Gunst, Susan J.

Muscle strips loaded with the Ca2+ indicator aequorin were studied in vitro to determine the effects of inhibitory stimuli on force and cytosolic free Ca2+. In muscles contracted isometrically with acetylcholine (ACh), 5-hydroxytryptamine (5-HT), carbachol, decreases in muscle force caused by isoproterenol (10(-5) M) or forskolin (10(-5) M) were accompanied by proportional decreases in aequorin luminescence. A similar relationship between decreases in muscle force and aequorin luminescence was observed when muscles were relaxed by stimulating Na+-K+-ATPase activity. These results suggest that the Ca2+ sensitivity of contractile proteins was not decreased during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent relaxation. However, aequorin luminescence did not decrease when muscles contracted by K+ depolarization were relaxed with isoproterenol. Incubation of muscles in forskolin depressed increases in both force and aequorin luminescence in response to 5-HT or ACh. Incubation of muscles in isoproterenol had a similar effect on responses to 5-HT but depressed increases in force without depressing increases in luminescence in response to ACh. Results indicate that under most conditions the reduction of cytosolic Ca2+ plays an important role in the cAMP-dependent relaxation of canine tracheal smooth muscle.

Canine tracheal smooth muscle strips were loaded with the bioluminescent Ca2+ indicator aequorin, mounted in a tissue bath, and attached to an electromagnetic lever to investigate the effect of changes in muscle length and load on cytosolic free Ca2+. The intracellular Ca2+ concentration ([Ca2+]i) was inversely correlated with muscle length and active force developed during isometric contractions elicited by electrical field stimulation. In addition, quick release to either constant length or constant load at any time point during an active contraction resulted in an increase in [Ca2+]i. These observations are consistent with the possibility that the binding of Ca2+ to contractile or regulatory proteins is decreased when muscle length or active force development is decreased, resulting in the release of bound Ca2+ into the cytoplasm. However, the possibility that changes in muscle length or load affect other mechanisms that regulate [Ca2+]i cannot be excluded.

Prejunctional and postjunctional muscarinic receptor subtypes were characterized in canine trachealis muscle strips. In vitro contractile responses of muscle strips to acetylcholine or electric field stimulation were determined in the absence and the presence of gallamine, pirenzepine, and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP). Gallamine had no effect on the contractile response to acetylcholine but enhanced the contractile response to electric field stimulation. Pirenzepine and 4-DAMP reduced the contractile response to acetylcholine and electric field stimulation. The pA2 value for pirenzepine vs. acetylcholine [7.18 +/- 0.59 (SD)] was consistent with the affinity of pirenzepine for M2 or M3-receptors; whereas the pA2 value for 4-DAMP vs. acetylcholine (8.92 +/- 0.42) and the extremely low affinity of gallamine indicated postjunctional muscarinic receptors of the M3 subtype. The enhancement of the contractile response to electric field stimulation by gallamine suggested the presence of M2-prejunctional receptors.

Intraparenchymal canine airway segments with branches tied off were mounted between two fluid-filled cannulas in an organ chamber. Airways were inflated to successive volumes ranging from 4 to 100% of the segment volume at 25 cmH2O. At each volume, pressure was monitored during isovolumetric contractions elicited by 10(-3) M acetylcholine. Small bronchi developed pressures greater than 30 cmH2O in response to acetylcholine at all volumes and were able to constrict to closure. Large bronchi developed pressures greater than 30 cmH2O only near maximal volumes and were able to constrict to only 30% of maximal volume. Maximal active pressures occurred at low volumes in small bronchi and at high volumes in large bronchi. However, maximal active circumferential tension and stress occurred at near-maximal volumes in both large and small bronchi. Circumferential length active-stress curves and maximal active-stress development for bronchi and trachealis muscle strips were similar. Similar length active-stress properties in different bronchi may produce significant differences in volume-pressure characteristics.

The effect of Na-K adenosinetriphosphatase (ATPase) on relaxation induced by isoproterenol, prostaglandin E2, sodium nitroprusside, and forskolin, a specific stimulant of adenylate cyclase, was investigated in canine tracheal smooth muscle strips. Relaxation in response to isoproterenol, prostaglandin E2, and forskolin was significantly decreased after inhibition of the Na-K ATPase by ouabain or a potassium-free medium, but relaxation to sodium nitroprusside was not affected. Relaxation to isoproterenol was greater in muscles contracted by 5-hydroxytryptamine than in those contracted by acetylcholine. The stimulation of Na-K ATPase activity with potassium also caused differences in relaxation between tissues contracted with 5-hydroxytryptamine or acetylcholine. Relaxation caused by isoproterenol by activation of the Na-K-ATPase was also decreased by the Ca2+-channel antagonists, verapamil and diltiazem. The results suggest 1) Na-K ATPase activity modulates relaxation caused by isoproterenol, prostaglandin E2, and forskolin in canine tracheal smooth muscle, 2) isoproterenol or activation of the Na-K ATPase may cause relaxation partly by reducing Ca2+ influx through potential-dependent Ca2+ channels, and 3) the differences in the inhibitory effects of isoproterenol and Na-K ATPase activity on muscles contracted by acetylcholine and 5-hydroxytryptamine could be due to differences between these contractile agents in their dependence on extracellular Ca2+ for activation.

In normal humans and dogs, the airways do not constrict to closure even when maximally stimulated. However, airway closure can be produced in isolated canine lobes and bronchial segments that are stimulated with maximal concentrations of bronchoconstrictors. These observations suggest that under normal conditions, physiological mechanisms to limit bronchoconstriction exist in vivo. In this investigation, we evaluated how mechanical factors that influence airway smooth muscle contractility contribute to the modulation of the pressure-volume characteristics of contracted canine intraparenchymal airways in vitro. Our results demonstrated that maximal and even submaximal contractile stimuli can produce airway closure in bronchi that are allowed to contract under isobaric conditions. However, the effectiveness of bronchoconstrictors is significantly reduced when the airways are subjected to tidal volume oscillations during contraction. In addition, airways contracted isovolumetrically at low volumes exhibit a markedly reduced sensitivity to submaximal concentrations of acetylcholine. This may limit bronchoconstriction at low lung volumes and transpulmonary pressures where the effectiveness of parenchymal stress in keeping the airways open is reduced. Together these factors could provide a mechanism by which bronchoconstriction is limited to low levels of airway resistance under normal conditions in vivo.

Tachyphylaxis to aerosolized histamine was studied in dogs anesthetized with thiamylal after pretreatment with prostaglandin synthesis inhibitors. Three consecutive histamine dose-response curves were obtained in nine dogs pretreated with 5 mg/kg indomethacin; two of these nine were also pretreated with 10 mg/kg indomethacin. Seven of the nine dogs were pretreated with 4 mg/kg sodium meclofenamate; four of these seven were also pretreated with 12 mg/kg. All dogs had tachyphylaxis at high concentrations of histamine regardless of inhibitor used. Pretreatment with indomethacin while the dogs were under alpha-chloralose-urethan anesthesia gave similar results. Histamine tachyphylaxis was also studied both in the presence and in the absence of indomethacin in tracheal smooth muscle strips obtained from seven additional dogs. A decrease in the median effective dose to histamine was observed in the indomethacin-treated strips, but tachyphylaxis to histamine remained. We conclude that prostaglandin synthesis inhibition does not reverse histamine tachyphylaxis either in vivo or in vitro. Thus the mechanism of histamine tachyphylaxis remains unexplained.

The objective of this investigation was to determine the minimum transpulmonary pressure (PL) at which the forces of interdependence between the airways and the lung parenchyma can prevent airway closure in response to maximal stimulation of the airways in excised canine lobes. We first present an analysis of the relationship between PL and the transmural pressure (Ptm) that airway smooth muscle must generate to close the airways. This analysis predicts that airway closure can occur at PL less than or equal to 10 cmH2O with maximal airway stimulation. We tested this prediction in eight excised canine lobes by nebulizing 50% methacholine into the airways while the lobe was held at constant PL values ranging from 25 to 5 cmH2O. Airway closure was assessed by comparing changes in alveolar pressure (measured by an alveolar capsule technique) and pressure at the airway opening during low-amplitude oscillations in lobar volume. Airway closure occurred in two of the eight lobes at PL = 10 cmH2O; in an additional five it occurred at PL = 7.5 cmH2O. We conclude that the forces of parenchymal interdependence per se are not sufficient to prevent airway closure at PL less than or equal to 7.5 cmH2O in excised canine lobes.

The possibility that differences in beta-adrenergic sensitivity among canine trachealis muscles contracted with different contractile agonists are related to differences in the receptor-occupancy characteristics of the contractile agonists was investigated. Relaxation to isoproterenol was compared in muscles contracted with the muscarinic agonists McN-A-343 and acetylcholine (ACh). The apparent dissociation constant (pKB) values for the M1-antagonist, pirenzepine, against ACh (6.96 +/- 0.18) and McN-A-343 (6.84 +/- 0.08) were similar. The pKB values for the M3-antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) against ACh (8.76 +/- 0.13) and McN-A-343 (8.71 +/- 0.10) were also similar, suggesting that these agonists were activating the same subtype of muscarinic receptor, probably M3. However, the contractile response to ACh was associated with a greater receptor reserve than that for McN-A-343. Isoproterenol relaxed muscles contracted with McN-A-343 much more effectively than those contracted with an equieffective concentration of ACh. The results suggest that the relative resistance of ACh-induced contractions to relaxation by isoproterenol may not be an inherent quality of muscarinic receptor stimulation. The large receptor reserve available to ACh may act to buffer the contractile response from the inhibitory effects of beta-adrenergic stimulation. Alternatively, ACh may be able to initiate subcellular mechanisms that are unavailable to agonists of lower efficacy.

Concentration-response curves for norepinephrine, acetylcholine, and 5-hydroxytryptamine were obtained in vitro alone and after precontraction with histamine, 5-hydroxytryptamine, or acetylcholine. Responses obtained to each agonist after precontraction were greater than responses to the agonist alone after subtraction of the force due to the precontracting stimulus. Augmentation of responses after precontraction was the greatest for norepinephrine, less for 5-hydroxytryptamine, and least for acetylcholine. Verapamil had no significant effect on the augmentation of responses to either 5-hydroxytryptamine or acetylcholine caused by precontraction. When the efficacy of acetylcholine was decreased by receptor alkylation with phenoxybenzamine, the augmentation of responses to acetylcholine caused by precontraction with histamine was significantly enhanced. Differences in the magnitude of the effect of precontraction on responses to different agonists may reflect differences in their efficiency of stimulus-response coupling in canine tracheal smooth muscle, or they may result from an increased expression of distinct receptors or receptor-mediated effects uncovered by the facilitory stimuli.