Chloride secretion by cultures of pig tracheal gland cells.

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.

Heart rate variability in hypertension caused by sleep disordered breathing and its modification by CPAP.

OBJECTIVES: 1) To analyze heart rate variability (HRV) changes, reflecting the sympathovagal balance with secondary hypertension caused by sleep disordered breathing (SDB), compared to healthy controls and essential hypertension without SDB; 2) to compare HRV changes between various degrees of SDB severity; and 3) to test the modification of HRV indices by continuous positive airway pressure (CPAP) in SDB patients. BACKGROUND: Differentiation of secondary hypertension caused by SDB from essential hypertension and healthy controls by ambulatory blood pressure measurement (ABPM) and its modification by CPAP, requires an analysis of HRV changes, as frequently used for the prediction of cardiovascular risk. METHODS: HRV changes were analyzed in 48 adults divided into six groups according to the apnoea/hypopnoea index (AHI), i.e. three groups with various degrees of SDB, a group with severe SDB after CPAP application, a group with essential hypertension without SDB, and a group of healthy controls. Night-time and daytime values of low frequency (LF) and high frequency (HF) bands and the LF/HF ratio were compared in the six groups. RESULTS: The night-time values of LF bands were higher in severe than in moderate and mild degrees of SDB, and the correlation of LF/HF ratio with AHI (r = 0.3511) suggests the gradual increase of sympathetic predominance with the severity of SDB. The high sympathetic activity substantially decreased after application of CPAP in severe SDB. CONCLUSION: The increased nocturnal values of the LF band and the LF/HF ratio, caused by frequent apnoea/ hypopnoea episodes, support the usefulness of HRV spectral analysis for the prediction of cardiovascular risk in patients with SDB (Tab. 1, Fig. 3, Ref. 36).

Voluntary and reflex cough and the expiration reflex; implications for aspiration after stroke.

Aspiration is a common result of stroke, and may lead to lung infections and pneumonia. Cough may prevent this aspiration and thus prevent the pneumonia. We review the four types of cough usually used to assess aspiration risk: voluntary cough (VC), reflex cough (RC), the laryngeal expiration reflex (LER), and cough on swallow (CoS). VC is easy to test but starts with an inspiration that may cause aspiration, and is controlled by cortico-brainstem pathways that may not be involved in influencing aspiration. RC also starts with an inspiration, and requires instrumental intervention, but is more relevant to protecting the lungs. The LER starts with an expiration, so is 'anti-aspiration', and is easy to test, but its neural mechanisms have not been fully analysed. CoS can be tested at the same time as direct observations of aspiration, but little is known about its neural mechanisms. Each method has its advocates, and the purpose of the review is to discuss how each may be applied and how the information from each may be assessed and valued.

Workshop: tuning the 'cough center'.

The Workshop considered the mechanisms whereby the 'cough center' could be tuned by various afferent inputs. There were particular presentations on the effects of inputs from the nose, mouth, respiratory tract and lungs, cerebral cortex, somatic tissues and the pharynx. From all these sites cough induced from the lungs could be increased or decreased in its strength or modified in its pattern. Thus 'tuning' of cough could be due to the interaction of afferent inputs, or to the sensitization or desensitization of brainstem neural pathways. The pattern of response depended on the 'type' of cough being studied and, in some instances, on the timing of the sensory input into the brainstem. Cough inputs could also affect various 'non-cough' motor outputs from the brain, although this was not the main theme of the Workshop. The main conclusion was that cough is not a stereotyped output from the medullary 'cough center', but that its pattern and strength depend on many afferent inputs acting on the 'cough center'.

Distinct generators for aspiration and expiration reflexes: localization, mechanisms and effects.

Re-evaluation of our earlier c-Fos-like immuno-reactive studies and brainstem transection/lesion experiments in over 40 anaesthetized, non-paralyzed cats allowed comparison of two distinct airway defensive reflexes with the distinct generators for inspiration (I) and expiration (E), described recently in juvenile rats. The spiration reflex (AspR) is characterized by solitary rapid and strong inspiratory effort with a reciprocal inhibition, preventing a subsequent active expiration, while the expiration reflex (ExpR) manifests by rapid and strong expiratory effort, starting without a preceding, inspiration, or reciprocal inhibition of occasional spontaneous inspiration. The retro-trapezoid nucleus/parafacial respiratory group neurones described as the distinct generator for active E in rats, are activated also during the ExpR in adult cats. Brainstem transection 5 mm above the obex eliminates the E generator and the ExpR, but preserves the I generator located in the pre-Bötzinger Complex, and also the AspR. This suggests the existence of a distinct I generator in cats as well as rats, and its contribution to the generation of the AspR. Persistence of the AspR in adult cats during asphyxic gasping, their similar character and the strong activation of I neurones at many places in the medulla and pons, suggest a common brainstem neuronal circuit contributing to generation of both the gasping and the gasp-like AspR. That the AspR and ExpR have distinct multilevel brainstem control mechanisms supports the dual theory of control and provides unique models for testing respiratory rhythm and pattern generation. The AspR may be compared with the powerful "auto-resuscitation effects of asphyxic gasping"; the ExpR may underly the effectiveness of the laryngeal chemoreflexes in prevention of lung diseases.

Clinical cough V: complementary and alternative medicine: therapy of cough.

We review the actions of complementary and alternative medicines (CAMs) in the treatment of cough and of the conditions associated with it; in particular asthma and upper respiratory tract infections. These therapies may work (1) peripherally, at the sites in the airways and lungs at which cough is being activated, (2) in the brainstem, where the neural "cough center" is situated, or (3) at the cerebral cortex, where cough can be initiated, suppressed or modified by conscious or unconscious controls. Of the large number of trials of CAMs against cough, most are inadequate in design. It may be difficult to randomize selection. Blinding is often impossible both for the patient and the therapist, and adequate placebo controls may be difficult to devise. The patient can usually identify the "active" treatment by the taste or smell of a medicine, or from the approach and apparatus being used. Pure chemicals can be extracted from many of the herbs used as antitussives, and can be shown to be effective in randomized, blind, and controlled trials, but it does not follow that the herb itself, used in the recommended formula and shown to be antitussive, acts by this agency unless a placebo effect is ruled out. A few herbs are identified where the evidence points to a true antitussive action. Of nonherbal treatments, the few positive results are usually outweighed by the larger number of negative ones. Thus, in general, CAMs for cough are welcomed enthusiastically by the patient but lack sound evidence for their efficacy. Antitussive chemicals can be extracted from many herbs, but it is no more than a reasonable hypothesis that the herb itself acts through this pathway.

Hormonal and purinergic stimulation of bicarbonate secretion in oviducts of rhesus monkey.

Because an increase in the HCO(3)(-) concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO(3)(-) across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO(3)(-). Purinergic agents potently stimulated short-circuit current (I(sc)) with an initial transient response declining within approximately 2 min to a sustained response. The potency sequence of ATP approximately UTP > ADP >> AMP suggested that the I(sc) response was mediated mainly by P2Y(2) receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline I(sc) or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO(3)(-)-CO(2) medium, and the pH of the unbuffered luminal medium was maintained at approximately 7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the I(sc) responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO(3)(-) secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO(3)(-) concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.

The expiration reflex from the trachea and bronchi.

The expiration reflex (ER) is a forced expiratory effort against a closed glottis that subsequently opens to eject laryngeal debris and prevent aspiration of material. It is distinct from the cough reflex. Its source is usually assumed to be restricted to the larynx and vocal folds, and its possible origin from the tracheobronchial (TB) tree has been suggested but never studied. The current authors re-analysed previous records with mechanical or chemical stimulation of the TB tree to see if an ER can consistently be elicited, and to see whether it has properties similar to that from the larynx and vocal folds. A random review was made of some of the extensive literature on TB "cough" to see if it confirmed the authors' conclusions. The TBER was consistently seen in cats and rabbits, either alone or followed by cough. These results are consistent with many studies in other species, including humans. It was enhanced, relative to cough, by inflation of the lungs and by general anaesthesia. Tracheobronchial expiration reflex occurs frequently with mechanical stimulation of the tracheobronchial tree. It differs fundamentally from many of the properties of "true" cough. Its features similar to the laryngeal expiration reflex suggest that both should be labelled "expiration reflexes" and not cough. Its existence should be taken into account in experimental, and possibly clinical, studies on tracheobronchial cough.

Differentiated structure and function of primary cultures of monkey oviductal epithelium.

We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Omega.cm(2) (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca(i). Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.

Cough: what's in a name?

The cough reflex (CR) and the expiration reflex (ER) are two defensive reflexes from the respiratory tract, the latter mainly from the larynx. Both are elicited by mechanical and chemical irritation of the airway mucosa, and are a characteristic of airway diseases, but they have different functions. The CR first draws air into the lungs, to accentuate the subsequent expulsive phase; the ER consists of a strong expiration, to prevent aspiration of material into the lungs. They have different sensory pathways, central nervous circuits, and physiological and pharmacological modulations. In practice, coughing often consists of a combination of the two reflexes, a cough bout, epoch or attack. Articles on cough usually do not distinguish between the two reflexes, or whether the coughs are single events or epochs; they usually only measure frequency of expiratory efforts, and neglect other aspects. Current methods for measuring and assessing cough are described, with indications of when the use of these methods may be important.

Cough as a symptom.

The Third International Symposium on Cough focused on acute and chronic cough, both clinical and basic science. Cough is a defensive and clearing mechanism, and can cause discomfort and nuisance; it is also an important symptom of many chronic airway diseases. In this Symposium, several issues concerning the relationship of cough to disease processes, different types of cough, and the concept of idiopathic cough will be discussed. Characterization of cough receptor(s) and identification of peripheral and central mechanisms for cough sensitization are current areas of investigation for delineating the cause of chronic cough. Peripheral mechanisms may be most important for acute cough such as after viral infections. The role of pathological changes at the level of the airway mucosa and of cortical pathways will be reviewed. Finally, therapeutic inhibition of the cough reflex remains an area of active research.

Secretion of lactoferrin and lysozyme by cultures of human airway epithelium.

Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.

Resistance of differentiated human airway epithelium to infection by rhinovirus.

Virtually all in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source.

Effects of media on differentiation of cultured human tracheal epithelium.

The purpose of the this study was to find media that supported high levels of differentiation in primary cultures of human tracheal epithelium. We tested six previously described, partially defined media and three nondefined media. Cells were grown with an air interface on porous-bottomed inserts, and differentiation was assessed from electrophysiological properties, levels of total protein and deoxyribonucleic acid, and histology. In all media, cells polarized and developed tight junctions, as assessed from transepithelial electrical resistance and were better differentiated at 14 d after plating than at 7 d. The partially defined media described previously by Gray et al. (Am. J. Respir. Cell. Mol. Biol. 14:104-112; 1996) and Matsui et al. (J. Clin. Invest. 102:1125-1131; 1998) and an undefined medium containing Ultroser G serum substitute produced the most highly differentiated epithelial cells, as revealed by a high short-circuit current (I(sc)) and a ciliated, pseudostratified appearance. In other media, cells tended to be either squamous or stratified squamous, with I(sc) levels <25% of those obtained with the three optimal media. Though no key factor in the composition of the partially defined media could be identified, two of the four media with high concentrations of retinoic acid produced good differentiation. In contrast, the two media with the lowest [Ca] (0.11 mM) produced poorly differentiated cells, as did the two partially defined media with low or no retinoic acid concentration.