Effects of hyperosmolarity on human isolated central airways.

1. We studied the effect of hyperosmolarity on human isolated airways because a better understanding of the effect of hyperosmolarity on the human airway wall may improve insight into the pathophysiology of hyperosmolarity-induced bronchoconstriction in asthma. 2. In cartilaginous bronchial rings dissected from fresh human lung tissue, hyperosmolar krebs-Henseleit buffer (450 mosM, extra sodium chloride added) evoked a biphasic response: a rapid relaxation phase (peak after 5.0 +/- 0.3 min) followed by a slow contraction phase (peak after 25.4 +/- 0.8 min). 3. With the histamine (H1) receptor antagonist mepyramine, the contraction phase was reduced to 41.2% of the control value (P less than 0.001), with atropine to 50.0% (P less than 0.01), with the local anaesthetic lignocaine to 48.7% (P less than 0.05) and with mepyramine together with atropine to 19.2% (P less than 0.001). 4. With the inhibitor of neutral metalloendopeptidase, phosphoramidon, the contraction phase increased to 128.0% of the control value (P less than 0.05) and after removal of the epithelium to 131.8% (P less than 0.05). 5. Indomethacin, the leukotriene C4/D4 (LTC4/D4) antagonist FPL 55712 or the blocker of nerve conduction, tetrodotoxin, had no effect on the contractile phase. 6. The relaxation phase was not altered by any of these drugs nor by epithelial denudation. The relaxation phase was also unchanged in the presence of alpha-chymotrypsin, which degrades muscle relaxing peptides such as vasoactive intestinal peptide. 7. Hyperosmolar buffer slightly increased the sensitivity and maximal response to methacholine as well as the cholinergic twitch to electric field stimulation. 8. We conclude that hyperosmolarity releases acetylcholine, histamine and neuropeptides in the human airway wall in sufficient quantities to contract airway smooth muscle. This release itself or its effect on airway muscle is modulated by the airway epithelium. The mechanism of the relaxation phase may be an unknown smooth muscle relaxing substance or a direct effect on the airway muscle, related to ion fluxes.

Effects of changes in osmolarity on isolated human airways.

The effects of hypo- and hyperosmolarity on the function of isolated human airways were studied. Changes in osmolarity induced an increasing bronchoconstriction that was proportional to the magnitude of the change in osmolarity. Hypertonicity-induced airway narrowing resulted when buffer was made hypertonic with sodium chloride or mannitol but not with urea. The airways showed no tachyphylaxis to repetitive exposure to hypo- and hypertonic buffer of 200 and 600 mosM, respectively. The bronchoconstriction was not secondary to stimulation of H1 or leukotriene C4/D4 receptors or the release of prostaglandins in the preparation. The bronchoconstriction in hypotonic buffer was totally dependent on extracellular calcium, whereas in hypertonic buffer the bronchoconstriction seemed partially dependent on intracellular calcium release. Isoprenaline prevented the bronchoconstriction in hyper- or hypotonic buffer of 450 and 250 mosM but not in buffer of 600 and 150 mosM. It is concluded that hypo- and hypertonic buffers lead to bronchoconstriction via different mechanisms, which relate to influx of extracellular calcium in hyposmolar buffer and probably to release of calcium from intracellular stores in hypertonic buffer. In strongly hypertonic buffer, part of the bronchoconstriction may be due to osmotic shrinkage. The relevance of our data for the mechanism of bronchoconstriction after inhalation of hypo- or hypertonic saline depends on whether changes in osmolarity around the airway smooth muscle occur in asthmatics but not in normal subjects, and this has not yet been established.

Treatment of non-malignant pain in the Dutch GP population: an analysis of treatment standards, prescription habits and basic attitudes.

The aim of this study was to evaluate whether dexibuprofen meets the basic requirements of Dutch General Practitioners (GPs) for acceptance as an analgesic in mild to moderate pain. The available GP treatment standards for mild to moderate pain were analysed. If no treatment standard was available, results from two semistructured round table group sessions were used. The available treatment standards are either diagnosis oriented (e.g. otitis media) or symptom oriented (e.g. lower backache). Prescription data (1988-2000) show that GPs usually prescribe the medication suggested by the standards. Paracetamol, diclofenac, ibuprofen and naproxen, all over 25 years old, have a combined marketshare greater than 84%. Most new drugs have been short-lived and/or little prescribed. Round tables show three basic dimensions to pain treatment: objective, subjective, and the action taken to relieve the pain. The objective dimension relates to anamnesis and physical examination to rule out serious causes. The subjective dimension relates to the doctor's assessment of the pain threshold of the patient, expectations towards the doctor, and previous action taken by the patient. The pain relief dimension relates to acceptance of the pain by the patient, the solution proposed by the doctor and the way this is presented. Medication prescribed should be exclusive for doctors. The GP must also have faith in the medication, and so previous experience is a dominant factor in the choice made. GPs are relatively insensitive to new products, as there are few unmet needs. As a new NSAID dexibuprofen meets two basic requirements. First, it is only available to doctors. Secondly, because it is a pure form of ibuprofen, doctors can relate to extensive previous experience when prescribing it. This might ensure its rapid acceptance compared to other new NSAIDs.

Control of airway caliber by autonomic nerves in asthma and in chronic obstructive pulmonary disease.

Autonomic nerves can influence airway caliber via their effects on airway smooth muscle, bronchial vessels, and mucous glands and may therefore contribute to airway narrowing in asthma or in chronic obstructive pulmonary disease (COPD). Human lungs receive cholinergic, noradrenergic, and peptidergic efferents and several types of afferents. Cholinergic nerve activity contributes to airway narrowing both in asthma and in COPD. Reflex vagal activity may be enhanced because of epithelial damage and exposition of sensory nerve endings to nonspecific irritants. Other possible mechanisms include defects in prejunctional receptors that inhibit acetylcholine release, several postjunctional factors that nonspecifically enhance the effect of a given degree of cholinergic muscle contraction on airway caliber, and interactions between inflammatory mediators and the cholinergic system. The main direct bronchodilating nerve activity in human lungs is nonadrenergic, and scanty data suggest that nonadrenergic inhibitory nerve activity may be variably reduced in asthmatics. Human airway muscle virtually lacks adrenergic innervation, but adrenergic nerves may influence airway caliber by acting on bronchial vessels, mucous glands, and parasympathetic nerves and ganglia. The response of asthmatic airways to beta-agonists seems intrinsically normal, but it may be reduced during severe asthma attacks. There are no convincing data that abnormal adrenergic control is present in the airways of patients with COPD. The physiologic relevance of excitatory neuropeptides in sensory nerves in human airways is uncertain. Tachykinins have proinflammatory and spasmogenic properties and are therefore of potential interest as a factor in the pathogenesis of obstructive airway disease. In conclusion, the data presently available support an abnormal autonomic control of the airways in asthma but not in COPD.

The increased responsiveness to inhaled methacholine in asthma: combination of causative factors.

In asthma the dose-response curve to inhaled methacholine (MCh) is shifted leftwards and shows an increased slope and maximum. Two factors might contribute to the different form and position of this curve: (1) mediator-induced airway muscle hypersensitivity and (2) thickening of the airway wall. We attempted to estimate the effect of a combination of these two factors on the increase in airway resistance of a single isolated human airway to increasing concentrations of MCh. The result suggests that mediator-induced hypersensitivity to cholinergic stimuli may explain a small part of the leftward shift of the asthmatic dose-response curve to MCh, especially at lower levels of bronchoconstriction, and that increased thickness of the airway wall may be more important for the increased slope and maximum of the asthmatic dose-response curve.

Activated human granulocytes contract isolated human airways.

Human granulocytes activated with serum treated zymosan (0.2 mg/ml) contract isolated human airways. The magnitude of the contraction depends on the number of granulocytes and the proportion of eosinophils among the granulocytes. The contraction is blocked by a leukotriene C4/D4 (LTC4/D4) receptor antagonist and by inhibition of lipoxygenase. This suggests that eosinophils rather than neutrophils are implicated in this response, which seems to be caused by LTC4/D4.

Additive effect of epithelial denudation and low levels of inflammatory mediators on the sensitivity of isolated human airways to methacholine.

The presence of low concentrations of histamine or the stable thromboxane analogue U46619 and the removal of the epithelium separately and addictively increase the sensitivity of isolated human airways to methacholine. This raises the possibility that these factors play a role in the pathogenesis of bronchial hyperresponsiveness to inhaled methacholine in asthma.

Electrically stimulated Krebs-Henseleit buffer does not relax precontracted human bronchi in vitro.

Electric field stimulation (EFS) causes a non-adrenergic relaxation of precontracted human airways. Only 10-20% of this response can be blocked with the nervous conductance blocker tetrodotoxin (TTX). Therefore, 80-90% of the non-adrenergic relaxation is not neurally mediated. The mechanism of this non-neural component is unclear. We examined whether EFS induces the generation of muscle relaxing substances in oxygenated Krebs-Henseleit buffer. Strips of central airways of 6 patients were precontracted with methacholine (10(-5) M) and exposed to Krebs buffer that had previously been stimulated with EFS (50 V, 0.3 ms, 30 s, 1-50 Hz). Results were compared to those obtained with unstimulated buffer. The contractile state of the airways did not change significantly with either EFS-stimulated or unstimulated buffer. We conclude that in oxygenated Krebs buffer EFS does not cause generation of substances that change the contractile state of prestimulated human airways.

Effects of zymosan-activated human granulocytes on isolated human airways.

In asthma a temporal association exists between the late allergic reaction (LAR), the influx of granulocytes into the airway wall, and an increase in bronchial responsiveness. We therefore tested the hypothesis that activated human granulocytes constrict isolated human airways and increase their sensitivity to cholinergic stimuli. Bronchial rings were dissected from 23 lung tissue specimens collected at thoracotomy and studied isotonically in organ baths. Airways were incubated with 1, 2, 5, 10, or 20 x 10(6) granulocytes from normal or atopic donors. Activation of the cells with serum-treated zymosan (STZ, 0.2 mg/ml), which itself did not alter baseline airway caliber, resulted in a bronchoconstriction proportional to the number of zymosan-activated granulocytes (ZAG) present (rs = 0.79, p less than 0.001). This contraction was reduced by about 70% with the leukotriene C4/D4 receptor antagonist FPL 55712 (11.5 microM; p less than 0.001) or with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM; p less than 0.001). The scavengers of activated oxygen molecules superoxide dismutase (300 U/ml) and bovine catalase (5,000 U/ml), the cyclooxygenase inhibitor indomethacin (10 microM), or the histamine (H1) receptor antagonist mepyramine (2.8 microM) had no effect. Granulocyte suspensions from atopic donors contained more eosinophils (p less than 0.001), and the magnitude of the contraction to 10 x 10(6) ZAG was related to the proportion of eosinophils (rs = 0.66, p less than 0.01). The sensitivity of the airways to methacholine was unchanged in the presence of 1, 2, or 5 x 10(6) ZAG and decreased with 10 or 20 x 10(6) ZAG (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cooling on responses of isolated human airways to pharmacologic and electrical stimulation.

We studied the effect of cooling on the responses of isolated human airways to the beta-agonist isoproterenol, the alpha/beta-agonist norepinephrine in the presence of the beta-blocker timolol, methacholine, leukotriene C4 (LTC4), and histamine. In addition, the effect of cooling on baseline airway tone and responses to electric field stimulation (EFS) was studied. At 27 degrees C the sensitivity (-logEC50) and maximal response to isoproterenol were unchanged. No measurable response was found to alpha-adrenergic stimulation with norepinephrine + timolol either before or during cooling. At 27 degrees C and 21 degrees C the sensitivity and maximal contraction to methacholine and LTC4 as well as the contraction to a single dose of histamine were reduced. Cooling diminished baseline airway tone. EFS produced a rapid cholinergic contraction followed by a deflection below baseline and a sustained noncholinergic contractile response, which was substantially reduced by the LTC4/D4 receptor antagonist FPL 55712 (11.5 microM) at all three temperatures. Cooling decreased the cholinergic response to EFS and increased the sensitivity to EFS-induced relaxation. In contrast, the sustained noncholinergic contractile response to EFS was not changed, suggesting that cooling facilitates the synthesis of LTC4/D4 that follows EFS and/or inhibits its inactivation. We conclude that in nonasthmatic, isolated human airways slow cooling of the airway wall down to 21 degrees C does not cause bronchoconstriction and does not increase the responsiveness to contractile or relaxing agonists. However, cooling increases the sensitivity to EFS-induced relaxation and might facilitate the accumulation of leukotriene C4/D4 in the airway wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of inflammatory mediators on the responsiveness of isolated human airways to methacholine.

Several studies have suggested that in asthmatics the quantities of inflammatory mediators such as histamine, thromboxane A2 (TxA2), prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF2 alpha), and leukotriene C4 (LTC4) that are present in the airway lumen are related to the degree of bronchial responsiveness to inhaled methacholine (MCh). Therefore, we studied the effect of these mediators on the cholinergic responsiveness of isolated human airway segments. Lung tissue collected at thoracotomy from 30 patients was studied. Dose-response curves to MCh were obtained from bronchial segments before, during, and after incubation with either a subthreshold or a threshold concentration of histamine (10(-10) or 10(-8) M), the stable TxA2 analogue U46619 (10(-11) or 10(-9) M), PGD2 (5 x 10(-9) or 5 x 10(-7) M), PGF2 alpha (10(-9) or 10(-7) M), or LTC4 (10(-11) or 10(-9) M). With the exception of LTC4, the presence of any of these mediators at either concentration increased the sensitivity to MCh by a factor of 1.1 to 2 (p less than 0.05, ANOVA). This increase did not depend on the dose of the mediator (p greater than 0.05, ANOVA). These data indicate that mediator-induced muscle hypersensitivity can explain a small part of the leftward shift of the dose-response curve to inhaled MCh as observed in asthma.

Effects of cooling on in vitro responses of human peripheral airways to inflammatory mediators and neurotransmitters.

In asthma, inhalation of cold dry air induces bronchoconstriction. It has been suggested that cooling of the airway wall might induce this bronchoconstriction. Therefore the effects of cooling on the contractility of human peripheral airways were studied in vitro before and during lowering of the temperature. Cooling relaxed human airways in vitro and reduced responsiveness to methacholine, histamine and LTC4 both in terms of -logEC50 and maximal effect. It also reduced the maximal effect of isoprenaline. There was no measurable alpha-adrenergic activity before or during cooling. We conclude that cooling does not sensitize human airways to any of these agonists and that cold-induced bronchoconstriction in vivo is not due to a direct effect of cooling on airway smooth muscle.

Measurement of human small airway smooth muscle function in vitro. Comparison of bronchiolar strips and segments.

We have compared isotonic responses to methacholine of human bronchiolar segments and spiral strips. Both types of preparations contracted dose-dependently to methacholine and had a stable intrinsic contractile activity, which was significantly higher in segments (p less than 0.001). ANOVA indicated that the total variation in responses of both strips and segments was similar and was mainly due to a significant between-preparations/within-patients variation. There was a small but significant trend towards a decrease of sensitivity (EC50) in time for both segments and strips. Net contraction, i.e. the difference between resting length and the length at maximal contraction, did not change in time. Limited length-active shortening experiments indicated that 250 mg was a suitable load for both strips and segments. We concluded that, although human bronchiolar strips and segments are functionally comparable, bronchiolar segments are preferable because of their practical and theoretical advantages over bronchiolar strips.

Eicosanoid levels in bronchoalveolar lavage fluid of young female smokers and non-smokers.

To evaluate indicators of inflammatory changes in the airways of young smokers we have measured the levels of several eicosanoids in bronchoalveolar lavage (BAL) fluid of 18 female smokers (age 33 +/- 2 years) and 9 female non-smokers (age 29 +/- 2 years) who were hospitalized for treatment not related to any pulmonary disease. In each BAL specimen the following eicosanoids were determined by radioimmunoassay: prostaglandin (PG) E2; PGF2 alpha; 9 alpha, 11 beta-PGF2, a metabolite of PGD2; 6-keto PGF1 alpha, a metabolite of prostacyclin; thromboxane (Tx) B2, a metabolite of TxA2; the 5-lipoxygenase products 5-hydroxy-eicosa-tetraenoic acid (HETE), leukotriene (LT) B4 and LTC4; the 12-lipoxygenase product 12-HETE; and the 15-lipoxygenase product 15-HETE. The concentrations of the cyclooxygenase products (pg ml-1) in the BAL fluid of the non-smokers were: PGE2 15.4 +/- 1.9, PGF2 alpha 7.6 +/- 1.0, 9 alpha, 11 beta-PGF2 8.7 +/- 1.8, TxB2 8.8 +/- 1.3, and 6-keto PGF1 alpha only 1.5 +/- 0.8. The concentration of the lipoxygenase products were: 15-HETE 781 +/- 200, 12-HETE 193 +/- 33, 5-HETE 14.0 +/- 3.1, LTC4 9.5 +/- 3.1, LTB4 6.2 +/- 1.4. BAL fluid from smokers contained two- to three-fold higher levels of TxB2 and PGF2 alpha (P less than 0.05). The levels of TxB2 and PGF2 alpha were positively correlated to the number of package years (rs = 0.55 and rs = 0.65, P less than 0.02). The concentrations of 5-, 12- and 15-HETE tended to be higher in BAL fluid from smokers, but this was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelium removal and peptidase inhibition enhance relaxation of human airways to vasoactive intestinal peptide.

In this study we evaluated the role of epithelial versus subepithelial peptidases in the responses of isolated peripheral and central human airways to VIP. Human airways were obtained at thoracotomies (n = 8) and studied in organ baths. Intact or epithelium-denuded strips of central and peripheral airways were incubated with or without a cocktail of peptidase inhibitors containing phosphoramidon (2.5 micrograms/ml), leupeptin, aprotinin, captopril, soybean trypsin inhibitor (all 20 micrograms/ml), and bestatin (2.8 micrograms/ml). After precontraction with histamine (5 x 10(-6) M), cumulative concentration-response curves to VIP (10(-10) to 10(-7) M) were obtained. Both intact central and peripheral airways showed only minor relaxations to VIP irrespective of the precontraction level. Removal of the epithelium and addition of peptidase inhibitors additively increased the sensitivity (> 20-fold) and maximal response to VIP in both central and peripheral airways. We conclude that (1) VIP relaxes both central and peripheral human airways but only in the absence of epithelium and/or the presence of peptidase inhibitors, and (2) both epithelial and subepithelial peptidases are important in the inactivation of VIP in human airways.