Virus induces airway hyperresponsiveness to tachykinins: role of neutral endopeptidase

We examined the effects of viral respiratory infection by Sendai virus on airway responsiveness to tachykinins in guinea pigs. We measured the change in total pulmonary resistance induced by substance P or capsaicin in the presence or absence of the neutral endopeptidase inhibitor, phosphoramidon, in infected and in noninfected animals. In the absence of phosphoramidon, the bronchoconstrictor responses to substance P and to capsaicin were greater in infected than in noninfected animals. Phosphoramidon did not further potentiate the responses to substance P and to capsaicin in the infected animals, whereas it did so in noninfected animals. Studies performed in vitro showed that nonadrenergic noncholinergic bronchial smooth muscle responses to electrical field stimulation were also increased in tissues from infected animals and that phosphoramidon increased the response of tissues from noninfected animals greatly but increased the responses of tissues from infected animals only slightly. Responses to acetylcholine were unaffected by viral infection. Neutral endopeptidase activity was decreased by 40% in the tracheal epithelial layer of the infected animals. We suggest that respiratory infection by Sendai virus causes enhanced airway responsiveness to tachykinins by decreasing neutral endopeptidase-like activity in the airway epithelium.     

Bronchial responses to substance P after antigen challenge in the guinea-pig: in vivo and in vitro studies

The effect of antigen challenge on the airway responses to substance P and on the epithelial neutral endopeptidase (NEP) activity was investigated in aerosol sensitized guinea-pigs. In vivo, bronchial responses to aerosolized substance P were similar to the responses observed in antigen-challenged guinea-pigs and in the control groups. In contrast, when the guinea-pigs were pretreated with the NEP inhibitor, phosphoramidon, a significant increase in the airway responses to substance P was observed after antigen challenge in vivo. However, in vitro, the contractile responses of the tracheal smooth muscle to substance P were similar between groups of guinea-pigs, in respect to the presence or absence of the epithelium and/or phosphoramidon. Histological studies showed an accumulation of eosinophils in the tracheal submucosa after antigen challenge and intact epithelial cells. These results show that in vivo bronchial hyperresponsiveness to substance P after antigen challenge in the guinea-pig is not associated with increased responses of the smooth muscle to exogenous SP in vitro. In addition, the results with phosphoramidon suggest that loss of NEP activity cannot account for the in vivo bronchial hyperresponsiveness to substance P presently observed.     

Contractile and relaxant responses of diabetic dog femoral arteries

Strips of femoral arteries of normal and alloxan-treated dogs were set up for isometric recording. The contractile response to phenylephrine and the relaxant response to acetylcholine were determined. Neither alloxan treatment nor mechanical removal of endothelium altered the EC50 value for phenylephrine. The slope of phenylephrine dose-response curves of diabetic and healthy vessels with intact endothelium was similar, whereas the slope of phenylephrine dose-response curves of endothelium-denuded diabetic arteries was significantly greater than that of the denuded healthy arteries. Removal of the endothelium completely abolished the relaxant effect of acetylcholine. The relaxant potency (IC50) of acetylcholine was not affected by alloxan treatment. The results suggest that in canine femoral arteries the relaxant activity of acetylcholine is unaffected in experimental diabetes and the damage of the arterial endothelium may play a role in the increased responsiveness of diabetic vessels to adrenergic agonists.     

Viral infection increases contractile but not secretory responses to substance P in ferret trachea.

Viral infection increases the airway smooth muscle response to substance P. This effect is due to decreased activity of neutral endopeptidase (EC 3.4.24.11), an enzyme that degrades substance P. Inhibition of neutral endopeptidase activity also potentiates substance P-induced 35SO4-labeled macromolecule secretion. Therefore we examined the in vitro effects of substance P on 35SO4-macromolecule secretion from the tracheae of influenza-infected ferrets. Despite a virus-induced loss of neutral endopeptidase activity (demonstrated in muscle bath experiments), there was no difference between control and infected tracheae in either baseline secretion [697 +/- 125 vs. 579 +/- 67 (SE) cpm/15 min; n = 15 tissues) or in the response to 10(-6) M substance P (increased by 218 +/- 63 and 195 +/- 51, respectively) or 10(-5) M substance P (increased by 416 +/- 95 and 354 +/- 54, respectively). Although phosphoramidon (10(-6) M) potentiated the secretory response to substance P, there was again no difference between control and infected tracheae. These data show that although viral infection decreases airway neutral endopeptidase activity, virus-induced hypersecretion is not due to a resulting increase in the secretory response to substance P.     

Phosphoramidon potentiates the bronchoconstriction induced by inhaled bombesin in guinea pigs

The effect of the neutral endopeptidase inhibitor, phosphoramidon, on the bronchoconstriction induced by aerosolized bombesin in the guinea pig was investigated. Administered by aerosol for 1 min, bombesin (0.01 or 0.1 mg/ml) induced a dose-dependent increase in pulmonary inflation pressure. Pretreatment of the guinea-pigs with phosphoramidon (0.1 mM), administered by aerosol for 15 min, 15 min prior to challenge, markedly potentiated the increase in pulmonary inflation pressure induced by bombesin (0.01 mg/ml) and substance P (0.1 mg/ml). This result suggests a local hydrolysis of bombesin by airway neutral endopeptidase reducing the activity of this peptide on smooth muscle.     

Airway hyperresponsiveness to bronchoconstrictor challenge after wood smoke exposure in guinea pigs.

Prior airway exposure to wood smoke induces an increase in airway responsiveness to subsequent smoke inhalation in guinea pigs (Life Sci. 63: 1513, 1998; 66: 971, 2000). To further characterize this airway hyperreactivity, we investigated and compared the airway responsiveness to bronchoconstrictor challenge before and 30 min after sham air exposure or wood smoke exposure in anesthetized and artificially ventilated guinea pigs. Various doses of substance P (0.8-6.4 microg/kg), capsaicin (0.2-3.2 microg/kg), prostaglandin F2alpha (30-3000 microg/kg), histamine (1-8 microg/kg), or acetylcholine (5-20 microg/kg) were intravenously injected at 2-min intervals in successively increasing doses to obtain the dose required to provoke a 200% increase in baseline total lung resistance (ED200). Wood smoke exposure significantly lowered the ED200 of substance P, capsaicin, and prostaglandin F2alpha whereas sham air exposure failed to do so. Furthermore, wood smoke exposure did not significantly alter the ED200 of histamine or acetylcholine. Pretreatment with phosphoramidon (2 mg/kg), an inhibitor of the neutral endopeptidase (the major degradation enzyme of substance P), before smoke exposure did not significantly affect the smoke-induced reduction in ED200 of substance P. Sectioning both cervical vagi before smoke exposure did not significantly alter the smoke-induced reduction in ED200 of capsaicin or prostaglandin F2alpha. These results suggest that airway exposure to wood smoke acutely produces airway hyperresponsiveness to substance P, capsaicin, and prostaglandin F2alpha, but not to histamine or acetylcholine. Since the combination of phosphoramidon and wood smoke exposure did not result in an additive potentiation of smoke-induced airway hyperresponsiveness to substance P, it is suggested that an inhibition of the degradation enzyme of substance P may contribute to this increase in airway reactivity. Furthermore, vagally-mediated bronchoconstriction does not play a vital role in enhanced airway responsiveness to capsaicin or prostaglandin F2alpha.     

O3-induced mucosa-linked airway muscle hyperresponsiveness in the guinea pig.

We investigated the effects of ozone exposure (3.0 ppm, 2 h) on the responsiveness of guinea pig airway muscle in vitro from animals developing bronchial hyperreactivity. Muscarinic reactivity in vivo was determined by measuring specific airway resistance (sRaw) in response to increasing concentrations of aerosolized acetylcholine (ACh) administered before and 30 min after exposure. Immediately after reactivity testing, multiple tracheal rings from ozone- and air-exposed animals were prepared and the contractile responses to increasing concentrations of substance P, ACh, or KCl were assessed in the presence of 10 microM indomethacin with or without 1 microM phosphoramidon, an inhibitor of neutral endopeptidase. Isometric force generation in vitro was measured on stimulation by cumulative concentrations of the agonists, and force generation (in g/cm2) was calculated after determination of muscle cross-sectional area. The smooth muscle of mucosa-intact airways from guinea pigs with ozone-induced bronchial hyper-reactivity proved to be hyperresponsive in vitro to substance P and ACh but not to KCl. Pretreatment with phosphoramidon abolished the increase in substance P responsiveness but had no effect on muscarinic hyperresponsiveness after ozone exposure. Furthermore, substance P responsiveness was not augmented in ozone-exposed airways in which the mucosa had been removed before testing in vitro. Likewise, muscarinic hyperresponsiveness was not present in ozone-exposed airways without mucosa. Our data indicate that airway smooth muscle responsiveness is increased in guinea pigs with ozone-induced bronchial hyperreactivity and suggest that this hyperresponsiveness may be linked to non-cyclooxygenase mucosa-derived factors.     

Neutral endopeptidase modulates neurotensin-induced airway contraction

To determine the role of endogenous neutral endopeptidase (NEP) (also called enkephalinase, EC 3.4.24.11) in regulating neurotensin-induced airway contraction, we used phosphoramidon, a specific NEP inhibitor, in the guinea pig. In studies in vitro, neurotensin and the COOH-terminal fragment neurotensin-(8-13) contracted strips of bronchial smooth muscle in a concentration-dependent fashion (P less than 0.001). In contrast, the NH2-terminal fragment neurotensin-(1-11) and the COOH-terminal fragment neurotensin-(12-13), the main fragments of neurotensin hydrolysis by NEP, had no effect. Phosphoramidon (10(-5) M) did not change resting tension but shifted the concentration-response curves to neurotensin to lower concentrations (P less than 0.001), whereas inhibitors of kininase II, aminopeptidases, serine proteases, and carboxypeptidase N were without effect. Removing the epithelium increased the contractile response to neurotensin (P less than 0.001), and phosphoramidon further increased the response to neurotensin in these tissues (P less than 0.001). Similar results were obtained in studies in vivo using aerosolized neurotensin and phosphoramidon. These results suggest that endogenous NEP in the airways modulates the effects of neurotensin on airway smooth muscle contraction by inactivating the peptide.     

Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase.

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.     

Neutral endopeptidase inhibitor potentiates endothelin-1-induced airway smooth muscle contraction.

To study the role of neutral endopeptidase (NEP) on endothelin-1-induced contraction of the airway smooth muscle, we examined the contractile effect of endothelin-1 in the isolated guinea pig trachea and human bronchus in the presence or absence of NEP inhibitor phosphoramidon. After incubation with phosphoramidon (10(-8) to 10(-5) M), we added endothelin-1 cumulatively from 10(-11) to 10(-7) M to the airway tissues in organ baths. Phosphoramidon significantly potentiated the endothelin-1-induced contraction in a concentration-dependent fashion in both guinea pig trachea and human bronchus, and it shifted the concentration-response curves to the left. Because NEP is known to cleave tachykinins, we next studied whether endothelin-1 contracts airway tissues by releasing endogenous tachykinins from bronchial C-fibers. After incubation with phosphoramidon (10(-5) M), we added endothelin-1 cumulatively from 10(-11) to 10(-7) M to the tissues that were treated with capsaicin to deplete the tachykinins. Phosphoramidon significantly potentiated the endothelin-1-induced contraction in the capsaicin-treated tissues, suggesting that endothelin-1 causes the contraction, at least in part, without releasing tachykinins. In contrast to the effect of phosphoramidon, captopril (an angiotensin-converting enzyme inhibitor), leupeptin (a serine protease inhibitor), and bestatin (an aminopeptidase inhibitor) did not modulate the effect of endothelin-1-induced contraction in both guinea pig trachea and human bronchus. From these results, we conclude that NEP plays an important role in regulating endothelin-1-induced contraction in the guinea pig trachea and human bronchus.     

Airway neutral endopeptidase-like enzyme modulates tachykinin-induced bronchoconstriction in vivo

To determine whether neutral endopeptidase (NEP), also called enkephalinase (EC 3.4.24.11), modulates the effects of exogenous and endogenous tachykinins in vivo, we studied the effects of aerosolized phosphoramidon, a specific NEP inhibitor, on the responses to aerosolized substance P (SP) and on the atropine-resistant response to vagus nerve stimulation (10 V, 5 ms for 20 s) in guinea pigs. SP alone (10(-7) to 10(-4) M; each concentration, 7 breaths) caused no change in total pulmonary resistance (RL, P greater than 0.5). Phosphoramidon (10(-4) M, 90 breaths) caused no change either in base-line RL (P greater than 0.5) or in the response to aerosolized acetylcholine (P greater than 0.5). However, in the presence of phosphoramidon, SP (7 breaths) produced a concentration-dependent increase in RL at concentrations greater than or equal to 10(-5) M (P less than 0.001). Phosphoramidon (10(-7) to 10(-4) M; each concentration, 90 breaths) induced a concentration-dependent potentiation of SP-induced bronchoconstriction (10(-4) M, 7 breaths; P less than 0.01). Vagus nerve stimulation (0.5-3 Hz), in the presence of atropine, induced a frequency-dependent increase in RL (P less than 0.001). Phosphoramidon potentiated the atropine-resistant responses to vagus nerve stimulation (P less than 0.001) at frequencies greater than 0.5 Hz. The tachykinin antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-substance P abolished the effects of phosphoramidon on the atropine-resistant response to vagus nerve stimulation (2 Hz, P less than 0.005). NEP-like activity in tracheal homogenates of guinea pig was inhibited by phosphoramidon with a concentration producing 50% inhibition of 5.3 +/- 0.8 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon

Neutral endopeptidase is a mammalian type II integral membrane zinc-containing endopeptidase, which degrades and inactivates a number of bioactive peptides. The range of substrates cleaved by neutral endopeptidase in vitro includes the enkephalins, substance P, endothelin, bradykinin and atrial natriuretic factor. Due to the physiological importance of neutral endopeptidase in the modulation of nociceptive and pressor responses there is considerable interest in inhibitors of this enzyme as novel analgesics and anti-hypertensive agents. Here we describe the crystal structure of the extracellular domain (residues 52-749) of human NEP complexed with the generic metalloproteinase inhibitor phosphoramidon at 2.1 A resolution. The structure reveals two multiply connected folding domains which embrace a large central cavity containing the active site. The inhibitor is bound to one side of this cavity and its binding mode provides a detailed understanding of the ligand-binding and specificity determinants.     

pH- and time-dependent inhibition of rat kidney neutral endoprotease 24.11 by thiorphan and phosphoramidon

The potencies of thiorphan and phosphoramidon as inhibitors of neutral endopeptidase 24.11 (NEP) are significantly affected by pH. Thiorphan and phosphoramidon are 10- and 150-fold more potent, respectively, when measured at pH 6.5 than at pH 8.5. The kinetic characteristics of these two inhibitors are different. NEP activity is readily inhibited by phosphoramidon upon mixing, while thiorphan becomes a more potent inhibitor only after preincubation with the enzyme.     

The effect of N-formyl-methionyl-leucyl-phenyl-alanine on cholinergic neurotransmission and its modulation by enkephalinase in rabbit airway smooth muscle

N-formyl-methionyl-leucyl-phenylalanine (FMLP), a synthetic analogue of bacterial chemotactic peptide, may play a role in airway hyperresponsiveness, and is cleaved by neutral endopeptidase-24.11 (enkephalinase). To determine the effect of FMLP on parasympathetic contraction of airway smooth muscle and its modulation by endogenous enkephalinase, we studied isolated rabbit tracheal ring segments under isometric conditions in vitro. FMLP did not cause muscle contraction, but it potentiated the contractile response to electrical field stimulation (EFS) in a dose-dependent fashion, with the maximal increase from the baseline response being 59.8 +/- 6.2% (mean +/- S.E.M., P less than 0.001), an effect that was abolished by t-Boc-Phe-Leu-Phe-Leu-Phe, partially inhibited by pyrilamine, but not by phentolamine or [D-Pro2,D-Trp7,9]substance P. In contrast, the contractile response to administered acetylcholine was not affected by FMLP. Pretreatment of tissues with thiorphan, an enkephalinase inhibitor, further potentiated the effect of FMLP on the EFS-induced contraction. These results suggest that FMLP facilitates cholinergic neurotransmission in rabbit airway smooth muscle probably by increasing acetylcholine release, and that this effect may be modulated by enkephalinase in the airway.     

Epithelium modulates the potency of vasoactive intestinal peptide in the guinea pig.

The purpose of the study was to evaluate the importance of the epithelium in determining the potency of exogenous vasoactive intestinal peptide (VIP) in inhibiting responses of isolated guinea pig trachea to vagal stimulation. Isolated innervated tracheal preparations (n = 56) were mounted in glass organ baths in Krebs-Henseleit (K-H) solution at 37 degrees C and gassed with 95% O2-5% CO2. The inside of the trachea was separately perfused with K-H solution at 1 ml/min. The vagal nerve trunks were stimulated (20 V, 1-ms pulses, 10-s trains) at low (0.5 Hz) and high frequency (15 Hz) alternately, and the contractile responses were measured as increases in intratracheal pressures. VIP (10(-8)-10(-7) M) inhibited responses to both high- and low-frequency stimulation. VIP was more potent in inhibiting contractions when administered to the outside than the inside surface of the trachea, and disruptionon of the epithelium abolished this difference. The endopeptidase inhibitors phosphoramidon and thiorphan (5 x 10(-6) M) potentiated the action of VIP. These data indicate that the epithelium reduces the efficacy of VIP. We suggest that the epithelium is a site of degradation of VIP by endopeptidase and may also be a diffusion barrier.     

感觉神经肽与支气管对肺动脉缺氧反应的影响

豚鼠离体肺动脉用辣椒素（Ｃａｐｓａｉｃｉｎ）耗竭感觉神经肽（ＳＮＰ）后,缺氧性收缩反应（ＨＰＶ）显著增高（Ｐ＜０．０１）。套有带完整上皮支气管的肺动脉的ＨＰＶ显著弱于不套或套有去上皮支气管肺动脉的ＨＰＶ（Ｐ＜０．０５）;支气管与肺动脉同时用Ｃａｐ处理后,此差异消失;只有外套的支气管先用Ｃａｐ预处理时,肺动脉ＨＰＶ仍显著强于溶剂预处理的对照组（Ｐ＜０．０５）;套有去上皮支气管的肺动脉缺氧反应显著强于不套支气管的肺动脉。结果提示：肺动脉Ｃ－感觉神经所释放的ＳＮＰ在肺动脉缺氧反应中具调节作用;支气管上皮层可释放舒血管物质调节ＨＰＶ,此物质与ＳＮＰ密切相关;支气管还可能释放缩血管物质,介导ＨＰＶ。     

Involvement of substance P in neutral endopeptidase modulation of carotid body sensory responses to hypoxia.

Previously, we showed that carotid bodies express neutral endopeptidase (NEP)-like enzyme activity and that phosphoramidon, a potent inhibitor of NEP, potentiates the chemosensory response of the carotid body to hypoxia in vivo. NEP has been shown to hydrolyze methionine enkephalin (Met-Enk) and substance P (SP) in neuronal tissues. The purpose of the present study is to determine whether NEP hydrolyzes Met-Enk and SP in the carotid body and if so whether these peptides contribute to phosphoramidon-induced potentiation of the sensory response to hypoxia. Experiments were performed on carotid bodies excised from anesthetized adult cats (n = 72 carotid bodies). The hydrolysis of Met-Enk and SP was analyzed by HPLC. The results showed that both SP and Met-Enk were hydrolyzed by the carotid body, but the rate of Met-Enk hydrolysis was approximately fourfold higher than that of SP. Phosphoramidon (400 microM) markedly inhibited SP hydrolysis ( approximately 90%) but had only a marginal effect on Met-Enk hydrolysis ( approximately 15% inhibition). Hypoxia (PO(2), 68 +/- 6 Torr) as well as exogenous administration of SP (10 and 20 nmol) increased the sensory discharge of the carotid body in vitro. Sensory responses to hypoxia and SP (10 nmol) were potentiated by approximately 80 and approximately 275%, respectively (P < 0.01), in the presence of phosphoramidon. SP-receptor antagonists Spantide (peptidyl) and CP-96345 (nonpeptidyl) either abolished or markedly attenuated the phosphoramidon-induced potentiation of the sensory response of the carotid body to hypoxia as well as to SP. These results demonstrate that SP is a preferred substrate for NEP in the carotid body and that SP is involved in the potentiation of the hypoxic response of the carotid body by phosphoramidon.     

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

Removal of the epithelium from preparations of guinea-pig airways 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.