Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Exercise may cause airway obstruction in asthmatics. This has been thought to be initiated by water loss causing hyperosmolarity of the airway hypophase, as well as airway cooling and the release of bronchoactive mediators (McFadden et al., 1986). Inhaled hypo-osmolar, hyperosmolar, and isotonic aerosols also can elicit pulmonary obstruction in asthmatics and in laboratory animals (Osborne et al., 1987; Eichler et al., 1992; Fujimura et al., 1997) through the release of mediators such as histamine, leukotrienes and bradykinin (Finnerty et al., 1985; Umeno et al., 1990; Makhdum and Pearce, 1993). The precise mechanisms responsible for the obstructive responses are unclear. Circulation through the mucosal vasculature of the airways also is affected by hyperosmolar (vasodilation involving nitric oxide) and hypo-osmolar (vasoconstriction) solutions applied to the mucosal surface (Smith et al., 1993; Prazma et al., 1994; Wells et al., 1994).

The airway epithelium is an important regulator of respiratory smooth muscle tone and reactivity (see Fedan et al., 1988 and Goldie and Hay, 1997 for review) because it is a diffusion barrier, a site of drug metabolism, and mediates the actions of some drugs. The epithelium also releases prostanoids and the nonprostanoid, nonnitric oxide inhibitory substance, epithelium-derived relaxing factor (EpDRF), which alters reactivity to contractile agonists, relaxant agonists, and allergen (Flavahan et al., 1985; Barnes et al., 1985; Hay et al., 1986a,b, 1987; Ilhan and Sahin, 1986; Grundström et al., 1992). Hyperosmolar solutions applied to the mucosal surface of guinea pig isolated, perfused trachea cause an epithelium-dependent relaxation of the smooth muscle via the release of EpDRF (Munakata et al., 1988; Fedan et al., 1990). The production of EpDRF and/or its inhibitory effects on the smooth muscle has been suggested to be linked to the Na⁺,K⁺-pump and Ca²⁺-dependent K⁺ channels (Raeburn and Fedan, 1989; Lamport and Fedan, 1990; Tamaoki et al., 1997).

In this study we hypothesized that the airway epithelium mediates or participates in responses of the airways to nonisotonic solutions and in the effects of nonisotonic solutions on airway reactivity to agonists. We employed the guinea pig isolated, perfused trachea preparation to examine the roles of epithelium in regulating smooth muscle diameter and reactivity to drugs because it allows separate delivery of agents to the mucosal (intraluminal) or serosal (extraluminal) surfaces (Munakata et al., 1989; Fedan et al., 1990; Fedan and Frazer, 1992; Kitano et al., 1992). Agents applied to the intraluminal perfusate affect the smooth muscle after having diffused across the epithelium, whereas agents applied to the serosal surface have direct access to the smooth muscle; consequently, reactivity to extraluminally applied contractile agonists is generally greater than after mucosal addition (Munakata et al., 1990; Fedan et al., 1990; Fedan and Frazer, 1992). With the regulatory role of the airway epithelium on smooth muscle reactivity in mind, the purposes of this study were to investigate 1) the relationship between luminal osmolarity and smooth muscle tone and reactivity to methacholine (MCh); 2) polarity across the epithelium in the effects of hyperosmolar solutions; 3) the effects of agents that inhibit prostanoid and nitric oxide formation, Na⁺ and Cl channels, and ion pumping and transport mechanisms; and 4) the effects of raised intraluminal osmolarity on postganglionic nerve-mediated mechanical responses of the smooth muscle. The companion article following this one (Dortch-Carnes et al., 1999) describes the relationships between epithelial bioelectric responses triggered by nonisotonic solutions and the ensuing smooth muscle mechanical events.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Guinea Pig Isolated, Perfused Trachea Preparation. The experimental protocols were approved by the institutional Animal Care and Use Committee. Male English short-hair SPF guinea pigs (457-742 g; Camm Research Institute, Wayne, NJ and Harlan Sprague-Dawley, Inc., Indianapolis, IN) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.). Four centimeters of the trachea was removed, placed in modified Krebs-Henseleit (MKH) solution, and cleaned. The segment was mounted onto a perfusion holder that contained indwelling side-hole catheters that were connected to the positive (inlet) and negative (outlet) sides of a differential pressure transducer, as described previously (Fedan and Frazer, 1992). The holder was placed into a 25-ml bath containing MKH solution (37°C), which is referred to as the serosal or extraluminal bath. The trachea was perfused (34 ml/min) with recirculating MKH solution (37°C) from a separate, 30-ml reservoir, which is referred to as the mucosal or intraluminal bath. Transmural pressure was adjusted to zero. Responses were measured as changes in the inlet minus outlet pressure difference (P), in cm of H₂O. A 1-h equilibration period was allowed before the experiment while washing the preparations at 15-min intervals by changing the MKH solution in both baths.

Epithelium Removal. To remove the epithelium from the trachea (>95%; Fedan and Frazer, 1992), before it was mounted to the perfusion apparatus, a 5- to 6-cm piece of trimmed pipe cleaner brush was advanced slowly into the lumen and withdrawn while rotating slowly.

MCh Concentration-Response Curves. MCh was added in stepwise-increasing, cumulative concentrations to the extra- or intraluminal baths. Two concentration-response curves were obtained, the first as a control and the second to examine the effect under study. The preparations were washed at 15-min intervals for 1.5 h between the concentration-response determinations. None of the effects on MCh concentration-response curves shown in Results are observed in two, consecutive control curves (Fedan and Frazer, 1992).

Concentration-Response Curves for Raised or Lowered Intraluminal Osmolarity. Concentration-response curves for relaxation responses to KCl or NaCl applied intraluminally to elevate osmolarity were generated after having obtained a stable contraction with extraluminally added MCh (3 × 10⁷ M; ~EC₅₀). Two such curves were obtained, the first as a control and the second, obtained after 1.5 h of washing, in the presence of the agent under study. The concentrations of the salts shown in the figures refer to the molar concentrations added to the MKH solution. In normosmolar MKH solution, [Na⁺], [K⁺] and [Cl] are 138, 6, and 122.8 mM, respectively.

Comparison of Responses to Extra- versus Intraluminal Hyperosmolarity. Responses to elevated extra- and intraluminal osmolarity in the same trachea were compared using a paired design protocol. The preparation was contracted with extraluminally added MCh (3 × 10⁷ M), after which 5.62, 13.3, or 80 mM NaCl was added to the extraluminal bath. After response stabilization, the same concentration of NaCl was applied to the intraluminal bath. The preparation was then washed repeatedly and allowed to equilibrate for 1.5 h. At the end of this period, MCh was added, and the procedure was repeated using KCl instead of NaCl. Only one concentration of NaCl and KCl was used in each experiment. These experiments were performed using intact and separate, epithelium-denuded tracheae.

Electric Field Stimulation of Perfused Trachea. After placement of the mounted trachea in the extraluminal bath, two platinum electrodes were aligned longitudinally on opposite sides of the trachea. The trachea was stimulated electrically with 10-s trains of square wave (120 V, 0.5 ms) pulses delivered at 7-min intervals. Two frequency-response curves, separated by 1.5 h of washing, were obtained; the first served as a control and the second was used to examine the effect under study. Neurogenic responses (contractile and relaxant phases, see Results) to electric field stimulation were blocked in the presence of the fast Na⁺ channel blocker, tetrodotoxin (10⁶ M; 30-min incubation), and contractions were antagonized by the muscarinic receptor blocker, atropine (10⁶ M; 30-min incubation; not shown).

Inhibitors. When examining the effects of inhibitors, control preparations were always run simultaneously to monitor possible alterations in time that were independent of the test agent. There usually were no differences between the first and second curves in the controls, but in some cases changes in reactivity, although not significant, occurred that affected interpretation of the effect under study; these results will be shown as appropriate. The following agents were examined for their effects 30 min after addition to the extra- and intraluminal baths unless otherwise indicated in Results: the cyclo-oxygenase inhibitor, indomethacin (3 × 10⁶ M); the Na⁺ channel blocker, amiloride (10⁴ M); the Cl channel blocker, 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS; 10⁴ M); the Na⁺-K⁺-2Cl cotransporter inhibitors, bumetanide and furosemide (10⁴ M); the cAMP phosphodiesterase inhibitor papaverine; and the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME; 10⁴ M). The effects of the Na⁺,K⁺-pump inhibitor, ouabain (10⁵ M), were examined 5 min after its addition to the extraluminal bath. This shorter incubation period was chosen to avoid the appearance of the slowly developing contraction that accompanies Na⁺-loading of the muscle.

Analysis of Results. Responses were quantified as P in centimeters of H₂O. Geometric mean EC₅₀ values were derived from least-squares analysis of a four-parameter logit curve fit and are presented with 95% confidence intervals (C.I.) in parentheses. Statistical comparisons of EC₅₀ values were done using normally distributed logEC₅₀ values. In Results, the EC₅₀ values for KCl and NaCl are given in terms of molar concentration added to MKH solution. The results were analyzed for differences using one-way ANOVA, ANOVA on ranks, or Student's t test for paired or nonpaired samples, as appropriate. Other results are expressed as means ± S.E.M.; n is the number of separate experiments. p < .05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Relaxation Responses to Hyperosmolar Intraluminal MKH Solutions. Addition of intraluminal KCl to MCh (3 × 10⁷ M)-contracted tracheae elicited relaxation. As little as 4.2 mM added KCl evoked the response (Fig. 1). The EC₅₀ was 15.5 (C.I., 12.6-19.2) mM added KCl. This value was not affected by the cyclo-oxygenase inhibitor, indomethacin (3 × 10⁶ M; not shown). Contraction to intraluminally added KCl never occurred in intact, epithelium-containing tracheae. In unstimulated tracheae, KCl added extraluminally resulted only in contraction; in epithelium-denuded tracheae, KCl added intra- or extraluminally resulted only in contraction (not shown). These findings indicate that the relaxant response to elevated intraluminal KCl concentration was dependent upon and mediated by the epithelium.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Relaxation of intact tracheae by intraluminal hyperosmolarity; cumulative osmolar concentration-response curves for relaxation of MCh (3 × 107 M)-contracted tracheae. Concentrations on abscissa refer to added molar concentrations; additions in osmolar concentration terms are twice molar values. Left, comparison of intraluminal KCl (n = 4) and intraluminal NaCl (n = 4) concentration-response curves obtained from epithelium-containing, intact trachea. Right, comparison of intraluminal NaCl concentration-response curves obtained from epithelium-containing ("intact", n = 4) and epithelium-free ("rubbed", n = 5) trachea.

Effect of Inhibitors on Responses to Intraluminal Hyperosmolarity. To circumvent the potential problem of KCl-induced contraction, NaCl was most often used to elevate intraluminal osmolarity, because the two salts had been found to be equipotent intraluminal relaxants (+epithelium). L-NAME (10⁴ M; Fig. 2) inhibited slightly the relaxation to intraluminal NaCl; this effect resembled the changes seen in the curves of control preparations examined in the absence of L-NAME (Fig. 2), but the effect of the inhibitor was significant, whereas the changes in the controls were not. Amiloride (10⁴ M) and DIDS (10⁴ M), alone and in combination (Fig. 3), inhibited significantly intraluminal NaCl-induced relaxation. Amiloride also inhibited intraluminal KCl-induced relaxation responses (p < .05, n = 7; not shown), whereas DIDS alone (n = 6, not shown) and amiloride together with DIDS (n = 6; not shown) inhibited relaxation at a nearly significant level (p < .06).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of L-NAME (104 M) on relaxation responses of MCh (3 × 107 M)-contracted, intact tracheae to intraluminally applied hyperosmolar NaCl. Left, NaCl concentration-response curves, the second obtained in the absence of L-NAME (n = 6). Right, NaCl concentration-response curves, the second obtained in the presence of L-NAME (n = 6; separate tracheae). *Maximum relaxation response significantly less than control. Note that, although not significant, a similar difference was seen between the two control curves.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Inhibitory effects of amiloride and DIDS, alone and together, on relaxation responses of MCh (3 × 107 M)-contracted, intact tracheae to intraluminally applied hyperosmolar NaCl. The second NaCl concentration-response curves were obtained in the presence of amiloride alone (left, 104 M; n = 4), DIDS alone (middle, 104 M; n = 6), or both agents (right, n = 6). *Maximum relaxation response significantly less than control.

4

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Inhibitory effects of bumetanide (104 M) on relaxation responses of MCh (3 × 107 M)-contracted, intact tracheae to intraluminally applied hyperosmolar KCl (left, n = 6) and intraluminally applied NaCl (right, n = 4). *Maximum relaxation response significantly less than control. Absence versus presence of bumetanide, p < .07.

5

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Potentiating effect of ouabain (105 M, 5-min incubation) on relaxation response of MCh (3 × 107 M)-contracted, intact tracheae to intraluminally applied hyperosmolar NaCl (n = 4). Maximum relaxation response in the absence versus presence of ouabain, p < .07.

Effects of Hyperosmolar Intraluminal MKH Solutions on Reactivity to MCh. Under control conditions, extraluminally applied MCh was appreciably and significantly more potent than intraluminally applied MCh (Table 1 and Fig. 6). Intraluminal hyperosmolarity was used to evoke EpDRF release, and the effects of released EpDRF on MCh concentration-response curves were assessed. Added KCl or NaCl concentrations were employed that approximated the EC₅₀ and EC₉₀ values for relaxation responses of intact trachea (i.e., 13.3 and 42.2 mM, respectively; Fig. 1). In both intact and rubbed preparations, the administration of intraluminal KCl or NaCl slightly decreased baseline in a few preparations, reflective of a small variable amount of spontaneous basal tone in the preparations. In the vast majority of tracheae there was no effect of the intraluminally administered salts on basal tone. The results presented in this section are, therefore, not attributable to effects of the salts on basal P. When added to the intraluminal bath, both concentrations of both salts decreased reactivity to intraluminal MCh (Fig. 6 and Tables 1 and 2). Intraluminal KCl and NaCl were less effective inhibitors of responses to extraluminal MCh, compared with those elicited by intraluminally added MCh. NaCl produced a greater antagonism of these responses than did KCl, irrespective of the bath to which MCh was added. The same inhibitory effects of hyperosmolar intraluminal KCl on intra- and extraluminal reactivity to MCh were observed in the presence of indomethacin (3 × 10⁶ M; Fig. 7 and Table 3), in support of previous observations that EpDRF is not a prostanoid.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Inhibitory effects of hyperosmolar intraluminal NaCl (A, C) or NaCl (B, D) on intraluminal (A, B) and extraluminal (C, D) MCh concentration-response curves of intact tracheae. n values were 4 to 7 for each group. *Maximum response significantly less than control. Note that maximum responses were reduced in all cases except C.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Lack of effect of indomethacin (3 × 106 M) on inhibitory effect of hyperosmolar intraluminal KCl on intraluminal MCh concentration-response curves. In this experiment using intact tracheae, indomethacin was present during both concentration-response determinations (C and D). MCh concentration-response curves were obtained before () and after () elevating intraluminal KCl concentration with 13.3 mM KCl (A and C; n = 4 for both) or 42.2 mM KCl (B and D; n = 4 for both). Osmolar concentration-dependence of inhibitory effect of KCl on reactivity to MCh also is shown.

View this table: [in this window] [in a new window]   TABLE 3 Effect of intraluminal KCl on reactivity to intra- and extraluminal MCh in presence of indomethacin (3 times 106 M)

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Lack of effect of hyperosmolar intraluminal NaCl solution on reactivity of rubbed tracheae to intraluminal MCh (A and B) and extraluminal MCh (C and D). Note that higher NaCl concentrations were intentionally employed than in Fig. 6. n = 4 to 5 for each group.

Responses to Hypo-Osmolar Intraluminal MKH Solutions. Because obstruction in human airways may be produced by hypo-osmolar as well as hyperosmolar aerosols (see Introduction), we reasoned that hypo-osmolar solutions might also affect airway diameter. Figure 9 illustrates that as little as a 1% reduction in the osmolarity of the perfusing Krebs' solution resulted in a measurable increase in P, and the response increased as tonicity decreased.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Elevation of P in response to intraluminal hypo-osmolarity. A response was seen with as little as a 1% reduction of the intraluminal MKH solution. This curve was generated with "cumulative" addition of water to the bath, each addition following the establishment of a stable, plateau response. n = 7.

4

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Elevation in P in response to intraluminal hypo-osmolarity: effects of papaverine (104 M; PAP) and epithelium removal. Epithelium-containing (intact, A and B) and epithelium-denuded (rubbed, C and D) preparations were perfused for 5 min with intraluminal water (IL H2O) to provide an extreme hypo-osmolar challenge. After returning normal MKH solution to perfusate and perfusing for 1 h, tracheae were contracted with extraluminal (EL) MCh (3.5 × 107 M). After 1 h of washing, preparations were incubated with papaverine (A and C) or with normal MKH solution (B and D), and challenged a second time with water and MCh. Intraluminal KCl (120 mM) was added at end of experiment to verify that epithelium was removed when desired, as demonstrated by contraction to intraluminal KCl in denuded tracheae or relaxation of intact tracheae. (In some cases elevation in P in response to water persisted beyond 5-min exposure period.) Tracings are representative of n = 2 to 4 separate experiments in each protocol.

Effects of Serosally Applied Hyperosmolar Solution: Is EpDRF Released Only in Response to Elevated Mucosal Osmolarity? When added to preparations that had been contracted with MCh, the addition of NaCl or KCl to the extraluminal bath gave rise to concentration-dependent relaxation responses (Fig. 11). The relaxation due to NaCl was reasonably well maintained but rose gradually to the initial level of MCh-induced tone. The response to KCl was very transient and was followed by a contraction to a level well above the value caused by MCh alone (not shown). Intraluminally added NaCl elicited significantly larger responses at 5.62 and 13.3 mM than were seen after extraluminal NaCl addition. Because of its transient nature, the relaxation phase of the response to all concentrations of extraluminally applied KCl was significantly smaller than those after intraluminal addition. The contraction to extraluminally added KCl accounted for the greater "efficacy" of extraluminally added NaCl compared with extraluminally added KCl. A comparison of relaxation responses to extraluminally added NaCl in separate intact and epithelium-denuded tracheae revealed that epithelium removal decreased significantly the hyperosmolarity-induced relaxation response at 80 mM added extraluminal NaCl (Fig. 12).

View larger version (17K): [in this window] [in a new window]   Fig. 11.   Comparison of relaxation responses of extraluminal MCh (3.5 × 107 M)-contracted perfused tracheae to elevated tonicity in the extraluminal (EL) and intraluminal (IL) baths. Left, tonicity elevated with NaCl; right, tonicity increased with KCl. *Significantly larger than value for extraluminal bath addition. n = 8 for 5.62 mM; n = 8 for 13.3 mM; n = 6 for 80 mM.

View larger version (22K): [in this window] [in a new window]   Fig. 12.   Comparison of relaxation responses of MCh (3.5 × 107 M)-contracted intact and epithelium-denuded (rubbed) tracheae to extraluminally applied NaCl. *Significantly smaller than value for intact tracheae. n = 8, 6 for 5.62 mM; n = 8, 9 for 13.3 mM; and n = 6, 6 for intact, rubbed, respectively.

Effects of Hyperosmolar Intraluminal MKH Solutions on Neurogenic Contractile Responses. Experiments were conducted to examine the effects of intraluminal hyperosmolarity on responses of the trachea elicited by endogenous transmitters released in response to electric field stimulation. Unlike responses to intraluminally applied exogenous agents, responses to endogenously released agonists would not be influenced by diffusion of the agent across the epithelium. Three patterns of response to electric field stimulation were observed in the tracheae of different animals: 1) rapid, transient, monophasic contraction only; 2) rapid, transient contraction followed by a slower developing and longer lasting contraction persisting beyond delivery of electrical impulses; and 3) rapid, transient contraction followed by relaxation below baseline. Often, but not always, transitions between the first response pattern to the second and/or third occurred with increasing stimulus frequency.

View larger version (24K): [in this window] [in a new window]   Fig. 13.   Effect of intraluminal hyperosmolar NaCl on electric field stimulation-induced contractile responses of intact tracheae. Two frequency-response curves were obtained from each trachea, first in the absence of NaCl (), and second () without any treatment (A, n = 4) or 30 min after addition of NaCl to perfusing solution as follows: B, 13.3 mM (n = 5); C, 30 mM (n = 8); and D, 120 mM (n = 8).

View larger version (23K): [in this window] [in a new window]   Fig. 14.   Effect of intraluminal hyperosmolar NaCl on electric field stimulation-induced contractile responses of epithelium-denuded (rubbed) tracheae. Protocol was similar to that in Fig. 13, except that denuded tracheae were used. A, no treatment (n = 6) or 30 min after the addition of NaCl to the perfusing solution, as follows: B, 13.3 mM (n = 6); C, 30 mM (n = 8); and D, 120 mM (n = 7). *Significantly less than control.

View larger version (16K): [in this window] [in a new window]   Fig. 15.   Comparison of effects of intraluminal hyperosmolar NaCl solution on electric field stimulation-induced contractile responses of intact and epithelium-free (rubbed) tracheae to maximal, 30 Hz stimulation. This figure depicts ratios of contractile responses to 30-Hz stimulation obtained in the absence of intraluminal hyperosmolar NaCl (Fig. 13) divided by responses obtained in the presence (Fig. 14) of intraluminal hyperosmolar NaCl, i.e., (curve 2)/(curve 1). This ratio provides a normalized index of relative magnitudes of inhibitory effect of NaCl on responses obtained from intact versus epithelium-free tracheae, irrespective of magnitudes of responses. n values are given in legends to Figs. 13 and 14. *Significantly less than ratio obtained in the absence of NaCl (None).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The perfused trachea responds to both increases and decreases in the osmolarity of the perfusing solution. Indeed, the trachea was sensitive to very small changes in osmolarity. The effects of mucosal hyperosmolarity involve EpDRF; an epithelial component may also exist in the response to intraluminal hypo-osmolarity. We also observed that the epithelium is involved in relaxation responses to extraluminally applied hyperosmolarity.

Relaxation Induced by Intra- and Extraluminal Hyperosmolarity. Our results indicate that the epithelium is an osmotic sensor which, upon elevation in osmolarity, brought about three effects that can affect airway diameter. The first effect was relaxation of the airway smooth muscle. Relaxation was produced equipotently by intraluminal KCl and NaCl, despite the fact that KCl is a powerful contractile agent when added to the extraluminal bath of intact tracheae or to the extra- or intraluminal baths of epithelium-denuded tracheae. This reiterates the conclusion by Munakata et al. (1988) that relaxation was stimulated by hyperosmolarity per se rather than by agent-specific mechanisms.

Inhibition of Reactivity to MCh by Stimulated Release of EpDRF. The second way in which the epithelium, acting as osmotic sensor, can regulate airway diameter is by affecting reactivity to contractile agents. Heretofore, the inhibitory effect of the epithelium on reactivity has been demonstrated by removing the epithelium. In the present study we used intraluminal hyperosmolarity to provoke EpDRF release from intact trachea and observed an osmolar concentration-dependent decrease in reactivity to intra- and extraluminally applied MCh, only in the presence of the epithelium. The fact that intraluminally added NaCl had no effect on reactivity of denuded tracheae to MCh is additional support for the conclusion that the smooth muscle is not the primary site of the relaxant effects of elevated mucosal tonicity. The inhibition was independent of the means used to increase osmolarity, because added KCl and NaCl were equieffective, and it occurred both in the absence and in the presence of indomethacin.

Inhibition of Neurotransmission by EpDRF. The third way that the epithelium, acting as an osmotic sensor, can affect airway diameter is by inhibiting neurotransmission. Raised intraluminal osmolarity produced a concentration-dependent inhibition of neurogenic contractile responses; the inhibition was substantially greater in the presence of the epithelium. In tracheae demonstrating two phases in the contractile response, both phases were inhibited. Because in intact trachea the effect of a given concentration of intraluminal NaCl would not reflect the direct effect of the salt seen in the absence of the epithelium, and because NaCl had no effect on concentration-response curves for either extra- or intraluminally administered MCh in the absence of the epithelium, these findings indicate that released EpDRF inhibited cholinergic postganglionic and excitatory nonadrenergic, noncholinergic neurotransmission in the trachea. A decrease in acetylcholine release after incubation with cultured epithelial cell supernatant has been observed in canine tracheal smooth muscle; reactivity to exogenous acetylcholine was not affected (Matsumoto et al., 1996). On the other hand, we found that responses to both exogenous and endogenous cholinergic agonists were inhibited by intraluminal hyperosmotic solution, which suggests that pre- and postjunctional mechanisms may operate in the guinea pig trachea. For technical reasons we could not determine whether EpDRF affected the neurogenic inhibitory phase of the responses. Nevertheless, our results agree with those of Flavahan et al. (1985), who observed that epithelium removal potentiated contractile responses of dog airways to electric field stimulation. Several approaches, therefore, have indicated that neural efferent function in the airways is modulated by EpDRF.

Hypotonic Intraluminal Solutions. Very small decrements in intraluminal osmolarity elevated P. Whether these responses involved epithelial swelling, a contractile factor from epithelium, and/or a direct effect on the airway smooth muscle, must be considered. Our findings suggest that the elevation of P in intact tracheae did not result primarily from swelling of the epithelium. First, responses were elicited by very small decreases in luminal osmolarity, i.e., ca. 1% reduction. Second, the pressor response to luminal hypo-osmolarity occurred both in the absence and presence of the epithelium. Third, responses to both hypo-osmolarity and MCh were inhibited by papaverine.