Pulmonary Pharmacology (1992) 5, 233-238

The Inhibitory Effect of Furosemide on the Contractile Responseof Equine Trachealis to Choline@ Nerve Stimulation M. Yu*, Z. Wang?, N. E. Robinson*$, F. J. Derksen* *Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824-1314, USA, and TDepartment of Physiology, Michigan State University, East Lansing, Michigan 48824-1101, USA

SUMMARY: The effects of furosemide on the responses of equine trachealis muscle with and without epithelium to electrical field stimulation (EFS) and exogenous acetylcholine (ACh) were investigated in organ baths. Tissues were pretreated with guanethidine and the parameters used for EFS were those previously demonstrated to activate postganglionic cholinergic neurons. In tissues with intact epithelium, furosemide (100~~) shifted the frequencyresponse curve to the right. In the preparations without epithelium, furosemide did not affect the response to EFS. Neither in epithelium-on nor in epithelium-off tissues was the ACh dose-response curve affected by the administration of furosemide. We conclude that furosemide (100 FM) decreases the equine tracheal smooth muscle responses to EFS through inhibition of cholinergic neurotransmission, and that this effect is dependent on epithelium. INTRODUCTION

of an inhibitory factor, modulates the response of the airway smooth muscle to cholinergic nerve stimulation” and may be involved in furosemide’s action on the airways. The present study was performed with isolated equine tracheal smooth muscle strips to determine: (1) the effect of furosemide on the response of isolated trachealis muscle to activation of muscarinic receptors by exogenously applied acetylcholine (ACh) and by electrical field stimulation (EFS); and (2) if removal of tracheal epithelium influenced furosemide’s action. The mechanism of furosemide’s action on the airways may be similar to that on blood vessels, with furosemide promoting the release of a dilator factor from the kidneys or other remote organ. This factor would then be transported to the lungs, where it may cause bronchodilation either directly or after further metabolism by epithelium or other tissue. If this is the case, no inhibitory effect of furosemide would be observed in this isolated smooth muscle study.

Furosemide is a potent natriuretic-diuretic agent that can also depress the response of smooth muscle to some agonists and nerve stimulation. In blood vessels, furosemide causes vasodilation when the vascular tone has been increased by agonists such as noradrenaline and vasopressin II or by sympathetic nerve stimulation. This vascular effect of furosemide requires the presence of the kidney and an intact endothelium, and is dependent on cyclooxygenase products.1.2~3 In the lungs, furosemide prevents asthma induced by exercise4 and by inhalation of an ultrasonic ‘fog’ of distilled waters and ameliorates early and late responses to antigen challenge.6 In ponies with heaves, a disease in which there is a major cholinergic component to airway obstruction,’ furosemide decreases pulmonary resistance, and increases dynamic compliance, indicating bronchodilation. These effects are only observed when the ponies have acute exacerbations of airway obstruction but are not evident when animals are in clinical remission.8 It is unclear how furosemide acts on the airways. Failure of furosemide to influence bronchoconstriction induced by histamine and methacholine suggests that its effect is not directly on airway smooth muscle.9Jo It may exert its effect by inhibition of neurotransmission in cholinergic and non-cholinergic constrictor pathways. Airway epithelium, by production

MATERIALS

Two horses and eight ponies (body weight 121358 kg) were used in this study. They had no history of respiratory disease and showed no clinical signs of respiratory tract disorder. Animals were killed by injection of an overdose of pentobarbital sodium. The mid-cervical portion of trachea was quickly removed and immersed in oxygenated Krebs-Henseleit solu-

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tion (composition in mu: NaCl 118.4, NaHCO, 25.0, dextrose 11.7, KC1 4.7, CaCl,.2H,O 2.6, MgSO,.7H,O 1.19, KH,PO, 1.16). The trachea was then transported a short distance to the laboratory where it was gassed with 95% 0,/5% CO, continuously. The trachea was opened longitudinally by cutting through the cartilages in the anterior aspect of the trachea. Tracheal smooth muscle strips were cut with a template along the muscle fiber direction. In one group of tissue, the epithelium was kept intact. In another group, the epithelium was peeled off.‘* Light microscopy demonstrated that all the epithelium and part of the sub-epithelial tissues were removed without damaging the smooth muscle. The epithelium-on and epithelium-off tissues were subjected to the same protocol. Each strip measuring 2 x 10 mm was suspended by 3-O silk surgical thread in a 15-ml organ bath containing Krebs-Henseleit solution bubbled with 95% 0,/5% CO, and maintained at 38°C. One end of the tissue was tied to a hook at the lower end of an electrode holder that was placed in the muscle bath. The other end was tied to a force transducer (Grass FT03) mounted on micromanipulators so that the tissue length could be changed. The isometric force generated by the muscle strip was recorded on a polygraph (Grass model 7D or 7E). Tissues were suspended between platinum electrodes for electrical stimulation. Electrical impulses were produced by a Grass S88 stimulator and passed through a stimulus power booster (Stimu-Splitter II, Med Lab Instrument, Ford Collins, CO, USA). The electrical impulses consisted of square waves. Tissues were equilibrated for approximately 100 min with a passive load of 2 g applied and maintained. In preliminary experiments, it was determined that 2 g is the optimal resting load to achieve optimal length for the trachealis strips. During the equilibration, tissues were electrically stimulated for 2-3 min at 8-10 min intervals (15 V, 0.5 ms pulse duration, 16 Hz) until the baseline was stable and the magnitude of the response to the same stimulus was consistent. The bath solution was changed every 15 min. At the end of the equilibration, the contractile response to 127 mM KC1 solution was determined. This response was used to normalize all the subsequent muscle forces developed in response to EFS and ACh. Tissues were then rinsed with Krebs-Henseleit solution until the muscle tension returned to baseline. After a 20-min incubation of tissues with 10 PM guanethidine, contractile responses to increasing frequencies (1, 2, 4, 8, 16, 32 Hz) of EFS (0.5 ms pulse duration, 15 V) were obtained. Each stimulus was applied until a plateau in induced force was achieved, namely, 2-3 min. The interval between consecutive stimuli was 6min, allowing the nerve and smooth muscle to recover from the previous excitation. Each

of six organ baths then received one of five concentrations of furosemide (10 nM to 0.1 mM in log increments) or its vehicle. Each ml of the vehicle contained 0.2 mg myristyl-gammapicolinium chloride, 1 mg EDTA sodium, 2 mg NaCl, and 1 mg sodium sulfite. The amount of vehicle administered to the control bath was equal to that used to dissolve 0.1 mM furosemide. After a 30-min incubation period with furosemide or its vehicle, a second frequency-response curve was obtained. Subsequently, ACh dose response curves were generated by cumulatively adding ACh to the muscle bath (10 nM to 0.1 mM in log increments). When induced force plateaued following addition of a concentration of ACh, the next concentration was added. At the end of the experiment, the tissues were removed from the bath, blotted with filter paper, and weighed. Drugs

Furosemide (injection 5%) and its vehicle were obtained from The Butler Company (Columbus, OH, USA): 10m~ furosemide solution was made with its vehicle and stored at room temperature protected from light. Before use, this furosemide solution was diluted with distilled water to the required concentrations. Guanethidine monosulfate and acetylcholine chloride (Sigma, St. Louis, MO, USA) were dissolved in distilled water and prepared daily. Drugs were added in a volume equal to 1% of the total volume of solution in the muscle bath. Concentrations of drugs were expressed as the final bath concentration. Data analysis

Responses to EFS and ACh were calculated as a percentage of the contraction produced by 127 mM KCl. The responses to EFS at each stimulus frequency, before and after furosemide administration, were compared by means of the paired r-test. Single factor randomized design ANOVA was used for the comparison of contractions induced by ACh in furosemide groups and in the vehicle group at each ACh concentration. P < 0.05 was considered statistically significant; all values were expressed as mean f SE; n is the number of animals.

RESULTS

Thirty-eight strips with epithelium (weight, 28.25 f 1.2 mg) and 38 without epithelium (weight, 25.35 f 1.2 mg) were used in this study. In epitheliumintact tissues, the force developed in response to EFS tended to decrease over time in the presence of furosemide vehicle. Although the decrement in force was significant at 8, 16, and 32 Hz, it was small, with

Inhibition of Contractile Response to Cholinergic Nerve Stimulation by Furosemide

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Fig. 1 Effect of furosemide (B: 10 nhf, C: 1 pM and D: 100 PM) and its vehicle (A: equivalent to the amount used to dissolve 100 pM furosemide) on contractile responses of equine trachealis strips with epithelium to electrical field stimulation (15 V, 0.5 ms pulse duration, l-32 Hz). Paired t-test was used for comparison between the pre- and post-furosemide contractions. * P< 0.05, comparison was made before the vehicle and/or time effect had teen corrected. A P-C 0.05, comparison was made after the vehicle and/or time effect had been corrected. Data are shown as mean f SE, n = 7 animals. A: ( x ) pre-vehicle; (0) post-vehicle. B, C, D: ( x ) pre-furosemide; (0) post-furosemide.

the largest mean force change being 5.4% at 32 Hz (Fig. 1A). Furosemide at 10 nM and 0.1 PM did not change the contractile response to EFS, but, from 1 PM to 100 PM, furosemide shifted the frequencyresponse curve to the right in a dose-dependent manner (Figs lB, C, and D). Data on 0.1 PM and 10 PM are not shown. This curve shift was in part due to the effect of time and/or vehicle. The results obtained with furosemide were therefore corrected for this effect based on the assumption that the loss of tissue sensitivity was the same in the vehicle- and furosemidetreated tissue.13 Following correction, 100 PM furosemide still significantly reduced the force resulting from EFS at 4, 8, 16, and 32 Hz. Removal of the epithelium did not significantly influence the force developed in response to 127 mM KC1 (26.5 f 3.3 and 29.9 f 1.2 g in epithelium-on and epithelium-off tissues, respectively). In the absence of epithelium, the EFS response curve was shifted significantly downward only at 32 Hz in the tissues treated with vehicle (Fig. 2A). Furosemide (10 nM to 10 J.LM) had no effect on the EFS response curve. Although furosemide at 100 PM was associated with a shift in the EFS response (Fig. 2D), when the data were adjusted

for the effects of time and/or vehicle, 100 PM furosemide had no significant effect on the response to EFS. Furosemide had no effect on the response to ACh in tissues with or without epithelium (Fig. 3).

DISCUSSION

In the presence of epithelium, 100 PM furosemide significantly inhibited the contractile response to EFS. Our previous experiments indicated that, given the stimulation parameters of the present study, equine trachealis muscle contractions induced by EFS are due to activation of postganglionic cholinergic nerves since they were eliminated completely by either 1 JAM atropine or 1 PM tetrodotoxin but unaffected by 10 PM hexamethonium. The effects of furosemide on the response to EFS could therefore be due either to direct actions on the smooth muscle or to inhibition of release of ACh from postganglionic parasympathetic nerves or both. The fact that furosemide did not influence the response to exogenous ACh suggests that its effects are primarily on postganglionic cholinergic nerves.

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Fig. 2 Effect of furosemide (B: 10 nM, C: 1 pM and D: 100 PM) and its vehicle (A: equivalent to the amount used to furosemide) on contractile responses of equine trachealis strips without epithelium to electrical field stimulation (15 duration, l-32 Hz). Paired r-test was used for comparison between the pre- and post-furosemide contractions. * P< was made before the vehicle and/or time effect had been corrected. No significant effect of furosemide was observed and/or time effect had been corrected. Data are shown as mean f SE; n = 7 animals. Symbols as for Figure 1.

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Fig. 3 Effect of furosemide (10 nM, 1 PM and 100 pM) on acetylcholine-induced contractions of equine trachealis strips with epithelium (A) and without epithelium (B). Single factor randomized design ANOVA was used to compare contractions in the furosemide groups with that in the vehicle group. No statistically significant effect of furosemide was observed. Data are shown as mean+ SE; n = 7 animals. ( x ) Vehicle; (0) furosemide 10 nM; (A) furosemide 1 PM; (Cl) furosemide 100 PM.

Although there appeared to be a dose-dependent response to furosemide, interpretation of the data was complicated by the fact that the response to EFS was significantly inhibited in the tissues treated with a concentration of vehicle equivalent to that used to

dissolve 100 JIM furosemide. This could have been a result either of addition of vehicle or a time-dependent effect or both. It is most likely that it was a result of the vehicle, because a similar inhibition was not observed with 10 nM furosemide, which contained

Inhibition of Contractile Response to Cholinergic Nerve Stimulation by Furosemide

considerably less vehicle. The vehicle effect was apparently not mediated through the epithelium, because it was present in tissues with and without epithelium. The effect of furosemide was abolished by the removal of epithelium. This result is analogous to that obtained in vascular smooth muscle. Furosemide inhibits the vasoconstrictor response to periarterial electrical stimulation in rat tail artery with intact endothelium, but this effect is lacking in endotheliumdenuded preparations.* Airway epithelium, by release of an epithelium-derived relaxing factor, is an important modulator of smooth muscle contractile response to a variety of factors including EFS.“J4 It is possible that furosemide’s action on equine tracheal smooth muscle is therefore due to the release of a relaxant factor from airway epithelium. In agreement with the results of the present study, an inhibitory effect of furosemide (100 PM and 1 mM) on cholinergic neurotransmission but not on AChinduced contractions has also been demonstrated by Ellwood et al.15 These investigators used guinea-pig trachealis pretreated with the cyclooxygenase inhibitor indomethacin. In the presence of indomethacin, the inhibitory effects of furosemide were apparently of greater magnitude than those observed in this study and were independent of the presence of the epithelium. Because of differences in experimental design and species used, it is not possible to explain the differences in the results of the two studies. A smooth muscle relaxant effect of furosemide in equids has also been observed in vivo. Administration of furosemide (1 mg/kg) intravenously or by aerosol causes a significant decrease in lung resistance and an increase in dynamic compliance in heavey ponies during acute exacerbations of airway obstruction but not in control ponies or in affected animals during disease remission.8 The inhibitory effect of furosemide on airway smooth muscle observed in tissues from normal equids in the present study is in contrast to the lack of effect of furosemide on control ponies observed in vivo. There are two possibilities for the discrepancy between the in vivo and in vitro results. In normal ponies, there is little resting bronchomotor tone,7 so it is not surprising that furosemide cannot dilate the airways any further. In the present study, furosemide’s effect was observed when the tissues received cholinergic stimulation, but furosemide did not influence the baseline tension. This experimental condition is more like the situation in heavey ponies during acute airway obstruction. Broadstone et al have shown that the major part of airway obstruction in these ponies is mediated through cholinergic nerves.’ These in-vivo and in-vitro observations and those of Ellwood et al suggest that the effects of furosemide are most prominent when there is increased smooth muscle force as a result of cholinergic nerve activity.

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Although the effects of furosemide on the response to EFS in tissues with epithelium were significant, they were not of large magnitude and were only achieved at a high concentration of the drug (100 PM). Significant in-vivo effects occur following injection of 1 mg/kg i.v. If this dose of furosemide is uniformly distributed throughout the body, the resulting concentration of furosemide would be approximately 3 pM. Because furosemide is highly bound to plasma albuminI it is unlikely that the in-vivo tissue concentration of furosemide could reach 100 FM. Therefore, the effect of furosemide in heavey animals may not be predominantly through direct actions on airway nerves. In rat mesenteric artery, furosemide inhibits the vasoconstrictor responses to angiotensin II and noradrenaline. After bilateral nephrectomy, this effect is completely prevented.’ Furosemide facilitates the release of arachidonic acid, PGE,, PGF,, and PGI, from the renal tubules.“-*’ These substances can affect the contractile response of airway smooth muscle to a number of stimulants.*’ Furosemide may utilize similar pathways to exert its action on both airway and vascular smooth muscle. In conclusion, this experiment demonstrates that 100 PM furosemide inhibited postganglionic cholinergic neurons innervating equine trachealis. This effect requires the presence of intact epithelium. Since the significant effective dose of furosemide in vitro (100 PM) is much higher than the estimated tissue concentration of furosemide in vivo (3 PM), the direct effect of furosemide on tracheal tissues is unlikely to be the main mechanism of furosemide’s bronchodilator action. References 1. Gerkens J F, Smith A J. Inhibition of vasoconstriction by furosemide in the rat. Br J Pharmacol 1984; 83: 363-371. 2. Gerkens J F. Inhibitory effect of furosemide on sympathetic vasoconstrictor responses: dependence on a renal hormone and the vascular endothelium. Clin Exp Pharmacol Physiol 1987; 14: 371-377. 3. Lundergan C F, Fitzpatrick TM, Rose J C, Ramwell P W, Kot PA. Effect of cyclooxygenase inhibition on the pulmonary vasodilator response to furosemide. J Pharmacol Exp Ther 1988; 246: 102-106. 4. Bianco S, Vaghi A, Robuschi M, Pasargiklian M. Prevention of exercise-induced bronchoconstriction by inhaled furosemide. Lancet 1988; 2: 252-255. 5. Moscato G, Dellabianca A, Falagiani P, Mistrello G, Rossi G, Rampulla C. Inhaled furosemide prevents both the bronchoconstriction and the increase in neutrophil chemotactic activity induced by ultrasonic “fog” of distilled water in asthmatics. Am Rev Respir Dis 1991; 143: 561-566. 6. Bianco S, Pieroni M G, Refini R M, Rottoli L, Sestini P. Protective effect of inhaled furosemide on allergen-induced early and late asthmatic reactions. N Engl J Med 1989; 321: 1069-1073. 7. Broadstone R V, Scott J C, Derksen F J, Robinson N E. Effects of atropine in ponies with recurrent airway obstruction. J Appl Physiol 1988; 65: 272&2725. 8. Broadstone R V, Robinson N E, Gray P R, Woods P S A, Derksen F J. Effects of furosemide on ponies with recurrent airway obstruction. Pulmon Pharmacol 1991; 4: 2033208.

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9. Nichol GM, Alton E W F W, Nix A, Geddes D M, Chung K F, Barnes P J. Effect of inhaled furosemide on metabisulfite- and methacholine-induced bronchoconstriction and nasal potential differences in asthmatic subjects. Am Rev Respir Dis 1990; 142: 576-580. 10. O’Connor B J, Chen-Worsdell Y M, Fuller R W, Chung K F, Barnes P J. Effect of inhaled furosemide on adenosine S-monophosphate- and histamine-induced bronchoconstriction in asthmatic subjects. Thorax 1990; 45: 333p. 11. Flavahan N A, Aarhus L L, Rimele T J, Vanhoutte PM. Respiratory epithelium inhibits bronchial smooth muscle tone. J Appl Physiol 1985; 58: 834838. 12. Gray P R, Derksen F J, Robinson N E, Slocombe R F, Peters-Golden M L. Epithelial strips: an alternative technique for examining arachidonate metabolism in equine tracheal epithelium. Am J Respir Cell Mol Biol 1992; 6: 29-36. 13. Kenakin T P. Analysis of dose-response data. In: Kenakin T P, ed. Pharmacologic analysis of drug-receptor interaction. New York: Raven Press, 1987: 129-162. 14. Stuart-Smith K, Vanhoutte P M. Epithelium-derived relaxing factors. In: Agrawal D K, Townley R G, eds. Airway smooth muscle: modulation of receptors and response. Boston: CRC Press, 1990: 129-143. 15. Ellwood W, Lotvall J 0, Barnes P J, Chung K F. Loop diuretics inhibit cholinergic and noncholinergic nerves in guinea-pig airway. Am Rev Respir Dis 1991; 143: 1340-1344.

16. Boles Ponto L L, Schoenwald R D. Furosemide (frusemide): a pharmacokinetic/pharmacodynamic review (part 1). Clin Pharmacokinet 1990; 18: 38 147 1. 17. Weber PC, Scherer B, Larsson C. Increase of free arachidonic acid by furosemide in man as the cause of prostaglandin and renin release. Eur J Pharmacol 1977; 41: 3299332. 18. Williamson H E, Bourland W A, Marchand G R, Farley D B, Van Orden D E. Furosemide induced release of prostaglandin E to increase renal blood flow. Proc Sot Exp Biol Med 1975; 150: 104106. 19. Sullivan J M, Patrick D R. Release of prostaglandin I,-like activity from the rat aorta: effect of captopril, furosemide, and sodium. Prostaglandins 1981; 22: 575-583. 20. Scherer B, Weber PC. Time-dependent changes in prostaglandin excretion in response to furosemide in man. Clin Sci 1979; 56: 77-81. 21. Robinson C, Holgate ST. Regulation by prostanoids. In: Crystal R G, West J B, eds. The lung. New York: Raven Press. 1991: 941-951.

Date received: 24 July 1991 Date accepted: 15 September 1991