Inhibition of Airway Constriction by Opioids Is Different down the Isolated Bovine Airway 

Bovine right or left diaphragmatic lung lobes from adult animals were obtained from the local abattoir. The lobes were removed immediately after death and immersed in chilled (4 degrees Celsius) and aerated physiologic salt solution (PSS). The lobes arrived in the laboratory within 1 or 2 h after the death of the animal. Bronchial rings of 3 or 4 mm length and 2-, 3-, 4-, and 5-mm internal diameter were dissected from the lung parenchyma. Care was taken to leave the bronchial epithelium intact. The bronchial rings were suspended between two vertical platinum electrodes (1 x 4 cm) parallel to each other in water-jacketed tissue baths containing PSS (37 degrees Celsius), which was continuously aerated with a gas mixture of 95% oxygen and 5% carbon dioxide and was of the following composition 0.8 mM MgSO⁴, 1.2 mM KH²PO⁴, 3.4 mM KCl, 2.4 mM CaCl², 110.5 mM NaCl, 25.7 mM NaHCO³, and 5.6 mM dextrose. The rings were mounted between two stainless steel triangular wires; the upper ring was connected using a silk string to a force transducer (model FT 03; Grass Medical Instruments, Quincy, MA) mounted on a micro-manipulator, allowing the airway to be gradually stretched. The lower triangular wire was connected via a silk string to a stationary hook at the bottom of the tissue bath. The bronchial rings were equilibrated with PSS for an average of 2 h and 40 min while they were supramaximally contracted every 5 min for 30 s by EFS (25 Hz, 25 V, 0.5 ms), provided by a DC amplifier (Department of Engineering, Mayo Clinic, Rochester, MN), triggered by a stimulator (model S44; Grass Medical Instruments).

Each ring was stretched after each stimulus until the contractile responses to EFS were maximal and constant (optimal length). They were maintained at this length throughout the study. Trachealis strips were prepared and treated as described previously. [9] The contractile response to EFS was completely abolished by 10 sup -6 M tetrodotoxin, suggesting that the EFS did not directly stimulate the smooth muscle. It was also completely abolished by 10 sup -6 M atropine, demonstrating that the contractile response was mediated by muscarinic receptors only.

At the end of each study, all rings and strips were blotted dry and weighed, and the inner diameters were measured with a ruler.

The airway generations corresponding to 2-, 3-, 4-, and 5-mm inner diameters were determined in the two diaphragmatic lobes from one additional animal by carefully fitting surgical probes of known diameters into the lumen of the airways.

Electrical Field Stimulation. Two rings each of 2-, 3-, 4, and 5-mm inner diameter from each of six animals were incubated with 10 sup -6 M propranolol to antagonize stimulation of beta-adrenoceptors and 10 sup -5 M indomethacin to block the effect of endogenously produced prostaglandins.

The eight rings from each animal were contracted for 30 s by EFS (25 V, 0.5 ms) at four stimulating frequencies (0.5 Hz, 2 Hz, 8 Hz, and 32 Hz) applied in random order (baseline frequency-response curves). Thereafter, all eight rings were washed with PSS containing 10 sup -6 M propranolol and 10 sup -5 M indomethacin. One ring from each of the four sizes was incubated with 10 sup -5 M D-Ala²-N-MePhe⁴-Gly-ol sup 5 -enkephalin (DAMGO); the other four rings not incubated with DAMGO served as time controls. After 10 min of incubation with DAMGO, all eight rings were contracted again by EFS at the four frequencies. After this second set of frequency-response curves, all rings were incubated for 8–10 min with naloxone (10 sup -5 M), and complete sets of frequency-response curves were obtained.

Acetylcholine. Two rings each of 3-mm inner diameters from another six animals were incubated for 30 min with 10 sup -6 M tetrodotoxin to block axonal conduction in postganglionic nerves, thereby preventing stimulation of prejunctional muscarinic receptors from contributing to the exogenous acetylcholine-induced contraction. Cumulative concentration-response curves to acetylcholine (10 sup -9 M to 10 sup -3 M, in half-log increments) were obtained in both rings (baseline concentration-response curves). The rings were washed with PSS containing 10 sup -6 M tetrodotoxin until the resting forces were reestablished. Thereafter one ring from each animal was incubated for 10 min with 10 sup -5 M DAMGO, and the other served as the time control. A second complete set of cumulative concentration-response curves to acetylcholine was obtained in both rings.

Electrical Field Stimulation. Two rings each of 2-, 3-, 4-, and 5-mm inner diameter from each of another seven animals were prepared similarly to those used for the DAMGO study. After completion of the baseline frequency-response curves, one ring from each of the four sizes was incubated with 10 sup -5 M trans-3,4-dichloro-N-methyl-N-(2–1-pyrrolidinyl) cyclohexyl benzene acetamide (U-50488 H). The other four rings not incubated with U-50488 H served as time controls. After 10 min of incubation with U-50488 H, a second complete set of frequency-response curves was obtained.

Two rings each of 3-mm inner diameter from another six animals were treated similarly. After the baseline set of frequency-response curves was obtained, one ring from each of the six animals was incubated with the selective kappa-agonist U-50488 H (10 sup -5 M) while the other rings served as time controls. A second complete set of frequency-response curves was then generated. Both rings from each animal were incubated for 10 min with the highly selective kappa-antagonist[15] 2,2'-[1,1'-biphenyl] 4,4'-diyl-bis [2-hydroxy-4,4-dimethyl]-morpholinium (nor-BNI)(10 sup -5 M), and a third complete set of frequency-response curves was obtained.

In two rings each (3-mm inner diameter) from another six animals, the effect of naloxone (10 sup -5 M) was studied in U-50488 H-incubated muscles (10 sup -5 M) by a similar protocol.

Electrical Field Stimulation. Two rings each of 2-, 3-, 4-, and 5-mm inner diameter from another four animals were prepared similarly to those used for the DAMGO study. After completion of the baseline frequency-response curves, one ring from each of the four sizes was incubated with 10 sup -5 M DPDPE. The other four rings served as time controls.

Inhibition by the three opioid agonists of contractile responses to EFS in bronchial rings (2, 3, 4, and 5 mm) was compared with inhibition in tracheal strips described in a previous study from this laboratory. [9] To confirm the conclusions from this “historical” comparison, inhibitory effects of DAMGO (10 sup -5 M) and U-50488 H (10 sup -5 M) on EFS-induced contractions were compared in eight trachealis strips and eight bronchial rings (3 mm). In these experiments, only trachealis strips and bronchial rings sampled from the same animals were compared.

Isometric forces were corrected for the effect of time:Equation 1where T^corris the time-corrected contractile response of the ring incubated with the test drug, and D¹and D²are the measured contractile responses before and after incubation of the ring with the test drug. C¹and C²are the corresponding measured contractile responses of the ring not incubated with the test drug (time controls).

The physical characteristics of the bronchial rings were tested for significant differences by repeated-measures analysis of variance, treating the inner diameter as a within-subject effect.

Inhibitions of contractile responses to EFS by each of the three opioids were calculated for each of the four stimulating frequencies and for each of the four inner diameters. Analysis of variance was then used to determine whether the inhibitions were significantly associated with the size of the inner diameter for each of the three opioid agonists.

To determine whether the inhibitions of contractile responses were significant, the mean inhibitions across the four inner diameters for each of the three opioid agonists were first calculated. One-sample t tests were used for each of the three opioids for the four stimulating frequencies.

Repeated-measures analysis of variance was used to determine whether naloxone or nor-BNI had antagonizing effects, whether DAMGO affected acetylcholine-induced contractions, and to test for differences in the effects of DAMGO, DPDPE, or U-50488 H in the trachealis strips and bronchial rings. Values are expressed as means +/- standard deviations.

We purchased DAMGO and DPDPE from Bachem Feinchemikalien A.G. (Bubendorf, Switzerland). The Upjohn Company (Kalamazoo, MI) provided U-50488 H. Indomethacin, naloxone hydrochloride, DL-propranolol hydrochloride, acetylcholine chloride, and tetrodotoxin were purchased from Sigma Chemical Company (St. Louis, MO) and nor-BNI dihydrochloride from Amersham, Milano, Italia. All drugs were dissolved in distilled water except indomethacin, which was dissolved in an alkaline phosphate buffer. Drugs were added to the PSS in 100-micro liter aliquots.

One hundred eighty-eight isolated bronchial rings and 16 trachealis strips from 43 animals were studied. Weights (P < 0.001), maximal forces (P < 0.001), and resting forces (P = 0.022) were significantly different among the four inner diameters (Table 1). Bronchi of 4-mm and 5-mm inner diameters were sublobar, and bronchi of 2-mm and 3-mm inner diameters were segmental. Neither DAMGO, U-50488 H, nor DPDPE had a discernible effect on the resting forces.

The average value for differences between measured and time-corrected forces was 2 +/- 26% of the measured force. In no study was the conclusion altered by using the time-corrected forces.

Electrical Field Stimulation. DAMGO (10 sup -5 M) did not result in significantly different inhibitions of EFS-induced contractions between rings of 2-, 3-, 4-, and 5-mm inner diameters (P = 0.65). Mean inhibitions across the four inner diameters of EFS-induced contractions produced by 10 sup -5 M DAMGO were significant at 0.5 Hz (P < 0.001), 2 Hz (P < 0.001), and 8 Hz (P = 0.001), but not at 32 Hz (P = 0.071;Figure 1). Naloxone (10 sup -5 M) significantly antagonized the effect of 10 sup -5 M DAMGO (P = 0.025) but had no effect on the contractile response in the muscles used as time controls.

Acetylcholine. DAMGO (10 sup -5 M) had no significant effect (P = 0.69) on acetylcholine-induced contractions.

Electrical Field Stimulation. No significant differences were produced by U-50488 H (10 sup -5 M) in inhibitions of EFS-induced contractions between rings of 2-, 3-, 4-, and 5-mm inner diameters (P = 0.28). Mean inhibitions across the four inner diameters of EFS-induced contractions produced by U-50488 H (10 sup -5 M) were small but significant at 32 Hz (P = 0.008) and 8 Hz (P = 0.045), but not at 2 Hz (P = 0.893) and 0.5 Hz (P = 0.145;Figure 2). Neither 10 sup -5 M nor-BNI nor 10 sup -5 M naloxone significantly antagonized the effect of 10 sup -5 M U-50488 H (P = 0.216 and P = 0.065, respectively;Figure 3).

Electrical Field Stimulation. We found that 10 sup -5 M DPDPE did not result in significant differences in contractile responses to EFS among rings of 2-, 3-, 4-, and 5-mm inner diameters. Mean inhibitions of EFS-induced contractions were not different at any of the four stimulating frequencies (P = 0.256).

Electrical Field Stimulation. Inhibitions of contractile responses to EFS produced by DAMGO (10 sup -5 M) and U-50488 H (10 sup -5 M) were significantly different between the 64 trachealis strips from a previous study [9] and the 68 bronchial rings from this study. Inhibitions by DAMGO were significantly (P < 0.001) smaller in the trachealis than the bronchial rings. In contrast, U-50488 H had a significantly (P < 0.001) larger inhibitory effect in trachealis strips than in bronchial rings (Figure 4). The effects of DPDPE (10 sup -5 M) were not significantly different.

In trachealis strips and bronchial rings sampled from the same animals, inhibition of contractile responses to EFS by U-50488 H (10 sup -5 M) was consistently and significantly larger (P < 0.001) in trachealis strips than in bronchial rings. In contrast, with DAMGO (10 sup -5 M), inhibitions in trachealis strips were larger than those in bronchial rings in only 1 of 16 observations, but this difference was not statistically significant.

The most important findings of this study are that 1) there were no significant differences in the effects of the three selective opioid agonists on EFS-induced contractions between isolated sublobar and segmental bovine bronchial rings, and 2) the inhibitory effects of DAMGO on EFS-induced contractions were larger, and those of U-50488 H smaller in bronchial rings compared with trachealis strips.

Stimulation of muscarinic receptors by EFS can cause synthesis and release of prostaglandins. [16] Endogenous prostaglandins may inhibit release of acetylcholine, resulting in a progressive reduction of contractile responses to EFS. [3,17] In this study, cyclooxygenase was blocked by 10 sup -5 M indomethacin, and no progressive reductions of contractile responses to repeated EFS were observed.

In isolated airways, contractile responses cannot be modulated by hormones or humoral substances circulating in the blood stream, nor can they be influenced by the central nervous system. Furthermore, EFS is a relatively nonspecific stimulation possibly resulting in a simultaneous unphysiologic release of several neurotransmitters. To achieve a more selective stimulus, bronchial rings and trachealis strips were stimulated at different frequencies ranging from 0.5 Hz to 32 Hz. Modulations of neurotransmission usually occur at lower frequencies. [18]

One must be careful in extrapolating from functional studies to the presence or absence of opioid receptors. Different levels of activities of peptidases in the trachealis strips (no epithelium) and bronchial rings (intact epithelium) and different rates in achieving the inhibitory effects could contribute to differences between trachealis strips and bronchial rings. To elucidate the combined effects of these two possibilities, eight bronchial rings sampled from eight animals and four trachealis strips sampled from four animals were incubated for 113 min with U-50488 H (10 sup -5 M). Contractile responses to EFS were determined at four frequencies after 10 min, 60 min, and again after 113 min of incubation. In the four trachealis strips, maximal inhibition (87 +/- 15%) was achieved at all frequencies after 10 min; it did not change significantly within the next 103 min. In contrast, a mean inhibition of 4 +/- 12% occurred after 10 min in bronchial rings; it increased to 19 +/- 22% after 60 min and to 31 +/- 29% after 113 min of incubation. These data suggest that different levels of activities of peptidases are not responsible for the differences between trachealis strips and bronchial rings. The slower rate of developing the inhibitory effect in bronchial rings, probably due to a diffusion barrier, results in an overestimation of the difference between bronchialis rings and trachealis strips. But, importantly, the difference in inhibition (82 +/- 21% vs. 31 +/- 29%) remained after 113 min of incubation. To reduce inactivation of opioids, several precautions were taken. The enkephalins were stored in sealed vials at -20 degrees Celsius, fresh solutions were prepared daily, tissues were incubated with opioids for only 10 min, and the tissue baths were wrapped with aluminum foil to protect against degradation by light.

In vivo, elastic-loaded shortening of the muscles rather than isometric contractions determines the patency of airways. Ideally, changes in elastic-loaded forces, which are more similar to isotonic contractions, should be measured. But comparative studies suggest that similar conclusions can be drawn from isotonic and isometric measurements. [19]

Inhibition of EFS-induced contractions by opioid agonists may result from inhibition of excitatory nonadrenergic-noncholinergic function. [7] However, this function did not contribute to the EFS-induced contractions in the bovine bronchial rings because atropine completely abolished EFS-induced contractions, showing that the contractions were cholinergic.

Electrical field stimulation may also stimulate inhibitory nonadrenergic-noncholinergic function and thereby reduce the contractile response. In 13 of 176 rings, EFS resulted in a transient relaxation below the resting force after the cholinergic contraction. This relaxation was unaffected by opioids, suggesting that opioids had no stimulatory effect. Because this relaxation occurred infrequently, no attempt was made to determine whether this relaxation was due to stimulation of inhibitory nonadrenergic-noncholinergic nerves.

From this, we conclude that opioids have an effect on the cholinergic nerves. Autoradiographic studies should verify whether the differences in inhibitory effects between the trachea and bronchi and between the type of specific opioid agonist were due to differences in the density of opioid receptors between trachea and bronchi and differences in the relative abundances of specific opioid receptors.

The inhibitory effect of DAMGO (10 sup -5 M) was frequency-dependent. It occurred only at the lower frequencies of 0.5–8 Hz and not at the higher frequency of 32 Hz, which is consistent with the results of other studies [6,8,9,20] and is typical for neuromodulators. [18] Naloxone had no effect on cholinergic neurotransmission in the rings used as time controls, suggesting that endogenous opioids did not affect the cholinergic neurotransmission. But naloxone completely antagonized the inhibitory effect of DAMGO, suggesting that the inhibitory effect was mediated by micro-opioid receptors.

The micro-opioid receptors are located prejunctionally, probably at the junction between the cholinergic nerve endings and the muscle cells. This latter conclusion is based on the failure of DAMGO to alter acetylcholine-induced contractions in rings incubated with tetrodotoxin. Stimulation of prejunctional opioid receptors apparently interferes with the release of cholinergic neurotransmitter(s), thus reducing EFS- but not acetylcholine-induced contractions. [4–9,20]

Like the effect of DAMGO, the inhibitory effect of U-50488 H (10 sup -5 M) was frequency-dependent. But in contrast to DAMGO, U-50488 H inhibited EFS-induced contractions by a small but significant amount only at the higher frequencies of 8 Hz and 32 Hz and not at the lower frequencies of 2 Hz and 0.5 Hz. This observation is in contrast to the inhibitions of contractile responses to EFS by U-50488 H in isolated bovine trachealis strips, where U-50488 H attenuated EFS induced contractions at low and high frequencies. [9] The failure of U-50488 H to inhibit in bronchial rings the contractile response to EFS at low frequencies suggests that U-50488 H may not act as a typical neuromodulator. [18] That the inhibitory effect of U-50488 H at 8 Hz and 32 Hz was not mediated via kappa-opioid receptors also is suggested by the observation that neither naloxone nor the highly selective antagonist nor-BNI [15] antagonized the inhibitory effect of U-50488 H. In contrast, in bovine trachealis strips nor-BNI did antagonize the effect of U-50488 H, [9] suggesting that kappa-opioid receptors were involved in the inhibition of EFS-induced contractions in the trachea. Perhaps U-50488 H interacted in the bronchi with other receptors. Interactions occur between opioid and beta-adrenergic receptors in canine bronchial smooth muscle. [21] In the current study, all rings were incubated with propranolol, making an interaction with beta-adrenergic receptors unlikely. However, interactions with other receptors cannot be excluded.

The selective delta-opioid agonist DPDPE (10 sup -5 M) had, like in isolated bovine trachealis, [9] no inhibitory effect on contractile responses to EFS in bronchial rings, suggesting that delta-opioid receptors do not mediate contractions in the bovine airways.

Comparison of the data from this study in bronchial rings with data from a previous study from this laboratory on trachealis strips [9] showed that inhibitions of EFS-induced contractions produced by DAMGO (10 sup -5 M) were significantly larger in bronchial rings than in trachealis strips. Conversely, U-50488 H (10 sup -5 M) had a significantly larger effect in trachealis strips. Neither in the trachealis strips nor bronchial rings did DPDPE affect the contractile responses to EFS. The conclusions from this comparison were confirmed in general when the tissues for trachealis strips and bronchial rings were sampled from the same animals.

Other authors found significant differences in inhibitory effects on contractile responses to EFS by DAMGO between upper and lower regions of the guinea pig trachea and main bronchi. [7] These differences in the inhibitory effects of DAMGO were thought to be due in part to an inhibitory effect of DAMGO on the facilitatory excitatory nonadrenergic-noncholinergic function. But the different effects of DAMGO and U-50488 H between bovine trachealis and bovine bronchial smooth muscle cannot be explained by an inhibitory effect of opioid agonists on excitatory nonadrenergic-noncholinergic function, because EFS-induced contractions were purely cholinergic. Thus it seems more likely that differences in the abundances of opioid receptors are responsible.

The normal function of opioid receptors in the airways remains unclear. Increased levels of enkephalins during stress may protect against neurogenic bronchoconstriction. [5] Opioids may also be released in the airways by immune cells that migrate to sites of inflammation. [18]

It has been suggested that opioids may have a therapeutic role in the management of asthma. [8] Opioids not only modulate cholinergic neurotransmission, but they are antitussive and inhibit neurogenic microvascular leakage [12,13] and mucus secretion. [14] However, they release histamine. Given their beneficial effects in bovine airways, it may be worthwhile to develop specific opioid agonists that are devoid of histamine release but maintain the other beneficial effects. Opioids can be administered by inhalation, [22] which should achieve adequate local concentrations in airways.

The authors thank D. R. Schroeder and C. Lange, Section of Biostatistics, Mayo Clinic, Rochester, Minnesota, for doing the statistical analyses, and J. Beckman and K. Street for their secretarial assistance and illustrations.