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.

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.

Bradykinin and B2 receptor antagonism in rat and human articular chondrocytes.

BACKGROUND AND PURPOSE: In osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis. EXPERIMENTAL APPROACH: BK receptor density and affinities of BK, its analogues and antagonists were measured in cultured human and rat chondrocytes by radioligand binding. Effects of BK were assessed by accumulation of inositol phosphates (IP) and release of interleukin (IL)-6 and IL-8. KEY RESULTS: Density of [³H]-BK binding sites was higher (13-30-fold) and BK evoked a greater (48-fold) IP production, in human than in rat chondrocytes. The BK B2 receptor antagonists MEN16132 and icatibant displayed similar binding affinity. MEN16132 was 40-fold more potent than icatibant in the IP assay. In human chondrocytes, BK increased release (over 24 h) of IL-6 and IL-8, effects blocked by MEN16132 but not by the B1 receptor antagonist Lys-[Leu8][desArg9]BK. BK-induced release of IL-6, but not of IL-8, was partially inhibited by indomethacin (10 µM) and nordihydroguaiaretic acid (10 µM). Antagonists for the prostanoid EP receptors (AH6809 10 µM; L-798,196, 200 nM; L-161,982, 1 µM) were ineffective. Dexamethasone (100 nM) partially inhibited release of both IL-6 and IL-8. Inhibitors of intracellular downstream signalling pathways (SB203580 10 µM; PD98059, 30 µM; SP600125, 30 µM; BAY-117085, 5 µM) indicated the involvement of p38 MAPK and the activation of NF-κB. CONCLUSION AND IMPLICATIONS: BK mediated inflammatory changes and cartilage degradation and B2 receptor blockade would, therefore, be a potential treatment for OA.

Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts.

BACKGROUND AND PURPOSE: Bradykinin (BK) and B2 receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B2 receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells. EXPERIMENTAL APPROACH: Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes. KEY RESULTS: [3H]-BK saturation studies indicated receptor density (Bmax) and K(d) values of 121,550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK(I) 8.9) was threefold more potent than icatibant (pK(I) 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC50 0.45 nM), with pK(B) values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC50 216 nM) and IL-8 (EC50 53 nM) release. Both MEN16132 (IL-6: pIC50 8.1; IL-8: pIC50 8.4) and icatibant (IL-6: pIC50 6.6; IL-8: pIC50 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C (U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase (LY294002), NF-kappaB (BAY-117085) and by the glucocorticoid dexamethasone. CONCLUSIONS AND IMPLICATIONS: Bradykinin via B2 receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B2 receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.

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.

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.

New conformationally homogeneous beta-turn antagonists of the human B2 kinin receptor.

We have designed and synthesized a conformationally homogeneous series of cyclic pentapeptides of the general structure c[Pro-aa(i)-D-Tic-Oic-aa(i + 3)] which adopt a type-II' beta-turn conformation believed important for high affinity antagonism of the bradykinin (BK) B2 receptor. We incorporated D-Tic and octahydroindole-2-carboxylic acid (Oic) residues (present in known active antagonists) in a cyclic pentapeptide that would place the D-aa in the i + 1 position of the beta-turn and a proline as a bridge between the C- and N-termini sides of the turn. In positions i and i + 3 alkyl, aromatic, polar or charged amino acids could be introduced without dramatically changing the overall structure. Ten analogues were studied using 1H nuclear magnetic resonance (NMR) and evaluated for their binding affinity for the human B2 receptor. The NMR data in dimethylsulfoxide (DMSO) confirmed the structural homogeneity within the class and, on the basis of this, one representative member of the series was chosen for a detailed structure determination using NMR data in sodium dodecylsulphate (SDS) micelles and molecular dynamics calculations. Despite the structural similarity, the binding affinity of the ten analogues was strongly influenced by the nature of the side-chains in positions i and i + 3, with the doubly charged analogue 49 (pKi = 6.2) proving best. This compound may serve as the starting point for the discovery of new non-peptide bradykinin B2 receptor antagonists.

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor.

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.

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.

Nonpeptide antagonists for kinin receptors.

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.

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.

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.

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).

Inflammation modifies the role of cyclooxygenases in the contractile responses of the rat detrusor smooth muscle to kinin agonists.

The contractile responses elicited by the selective kinin B1 and B2 receptor agonists [desArg9]-bradykinin ([desArg9]-BK) and [Hyp3, Tyr(Me)8]-bradykinin ([Hyp3, Tyr(Me)8]-BK) (1 nM-10 microM), respectively, were evaluated in control vs. inflamed (cyclophosphamide 150 mg kg-1 i.p., 48 h before the sacrifice) rat isolated urinary bladder strips. The contractile responses to the B2 receptor agonist did not differ in control vs. inflamed bladders, whereas the contractile responses to [desArg9]-BK were potentiated in inflamed bladders. The selective B1 and B2 receptor antagonists B 9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-OH) and Hoe 140 (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH), both at 1 microM, inhibited the response to the B1 and B2 receptor agonists, respectively, in both control and inflamed bladders. In addition, the concentration-response curve to [Hyp3, Tyr(Me)8]-BK was shifted to the right and depressed by B 9858 in inflamed bladders. The nonselective cyclooxygenase (COX) inhibitors S-(-)-ketoprofen (10 microM) and piroxicam (30 microM) markedly depressed the concentration-response curves to [desArg9]-BK and [Hyp3, Tyr(Me)8]-BK in control bladders, but neither drug affected the B1 or B2 receptor agonist-mediated responses in inflamed bladders. The selective inhibitor of the inducible COX-2 isoenzyme, NS-398 (1 microM), did not inhibit the contractile responses to [desArg9]-BK and [Hyp3, Tyr(Me)8]-BK in either control or inflamed bladders, whereas it significantly potentiated the response to the B1 receptor agonist in inflamed bladders. The exogenous administration of prostaglandin E2 (PGE2) induced S-(-)-ketoprofen-resistant contractile responses that were depressed in inflamed bladders. Pretreatment with S-(-)-ketoprofen restored the PGE2-mediated contractile responses of inflamed bladders to control values. PGE2 assay revealed that the basal production of PGE2 is significantly higher after inflammation than in control conditions. [desArg9]-BK and [Hyp3, Tyr(Me)8]-BK (1 microM each) both stimulated PGE2 production, and their effect was larger in inflamed than in control bladders. Piroxicam (30 microM) prevented the PGE2 production evoked by [desArg9]-BK in both control and inflamed bladders and likewise abolished that produced by [Hyp3, Tyr(Me)8]-BK. NS-398 (1 microM) reduced the PGE2 production elicited by [desArg9]-BK in control and inflamed bladders. When NS-398 was tested on the [Hyp3, Tyr(Me)8]-BK-induced PGE2 production, it inhibited PGE2 production in the inflamed bladders only, without significantly modifying the response obtained in controls. These findings demonstrate that 1) in normal bladders, the activation of B1 and B2 receptors evokes contraction that is largely mediated by COX-1 metabolites, whereas the COX-2 appears to be involved in PGE2 production after the activation of B1 receptor only, without interfering with contraction, and 2) in inflamed bladders, the activation of B1 and B2 receptors still produce PGE2, but the contractile response is not reduced by COX inhibitors, a result that indicates that additional mechanisms play a compensatory role.

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.

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.

Effect of longitudinal muscle-myenteric plexus removal and indomethacin on the response to tachykinin NK-2 and NK-3 receptor agonists in the circular muscle of the guinea-pig ileum.

1. The effect of removal of the longitudinal muscle-myenteric plexus (LM-MP) and/or indomethacin (10 microM) on the response to the tachykinin NK-2 receptor selective agonist, [beta Ala8]NKA(4-10), or to the NK-3 receptor selective agonist, senktide, was investigated by measuring mechanical activity (isotonic recording) of circular muscle (ring preparation) of the guinea-pig ileum. 2. Indomethacin (10 microM) increased the percentage of ileal rings displaying spontaneous activity, either intact or LM-MP-free. The response to senktide (10 nM and 1 microM) was lower in LM-MP-free than in intact ileal rings, either in the absence or presence of indomethacin. The response to a low concentration (10 nM) of [beta Ala8] NKA (4-10) was enhanced in LM-MP-free rings and by indomethacin. 3. In intact ileal rings, the response to senktide was unaffected by atropine (3 microM) alone or by the tachykinin NK-2 receptor antagonist MEN 10,376 (10 microM) alone while it was reduced by the combined administration of the two antagonists. The response to senktide was greatly reduced by tetrodotoxin (TTX, 1 microM). Senktide-induced contractions (10 nM) were also reduced by the blocker of N-type voltage-sensitive calcium channels, omega-contoxin (CTX, 0.1 microM). 4. In about 30% of preparations tested, an inhibitory response (decrease in spontaneous activity) to 10 nM senktide, was disclosed in CTX-treated intact ileal rings. This inhibitory effect was TTX-sensitive. 5. In LM-MP-free ileal rings, the response to senktide was abolished or reduced by atropine and MEN 10,376, alone or in combination, and was also reduced or abolished by TTX and CTX. 6. The response to [beta Ala8]NKA (4-10) was inhibited by MEN 10,376, in both intact and LM-MP-free ileal rings while it was unaffected by atropine, TTX or CTX. 7. These results indicate that indomethacin pretreatment induces a regular background activity for studying the motor response to tachykinins in the circular muscle of the ileum, probably by blocking the formation of relaxant prostanoids. A further increase in sensitivity to direct smooth muscle stimulation (NK-2 receptor agonist) can be obtained by removal of the LM-MP. The response to NK-3 receptor stimulation is diminished but not abolished by removal of the LM-MP, suggesting that NK-3 receptors are located on neuronal bodies of myenteric neurons, but possibly also at other sites (possibly, nerve terminals).(ABSTRACT TRUNCATED AT 400 WORDS)

Nitric oxide is the mediator of tachykinin NK3 receptor-induced relaxation in the circular muscle of the guinea-pig ileum.

The tachykinin NK3 receptor agonist, senktide, produces concentration-dependent contraction of the circular muscle of the guinea-pig ileum (EC50 2.59 nM). In the presence of the blocker of neuronal type of voltage-sensitive calcium channels, omega-conotoxin (0.1 microM), the contractile response to a low concentration of senktide was converted to an inhibitory effect on spontaneous activity of the ileum. This inhibitory effect was further enhanced in the presence of atropine (1 microM) and was abolished by tetrodotoxin (1 microM), indicating its neural origin. In the presence of atropine and omega-conotoxin, the inhibitory response to senktide (1 nM) was greatly inhibited or even abolished by L-nitroarginine (30 microM), its effect being prevented by L-arginine but not by D-arginine (300 microM in each case). Apamin (0.1 microM) failed to significantly affect the inhibitory response to senktide. Apamin enhanced spontaneous activity of the preparation while L-nitroarginine had no effect. Neither apamin nor L-nitroarginine affected the inhibitory response to isoprenaline. These findings indicate that inhibition of circular muscle activity produced through NK3 receptor stimulation in the guinea-pig ileum is mediated through a neuronal pathway involving nitric oxide or a nitric oxide-like substance(s) generation.

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.