piperidines and phosphoramidon

Toluene is a representative airborne occupational and domestic pollutant that causes eye and respiratory tract irritation. We investigated whether a single inhalation of toluene elicits microvascular leakage in the rat airway. We also evaluated the effects of CP-99,994, a tachykinin NK(1) receptor antagonist, and ketotifen, a histamine H1 receptor antagonist with mast cell-stabilizing properties, on the airway response. The content of Evans blue dye that extravasated into the tissues was measured as an index of plasma leakage. Toluene (18-450 ppm, 10 min) concentration-dependently induced dye leakage into the trachea and main bronchi of anesthetized and mechanically ventilated rats. Toluene at concentrations of ≥ 50 and ≥ 30 ppm caused significant responses in the trachea and main bronchi, respectively, which both peaked after exposure to 135 ppm toluene for 10 min. This response was abolished by CP-99,994 (5 mg/kg i.v.), but not by ketotifen (1mg/kg i.v.). Nebulized phosphoramidon (1 mM, 1 min), a neutral endopeptidase 24.11 inhibitor, significantly enhanced the response induced by toluene (135 ppm, 10 min) compared with nebulized 0.9% saline (1 min). These results show that toluene can rapidly increase airway plasma leakage that is predominantly mediated by tachykinins endogenously released from airway sensory nerves. However, mast cell activation might not be important in this airway response.

Topics: Animals; Bronchi; Dose-Response Relationship, Drug; Glycopeptides; Inhalation Exposure; Ketotifen; Male; Mast Cells; Microvessels; Neurogenic Inflammation; Piperidines; Rats; Rats, Wistar; Solvents; Tachykinins; Time Factors; Toluene; Trachea

Using perfused hearts from streptozotocin-induced long-term diabetic rats, we studied the coronary vasoconstrictor effect of the endothelin-1 (ET-1) precursor big ET-1 and also whether this response was modulated by N(epsilon)-(carboxymethyl)lysine (CML; a representative advanced glycation end product that is implicated in the pathogenesis of diabetic vasculopathy). The big ET-1-induced vasoconstriction (a) developed more rapidly (i.e., was greater in the first 30 min) in the diabetic group than in the age-matched controls, and (b) in each group was largely suppressed by phosphoramidon [nonselective endothelin-converting enzyme (ECE)/neutral endopeptidase (NEP) inhibitor] or CGS35066 (selective ECE inhibitor), but not by thiorphan (selective NEP inhibitor). The ET-1 release occurring after treatment with big ET-1, which was greater in diabetic coronary arteries than in the controls, was reduced by CGS35066. The dose-response curve for ET-1 was shifted to the left in the diabetics, so that at some lower doses of ET-1 the vasoconstriction was greater than in the controls. CML enhanced big ET-1- or ET-1-induced vasoconstriction in the controls, but not in the diabetics. Finally, the plasma level of CML was higher in diabetic than in control rats. These findings suggest (a) that the increased responsiveness to big ET-1 shown by diabetic coronary arteries may be attributable both to a more rapid conversion of big ET-1 to ET-1 (by ECE), allowing it to exert its contractile activity, and to an increased vascular sensitivity to ET-1, and (b) that CML may be at least partly responsible for the diabetes-associated enhancement of big ET-1-mediated coronary vasoconstriction.

Topics: Animals; Aspartic Acid Endopeptidases; Benzofurans; Blood Glucose; Body Weight; Coronary Vessels; Diabetes Mellitus, Experimental; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Heart; Lipids; Lysine; Male; Metalloendopeptidases; Myocardium; Oligopeptides; Organ Size; Organophosphonates; Peptides, Cyclic; Perfusion; Piperidines; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Thiorphan; Vasoconstriction

Leptin, a multifunctional hormone that regulates food intake and energy expenditure, has emerged recently as an important modulator of gastric mucosal responses to Helicobacter pylori infection. We applied the animal model of H. pylori LPS-induced gastritis to investigate the role of endothelin-1 (ET-1) in the mucosal leptin production. We show that the histologic pattern of inflammation reached a maximum on the fourth day following the LPS and was reflected in a marked increase in the mucosal level of ET-1 and leptin. Therapeutic administration of phosphoramidon, an inhibitor of ECE-1 activity, led to a 61.2% decline in the mucosal ET-1 level and a 64.1% reduction in leptin, while the severity of mucosal inflammatory involvement increased by 28.6%. A drop in the level of leptin and the increase in severity of the inflammatory involvement elicited by the LPS was also attained in the presence of ET(A) receptor antagonist BQ610, but not the ET(B) receptor antagonist BQ788. Moreover, administration of ERK inhibitor, PD98059, in the presence of ET(B) receptor antagonist, but not the ET(A) receptor antagonist, caused reduction in the mucosal leptin level. Our findings are the first to implicate ET-1 as a key factor in up-regulation of gastric mucosal leptin-associated H. pylori infection. We also show that the effect of ET-1 on leptin production is a consequence of ET(A) receptor activation.

Topics: Animals; Aspartic Acid Endopeptidases; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gastric Mucosa; Gastritis; Glycopeptides; Helicobacter pylori; Leptin; Lipopolysaccharides; Metalloendopeptidases; Oligopeptides; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Up-Regulation

Leptin, a multifunctional hormone that regulates food intake and energy expenditure, has emerged recently as an important modulator of inflammatory cascades associated with wound healing. In this study, we applied the animal model of buccal mucosal ulcer to investigate the role of endothelin-1 (ET-1) and leptin in soft oral tissue repair. Using groups of rats with experimentally induced buccal mucosal ulcers we show that ulcer onset was characterized by a marked increase in the mucosal level of ET-1 and leptin. However, while the ET-1 level gradually declined with healing, the mucosal level of leptin increased reaching maximum expression on the 4th day of healing. Therapeutic administration of phosphoramidon, an inhibitor of ECE-1 activity, not only led to a 53.2% drop in the ET-1, but also produced a dose-dependent reduction (up to 50.9%) in the mucosal level of leptin and up to 42.3% decline in the rate of ulcer healing. A marked drop (54.2%) in the mucosal level of leptin and the reduction (46.8%) in the rate of ulcer healing was also attained in the presence of ETA receptor antagonist BQ610 administration, but not the ETB receptor antagonist BQ788. Moreover, administration of ERK inhibitor, PD98059 in the presence of ETB receptor antagonist, but not the ETA receptor antagonist, caused the reduction the mucosal leptin level as well as a decline in the rate of ulcer healing. Our findings are the first to implicate the requirement for both ET-1 and leptin in orderly progression of the events of soft oral tissue repair. We also show that ET-1 is a key factor in up-regulation of leptin production associated with oral mucosal ulcer healing , and that the effect of ET-1 on leptin production is a consequence of ETA receptor activation and subsequent signaling through MAPK/ERK.

Topics: Acetic Acid; Animals; Aspartic Acid Endopeptidases; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Flavonoids; Glycopeptides; Leptin; Metalloendopeptidases; Mitogen-Activated Protein Kinase Kinases; Mouth Mucosa; Oligopeptides; Oral Ulcer; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Time Factors; Up-Regulation; Wound Healing

Substance P (SP) and its preferred NK1 receptor are widely expressed throughout the fear-processing pathways of the brain and its role in the modulation of experimental anxiety has been demonstrated. SP, like other peptides, are cleaved by peptidases in two fragments: C-terminal (SP 6-11) and N-terminal (SP 1-7) that could be responsible for its anxiogenic-like response. In this study we investigate the effects of i.c.v. micro-injections of SP free acid (SPfa), which is resistant to enzymatic cleavage, the influence of the pretreatment with peptidase inhibitors (PIs), thiorphan and/or phosphoramidon, as well as the effects of SP 6-11 and SP 1-7 and the participation of NK1 and NK2 receptors on their behavioral effects. Adult male Wistar rats were treated with 10 pmol solutions of SP 6-11, SP 1-7 or 1 and 10 pmol of SPfa and evaluated in the elevated plus maze (EPM) test. Other experimental groups received thiorphan 0.2 pmol, phosphoramidon 2 pmol or both PIs 30 min prior SP 1-11, 10 pmol i.c.v. The C-terminal fragment (SP 6-11, 10 pmol) and SPfa (1 pmol) promoted an anxiogenic-like profile of action similar to 10 pmol of SP 1-11, i.e., a decrease of entries and time spent on the open arms, whereas the N-terminal fragment (SP 1-7) was inactive at the EPM. The effect of SP 6-11 was inhibited by pretreatment (100 pmol) with NK1 (FK 888) and NK2 (SR 48968) antagonists. Moreover, both PIs enhanced the SP effect when used alone, but their combination produced an apparent reversion of anxiogenic-like effect produced by SP. Altogether, our results give further support to the SP role in the modulation of experimental anxiety in rats.

Topics: Analysis of Variance; Animals; Behavior, Animal; Benzamides; Dipeptides; Dose-Response Relationship, Drug; Drug Interactions; Glycopeptides; Indoles; Injections, Intraventricular; Male; Maze Learning; Microinjections; Motor Activity; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Protease Inhibitors; Pyrrolidonecarboxylic Acid; Rats; Rats, Wistar; Receptors, Neurokinin-2; Substance P; Thiorphan; Time Factors

Human chymase produces not only angiotensin II but also endothelin(ET)-1(1-31). We previously reported that ET-1(1-31) had several biological activities in vascular smooth muscle cells. In this study, we investigated the vasoconstrictor effect of ET-1(1-31) on the renal resistance vessels using in vitro microperfused rabbit afferent and efferent arterioles. ET-1(1-31) decreased the lumen diameter of the afferent and efferent arterioles dose-dependently. ET-1(1-31)-induced afferent arteriolar vasoconstriction was not affected by phosphoramidon, an ET converting enzyme inhibitor. ET-1(1-31)-induced renal arteriolar vasoconstriction was inhibited by BQ123, an ETA receptor inhibitor, but not by BQ788, an ETB receptor inhibitor. These results suggest that ET-1(1-31)-induced renal arteriolar vasoconstriction may be mediated by ETA-like receptors.

Topics: Animals; Arterioles; Aspartic Acid Endopeptidases; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Glycopeptides; Humans; Kidney; Male; Metalloendopeptidases; Muscle, Smooth, Vascular; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Perfusion; Piperidines; Rabbits; Receptor, Endothelin A; Receptors, Endothelin; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents

Endothelin-1-(1-31) is a new bioactive 31-amino-acid-length peptide generated from big endothelin-1 by chymase or other chymotrypsin-type proteases with various pathophysiologic functions. In this study, we have detected the specific and monophasic binding of [125I]endothelin-1-(1-31) in porcine lung membranes. Competition studies of [125I]endothelin-1-(1-31) binding by unlabeled endothelin-1-(1-31), endothelin-1, endothelin-3, and antagonists and agonists of endothelin ET(A) and ET(B) receptors suggest that the binding protein is an endothelin ET(B) or ET(B)-like receptor rather than an endothelin ET(A) receptor in porcine lungs. Kinetic studies showed that the affinity of endothelin-1-(1-31) to its receptor was approximately one order of magnitude lower than that of endothelin-1, and that the specific binding of endothelin-1-(1-31) was about 19% of endothelin-1 binding. The binding of [125I]endothelin-1-(1-31) was extremely slow, slower even than that of endothelin-1, and nearly irreversible. This unique quasi-irreversibility may explain the slow-onset and long-lasting biologic effects of this peptide in vivo.

Topics: Animals; Binding, Competitive; Cell Membrane; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-3; Enzyme Inhibitors; Glycopeptides; Humans; Iodine Radioisotopes; Lung; Metalloendopeptidases; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Radioligand Assay; Receptor, Endothelin B; Receptors, Endothelin; Swine

1. This study was designed to investigate the mechanisms for the contractions induced by tachykinins (substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)) in the rabbit corpus cavernosum strips, using fura-PE3 fluorimetry and alpha-toxin permeabilization. 2. Tachykinins induced contractions in the rabbit corpus cavernosum in a concentration-dependent manner. The potency order was SP>NKA>NKB. 3. The tachykinin-induced contractions were enhanced by phosphoramidon (PPAD), an endopeptidase inhibitor, but not by N(omega)-nitro-L-arginine methylester (L-NAME). 4. The NK(1) receptor selective antagonist, SR 140333 significantly inhibited the tachykinin-induced contractions. Although the NK(2) receptor selective antagonist, SR 48968 alone did not influence the effects of tachykinins, it potentiated the inhibitory effect of SR 140333. The NK(3) receptor selective antagonist, SR142801 had no effect. 5. In the rabbit corpus cavernosum, tachykinins induced sustained increases in [Ca(2+)](i) and tension in normal PSS, while only small transient increases in [Ca(2+)](i) and tension were observed in Ca(2+)-free solution. 6. In alpha-toxin permeabilized preparations, tachykinins induced an additional force development at a constant [Ca(2+)](i). 7. These results indicated that in the rabbit corpus cavernosum: (1) Tachykinins induced contractions by increasing both the [Ca(2+)](i) and myofilament Ca(2+) sensitivity; (2) The tachykinin-induced [Ca(2+)](i) elevations were mainly due to the Ca(2+) influx; (3) Tachykinin-induced contractions were mainly mediated through the activation of NK(1) receptor expressed in the rabbit corpus cavernosum smooth muscle, and affected by the endopeptidase activity and (4) Tachykinins may thus play a role in controlling the corpus cavernosum tone.

Topics: Animals; Benzamides; Calcium; Dose-Response Relationship, Drug; Glycopeptides; In Vitro Techniques; Male; Muscle Contraction; Neurokinin A; Neurokinin B; Neurokinin-1 Receptor Antagonists; NG-Nitroarginine Methyl Ester; Penis; Permeability; Piperidines; Quinuclidines; Rabbits; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Substance P; Tachykinins; Type C Phospholipases

Endothelin (ET)-1[1-21] stimulates steroid secretion and zona glomerulosa growth and is expressed in the human and rat adrenal cortex together with its receptor subtypes A and B (ETA and ETB). Although ET-1[1-21] is generated from bigET-1 by an ET-converting enzyme (ECE-1), there is evidence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, the role of which in adrenal pathophysiology is largely unknown. Gene expression and immunohistochemical studies allowed localization of chymase in the normal human adrenal cortex. Sizable amounts, not only of ET-1[1-21] but also of ET-1[1-31], were found in the adrenal vein plasma of three patients. ET-1[1-21] and ET-1[1-31] elicited a clear-cut secretory response by dispersed human adrenocortical cells, ET-1[1-31] being significantly less potent than ET-1[1-21]. The secretagogue effect of ET-1[1-31] was abolished by the ETA receptor antagonist BQ-123 and was unaffected by the ETB receptor antagonist BQ-788. Because, in humans, the secretagogue effect of ET-1[1-21] involves both ETA and ETB receptors, the weaker action of ET-1[1-31] could be attributable to a selective ETA receptor activation. Two lines of evidence support this contention: 1) ET-1[1-31] was more effective than ET-1[1-21] in stimulating ETA-mediated cell proliferation of human adrenocortical cells cultured in vitro; and 2) autoradiography showed that a) ET-1[1-31] displaced in vitro [(125)I]ET-1[1-21] binding to the ETA, but not ETB receptors, in human internal thoracic artery rings; and b) BQ-123, but not BQ-788, eliminated [(125)I]ET-1[1-31] binding in the rat adrenal cortex.

Topics: Adrenal Cortex; Aldosterone; Autoradiography; Cell Division; Cells, Cultured; Chymases; Endothelin-1; Endothelins; Glycopeptides; Humans; Hydrocortisone; Immunohistochemistry; In Vitro Techniques; Muscle, Smooth, Vascular; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine Endopeptidases

Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.

Topics: Administration, Cutaneous; Animals; Anti-Inflammatory Agents, Non-Steroidal; Capillary Permeability; Capsaicin; Croton Oil; Dermatitis, Allergic Contact; Dermatitis, Irritant; Enzyme Inhibitors; Female; Glycopeptides; Injections, Intravenous; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neprilysin; Neurokinin-1 Receptor Antagonists; Piperidines; Quinuclidines; Skin; Substance P

In the mammalian female reproductive tract, tachykinin neuropeptides, such as substance P (SP), are localized to a population of sensory fibers and their precise physiological role is still unknown. The aim of the present study was to characterize the population of tachykinin receptors in the pregnant rat uterus and to assess their regulation during the course of pregnancy and after delivery. The expression of the tachykinin NK(1) receptor (NK(1)R), the tachykinin NK(2) receptor (NK(2)R), and the tachykinin NK(3) receptor (NK(3)R) in uteri from rats at different stages of pregnancy and on Day 1 postpartum was investigated by using a semiquantitative reverse transcription-polymerase chain reaction. The contractile effect of tachykinin receptor agonists acting selectively on the NK(1)R, the NK(2)R, or the NK(3)R was investigated by conventional organ bath techniques. Serum levels of estrogen and progesterone were measured by RIA. Our data show that the expression and function of NK(1)R and NK(3)R varied along the course of pregnancy and at postpartum. Uterine NK(2)R mRNA levels remain stable during the course of pregnancy and at Day 1 postpartum; and the contractions elicited by activating selectively the NK(2) receptor in the presence of the neutral endopeptidase inhibitor phosphoramidon (1 microM) were similar in early, mid, or late pregnancy. These results show that the expression and function of tachykinin receptors within the uterus vary with reproductive state and length of gestation, supporting a role for tachykinins in pregnancy and/or parturition in the rat.

Topics: Animals; Estradiol; Female; Gene Expression; Glycopeptides; Piperidines; Pregnancy; Progesterone; Quinuclidines; Rats; Rats, Wistar; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Tachykinin; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Uterine Contraction; Uterus

We investigated whether the polycation poly-L-arginine elicited cutaneous vascular hyperpermeability and scratching behavior and, if so, whether these responses involved mast cells and sensory nerves in rats. Intradermal injections of poly-L-arginine induced vascular hyperpermeability and scratching behavior. Combined treatment with chlorpheniramine and methysergide almost completely suppressed the poly-L-arginine (50 microg/site)-induced plasma leakage. Capsaicin desensitization and the tachykinin NK(1) receptor antagonist LY303870, (R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane, partially inhibited the leakage. In mast cell-deficient rats, poly-L-arginine only minimally induced plasma leakage. On the other hand, capsaicin desensitization and LY303870, but not chlorpheniramine or methysergide, suppressed the poly-L-arginine (200 microg/site)-induced scratching. Moreover, poly-L-arginine elicited the scratching even in mast cell-deficient rats. These results suggest that substance P is at least partly involved in both the cutaneous plasma leakage and the scratching behavior induced by poly-L-arginine. Moreover, mast cell-derived amines are suggested to be involved in the plasma extravasation but scarcely, if any, in the scratching behavior.

Topics: Animals; Behavior, Animal; Capillary Permeability; Capsaicin; Chlorpheniramine; Dose-Response Relationship, Drug; Glycopeptides; Histamine; Histamine H1 Antagonists; Indoles; Injections, Intradermal; Male; Mast Cells; Methysergide; Neurons, Afferent; Peptides; Piperidines; Rats; Rats, Wistar; Serotonin; Serotonin Antagonists; Skin; Substance P

We identified the predominance of neurokinin-2 receptors and evaluated the inhibition of spontaneous contraction via the blockade of neurokinin-2 receptors in human ureteral segments.. Excess ureteral segments from human subjects undergoing donor nephrectomy or reconstructive procedures were suspended in tissue baths containing Krebs buffer. After spontaneous contractions were recorded, tissues were incubated with 1 microM. solutions of phosphoramidon and captopril (to inhibit peptide degradation) and either the neurokinin-1 receptor antagonist CP 99,994, the neurokinin-2 receptor antagonist SR 48,968, the neurokinin-3 receptor antagonist SR 142,801 or dimethyl sulfoxide (control) for 1 hour. Contraction magnitude and frequency were again recorded and compared with spontaneous levels. Concentration-response curves to the tachykinins substance P, and neurokinins A and B were determined in the presence and absence of antagonists.. Neurokinin A increased contractility at lower concentrations than substance P or neurokinin B (p <0.013). Neurokinin-2 receptor blockade produced a 100-fold rightward shift of the concentration-response curves (p <0.013), while neurokinins 1 and 3 receptor blockade had no effect. SR 48,968 significantly reduced contractility during the 1-hour incubation period, causing a 97% reduction in spontaneous rates compared with a 29% reduction in control tissues. CP 99,994 and SR 142,801 had no significant effect.. Neurokinin-2 is the predominant receptor subtype responsible for tachykinin induced contraction of human ureteral smooth muscle. In vitro treatment with the neurokinin-2 antagonist SR 48,968 reduces the spontaneous contraction rate by 97% in vitro. Neurokinin-2 receptor antagonists may have clinical applications for ureteral disease.

Topics: Angiotensin-Converting Enzyme Inhibitors; Benzamides; Captopril; Dose-Response Relationship, Drug; Glycopeptides; Humans; Metalloendopeptidases; Muscle Contraction; Piperidines; Receptors, Neurokinin-2; Tachykinins; Ureter

To evaluate whether neutral endopeptidase (NEP) inhibitors have adverse respiratory effects, the influence of a NEP inhibitor on bradykinin (BK)-induced bronchoconstriction was investigated. In anesthetized and artificially ventilated guinea pigs, changes in airway opening pressure (Pao) were measured as an index of bronchoconstriction. An infusion of phosphoramidon (3 mg kg(-1) h(-1)), a NEP inhibitor, significantly enhanced the bronchoconstriction induced by high-dose BK (30 nmol kg(-1), i.v.). Capsaicin (0.1 mg kg(-1), i.v.) and SR48968 (0.3 mg kg(-1), i.v.), an NK2 receptor antagonist, significantly inhibited the phosphoramidon-induced enhancement of BK-induced bronchoconstriction, although FK888 (3 mg kg(-1), i.v.), an NK1 receptor antagonist, did not. Both neurokinin A (NKA) (0.1-3 nmol kg(-1), i.v.) and substance P (SP) (0.1-3 nmol kg(-1), i.v.) induced dose-dependent bronchoconstriction which was enhanced by phosphoramidon infusion, although these enhancements were more prominent in the NKA series. Phosphoramidon partially inhibited BK degradation in lung homogenate, and both NKA and SP degradation in the lung homogenate were significantly suppressed by phosphoramidon. In bronchoalveolar lavage fluid (BALF), levels of NKA and SP were significantly elevated after a bolus of BK with a phosphoramidon infusion. These results suggest that NEP inhibitors may have adverse respiratory effects resulting from inhibition of the degradation of neurokinins, but mainly of NKA, when a large amount of BK is generated.

Topics: Animals; Benzamides; Bradykinin; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Capsaicin; Dipeptides; Dose-Response Relationship, Drug; Drug Antagonism; Drug Interactions; Glycopeptides; Guinea Pigs; Indoles; Lung; Male; Metalloendopeptidases; Neurokinin A; Piperidines; Substance P

We investigated the role of endogenous endothelins in catecholamine secretion in response to transmural electrical stimulation in the retrogradely perfused rat adrenal gland. (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-++ +methy l-pentanoyl]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-+ ++pyridyl) propionic acid (FR139317; 0.03-3 microM), an endothelin ET(A) receptor antagonist, inhibited the electrical stimulation-induced epinephrine and norepinephrine output. Neither N-cis-2, 6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1- methoxycarbonyl tryptophanyl-D-norleucine (BQ-788; 0.03-3 microM), an endothelin ET(B) receptor antagonist, nor phosphoramidon (1-100 mM), an endothelin-converting enzyme inhibitor, affected the catecholamine output responses. However, the inhibition by FR139317 of the catecholamine output responses was abolished by pretreatment with phosphoramidon (100 mM) or BQ-788 (3 microM). These results indicate that activation of endothelin ET(B) receptors by endogenous endothelins inhibits the catecholamine output responses under the condition in which endothelin ET(A) receptors are blocked. Exogenous endothelin-1 (1-100 nM) did not affect the catecholamine output responses, but it inhibited the responses under treatment with phosphoramidon and FR139317. Activation of endothelin ET(A) receptors may interfere with the endothelin ET(B) receptor-mediated inhibitory action on the neuronally evoked secretion of adrenal catecholamines.

Topics: Adrenal Glands; Animals; Azepines; Catecholamines; Dose-Response Relationship, Drug; Electric Stimulation; Endothelin-1; Endothelins; Epinephrine; Glycopeptides; In Vitro Techniques; Indoles; Male; Norepinephrine; Oligopeptides; Piperidines; Rats; Rats, Wistar

1. This study sought to determine whether neurogenic inflammation occurs in the airways by examining the effects of capsaicin or substance P on microvascular plasma leakage in the trachea and lungs of male pathogen-free C57BL/6 mice. 2. Single bolus intravenous injections of capsaicin (0.5 and 1 micromol kg(-1), i.v.) or substance P (1, 10 and 37 nmol kg(-10, i.v.) failed to induce significant leakage in the trachea, assessed as extravasation of Evans blue dye, but did induce leakage in the urinary bladder and skin. 3. Pretreatment with captopril (2.5 mg kg(-1), i.v.), a selective inhibitor of angiotensin converting enzyme (ACE), either alone or in combination with phosphoramidon (2.5 mg kg(-1), i.v.), a selective inhibitor of neutral endopeptidase (NEP), increased baseline leakage of Evans blue in the absence of any exogenous inflammatory mediator. The increase was reversed by the bradykinin B2 receptor antagonist Hoe 140 (0.1 mg kg(-1), i.v.). 4. After pretreatment with phosphoramidon and captopril, capsaicin increased the Evans blue leakage above the baseline in the trachea, but not in the lung. This increase was reversed by the tachykinin (NK1) receptor antagonist SR 140333 (0.7 mg kg(-1), i.v.), but not by the NK2 receptor antagonist SR 48968 (1 mg kg(-1), i.v.). 5. Experiments using Monastral blue pigment as a tracer localized the leakage to postcapillary venules in the trachea and intrapulmonary bronchi, although the labelled vessels were less numerous in mice than in comparably treated rats. Blood vessels of the pulmonary circulation were not labelled. 6. We conclude that neurogenic inflammation can occur in airways of pathogen-free mice, but only after the inhibition of enzymes that normally degrade inflammatory peptides. Neurogenic inflammation does not involve the pulmonary microvasculature.

Topics: Adrenergic beta-Antagonists; Angiotensin-Converting Enzyme Inhibitors; Animals; Benzamides; Blood Vessels; Bradykinin; Bradykinin Receptor Antagonists; Capillary Permeability; Capsaicin; Captopril; Dose-Response Relationship, Drug; Evans Blue; Glycopeptides; Lung; Male; Mice; Mice, Inbred C57BL; Neurokinin-1 Receptor Antagonists; Piperidines; Protease Inhibitors; Quinuclidines; Receptors, Neurokinin-2; Skin; Specific Pathogen-Free Organisms; Substance P; Trachea; Urinary Bladder

The current studies were undertaken to investigate the role of endothelin-1 (ET-1) and its receptors in contractions of isolated pulmonary vessels of the pig induced by diaspirin cross-linked hemoglobin (DCLHb). Second-order pulmonary arteries (PAs) and veins (PVs) were isolated from pigs, cut into rings (4 to 5 mm), and mounted at optimal passive tension in 37 degrees C Krebs-filled tissue baths bubbled with 95% O2/5% CO2. Isometric tension was recorded continuously. In paired rings, concentration responses to ET-1 (10(-10) to 10(-7) mol/L), DCLHb (10(-9) to 3x10(-6) mol/L), and N-nitro-L-arginine (LNA) (10(-6) to 5x10(-5) mol/L) in the presence and absence of the ET(A) receptor antagonist BQ123 (3x10(-5) mol/L) were determined. PVs and PAs with intact endothelium and rings from which the endothelium was removed (denuded) were pretreated with the ET(B) receptor antagonist BQ788 to determine the contribution of ET(B) receptors to ET-1, DCLHb, and LNA responses. ET-1, DCLHb, and LNA caused concentration-dependent increases in tension in all vessels. In the presence of BQ123, the 50% effective concentration (EC50) of ET-1 was significantly increased (by 5-fold to 10-fold) in all vessels. DCLHb concentration responses were significantly attenuated-in the PVs by 45% and in the PAs by 79%-during treatment with BQ123. BQ123 attenuated LNA responses in PVs by 35% and in PAs by 87%. Treatment with BQ788 had no effect on endothelium-intact PVs or PAs but significantly increased ET-1 EC50 (log of the molar concentration) from -9.0+/-0.22 to -7.8+/-0.05 in denuded PAs. The ET-1 EC50 was significantly decreased in denuded PAs (-9.0+/-0.22) as compared with responses in endothelium-intact PAs (-8.1+/-0.18). DCLHb concentration responses were attenuated by 71% and LNA responses by 80% during antagonism with BQ788 in the intact PAs only. These data demonstrate that ET-1 plays a role in DCLHb-induced contractions in the PA and PV. The contributions of ET are mediated by both ET(A) and ET(B) receptors in the PA but only by ET(A) receptors in the PV. These results suggest that the vasoconstrictor actions of DCLHb, which have previously been shown to depend on its interference with endothelium-generated NO, may also involve ET. This may reflect the importance of the interaction of these two endothelium-generated physiologic antagonists in the pulmonary circulation.

Topics: Animals; Aspirin; Endothelin Receptor Antagonists; Endothelin-1; Glycopeptides; Hemoglobins; Isometric Contraction; Muscle, Smooth, Vascular; Nitroarginine; Oligopeptides; Piperidines; Pulmonary Artery; Pulmonary Veins; Receptor, Endothelin A; Receptor, Endothelin B; Swine; Vasoconstrictor Agents

The effects of endothelin (ET)-1(1-31) and ET-2(1-31), human chymase products of the corresponding big ETs, on the intracellular free Ca2+ concentration ([Ca2+]i) and [125I]-ET-1 binding were investigated using human cultured bronchial smooth muscle cells (BSMC). ET-1(1-31) (10(-8)M - 3 x 10(-7)M) and ET-2(1-31) (3 x 10(-8)M - 3 x 10(-6) M) caused an increase in [Ca2+]i in a concentration-dependent manner. Big ET-1 (3 x 10(-8)M - 10(-6)M) also caused this increase, but not big ET-2 at concentrations up to 10(-6)M. The [Ca2+]i increase induced by ET-1 was inhibited by both BQ123, an ET(A)-receptor antagonist, and BQ788, an ET(B)-receptor antagonist, whereas that induced by ET-3 was inhibited by BQ788 but not by BQ123. Increases in [Ca2+]i caused by ET-1(1-31), big ET-1 and ET-2(1-31) were completely inhibited by 10(-4)M phosphoramidon, a dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor, and 10(-5)M thiorphan, a NEP inhibitor. Scatchard plot analyses of the saturation curves of [125I]-ET-1 and [125I]-ET-3 showed that both ET(A)- and ET(B)- receptors at the ratio of 4:1 were expressed on BSMC. ET-1(1-31), big ET-1 and ET-2(1-31) inhibited [125I]-ET-1 binding in a concentration-dependent manner, and these effects were attenuated by treatment with thiorphan. On the other hand, big ET-2 slightly inhibited the binding at a high concentration and this was not affected by thiorphan. These results suggest that ET-1(1-31), big ET-1 and ET-2(1-31) cause an increase in [Ca2+]i by being converted into the corresponding ET-1 and ET-2 by NEP, but this did not occur with big ET-2 in human BSMC. ET-2(1-31), produced by human chymase from big ET-2 might be important for the generation of ET-2 in human bronchial tissue.

Topics: Adult; Binding, Competitive; Bronchi; Calcium; Cells, Cultured; Endothelin-1; Endothelin-3; Endothelins; Glycopeptides; Humans; Iodine Radioisotopes; Male; Muscle, Smooth; Oligopeptides; Peptide Fragments; Peptides, Cyclic; Piperidines; Protease Inhibitors; Radioligand Assay; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin

Coronary dysfunctions identified in the presence of chronic heart failure are an important pathophysiologic abnormality that influences the prognosis of the disease. Because the endothelin pathway plays a significant role in the increased peripheral vascular tone associated with heart failure, we hypothesized that the endothelin pathway may be involved in the abnormal coronary vasomotion associated with this pathologic condition. Experiments were carried out in failing hearts (UM-X7.1 cardiomyopathic hamsters, aged 225-250 days) and normal hearts (Syrian LVG hamsters, also aged 225-250 days). Isolated hearts were perfused at constant flow and exposed to the blocker of the generation of endothelin-1 (ET-1), phosphoramidon (10 microM infusion), as well as to the selective ET(A)-receptor antagonist BQ 123 (10 microM infusion) and to a selective ET(B)-receptor antagonist BQ 788 (1 microM infusion). Coronary and cardiac effects of exogenous ET-1 (0.01-100 pmol) were also studied. Phosphoramidon, BQ 788, and BQ 123 did not altered coronary perfusion pressure either in normal or in failing hearts, whereas cardiac contractility was significantly impaired in the presence of phosphoramidon and BQ 123. Coronary sensitivity to exogenous ET-1 did not demonstrate a significant difference between normal and failing hearts [median effective concentration (EC50), 7 pmol in failing hearts vs. 12 pmol in normal hearts; p = NS]. In the presence of exogenous ET-1, cardiac contractility was significantly increased in both groups. In normal hearts, the exogenous ET-1-induced increase in coronary perfusion pressure was completely antagonized by BQ 123, whereas combined administration of BQ 788 and BQ 123 was necessary to induce complete inhibition in failing hearts. The positive inotropic effect elicited by exogenous ET-1 (EC50) was completely abolished in the presence of BQ 123, whereas BQ 788 had no significant effect. Results indicate that the endothelin pathway does not play a significant role in the altered coronary vasomotion observed in this model of chronic heart failure. On the contrary, the endothelin pathway appears to participate in the maintenance of myocardial contractility. According to these observations, administration of an inhibitor of ET-1 synthesis, as well as the use of an ET(A)-receptor antagonist, may be contraindicated in the presence of poor left ventricular function because the endothelin pathway contributes significantly to the maintenance of cardiac

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Coronary Circulation; Coronary Vessels; Cricetinae; Electrocardiography; Endothelin Receptor Antagonists; Endothelin-Converting Enzymes; Glycopeptides; Heart; Heart Failure; Mesocricetus; Metalloendopeptidases; Oligopeptides; Peptides, Cyclic; Piperidines; Protease Inhibitors; Receptor, Endothelin A; Receptor, Endothelin B

The effects of i.v. injections of two endogenous tachykinins, substance P (SP) and neuropeptide gamma and the highly selective tachykinin agonists [Sar9,Met(O2)11]-SP, [Lys5,MeLeu9, Nle10]-NKA(4-10) and senktide, on total lung resistance (RL), dynamic lung compliance (Cdyn) and systemic blood pressure, were compared in the anaesthetized rabbit. Senktide, the NK-3 receptor selective agonist, had no effect on RL, Cdyn or blood pressure. The other four agonists caused dose-dependent increases in RL and Cdyn, with [Sar9,Met(O2)11]-SP being the most potent agonist in producing changes in the absence of phosphoramidon. This suggested that NK-1 receptors play an important role in these responses. [Sar9, Met(O2)11]-SP, SP and neuropeptide gamma also decreased blood pressure. Phosphoramidon (1 mg/kg) potentiated the changes in RL and Cdyn evoked by [Sar9,Met(O2)11]-SP and SP, with very marked enhancement of responses to neuropeptide gamma. Responses to [Lys5, MeLeu9,Nle10]-NKA(4-10) were unaffected, suggesting that this NK-2 selective agonist may not be catabolized by neutral endopeptidase (NEP). In the presence of phosphoramidon, the non-peptide tachykinin NK-1 receptor selective antagonist CP 96345 (80 nmol/kg) reduced all responses to [Sar9,Met(O2)11]-SP and SP, whereas the NK-2 selective antagonist SR 48968 (40 nmol/kg) inhibited the bronchomotor but not the vasodepressor responses to neuropeptide gamma and [Lys5,MeLeu9, Nle10]-NKA(4-10). The fall in blood pressure induced by neuropeptide gamma was diminished by CP 96345, whereas bronchoconstriction was unaffected, indicating possible differences in NK-1 receptors in the vasculature and airways. Electrical stimulation of the distal ends of vagus nerves caused increases in RL which were abolished by atropine (1 mg/kg).

Topics: Anesthesia; Animals; Benzamides; Biphenyl Compounds; Bronchoconstriction; Drug Interactions; Glycopeptides; Neprilysin; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Protease Inhibitors; Rabbits; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P; Tachykinins; Vagus Nerve; Vasoconstriction

The smoke-induced airway hyperresponsiveness (SIAHR) to inhaled wood smoke was investigated in anesthetized guinea pigs. Two smoke challenges (each 10 ml) separated by 30 min were delivered into the lungs by a respirator. In control animals, SIAHR was evidenced by an average bronchoconstrictive response (an increase in total lung resistance) to the second smoke challenge (SM2) that was approximately 4.3-fold greater than that to the first challenge (SM1). Pretreatment with CP-96,345 and SR-48,968 (neurokinin-1 and -2 receptor antagonists; each 1 mg/kg) in combination totally prevented this SIAHR, while pretreatment with CP-96,344 and SR48,965 (inactive enantiomers of CP-96,345 and SR-48,968, each 1 mg/kg) in combination failed to do so. Pretreatment with CP-96,345 (1 mg/kg), SR48,968 (1 mg/kg), or atropine (50 microg/kg) significantly alleviated this SIAHR. Pretreatment with phosphoramidon [an inhibitor of neutral endopeptidase (NEP); 2 mg/kg], which suppresses the degradation of tachykinins, induced an increase in airway reactivity that largely mimicked this SIAHR. The NEP activity measured in airway tissues excised 30 min after SM1 was significantly lower than that in air control value. These results suggest that 1) a prior wood smoke exposure induces an airway hyperresponsiveness to the subsequent wood smoke inhalation, 2) a tachykininergic mechanism involving both neurokinin-1 and -2 receptors is essential for, and a cholinergic mechanism is also involved in the development of this SIAHR, and 3) inactivation of airway NEP by wood smoke may contribute to this SIAHR.

Topics: Airway Resistance; Animals; Atropine; Benzamides; Biphenyl Compounds; Bronchi; Bronchial Hyperreactivity; Cholinergic Antagonists; Drug Combinations; Glycopeptides; Guinea Pigs; Male; Neprilysin; Piperidines; Protease Inhibitors; Receptors, Cholinergic; Receptors, Tachykinin; Smoke; Wood

Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness and airway inflammation.. This study was designed to investigate the effects of the tachykinin NK1 receptor antagonist SR 140333 (Nolpitantium) and the NK2 receptor antagonist SR 48968 (Saredutant) on the activation of alveolar macrophages in the guinea-pig.. Guinea-pigs sensitized and challenged by ovalbumin administered by aerosol or naive guinea-pigs were exposed by aerosol to the neutral endopeptidase, phosphoramidon and, 15 min later, to substance P. Twenty-four hours later, bronchoalveolar lavages were performed and the cell composition of bronchoalveolar lavage fluids and the arachidonate release from alveolar macrophages stimulated in vitro with fMLP were evaluated.. Antigen challenge in sensitized guinea-pigs induced an increase in the total number of cells and granulocytes in the bronchoalveolar lavage fluids that was not reduced by pre-treatment of guinea-pigs with a single dose of SR 140333 or SR 48968 (1 mg/kg). Substance P exposure in phosphoramidon-pretreated guinea-pigs did not induce an increase in the total number of cells. In contrast, antigen or substance P exposure induced a significant increase in the in vitro fMLP-induced arachidonate release from alveolar macrophages. Pre-treatment of the guinea pigs with SR 140333 or SR 48968 did not reduce the increase in arachidonate release from fMLP-stimulated alveolar macrophages from sensitized and challenged guinea-pigs. Pre-treatment of the animals by SR 140333 and SR 48968 reduced the enhanced arachidonate release induced by fMLP from substance P-exposed guinea-pigs.. The present data demonstrate the importance of NK1- and NK2-receptor stimulation in the development of substance P-induced increased reactivity of alveolar macrophages.

Topics: Animals; Antigens; Arachidonic Acid; Benzamides; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Endopeptidases; Glycopeptides; Guinea Pigs; Macrophage Activation; Macrophages, Alveolar; Male; Neurokinin-1 Receptor Antagonists; Ovalbumin; Piperidines; Quinuclidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P

Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness (AHR) and airway inflammation. However, it is unknown which tachykinin receptors mediate these biological activities. The effects of two antagonists of tachykinin neurokinin-1 (NK1) and tachykinin neurokinin-2 (NK2) receptors, SR 140333 and SR 48968, respectively, were investigated on substance P (SP)-induced airway hyperresponsiveness and potentiation of the histamine-induced increase in microvascular leakage, in phosphoramidon-pretreated guinea-pigs. Guinea-pigs were pretreated with phosphoramidon (0.1 mM aerosol for 15 min) and exposed 15 min later to saline solution alone or to saline solution containing SP (0.1 mg.mL-1 for 30 min). Twenty four hours later, the animals were anaesthetized and prepared for the recording of the pulmonary inflation pressure (PIP) to acetylcholine or for the investigation of microvascular leakage to histamine. Pretreatment of the guinea-pigs with a single dose of SR 48968 (1 mg.kg-1, i.p.) 30 min before SP exposure, significantly prevented the development of AHR, whereas SR 140333 (1 mg.kg-1, i.p.) did not. In a second set of experiments, phosphoramidon-pretreated guinea-pigs exposed to SP presented a significant potentiation of the histamine-induced increase in microvascular leakage in pulmonary airways. When the guinea-pigs were pretreated with SR 140333, an inhibition of the increased microvascular leakage to histamine was observed. In contrast, no significant inhibitory activity was noted when the guinea-pigs were pretreated with SR 48968. The present data demonstrate the importance of tachykinin NK2 receptor stimulation in the development of airway hyperresponsiveness and that of tachykinin NK1 receptor stimulation in microvascular leakage hypersensitivity in phosphoramidon-pretreated and substance P-exposed guinea-pigs. The results also suggest a dissociation between the presence of microvascular leakage and the occurrence of airway hyperresponsiveness.

Topics: Animals; Benzamides; Bronchial Hyperreactivity; Capillary Leak Syndrome; Female; Glycopeptides; Guinea Pigs; Male; Metalloendopeptidases; Neurokinin-1 Receptor Antagonists; Piperidines; Premedication; Quinuclidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Stereoisomerism; Substance P

The involvement of tachykinin receptors in skin inflammation induced by substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) was investigated in mouse ears. Intradermal injection of tachykinins (0.1-100 pmol/site) into the ear skin produced oedema formation. RP 67580 (ED50: 0.34 mg/kg, i.v.) and SR 140333 (ED50: 0.19 mg/kg, i.v.), the non-peptide NK1 receptor antagonists, inhibited SP-induced oedema. SR 140333 was also effective in preventing NKA- and NKB-induced oedema. SR 48968 (1 mg/kg, i.v.), a non-peptide NK2 antagonist, induced a significant inhibition of NKA-induced oedema but had no effect on the response to SP and NKB. SR 142801 (3 mg/kg, i.v.), a non-peptide NK3 antagonist, prevented only NKB-induced oedema. In contrast, phosphoramidon (0.1 and 0.5 mg/kg, i.v.), an endopeptidase inhibitor, enhanced the oedema response to tachykinins. SR 140333, SR 48968, and SR 142801 blocked the enhancement by phosphoramidon of the response to SP, NKA, and NKB, respectively. Plasma extravasation in ear skin was induced by i.v. injection of tachykinins (0.7-17.6 nmol/kg). RP 67580 (ED50: 0.15 mg/kg, i.v. for SP) and SR 140333 (ED50: 14.3 micrograms/kg, i.v. for SP) inhibited tachykinin-induced plasma extravasation in ear skin. However, SR 48968 and SR 140281 had no effect on the vascular response to tachykinins. Chlorpheniramine (4 mg/kg, i.v.), a histamine H1 blocker, inhibited the response to local SP but not to i.v. SP. These results suggest that in addition to the NK1 receptors, functional NK2 and NK3 receptors may participate in the oedema response to local NKA and NKB in the ear skin. However, it appears that NK1 receptors on blood vessels are involved predominantly in plasma extravasation induced by i.v. tachykinins in the ear.

Topics: Animals; Capillary Permeability; Edema; Glycopeptides; Male; Mice; Neurokinin A; Neurokinin B; Piperidines; Quinuclidines; Receptors, Tachykinin; Substance P

We investigated the potential of corticotropin-releasing factor (CRF) to reduce neurogenic plasma extravasation in sensitised guinea pig airways evoked by antigen challenge. Inhalation of 5% ovalbumin for 2 min in the presence of phosphoramidon (2.5 mg/kg, i.v.) increased extravasation of Evans blue dye in the trachea and main bronchi. The increase in plasma extravasation induced by antigen challenge was significantly reduced by pretreatment with CRF (30 nmol/kg, i.v.) (73% in the trachea and 42% in the main bronchi). The inhibition of plasma extravasation by CRF (30 nmol/kg, i.v.) alone was not different from the inhibition induced by the combination of CRF and the tachykinin NK1 receptor antagonist, CP-99,994 (4 mg/kg, i.v.) (73% in the trachea and 38% in the main bronchi). CRF (30 nmol/kg, i.v.) inhibited by 32% in the trachea and by 43% in the main bronchi plasma extravasation induced by aerosolised bradykinin but did not reduce the plasma extravasation caused by aerosolised substance P in the presence of phosphoramidon. These findings suggest that CRF reduces ovalbumin-induced plasma extravasation in guinea pig airways by inhibiting the release of tachykinins from primary sensory nerves.

Topics: Animals; Capillary Permeability; Corticotropin-Releasing Hormone; Glycopeptides; Guinea Pigs; Hemodynamics; In Vitro Techniques; Male; Neurokinin-1 Receptor Antagonists; Piperidines; Protease Inhibitors; Respiratory Physiological Phenomena; Respiratory System; Substance P; Tachykinins

Our previous report suggests the important role that ETB receptors play in the clearance of circulating endothelin (ET)-1. The present study confirmed this finding by measuring the extracellular levels of immunoreactive (ir) ET-1 and intracellular contents of prepro (pp) ET-1 mRNA in the presence and absence of ETA- and ETB-selective antagonists (i.e., BQ-123 and BQ-788, respectively) in cultured human umbilical vein endothelial cells (HUVECs). ET-1 was secreted into the culture medium of HUVECs in a time-dependent manner with a plateau reached after incubation for more than 36 hr. In the presence of 10 microM BQ-788, the irET-1 level was enhanced significantly (i.e., up to 180% of control at 48 hr) whereas BQ-123 had no such effect. Specific binding of [125I]ET-1 to HUVECs was inhibited strongly by BQ-788 (IC50 = 2.4 nM) but very weakly by BQ-123 (IC50 = 1.4 microM), indicating that BQ-788 has a potent affinity for ETB receptors in HUVECs. The expression of ppET-1 mRNA was not changed by BQ-788. Extracellular levels of ET-1 decreased gradually after cellular treatment with cycloheximide. This decrease was significantly inhibited by BQ-788 but not by BQ-123 and was non-existent when the cells were incubated at 4 degrees C (where internalization of the receptor protein is not likely). In conclusion, HUVECs secrete ET-1 which, in turn, is internalized after binding to ETB receptors on HUVECs.

Topics: Amino Acid Sequence; Cells, Cultured; Cycloheximide; Endothelin Receptor Antagonists; Endothelins; Endothelium, Vascular; Gene Expression; Glycopeptides; Humans; In Vitro Techniques; Lung; Molecular Sequence Data; Oligopeptides; Peptides, Cyclic; Piperidines; Receptors, Endothelin; RNA, Messenger

1. In the present study, we have investigated the role of kinins in allergen-induced bronchoconstriction. 2. Anaesthetized guinea-pigs were sensitized to ovalbumin, ventilated artificially, pretreated with atropine (1.4 mumol kg-1, i.v.) and total pulmonary resistance (RL) measured. In preliminary studies in the presence of the neutral endopeptidase inhibitor, phosphoramidon (4.5 mumol kg-1, i.v.), the bradykinin B2 receptor antagonist Hoe 140 (0.1 mumol kg-1, i.v.) completely abolished the increase in RL following aerosolized bradykinin (1 mM, 40 breaths), but had no effect on the increase in RL following aerosolized neurokinin A (NKA, 10 microM, 40 breaths). On the other hand, a combination of the NK1 (CP-96,345, 2 mumol kg-1, i.v.) and NK2 (SR 48968, 0.3 mumol kg-1, i.v.) tachykinin receptor antagonists abolished completely the increase in RL produced by NKA and partially inhibited the increase in RL produced by bradykinin. These results confirm previous studies that suggest that bradykinin induces the release of tachykinins from sensory nerves in guinea-pig airways. 3. Aerosolized ovalbumin (0.5%, 5 breaths) increased RL in sensitized guinea-pigs pretreated with atropine (1.4 mmol kg-1, i.v.), an effect that began within 2 min and reached a maximum within 5 min; RL remained above baseline at 20 min. Pretreatment with the bradykinin B2 receptor antagonist, Hoe 140, decreased the bronchoconstrictor effect of ovalbumin markedly at 10 to 20 min. In the presence of phosphoramidon (4.5 mumol kg-1, i.v.) the inhibition induced by Hoe 140 was apparent earlier and remained over the 20 min period of study. 4. Pretreatment with a combination of NK1 (CP-96,345) and NK2 (SR 48968) tachykinin receptor antagonists also markedly inhibited ovalbumin-induced bronchoconstriction; addition of the bradykinin B2 receptor antagonist to the NK1 and NK2 tachykinin receptor antagonists had no additional inhibitory effect on antigen-induced bronchoconstriction.5. These findings confirm that activation of sensory nerves to release tachykinins in guinea-pig airways contribute to antigen-induced bronchoconstriction, and provide evidence that tachykinin release is due to kinins generated during the allergic response.

Topics: Adrenergic beta-Antagonists; Airway Resistance; Anaphylaxis; Animals; Benzamides; Biphenyl Compounds; Bradykinin; Bronchoconstriction; Glycopeptides; Guinea Pigs; Hypnotics and Sedatives; Kinins; Male; Neprilysin; Neurokinin-1 Receptor Antagonists; Piperidines; Receptors, Neurokinin-2; Tachykinins

We characterized the endothelin (ET) receptor subtypes responsible for signal transduction in cultured porcine kidney epithelial LLC-PK1 cells. Both ET-1 (IC50, 43 pM) and ET-3 (IC50, 46 pM) inhibited the binding of [125I]ET-1 to LLC-PK1 cells to a similar extent. The binding affinity of LLC-PK1 cells was about 10,000 times higher for the ETB antagonist BQ-788 [N-cis-2,6-dimethyl-piperidinocarbonyl-L-tau-metylleucyl-D-+ ++Nin- methoxycarbonyltryptophanyl-D-norleucine] (IC50, 1.3 nM) than for the ETA antagonist BQ-123 [cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu)] (IC50, 14 microM). ET-1 enhanced cyclic GMP (cGMP) production, but reduced vasopressin- and forskolin-stimulated cyclic AMP (cAMP) production. Both effects of ET-1 were antagonized by BQ-788, but not by BQ-123. The cAMP decrease, but not the cGMP increase, in response to ET-1 was inhibited by pertussis toxin, suggesting that the former response is mediated by pertussis toxin-sensitive Gi, whereas the latter is mediated by a pertussis toxin-insensitive G-protein. Therefore, the ETB receptors in LLC-PK1 cells couple to the two types of signal transduction cascades to reduce cAMP production and stimulate cGMP production via distinct G-proteins. ET-1 and probably also ET-3 may play a role in the regulation of renal epithelial transport by decreasing cAMP and increasing cGMP.

Topics: Amino Acid Sequence; Animals; Arginine Vasopressin; Colforsin; Cyclic AMP; Cyclic GMP; Endothelin Receptor Antagonists; Endothelins; Epithelium; Glycopeptides; Kidney Tubules; LLC-PK1 Cells; Molecular Sequence Data; Oligopeptides; Peptides, Cyclic; Piperidines; Protein Binding; Receptor, Endothelin B; Receptors, Endothelin; Signal Transduction; Swine

Neuropeptide gamma (NP gamma) induced a contractile response of the human isolated bronchus which was potentiated by the neutral endopeptidase inhibitor, phosphoramidon, but was not modified by atropine and indomethacin. NP gamma was 3.31-fold more potent than NKA. Contractile response curves to NP gamma were shifted to the right and maximal responses reduced by the non-peptide NK2-receptor antagonist, SR 48968. The pKB of SR 48968 (8.94 +/- 0.18, n = 15), calculated according to Kenakin (1987) was very close to that reported for [Nle10]-NKA (4-10), a specific agonist of neurokinin NK2-receptors (8.86 +/- 0.13, n = 13), suggesting that the contractile effects of NP gamma on the human isolated bronchus were mediated through NK2A-receptors.

Topics: Benzamides; Bronchi; Bronchoconstriction; Culture Techniques; Dose-Response Relationship, Drug; Drug Interactions; Glycopeptides; Humans; Peptide Fragments; Piperidines; Receptors, Tachykinin; Tachykinins; Thermolysin

The action of tachykinins in the circular muscle of the human esophageal body is not known. The present study aimed to determine the response to tachykinins and the receptor type mediating this response.. Specimen were obtained from organ donors or patients undergoing esophagectomy for cancer, and isometric tension in response to tachykinins was measured.. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) evoked a concentration-dependent contraction with the following order of potency: NKA > NKB > SP. The neutral endopeptidase inhibitor, phosphoramidon, increased only the response to SP. [beta Ala8]NKA(4-10), a selective agonist of the NK2 receptor, produced a concentration-dependent contraction, whereas [Sar9,Met(O2)11]SP and [MePhe7]NKB, selective agonists of NK1 and NK3 receptors, respectively, had no effect. Contraction evoked by NKA was inhibited by the nonpeptide NK2 antagonist SR 48968 but not by the nonpeptide NK1 receptor antagonist CP-96,345, tetrodotoxin, or atropine. SR 48968 did not affect the response to carbachol.. Tachykinins contract the circular muscle of human esophageal body by activation of NK2 receptors without involvement of neural mechanisms. Response to SP is modulated by a phosphoramidon-sensitive enzymatic activity.

Topics: Adolescent; Adult; Aged; Atropine; Benzamides; Biphenyl Compounds; Carbachol; Dose-Response Relationship, Drug; Esophagus; Glycopeptides; Humans; Isometric Contraction; Middle Aged; Muscle Contraction; Muscle, Smooth; Neprilysin; Neurokinin A; Neurokinin B; Piperidines; Receptors, Tachykinin; Substance P; Tachykinins; Tetrodotoxin

Tachykinin receptors mediating uterotonic effects were examined in preparations from oestrogen-primed rats. In the absence of peptidase inhibitors [Lys5-MeLeu9-Nle10] NKA (4-10) was 14-fold more potent than neurokinin A (NKA), but the two peptides were equipotent in the presence of phosphoramidon alone and in combination with amastatin. The NK-2 receptor antagonist SR 48968 antagonised responses to the tachykinins. These findings indicate that an NK-2 receptor is present in the oestrogen-primed rat uterus and that endopeptidase 24.11 plays a major role to inactivate NKA in this tissue.

Topics: Animals; Anti-Bacterial Agents; Benzamides; Carbachol; Estradiol; Female; Glycopeptides; Kinetics; Neurokinin A; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Peptides; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Uterus

The contractile response to natural tachykinins and selective peptide agonists for tachykinin receptors was studied in strips of circular smooth muscle of human lower esophageal sphincter in vitro. The effects of phosphoramidon, which inhibits neutral endopeptidase (EC.3.4.24.11) and of the non-peptide compounds, SR 48968 and CP-96,345, which selectively block NK1 and NK2 receptors, respectively, were also investigated. Substance P, neurokinin A and neurokinin B produced a concentration-dependent contractile response. The rank order of potency was neurokinin A > neurokinin B > substance P. Phosphoramidon (1 microM) potentiated the response to substance P without changing the order of potency of natural tachykinins. The NK2-selective agonist, ([ beta Ala8]neurokinin A-(4-10)), produced a concentration-dependent contraction. The NK1 ([Sar9,Met(O2)11]substance P, 1 microM) and NK3 ([MePhe7]neurokinin B, 1 microM) selective agonists, however, did not exert any contractile effect. The selective NK2 antagonist, SR 48968, potently inhibited in a concentration-dependent (10 nM-1 microM) manner the response to neurokinin A, without affecting the response to carbachol. The selective NK1 antagonist, CP-96,345 (1 microM), did not affect the response to neurokinin A. These results indicate that tachykinins contract the circular muscle of human lower esophageal sphincter, and that this effect is mediated by NK2 receptor stimulation. Moreover, a phosphoramidon-sensitive mechanism plays a role in the regulation of the response to substance P.

Topics: Benzamides; Biphenyl Compounds; Dose-Response Relationship, Drug; Esophagogastric Junction; Glycopeptides; Humans; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Neprilysin; Neurokinin A; Neurokinin B; Piperidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Tachykinins

The role of tachykinins released from sensory nerves in bronchoconstriction induced by antigen was studied in sensitized guinea pigs anesthetized with pentobarbital sodium and pretreated with atropine. The combination of NK2 (SR-48968) and NK1 (CP-96,345) tachykinin-receptor antagonists abolished the increase in total pulmonary resistance (RL) evoked by intravenous capsaicin but did not affect the response evoked by intravenous histamine. A small dose of aerosolized ovalbumin (OVA, 0.1%) produced a small increase in RL that was further increased and markedly prolonged by the neutral endopeptidase (NEP) inhibitor phosphoramidon; this bronchoconstrictor effect of OVA was markedly reduced by the NK2-receptor antagonist and was abolished by the combination of the NK1 and NK2-receptor antagonists together. When a larger dose of OVA (0.5%) was used, a maximal bronchoconstrictor response was obtained. Phosphoramidon did not potentiate this response significantly. The combination of NK1- and NK2-receptor antagonists blunted the response at 5 min only slightly but markedly attenuated the later (10-20 min) response. These results show that tachykinins released from sensory nerves play a significant role in antigen-induced bronchoconstriction in guinea pigs. This effect is exaggerated when the normal modulation of neuropeptides by NEP is inhibited and is mediated predominantly by NK2-receptor activation, with a smaller contribution by NK1 receptors.

Topics: Aerosols; Animals; Atropine; Benzamides; Biphenyl Compounds; Bronchoconstriction; Capsaicin; Glycopeptides; Guinea Pigs; Histamine; Immunization; Inflammation; Lung; Male; Neurokinin-1 Receptor Antagonists; Ovalbumin; Piperidines; Pulmonary Circulation; Receptors, Neurokinin-2; Vascular Resistance