Jeffrey S. Fedan

Contribution by purines to the neurogenic response of the vas deferens of the guinea pig

The specific purine-receptor antagonist, arylazido aminopropionyl ATP (ANAPP3), was used to determine if purines released from nerves of the guinea-pig was deferens contribute to the neurogenic response. ANAPP3, which is a photoaffinity label, antagonized contractile responses of the in vitro vas deferens to transmural stimulation, reversibly in the presence of the compound and irreversibly after its photoactivation in the presence of the tissues. The antagonism by ANAPP3 was augmented by depletion of norepinephrine produced by reserpine pretreatment. Responses of untreated tissues were only slightly antagonized by the alpha-adrenoceptor blocker prazosin. However, neurogenic responses were markedly reduced in the combined presence of ANAPP3 and prazosin. ANAPP3 did not affect the release of tritium from tissues prelabeled with [3H]norepinephrine. The initial phasic component of the neurogenic response was preferentially antagonized by ANAPP3 whereas the secondary more tonic component of the response was preferentially antagonized by prazosin and reserpine pretreatment. Small, residual responses remaining after chemical sympathectomy produced by 6-hydroxydopamine pretreatment were potentiated, rather than inhibited, by ANAPP3 and were, unlike untreated tissues, sensitive to atropine. These and previous findings indicate that ATP or a related purine, originating from adrenergic neurons, acts as a co-transmitter with norepinephrine in this tissue.

Airway epithelium modulates the reactivity of guinea-pig respiratory smooth muscle.

The influence of the epithelium on the reactivity of guinea-pig tracheal smooth muscle in vitro was investigated. Mechanical removal of the epithelium produced contrasting effects on methacholine-, histamine- and K+-induced contractions of tracheal strips. Epithelium removal resulted in 2.1-fold shifts to the left of histamine and methacholine concentration-response curves, and an increase in the maximum response to histamine; the maximum response to methacholine was unaffected. Epithelium removal had little effect on the sensitivity, but decreased the maximum response to K+. The results suggest that inhibitory and excitatory factor(s) are released from epithelial cells, and that these factors modulate the reactivity of the smooth muscle. Indomethacin (1 microM) produced in epithelium-containing preparations qualitatively identical effects on the sensitivity to methacholine and on the maximum response to histamine as removal of the epithelium. However, indomethacin was without effect on the sensitivity to histamine in the presence or absence of the epithelium. Alterations in the production or release of epithelial cell-derived factors may contribute to the airway hyperreactivity observed in respiratory disorders.

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

1 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. 2 Preincubation of tracheal strips with an inhibitor of extraneuronal uptake, corticosterone (50 μM), 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. 3 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. 4 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. 5 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. 6 These results provide further evidence that the epithelium has an important role in modulating the sensitivity of guinea‐pig trachealis to drugs.

Epithelium removal increases the reactivity of human isolated tracheal muscle to methacholine and reduces the effect of verapamil.

Removal of the epithelium increased the sensitivity of human isolated tracheal smooth muscle to methacholine, producing a greater than 2-fold leftward shift in the concentration-response curve. Concomitantly, the ability of verpamil to lower the maximum contractile response was reduced in tissues without an intact epithelium. These findings suggest a role for the epithelium in modulating the reactivity of human and extend the findings of similar studies in experimental animals.

Evidence for a contribution by purines to the neurogenic response of the guinea-pig urinary bladder

In order to determine if ATP contributes as an excitatory transmitter in the guinea-pig bladder, experiments were conducted with ANAPP3, a photoaffinity analogue of ATP, which is an antagonist of adenine nucleotides in several other smooth muscles. With or without photoactivation with visible light, ANAPP3 antagonized contractile responses of in vitro strips of bladder to exogenous ATP. The antagonism was specific in that responses to acetylcholine and KCl were not affected by ANAPP3. Responses of strips of bladder to transmural electrical stimulation were not antagonized by ANAPP3 and were relatively insensitive to atropine. However, combined treatment with ANAPP3 and atropine produced a marked antagonism of the neurogenic response. In experiments with bladders obtained from animals pretreated with 6-hydroxydopamine, the ANAPP3-sensitive component of the neurogenic response was absent. These results suggest that acetylcholine, released from cholinergic nerves, and a purine, released from 6-hydroxy-dopamine-sensitive nerves, are both involved in motor transmission in this tissue.

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.

A pharmacological investigation of the biphasic nature of the contractile response of rabbit and rat vas deferens to field stimulation.

It has been demonstrated previously with the vas deferens of the guinea-pig that the first and second phases of the contractile response to motor nerve stimulation are preferentially antagonized by the P2-purinoceptor antagonist arylazido aminopropionyl ATP (ANAPP3), and the alpha 1-adrenoceptor antagonist prazosin, respectively. We have now investigated the effect of the two antagonists on the biphasic contraction in the vas deferens of two other species; rabbit and rat. ANAPP3, in a concentration which antagonized responses to exogenously applied ATP but not those to exogenous norepinephrine, preferentially reduced the initial phasic response of the rabbit vas deferens to motor nerve stimulation without significantly reducing the secondary, tonic phase of the response. Prazosin had the opposite effect; antagonizing the response to norepinephrine but not to ATP and reducing the tonic response to motor nerve stimulation without significantly reducing the initial phasic response. Results obtained with the rat vas deferens were similar. The present results combined with previous findings suggest that ATP and norepinephrine act as cotransmitters in the vas deferens of several species.

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 μm) caused relaxation of epithelium‐intact preparations, but following epithelium removal, the response to arachidonate was converted to contraction. In the presence of indomethacin (1 μm), 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; 11 μm) 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.

Epithelium modulates the reactivity of ovalbumin-sensitized guinea-pig airway smooth muscle

Mechanical removal of the airway epithelium alters the in vitro reactivity of airway smooth muscle. The modulation of reactivity may involve the release of inhibitory and excitatory factors from epithelial cells. Guinea pigs sensitized with ovalbumin have been used as an animal model of airway hyperreactivity. We evaluated the influence of the epithelium on the reactivity of in vitro tracheal smooth muscle from control and ovalbumin-sensitized guinea pigs, and the extent to which the presence of the epithelium affects the contractile response to in vitro challenge with ovalbumin. In both control and ovalbumin-sensitized tissues, epithelium removal increased the sensitivity of the preparations to histamine, methacholine and isoproterenol to a similar extent, i.e., 2- to 2.5-fold. Epithelium removal resulted in an 8.1-fold increase in sensitivity to ovalbumin in sensitized tissues. The epithelium appears not only to modulate the reactivity of the tissues to bronchoactive agents, but it also influences the magnitude of the contractile response following antigen challenge.

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.