Nerve growth factor secretion by human lung epithelial A549 cells in pro- and anti-inflammatory conditions.

Nerve growth factor (NGF) has recently been presented as a possible effector of inflammation and bronchial hyperresponsiveness. However, the production of NGF in human airways as well as the regulation of its expression by inflammatory cytokines and glucocorticoids have received little attention. A549 epithelial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, and starved for 24 h. The effect of the pro-inflammatory cytokine interleukin-1beta (1-30 U/ml), and of the glucocorticoid dexamethasone (1 microM) on NGF secretion was studied and quantified by enzyme-linked immunosorbent assay (ELISA). In addition, NGF production within the cells was visualized by immunocytochemistry. Under basal conditions, A549 cells produced and secreted NGF (12.6+/-2.0 pg/ml). Stimulation by interleukin-1beta for 24 h induced a dose-dependent increase in NGF production (maximal at 10 U/ml with 59.6+/-3.5% increase, P<0.05). Dexamethasone (1 microM) markedly reduced the constitute NGF secretion by 44.9% (7.0+/-2.1 pg/ml, P<0.001). In addition, the interleukin-1beta-stimulated NGF secretion was inhibited to approximately the same low level (8.5+/-2.5 pg/ml, P<0.001). In conclusion, we here report that human airway A549 epithelial cells are capable of producing NGF. This production is positively regulated by the pro-inflammatory interleukin-1beta, and negatively regulated by dexamethasone.

Local increase in the number of mast cells and expression of nerve growth factor in the bronchus of asthmatic patients after repeated inhalation of allergen at low-dose.

BACKGROUND: Repeated inhalation of allergen at low-dose induces an increase in bronchial hyper-responsiveness, without any associated symptom. The concomitant events in the bronchus have not been described. OBJECTIVE: We have studied the dynamic number of mast cells in the airways of patients with mild asthma before and after repeated inhalation of allergen at low-dose and the expression of nerve growth factor (NGF), which is reported to promote growth and survival of mast cells. METHODS: Twelve patients with mild asthma to cat allergen were enrolled at random in a blind placebo-controlled study, and submitted to repeated low-dose allergen exposure (1/5 of the provocative dose). Mast cells were immunolocalized using an antibody against mast cell tryptase. NGF and its high affinity receptor, TrkA, were immunolocalized using anti-NGF and anti-TrkA antibodies, respectively. NGF mRNA was quantified by competitive polymerase chain reaction (PCR) after reverse transcription of total RNA extracted from bronchial biopsy. NGF protein levels were measured by ELISA in bronchoalveolar lavage (BAL) fluid. RESULTS: Bronchial mast cell number was increased significantly after allergen exposure as compared with before. NGF expression in the bronchus was immunolocalized mainly to epithelial cells, but also to fibroblasts, blood vessels, and a few infiltrated cells. NGF mRNA levels in bronchial biopsies were increased significantly after allergen exposure. The high affinity receptor for NGF, TrkA, was immunolocalized to the infiltrated mast cell membrane. CONCLUSION: Our study shows that the increase in the number of mast cells and in the expression of NGF induced by allergen exposure in the bronchus of asthmatic patients is occurring before the onset of symptoms. In addition, our finding of the presence of the TrkA receptor on the membrane of the infiltrated mast cell in situ brings evidence of the mast cell as a target cell for the growth factor activity of NGF in the airways in asthma.

Human lung fibroblasts secrete nerve growth factor: effect of inflammatory cytokines and glucocorticoids.

Nerve growth factor (NGF) has recently been suggested to contribute to inflammation and bronchial hyperresponsiveness in asthma. However, the cell types capable of NGF production in the human lung and airways, as well as the regulatory role of pro-inflammatory cytokines and of glucocorticoids on NGF secretion in pulmonary cells, have not been described. Human pulmonary fibroblasts were cultured in the presence or absence of either interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and/or glucocorticoids. NGF secretion was measured by enzyme-linked immunosorbent assay. The human pulmonary fibroblasts constitutively secreted NGF in vitro. The rate of NGF secretion was shown to be cell density-dependent, since higher NGF secretion was detected in preconfluent cells, i.e. ones with less established cell-to-cell contact (41.0+/-5.0 pg x 10(-6) cells at 80% confluence), than cells in higher densities (8.2 +/- 3.4 pg x 10(-6) cells at 100% confluence). Stimulation with the pro-inflammatory cytokines IL-1beta (0.3-30 U x mL(-1)) or TNF-alpha (0.1-30 ng x mL(-1)) dose- and time-dependently (8-72 h) elevated the NGF secretion (effective concentration causing 50% of the maximum response (EC50)=2.9 U x mL(-1) and 1.0 ng x mL(-1), respectively). Treatment with the glucocorticoid budesonide (10(-7) M) markedly reduced the constitutive secretion of NGF by 42%, and attenuated the cytokine-stimulated NGF secretion to the same level. In conclusion, human lung fibroblasts may serve as a source of nerve growth factor in the lung, positively regulated by the asthma-associated and pro-inflammatory cytokines interleukin-1beta and tumour necrosis factor-alpha, and negatively regulated by the anti-inflammatory glucocorticoids.

Modulation of neuronal nitric oxide release by soluble guanylyl cyclase in guinea pig colon.

Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexus-containing smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO(-)(2). In the presence of 8-Br-cGMP, YC-1 and zaprinast (all at 10(-4) M) the NO/NO(-)(2)-release increased to 152 +/- 16% (P < 0.05), 164 +/- 37% (P < 0.05) and 290 +/- 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10(-5) M) decreased the evoked release of NO/NO(-)(2) to 49 +/- 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones.

Evidence for nonvesicular nitric oxide release evoked by nerve activation.

The gaseous nature of nitric oxide (NO) has led to the general assumption that its release from neurons during nerve stimulation is independent of vesicular storage. However, recent findings have shown that NO can exist intracellularly as part of more stable bioactive molecules, suggesting that the role of vesicular exocytosis for NO release cannot be excluded simply based on the chemical nature of NO itself. We have used botulinum toxin B (BTX B) to directly address the role of vesicular exocytosis for NO release. BTX B cleaves the synaptic vesicle protein synaptobrevin/VAMP, and by this inhibits Ca++-mediated exocytic release of neurotransmitters. As a target organ we used the guinea-pig enteric nervous system, which innervates the gastrointestinal tract, and in which both classical neurotransmitters as well as NO are released and influence smooth muscle activity. As expected, BTX B (0.1 microM) blocked the nerve stimulation-induced cholinergic and tachykininergic smooth muscle contractions, and markedly inhibited the nerve stimulation-evoked release of [3H]-choline. In contrast, BTX B (0.1 microM) had no effect on nerve stimulation-evoked relaxations, which were equally inhibited by an NO-synthase inhibitor as well as by a selective inhibitor of soluble guanylyl cyclase. In addition, nerve stimulation-evoked NO synthase-dependent outflow of NO/NO2- was unaffected by BTX B (0.1 microM). These findings suggest that the neuronal release of endogenous NO is independent of intact synaptobrevin/VAMP, and therefore provide further evidence that nerve-mediated release of further NO is nonvesicular.

Nitric oxide-dependent relaxation induced by M1 muscarinic receptor activation in the rat small intestine.

The aim of the present study was to investigate whether muscarinic M1 receptor activation induces intestinal relaxation via nerve-dependent nitric oxide formation. Mechanical activity in longitudinal segments of rat jejunum was recorded isotonically in organ baths. The muscarinic M1 receptor agonist 4-[[[(3-Chlorophenyl)amino]carbonyl]oxy]-N,N,N,-trimethyl-2-butyn- 1-ammonium chloride (McN-A-343, 10(-7)-10(-4) M) induced a concentration-dependent relaxation of rat jejunum. Relaxations induced by McN-A-343 (10(-5) M) were inhibited by the M1 receptor antagonist telenzepine (10(-8) M), and enhanced by the M3 receptor antagonist para-fluorohexahydrosiladifenidol (p-F-HHSiD; 3x10(-7) M). The inhibitory responses induced by McN-A-343 were abolished by the nitric oxide synthase inhibitors Nomega-nitro-L-arginine (L-NOARG; 10(-4) M) and Nomega-monomethyl-L-arginine (L-NMMA; 3x10(-5) M), the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 10(-5) M), and by tetrodotoxin (TTX; 3x10(-7) M). Guanethidine or hexamethonium did not affect inhibitory responses induced by McN-A-343. In conclusion, McN-A-343 induces nerve-dependent, nitrergic relaxations in rat jejunum, via activation of muscarinic M1 receptors. Hence, selective muscarinic M1 receptor agonists or antagonists might offer possibilities for pharmacological manipulation of the NO system.

Blockade of nitrergic neuroeffector transmission in guinea-pig colon by a selective inhibitor of soluble guanylyl cyclase.

The role of soluble guanylyl cyclase in nitrergic inhibitory neuroeffector transmission was investigated in the longitudinal muscle from guinea-pig colon, by using an inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). In preparations precontracted with histamine, electrical field stimulation (EFS) or exogenous nitric oxide (NO) induced relaxations. The relaxation induced by NO-application was abolished by ODQ. Both ODQ and the NO-synthase inhibitor N omega-nitro-L-arginine (L-NOARG) partially inhibited the EFS-evoked relaxation to a similar extent. These effects were dose-dependent. The inhibition was more pronounced in the late phase of the EFS-induced relaxation. The inhibitory effect of ODQ on EFS-induced relaxation was not affected by additional application of L-NOARG. When NO-formation was blocked by L-NOARG, a subsequent addition of ODQ gave no further inhibition of the relaxation. These findings suggest that inhibitory non-adrenergic, non-cholinergic neurotransmission in guinea-pig colon is dependent on endogenous formation of NO, and that the NO-effect is exclusively mediated via the soluble guanylyl cyclase pathway. The existence of an NO-independent inhibitory transmission, which is not mediated through the cyclic GMP pathway, is also indicated. Furthermore, it is demonstrated that the NO/soluble guanylyl cyclase-independent transmission has an earlier onset as compared with the NO/soluble guanylyl cyclase-dependent pathway.

Blockade of nitric oxide-evoked smooth muscle contractions by an inhibitor of guanylyl cyclase.

Nitric oxide-induced contractile responses of smooth muscle were studied in vitro in guinea-pig small intestine. Application of nitric oxide (NO; 0.3-30 microM) evoked a small initial relaxation followed by a marked contractile response in plexus-containing longitudinal smooth muscle preparations from small intestine. The extent of the NO-evoked contractile response was dose-dependent and the response was blocked by tetrodotoxin. Atropine significantly reduced the NO-evoked contraction and the remaining part was abolished by the NK1-receptor antagonist CP 96,345. An inhibitor of soluble guanylyl cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), abolished the NO-evoked contractile response. The results suggest that NO, in addition to the classical direct smooth muscle relaxing effect, causes activation of excitatory neurones, via a pathway utilizing soluble guanylyl cyclase, which leads to a smooth muscle contraction.

Nerve stimulation-induced nitric oxide release as a consequence of muscarinic M1 receptor activation.

The aim of the present study was to investigate whether nerve stimulation-induced nitric oxide (NO) release in the guinea-pig colon is affected by acetylcholine and to identify the muscarinic receptor subtype involved. Nerve-smooth muscle preparations were suspended in a superfusion chamber and NO/NO2- overflow in the superfusate was detected by chemiluminescence analysis. Transmural nerve stimulation evoked a significant increase in NO/NO2- release, which was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME) and abolished by tetrodotoxin. Exogenous acetylcholine concentration-dependently increased NO/NO2- release and atropine reduced nerve stimulation-evoked NO/NO2- release. The muscarinic M1 receptor selective antagonist telenzepine (10(-8) M) was as effective as atropine (10(-6) M) in inhibiting NO/NO2- release. The muscarinic M3 receptor antagonists 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and para-fluoro-hexahydrosila-difenidol (p-F-HHSiD) markedly inhibited cholinergic contractions at 3 x 10(-8) M and 3 x 10(-7) M respectively, but did not affect NO/NO2- release. In conclusion, nerve-induced NO/NO2- release in the guinea-pig colon is to a substantial part due to muscarinic M1 receptor activation. Thus acetylcholine, a major contractile neurotransmitter in the gut, can release NO which could act as a negative feedback mechanism on intestinal smooth muscle or neuronal activity.

Modulation of cholinergic and substance P-like neurotransmission by nitric oxide in the guinea-pig ileum.

1. The role of endogenous nitric oxide (NO) as a modulator of enteric neurotransmission was investigated in longitudinal muscle myenteric plexus (LMMP) preparations of guinea-pig isolated ileum. 2. In tissues previously incubated with [3H]-choline, exogenous NO inhibited electrically-evoked [3H]-choline overflow as well as responses to exogenous agonists, indicating that NO has the potential of neuromodulation both pre- and postjunctionally. 3. A series of NO synthase inhibitors enhanced contractile responses to nerve stimulation indicating inhibitory neuromodulation by endogenous NO. 4. The potency order of the NO synthase inhibitors and their consistent effects after dexamethasone, on responses to nerve stimulation, indicate action on a constitutive NO synthase. 5. Responses enhanced by NO synthase inhibitors were inhibited by the substance P receptor antagonist, spantide, suggesting a neuromodulatory influence on substance P-like neurotransmission by the endogenous NO. 6. NO synthase inhibition did not modify contractile responses to application of acetylcholine or substance P, or [3H]-choline overflow, indicating that endogenous NO mainly has a prejunctional inhibitory action on substance P-like neurotransmission. Nor did it modify responses to direct electrical muscle stimulation in the presence of tetrodotoxin. This suggests a prejunctional enhancing effect by NO synthesis inhibition. 7. Evidence for endogenous NO modulation of acetylcholine release was obtained when NO synthase inhibition modified atropine-sensitive, nerve-mediated contractile responses. However, [3H]-choline overflow was unaltered by NO synthase inhibition. 8. NO synthase inhibition did not modify responses to inhibitory neurotransmission. 9. The findings suggest that endogenous NO inhibits substance P-like motor neurotransmission, probably via prejunctional mechanisms. Cholinergic transmission may also be reduced by endogenous NO, acting prejunctionally.