Cotransmitters in the motor nerves of the guinea pig vas deferens: electrophysiological evidence.

The contractile response of the guinea pig vas deferens to motor nerve stimulation is biphasic. The first phase is antagonized by the specific adenosine triphosphate-receptor antagonist arylazido aminoproprionyl adenosine triphosphate (ANAPP3), and the second by the alpha-receptor antagonist prazosin. The underlying electrical event, the excitatory junction potential, is also blocked by ANAPP3, but not by prazosin.

Purinergic receptors: photoaffinity analog of adenosine triphosphate is a specific adenosine triphosphate antagonist.

Arylazido aminopropionyl adenosine triphosphate (ANAPP3), a photoaffinity label, antagonized specifically adenine nucleotide-induced contractions of the guinea pig vas deferens. Irradiation of tissues with visible light in the presence of ANAPP3 resulted in an irreversible antagonism, which was prevented when adenosine triphosphate was present. In the absence of light, the antagonism was reversible and may have resulted from an autoinhibition phenomenon. Responses to acetylcholine, histamine, norepinephrine, and potassium chloride were not affected by ANAPP3.

Spermine decreases peripheral vascular resistance in the dog and relaxes the isolated aorta of the guinea pig.

Polyamines, found in all nucleated cells, have been shown to relax uterine, gastrointestinal, and respiratory tract smooth muscle. When parenterally administered to the rat or dog they cause hypotension. To determine the range of the relaxing effect of polyamines on smooth muscle and the mechanism of the hypotensive effect anaesthetised dogs were infused with increasing intravenous bolus doses of spermine while cardiovascular measurements were made. At doses of greater than or equal to 2 mg X kg-1 spermine a reversible hypotensive effect was produced that is accounted for by changes in peripheral vascular resistance. It is unlikely that this effect was due to the known propensity of polyamines to release histamine because the hypotensive action of 2 mg X kg-1 spermine was unaltered by pretreatment with the H1 and H2 blockers, diphenhydramine and cimetidine. Evidence that the spermine effect is not mediated by circulating agents was shown by the ability of spermine to relax the precontracted guinea pig isolated aorta. These results in vascular smooth muscle may be added to those in other types of smooth muscle shown to be relaxed by polyamines; the polyamine effect on smooth muscle may be ubiquitous. These data also raise the possibility that changing concentrations of tissue polyamines may play a role in the regulation of tissue blood flow.

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

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

Potentiation of adenosine triphosphate-induced contractile responses of the guinea-pig isolated vas deferens by adenosine monophosphate and adenosine 5'-monophosphorothioate.

The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (300 microM) concentration of ATP to obtain control responses, one vas deferens of a pair was incubated for 5 min with one of the adenine nucleotides, while the contralateral preparation was incubated with the corresponding phosphorothioate analogue. At the conclusion of the incubation the preparations were challenged again with ATP. Incubation with AMP or AMP alpha S resulted in a transient potentiation of responses to 1 microM and 300 microM ATP. The potentiation following incubation with AMP alpha S was larger than that produced by AMP. After incubation with ADP, ADP beta S, ATP and ATP gamma S, responses to 1 microM ATP were decreased, while those to 300 microM ATP were unaffected. Thus, incubation with AMP and AMP alpha S results in potentiation, rather than inhibition, of ATP-induced responses. On the other hand, 5'-diphosphate, 5'-triphosphate, 5'-O-(2-thiodiphosphate) and 5'-O-(3-thiotriphosphate) moieties on adenosine have no effect or cause autoinhibition. These results indicate that AMP exerts a potentiating effect on reactivity to exogenous ATP. AMP arising from the enzymatic degradation of ATP might modulate the level of response to ATP released endogenously as a cotransmitter.

Modulation of the reactivity of the guinea-pig isolated trachealis by respiratory epithelium: effects of cooling.

1. Examination has been made of the effects of epithelium removal on the reactivity of guinea-pig trachealis to methacholine at 37 degrees C and 22 degrees C, and on responses to activation of the Na+/K(+)-pump by abrupt temperature increase from 22 degrees C to 37 degrees C. 2. At 37 degrees C, epithelium removal increased the sensitivity of isolated tracheal strips to methacholine without affecting the maximum isometric contractile response. Epithelium removal resulted at 22 degrees C in a decrease in sensitivity to methacholine, i.e. an effect opposite to that seen at 37 degrees C. While the maximum response of intact strips to methacholine was enhanced at 22 degrees C, the maximum response of denuded preparations was decreased. 3. The increase in sensitivity to methacholine at 37 degrees C after epithelium removal was mimicked in intact preparations by indomethacin (1 microM). Indomethacin did not mimic the decrease in methacholine sensitivity and maximum response caused by epithelium removal at 22 degrees C. 4. Following incubation at 22 degrees C, abrupt increase in temperature to 37 degrees C elicited relaxation in both epithelium-containing and epithelium-denuded tracheal strips. In epithelium-containing preparations the relaxation was more pronounced and followed by contraction. Ouabain (1 microM) converted the relaxation of denuded preparations to contraction, but was ineffective in intact strips. The relaxation of intact strips was, however, inhibited by a greater ouabain concentration (10 microM). 5. These findings indicate that the modulatory effect of the epithelium is temperature-dependent. In cooled preparations, the epithelium enhances reactivity. At 37 degrees C, an epithelium-derived factor reduces reactivity, and this may partially be due to activation of the electrogenic Na+/K+-pump.

The effects of alterations in electrogenic Na+/K+ -pumping in guinea-pig isolated trachealis: their modulation by the epithelium.

1 An examination was made of the effect of epithelium removal on mechanical responses of guinea-pig isolated tracheal strips after inhibition or activation of electrogenic Na+/K+ -pumping. 2 The Na+/K+ -pump inhibitor ouabain (0.1-10 microM) evoked concentration-dependent contractions which were potentiated by epithelium removal. 3 K+-free solution, which inhibits Na+/K+-pumping, produced a slow, sustained relaxation in intact preparations. In epithelium-free preparations the relaxation was transient and of lesser magnitude. 4 The addition of K+ (10 or 30 mM), which activates Na+/K+-pumping, to preparations bathed in K+-free solution caused a relaxation of preparations under spontaneous tone or contracted with methacholine; the magnitude and duration of relaxation was greater in the epithelium-free preparations. Ouabain (0.1 microM) attenuated the relaxation to K+ in intact preparations and converted the response of epithelium-free preparations to a contraction. In the presence of a higher concentration of ouabain (1 microM), intact preparations contracted in response to K+. 5 In normal K+ solution, ouabain (0.1 microM) increased the sensitivity of intact preparations to methacholine but reduced their sensitivity to K+. Ouabain was without these effects in epithelium-free preparations. 6 Thus, responses of intact preparations to perturbations which affect electrogenic Na+/K+-pumping in trachealis are influenced by an epithelium-derived factor. The production of the factor may be linked to an epithelial Na+/K+-pump, or the factor may modulate the activity of an electrogenic Na+/K+-pump in the muscle.

Tetrodotoxin-sensitive, K+-induced relaxation of guinea-pig isolated trachealis in the presence of Ca2+-entry blocking drugs, Ca2+-free solution and after polyamine exposure.

We have previously observed a paradoxical relaxant effect of K+ on guinea-pig isolated trachealis after exposure to polyamines. The purpose of the present study was to evaluate whether the relaxation involved a reduction in the entry of extracellular Ca2+. We therefore investigated the effect of K+ in the presence of Ca2+-entry blocking drugs and in the presence of Ca2+-free solution. In the presence of nifedipine (10(-5) M), verapamil (10(-5) M) or diltiazem (10(-5) M), K+ (30 mM) induced relaxation of the trachealis muscle. The relaxation to K+ was not blocked by ouabain (10(-6) M), propranolol (10(-6) M), or indomethacin (10(-6) M). A relaxation in response to K+ was also observed in Ca2+-free solution, (with tone induced by methacholine), an effect not blocked by propranolol or ouabain. Tetraethylammonium (30 mM) (TEA), which ordinarily evokes contractile responses, induced trachealis relaxation in the presence of verapamil or nifedipine. The relaxation was unaltered by ouabain or propranolol. Tetrodotoxin (10(-6) M) (TTX) blocked 65% of the K+-induced relaxation in the presence of nifedipine and 100% of K+-induced relaxation either in a Ca2+-free solution or after polyamine exposure. TTX was without effect on TEA-induced relaxation after Ca2+-entry blocking drugs. Atropine (10(-6) M) or hexamethonium (10(-6) M) did not affect K+-induced relaxation after polyamine exposure. The concentration-response curve for K+-induced contraction in normal modified Krebs-Henseleit solution was shifted to the left by TTX. 8 It is concluded: (a) K+ has a direct effect on the trachealis causing contraction and an indirect effect, mediated by neurotransmitter release, causing relaxation. This latter effect is exposed when the direct effect is inhibited by Ca2+-entry blocking drugs, Ca2+-free solution or polyamine exposure; the indirect effect is non-adrenergic, non-cholinergic and not via ganglionic transmission; (b) the TEAinduced relaxation and a component of the K+-induced relaxation after Ca2+ blocking drugs cannot be explained by neurotransmitter release; (c) polyamines may act as naturally occurring Ca2+ antagonists.

The effects of epithelium removal on the sensitivity of guinea-pig isolated trachealis to bronchodilator drugs.

Mechanical removal of the epithelium increased the sensitivity of tracheal strips to isoprenaline, sodium nitroprusside, and to adenosine (only in the presence of inhibitors of its uptake and metabolism). Epithelium removal was without effect on sensitivity to salbutamol or papaverine. Preincubation of tracheal strips with an inhibitor of extraneuronal uptake, corticosterone (50 microM), had no effect on tissue sensitivity to either salbutamol or papaverine. However, the steroid both increased sensitivity to isoprenaline, and abolished the effect of epithelium removal on sensitivity to this catecholamine. These results suggest that in the guinea-pig, the tracheal epithelium is a major source of extraneuronal uptake for catecholamines. Furthermore, the increase in trachealis sensitivity to isoprenaline following epithelium removal is probably due to loss of these sites of extraneuronal uptake. The fact that sensitivity to salbutamol, papaverine and adenosine (in the absence of metabolic inhibitors) was not increased by denuding the epithelium indicates that loss of a diffusion barrier to drugs is not the mechanism of increased sensitivity. Adenosine (and possibly nitroprusside) may cause the epithelium to release a smooth muscle excitatory factor. Thus, removal of the epithelium attenuates this excitatory influence and enhances smooth muscle responsiveness to adenosine. These results provide further evidence that the epithelium has an important role in modulating the sensitivity of guinea-pig trachealis to drugs.

The effect of ouabain on tension in isolated respiratory tract smooth muscle of humans and other species.

1. The Na+,K+-pump has been implicated in animal models of airway hyperreactivity. We examined the effects of inhibiting the Na+,K+-pump and Na+,Ca2+-exchange on isometric tone of isolated trachealis from humans and other species. 2. In preparations from 5 out of 9 humans, strong spontaneous contractions (36-48 h-1; up to 1.8 g) developed within 25 min. 3. Ouabain (10(-7)-10(-5) M) caused an immediate and sustained contraction. This response was not blocked by atropine, diphenhydramine, or cimetidine. 4. Contractions were also elicited when the normal physiological solution was changed to a K+-free solution, a procedure which inhibits the Na+,K+-pump, and in reduced (15 mM) Na+ solution, which inhibits Na+,Ca2+ exchange. 5. In preparations of dog and guinea-pig isolated trachea, ouabain (10(-5) M) caused a multiphasic response; in the rabbit, ouabain was without effect. K+-free solution was without effect in the dog preparations and produced relaxation of the guinea-pig trachea. Guinea-pig tracheae responded to a low Na+ solution with a strong contraction. 6. Our findings indicate that: (a) human airway smooth muscle may be a spontaneously contracting muscle, at least in vitro, (b) a prolonged contraction to ouabain is unique for the human airway smooth muscle among the animals tested, as is the contraction in a K+-free medium, and (c) the contractile response does not involve acetylcholine or histamine release, but may involve a Na+,Ca2+-exchange mechanism. These results suggest that the level of Na+,K+-pump activity could play a role in determining the degree of bronchomotor tone in humans.

Relaxation of guinea-pig tracheal smooth muscle to arachidonate is converted to contraction following epithelium removal.

1 The effect of epithelium removal on responses of guinea-pig isolated trachealis to sodium arachidonate has been examined. 2 Arachidonate (100 microM) caused relaxation of epithelium-intact preparations, but following epithelium removal, the response to arachidonate was converted to contraction. In the presence of indomethacin (1 microM), arachidonate caused contraction in intact and denuded trachea. 3 Arachidonate also produced concentration-dependent effects, the qualitative nature of which varied with the presence or absence of the epithelium. In the presence of indomethacin, tracheal strips contracted in a concentration-dependent manner whether or not the epithelium had been removed. 4 Nordihydroguaiaretic acid (NDGA; 10 microM) markedly inhibited the contractile response of denuded strips to arachidonate. In intact tissues this lipoxygenase inhibitor converted the arachidonate-induced relaxation to a concentration-dependent contraction. The contraction to arachidonate, in the presence of NDGA, was epithelium-dependent. In the presence of both indomethacin and NDGA, responses to arachidonate were abolished. 5 It is concluded that the relaxation of guinea-pig trachea to arachidonic acid is epithelium-dependent and is mediated by an inhibitory product of the cyclo-oxygenase metabolic pathway. The contraction in denuded trachea, and trachea in the presence of indomethacin, may be mediated by lipoxygenase products of arachidonic acid metabolism, i.e. peptidoleukotrienes. The mediator of the epithelium-dependent contraction in NDGA-treated tissues is unknown.

Differential effects of epithelium removal on the responsiveness of guinea-pig tracheal smooth muscle to bronchoconstrictors.

1 The influence of the epithelium on contractions produced by the peptidoleukotrienes, 5-hydroxytryptamine (5-HT) and the thromboxane mimetic, U-44069, was examined in trachea from control and ovalbumin-sensitized guinea-pigs. 2 In control tissues removal of the epithelium produced an approximately 2 to 4 fold leftward shift in leukotriene C4 (LTC4) and LTD4 concentration-response curves, but no effect on LTE4-induced contractions. Similar results were obtained in preparations from ovalbumin-sensitized animals. 3 Responses produced by 5-HT or U-44069 were similar in the presence and absence of the epithelium in control guinea-pigs. 4 Indomethacin produced contrasting effects on leukotriene-induced contractions in control guinea-pigs: an increase in sensitivity to LTC4 in the presence but not absence of the epithelium, no effect on LTD4-induced contractions and a decrease in sensitivity to LTE4 in both epithelium-containing and epithelium-free preparations. 5 These results indicate that there is selectivity in the effects of epithelium removal on agonist-induced contractions of the guinea-pig trachea. This provides further evidence for the modulatory influence of the epithelium on the reactivity of mammalian airway smooth muscle and supports the postulated existence of an epithelium-derived inhibitory factor. The observation that in intact trachea indomethacin mimics the effects of epithelium removal on LTC4-induced responses, suggests the involvement of a prostanoid(s) in this phenomenon.

An overview of species differences in the effects of a water extract of cotton bract on isolated airway smooth muscle, and effects of E. coli lipopolysaccharide.

Our laboratory has been comparing the activity of a water extract of cotton bract (CBE) with the isolated trachealis smooth muscle of the dog, guinea pig, and cat. CBE induced contractions that were not mediated by 5-hydroxytryptamine (5-HT), histamine, or muscarinic receptors. The active agent(s) in CBE was dialyzable (less than 14,000 molecular weight), and substantial activity was retained after low-temperature ashing. CBE potentiated contractions of dog trachealis to histamine and 5-HT and relaxation responses to isoproterenol, whereas it had no effect on responses to methacholine and KCl. In the guinea pig trachealis, CBE reduced responsiveness to KCl, potentiated relaxations to adenosine and ATP, and did not alter the responses to the remaining agents. Responses of cat trachealis to KCl and isoproterenol were potentiated by CBE, while those to 5-HT were unaffected. Neurogenic cholinergic contractile responses were potentiated by CBE in the trachealis of the dog, but not of the guinea pig, while neurogenic relaxations were potentiated by CBE in guinea pig trachealis but not in the dog trachealis. There are thus marked species differences in the acute effects of CBE on airway smooth muscle. Due to recent interest in the possible involvement of bacterial endotoxins in the etiology of byssinosis, we examined the effects of E. coli lipopolysaccharide (LPS) in guinea pig trachealis. An initial examination revealed that LPS potentiated responses to histamine, but not those to methacholine and isoproterenol. This effect vanished upon a second appraisal with a different batch of LPS. The effect of LPS in airway smooth muscle is thus, at present, equivocal.

Role of intrinsic airway neurons in ozone-induced airway hyperresponsiveness in ferret trachea.

Exposure to ozone (O(3)) enhances airway responsiveness, which is mediated partly by the release of substance P (SP) from airway neurons. In this study, the role of intrinsic airway neurons in O(3)-induced airway responses was examined. Ferrets were exposed to 2 ppm O(3) or air for 1 h. Reactivity of isolated tracheal smooth muscle to cholinergic agonists was significantly increased after O(3) exposure, as were contractions to electrical field stimulation at 10 Hz. Pretreatment with CP-99994, a neurokinin type 1 receptor antagonist, partially abolished the O(3)-induced reactivity to cholinergic agonists and electrical field stimulation. The O(3)-enhanced airway responses were present in tracheal segments cultured for 24 h, a procedure shown to deplete sensory nerves while maintaining viability of intrinsic airway neurons, and all the enhanced smooth muscle responses were also diminished by CP-99994. Immunocytochemistry showed that the percentage of SP-containing neurons in longitudinal trunk and the percentage of neurons innervated by SP-positive nerve fibers in superficial muscular plexus were significantly increased at 1 h after exposure to O(3). These results suggest that enhanced SP levels in airway ganglia contribute to O(3)-induced airway hyperresponsiveness.

Potentiating and inhibitory effects of periodate-oxidized ATP analogs on contractions of vas deferens to ATP.

Previous studies have shown that treatment of guinea-pig isolated vas deferens with the affinity label periodate-oxidized ATP (2',3'-dialdehyde ATP), results in two irreversible effects on biphasic contractile responses to ATP, i.e., potentiation of the P2X purinoceptor-mediated first phase and inhibition of the ecto-kinase-mediated second phase. The present experiments were designed to evaluate whether periodate-oxidized ADP, periodate-oxidized AMP, and periodate-oxidized adenosine, produce similar effects. Periodate-oxidized ATP and periodate-oxidized ADP (10(-2) M) elicited contraction of the vas deferens (periodate-oxidized ATP > periodate-oxidized ADP; periodate-oxidized AMP and periodate-oxidized adenosine had no agonist activity. After incubation of the preparations for 5 min with 10(-2) M periodate-oxidized ATP, periodate-oxidized ADP, periodate-oxidized AMP or periodate-oxidized adenosine, the first phase of contraction to submaximal ATP concentrations was potentiated. Simultaneously, periodate-oxidized ATP, periodate-oxidized ADP and periodate-oxidized AMP inhibited the second contractile phase, whereas periodate-oxidized adenosine did not. The results indicate that the requirement for 5'-phosphate to produce potentiation and inhibition is different: 5'-phosphate is not needed to potentiate the first phase of contraction to ATP, but at least one 5'-phosphate is required to inhibit the second phase of contraction.

The effect of pretreatment with 6-hydroxydopamine on the norepinephrine concentration and sensitivity of the rat vas deferens.

Injection of 6-hydroxydopamine via the dorsal vein of the penis results in a marked depletion of the endogenous norepinephrine of the vas deferens. Seven days after pretreatment with 6-hydroxydopamine there is a shift to the left and increase in maxima of the dose-response curves for norepinephrine and methoxamine. The results indicate that pretreatment with 6-hydroxydopamine produces denervation of the vas deferens and that the in vitro tissue exhibits both prejunctional and postjunctional supersensitivity.

Regional differences in reactivity and in the influence of the epithelium on canine intrapulmonary bronchial smooth muscle responsiveness.

Differences in the reactivity and in the influence of the epithelium on responsiveness of canine 2nd and 3rd generation airway smooth muscle were examined. Epithelium-containing 3rd generation airways produced a greater maximum contraction and were more sensitive to methacholine and histamine, but not to KCl, than corresponding 2nd generation airways. Mechanical removal of the epithelium increased the sensitivity to methacholine and histamine in 2nd generation airways; there was also an increase in the maximum response elicited by histamine, but not by methacholine, in epithelium-free preparations. In contrast, there was no significant difference in the sensitivity to or the maximum response elicited by histamine or methacholine in epithelium-containing and epithelium-free 3rd generation airways. Epithelium removal had no effect on KCl-induced responses in either airway region. The inhibitory effects of verapamil (1 microM) against KCl- and methacholine-induced responses were identical in preparations containing and lacking the epithelium. The results support the postulate of an epithelium-derived inhibitory factor modulating airway smooth muscle reactivity. Furthermore, the influence of the epithelium exhibits regional differences, being greater in larger airways.

Polyamines and putreanine relax respiratory tract smooth muscle in the guinea-pig.

Spermidine, spermine, and putreanine, a metabolite of spermidine, caused transient relaxation of the isolated guinea-pig trachealis. The spermidine effect was not blocked by ouabain, propranolol or indomethacin. Spermidine treatment was shown to desensitize the tissue to subsequent additions of spermidine. After exposure to spermidine and repeated washes with spermidine-free solution, trachealis responded to KCl (30 mM) with a paradoxical relaxation. This relaxation was not blocked by propranolol, ouabain or indomethacin.

Investigation of relaxations of the rabbit anococcygeus muscle by nerve stimulation and ATP using the ATP antagonist ANAPP3.

When tone was raised by histamine (10(-6) M), field stimulation (0.2-8 Hz) induced relaxation of the rabbit anococcygeus muscle in the continuous presence of guanethidine (10(-5) M) and atropine (10(-6) M). Similar relaxations could be induced by ATP and adenosine, which were approximately equipotent, but the non-hydrolyzable analogue beta-gamma-methylene ATP was less potent and produced relaxations which were slower. Although PGE2 was a potent relaxant in this muscle, release of endogenous prostaglandins does not appear to mediate the response to ATP since indomethacin (2 x 10(-5) M) pretreatment did not reduce responses to ATP. The specific ATP receptor antagonist, ANAPP3 (10(-4) or 10(-3) M) did not reduce responses to nerve stimulation and only slightly reduced those of exogenous ATP. The results indicate that responses to ATP could be mediated partly by the products of its hydrolysis and do not support the proposal that ATP is the inhibitory transmitter in this muscle.

Reactivity of guinea-pig isolated trachea to methacholine, histamine and isoproterenol applied serosally versus mucosally.

Guinea-pig tracheas were perfused with recirculating modified Krebs-Henseleit solution while monitoring changes in inflow-outflow pressure difference, which is an index of trachealis muscle tone. The reactivities of the trachealis muscle to methacholine, histamine and isoproterenol applied separately to the mucosal (intraluminal, IL) or serosal (extraluminal, EL) compartments were compared, and evidence for the agonist-induced release of epithelium-derived relaxing factor (EpDRF) was sought. All agents were more potent when added to the EL compartment, but the IL/EL EC50 ratios were different: 100 for methacholine, 41 for histamine and 25 for isoproterenol. Methacholine or histamine added to the IL compartment, after the preparations were pre-contracted with the same concentration of the agonist or 30 mM KCl added EL, did not result in relaxation. Likewise, IL isoproterenol did not evoke contraction. IL KCl evoked relaxation. The results indicate that the epithelium reduces access of bronchoactive agents to the muscle, while an immediate relaxant effect of EpDRF released by agonists could not be demonstrated.