Douglas W.P. Hay

The effects of epithelium removal on the sensitivity of guinea-pig isolated trachealis to bronchodilator drugs

1 Mechanical removal of the epithelium increased the sensitivity of tracheal strips to isoprenaline, sodium nitroprusside, and to adenosine (only in the presence of inhibitors of its uptake and metabolism). Epithelium removal was without effect on sensitivity to salbutamol or papaverine. 2 Preincubation of tracheal strips with an inhibitor of extraneuronal uptake, corticosterone (50 μM), had no effect on tissue sensitivity to either salbutamol or papaverine. However, the steroid both increased sensitivity to isoprenaline, and abolished the effect of epithelium removal on sensitivity to this catecholamine. 3 These results suggest that in the guinea‐pig, the tracheal epithelium is a major source of extraneuronal uptake for catecholamines. Furthermore, the increase in trachealis sensitivity to isoprenaline following epithelium removal is probably due to loss of these sites of extraneuronal uptake. 4 The fact that sensitivity to salbutamol, papaverine and adenosine (in the absence of metabolic inhibitors) was not increased by denuding the epithelium indicates that loss of a diffusion barrier to drugs is not the mechanism of increased sensitivity. 5 Adenosine (and possibly nitroprusside) may cause the epithelium to release a smooth muscle excitatory factor. Thus, removal of the epithelium attenuates this excitatory influence and enhances smooth muscle responsiveness to adenosine. 6 These results provide further evidence that the epithelium has an important role in modulating the sensitivity of guinea‐pig trachealis to drugs.

Epithelium removal increases the reactivity of human isolated tracheal muscle to methacholine and reduces the effect of verapamil.

Removal of the epithelium increased the sensitivity of human isolated tracheal smooth muscle to methacholine, producing a greater than 2-fold leftward shift in the concentration-response curve. Concomitantly, the ability of verpamil to lower the maximum contractile response was reduced in tissues without an intact epithelium. These findings suggest a role for the epithelium in modulating the reactivity of human and extend the findings of similar studies in experimental animals.

Relaxation of guinea-pig tracheal smooth muscle to arachidonate is converted to contraction following epithelium removal

1 The effect of epithelium removal on responses of guinea‐pig isolated trachealis to sodium arachidonate has been examined. 2 Arachidonate (100 μm) caused relaxation of epithelium‐intact preparations, but following epithelium removal, the response to arachidonate was converted to contraction. In the presence of indomethacin (1 μm), arachidonate caused contraction in intact and denuded trachea. 3 Arachidonate also produced concentration‐dependent effects, the qualitative nature of which varied with the presence or absence of the epithelium. In the presence of indomethacin, tracheal strips contracted in a concentration‐dependent manner whether or not the epithelium had been removed. 4 Nordihydroguaiaretic acid (NDGA; 11 μm) markedly inhibited the contractile response of denuded strips to arachidonate. In intact tissues this lipoxygenase inhibitor converted the arachidonate‐induced relaxation to a concentration‐dependent contraction. The contraction to arachidonate, in the presence of NDGA, was epithelium‐dependent. In the presence of both indomethacin and NDGA, responses to arachidonate were abolished. 5 It is concluded that the relaxation of guinea‐pig trachea to arachidonic acid is epithelium‐dependent and is mediated by an inhibitory product of the cyclo‐oxygenase metabolic pathway. The contraction in denuded trachea, and trachea in the presence of indomethacin, may be mediated by lipoxygenase products of arachidonic acid metabolism, i.e. peptidoleukotrienes. The mediator of the epithelium‐dependent contraction in NDGA‐treated tissues is unknown.

Differential effects of epithelium removal on the responsiveness of guinea‐pig tracheal smooth muscle to bronchoconstrictors

1 The influence of the epithelium on contractions produced by the peptidoleukotrienes, 5‐hydroxytryptamine (5‐HT) and the thromboxane mimetic, U‐44069, was examined in trachea from control and ovalbumin‐sensitized guinea‐pigs. 2 In control tissues removal of the epithelium produced an approximately 2 to 4 fold leftward shift in leukotriene C4 (LTC4) and LTD4 concentration‐response curves, but no effect on LTE4‐induced contractions. Similar results were obtained in preparations from ovalbumin‐sensitized animals. 3 Responses produced by 5‐HT or U‐44069 were similar in the presence and absence of the epithelium in control guinea‐pigs. 4 Indomethacin produced contrasting effects on leukotriene‐induced contractions in control guinea‐pigs: an increase in sensitivity to LTC4 in the presence but not absence of the epithelium, no effect on LTD4‐induced contractions and a decrease in sensitivity to LTE4 in both epithelium‐containing and epithelium‐free preparations. 5 These results indicate that there is selectivity in the effects of epithelium removal on agonist‐induced contractions of the guinea‐pig trachea. This provides further evidence for the modulatory influence of the epithelium on the reactivity of mammalian airway smooth muscle and supports the postulated existence of an epithelium‐derived inhibitory factor. The observation that in intact trachea indomethacin mimics the effects of epithelium removal on LTC4‐induced responses, suggests the involvement of a prostanoid(s) in this phenomenon.

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD4, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE2, PGF2 alpha and PGI2 (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD4, methacholine and histamine. LTD4 (3-100 nM), methacholine (0.1-10 microM) or histamine (0.3-30 microM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE2 and PGF2 alpha, but not PGI2, was decreased in tissues lacking epithelium. Indomethacin (1 microM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI2 and is unlikely to be PGF2 alpha or PGE2. However, the possibility remains that the basal release of PGE2 and/or PGF2 alpha derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE2 and PGF2 alpha which may be involved in the maintenance of baseline tone.

Endothelin receptors and calcium translocation pathways in human airways.

Tension and phosphatidyl inositol (PI) turnover experiments were conducted to investigate the receptors and signal transduction pathways responsible for contractions elicited by endothelin (ET) ligands in human bronchus. Nicardipine (1 µM), the L-type calcium channel inhibitor, or incubation in Ca2+-free medium, produced marked inhibition of contractions to the ETB receptor-selective agonist, sarafotoxin S6c, and especially those induced by KCl. In contrast, Ca2+-free medium was without appreciable effect against contraction produced by endothelin-1 (ET-1), the non-selective ETA and ETB receptor agonist. In Ca2+-free medium, ryanodine (10 µM), which inhibits intracellular calcium mobilization, reduced sarafotoxin S6c- and ET-1-induced responses, but was without effect on responses to KCl. Similarly, nickel chloride (Ni2+; 1 mM) caused marked inhibition of contractions induced by sarafotoxin S6c or ET-1, but had no significant effect on KCl concentration-response curves. The mixed ETA/ETB receptor antagonist SB 209670 (3 µM) inhibited responses to sarafotoxin S6c and ET-1 such that concentration-response curves were shifted rightward, at the 30% maximum response level, by 10.0- and 3.8-fold, respectively, whereas BQ-123 (3 µM), the ETA receptor antagonist, was without effect on responses induced by either agonist. ET-1 (1 nM–0.3 µM) caused a concentration-dependent stimulation of PI turnover, whereas sarafotoxin S6c (0.3 nM–0.1 µM) induced only small and variable increases, except at the highest concentration. The increase in PI turnover evoked by ET-1 was inhibited by SB 209670 (3 µM), and also by BQ-123 (3 µM). This is consistent with linkage of ETA receptors to activation of inositol phosphate generation in human bronchial smooth muscle cells. Collectively, the data suggest that differences exist in the relative contributions of intracellular and extracellular Ca2+ mobilization mechanisms elicited by ETA and ETB receptor activation. Thus, sarafotoxin S6c-induced, ETB receptor-mediated contraction in human bronchial smooth muscle appears to be dependent, in part, upon extracellular Ca2+, although a significant component of the response was also mediated by intracellular Ca2+ release, including from ryanodine-sensitive stores. ETA receptor-mediated contraction of human airway smooth muscle was activated largely via the release of intracellular Ca2+.

The development of tone in the smooth muscle of guinea-pig isolated tracheal preparations may be influenced by prostanoids released from the adjacent airway cartilage

Guinea-pig tracheal strip preparations containing cartilage, placed under an applied load in vitro, develop tone spontaneously. The finding that spontaneous tone is reduced by indomethacin suggests that one or more prostanoids are involved in the development of spontaneous tone in this species. In this study we examined the effects of removing the cartilage component of the preparations on changes in tone induced by indomethacin and isoproterenol. In contrast to preparations containing cartilage, tissues devoid of cartilage, did not develop tone after the application of an initial 1 g resting load. Indomethacin (1 microM) reduced resting tone by 0.62 +/- 0.14 g in cartilage-containing tissues but, in contrast, reduced tone by only 0.03 +/- 0.01 g in tissues devoid of cartilage. Furthermore, relaxation responses (0.38 +/- 0.05 g) to isoproterenol (1 microM) could be produced in cartilage-containing preparations but not in cartilage-free preparations. Radioimmunoassays indicated that the release of PGE2, PGF2 alpha and 6-keto PGF1 alpha, the end-product of PGI2 breakdown, was diminished in preparations lacking cartilage. Thus, in guinea-pig airway preparations cartilage is apparently a source of sufficient prostanoids to induce spontaneous tone.

Calcium uptake into guinea-pig trachealis: The effect of epithelium removal

Removal of the epithelium from preparations of guinea-pig airways in vitro increases the responsiveness of the smooth muscle of normal and ovalbumin-sensitized animals to a number of contractile agents. To determine if epithelium removal results in an increase in Ca2+ entry into the smooth muscle, the effect of removing the epithelium on Ca2+ uptake into the trachealis smooth muscle was studied using a modified La3+-technique. KCl increased Ca2+ uptake in the presence and absence of the epithelium in control and sensitized animals. Methacholine did not promote Ca2+ uptake, whether or not the epithelium was present, in either control or sensitized animals. Ovalbumin did not stimulate Ca2+ uptake into the trachealis of sensitized animals. These results indicate that the increase in responsiveness of airway smooth muscle seen on epithelium removal is not a consequence of a facilitation of Ca2+ entry into the muscle. The increased responsiveness to methacholine in control animals, and to ovalbumin in preparations in tension studies in epithelium-free tissues from sensitized animals, cannot be explained by an increased availability of extracellular Ca2+ into the muscle, but, rather may reflect some other effect of the epithelium-derived modulatory factor.

Differences in the ability of salbutamol to prevent and reverse LTC4-induced contractions of the guinea-pig isolated trachea: Influence of l-serine borate

In the presence of l-serine borate, salbutamol was much more effective in reversing rather than preventing LTC4-induced contractions of guinea-pig trachea. This suggests that different mechanisms are involved in initiating versus maintaining LTC4-induced contractions. In addition, the ability of salbutamol pretreatment to prevent LTC4-induced contractions was reduced substantially in the presence of l-serine borate, suggesting that the metabolites of LTC4 (LTD4 and LTE4) are more sensitive to inhibition by beta-adrenoceptor agonists than is LTC4 itself.

Postulated Mechanisms Underlying Airway Hyperreactivity

Airway hyperreactivity is defined as a marked nonspecific increase in sensitivity of the respiratory tract to a variety of stimuli that results acutely in an enhanced bronchospastic response (Benson, 1975; Boushey et al., 1980; Simonsson, 1983). The diverse physical, chemical, and pharmacological stimuli include various smooth muscle spasmogens (Benson, 1975; Boushey et al., 1980), cold air (Wells et al., 1960; Simonsson et al., 1967), dust (Simonsson et al., 1967), exercise (Lee and Anderson, 1985), environmental irritants (Simonsson, 1983), and forced breathing (Simonsson et al., 1967). This phenomenon is a hallmark of asthma and is considered the underlying abnormality in the pathophysiology of this disorder. In addition, airway hyperreactivity is commonly manifest in other pulmonary disorders, including chronic bronchitis (Klein and Salvaggio, 1966; Laitinen, 1974), cystic fibrosis (Mellis, 1978), allergic rhinitis (Laitinen, 1974; Cockcroft et al., 1977), and emphysema (Klein and Salvaggio, 1966; Laitinen, 1974), and also occurs during viral infections (Empey et al., 1976). This chapter deals predominantly with the hyperreactivity of airways associated with asthma.