) > GR 82,334 (7.01) > (+/-)-CP 96,345 (6.80). The same antagonists were significantly less potent when tested against [Sar9]SP sulphone, while maintaining the same rank order of potency: RP 67,580 (7.00) > GR 82,334 (5.93) > (+/-)-CP 96,345 (< 6). The antagonists did not affect the concentration-response curve to bombesin. 4. To exclude the involvement of the NK2 receptor, a second series of experiments was performed in the presence of the potent nonpeptide NK2 receptor antagonist, SR 48,968. SR 48,968 (1 microM) produced a rightward shift of the concentration-response curve to the NK2 receptor selective agonist, [beta Ala8]neurokinin A (NKA) (4-10). SR 48,968 did not significantly modify the response to SP, NKA, neurokinin B (NKB), neuropeptide K (NPK), neuropeptide gamma (NP gamma), SP(4-11), SP(6-11), septide or [Sar9]SP sulphone. 5. In the absence or presence of SR 48,968, RP 67,580 antagonized in a competitive manner the response to septide, [Sar9]SP sulphone, SP(4-11) and SP(6-11): pKB values obtained in the absence and presence of SR 48,968 were not significantly different for any of these four agonists.6. RP 67,580 antagonized the response to SP and NKA both in the absence and presence of SR 48,968.In both cases, the slopes of the Schild plots were significantly different from unity. Mean dose-ratios produced by RP 67,580 in the presence of SR 48,968 were larger than those measured without NK2receptor blockade for both SP and NKA.7. RP 67,580 (3 MicroM) did not antagonize the response to NKB in the absence of SR 48,968. In the presence of SR 48,968, RP 67,580 acted as a competitive antagonist of NKB-induced contractions with apKB value (7.63) not significantly different from that measured towards septide. In the present of SR48,968, RP 67,580, GR 82,334 and (+/-)-CP 96,345 antagonized the response to NKB with a rank order of potency identical to that measured towards septide or [Sar9]SP sulphone.8. In the absence of SR 48,968, RP 67,580 (3 MicroM) produced a small shift of the concentration-response curve to neuropeptide K and was ineffective toward neuropeptide T. In the presence of SR 48,968 a clear shift of the curve to both agonists was observed.9. These findings are compatible with the idea that a septide-sensitive tachykinin receptor may exist in the rat urinary bladder. The septide-sensitive receptor is recognized by NK1 receptor antagonists with higher affinity than the 'classical' NK1 receptor recognized by [Sar9]SP sulphone. Our data suggest that NKB, after NK2 receptor blockade, is a more suitable ligand than SP for activation of the 'septidesensitive'receptor. While the final proof for the existence of possible NK1 receptor subtypes must await confirmation at the molecular level, the present findings provide strong pharmacological evidence that either NK, receptor subtypes or a novel type of tachykinin receptor exist in the rat urinary bladder.

The longitudinal muscle of rat ileum as a sensitive monoreceptor assay for bradykinin B1 receptors.

1. Various bradykinin derivatives, acting preferentially at B1 or B2 receptors, were tested in the isolated longitudinal smooth muscle of rat ileum. Experiments were carried out in the presence of chlorpheniramine and atropine (both 1 microM), guanethidine and indomethacin (both 3 microM) and of the peptidase inhibitors (captopril, bestatin and thiorphan, all 1 microM). 2. The rank order of potency was (pD2 values +/- s.e.mean, n = 5 in parentheses, at 5 h from set-up): [des-Arg9]-BK (8.27 +/- 0.11) > or = [des-Arg10]-kallidin (7.67 +/- 0.24) > bradykinin (6.69 +/- 0.25). The B2 receptor selective agonist, [Hyp3,Tyr(Me)8]-BK, was approximately 10 fold less active than bradykinin. Contractile responses to all agonists increased with time. The maximal response to the B1 receptor agonist, [desArg9]-BK at 5 h (94 +/- 2%) was significantly (P < 0.05) greater than that measured at 2 h (74 +/- 2%). 3. The B2 receptor antagonist, D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK (Hoe 140, 0.1 microM) did not affect responses to the B1 receptor agonist [des-Arg9]-BK (0.1 nM--1 microM) nor those to the B2 receptor agonist, [Hyp3,Tyr(Me)8]-BK (1 nM--10 microM). In control experiments performed in the longitudinal smooth muscle of guinea-pig ileum and rat isolated urinary bladder as bioassays for B2 receptors, the B2 receptor antagonist Hoe 140 (0.1 microM) antagonized bradykinin-induced contractions. 4. In the rat isolated ileum the B1 receptor antagonist, D-Arg[Hyp3, Thi5, D-Tic7, Oic8, des-Arg9]-BK ([des-Arg10]-Hoe 140, 0.3 - 10 microM) competitively antagonized contractile responses to [des-Arg9]-BK with an estimated pKB of 6.74 +/- 0.08 (Schild plot slope with confidence limits 1.22, (0.70 - 1.73) n = 13). In control experiments in the guinea-pig isolated ileum and rat isolated urinary bladder, [des-Arg10]-Hoe 140 (1 - 10 microM) did not inhibit B2 receptor-mediated contractile responses. 5. The putative B1 receptor antagonist, [Leu8,des-Arg9]-BK, behaved as a partial agonist when responses were determined 2 h from set-up (pD2 6.43 +/- 0.21, n = 5; Emax 30% of that evoked by [des-Arg9]-BK); at 5 h from set-up it behaved as a full agonist (pD2 7.48 +/- 0.12, n = 5; Emax 90% of that evoked by [des-Arg9]-BK). At this time the response to [Leu8,des-Arg9]-BK was antagonized in a concentration-dependent manner by [des-Arg10]-Hoe 140, which at 1 microM and 10 microM, produced dose-ratios of 6.33 +/- 3.66 (n = 4) and 103 +/- 40 (n = 4). 6. In view of the rank order of potency of agonists, the antagonist activity by [des-Arg10]-Hoe 140 and the lack of antagonist activity of Hoe 140, we conclude that the longitudinal smooth muscle of rat ileum, after histamine, acetylcholine, noradrenaline, and prostanoid production blockade, is a sensitive monoreceptor assay for studying the pharmacology of bradykinin B1 receptors. Further the preparation can also be used as a sensitive bioassay to identify partial agonist activity of B1 receptor antagonists such as [Leu8,desArg9]-BK.

Pharmacological analysis of the local and reflex responses to bradykinin on rat urinary bladder motility in vivo.

1. The topical application of bradykinin (BK) (0.05-5000 pmol/rat) onto the serosal surface of the urinary bladder in urethane-anaesthetized rats, evoked low amplitude tonic contractions (not exceeding 25 mmHg) or high amplitude (about 50 mmHg), phasic reflex contractions (chemoceptive micturition reflex) which were abolished by bilateral ablation of the pelvic ganglia. In ganglionectomized rats, BK induced only a local, tonic-type contraction. 2. Systemic capsaicin pretreatment (164 mumol kg-1, 4 days before) reduced the incidence of chemoceptive reflex induced by BK (500 pmol/rat) but had no effect on the magnitude of the tonic-type contraction elicited by BK in ganglionectomized rats. Indomethacin (11 mumol kg-1, 20 min before) reduced the incidence but not the amplitude of the reflex contractions induced by topical application of BK (500 pmol/rat). In ganglionectomized rats, indomethacin (11 mumol kg-1, 20 min before) decreased the amplitude of the tonic contraction evoked by BK. Indomethacin did not affect the chemoceptive reflex induced by topical application of capsaicin (15 nmol/rat) onto the bladder. 3. Intrathecal administration of the tachykinin NK1 receptor antagonists, RP 67,580 (10 nmol/rat) or SR 140,333 (10 nmol/rat), abolished the chemoceptive reflex induced by BK without modifying the magnitude of the tonic contraction. SR 140,333 (10 nmol/rat) also abolished the occurrence of the chemoceptive reflex induced by capsaicin. 4. Intravenous administration of the B2 receptor antagonist, Hoe 140 (35 nmol kg-1, 10 min before) abolished the reflex and local effects induced by BK on bladder motility but failed to modify the chemoceptive reflex induced by topical application of capsaicin (15 nmol/rat). 5. Intrathecal administration of Hoe 140 (10 nmol/rat) reduced the incidence of the chemoceptive reflex induced by BK but had no effect on the amplitude of the local motor response. Likewise, Hoe 140(10 nmol/rat, i.t.) reduced the incidence of reflex bladder contractions induced by topical application of capsaicin (15 nmol/rat) without affecting the magnitude of the tonic-type contraction.6. These findings indicate that BK stimulates motility through B2 receptors in the rat urinary bladder.BK activates the reflex response by stimulating capsaicin-sensitive afferent nerves with a contribution from prostanoids. At the spinal cord level, tachykinin NK1 and BK B2 receptors could also be involved in the chemoceptive reflex induced by BK or capsaicin.

Characterization of the tachykinin NK2 receptor in the human bronchus: influence of amastatin-sensitive metabolic pathways.

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a 'NK2A' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A.