Functional identification of epithelial and smooth muscle histamine-dependent relaxing mechanisms in the bovine trachea,but not in bronchi

Theoretically, the overall effect of histamine on respiratory smooth muscle is the result of a subtle balance of contraction and relaxation. The aim of the study was to identify histamine type 2 (H2) and 3 (H3) receptor-dependent relaxing mechanisms in the contractile elements of the bovine tracheobronchial tree. In bronchial preparations, histamine induced very weak contractions, which were not exacerbated with the H2-antagonist cimetidine. Moreover, precontracted bronchial rings never relaxed in response to cumulative doses of histamine or amthamine (H2-agonist). In intact tracheal preparations, histamine induced strong contractions that were exacerbated by cimetidine (E(max): +17.2+/-6.6%) but not by thioperamide (H3-antagonist). Precontracted tracheal bundles did not relax in response to cumulative doses of the H3-agonist R-alpha-methylhistamine. The tracheal contractile response was higher in denuded compared to intact preparations (11.0+/-1.2 vs. 6.0+/-1.7 g). Cimetidine effect was dramatically potentiated in denuded tracheal strips (+40.0+/-11.7%). It is concluded that the weak response of bovine bronchi to histamine is due to a relative scarcity of H1 receptors on bronchial smooth muscle rather than to H2- or H3-dependent relaxation. In the bovine trachea, the smooth muscle possesses relaxing H2 but no H3 receptors. The epithelium exercises a relaxation, which is independent from H2 and H3 receptors.     

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.     

Epithelial modulation of allergen and drug effects in guinea pig airways.

The effect of egg albumin (EA) challenge on tracheal tube preparations from sensitized guinea pigs was studied with regard to EA permeability, histamine release and penetrability, and the contractile response of the preparation. We used a plethysmographic method that allowed simultaneous measurement of changes in smooth muscle tension and collection of samples for determination of mediators. Our results clearly show that epithelial damage potentiates the contractile response to histamine, potassium ions, and acetylcholine. Epithelial damage did not alter the maximal contractile response in preparations challenged with high antigen concentrations (EA, 1 mg/ml), but histamine release measured in the perfusate increased substantially. The permeability of the preparations to EA was greater when the epithelium was damaged. No increase in the permeability in response to the EA challenge was observed. The present study has demonstrated that guinea pig airway epithelium constitutes a barrier for both antigen and drugs. We also present a method for recording contractile responses from intact whole tracheal preparations, in which the epithelium can still act as a barrier, as is the case in vivo.     

Modulation of the contractile responses of guinea pig isolated tracheal rings after chronic intermittent hypobaric hypoxia with and without cold exposure.

Previous studies have documented that repetitive exposure to intermittent hypoxia, such as that encountered in preparation to high-altitude ascent, influences breathing. However, the impact of intermittent hypoxia on airway smooth muscle has not been explored. Ascents to high altitude, in addition to hypoxia, expose individuals to cold air. The objective of the present study is to examine the effect of chronic intermittent hypobaric hypoxia (CIH) and CIH combined with cold exposure (CIHC) on tracheal smooth muscle responses to various contractile and relaxant agonists. Experiments were performed on tracheal rings harvested from adult guinea pigs exposed either to CIH or CIHC [14 days (6 h/day) at barometric pressure of 350 mmHg with and without cold exposure of 5 degrees C] or to room air (normoxia). CIH and CIHC attenuated maximum contractile responses to ACh compared with normoxia. The maximum contractile response to histamine decreased with CIH, whereas CIHC restored the response back to normoxia. Both CIH and CIHC attenuated maximum contractile responses to 5-HT. Altered contractile responses after CIH and CIHC were independent of epithelium. Isoproterenol-induced relaxation was not altered by CIH, whereas it was enhanced after CIHC, and these responses were independent of the epithelium. The data demonstrate that intermittent exposure to hypoxia profoundly influences contractile response of tracheal smooth muscle, and cold exposure can further modulate the response, implying the importance of cold at high altitude.     

Epithelium modulates the reactivity of ovalbumin-sensitized guinea-pig airway smooth muscle

Mechanical removal of the airway epithelium alters the in vitro reactivity of airway smooth muscle. The modulation of reactivity may involve the release of inhibitory and excitatory factors from epithelial cells. Guinea pigs sensitized with ovalbumin have been used as an animal model of airway hyperreactivity. We evaluated the influence of the epithelium on the reactivity of in vitro tracheal smooth muscle from control and ovalbumin-sensitized guinea pigs, and the extent to which the presence of the epithelium affects the contractile response to in vitro challenge with ovalbumin. In both control and ovalbumin-sensitized tissues, epithelium removal increased the sensitivity of the preparations to histamine, methacholine and isoproterenol to a similar extent, i.e., 2- to 2.5-fold. Epithelium removal resulted in an 8.1-fold increase in sensitivity to ovalbumin in sensitized tissues. The epithelium appears not only to modulate the reactivity of the tissues to bronchoactive agents, but it also influences the magnitude of the contractile response following antigen challenge.     

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 PGE_2α 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), methacoline (0.1–10 μM) or histamine (0.3–30 μM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE₂ and PGF_2α but not PGI₂, was decreased in tissues lacking epithelium. Indomethacin (1 μM) 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 PGF_2α or PGE₂. However, the possibility remains that the basal release of PGE₂ and/or PGF_2α 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 PGF_2α which may be involved in the maintenance of baseline tone.     

The effect of verapamil is reduced in isolated airway smooth muscle preparations lacking the epithelium

The effect of epithelium removal on the reactivity of rabbit airway smooth muscle to bronchoactive agents and on the effect of verapamil was studied in vitro using preparations from several levels within the respiratory tree, i.e., trachea, primary (10) and secondary (20) bronchus. Methacholine contracted tissues from all three levels of airway. Histamine contracted strips from 20 bronchus, had an inconsistent action in strips from 10 bronchus and was without effect in tracheal preparations. K+ contracted tissues from the trachea and 10 bronchus, and had a mixed action in 20 bronchial strips. Removal of the epithelial cell layer variably affected the reactivity of the smooth muscle to the three agents studied. In 20 bronchus, epithelium removal potentiated responses to histamine and methacholine. In 10 bronchus, only responses to methacholine were consistently augmented. In tracheal preparations epithelium removal did not alter the reactivity of the tissue to any agent examined. Verapamil (1 microM) attenuated responses to all agents and increased in its potency from tracheal through 10 to 20 bronchial preparations. Following epithelium removal, verapamil was substantially less effective in 20 bronchi, yet its effects were unchanged in the trachea. The results indicate that the epithelial cell layer modulates airway smooth muscle reactivity; this phenomenon is apparently widespread in mammals, the modulatory effect is more prominent in the smaller airways, and the magnitude of the effect of verapamil on airway smooth muscle is, in part, related to the presence of the epithelium.     

Epithelium-dependent regulation of airways smooth muscle function. A histamine-nitric oxide pathway.

The airway epithelium is responsible for the production of a number of arachidonic acid and non-prostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM-100 microM) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KC1 in the presence or not of 10(-5) Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)-monomethyl L-arginine (L-NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 microM) on KC1 contraction induced a relaxation of 10% of the initial contraction. The additional use of L-NMMA decreased the relaxation to 5% of initial contraction. MB rather than L-NMMA increased the contraction significantly (p<0.01). Epithelium removal increased the contraction induced by KC1 (80 mM) and histamine (1 microM) by about 30% (p<0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma.     

Respiratory epithelium inhibits bronchial smooth muscle tone

The aim of the present study was to determine whether or not the respiratory epithelium can modulate the responsiveness of bronchial smooth muscle. Paired rings of canine bronchi (4-6 mm OD), in some of which the epithelium had been removed mechanically (by rubbing the luminal surface), were mounted in physiological saline solution, gassed with 95% O2-5% CO2, and maintained at 37 degrees C. The presence or absence of the epithelium was confirmed by histological examination. Removal of the epithelium increased the contractile responses evoked by acetylcholine, histamine, and 5-hydroxytryptamine. Transmural nerve stimulation evoked similar peak responses in the presence and absence of epithelium. In unrubbed preparations, the peak response was followed by a gradual decrease when the stimulation was continued. This decrease, which persisted in the presence of propranolol, was not observed in epithelium-denuded preparations. In bronchial rings contracted with acetylcholine, isoproterenol produced concentration-dependent relaxations which were significantly greater in rings with epithelium compared with denuded rings. These results suggest that respiratory epithelial cells may generate an inhibitory signal to decrease the responsiveness of bronchial smooth muscle to contractile agonists and augment the effectiveness of inhibitory stimuli.     

Differential effects of prostaglandin E2 on contractions of airway smooth muscle

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3',5'-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.     

Contraction of guinea pig trachea by epidermal growth factor--urogastrone

To evaluate further the action of epidermal growth factor - urogastrone (EGF-URO) in smooth muscle systems, we examined the effect of the peptide on guinea pig tracheal strips. The cumulative addition of EGF-URO to the organ bath resulted in a concentration-dependent tonic contraction without tachyphylaxis. The half-maximal contraction was obtained at 13 +/- 3 ng/mL EGF-URO (2 nM). The maximum contraction at 100 ng/mL approached 60% of that induced by 1 microM histamine. No significant difference in the EGF-URO-induced contraction was observed in the presence or absence of a functional epithelium. Preincubation with 1 microM indomethacin for 20 min abolished the action of EGF-URO. The contractile effect of EGF-URO was not affected by yohimbine, propranolol, atropine, tetrodotoxin, and esculetin. However, mepacrine caused inhibition by 37 +/- 7% (mean +/- SEM for n = 3). Verapamil (10 microM) inhibited the EGF-induced response by 62 +/- 5% (mean +/- SEM for n = 4); the response was also absent in Ca-free (1 mM EGTA) buffer. However, the response was restored after the readdition of calcium. Our results suggest that EGF-URO can modulate tracheal smooth muscle contractility via a cyclooxygenase product and raise the possibility that EGF-URO might play a role in controlling pulmonary smooth muscle tone in vivo.     

Some Characteristic Relaxant Effects of Aqueous Leaf Extract of Andrographis paniculata and Andrographolide on Guinea pig Tracheal Rings

The ethnomedicinal uses of the aqueous leaf extract of Andrographis paniculata Nees (AP) include treatment of pain and inflammation, malaria, asthma and common cold. We designed this study to characterize some effects of AP and those of its andrographolide constituent. Guinea pig tracheal rings suspended in organ baths containing PSS were precontracted with histamine or carbachol and then exposed to cumulative concentrations of AP, andographolide or theophylline. The effect of AP was tested in Ca2+-depleted tracheal rings stimulated with the EC50 of histamine in Ca2+-free PSS. IC50 and Emax values were calculated for each relaxant. Results showed that both AP and andrographolide possessed relaxant effects on the tracheal smooth muscle. While AP was more effective on histamine-induced contraction, andrographolide and theophylline were more effective on carbachol-induced contraction. The IC50 values of andrographolide were significantly higher than those of theophylline in the two contractile agents. The presence of AP significantly attenuated the contractile force produced by 6.4 x 10-3 M Ca2+ in Ca2+-depleted rings. It is concluded that andographolide contributes at least in part to the relaxant action of AP on tracheal smooth muscles. The mechanism of action is related to inhibition of Ca2+ influx into tracheal smooth muscle cells.     

The influence of L-NG-nitro-arginine on field stimulation induced contractions and acetylcholine release in guinea pig isolated tracheal smooth muscle

The interaction between parasympathetic and inhibitory non-adrenergic, non-cholinergic nerves in tracheal smooth muscle was investigated by determining the effects of the NO-synthase inhibitor L-NG-nitro-arginine (L-NOARG) on contractions and the associated acetylcholine release elicited by field stimulation of the muscle. At frequencies above 2Hz contractile responses to field stimulation were potentiated by L-NOARG (50 microM). alpha-chymotrypsin pre-treatment potentiated contractile responses at all frequencies, but the effects of L-NOARG were unaltered. The effect of L-NOARG on responses to 5Hz electrical stimulation was not mimicked by D-NOARG, was reversed by L-, but not D-arginine and was unaffected by epithelium removal. L-NOARG did not affect responses to exogenous acetylcholine nor the overflow of 3H from tissues previously loaded with [3H]-choline. It is therefore concluded that field stimulation of tracheal smooth muscle induces the release of an endogenous nitrate, which, by an inhibitory action on smooth muscle, functionally antagonises the concomitantly released parasympathetic neurotransmitter.     

Age- and body-weight-dependent changes in the reactivity to histamine of the airways smooth muscles in cats

The relation between age and body weight and the reactivity to histamine of the tracheopulmonary contractile tissues was studied in cats in vitro. The reactivity of the tracheal smooth muscle to histamine decreased with increasing age from the newborn (100%) and one-month-old (57%) to the adult (0%) cat, whereas the amplitudes of contractions of lung strips increased. The amplitudes in adult animals also increased moderately with increasing body weight.     

Ontogeny of neurokinin-1 receptors in the porcine respiratory system

We characterized the ontogeny of NK-1 receptor agonist affinity (Kd) and density (Bmax) in membranes from tracheal epithelium, smooth muscle, and lung of pigs aged 1-7 days, 8-21 days, and adult in comparison to contractile responses in vitro. Affinity of [125I] Bolton-Hunter substance P ([125I]BH-SP) in epithelium and smooth muscle was three- to fourfold lower in young piglets than in adults. The Bmax of NK-1 sites in epithelium was elevated by more than twofold at 8-21 days relative to 1-7 days piglets and adults. In the lung, NK-1 density as well as affinity was lower than in trachea, regardless of age. In all three groups, [125I]BH-SP binding was potently inhibited by Gpp(NH)p, in both trachea and lung, implying coupling to G-proteins. Inhibition by Gpp(NH)p was most potent in the adult relative to younger animals, in both tracheal epithelium and smooth muscle. Functional sensitivity to the NK-1 agonists substance P and septide was reduced in neonates, as shown by the higher concentration of agonist required to elicit contractile responses. We conclude that the reduced sensitivity of newborn piglet airways to substance P reflects immaturity of G-protein coupling to NK-1, independent of receptor density.     

Isolation and characterization of guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture

Summary A simple 30-min enzyme digestion procedure has been used to release guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture. Primary cell cultures initially consist of numerous epithelial colonies and 70–1000 morphologically differentiated smooth muscle cells per 600 mg (wet weight) tracheal tissue depending on the age of the animal. Both cell types proliferate to form a confluent monolayer within 5–17 days. Pure subcultures of tracheal smooth muscle cells are obtained by limited trypsin digestion of the primary culture. Eighty percent of these subcultured smooth muscle cells retain the ability to contract in response to histamine (10^-6 M) and to form reaggregates even after 20 or more passages. Examination of these cells by electron microscopy reveals both biosynthetic and contractile components of smooth muscle. Analysis of this dual phenotype may provide valuable information about the regulation of tracheal smooth muscle cell growth and differentiation.This research was supported in part by a grant from the Foundation for Research in Bronchial Asthma and Related Diseases, Worcester, Massachusetts 01604, USA     

Pharmacological analysis of reactivity changes in airways due to acute inflammation in cats

Our recent in vitro studies on airways smooth muscles of the cat with turpentine oil inflammation showed the occurrence of a contractile response of tracheal preparations and a significant increase in the isometric tension of lung strips to histamine application. This study was aimed to establish whether histamine H2-receptors participated in the changed in vitro reactivity of the airways smooth muscles of cats suffering from experimentally induced airway inflammation. Pretreatment of control tracheal preparations, control and experimental groups of the lung strips by cimetidine did not change the character of the histamine response. Similarly, the amplitude of histamine relaxation, of the tracheal preparations partially contracted by carbachol was unchanged by experimental inflammation. Clemastine significantly shifted the histamine dose-response curves to the right in both groups of lung strips. However, significant differences in lung strip reactivity between control and experimental groups of cats were not eliminated. Our results do not support the role of histamine H2-receptors in the pathologically increased airway reactivity to histamine in vitro.     

Enalapril and diltiazem co-administration and respiratory side effects of enalapril

A persistent, chronic dry cough is the most common adverse effect of angiotensin converting enzyme (ACE) inhibitors therapy. The mechanism of this respiratory adverse effect is related to the inhibition of ACE and the accumulation of bradykinin, substance P, prostanoids and other inflammatory neuropeptides in the airways. The aim of this study was to follow the relationship between 15-day administration of enalapril and the defense reflexes (cough and bronchoconstriction) of the airways in experimental animals, as well as the possibility of their pharmacological restriction with simultaneous diltiazem administration. Cough reflex was investigated by the method of mechanical irritation of laryngopharyngeal and tracheobronchial area in non-anesthetized cats. The reactivity of tracheal smooth muscles of the airways to bronchoconstrictor mediators (histamine 10 nM - 1 mM, acetylcholine 10 nM - 1 mM and KCl 1 mM - 100 mM) was evaluated by an in vitro method in guinea pigs. Enalapril 5 mg/kg/day and diltiazem 30 mg/kg/day were administered perorally for 15 days. The results showed that long-lasting administration of enalapril resulted in a significant increase of measured cough parameters and increased reactivity of tracheal smooth muscle to histamine and KCl. Simultaneous administration of enalapril together with diltiazem significantly decreased the enalapril induced cough, and decreased enalapril induced hyperreactivity of tracheal smooth muscles to KCl. The results showed a partially protective effect of diltiazem and enalapril co-administration on the respiratory adverse effects induced by enalapril therapy.     

Potentiation of contraction of rabbit airway smooth muscle by some cyclooxygenase products

An alteration in smooth muscle sensitivity may be one of the mechanisms of the airway hyperresponsiveness observed in asthma. Indomethacin inhibits experimentally induced airway hyperresponsiveness. We thus examined the effects of the cyclooxygenase products PGD2, PGF2 alpha and a thromboxane A2 analogue U46619 on contractile responses of rabbit airway smooth muscle to histamine, carbachol and electrical field stimulation (EFS). PGD2 did not potentiate any contractile responses. When PGF2 alpha (1 microM) was administered 30 min before cumulative concentration-response curves to histamine and carbachol, no potentiation was observed. However, PGF2 alpha (1 microM) added immediately before EFS and bolus doses of histamine potentiated the contractile responses. U46619 increased the cumulative concentration-responses to both histamine and carbachol. The fact that we could alter smooth muscle sensitivity in vitro with PGF2 alpha and a thromboxane analogue suggests that these mediators may be involved in the airway hyperresponsiveness observed in asthma.     

Neuropeptide Y: prejunctional inhibition of vagally induced contractions in the guinea pig trachea

The effects of neuropeptide Y (NPY) on the contractile response to vagus nerve stimulation at different frequencies was studied in an isolated tracheal tube preparation from guinea pig. NPY had no effect on basal smooth muscle tension or on the contractile effect of carbachol, but inhibited vagally induced contractions in a concentration-dependent manner with a greater inhibition at low frequencies than at high. We suggest that the effect is exerted prejunctionally.