Noble gas (argon and xenon)-saturated cold storage solutions reduce ischemia-reperfusion injury in a rat model of renal transplantation.

BACKGROUND: Following kidney transplantation, ischemia-reperfusion injury contributes to adverse outcomes. The purpose of this study was to determine whether a cold-storage solution saturated with noble gas (xenon or argon) could limit ischemia-reperfusion injury following cold ischemia. METHODS: Sixty Wistar rats were randomly allocated to 4 experimental groups. Kidneys were harvested and then stored for 6 h before transplantation in cold-storage solution (Celsior®) saturated with either air, nitrogen, xenon or argon. A syngenic orthotopic transplantation was performed. Renal function was determined on days 7 and 14 after transplantation. Transplanted kidneys were removed on day 14 for histological and immunohistochemical analyses. RESULTS: Creatinine clearance was significantly higher and urinary albumin significantly lower in the argon and xenon groups than in the other groups at days 7 and 14. These effects were considerably more pronounced for argon than for xenon. In addition, kidneys stored with argon, and to a lesser extent those stored with xenon, displayed preserved renal architecture as well as higher CD-10 and little active caspase-3 expression compared to other groups. CONCLUSION: Argon- or xenon-satured cold-storage solution preserved renal architecture and function following transplantation by reducing ischemia-reperfusion injury.

Mechanisms of interleukin 1beta-induced human airway smooth muscle hyporesponsiveness to histamine. Involvement of p38 MAPK NF-kappaB.

We have investigated the effect of IL-1beta on histamine H(1)-receptor (H(1)R)-mediated inositol phosphate (IP) accumulation in human airway smooth muscle cells (HASMC) and on histamine-induced contraction of human bronchial rings. Stimulation of HASMC for 24 h with IL-1beta resulted in significant loss of histamine-induced IP formation, which was associated with a reduction of histamine- induced contraction of IL-1beta-treated human bronchial rings. An inhibitor of NF-kappaB activation, pyrrolidine dithiocarbamate, and a p38 MAPK inhibitor, blocked the IL-1beta-induced H(1)R desensitization, whereas anisomycin, an SAPK/JNK and p38 MAPK activator, mimicked the effect of IL-1beta. IL-1beta has been demonstrated to induce cox-2 expression and PGE(2) synthesis. In our study, indomethacin a cox antagonist, completely inhibited the effect of IL-1beta on H(1)R, whereas exogenously added PGE(2) was able to desensitize H(1)R. Furthermore, H-89, a selective PKA inhibitor, antagonized the effect of IL-1beta. Here, we have demonstrated that IL-1beta desensitizes H(1)R, which involves the activation of p38 MAPK and NF-kappaB, leading to the expression of cox-2 and the synthesis of PGE(2). PGE(2) increases intracellular cAMP resulting in PKA activation, which phosphorylates and functionally uncouples H(1)R. Our results suggest that IL-1beta protects airway smooth muscle against histamine-induced contractile responses and that bronchial hyperreactivity to histamine is not associated with proinflammatory cytokine-induced enhancement in H(1)R signaling.

Epinastine (WAL 801CL) inhibits the electrical field stimulation-induced cholinergic contraction in guinea pig and human airways in vitro.

Epinastine is an antihistamine drug with binding affinities at 5-hydroxytryptamine (5-HT) receptors. The current study was performed to investigate whether epinastine could modulate the cholinergic contraction in guinea pig and human airways in vitro. Isolated guinea pig and human airway preparations were suspended in organ baths containing modified Krebs-Henseleit solution. Electrical field stimulation was applied to elicit cholinergic contractions. Epinastine produced a concentration-dependent inhibition of the cholinergic contraction in guinea pig airways and pretreatment with methysergide (5-HT1/2/7 antagonist) significantly attenuated these inhibitory effects of epinastine. Pretreatment with tropisetron (5-HT3/4 antagonist), ketanserin (5-HT2 antagonist), SDZ216-525 (5-HT1A antagonist) or phentolamine (alpha-adrenergic antagonist) had no effect. Epinastine did not displace the concentration-response curve to acetylcholine. These results suggest that epinastine inhibits the cholinergic contraction in guinea pig airways through stimulation of prejunctional 5-hydroxytryptamine receptors, located to postganglionic cholinergic nerves. Inhibitory effects of epinastine on the cholinergic contraction in human airways in vitro were also demonstrated, which suggests that a similar mechanism might be present in human airways. The pharmacological profile of epinastine, which shows binding affinity at the 5-hydroxytryptamine7 receptor but not at the 5-hydroxytryptamine1 receptor subtypes corroborates the hypothesis that the inhibitory prejunctional 5-hydroxytryptamine receptor on cholinergic nerves is of the 5-hydroxytryptamine7 subtype.

The effects of 5-HT on cholinergic contraction in human airways in vitro.

Inhaled 5-hydroxytryptamine (5-HT) causes bronchoconstriction in asthmatics, and 5-HT plasma levels are elevated in asthma. Electrical field stimulation (EFS) of human airways, in vitro, evokes cholinergic contraction mediated by the release of acetylcholine (Ach) from postganglionic cholinergic nerves. The present study investigates whether selective 5-HT agonists and antagonists can modulate EFS-induced cholinergic contraction in human airways in vitro. Human airways, obtained from resections for bronchial carcinoma or organ transplant donors, were suspended under 2-g tension, between two platinum wire electrodes, in carbogenated Krebs solution at 37 degrees C and EFS was applied (1-32 Hz, 50 V, 0.5 ms, 15 s every 4 min) to elicit cholinergic contractions. 5-HT (10 microM-0.3 mM) produced frequency- and concentration-dependent facilitation of cholinergic contraction, but did not displace the concentration/response curve to Ach. Tropisetron (1 microM), a 5-HT3 and 5-HT4 antagonist, completely blocked the facilitatory effect of 5-HT (100 microM), whereas both ondansetron (1 microM) and GR 125478D (1 microM), a selective 5-HT3 and 5-HT4 antagonist, respectively, also attenuated the 5-HT-induced enhancement of cholinergic contraction. This facilitatory effect of 5-HT was partially mimicked by both selective 5-HT3 (2-methyl-5-HT) and 5-HT4 (RS 67333 and 5-methoxytryptamine) agonists. Fluoxetine (10 microM), a 5-HT uptake inhibitor, had no effect on the 5-HT (10-100 microm) induced potentiation of cholinergic contraction. These findings suggest that 5-HT facilitates cholinergic contraction in human airways in vitro through stimulation of both prejunctional 5-HT3 and 5-HT4 receptors. This may implicate a role of 5-HT in asthma.

Expression of monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 by human airway smooth-muscle cells. Modulation by corticosteroids and T-helper 2 cytokines.

We have demonstrated that, in addition to their contractile function, human airway smooth-muscle cells (HASMC) are able to express and to secrete chemokines of the monocyte chemotactic protein (MCP)/ eotaxin subfamily. This group of chemokines is believed to play a fundamental role in the development of allergic airway diseases such as asthma. The expression levels of MCP (MCP-1, -2, and -3) messenger RNA (mRNA) were compared with those of regulated on activation, normal T cells expressed and secreted (RANTES) mRNA in HASMC in culture. HASMC express MCP and RANTES mRNA after stimulation with interleukin (IL)-1beta, tumor necrosis factor-alpha, and interferon-gamma. MCP mRNA was maximal at 8 h, whereas RANTES mRNA expression was delayed to 24 h after stimulation. Further, significant differences were observed in the induction patterns of MCP and RANTES mRNA expression after stimulation with the individual cytokines. Dexamethasone (DEX) significantly inhibited cytokine-induced accumulation of MCP and RANTES mRNA, in contrast to IL-4, IL-10, and IL-13, which had no inhibitory effect on cytokine-induced chemokine expression. The cytokine-induced MCP mRNA expression in HASMC was associated with MCP release, which was inhibited by DEX and post-translationally by IL-4. HASMC can actively participate in the pathogenesis of asthma by the expression and release of chemokines, which are likely to play a critical role in the generation and regulation of the inflammatory response characteristic of allergic airway diseases.

Protective effects of the lazaroid U-74389G against hyperoxia in rat type II pneumocytes.

The aims of this study were to investigate the effect of hyperoxia on O2(-.), H2O2 and .NO generation and iNOS mRNA levels in rat type II pneumocytes in vitro and the possible protective effect of the lazaroid U-74389G. Rat type II pneumocytes were exposed, 36 h after isolation, to air, 60% or 85% O2 for 48 h. At the beginning of the experiment and 24 h later, the cells were exposed for 30 min to either 30 microM U-74389G or only the vehicle for the lazaroid (control). Exposure to 60% and 85% O2 decreased nitrite production 2.9-fold and 3.9-fold, and increased O2(-.) and H2O2 generation 4.6-fold and 6.7-fold, respectively. In the 85% O2-exposed cells, hyperoxia increased lipid peroxidation (thiobarbituric acid reactive substances, TBARS production) 2-fold and iNOS mRNA production 5.4-fold. U-74389G prevented the decrease in nitrite and the rise in O2(-.) and H2O2 production, the increase in TBARS and the rise in iNOS mRNA after hyperoxia. We conclude that exposure of type II pneumocytes in vitro to subtoxic oxygen levels leads to a disturbance in the .NO-O2(-.) balance despite increased iNOS mRNA levels. The lazaroid U-74389G appears to be a useful compound in the protection of hyperoxic lung injury by restoration of this .NO-O2(-.) balance and prevention of TBARS formation.

The effects of 8-hydroxy-2-(di-n-propylamino)tetralin on the cholinergic contraction in guinea pig and human airways in vitro.

Electrical field stimulation of guinea pig tracheal strips and human bronchial rings, in vitro, evokes a cholinergic contraction mediated by the release of acetylcholine. 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) is a 5-HT1A and 5-HT7 agonist. In this study, we have investigated whether 8-OH-DPAT could modulate the cholinergic contraction in guinea pig and human airways in vitro. 8-OH-DPAT (1 to 30 microM) produced a concentration-dependent inhibition of the cholinergic contraction in guinea pig tracheal strips with a maximal inhibition of 75.8% +/- 4. 7% (30 microM, 0.5 Hz). Pretreatment of the tissues with the 5- HT1/2/7 antagonist methysergide (10 to 30 microM) significantly attenuated the inhibitory effects of 8-OH-DPAT (10 to 30 microM) on the cholinergic contraction. Pretreatment with ketanserin (10 microM), a 5-HT2 antagonist, tropisetron (1 microM), a 5-HT3/4 antagonist, SDZ 216-525 (1 to 10 microM) and pindobind (10 microM), both selective 5-HT1A antagonists, or capsaicin (10 microM), which depletes sensory nerves from neuropeptides, had no effect on the inhibition of the cholinergic contraction by 8-OH-DPAT (10 to 30 microM). 5-carboxamidotryptamine (5-CT) (10 to 100 microM), a 5-HT1/2/7 agonist, partially mimicked the inhibitory effects of 8-OH-DPAT on the cholinergic contraction. 8-OH-DPAT (10 to 30 microM) also inhibited the cholinergic contraction in human bronchial rings in vitro with a maximal inhibition of 46.2% +/- 7.2% (30 microM, 1 Hz). SDZ 216-525 (10 microM) had no effect, whereas methysergide (30 microM) partially prevented the effect of 8-OH-DPAT in human airways. 8-OH-DPAT (30 microM) did not displace the concentration-response curve to acetylcholine (10 nM-30 mM) in guinea pig and human airways in vitro. These results suggest that 8-OH-DPAT inhibits the cholinergic contraction in guinea pig and human airways in vitro through stimulation of prejunctional atypical 5-HT receptors, possibly of the 5-HT7 subtype, located on postganglionic cholinergic nerves.

Regulation of H1-receptor coupling and H1-receptor mRNA by histamine in bovine tracheal smooth muscle.

1. Pretreatment of bovine tracheal smooth muscle (BTSM) with histamine (1-100 microM, 1 h) induced a concentration-dependent desensitization of the contractile response to subsequently administered histamine, with a reduction of the maximum response of 72 +/- 8% (n = 5) following pre-exposure to 100 microM histamine. In contrast, concentration-response curves to the muscarinic agonist, methacholine were not affected following histamine pretreatment, indicating a homologous desensitization. Furthermore, concentration-response curves to NaF, a G-protein activator, were not altered following histamine pre-incubation. 2. The histamine H1-receptor (H1R) desensitization could be antagonized by mepyramine (an H1-receptor antagonist, 1 microM) but not by cimetidine (an H2-receptor antagonist, 10 microM), indicating that the desensitization occurred via stimulation of histamine H1-receptors, without evidence for the involvement of histamine H2-receptors. 3. Indomethacin (10 microM) did not block the H1R desensitization, suggesting no involvement of prostaglandins. Furthermore, histamine pre-incubation in calcium free medium still induced a functional uncoupling of H1R. 4. GF 109203X, a protein kinase C (PKC) inhibitor, and H-7, a non-selective kinase inhibitor, did not antagonize the homologous H1R desensitization. 5. The steady-state level of H1R mRNA, assessed by Northern blot analysis, was not affected by prolonged histamine exposure (100 microM, 0.5, 1, 2, 4, 16 and 24 h). 6. These results suggest that histamine induces desensitization of the H1R at the level of the receptor protein, which involves a mechanism independent of PKC, PKA, PKG and calcium influx, suggesting the involvement of a receptor-specific kinase.

Desensitization of the histamine H1-receptor and transcriptional down-regulation of histamine H1-receptor gene expression in bovine tracheal smooth muscle.

We have investigated the role of protein kinase C (PKC) in the desensitization of histamine H1-receptors and in the expression of the histamine H1-receptor gene in airway smooth muscle. Prolonged 4beta-phorbol 12,13 dibutyrate (PDBu) pretreatment (4 h, 100 nM-1 microM) of bovine trachealis caused a concentration-dependent loss of contraction in response to histamine H1-receptor stimulation, which was associated with a concentration-dependent decrease in histamine-induced total [3H]-inositol phosphates accumulation. In contrast, the responses to sodium fluoride, a direct G-protein activator, were unalterd by PDBu (100-300 nM) pre-incubation and only slightly reduced following incubation with 1 microM PDBu. A selective PKC inhibitor, GF 109203X, partially blocked the PDBu (1 microM)-induced desensitization and completely blocked the effect of 100 nM PDBu, confirming the involvement of PKC. Binding experiments using [3H]-pyrilamine revealed a class of high-affinity binding sites within the range for the histamine H1 receptor in airway smooth muscle. PDBu (1 microM) pretreatment for 4 h did not change the number of histamine H1 receptors. PDBu (1 microM) exposure caused a time-dependent reduction in the steady-state levels of histamine H1-receptor mRNA, which was inhibited by pre-incubation with GF 109203X and by cycloheximide, a protein synthesis inhibitor. Nuclear run-on assays revealed a 50% reduction in the rate of histamine H1-receptor gene transcription after 17 h PDBu pretreatment, whereas mRNA stability was not affected by PDBu pretreatment (17 h). In conclusion, we have shown a PKC-mediated desensitization of the histamine H1-receptor in BTSM and a transcriptional down-regulation of the histamine H1-receptor gene expression, which requires new protein synthesis.

Changes in gamma-glutamyltransferase activity in rat lung tissue, BAL, and type II cells after hyperoxia.

The effect of hyperoxia on gamma-glutamyltransferase (gamma-GT), an important enzyme for the uptake of precursor molecules for intracellular synthesis of glutathione (GSH), has not been established. Our aim was to investigate the effects of prolonged subtoxic levels of hyperoxia on gamma-GT activity and GSH levels in lung tissue, epithelial lining fluid (ELF), and isolated rat type II cells immediately after their isolation and 48 h later when kept in culture in normoxia. Seventeen male Wistar rats were divided in three groups (n = 5-7) and were exposed to air or to 60 or 85% O2 for 7 days. Pulmonary gamma-GT activity increased in the 60 and 85% O2-exposed animals (1.6- and 3.2-fold, respectively), and tissue GSH levels increased only in the 60% O2 group (1.3-fold). In isolated type II cells from 60 and 85% O2-exposed animals, gamma-GT activity decreased by -70 and -88%, respectively, which was supported by cytochemical staining. Type II cell gamma-GT mRNA expression tended only to decrease after 85% O2. Type II cell gamma-GT activity strongly correlated with ELF gamma-GT (r = 0.60, P < 0.001), and ELF gamma-GT strongly correlated with ELF GSH (r = 0.75, P < 0.0001). When in culture, type II cell gamma-GT activity and GSH levels remained, respectively, 2.5- and 1.9-fold lower in the 60% O2-exposed group, but, in the 85% O2-exposed group, gamma-GT activity increased 2.1-fold, and GSH levels dropped to the levels of the control cells. Hyperoxia led to a concentration-dependent decrease in gamma-GT activity in rat type II cells, possibly by direct inactivation, but led to an increase in whole lung tissue gamma-GT. There seemed to be a negative feedback between intracellular GSH levels and type II cell gamma-GT activity. gamma-GT levels in the ELF were correlated with type II cell gamma-GT activity, but ELF gamma-GT did not seem to play an active role in the regulation of the ELF GSH pool. Hyperoxia decreased ELF GSH levels, possibly by increased degradation of GSH in the parenchymal lung tissue as a result of the increased gamma-GT activity.

N-acetylcysteine does not protect against type II cell injury after prolonged exposure to hyperoxia in rats.

Although the antioxidant properties of N-acetylcysteine (NAC) in vitro are widely accepted, the efficacy of NAC in the prevention of O2 toxicity in vivo is poorly documented. The aim of our study was to investigate the presumed protective effect of NAC on hyperoxic lung injury, focusing on gamma-glutamyltransferase (gamma-GT) activity and glutathione (GSH) levels in lung tissue, epithelial lining fluid (ELF), and isolated rat type II cells immediately after their isolation and 48 h later when kept in culture in normoxia. Thirty-four male Wistar rats were divided in three groups (n = 10-14) and were exposed to air or to 60 or 85% O2 for 7 days. One-half of the rats in each group received 200 mg/kg NAC intraperitoneally one time per day from 3 days before exposure until the end of the experiment, and the other one-half received the vehicle. In the 85% O2-exposed animals, NAC led to more respiratory distress and weight loss. NAC did not prevent the rise in bronchoalveolar lavage lactate dehydrogenase and alkaline phosphatase, but it did prevent the rise in calculated ELF volume. NAC decreased GSH levels (1.4-fold) and gamma-GT activity (1.8-fold) in the air-exposed type II cells. In the 60% O2-exposed group, no effects of NAC were seen (except for a decrease in gamma-GT mRNA expression), but, in the 85% O2-exposed group, NAC gave rise to higher GSH (2.6-fold) and higher gamma-GT activity (2.9-fold) in the ELF and lower GSH (6.9-fold) and higher gamma-GT activity (3.6-fold) in the type II cells. Even in culture, GSH levels remained 1.5-fold lower than in the cells from the air-exposed animals and 2-fold lower than in the cells from the 85% O2-exposed animals. There was increased DNA damage (as assessed by thymidine incorporation) and apoptosis after hyperoxia, especially after 60% O2, and this effect was amplified after NAC treatment. Although protective at the endothelial side, NAC treatment led to adverse effects at the epithelial side, despite, or probably because of, restoration of the ELF GSH levels in the presence of high O2 levels. Because NAC is rapidly metabolized to cysteine, it is plausible that the effects of NAC are manifested through the toxic effects of cysteine.

Decrease in gamma-glutamyltransferase activity in rat type II cells exposed in vitro to hyperoxia: effects of the 21-aminosteroid U-74389G.

Although the effect of hyperoxia on antioxidant enzymes is well known, the effect of subtoxic levels of hyperoxia on gamma-glutamyltransferase (gamma-GT), involved in the degradation and uptake of extracellular GSH for intracellular GSH synthesis, is unknown. The aim of the study was to investigate (1) the effects of in vitro hyperoxia on gamma-GT activity of type II cells and (2) the effects of the lazaroid U-74389G and N-acetylcysteine (NAC) on the hyperoxia-induced changes in gamma-GT and antioxidant enzyme activities. At 48 h after isolation, rat type II cells were exposed for 2 days to air, 60% O2 or 85% O2 with or without 30 microM U-74389G or 100 microM NAC. After the exposure, the cells were harvested and assayed for superoxide dismutase (SOD), glutathione peroxidase (GPx), gamma-GT activity, and GSH levels. In another series of experiments 85% O2-exposed cells, with or without U-74389G, were used for Northern blotting of gamma-GT mRNA. Exposure to 60% O2 decreased gamma-GT and GSH by -47 and -34%, respectively, while SOD and GPx activities remained unchanged. After 85% O2-exposure gamma-GT decreased by -55%, SOD and GPx increased by +55 and +87%, respectively, while GSH decreased by -35%. NAC treatment decreased gamma-GT activity by -42% in the air-exposed cells. After 60% O2, U-74389G led to significantly higher gamma-GT (+117%) and GSH (+26%) while NAC only led to higher GSH (+28%) compared to the oxygen-exposed cells not treated with NAC or U-74389G. After 85% O2 U-74389G increased gamma-GT, SOD, and GSH by +72, +58, and +68%, respectively, while NAC only increased SOD (+49%) and GSH (+26%) compared to the oxygen-exposed cells not treated with NAC or U-74389G. The 85% O2 exposure, with or without U-74389G, had no effect on gamma-GT mRNA levels. The results show that hyperoxia decreases rat type II cell gamma-GT activity in vitro. This effect was not related to an altered regulation at mRNA level and it was not associated with the hyperoxia-induced decrease in intracellular GSH, since restoration of the GSH levels by NAC did not restore gamma-GT activity. The lazaroid U-74389G with vitamin E-like properties effectively prevented the decrease in gamma-GT and GSH, so that direct inactivation of the membrane-bound gamma-GT by hyperoxia is the most likely mechanism.

Increase in gamma-glutamyltransferase by glutathione depletion in rat type II pneumocytes.

The purpose of our study was to investigate the effect of oxidative stress or intracellular glutathione (GSH) depletion on gamma-glutamyltransferase (gamma-GT) activity in cultured type II pneumocytes. Twenty-four hours after isolation, primary cultures of rat type II pneumocytes were preincubated with one of four compounds: 15, 30, 60, 125, 250 microM L-buthionine-[SR]-sulfoximine (BSO) for 3 h; 100, 200, 400, 800 microM tertiary-butylhydroperoxide (t-BOOH) for 45 min; 10, 25, 50, 100 microM menadione for 15 min; 100, 1000 microM paraquat for 1 h. GSH levels, H2O2 and O2.- generation were measured immediately after the incubation, gamma-GT activity and GSH levels also up to 24 h or 48 h later. Exposure to BSO led to a persistent GSH depletion without increase in H2O2 or O2.- production, together with a dose and time-dependent increase (doubling) of gamma-GT activity with a nonsignificant increase in gamma-GT mRNA expression 24 h after exposure to BSO. Exposure to 100 microM menadione, which increased H2O2 production, decreased gamma-GT activity. t-BOOH or paraquat did not give rise to a measurable increase in H2O2 or O2.-. Paraquat did not affect initial GSH levels, but increased GSH and decreased gamma-GT activity 24 h later. t-BOOH (400 and 800 microM) initially decreased GSH, and tended to increase GSH 24 h later, 100 and 200 microM increased gamma-GT activity 24 h later, but 800 microM decreased it. Restoration of intracellular GSH levels by addition of GSH to the culture medium completely prevented the increase in gamma-GT activity by BSO, while the addition of catalase or DMTU had no effect. We conclude that at least two effects are operating upon gamma-GT activity: GSH depletion seems to increase gamma-GT activity, while exposure to compounds generating oxidative stress correlates with a decrease in gamma-GT activity.

Opioids modulate the cholinergic contraction but not the nonadrenergic relaxation in guinea-pig airways in vitro.

(D-ALa2, NMePhe4, Gly-ol5) encephalin (DAMGO), a selective mu-opioid receptor agonist, has previously been demonstrated to inhibit the cholinergic and the noncholinergic contraction in guinea-pig airways. In contrast, opioids had no inhibitory effect on cholinergic neurotransmission in the upper trachea when stimulated at 8 Hz. We investigated whether DAMGO, a selective mu-opioid receptor agonist, [D-Pen 2,5] encephalin (DPDPE), a selective delta-opioid receptor agonist, and U-69593, a selective kappa-opioid receptor agonist could modulate the cholinergic contraction in the upper trachea at different frequencies of stimulation. Moreover, we have investigated whether DAMGO, DPDPE and U-69593 could also modulate the iNANC relaxation. DAMGO (1-100 microM) inhibited the cholinergic contraction in the upper trachea with a maximum inhibition of 57+/-15% at 1 Hz (n=4; p<0.05). On the other hand, DPDPE (10 microM) and U69593 (10 microM) did not produce any significant inhibition of the cholinergic contraction. Naloxone, an opioid receptor antagonist (100 microM), was able to antagonize the inhibitory effect of DAMGO (n=5; p<0.01) on the cholinergic contraction at a frequency of 2 Hz. DAMGO (10 microM) did not displace the cumulative concentration-response relationship to acetylcholine (10 nM-10 mM), (n=4; NS). This provides evidence that prejunctional mu-opioid receptors (and not delta-opioid or kappa-opioid receptors) modulate cholinergic contraction in the upper trachea. In contrast, DAMGO (10 microM) had no significant inhibitory effect on the nonadrenergic relaxation (n=4; NS) in the upper trachea. Neither DPDPE nor U-69593 had any effect on the nonadrenergic relaxation. These findings suggest that DAMGO directly inhibits the cholinergic contraction and that the opioid receptor involved in the inhibition of the cholinergic contraction in the upper trachea is of the mu-opioid type. The finding that opioids inhibit cholinergic contraction without altering NANC relaxations suggests that distinct populations of nerves mediate these two effects.

5-HT modulates noncholinergic contraction in guinea pig airways in vitro by prejunctional 5-HT1-like receptor.

5-Hydroxytryptamine (5-HT) has been demonstrated to cause both constriction and relaxation of guinea pig airways, partly through direct action on airway smooth muscle and partly through postganglionic facilitation of cholinergic neurotransmission. We performed an in vitro study to investigate whether 5-HT can modulate the noncholinergic contraction in guinea pig airways due to release of neuropeptides from airway sensory nerves. In the presence of atropine (1 microM), ketanserin (10 microM), and indomethacin (10 microM), 5-HT (0.1-100 microM) produced concentration-dependent inhibition of electrical field stimulation-induced noncholinergic contraction with maximal inhibition of approximately 72 +/- 4%. Tropisetron (ICS-205-930, 1 microM), a 5-HT3 and 5-HT4 receptor antagonist, was unable to prevent the inhibition produced by 5-HT. Methiothepin (1-100 nM), a 5-HT1 and 5-HT2 receptor antagonist, produced a concentration-dependent inhibition of the effect of 5-HT (1 microM) with a 50% inhibition concentration value of 66 nM. 5-HT (100 microM) had no effect on the cumulative concentration-response relationship to exogenous substance P (10 nM-10 microM). The concentration of agonist causing 35% inhibition of the noncholinergic contraction (EC35) was calculated, and a rank order of potency was established: 5-carboxamidotryptamine (EC35 = 0.24 microM) > 5-HT (EC35 = 0.77 microM) > 8-hydroxy-2-(dipropylamino)tetralin (EC35 = 8.1 microM) > sumatriptan (EC35 = 18 microM). We conclude that 5-HT concentration dependently modulates noncholinergic contraction in guinea pig airways in vitro by a prejunctional mechanism. This effect is probably mediated through a 5-HT1-like receptor; however, the exact subtype remains to be elucidated.

Ketotifen modulates noncholinergic contraction in guinea pig airways in vitro by a prejunctional nonhistamine receptor.

In guinea pig airways, electrical field stimulation (50 V, 0.5 msec, 8 Hz for 20 seconds) produces a rapid contraction, which is followed by a long-lasting contraction, at least in the lower part of the trachea and in the bronchi. The latter contraction is due to the release of neuropeptides from airway sensory nerves. Ketotifen fumarate has been demonstrated to inhibit the noncholinergic contraction in guinea pig airways in vitro, but no attempt has been made to identify the receptor type. Therefore we have performed an in vitro study to investigate which receptor is responsible for the inhibitory effects of ketotifen on noncholinergic contraction in guinea pig airways. Ketotifen (3 to 100 mumol/L) produced a concentration-dependent inhibition of the noncholinergic contraction, with a maximum inhibition of 74% +/- 7% at 8 Hz stimulation (p < 0.001; n = 5). Pretreatment of the tissues with either cimetidine (10 mumol/L) or thioperamide (10 mumol/L) or phentolamine (10 mumol/L) did not prevent the inhibitory effect of ketotifen (10 mumol/L). Cetirizine (10 mumol/L), on the other hand, produced no inhibition of the noncholinergic contraction at all. Metitepine (0.1 mumol/L) and methysergide (1 mumol/L), both 5-HT1 antagonists, attenuated the inhibitory effect of ketotifen (10 mumol/L). Ketanserin (a 5-HT2 antagonist, 10 mumol/L) and tropisetron (a 5-HT3 antagonist, 1 mumol/L) had no effect. Ketoifen (100 mumol/L) did not affect the cumulative dose-response relationship to exogenous substance P (0.01 mumol/L to 10 mmol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of loop diuretics on cholinergic neurotransmission in human airways in vitro.

BACKGROUND: Frusemide can inhibit various indirectly acting bronchoconstrictor stimuli in asthmatic patients. Both frusemide and bumetanide also modulate airway neurotransmission in some species but there are no data on the effect of loop diuretics on neurotransmission in man. An in vitro study was performed in human airways to investigate the possible neuromodulatory action of two loop diuretics, frusemide and bumetanide, and to elucidate whether a cyclooxygenase inhibitor such as indomethacin could modulate the effect of frusemide. The effect of acetazolamide, a carbonic anhydrase inhibitor, was also investigated. METHODS: Electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5-32 Hz for 15 seconds) in human airways with or without epithelium was used to induce a cholinergic contraction (n = 5 in all experiments). Indomethacin was present throughout. After obtaining a control frequency-response curve, different concentrations of diuretic were added to the organ bath and another frequency-response curve was constructed. To determine whether the effect of the diuretic was prejunctional or postjunctional a cumulative concentration-response curve to exogenous acetylcholine (Ach, 0.3 mumol/l to 10 mmol/l) was constructed in the presence of a diuretic (frusemide 1 mmol/l or bumetanide 0.1 mmol/l) or its vehicle. In some experiments indomethacin was omitted from the organ bath to investigate the possible involvement of cyclooxygenase products. RESULTS: Both frusemide (10 mumol/l to 1 mmol/l) and bumetanide (1 mumol/l to 0.1 mmol/l) produced a concentration-dependent inhibition of the EFS-induced cholinergic contraction in human airways in vitro but only in epithelium denuded tissues. Frusemide (1 mmol/l) produced a maximum inhibition of 46.3% (SE 9.9%) at 0.5 Hz and bumetanide (0.1 mmol/l 39.6 (6.2)% at 0.5 Hz. Without indomethacin in the organ bath the frusemide-induced inhibition was enhanced at 4, 8, and 16 Hz, but bumetanide-induced inhibition was not enhanced at any frequency when indomethacin was omitted. Frusemide (1 mmol/l) and bumetanide (0.1 mmol/l) had no effect on the cumulative concentration-response curve to exogenous Ach (0.3 mumol/l to 10 mmol/l). Acetazolamide (100 mumol/l) had no effect on the EFS-induced cholinergic contraction in tissues with or without epithelium. CONCLUSIONS: In human airways in vitro both frusemide and bumetanide produced a concentration-dependent inhibition of the EFS-induced cholinergic contraction. This inhibition is mediated through a prejunctional mechanism. Epithelium removal was necessary to achieve this effect. The mechanism of action of frusemide and bumetanide on airway nerves remains unclear: inhibition of the Na-K-Cl cotransporter is a possibility and, for frusemide, release of endogenous cyclooxygenase products may be involved. Carbonic anhydrase inhibition, on the other hand, is unlikely to be a factor.

Furosemide and bumetanide, but not nedocromil sodium, modulate nonadrenergic relaxation in guinea pig trachea in vitro.

Furosemide has recently been shown to be effective in inhibiting various indirect challenges in asthmatic patients, but its mode of action is not yet clear. There is some evidence that furosemide has an inhibitory effect on sensory and cholinergic nerves in the airways. We have investigated the effects of furosemide, bumetanide, and nedocromil sodium on inhibitory nonadrenergic, noncholinergic (iNANC) responses in guinea pig trachea in vitro using electrical field stimulation (50 V, 0.5 ms, 2 to 32 Hz for 30 s) and exogenously applied vasoactive intestinal peptide (VIP) or nitroprusside. In the presence of atropine (1 microM), indomethacin (10 microM), and propranolol (1 microM), both furosemide and bumetanide but not nedocromil sodium produced a concentration-dependent inhibition of the iNANC response (maximum inhibition, 31.2 +/- 5.6% with 100 microM furosemide at 16 Hz and 44.2 +/- 4.1% with 10 microM bumetanide at 4 Hz). Furthermore, after pretreatment of the tissues with L-NG-monomethyl arginine (90 microM), alpha-chymotrypsin (2 U/ml), or both, furosemide and bumetanide produced a further inhibition of the iNANC relaxation. Neither loop diuretic had any effect on the concentration-response curves to exogenous VIP (10(-9) to 10(-7) M) or nitroprusside (10(-8) to 10(-6) M). These results indicate that loop diuretics may inhibit nonadrenergic relaxation in guinea pig trachea in vitro by a prejunctional mechanism, probably through inhibition of nerve activation, the exact mechanism of which is still undefined.