Metabolism and formation of DNA adducts of benzo(a)pyrene in human diploid fibroblasts.

Cultured human diploid skin fibroblasts incubated with [G-3H]benzo(a)pyrene yielded about 10 times more H2O-=soluble benzo(a)pyrene metabolites and DNA adducts of stationary growth phase than did proliferating cultures. This increased formation could be blocked by alpha-naphthoflavone. Trichloropropenoxide and cyclohexenoxide, inhibitors of the epoxide hydratase, inhibited predominantly the formation of DNA adducts. Cultures from older individuals formed significantly more benzo(a)pyrene metabolites and DNA adducts, but control cultures from patients with either lung cancer or melanoma did not. The age influence was not apparent when the ratio of DNA adducts to H2O-soluble metabolites was determined for each individual cell line. However, the proportion of DNA-bound material in the cells from patients with lung cancer was significantly increased compared to cells from melanoma patients or healthy individuals.

Effect of nitrogen dioxide inhalation on surfactant phosphatidylcholine synthesis in rat alveolar type II cells.

After exposure of rats to NO2 (10 ppm, 72 h) type II pneumocytes were isolated and compared to cells from control animals in order to determine whether nitrogen dioxide inhalation affects surfactant phospholipid synthesis. (1) Exposed cells contained more DNA, protein and phospholipid than type II cells from controls. (2) Choline kinase, CTP: cholinephosphate cytidylyltransferase, and cholinephosphotransferase showed higher specific activities in the exposed cells. (3) In correspondence with this finding, the incorporation rates of choline into intermediate metabolic products were also higher in the NO2-exposed cells. (4) The pool sizes of the intermediate metabolic products of the CDP-choline-pathway for the synthesis of phosphatidylcholine were also higher in the cells isolated from exposed animals. This suggests that acute nitrogen dioxide exposure leads to an enhanced phospholipid synthesis that may be responsible for the higher amount of phospholipid detectable in lung lavage.

Influence of septic shock upon phosphatidylcholine remodeling mechanism in rat lung.

Septic shock in rats lead to pulmonary disorders associated with alterations of phospholipid metabolism. The ratio between phosphatidylcholine and lysophosphatidylcholine is lowered both in lung tissue and in pulmonary surfactant because enzymes of phosphatidylcholine remodeling mechanism are distinctly affected by septic shock. Specific activity of phospholipase A2 is enhanced 5-fold while specific activities of lysolecithin acyltransferase and lysolecithin : lysolecithin acyltransferase are only slightly increased or remain unchanged. Beyond that, palmitic acid content of lung tissue phosphatidylcholine is significantly reduced and replaced mainly by arachidonic acid. The release of this fatty acid by action of phospholipase A2 may lead via intermediates to the generation of potent mediators such as prostaglandins, thromboxane or slow-reacting substance.

A cytofluorometric method to quantify membrane antigens on individual alveolar macrophages.

An immunofluorescence method for assaying membrane-bound antigens on individual alveolar macrophages (AM) collected by bronchoalveolar lavage (BAL) is described. Cells were labelled with FITC-conjugated anti-HLA-DR antibodies in a single (direct) step. Quantification of the fluorescence was performed by computer-assisted cytophotometry. Alveolar macrophages, especially when obtained from cigarette smokers, exhibited an autofluorescence which interfered with the measurement of specific fluorescence. The specific fluorescence was calculated by determination of total fluorescence in the wave-length optimal for FITC and the non-specific fluorescence in a different wavelength during a second measurement. Specificity and reproducibility testing confirmed the reliability of the method.

Effects of cilazapril on hypertension, sleep, and apnea.

Epidemiologic studies revealed that up to 10 percent of middle-aged men show more than 10 cessations of breathing of more than 10 seconds' duration. In these patients, increased morbidity and mortality rates have been proved. More than 50 percent of apnea patients exhibit arterial hypertension, and up to 50 percent of hypertensive patients experience sleep apnea. Patients with sleep apnea and essential hypertension need special attention paid to their antihypertensive therapy because the following side effects of drugs have to be avoided: increases of cardiac insufficiency, hyperviscosity of the blood, intensification of the hypersomnia by central sedation, intensification of a pre-existing tendency towards arrhythmias, and deprivation of deep and rapid eye movement sleep. In this study, the effects of angiotensin-converting enzyme inhibitors in patients with sleep apnea and hypertension are examined. An interim evaluation of six patients (aged 50 to 57) yielded the following results: Average Broca index, 124; average blood pressure before therapy, 159/102 mm Hg; average blood pressure after therapy, 132/78; a decrease of the apnea and hypopnea index from x = 31 (range, 12 to 77) to x = 20 (range, two to 54). Therapy did not influence sleep structure: before therapy, an average of 19 percent of sleep episodes were of the rapid eye movement type (range, 11 to 32 percent); after therapy, 23 percent were of this type on average (range, 21 to 25 percent). A final evaluation will be carried out after the second study phase for 12 patients who have been treated in a double-blind scheme with metropolol versus cilazapril.

MESAM: a heart rate and snoring recorder for detection of obstructive sleep apnea.

The high prevalence of sleep-related breathing disorders demands the development of ambulatory recording devices that can handle data with a high degree of selectivity and are easy to use and to interpret. A digital device based on the recording of heart rate and breathing sounds was developed. Patients with sleep-related breathing disorders can be preselected before they undergo sleep laboratory investigations. Treatment control can be achieved ambulatory, having an initial recording.

Reduced catecholamine response of lymphocytes from patients with rheumatoid arthritis.

Catecholamines modulate lymphocyte function via stimulation of beta2-adrenergic receptors (beta2R). Previous investigations revealed a decreased density of beta2R on peripheral blood mononuclear cells (PBMC) in patients with chronic rheumatic diseases. Aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 12 healthy blood donors (HD) were investigated. Beta2R were determined by a radioligand binding assay. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with pokeweed mitogen (PWM) and monoclonal anti-CD3-antibodies (OKT3), respectively. In parallel, alpha1- or beta-receptor antagonist were added to the culture to determine the specificity of the catecholaminergic effects. The results showed that depending on the stimulus and the catecholamine concentration employed E and NE exert inhibitory (OKT3) or stimulatory signals (PWM) on lymphocyte proliferation. Inhibitory effects could be abolished by adding beta-antagonist, while stimulatory signals were diminished after addition of alpha1- of beta-antagonist. Patients with RA showed a significantly reduced density of beta2R compared to HD paralleled by a significantly reduced influence of catecholamines on lymphocyte function. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that are mediated via alpha1- and beta-adrenergic receptors. In this respect the reduced catecholamine response of PBMC from RA patients may contribute to the pathogenic process of RA.

Central alveolar hypoventilation and sleep. Treatment by intermittent positive-pressure ventilation through nasal mask in an adult.

Idiopathic central alveolar hypoventilation, uncommonly seen in middle-aged adults, has often been treated by tracheostomy and assisted ventilation during sleep or by implantation of a diaphragmatic pacemaker with or without tracheostomy. We report the successful treatment of a middle-aged man by the easy application of intermittent positive-pressure ventilation through a nose mask.

Proliferation and number of Clara cell 10-kDa protein (CC10)-reactive epithelial cells and basal cells in normal, hyperplastic and metaplastic bronchial mucosa.

Clara cell 10-kDa protein (CC10) is an inhibitor of phospholipase A2 and binds to phosphatidylinositol. It may therefore interfere with intracellular signal transduction. Bronchial CC10-reactive cells have been described by several authors. In contrast to the bronchiolar CC10-containing Clara cell, which is a progenitor cell of terminally differentiated airway epithelium, the role of bronchial CC10-reactive cells remains to be elucidated. We assessed the number of bronchial CC10-reactive cells in relation to cytokeratin (CK) expression and proliferative activity in normal, hyperplastic and squamous metaplastic epithelium. Sixty-five human bronchial mucosal specimens were investigated immunohistochemically for CK expression (CK7, CK13 and CK5/6), proliferative activity (MIB-1) and number of CC10-reactive epithelia. The proliferation fraction of CC10-reactive cells was assessed with double staining for MIB-1 and CC10. The proliferation index of the epithelium differed significantly between normal, hyperplastic and metaplastic epithelium. The number of CC10-reactive cells was inversely related to the epithelial proliferation. Bronchial CC10-reactive cells showed no proliferative activity as assessed using immunohistochemical double staining for CC10 and MIB-1. In contrast to normal and hyperplastic epithelium, squamous metaplasia disclosed CK5/6 in all epithelial layers, a loss of CK7 and a gain of CK13. We conclude that CC10-reactive cells have no progenitor role in the bronchial mucosa. However, because the proliferative activity is inversely related to the number of CC10-reactive cells, the CC10 protein may play a role in the regulation of epithelial repair. Squamous metaplasia most likely originates from basal cells.

Gallium-67 activity in bronchoalveolar lavage fluid in sarcoidosis.

Roentgenograms and gallium-67 scans and gallium-67 counts of BAL fluid samples, together with differential cell counts, have proved to be useful in assessing activity and lung involvement in sarcoidosis. In active pulmonary sarcoidosis gallium-67 scans are usually positive. Quantitation of gallium-67 uptake in lung scans, however, may be difficult. Because gallium-67 uptake and cell counts in BAL fluid may be correlated, we set out to investigate gallium-67 activity in BAL fluid recovered from patient of different groups. Sixteen patients with recently diagnosed and untreated sarcoidosis, nine patients with healthy lungs, and five patients with CFA were studied. Gallium-67 uptake of the lung, gallium-67 activity in the lavage fluid, SACE and LACE levels, and alpha 1-AT activity were measured. Significantly more gallium-67 activity was found in BAL fluid from sarcoidosis patients than in that from CFA patients (alpha = .001) or patients with healthy lungs (alpha = .001). Gallium-67 activity in BAL fluid could be well correlated with the number of lymphocytes in BAL fluid, but poorly with the number of macrophages. Subjects with increased levels of SACE or serum alpha 1-AT showed higher lavage gallium-67 activity than did normals, but no correlation could be established. High gallium-67 activity in lavage fluid may be correlated with acute sarcoidosis or physiological deterioration; low activity denotes change for the better. The results show that gallium-67 counts in BAL fluid reflects the intensity of gallium-67 uptake and thus of activity of pulmonary sarcoidosis.

Disease activity related catecholamine response of lymphocytes from patients with rheumatoid arthritis.

In patients with chronic rheumatic diseases, a decreased density of beta 2-adrenergic receptors (beta 2R) on peripheral blood mononuclear cells (PBMC) could be demonstrated negatively correlating with various disease activity parameters. The aim of the present study was to determine the impact of this decrease on catecholamine response of PBMC from patients with rheumatoid arthritis (RA) in vitro. PBMC from 17 patients with RA and 6 healthy blood donors (HD) were investigated. The effects of epinephrine (E) and norepinephrine (NE) on PBMC proliferation were studied using cells activated with phytohemagglutinin (PHA) and monoclonal anti-CD3-antibodies (OKT3), respectively. The results revealed that lymphocytes of patients with RA showed a significantly reduced influence of catecholamines on PBMC function. In RA patients with high disease activity only, a shift to alpha 1-adrenergic-mediated catecholamine effects upon PBMC reactivity could be observed. The study demonstrates the intricate relationship between PBMC reactivity and catecholamine effects that is mediated via alpha- and beta-adrenergic receptors due to disease activity. In this respect the altered catecholamine response of PBMC from patients with RA may contribute to the pathogenic process of RA.

Expiratory flow limitation and intrinsic positive end-expiratory pressure in obesity.

Breathing at very low lung volumes might be affected by decreased expiratory airflow and air trapping. Our purpose was to detect expiratory flow limitation (EFL) and, as a consequence, intrinsic positive end-expiratory pressure (PEEPi) in grossly obese subjects (OS). Eight OS with a mean body mass index (BMI) of 44 +/- 5 kg/m2 and six age-matched normal-weight control subjects (CS) were studied in different body positions. Negative expiratory pressure (NEP) was used to determine EFL. In contrast to CS, EFL was found in two of eight OS in the upright position and in seven of eight OS in the supine position. Dynamic PEEPi and mean transdiaphragmatic pressure (mean Pdi) were measured in all six CS and in six of eight OS. In OS, PEEPi increased from 0.14 +/- 0.06 (SD) kPa in the upright position to 0.41 +/- 0.11 kPa in the supine position (P < 0.05) and decreased to 0.20 +/- 0.08 kPa in the right lateral position (P < 0.05, compared with supine), whereas, in CS, PEEPi was significantly smaller (<0.05 kPa) in each position. In OS, mean Pdi in each position was significantly larger compared with CS. Mean Pdi increased from 1.02 +/- 0.32 kPa in the upright position to 1.26 +/- 0.17 kPa in the supine position (not significant) and decreased to 1. 06 +/- 0.26 kPa in the right lateral position (P < 0.05, compared with supine), whereas there were no significant changes in CS. We conclude that in OS 1) tidal breathing can be affected by EFL and PEEPi; 2) EFL and PEEPi are promoted by the supine posture; and 3) the increased diaphragmatic load in the supine position is, in part, related to PEEPi.

Effects of VIP and related peptides on airway mucus secretion from isolated rat trachea.

Vasoactive intestinal polypeptide (VIP) is known as an important regulator of airway function. It has been suggested that VIP is involved in the pathogenesis of asthma due to its relaxant effects on smooth muscles. The present study was designed to characterize the effects of the peptides of the VIP family on airway mucus secretion. The peptides VIP, PHI, PACAP-27, PACAP-38, GLP-I, exendin-4, helodermin, helospectin I and helospectin II were investigated using isolated rat trachea. Data show that PACAP-27 is the most potent stimulator of airway mucus secretion (225% stimulation). The rank order of potency was PACAP-27 > VIP > helospectin II > PHI > exendin-4 = helodermin = helospectin I = PACAP-38. The addition of the protease inhibitor thiorphan enhanced the effects of PHI and helodermin, but not of the other peptides. These data show that the peptides of the VIP family stimulate airway mucus secretion differently.

Galanin and somatostatin inhibition of substance P-induced airway mucus secretion in the rat.

Substance P is present in several neurons innervating the lung. Tachykinin receptors are expressed on submucosal gland cells. Substance P is known to be a potent stimulator of airway mucus secretion. In the present study we characterized the effects of galanin and somatostatin on basal and substance P-induced mucus secretion. The stimulatory effect of substance P was concentration-dependent (100 pmol/l: 112%, 1 nmol/l: 120%, 10 nmol/l: 153%, 100 nmol/l: 223%, 1 mumol/l: 275%, 10 mumol/l: 172%) and was inhibited by galanin and somatostatin (1 mumol/l substance P: 277%; 1 mumol/l substance P + 1 mumol/l somatostatin: 190%, p < 0.01; 1 mumol/l substance P + 1 mumol/l galanin: 206%, p < 0.05). In the presence of lower concentrations of substance P 1 mumol/l somatostatin and 1 mumol/l galanin did not modify mucus secretion. Lower concentrations of galanin and somatostatin did not significantly change mucus secretion stimulated by 1 mumol/l substance P. Both, galanin and somatostatin at 1 mumol/l left basal airway mucus secretion unaltered. These data suggest that mucus secretion into airways is regulated by a complex network of peptidergic stimulators and inhibitors including substance P, somatostatin and galanin.

Effects of selective and non-selective phosphodiesterase inhibitors on tracheal mucus secretion in the rat.

The present study was designed to characterize the effects of unselective and isoenzyme-selective phosphodiesterase inhibitors on airway mucus secretion. The isolated rat trachea was incubated in a modified Ussing chamber. Mucus macromolecules were metabolically labelled with 35S. The inhibitors were applied at the luminal side. The unselective phosphodiesterase inhibitors theophylline, enprofylline and 3-isobutyl-methylxanthine stimulated mucus secretion in a concentration-dependent manner with half-maximum effects (EC50 values) at 690 microM, 400 microM and 46 microM, respectively. The adenosine antagonist 8-phenyltheophylline did not significantly stimulate mucus output, suggesting a negligible role of adenosine in the cellular mechanisms of mucus secretion. Adenosine itself did not increase radiolabel output. Rolipram, an inhibitor of phosphodiesterase isoenzyme IV, and zardaverine, which inhibits the isoenzymes III and IV, increased potently macromolecule output with EC50 values of 40 nM and 6 microM, respectively. The selective inhibitors of phosphodiesterase isoenzymes III and V, motapizone and zaprinast, did not influence airway mucus release, suggesting a relatively low activity of isoenzymes III and V in glands of rat trachea. The stimulatory effect of theophylline on airway mucus secretion may contribute to its beneficial action in chronic obstructive airway disease. Our data suggest that this effect is mediated predominantly by phosphodiesterase isoenzyme IV.

Effects of the nitric oxide/cGMP system compared with the cAMP system on airway mucus secretion in the rat.

Mucus secretion of the airways is under the control of a variety of intracellular second messenger systems. Cyclic nucleotides such as cGMP, coupled to the recently discovered nitric oxide system, and cAMP are of outstanding interest in this respect. The present study used the modified Ussing chamber technique and mucins labelled with (35)SO(4) to investigate mucus secretion in the rat trachea to clarify the contribution of these different second messenger systems to the control of mucin secretion.A variety of drugs affecting either the generation or the breakdown of the respective cyclic nucleotides were used. Neither drugs interfering with nitric oxide synthase nor the phosphodiesterase isoenzyme responsible for cGMP breakdown nor cGMP analogues were able to affect mucus secretion. In contrast, stimulation of adenylate cyclase or inhibition of the respective phosphodiesterase resulted in a potent increase of mucus secretion. In conclusion, we failed to show the involvement of the nitric oxide/cGMP system, whereas the cAMP system seems to be a very efficient regulator of mucus secretion in the rat trachea.

Arterial hypertension and sleep apnoea: effect of the angiotensin-converting enzyme (ACE) inhibitor cilazapril on continuously measured blood pressure during sleep and wakefulness.

Male patients with arterial hypertension and obstructive sleep-related breathing disorders (mean age 50 y, Body Mass Index (BMI) 32.4 kg m-2, Respiratory Disturbance Index (RDI) 47.2 and systolic/diastolic blood pressure (SBD/DBD) 162/103 mmHg) were examined before and after 8 days of treatment with the long-acting angiotensin-converting-enzyme (ACE) inhibitor cilazapril 2.5 mg vs. placebo in a double-blind design with parallel groups. Cardiorespiratory polysomnography was carried out at night; during daytime wakefulness patients submitted to examinations of physical and mental exertion. Cilazapril reduced the mean pressure during the entire examination period (day and night) by 9.55 (SD +/- 7.13) mmHg, compared to 4.57 (SD +/- 7.20) mmHg for placebo (P < 0.006), independently from systematic changes of heart rate (x = -3.3 and -3.5 bpm, respectively). During REM sleep, mean arterial pressure was significantly reduced by 8.63 (SD +/- 10.1) mmHg, compared to a reduction on placebo of 3.17 (SD 9.6) mmHg (P = 0.023). Under psychometric strain, the mean arterial pressure was reduced by 15.31 (SD +/- 8.7) mmHg with cilazapril; under placebo medication by 6.19 (SD +/- 7.3) mmHg (P < 0.0001). Heart rate was not significantly changed.

Invasive electrophysiological evaluation of patients with sleep apnoea-associated ventricular asystole-methods and preliminary results.

Twelve patients (aged 48 +/- 12 y) with ventricular asystole of >3 s due to complete atrioventricular (AV) block (n = 8), sinoatrial (SA) block or sinus node arrest (n = 3) or both (n = 1) associated with obstructive sleep apnoea underwent invasive electrophysiological evaluation of sinus node function and AV conduction properties before and after administration of atropine (0.02 mg kg-1). Ventricular asystole lasted for 5.9 +/- 2.8 s (range 3.1-13 s). Sinus node function was assessed by measurement of sinus node recovery time, sinoatrial conduction time, and the response of sinus rate to atropine. Parameters of AV-conduction assessment included AH- and HV-intervals, AV- and VA-Wenckebach periods, and effective refractory period of the AV node before and after atropine. Sinus node function was normal in 11 of the 12 study patients and moderately abnormal in 1 patient. AV-nodal function was normal in 8 patients and moderately abnormal in 4 patients. A slightly prolonged HV-interval (59-63 ms) was present in 6 patients. Intra- or infra His block was not observed in any patient. In conclusion, normal or only moderately abnormal electrophysiological findings in patients with sleep apnoea-associated ventricular asystole suggest that a neurally mediated cardioinhibitory reflex may cause ventricular asystole in these patients. This sleep apnoea-triggered 'vasovagal' reflex may unmask pre-existing mild to moderate structural abnormalities of the AV conduction system.

Increased incorporation of fatty acids into phospholipids of lungs and livers of rabbits under the influence of bromhexine and ambroxol.

The incorporation of lauric acid, palmitic acid and oleic acid into phospholipids of lung and liver has been studied in tissue slices of control rabbits and of rabbits treated with bromhexine or ambroxol in doses of 10 mg/kg. A marked increase (up to 200% of the controls) of palmitic acid incorporation into phosphatidylcholine (lecithin) and phosphatidylethanolamine of the lung was found whereas the incorporation rate of palmitic acid into lecithin and phosphatidylethanolamine of the liver displayed no significant change. The incorporation of lauric acid and oleic acid into lung phospholipids was not accelerated. The observed effects were more marked in short time experiments (analysis 2 h after drug injection) than after treatements for 7 days. It is concluded that the phospholipid synthesis is stimulated by the drugs especially in the lungs. This seems to be of particular interest with respect to the surfactant system of the lung and might have some therapeutic relevance.

Galanin and somatostatin inhibition of neurokinin A and B induced airway mucus secretion in the rat.

Neurokinin A and B are present in neurons situated in lung and NK-1 receptors have been described on tracheal submucosal gland cells. In the present study we compared the ability of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) to stimulate airway mucus secretion. Furthermore, we characterized the interaction of NKA and NKB with galanin and somatostatin. The rank order of the tachykinins to stimulate airway mucus secretion was SP > NKA > NKB suggesting that NK-1 receptors mediate these effects(EC50:SP: 50 nmol/l, NKA: 200 nmol/l, NKB: 400 nmol/l). Galanin and somatostatin were equally potent to inhibit NK-A and NK-B stimulated airway mucus release. These results suggest that NK-A and NK-B are potent stimulators of airway macromolecule secretion. Galanin and somatostatin potently inhibit these actions of the tachykinins. Therefore, airway mucus secretion is controlled by a complex network of several different mediators.